(12) Patent Application Publication (10) Pub. No.: US 2010/0247552 A1 Tonegawa Et Al

US 2010O247552A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0247552 A1 Tonegawa et al. (43) Pub. Date: Sep. 30, 2010

(54) PAK MODULATORS Publication Classification (75) Inventors: Susumu Tonegawa, Chestnut Hill, (51) Int. Cl. MA (US); Mansuo L. Hayashi, A 6LX 39/395 (2006.01) CI2O 1/48 (2006.01) Winchester, MA (US); Bridget A638/17 (2006.01) Dolan, Boston, MA (US) A6IR 48/00 (2006.01) Correspondence Address: A6II 3L/22 (2006.01) CHOATE, HALL & STEWART LLP A63L/45 (2006.01) TWO INTERNATIONAL PLACE A6IP 25/00 (2006.01) BOSTON, MA 02110 (US) A6IP 5/00 (2006.01) GOIN 33/00 (2006.01) (73) Assignee: MASSACHUSETTS INSTITUTE GOIN 33/573 (2006.01) OF TECHNOLOGY, Boston, MA AOIK 67/027 (2006.01) (US) (52) U.S. Cl...... 424/172.1; 435/15: 514/17.7: 514/44 A: 514/680: 514/406; 800/3:435/7.4: 800/18 (21) Appl. No.: 12/514,288 (22) PCT Fled: Nov. 9, 2007 (57) ABSTRACT The present invention provides methods for treating fragile X (86) PCT NO.: PCT/US2007/084325 syndrome and/or other neurodevelopmental disorders by administering p21-activated kinase (PAK) modulators to a S371 (c)(1), patient Suffering from, Susceptible to, and/or exhibiting one (2), (4) Date: May 24, 2010 or more symptoms of FXS and/or other neurodevelopmental disorders. The present invention provides PAK modulators Related U.S. Application Data and pharmaceutical compositions comprising PAK modula (60) Provisional application No. 60/858,108, filed on Nov. tors. The present invention further provides methods for iden 10, 2006. tifying and/or characterizing PAK modulators. Patent Application Publication Sep. 30, 2010 Sheet 1 of 22 US 2010/024.7552 A1

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Patent Application Publication Sep. 30, 2010 Sheet 2 of 22 US 2010/024.7552 A1

Figure 1B

GenBank Accession Number NM 008031 (mouse FMR1): MEELVVEVRGSNGAFYKAFVKDVHEDSITVAFENN WQPERQPFHDVRFPPPVGYNKDNESDEVEVYSR ANEKEPCCWWLAKVRMIKGEFYVEYAACDATYNE VTERLRSVNPNKPATKDTFHKKLEVPEDLROMCAK ESAHKDFKKAVGAFSVTYDPENYOLVLSINEVTSKR AHMLDMHFRSLRTKLSLILRNEEASKOLESSROLAS RFHEORFIVREDLMGLAGTHGANICROARKVPGVTAD LDEDTCTFHYGEDODAVKKARSFLEFAEDVOVPR NLVGKVGKNGKLIGREVDKSGVWRVREAENEKSVP GREEE IMPPSSLPSNNSRVGPNSSEEKKHLDTKENTH FSQPNSTKVORVLVVSSIVAGGPCRKPEPKAWOGMV PFVFVGTKDSIANATVLLDYHLNYLKEVDOLRERLO DEQLROGASSRPPPNRTDKEKGYVTDDGOGMGR GSRPYRNRGHGRRGPGYTSGTNSEASNASETESD HRDELSDWSLAPTEEERESFLRRGDGRRRGGGGR GQGGRGRGGGFKGNDDHSRTDNRPRNPREAKGR TADGSLOSASSEGSRLRTGKDRNOKKEKPDSVDGL OPLVNGVP Patent Application Publication Sep. 30, 2010 Sheet 3 of 22 US 2010/024.7552 A1

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CAS Registry Na, 742112-33-0 Erina Nate: 2-amino-N-(4-5-(2-phenanthrenyl)- gure (a 3-(trifluoromethyl-1 fi-yrazol-3- yl)phenyl-acetinicle F: CHFNC W 30S Eity: 38% Siahility: a2 years at -2°C ill-title Supplied as: A crystallinic solid l Laboratory Procedures iot log term storage, we stigest that OSUO3Cl2 be stored as supplied at -20°C, it will be stable for a least two e: rs, OSU(302 is supplied as a crystallirie 3 clid. A stock solution fray be rade by dissolving the OStJ05012 in air orgari: solvent purged with an inert gas. CSUC3012 is soluble in organic solvents such as ethaici, DMSO, and direchyi iotmanids (DMF). The solubility of CS 03012 in these solvents is 2 raginal ill ethanol and 30 mgal in DMSO and DM OSGC3013 is spasingly soluks in aqueous suffers, If aqueous stock solutions are required for biological experiments, they can best be prepared by dilutisig the organic sufvent soiulio into aqueous affers or isotonic sain, Ensure that the residual &mount of organic solent is insignificant, since organic solvents may have physiological effects at low &oisix rations, We clo is reco, thriend storing the aqueous solution for more than one day. Cyclooxygenase-2 (COX-2) appears to play a significa fit role in the development and progression of cateer and COX 2 inhibitors such is Celecoxib exhibit atti-cancer activity. OSU03.012 is an analog of Celecoxib" that exhibits Anti-cancer activity in a COX-2-independent manner via inhibition of the phosphatidy inositol-3-kinase (PEBKAkt pathway. It has a ICs of 5 M for inhibition of 3-phosphain.csirida-depellant kinase-l. (PDK-I), and therefore Akt activation, with no measurable COX-2 inhibitiofi, p to 50 M. CSJ 3012 is a roceri inhibitor of Emor cell growth with an average inhibitory concent Tattoh of 1,1 M across a partei of 30 cance cell lines. It does no inhibit signal transduction through the initogen-activated protein kinase (MAPK) pathway. CSU03012 induces apoptosis of chronic iynpokocytic leukemia (CLL) cells independent of bcl-2 overexpression tising both caspase-dependent and independent pathways. References I. Sibbarximaiah, K. and Dannenberg A.J. Cyclooxygenase 2: A tolecular target for cance; prevetition ind treatisciut, Teisharitical Sci. 2;(2), 96.02 (2003). 2, Johnson, Aj, Smith, ..., 2.hu, J., et al. A novel celecoxii derivative, SE312, induces cytotoxicity in primary CLL cells and transformed 8-cellynaphotta cell lic pia caspasc. and Bcl-2-independent mechanism. BoudiOS, 2504-2508 (2005). 3, Zhu, J., Huang, J.-W., Tseng, P-Fi, e: it. From the cyclooxygenase-2 inhibitor celecoxib to a hotel class of 3 phosphoir.ositide-dependent protein kinase-E inhibiters. Cineer Res, 64, 4309-4318 (2004). 4. Kuxtab, J.E., i.ec, C. Cher, C.-S., iii, Celecoxib analogus disfupi. Aict signaling, which is grillonly 3ciated in

primary breast tainois. Bear Cancer Rajearch 7(5), R796-R807 (2004).

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Patent Application Publication Sep. 30, 2010 Sheet 12 of 22 US 2010/024.7552 A1

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PAK MODULATORS synaptic morphology and/or function, such as Rac1, micro tubule-associated protein 1B, activity-regulated cytoskel RELATED APPLICATIONS eton-associated protein, and alpha-calcium/calmodulin-de 0001. This application claims priority under 35 U.S.C. pendent protein kinase II (Zhang et al., 2001, Cell, 107:591; S119(e) to U.S. provisional patent application, U.S. Ser. No. Lee et al., 2003, Development, 130:5543; and Zalfa et al., 60/858,108, filed Nov. 10, 2006 (“the 108 application”). The 2003, Cell, 112:317; all of which are incorporated herein by entire contents of the 108 application are incorporated herein reference). by reference. SUMMARY OF THE INVENTION GOVERNMENT SUPPORT 0005. The present invention encompasses the recognition 0002 The United States Government has provided grant that signaling pathways mediated by p21-activated kinase support utilized in the development of the present invention. (PAK) and FMRP may antagonize each other to regulate In particular, the National Institutes of Health (contract num synaptic morphology and/or function. The present invention ber MH78821) and the National Institute of Mental Health encompasses the recognition that abnormalities in cortical Center (contract number MH58880) have supported develop spine morphology of FXS patients and FMR1 knockout mice ment of this invention. The United States Government has are opposite to those we found in transgenic mice in which PAK activity is inhibited by its dominant negative form (dn certain rights in the invention. PAK). The present invention encompasses the recognition BACKGROUND OF THE INVENTION that FMRP binds to PAK. 0006. The present invention provides systems, including 0003 Fragile X syndrome (FXS) is the most commonly methods, reagents, and/or compositions, for treating fragile X inherited form of mental retardation, with symptoms of syndrome (FXS) and/or other neurodevelopmental disorders. hyperactivity, Stereotypy, anxiety, seizure, impaired social For example, according to the present invention, administra behavior, and/or cognitive delay. FXS results from the loss of tion of PAK modulators may be used to treat patients suffer expression of the fragile X mental retardation 1 (FMR1) gene, ing from, Susceptible to, and/or exhibiting symptoms of FXS which encodes the fragile X mental retardation protein and/or other neurodevelopmental disorder. (FMRP). FMR1 knockout (FMR1 KO) mice and FXS 0007. In some embodiments, the present invention pro patients show similar behavioral phenotypes, as well as simi vides modulators of PAK activity and/or levels. In some lar abnormalities in synaptic morphology in the brain embodiments, a modulator of PAK stimulates PAK activity (O’Donnell et al., 2002, Annu. Rev. Neurosci., 25:315; incor and/or increases its levels. In some embodiments, a modula porated herein by reference). Their brains have more den tor of PAK inhibits PAK activity and/or decreases its levels. In dritic spines and/or a higher proportion of longer and/or thin accordance with some embodiments, compositions are pro ner spines compared to normal individuals (Hinton et al., vided comprising at least one PAK modulator and a pharma 1991, Am. J. Med. Genet., 41:289; Comery et al., 1997, Proc. ceutically acceptable excipient. In certain embodiments, Natl. Acad. Sci., USA, 94:5401; and Irwin et al., 2001, Am. J. PAK modulators function by modulating the interaction Med. Genet., 98:161; all of which are incorporated herein by between PAK and the fragile X mental retardation protein reference). Dendritic spines are the protrusions from den (FMRP). dritic shafts that serve as postsynaptic sites of the majority of 0008. The present invention provides systems, including excitatory synapses in mammals. Correlated with altered methods, reagents and/or compositions, for identifying PAK spine morphology, FMR1 KO mice display abnormal synap modulators. In some embodiments, such methods include tic function, including enhanced long-term depression (LTD) high throughput screening methods. In some embodiments, mediated by metabotropic glutamate receptor in the hippoc the methods include in vitro, in cyto, and/or in Vivo assays. ampus and/or impaired long-term potentiation (LTP) in the 0009. The present invention provides specific PAK modu cortex, compared to wild type mice (Huber et al., 2002, Neu lators. In some embodiments, specific PAK modulators ropharmacology, 37:571; and Li et al., 2002, Mol. Cell Neu include Small molecule therapeutics. In some embodiments, rosci., 19:138; both of which are incorporated herein by ref small molecule modulators of PAK include Emodin, OSU erence). Thus, these findings demonstrate that FMRP 03012, combinations thereof, and/or derivatives thereof The functions in regulating spine morphology, synaptic function, present invention provides pharmaceutical compositions and/or animal behavior. comprising Emodin, OSU-03012, combinations thereof, and/ 0004 FMRP is a selective RNA-binding protein that asso orderivatives thereof The present invention provides methods ciates with polyribosomes (Corbin et al., 1997, Hum. Mol. of treating FXS and/or other neurodevelopmental disorders Genet., 6:1465; and Stefani et al., 2004, J. Neurosci., comprising administering Emodin, OSU-03012, combina 24.7272; both of which are incorporated herein by reference) tions thereof, and/or derivatives thereof to a patient suscep and with Argonaute2 (AGO2) and Dicer, two members of tible to, suffering from, and/or exhibiting symptoms of FXS RISC, a complex that is required for RNAi-mediated gene and/or other neurodevelopmental disorder. silencing (Ishizuka et al., 2002, Genes Dev., 16:2497; incor 0010. This application refers to various patent publica porated herein by reference). FMRP is thought to regulate tions and non-patent publications, the contents of all of which synaptic morphology and/or function because of its ability to are incorporated herein by reference. repress translation of its RNA binding partners (Laggerbauer et al., 2001, Hum. Mol. Genet., 10:329; Liet al., 2001, Nucleic BRIEF DESCRIPTION OF THE DRAWING Acids Res., 29:2276; and Mazroui et al., 2002, Hum. Mol. Genet., 11:3007: all of which are incorporated herein by (0011 FIG. 1. Representative FMR and FXR amino acid reference), perhaps using an RNAi-mediated mechanism. sequences. FIG. 1A shows an alignment of representative Some of these RNAs encode proteins that are involved in examples of Drosophila melanogaster FMR1 (dFMR1; Gen US 2010/0247552 A1 Sep. 30, 2010

Bank AAG22045), human FMR1 (hFMR1; GenBank 0017 FIG. 7. Chemical structure of OSU-03012. AAB18829), human FXR1 (hFXR1; GenBank AAC50155), 0018 FIG. 8. PAK is involved in multiple signaling path and human FXR2 (hEXR2: GenBank AAC50292) amino ways. PAK participates upstream of Raf mitogen-activated acid sequences. Dark gray boxes mark identical amino acids, protein kinase kinase (MEK), extracellular signal-regulated and light gray boxes denote conservative amino acid Substi kinase (ERK), and MAPK-interacting serine/threonine tutions. Insertions are denoted by a dash. The FMR1/FXR kinase 1 (Mink1) in the ERK signaling pathway. PAK partici interaction domain is depicted. “KH1' and “KH2: KH pates downstream of phosphoinositide-dependent kinase 1 domains: “NES: nuclear export signal: “RGG’: domain that (PDK1), PDK2, and phosphatidylinositol 3-kinase (PI3K) in mediates protein-protein interactions, RNA-binding, and/or the PI3K signaling pathway. Figure modified from Klann and may be methylated. Figure modified from Wan et al., 2000, Dever (2004, Nat. Rev. Neurosci., 5:931). Mol. Cell. Biol., 20:8536 (incorporated herein by reference). (0019 FIG. 9. Schematic representation of the extracellu FIG. 1B shows an amino acid sequence for mouse FMR1 lar signal-regulated kinase (ERK) pathway. The mitogen (GenBank NM 008031). activated protein (MAP) kinase cascade comprises three 0012 FIG. 2. Representative human PAK1, PAK2, and sequential kinases: MAP kinase kinase kinase (MAPKKK), PAK3 amino acid sequences. Shown is an alignment of rep MAP kinase kinase (MAPKK), and MAP kinase (MAPK). resentative examples of human PAK1 (GenBank ERK 1/2 phosphorylate a variety of nuclear, cytosolic and AAA65441), human PAK2 (GenBank AAA65442), and cytoskeletal targets. Integrin-focal adhesion kinase (FAK) human PAK3 (GenBank AAC36097) amino acid sequences. pathway, which is activated by adhesion of integrins to spe Asterisks below the sequences mark identical amino acids, cific extracellular matrix (ECM) molecules, is involved in and dots below the sequences denote conservative amino acid activating the ERK pathway. Activation of the ERK pathway substitutions. Insertions are denoted by a dash. Blackboxes is often associated with cell proliferation, cell survival and mark proline-rich regions containing putative SH3-binding cell migration. Exemplary inhibitors of the ERK pathway are PXXP motifs. The open box marks the noncanonical PIX shown. Several negative regulators of the ERK pathway exist, binding site. Gray boxes indicate highly charged basic or such as MAP kinase phosphatases (MKPs/DUSPs) and acidic tracts. The dark overhead lines indicate the Sprouty proteins. Expression of MKPs and Sprouty proteins homodimerization domain (amino acids 78-87), CRIB motif is induced in an ERK-dependent manner, and thus these pro (amino acids 75-90), p21-binding domain (PBD; amino acids teins participate in the negative feedback regulatory loop of 67-113), and autoinhibitory switch domain (amino acids the ERK pathway. 83-139) for PAK1. Diagnostic kinase motifs in the catalytic 0020 FIG. 10. Golgi analysis. Representative dendritic domain are boxed and numbered per convention. Figure segments of layer II/III pyramidal neurons from wild-type modified from Bokoch et al., 2003, Annu. Rev. Biochem., (WT: n=20 neurons, 2 mice), dnPAKTG mice (n=30 neurons, 72:743 (incorporated herein by reference). 3 mice). FMR1 KO mice (n=20 neurons, 2 mice), and double 0013 FIG.3. Representative human PAK4, PAK5, PAK6, mutant dnPAKTG: FMR1 KO mice (dMT; n=40 neurons, 4 and PAK7 amino acid sequences. Shown is an alignment of mice). On each primary apical dendritic branch, ten consecu representative examples of human PAK4 (GenBank tive 10um-long dendritic segments were analyzed to quantify NP 005875), human PAK5 (GenBank CAC18720), human spine density per 10um-long dendritic segment (FIG. 11) and PAK6 (GenBankNP 064553), and human PAK7 (GenBank mean spine density (FIG. 12). Q9P286) amino acid sequences. Asterisks below the 0021 FIG. 11. Quantification of spine density. On each sequences mark identical amino acids, and dots below the primary apical dendritic branch, ten consecutive 10um-long sequences denote conservative amino acid Substitutions. dendritic segments were analyzed to quantify spine density Insertions are denoted by a dash. per 10 um-long dendritic segment. Spine density in dMTs 0014 FIG. 4. Representative human PAK4, PAK5, and was comparable to wild-type controls in all dendritic seg PAK6 amino acid sequences. Shown is an alignment of rep ments except segment 7 and 8 (p-0.05 in segments 1-6, 9, and resentative examples of human PAK4 (GenBank 10; p <0.01 in segments 7 and 8). CAAO9820), human PAK5 (GenBank BAA94194), and 0022 FIG. 12. Quantification of mean spine density. (A) human PAK6 (GenBank AAF82800) amino acid sequences. On each primary apical dendritic branch, ten consecutive 10 Black boxes mark identical amino acids. Insertions are um-long dendritic segments were analyzed to quantify mean denoted by a dash. Diagnostic kinase motifs in the catalytic spine density per 10 um-long dendritic segment. Mean spine domain are boxed and numbered per convention. Figure density in dMTs (1.28+0.02) was significantly lower than that modified from Dan et al., 2002, Mol. Cell. Biol., 22:567 in FMK1 KO mice (1.60+0.02; p<0.001) and significantly (incorporated herein by reference). higher than that in dnPAKTG mice (1.06+0.01; p-0.001). 0015 FIG. 5. Representative Caenorhabditis elegans, ANOVA, p<0.0001.***:p<0.001. Drosophila melanogaster; and rat PAK1 amino acid (0023 FIG. 13. Quantification of spine length. FMR1 KO sequences. Shown is an alignment of representative examples neurons (444 spines) exhibited a significant shift in the over of PAK1 amino acid sequences from C. elegans (CEPAK; all spine distribution towards spines of longer length com GenBank BAA 11844), D. melanogaster (DPAK; GenBank pared to wild-type neurons (406 spines; Kolmogorov AAC47094), and rat (PAK; GenBank AAB95646). Black Smirnov test: p-0.05), whilednPAKTG neurons (630 spines) boxes mark identical amino acids. Insertions are denoted by a exhibited the opposite shift to shorter spines (p<0.01). In dash. The N-terminal box marks the p21-binding domain, and contrast, spine length distribution in dMT neurons (785 the C-terminal box denotes the C-terminal kinase domain. spines) overlapped well with wild-type neurons and is sig Figure modified from Chen et al., 1996, J. Biol. Chem., 271: nificantly different from FMR1 KO neurons (p<0.01). 26362 (incorporated herein by reference). 0024 FIG. 14. PAK inhibition rescues reduced cortical 0016 FIG. 6. OSU-03012 product information from Cay LTP in FMR1 KO mice. (A) Input-output curves plotting the man Chemical catalog (May 2006). changes in field excitatory post-synaptic potential (fEPSP) US 2010/0247552 A1 Sep. 30, 2010 amplitude and their corresponding presynaptic stimulus dnPAKTG mice exhibited a significant reduction compared intensity in wild-type (n=45 slices, 16 mice), dnPAKTG to wild-type controls (ANOVA for each tone session, p<0.05; (n=30 slices, 10 mice), FMR1 KO (n=57 slices, 19 mice) and for FMR1 KO versus WT, p<0.05 for session 1 and p<0.01 for dMT mice (n=24 slices, 8 mice). (B) Cortical LTP induced by sessions 2 to 7; for dnPAKTG versus WTip-0.05 for session TBS was enhanced indnPAKTG (n=13 slices, 11 mice), but 1 and p-0.01 for sessions 2 to 7). dMT mice also showed reduced in FMR1 KO (n=17 slices, 11 mice), relative to freezing deficits during the first several tone sessions (ses wild-type controls (n=17 slices, 11 mice; for responses at 55 sions 1 to 4) compared to wild-type controls (p<0.05). How minutes post stimulation, ANOVA, p<0.05; for both dnPAK ever, with additional tone sessions (sessions 5 to 7), freezing TG versus WT and FMR1 KO versus WT, p<0.04). By con by dMT mice caught up to that of wild-type controls (p-0.05). trast, the magnitude of LTP was indistinguishable between “n.s.”: not statistically different. *: p<0.05; **: p<0.01: ***: dMT slices (n=13 slices, 9 mice) and wild-type controls (p-0. p<0.001. 05 for responses at 55 minutes post stimulation). An overlay 0027 FIG. 17. Quantification of meantone-induced freez of representative field potential traces taken during baseline ing. Average freezing for tone sessions 1 to 4: ANOVA p<0. of recording and at 55 minutes post stimulation is shown for 05. dMT mice showed freezing deficits compared to wild each genotype. type controls (p<0.05), but the deficits in dMT mice were less 0025 FIG. 15. Open field test. Mice of different genotypes pronounced compared to dnPAKTG (p<0.01) or FMK1 KO were Subjected to the open field test according to standard mice (p<0.01). Average freezing for tone sessions 5 to 7: procedures. Each mouse ran for 10 minutes in an activity ANOVA p-0.05. Freezing level in dMT mice was not signifi monitor chamber. Open field activity was detected by photo cantly different from wild-type controls (p-0.05) and there beam breaks and analyzed by VersaMax software. Activities were trends in its difference from dnPAKTG (p=0.12) or measured were the amount of time the mouse spent in the FMR1 KO mice (p=0.07). center of the field, the number of times the mouse exhibited 0028 FIG. 18. Immunoprecipitation. Immunoprecipita repetitive behaviors (“stereotypy), and the total distance tions were performed using either rabbit serum (negative traveled by the mouse. Genotypes are as follows: (1) wild control), C-PAK1, or C-PAK1 plus a blocking peptide. West type (n=10 mice); (2) dnPAKTG (n=10 mice); (3) FMR1 KO ern blots were probed foreither PAK1 or FMRP. C-PAK1, but (n=11 mice); and (4) dnPAKTG: FMR1 KO (“dMT mice' not rabbit serum, immunoprecipitates FMRP in this assay. n=11 mice). “n.s.”: not statistically different. *: p<0.05: ***: The specificity of the interaction was tested by including a p-0.001. (A) FMR1 KO traveled a longer distance compared blocking peptide specific for O-PAK1 in the immunoprecipi to wild-type mice (ANOVA, p<0.01. WT: 15.29+0.92 m; tation reaction. Input: 2% of the extract used for a single FMR1 KO: 20.99+1.10 m, p<0.001). (B) FMR1 KO exhib immunoprecipitation was loaded on the gel. ited a higher number of repetitive behaviors than wild-type (0029 FIG. 19. GST pull down. FMRP was produced by in mice (stereotypy counts: ANOVA, p<0.05. WT: 1636+119: vitro-translation. GST and GST-tagged PAK1 were purified FMR1 KO: 2049+125.p<0.05). (C) FMR1 KO stayed a from a bacterial expression system. Wild type FMRP and the longer period of time in the center of the open field than FMRP mutants depicted in FIG.21 were used in the GST-pull wild-type mice (ANOVA, p<0.001. WT: 79.8+8.5 sec; FMR1 down assay. “Input': in vitro translated FMRP sample before KO: 143.1+12.0 sec, p<0.001). In all of these three behaviors, the reaction was carried out: “MW’: molecular weight stan dMT mice exhibited comparable performance to wild-type dard. controls (p-0.05 for all of the following parameters: distance 0030 FIG. 20. Characterization of the interaction between traveled: 17.76+0.91 m; stereotypy counts: 1756+102; center PAK1 and various FMRP variants in vitro. In vitro-translated time: 108.8+14.6 sec). FMRP variants were incubated with GST or GST-PAK1 and 0026 FIG. 16. Trace fear conditioning task. Mice of dif glutathione Sepharose beads. The complexes isolated by this ferent genotypes (WT. n=15 mice: dnPAKTG, n=12 mice; method were subjected to SDS-PAGE and Western blotted for FMR1 KO, n=15 mice; dMT, n=9 mice) were subjected to the FMRP. “Input: 10% of in vitro-translated FMRP sample trace fear conditioning task according to standard procedures. before the binding reaction was carried out was loaded on the On day 1 ("conditioning), mice were placed into a training gel. chamber for 60 seconds before the onset of a 15-second white 0031 FIG. 21. Wildtype and mutant FMRP domainstruc noise tone. Another 30 seconds later, mice received a 1-sec ture. FIG. 21 (adapted from Mazroui et al., 2003, Hum. Mol. ond shock (0.7 mA intensity). Thus, one trial is composed of Genet. 12:3087; incorporated herein by reference) shows a tone, 30 seconds blank time (also called “trace'), and then schematic structure of FMRP, highlighting various functional shock. Seven trials with an intertrial interval (ITI) of 210 domains including three RNA-binding motifs (RGG, KH1, seconds were performed. To examine whether mice remem and KH2) and the phosphorylation site (S499, represented by ber this association, on day 2 (“tone test'), mice were placed a white asterisk). The constructs used for in vitro binding into a new chamber with a different shape and smell from the included full length (WT), truncated (ARGG, AS499 and first chamber. After 60 seconds, a 15-second tone was AKH), or mutated (1304N) FMRP. ARGG refers to the FMRP repeated for seven times with an ITI of 210 seconds. Video variant with a deletion of the RGG box at amino acids 526 images were digitized and the percentage of freezing time 555. The deleted area in AS499 spans amino acids 443-527 during each ITI was analyzed by Image FZ program. Freezing and includes the phosphorylation site, was defined as the absence of all but respiratory movement for 0032 S499, as well as putative phosphorylation sites. The a 1-second period. On day 1 (“Conditioning'), the four geno isoleucine to asparagine missense mutation in the KH2 types of mice exhibited comparable amounts of freezing pre domain mimics that previously reported in a human FXS conditioning (“Baseline') and post-conditioning in all trials. patient (1304N, represented by a black asterisk). The AKH At the 24-hour tone test, the four genotypes exhibit compa deletion mutant lacks both KH domains in tandem corre rable amounts of pre-tone freezing (ANOVA p-0.05). How sponding to amino acids 207-425. Adapted from Mazrouiet ever, for tone-dependent freezing. FMR1 KO mice and al., 2003, Hum. Mol. Genet., 12:3087; incorporated herein by US 2010/0247552 A1 Sep. 30, 2010

reference; numbering refers to the amino acid positions des mentation of an intact antibody and/or it may be recombi ignated by the SwissProt Q06787 entry. nantly produced from a gene encoding the partial antibody sequence. Alternatively or additionally, an antibody fragment DEFINITIONS may be wholly or partially synthetically produced. An anti body fragment may optionally comprise a single chain anti 0033 Amino acid: As used herein, term “amino acid.” in body fragment. Alternatively or additionally, an antibody its broadest sense, refers to any compound and/or Substance fragment may comprise multiple chains which are linked that can be incorporated into a polypeptide chain. In some together, for example, by disulfide linkages. An antibody embodiments, an amino acid has the general structure H.N- fragment may optionally comprise a multimolecular com C(H)(R)—COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an plex. A functional antibody fragment typically comprises at amino acid is a synthetic amino acid; in some embodiments, least about 50 amino acids and more typically comprises at an amino acid is a D-amino acid; in Some embodiments, an least about 200 amino acids. amino acid is an L-amino acid. “Standard amino acid refers 0036) Approximately: As used herein, the term “approxi to any of the twenty standard L-amino acids commonly found mately” or “about,” as applied to one or more values of in naturally occurring peptides. "Nonstandard amino acid interest, refers to a value that is similar to a stated reference refers to any amino acid, other than the standard amino acids, value. In certain embodiments, the term “approximately” or regardless of whether it is prepared synthetically or obtained “about” refers to a range of values that fall within 25%, 20%, from a natural source. As used herein, “synthetic amino acid 19%. 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, encompasses chemically modified amino acids, including but 8%, 7%, 6%. 5%, 4%,3%, 2%, 1%, or less in either direction not limited to salts, amino acid derivatives (such as amides), (greater than or less than) of the stated reference value unless and/or Substitutions Amino acids, including carboxy- and/or otherwise stated or otherwise evident from the context (ex amino-terminal amino acids in peptides, can be modified by cept where such number would exceed 100% of a possible methylation, amidation, acetylation, and/or Substitution with value). other chemical groups that can change the peptide's circulat 0037 Biologically active: As used herein, the phrase “bio ing half-life without adversely affecting their activity. Amino logically active' refers to a characteristic of any Substance acids may participate in a disulfide bond. The term "amino that has activity in a biological system and/or organism. For acid is used interchangeably with “amino acid residue.” and instance, a Substance that, when administered to an organism, may refer to a free amino acid and/or to an amino acid residue has a biological effect on that organism, is considered to be of a peptide. It will be apparent from the context in which the biologically active. In particular embodiments, where a pro term is used whether it refers to a free amino acid or a residue tein or polypeptide is biologically active, a portion of that of a peptide. protein or polypeptide that shares at least one biological activ 0034. Animal: As used herein, the term “animal' refers to ity of the protein or polypeptide is typically referred to as a any member of the animal kingdom. In some embodiments, “biologically active' portion. "animal' refers to humans, at any stage of development. In 0038 Candidate substance: As used herein, the term “can Some embodiments, “animal' refers to non-human animals, didate Substance' refers to any Substance that may potentially at any stage of development. In certain embodiments, the inhibit FXS and/or modulate PAK activity. A candidate sub non-human animal is a mammal (e.g., a rodent, a mouse, a rat, stance may be a protein, a nucleic acid, a small molecule, a a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, lipid, a carbohydrate, a glycoprotein, a proteoglycan, combi and/or a pig). In some embodiments, animals include, but are nations thereof, and/or characteristic portions thereof It may not limited to, mammals, birds, reptiles, amphibians, fish, prove to be the case that the most useful candidate Substances insects, and/or worms. In some embodiments, an animal may will be substances that are structurally related to FMRP. PAK, be a transgenic animal, genetically-engineered animal, and/or their binding partners, their upstream effectors, and/or their a clone. downstream effectors (e.g., mimics). However, it may be the 0035 Antibody: As used herein, the term “antibody” case that the most useful candidate substances will have little refers to any immunoglobulin, whether natural or wholly or or no structural relationship to FMRP. PAK, their binding partially synthetically produced. All derivatives thereof partners, their upstream effectors, and/or their downstream which maintain specific binding ability are also included in effectors. In some embodiments, candidate Substances are the term. The term also covers any protein having a binding provided as individual Substances. In some embodiments, domain which is homologous or largely homologous to an candidate substances are provided in the form of a library, immunoglobulin binding domain. Such proteins may be collection, and/or set of candidate Substances. In some derived from natural sources, or partly or wholly syntheti embodiments, candidate Substances can be screened for their cally produced. An antibody may be monoclonal or poly ability to modulate PAK. clonal. An antibody may be a member of any immunoglobu 0039 Characteristic portion: As used herein, the term a lin class, including any of the human classes: IgG, IgM, IgA, “characteristic portion of a substance, in the broadest sense, Igl), and IgE. As used herein, the terms “antibody fragment' is one that shares some degree of sequence and/or structural or “characteristic portion of an antibody' are used inter identity and/or at least one functional characteristic with the changeably and refer to any derivative of an antibody which is relevant intact Substance. For example, a “characteristic por less than full-length. In general, an antibody fragment retains tion of a protein or polypeptide is one that contains a con at least a significant portion of the full-length antibody's tinuous stretch of amino acids, or a collection of continuous specific binding ability. Examples of antibody fragments stretches of amino acids, that together are characteristic of a include, but are not limited to, Fab, Fab', F(ab')2, sclv, Fv, protein or polypeptide. In some embodiments, each Such dsFv diabody, and Fd fragments. An antibody fragment may continuous stretch generally will contain at least 2, 5, 10, 15, be produced by any means. For example, an antibody frag 20, 50, or more amino acids. A “characteristic portion of a ment may be enzymatically or chemically produced by frag nucleic acid is one that contains a continuous stretch of nucle US 2010/0247552 A1 Sep. 30, 2010

otides, or a collection of continuous stretches of nucleotides, embodiments, FXS refers to a disease, disorder, and/or con that together are characteristic of a nucleic acid. In some dition caused by and/or associated with one or more of the embodiments, each Such continuous stretch generally will following: (1) a mutation in FMR1 (the nucleotide sequence contain at least 2, 5, 10, 15, 20, 50, or more nucleotides. In encoding FMRP); (2) defective FMR1 expression; (3) general, a characteristic portion of a Substance (e.g. of a increased and/or decreased expression of FMRP; (4) defec protein, nucleic acid, Small molecule, etc.) is one that, in tive FMRP function; (5) increased and/or decreased expres addition to the sequence and/or structural identity specified sion of FMRP's natural binding partners; (6) an increased above, shares at least one functional characteristic with the and/or decreased ability of FMRP to bind to its natural bind relevant intact Substance. In some embodiments, a character ing partners; (7) decreased or absent arginine methylation of istic portion may be biologically active. FMRP; (8) the increased methylation of FMR1 CpG repeats 0040 Expression: As used herein, “expression of a in the 5' UTR of exon 1; (9) the mislocalization or misexpres nucleic acid sequence refers to one or more of the following sion of FMRP within the cellor within the organism; (10) the events: (1) production of an RNA template from a DNA modulation of function of FMR1 transcription factors Sp1 sequence (e.g., by transcription); (2) processing of an RNA and/or NRF 1: (11) an increased and/or decreased ability of transcript (e.g., by splicing, editing, 5' cap formation, and/or FMRP to associate with polysomes; (12) the loss of function 3' end formation); (3) translation of an RNA into a polypep of FXR1 and/or FXR2, which are homologous to FMR1 and tide or protein; (4) post-translational modification of a may compensate for loss of FMR1 function; (13) the polypeptide or protein. decreased ability of FMRP to recognize RNA secondary 004.1 FMR1: As used herein, the “FMR1 gene refers to structures that FMRP normally recognizes (such as intramo any nucleotide sequence that encodes the fragile X mental lecular G quartet and/or FMRP “kissing complex'); (14) an retardation protein (FMRP) and/or a characteristic portion increased and/or decreased ability of FMRP to interact with thereof Representative examples of FMR1 nucleotide miRNAs and/or members of the miRNA pathway; (15) an sequences depicted in FIG. 1 include GenBank Accession increased and/or decreased ability of FMRP to interact with Number AF305881 and GenBank Accession Number its known target RNAS, such as RNAS encoding Rac1, micro L29074. The “FXR gene is homologous to FMR1 and may tubule-associated protein 1B, activity-regulated cytoskel compensate for loss of FMR1 function. Representative eton-associated protein, and/or alpha-calcium/calmodulin examples of FXR nucleotide sequences include, but are not dependent protein kinase II; (16) an increased and/or limited to, FXR1 (GenBank Accession Number U25165) and decreased ability of phosphatase PP2A to act on FMRP FXR2 (GenBank Accession Number U31501). FXR1 and (PP2A is thought to bring about translational repression activ FXR2 genes depicted in FIG. 1 are homologous to FMR1 and ity of FMRP); (17) the increased and/or decreased activity of may compensate for loss of FMR1 function. In some embodi mGluR5, which is known to decrease activity of phosphatase ments, an FMR1 gene comprises any nucleotide sequence PP2A (18) exaggerated signaling in mGluR pathways (Bear that shares about 70%, about 80%, about 90%, about 95%, or et al., 2004, Trends Neurosci., 27:370; incorporated herein by greater than 95% sequence identity with the sequences of reference); (19) disruption of a KH domain of FMRP, which GenBank Accession Numbers AF305881, L29074, U25165, decreases the ability of FMRP to interact with PAK; and/or and/or U31501. (20) an I304N mutation in FMRP, which decreases the ability 0042. Fragile X mental retardation protein (FMRP): As of FMRP to interact with PAK. Those of ordinary skill in the used herein, “fragile X mental retardation protein,” or art will appreciate that the teachings of the present invention “FMRP” refers to any protein product of the fragile X mental described herein with respect to FXS are in fact applicable to retardation (FMR1) nucleotide sequence, and/or a character other neurodevelopmental disorders including, for example, istic portion thereof Representative examples of FMRP premature ovarian failure (POF), fragile X-associated tremor amino acid sequences depicted in FIG. 1 include, but are not ataxia (FXTAS), and/or other neurodevelopmental disorders, limited to, GenBank Accession Number AAG22045 and including, but not limited to, various forms of mental retar GenBank Accession Number AAB18829. The “FXR pro dation and/or autism spectrum disorders (ASD). Further tein is homologous to FMRP and may compensate for loss of more, those of ordinary skill in the art will appreciate that the FMRP function. Representative examples of FXRamino acid teachings of the present invention described herein with sequences include, but are not limited to, FXR1 (GenBank respect to FXS are applicable to any disease, disorder, and/or Accession Number AAC50155) and FXR2 (GenBank Acces condition caused by and/or associated with one or more of the sion Number AAC50292). In some embodiments, FMRP following: (1) a mutation in FMR1 (the nucleotide sequence comprises any amino acid sequence that shares about 60%, encoding FMRP); (2) defective FMR1 expression; (3) about 70%, about 80%, about 90%, about 95%, or greater increased and/or decreased expression of FMRP; (4) defec than 95% sequence identity with the sequences of GenBank tive FMRP function; (5) increased and/or decreased expres Accession Numbers AAG.22045, AAB18829, AAC50155, sion of FMRP's natural binding partners; (6) an increased and/or AAC50292. and/or decreased ability of FMRP to bind to its natural bind 0043 Fragile X Syndrome (FXS): As used herein, in some ing partners; (7) decreased or absent arginine methylation of embodiments “fragile X syndrome,” or “FXS. refers to a FMRP; (8) the increased methylation of FMR1 CpG repeats disease, disorder, and/or condition characterized by one or in the 5' UTR of exon 1; (9) the mislocalization or misexpres more of the following symptoms: (1) behavioral symptoms, sion of FMRP within the cellor within the organism; (10) the including but not limited to hyperactivity, Stereotypy, anxiety, modulation of function of FMR1 transcription factors Sp1 seizure, impaired social behavior, and/or cognitive delay; (2) and/or NRF 1: (11) an increased and/or decreased ability of defective synaptic morphology, such as an abnormal number, FMRP to associate with polysomes; (12) the loss of function length, and/or width of dendritic spines; and/or (3) defective of FXR1 and/or FXR2, which are homologous to FMR1 and synaptic function, such as enhanced long-term depression may compensate for loss of FMR1 function; (13) the (LTD) and/or reduced long-term potentiation (LTP). In some decreased ability of FMRP to recognize RNA secondary US 2010/0247552 A1 Sep. 30, 2010

structures that FMRP normally recognizes (such as intramo duced in one or both of a first and a second nucleic acid lecular G quartet and/or FMRP “kissing complex'); (14) an sequences for optimal alignment and non-identical sequences increased and/or decreased ability of FMRP to interact with can be disregarded for comparison purposes). In certain miRNAs and/or members of the miRNA pathway; (15) an embodiments, the length of a sequence aligned for compari increased and/or decreased ability of FMRP to interact with son purposes is at least 30%, at least 40%, at least 50%, at its known target RNAS, such as RNAS encoding Rac1, micro least 60%, at least 70%, at least 80%, at least 90%, at least tubule-associated protein 1B, activity-regulated cytoskel 95%, or substantially 100% of the length of the reference eton-associated protein, and/or alpha-calcium/calmodulin sequence. The nucleotides at corresponding nucleotide posi dependent protein kinase II; (16) an increased and/or tions are then compared. When a position in the first sequence decreased ability of phosphatase PP2A to act on FMRP is occupied by the same nucleotide as the corresponding (PP2A is thought to bring about translational repression activ position in the second sequence, then the molecules are iden ity of FMRP): (17) the increased and/or decreased activity of tical at that position. The percent identity between the two mGluR5, which is known to decrease activity of phosphatase sequences is a function of the number of identical positions PP2A; (18) exaggerated signaling in mGluR pathways (Bear shared by the sequences, taking into account the number of et al., 2004, Trends Neurosci., 27:370; incorporated herein by gaps, and the length of each gap, which needs to be introduced reference); (19) disruption of a KH domain of FMRP, which for optimal alignment of the two sequences. The comparison decreases the ability of FMRP to interact with PAK; and/or of sequences and determination of percent identity between (20) an 1304N mutation in FMRP, which decreases the ability two sequences can be accomplished using a mathematical of FMRP to interact with PAK. algorithm. For example, the percent identity between two 0044) Functional: As used herein, a “functional biologi nucleotide sequences can be determined using the algorithm cal molecule is a biological molecule in a form in which it of Meyers and Miller (CABIOS, 1989, 4:11-17), which has exhibits a property and/or activity by which it is character been incorporated into the ALIGN program (version 2.0) ized. using a PAM120 weight residue table, a gap length penalty of 0045 Gene: As used herein, the term “gene' has its mean 12 and a gap penalty of 4. The percent identity between two ing as understood in the art. It will be appreciated by those of nucleotide sequences can, alternatively, be determined using ordinary skill in the art that the term “gene' may include gene the GAP program in the GCG Software package using an regulatory sequences (e.g., promoters, enhancers, etc.) and/or NWSgapdna. CMP matrix. intron sequences. It will further be appreciated that defini 0049 Isolated: As used herein, the term "isolated” refers tions of gene include references to nucleic acids that do not to a substance and/or entity that has been (1) separated from encode proteins but rather encode functional RNA molecules at least Some of the components with which it was associated Such as thrNAS, RNAi-inducing agents, etc. For the purpose when initially produced (whether in nature and/or in an of clarity we note that, as used in the present application, the experimental setting), and/or (2) produced, prepared, and/or term “gene' generally refers to a portion of a nucleic acid that manufactured by the hand of man. Isolated Substances and/or encodes a protein; the term may optionally encompass regu entities may be separated from at least about 10%, about 20%, latory sequences, as will be clear from context to those of about 30%, about 40%, about 50%, about 60%, about 70%, ordinary skill in the art. This definition is not intended to about 80%, about 90%, or more of the other components with exclude application of the term 'gene' to non-protein-coding which they were initially associated. In some embodiments, expression units but rather to clarify that, in most cases, the isolated agents are more than about 80%, about 85%, about term as used in this document refers to a protein-coding 90%, about 91%, about 92%, about 93%, about 94%, about nucleic acid. 95%, about 96%, about 97%, about 98%, about 99%, or more 0046 Gene productor expression product: As used herein, than about 99% pure. As used herein, a substance is “pure' if the term “gene product” or “expression product' generally it is substantially free of other components. refers to an RNA transcribed from the gene (pre-and/or post 0050 Natural binding partner: As used herein, the term processing) or a polypeptide (pre- and/or post-modification) “natural binding partner refers to any substance that binds to encoded by an RNA transcribed from the gene. PAK and/or FMRP. In some embodiments, the substance 0047 Homology: As used herein, the term “homology’ binds directly, and in Some embodiments, the Substance binds refers to the overall relatedness between polymeric mol indirectly. A natural binding partner Substance may be a pro ecules, e.g. between nucleic acid molecules (e.g. DNA mol tein, nucleic acid, lipid, carbohydrate, glycoprotein, pro ecules and/or RNA molecules) and/or between polypeptide teoglycan, and/or small molecule that binds to either PAK molecules. In some embodiments, polymeric molecules are and/or FMRP. A change in the interaction between PAK and/ considered to be “homologous' to one another if their or FMRP and a natural binding partner may manifest itself as sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, an increased and/or decreased probability that the interaction 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% iden forms. A change in the interaction between PAK and/or tical. In some embodiments, polymeric molecules are con FMRP and a natural binding partner may manifest itself as an sidered to be “homologous' to one another if their sequences increased and/or decreased concentration of PAK and/or are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, FMRP/natural binding partner complex within the cell. This 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar. can result in an increased and/or decreased activity of PAK 0048 Identity: As used herein, the term “identity” refers to and/or FMRP. The present invention identifies FMRP as a the overall relatedness between polymeric molecules, e.g. novel natural binding partner of PAK. Other natural binding between nucleic acid molecules (e.g. DNA molecules and/or partners of PAK include PAK substrates and/or other sub RNA molecules) and/or between polypeptide molecules. Cal stances that interact with PAK. Examples of natural binding culation of the percent identity of two nucleic acid sequences, partners of PAK include; but are not limited to; Myosin light for example, can be performed by aligning the two sequences chain kinase (MLCK); regulatory Myosin light chain for optimal comparison purposes (e.g., gaps can be intro (R-MLC); Myosin I heavy chain; Myosin II heavy chain; US 2010/0247552 A1 Sep. 30, 2010

Myosin VI; Caldesmon; Desmin, Op.18/stathmin: Merlin; 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hex Filamin A; LIM kinase (LIMK); Ras; Rafi Mek: p.47P': ose); and/or modified phosphate groups (e.g., phosphorothio BAD; caspase 3: estrogen and/or progesterone receptors; ates and 5'-N-phosphoramidite linkages). In some embodi RhoGEF: GEF-H1; NET1: Gaz; phosphoglycerate mutase ments, the present invention is specifically directed to B: RhoGDI; prolactin: p41": Aurora-A: Rac/Cdc42: CIB: “unmodified nucleic acids.” meaning nucleic acids (e.g. poly sphingolipids; G-protein B and/or Y subunits; PIX/COOL: nucleotides and residues, including nucleotides and/or GIT/PKL: Paxillin: Nef: NESH; SH3-containing proteins nucleosides) that have not been chemically modified in order (e.g.Nck and/or Grb2); kinases (e.g. Akt, PDK1, PI 3-kinase/ to facilitate or achieve delivery. p85, Cdk5, Cdc2, Src kinases, Ab1, and/or protein kinase A 0052 PAK activity: As used herein, the term “PAK activ (PKA)); and/or phosphatases (e.g. phosphatase PP2A, ity” refers to any activity and/or function of p21-activated POPX1, and/or POPX2) (Bokoch et al., 2003, Annu. Rev. kinase. Examples of “PAK activity” include, but are not lim Biochem., 72:743; and Hofmann et al., 2004, J. Cell Sci., ited to, PAK binding to other substances, PAK kinase activity, 117:4343; both of which are incorporated herein by refer autophosphorylation, translocation, etc. “PAK activity” is ence). Prominent upstream effectors of PAK include, but are used interchangeably with “PAK function.” not limited to, PDK1, PDK2, PI3K, and/or NMDARs. 0053 PAK Modulator: As used herein, the term “PAK 0051 Nucleic acid: As used herein, the term “nucleic modulator” refers to any substance that directly and/or indi acid, in its broadest sense, refers to any compound and/or rectly changes, affects, alters, increases, and/or decreases the Substance that is or can be incorporated into an oligonucle activity and/or levels of PAK. PAK modulators may modulate otide chain. In some embodiments, a nucleic acid is a com the level of PAK mRNA and/or protein; an activity of PAK; pound and/or Substance that is or can be incorporated into an the half-life of PAK mRNA and/or protein; and/or the inter oligonucleotide chain via a phosphodiester linkage. In some action between PAK and its natural binding partners (e.g., a embodiments, “nucleic acid refers to individual nucleic acid Substrate for a PAK kinase, a Rac protein, a cdc42 protein, residues (e.g. nucleotides and/or nucleosides). In some and/or FMRP), as measured using standard methods. “PAK embodiments, “nucleic acid refers to an oligonucleotide inhibitors' may inhibit, decrease, and/or abolish the level of chain comprising individual nucleic acid residues. As used PAK mRNA and/or protein; an activity of PAK; the half-life herein, the terms "oligonucleotide' and “polynucleotide' can of PAK mRNA and/or protein; and/or the interaction between be used interchangeably. In some embodiments, “nucleic PAK and its natural binding partners (e.g., a Substrate for a acid encompasses RNA as well as single and/or double PAK kinase, a Rac protein, a cdc42 protein, and/or FMRP), as stranded DNA and/or cDNA. Furthermore, the terms "nucleic measured using standard methods. "PAK activators’ may acid, “DNA “RNA and/or similar terms include nucleic activate and/or increase the level of PAK mRNA and/or pro acid analogs, i.e. analogs having other than a phosphodiester tein; an activity of PAK; the half-life of PAK mRNA and/or backbone. For example, the so-called “peptide nucleic acids.” protein; and/or the interaction between PAK and its natural which are known in the art and have peptide bonds instead of binding partners (e.g., a Substrate for a PAK kinase, a Rac phosphodiester bonds in the backbone, are considered within protein, a cdc42 protein, and/or FMRP), as measured using the scope of the present invention. The term “nucleotide standard methods. mRNA expression levels may be deter sequence encoding an amino acid sequence' includes all mined using standard RNase protection assays and/or in situ nucleotide sequences that are degenerate versions of each hybridization assays, and/or the level of protein may be deter other and/or encode the same amino acid sequence. Nucle mined using standard Westernand/or immunohistochemistry otide sequences that encode proteins and/or RNA may analysis. The phosphorylation levels of signal transduction include introns. Nucleic acids can be purified from natural proteins downstream of PAK activity may be measured using Sources, produced using recombinant expression systems and standard assays. PAK modulators may modulate binding optionally purified, chemically synthesized, etc. Where between PAK and FMRP. “PAK inhibitors' may reduce, appropriate, e.g., in the case of chemically synthesized mol abolish, and/or remove the binding between these two enti ecules, nucleic acids can comprise nucleoside analogs such as ties, and “PAKactivators' may increase and/or strengthen the analogs having chemically modified bases or Sugars, back binding between these two entities. Thus, binding between bone modifications, etc. A nucleic acid sequence is presented PAK and FMRP is stronger in the absence of the inhibitor than in the 5' to 3' direction unless otherwise indicated. The term in its presence and is weaker in the absence of the activator “nucleic acid segment' is used hereinto refer to a nucleic acid than in its presence. Put another way, a PAK inhibitor sequence that is a portion of a longer nucleic acid sequence. In increases the K of binding between PAK and FMRP, and a many embodiments, a nucleic acid segment comprises at least PAK activator decreases the K of binding between PAK and 3, 4, 5, 6,7,8,9, 10, or more residues. In some embodiments, FMRP. Alternatively or additionally, PAK modulators may a nucleic acid is or comprises natural nucleosides (e.g. inhibit and/or activate the kinase activity of PAK. In particu adenosine, thymidine, guanosine, cytidine, uridine, deoxyad lar, PAK inhibitors and/or activators may modulate the ability enosine, deoxythymidine, deoxyguanosine, and deoxycyti of PAK to phosphorylate FMRP. PAK modulators may be dine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thio inorganic and/or organic. PAK modulators may comprise one thymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, or more of the following: proteins, peptides, antibodies, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uri nucleic acids, RNAi-inducing entities, antisense oligonucle dine, 2-aminoadenosine, C5-bromouridine, C5-fluorouri otides, ribozymes, Small molecules, glycoproteins, pro dine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cy teoglycans, viruses, lipids, and/or carbohydrates. PAK modu tidine, C5-methylcytidine, 2-aminoadenosine, lators may be in the form of monomers, dimers, oligomers, 7-deaZaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-OX and/or in a complex. In some embodiments, as used herein, a oguanosine, O(6)-methylguanine, and 2-thiocytidine); “PAK modulator” is a substance that modulates all isoforms chemically modified bases; biologically modified bases (e.g., of PAK. In some embodiments, as used herein, a "PAK modu methylated bases); intercalated bases; modified Sugars (e.g., lator” is a substance that modulates a single isoform of PAK. US 2010/0247552 A1 Sep. 30, 2010

In some embodiments, as used herein, a "PAK modulator” is appreciate that a protein can sometimes include more than a substance that modulates two or more isoforms of PAK. In one polypeptide chain, for example linked by one or more some embodiments, as used herein, a "PAK modulator” is a disulfide bonds or associated by other means. Polypeptides substance that modulates a subset of PAK isoforms. In some may contain L-amino acids, D-amino acids, or both and may embodiments, as used herein, a "PAK modulator” is a sub contain any of a variety of amino acid modifications orana stance that modulates any of the isoforms of PAK. logs known in the art. Useful modifications include, e.g., 0054 PAK protein: As used herein the term “PAK protein' terminal acetylation, amidation, etc. In some embodiments, refers to a protein that belongs in the family of PAK serine/ proteins may comprise natural amino acids, non-natural threonine protein kinases. These include mammalian isoform amino acids, synthetic amino acids, and combinations thereof identified, e.g., PAK1, PAK2, PAK3, PAK4, PAK5, PAK6, The term "peptide' is generally used to refer to a polypeptide and/or PAK7; and/or lower eukaryotic isoforms, such as the having a length of less than about 100 amino acids. yeast Ste20 (Leberter et al., 1992, EMBO.J., 11:4805; incor 0056 Small Molecule: In general, a “small molecule' is porated herein by reference) and/or the Dictyostelium single understood in the art to be an organic molecule that is less than headed myosin I heavy chain kinases (Wu et al., 1996, J. Biol. about 5 kilodaltons (Kd) in size. In some embodiments, the Chem., 271:31787; incorporated herein by reference). Rep small molecule is less than about 4 Kd, 3 Kid, about 2 Kid, or resentative examples of PAKamino acid sequences depicted about 1 Kid. In some embodiments, the small molecule is less in FIGS. 2, 3, 4, and 5 include, but are not limited to, human than about 800 daltons (D), about 600 D, about 500 D, about PAK1 (GenBank Accession Number AAA65441), human 400 D, about 300 D, about 200 D, or about 100 D. In some PAK2 (GenBank Accession Number AAA65442), human embodiments, a small molecule is less than about 2000 g/mol, PAK3 (GenBank Accession Number AAC36097), human less than about 1500 g/mol, less than about 1000 g/mol, less PAK 4 (GenBank Accession Numbers NP 005875 and than about 800 g/mol, or less than about 500 g/mol. In some CAA09820), human PAKS (GenBank Accession Numbers embodiments, Small molecules are non-polymeric. In some CAC18720 and BAA94194), human PAK6 (GenBankAcces embodiments, Small molecules are not proteins, peptides, or sion Numbers NP 064553 and AAF82800), human PAK7 amino acids. In some embodiments, Small molecules are not (GenBank Accession Number Q9P286), C. elegans PAK nucleic acids or nucleotides. In some embodiments, Small (GenBank Accession Number BAA 11844), D. melanogaster molecules are not saccharides or polysaccharides. PAK (GenBank Accession Number AAC47094), and rat 0057 Subject: As used herein, the term “subject” or PAK1 (GenBank Accession Number AAB95646). Represen "patient” refers to any organism to which a composition of tative examples of PAK genes encoding PAK proteins this invention may be administered, e.g., for experimental, include, but are not limited to, human PAK1 (GenBank diagnostic, prophylactic, and/or therapeutic purposes. Typi Accession Number U24152), human PAK2 (GenBank cal Subjects include animals (e.g., mammals such as mice, Accession Number U24153), human PAK3 (GenBank rats, rabbits, non-human primates, and humans; insects; Accession Number AF068864), human PAK4 (GenBank worms; etc.). Accession Number AJO11855), human PAK5 (GenBank 0.058 Substantially: As used herein, the term “substan Accession Number AB040812), and human PAK6 (GenBank tially” refers to the qualitative condition of exhibiting total or Accession Number AF276893). In some embodiments, a near-total extent or degree of a characteristic or property of PAK protein comprises any amino acid sequence that shares interest. One of ordinary skill in the biological arts will under about 60%, about 70%, about 80%, about 90%, about 95%, or stand that biological and chemical phenomena rarely, if ever, greater than 95% sequence identity with the sequences of go to completion and/or proceed to completeness or achieve GenBank Accession Numbers AAA65441, AAA65442, or avoid an absolute result. The term “substantially is there AAC36097, NP 005875, CAA09820, CAC18720, fore used herein to capture the potential lack of completeness BAA94194, NP 064553, AAF82800, Q9P286, BAA 11844, inherent in many biological and chemical phenomena. AAC47094, and/or AAB95646. In some embodiments, a 0059 Suffering from: An individual who is “suffering PAK gene comprises any nucleotide sequence that shares from FXS and/or other neurodevelopmental disorders has about 60%, about 70%, about 80%, about 90%, about 95%, or been diagnosed with or displays one or more symptoms of greater than 95% sequence identity with the sequences of FXS and/or other neurodevelopmental disorders. GenBank Accession Numbers U24152, U24153, AF068864, 0060 Susceptible to: An individual who is “susceptible AJO11855, AB040812, and/or AF276893. In some embodi to' FXS has not been diagnosed with FXS and/or may not ments, as used herein, "PAK’refers to all isoforms of PAK. In exhibit symptoms of FXS but may be characterized by one or some embodiments, as used herein, “PAK’ refers to a single more of the following: (1) a mutation in FMR1 (the nucle isoform of PAK. In some embodiments, as used herein, otide sequence encoding FMRP); (2) defective FMR1 expres “PAK’ refers to two or more isoforms of PAK. In some sion; (3) increased and/or decreased expression of FMRP; (4) embodiments, as used herein, "PAK’ refers to a subset of defective FMRP function; (5) increased and/or decreased PAK isoforms. In some embodiments, as used herein, "PAK expression of FMRP's natural binding partners; (6) an refers to any of the isoforms of PAK. increased and/or decreased ability of FMRP to bind to its 0055 Protein: As used herein, the term “protein” refers to natural binding partners; (7) decreased or absent arginine a polypeptide (i.e., a string of at least two amino acids linked methylation of FMRP; (8) the increased methylation of to one another by peptide bonds). Proteins may include moi FMR1 CpG repeats in the 5' UTR of exon 1; (9) the mislo eties other than amino acids (e.g., may be glycoproteins, calization or misexpression of FMRP within the cellor within proteoglycans, etc.) and/or may be otherwise processed or the organism; (10) the modulation of function of FMR1 tran modified. Those of ordinary skill in the art will appreciate that scription factors Sp1 and/or NRF 1: (11) an increased and/or a “protein’ can be a complete polypeptide chain as produced decreased ability of FMRP to associate with polysomes: (12) by a cell (with or without a signal sequence), or can be a the loss of function of FXR1 and/or FXR2, which are characteristic portion thereof Those of ordinary skill will homologous to FMR1 and may compensate for loss of FMK/ US 2010/0247552 A1 Sep. 30, 2010 function; (13) the decreased ability of FMRP to recognize only early signs of the disease for the purpose of decreasing RNA secondary structures that FMRP normally recognizes the risk of developing pathology associated with the disease. (such as intramolecular G quartet and/or FMRP “kissing 0065. Vector: As used herein, “vector refers to a nucleic complex'); (14) an increased and/or decreased ability of acid molecule capable of transporting another nucleic acid to FMRP to interact with miRNAS and/or members of the which it has been linked. In some embodiment, vectors are miRNA pathway; (15) an increased and/or decreased ability capable of extra-chromosomal replication and/or expression of FMRP to interact with its known target RNAs, such as of nucleic acids to which they are linked in a host cell such as RNAs encoding Rac1, microtubule-associated protein 1B, a eukaryotic and/or prokaryotic cell. Vectors capable of activity-regulated cytoskeleton-associated protein, and/or directing the expression of operatively linked genes are alpha-calcium/calmodulin-dependent protein kinase II; (16) referred to herein as “expression vectors.” an increased and/or decreased ability of phosphatase PP2A to DETAILED DESCRIPTION OF CERTAIN act on FMRP (PP2A is thought to bring about translational PREFERRED EMBODIMENTS OF THE repression activity of FMRP); (17) the increased and/or INVENTION decreased activity of mGluR5, which is known to decrease activity of phosphatase PP2A; (18) exaggerated signaling in 0066. In some embodiments, the present invention pro mGluR pathways (Bear et al., 2004, Trends Neurosci., vides methods for treating fragile X syndrome (FXS) and/or 27:370; incorporated herein by reference); (19) disruption of other neurodevelopmental disorders. In certain embodi a KH domain of FMRP, which decreases the ability of FMRP ments, treatment of patients with one or more PAK modula to interact with PAK; and/or (20) an I304N mutation in tors may be used to treat FXS. FMRP, which decreases the ability of FMRP to interact with 0067. In some embodiments, the present invention pro PAK. In some embodiments, an individual who is susceptible vides modulators of PAK activity and/or levels. In some to FXS will develop FXS and/or other neurodevelopmental embodiments, a modulator of PAK increases PAK activity disorder. In some embodiments, an individual who is suscep and/or levels. In some embodiments, a modulator of PAK tible to FXS will not develop FXS and/or other neurodevel inhibits PAK activity and/or levels. In accordance with some opmental disorder. embodiments, compositions are provided comprising at least one PAK modulator and a pharmaceutically acceptable 0061 Test substance: As used herein, the phrase “test sub excipient. In certain embodiments, PAK modulators function stance” refers to any substance that may be utilized in the by modulating the interaction between PAK and the fragile X systems, methods, assays, and/or compositions described mental retardation protein (FMRP). herein. A “test substance' may refer to one or more of the 0068. In some embodiments, the present invention pro following: (1) a PAK protein, a nucleic acid encoding PAK, vides methods for identifying PAK modulators. In some and/or homolog, portion, variant, mutant, and/or derivative embodiments, such methods include high throughput screen thereof; (2) a natural binding partner of PAK, a nucleic acid ing methods. In some embodiments, the methods include in encoding a natural binding partner of PAK, and/or a homolog, vitro, in cyto, and/or in Vivo assays. portion, variant, mutant, and/or derivative thereof, and/or (3) p21-Activated Kinase (PAK) an FMRP protein, a nucleic acid encoding FMRP, and/or a 0069 PAK, a family of serine-threonine kinases that is homolog, portion, variant, mutant, and/or derivative thereof. composed of at least three members, PAK1, PAK2 and/or (4) a Substrate of PAK kinase, a nucleic acid encoding a PAK3, functions downstream of the small GTPases Rac and/ Substrate of PAK, and/or a homolog, portion, variant, mutant, or Cdc42 to regulate multiple cellular functions, including and/or derivative thereof; and/or (5) a substance related to motility, morphogenesis, angiogenesis, and/or apoptosis PAK signal transduction, and/or a homolog, portion, variant, (Bokochet al., 2003, Annu. Rev. Biochem., 72:743; and Hof mutant, and/or derivative thereof. In some embodiments, a mann et al., 2004, J. Cell Sci., 117:4343; both of which are test Substance is a protein, nucleic acid; Small molecule: incorporated herein by reference). GTP-bound Rac and/or carbohydrate; lipid; library, collection, and/or set of any of Cdc42 bind to inactive PAK, releasing steric constraints these; and/or combination of any of these. imposed by a PAK autoinhibitory domain and/or permitting 0062. Therapeutically effective amount: As used herein, PAK auto-phosphorylation and/or activation. Numerous the term “therapeutically effective amount’ means an amount autophosphorylation sites have been identified that serve as of inventive PAK modulator that is sufficient, when adminis markers for activated PAK. tered to a subject suffering from or susceptible to FXS and/or 0070. In some embodiments, as used herein, “PAK’ refers other neurodevelopmental disorders, to treat, diagnose, pre to all isoforms of PAK (e.g. PAK1, PAK2, PAK3, PAK4, vent, and/or delay the onset of the FXS and/or other neurode PAK5, PAK6, PAK7, etc.). In some embodiments, as used velopmental disorders symptom(s) and/or condition(s). herein, “PAK’ refers to a single isoform of PAK. In some 0063. Therapeutic agent: As used herein, the phrase embodiments, as used herein, "PAK’ refers to two or more “therapeutic agent” refers to any agent that, when adminis isoforms of PAK. In some embodiments, as used herein, tered to a subject, has a therapeutic effect and/or elicits a “PAK’ refers to a subset of PAK isoforms. In some embodi desired biological and/or pharmacological effect. ments, as used herein, “PAK’ refers to any of the isoforms of 0064 Treating: As used herein, the term “treat,” “treat PAK. In some embodiments, as used herein, "PAK is a ment,” or “treating” refers to any method used to partially or substance that is at least 60%, at least 70%, at least 80%, at completely alleviate, ameliorate, relieve, inhibit, prevent, least 90%, at least 95%, or at least 98% identical to any and/or delay onset of reduce severity of and/or reduce incidence of all of PAK1, PAK2, PAK3, PAK4, PAK5, PAK6, and/or one or more symptoms or features of a particular disease, PAK7. disorder, and/or condition (e.g., FXS and/or other neurode 0071 Prominent downstream targets of mammalian PAK velopmental disorders). Treatment may be administered to a include, but are not limited to, substrates of PAK kinase, such subject who does not exhibit signs of a disease and/or exhibits as Myosin light chain kinase (MLCK), regulatory Myosin US 2010/0247552 A1 Sep. 30, 2010 light chain (R-MLC), Myosins I heavy chain, myosin II heavy enzyme, a cytokine (such as an interferon and/or an interleu chain, Myosin VI, Caldesmon, Desmin, Op.18/stathmin, Mer kin), an antibiotic, a polyclonal or monoclonal antibody and/ lin, FilaminA, LIM kinase (LIMK), Ras, Raf, Mek, p47", or an effective part thereof, such as an Fv fragment, which BAD, caspase 3, estrogen and/or progesterone receptors, antibody or part thereof may be natural, synthetic, or human RhoGEF, GEF-H1, NET1, Goz, phosphoglycerate mutase ized, a peptide hormone, a receptor, a signaling molecule B, RhoGDI, prolactin, p41', and/or Aurora-A (Bokochet and/or other protein; a nucleic acid, as defined below, includ al., 2003, Annu. Rev. Biochem., 72:743; and Hofmann et al., ing, but not limited to, an oligonucleotide and/or modified 2004, J. Cell Sci., 117:4343; both of which are incorporated oligonucleotide, an RNAi-inducing entity, an antisense oli herein by reference). Other substances known to bind to PAK gonucleotide and/or modified antisense oligonucleotide, in cells include CIB. sphingolipids; G-protein B and/or Y cDNA, genomic DNA, an artificial and/or natural chromo subunits; PIX/COOL: GIT/PKL: Nef: Paxillin; NESH; SH3 Some (e.g. a yeast artificial chromosome, bacterial artificial containing proteins (e.g. Nck and/or Grb2); kinases (e.g. Akt, chromosome, etc.) and/or portion thereof, RNA, including PDK1, PI 3-kinase/p85, Cdk5, Cdc2, Src kinases, Abl, and/or mRNA, tRNA, rRNA and/or a ribozyme, and/or a peptide protein kinase A (PKA)); and/or phosphatases (e.g. phos nucleic acid (PNA); a virus or virus-like particle; a nucle phatase PP2A, POPX1, and/or POPX2) otide, ribonucleotide, and/or synthetic analogue thereof, 0072 Prominent upstream effectors of PAK include, but which may be modified or unmodified; an amino acid and/or are not limited to, PDK1, PDK2, PI3K, and/or NMDARs. analogue thereof, which may be modified or unmodified; a 0073. The present invention encompasses the recognition non-peptide (e.g., Steroid) hormone; a glycoprotein; a pro that the abnormalities in cortical spine morphology of FXS teoglycan; a lipid; and/or a carbohydrate. Small molecules, patients and/or FMR1 KO mice are substantially opposite of including inorganic and/or organic chemicals, which bind to those found in transgenic mice in which activity of p21 and/or occupy the active site of the polypeptide thereby mak activated kinase (PAK) is inhibited by its dominant negative ing the catalytic site inaccessible to Substrate Such that normal form (dnPAKTG mice), for example, in the postnatal fore biological activity is prevented, are included. A PAK modu brain. lator may be in solution and/or in Suspension (e.g., in crys talline, colloidal, or other particulate form). A PAK modula PAK Modulators tor may be in the form of a monomer, dimer, oligomer, etc., 0074 PAK inhibitors have been described in the art as and/or in a complex. possible Substances for use in the treatment of cancer, 0078. In some embodiments, as used herein, a “PAK endometriosis, urogenital disorders, macropinocytosis, viral modulator” is a substance that modulates all isoforms of PAK infection, vascular permeability, joint disease, lymphocyte (e.g. PAK1, PAK2, PAK3, PAK4, PAK5, PAK6, PAK7, etc.). activation, muscle contraction, and/or diabetes (see, for In some embodiments, as used herein, a "PAK modulator” is example, U.S. Pat. Nos. 5,863,532, 6,191,169, and 6.248, a Substance that modulates a single isoform of PAK. In some 549; U.S. Patent Applications 2002/0045564, 2002/086390, embodiments, as used herein, a "PAK modulator” is a sub 2002/106690, 2002/142325, 2003/124107, 2003/166623, stance that modulates two or more isoforms of PAK. In some 2004/09 1992, 2004/102623, 2004/208880, 2005/0203114, embodiments, as used herein, a "PAK modulator” is a sub 2005/037965, 2005/080002, and 2005/233.965; EP Patent stance that modulates a subset of PAK isoforms. In some Publication 1492871; Kumar et al., 2006, Nat. Rev. Cancer, embodiments, as used herein, a "PAK modulator” is a sub 6:459; Eswaren et al., 2007, Structure, 15:201; all of which stance that modulates any of the isoforms of PAK. In some are incorporated herein by reference). embodiments, as used herein, a "PAK modulator” is a sub 0075 PAK inhibitors have also been described in the art as stance that modulates any of PAK1, PAK2, PAK3, PAK4, possible Substances for use in the treatment of neurological PAK5, PAK6, and/or PAK7. In some embodiments, as used disorders characterized by nerve damage and/or neurodegen herein, a “PAK modulator” is a substance that modulates any eration. Such disorders include Parkinson's disease, Alzhe subset of PAK1, PAK2, PAK3, PAK4, PAK5, PAK6, and/or imer's disease, prion-related diseases, neurofibromatosis, PAK7. In some embodiments, as used herein, a "PAK modu stroke-induced nerve damage, and/or diseases associated lator” is a substance that modulates all of PAK1, PAK2, with nerve injury due to ischaemia and/or anoxia (see, for PAK3, PAK4, PAK5, PAK6, PAK7. In some embodiments, as example, U.S. Pat. Nos. 5,952,217 and 6,046,224; U.S. Patent used herein, a "PAK modulator” is a substance that modulates Application 2006/088897; and PCT applications WO any entity that is at least 60%, at least 70%, at least 80%, at 99/02701 and WO 04/07507; all of which are incorporated least 90%, at least 95%, or at least 98% identical to any and/or herein by reference). all of PAK1, PAK2, PAK3, PAK4, PAK5, PAK6, and/or 0076. However, PAK modulators have not previously been PAK7. described in the art as possible substances for use in the 0079 Candidate PAK modulators may be isolated from treatment of neurodevelopmental disorders, including but not natural sources, such as animals, bacteria, fungi, plants, and/ limited to FXS, premature ovarian formation (POF), fragile or marine samples. It will be understood that candidate PAK X-associated tremorataxia (FXTAS), various forms of men modulators can be derived and/or synthesized from chemical tal retardation, and/or autism spectrum disorders (ASD). The compositions and/or man-made substances. present invention encompasses the use of PAK modulators in 0080 Rational Drug Design the treatment of FXS, POF, FXTAS, and/or other neurode 0081. As used herein the term “candidate substance' velopmental disorders. refers to any substance that may potentially inhibit FXS and/ 0077. In some embodiments, a PAK modulator refers to a or modulate PAK activity. A candidate substance may be a Substance, wherein the Substance may be inorganic and/or protein, a nucleic acid, a small molecule, a lipid, a carbohy organic; a biological effector and/or a nucleic acid encoding drate, a glycoprotein, a proteoglycan, combinations thereof, an agent such as a biological effector, a protein, polypeptide, and/or characteristic portions thereof It may prove to be the or peptide including, but not limited to, a structural protein, an case that the most useful candidate substances will be sub US 2010/0247552 A1 Sep. 30, 2010

stances that are structurally related to FMRP, PAK, their 0088. In some embodiments, libraries of candidate sub binding partners, their upstream effectors, and/or their down stances may include, for example, proteins (e.g. peptides, stream effectors (e.g., mimics). Using lead compounds to polypeptides, antibodies, amino acids, etc.), nucleic acids help develop improved compounds is known as “rational drug (e.g., DNA, RNA, oligonucleotides, RNAi-inducing entities, design' and includes not only comparisons with known antisense nucleic acids, ribozymes, peptide nucleic acids, inhibitors and/or activators, but predictions relating to the aptamers, etc.), carbohydrates (e.g. monosaccharides, disac structure of target Substances. charides, polysaccharides, etc.), Small molecules (e.g. 0082 In some embodiments, rational drug design may be organic Small molecules, inorganic Small molecules, etc.), used to predict and/or produce structural analogs of known combinations thereof, and/or characteristic portions thereof. biologically-active PAK modulators (i.e. “PAK modulator Each member of a library may be singular and/or may be a starting materials”). By creating Such analogs, it is possible to part of a mixture (e.g., a “compressed library'). A library may fashion therapeutic agents which may be more active and/or comprise Substantially purified Substances and/or may be stable than the PAK modulator starting material, may have 'dirty (i.e., containing a significant quantity of impurities). different susceptibility to alteration, and/or may affect the I0089. In some embodiments, candidate substances may be function of various the PAK modulator starting material. In used in an initial screen in batches (e.g. batches of types of Some embodiments, a three-dimensional structure for a substances). The substances of those batches which show known candidate Substance and/or characteristic portion enhancement and/or reduction of the activity being assayed thereof could be generated. In some embodiments, this is may Subsequently be tested individually. accomplished by X-ray crystallography, computer modeling, 0090. In some embodiments, libraries are acquired from and/or by a combination of these approaches. various commercial sources in an effort to "brute force' the 0083. In some embodiments, antibodies are used to ascer identification of useful Substances. In some embodiments, tain the structure of a PAK modulator starting material. In commercially available libraries are obtained from Affyme principle, this approach yields a pharmacore upon which trix, ArCRule, Neose Technologies, Sarco, Ciddco, Oxford Subsequent drug design can be based. It is possible to bypass Asymmetry, Maybridge, Aldrich, Panlabs, Pharmacopoeia, protein crystallography altogether by generating anti-idio Sigma, and/or Tripose. A comprehensive review of combina typic antibodies to a functional, pharmacologically active torial libraries, in particular their construction and/or uses is antibody. As a mirror image of a mirror image, the binding provided in Dolle et al. (1999, J. of Comb. Chem., 1:235: site of anti-idiotype would be expected to be an analog of the incorporated herein by reference). Reference is made to com original antigen. The anti-idiotype could then be used to binatorial peptide library protocols (Cabilly, ed., Methods in identify and/or isolate peptides from banks of chemically Molecular Biology, Humana Press, Totowa, N.J., 1998; incor and/or biologically-produced peptides. Selected peptides porated herein by reference). would then serve as pharmacores. Anti-idiotypes may be 0091. Further references describing combinatorial librar generated using the methods described herein for producing ies, their production and/or use include those available from antibodies, using an antibody as the antigen. the URL http://www.netsci.org/Science/Combichem/, 0084. Alternatively or additionally, the present invention including The Chemical Generation of Molecular Diversity. encompasses the recognition that other sterically similar Sub Michael R. Pavia, Sphinx Pharmaceuticals, A Division of Eli stances may be formulated to mimic the key portions of the Lilly (Published July, 1995); Combinatorial Chemistry: A structure of rationally-designed PAK modulators. Such sub Strategy for the Future MDL Information Systems dis stances may be used in the same manner as or in a different cusses the role its Project Library plays in managing diversity manner from the PAK modulator starting material. libraries (PublishedJuly, 1995); Solid Support Combinatorial 0085 Libraries Chemistry in Lead Discovery and SAR Optimization, Adnan 0.086. In some embodiments, libraries of candidate sub M. M. Malli and Barry E. Toyonaga, Ontogen Corporation stances may be employed in the methods and/or compositions (Published July, 1995): Non-Peptidic Bradykinin Receptor described herein. The phrase “library of candidate sub Antagonists From a Structurally Directed Non-Peptide stances, as used herein, refers to a collection and/or set of Library. Sarvajit Chakravarty, Babu. J. Mavunkel, Robin multiple species of Substances that consist of randomly- and/ Andy, Donald J. Kyle, Scios Nova Inc. (Published July, or systematically-selected Subunits and/or members. Screen 1995); Combinatorial Chemistry Library Design using Phar ing libraries of candidate Substances is a rapid and/or efficient macophore Diversity Keith Davies and Clive Briant, Chemi way to screen large number of related and/or unrelated Sub cal Design Ltd. (Published July, 1995): A Database System stances for activity. Combinatorial approaches lend them for Combinatorial Synthesis Experiments-Craig James and selves to rapid evolution of potential drugs by the creation of David Weininger, Daylight Chemical Information Systems, second, third, fourth, etc. generation Substances modeled of Inc. (Published July, 1995); An Information Management active, but otherwise undesirable substances. Architecture for Combinatorial Chemistry, Keith Davies and 0087. In certain embodiments, combinatorial libraries Catherine White, Chemical Design Ltd. (Published July, (also known as “combinatorial chemical libraries'), small 1995); Novel SoftwareTools for Addressing Chemical Diver molecule libraries, peptides and/or peptide mimetics, defined sity, R. S. Pearlman, Laboratory for Molecular Graphics and chemical entities, oligonucleotides, and/or natural product Theoretical Modeling, College of Pharmacy, University of libraries are screened for activity. In some embodiments, a Texas (Published June/July, 1996); Opportunities for Com library of candidate Substances may comprise a synthetic putational Chemists Afforded by the New Strategies in Drug combinatorial library (e.g., a combinatorial chemical library), Discovery: An Opinion, Yvonne Connolly Martin, Computer a cellular extract, a bodily fluid (e.g., urine, blood, tears, Assisted Molecular Design Project, Abbott Laboratories Sweat, and/or saliva), or other mixture of synthetic and/or (Published June/July, 1996); Combinatorial Chemistry and natural Substances (e.g., a library of small molecules and/or a Molecular Diversity Course at the University of Louisville: A fermentation mixture). Description, Arno F. Spatola, Department of Chemistry, Uni US 2010/0247552 A1 Sep. 30, 2010 versity of Louisville (PublishedJune/July, 1996); Chemically systems, which may be screened directly as bacteriophage Generated Screening Libraries: Present and Future. Michael plaques and/or as colonies of lysogens, as previously R. Pavia, Sphinx Pharmaceuticals, A Division of Eli Lilly described (Huse et al., 1989, Science, 246:1275; Caton et al., (Published June/July, 1996); Chemical Strategies For Intro 1990, Proc. Natl. Acad. Sci., USA, 87; Mullinax et al., 1990, ducing Carbohydrate Molecular Diversity Into The Drug Dis Proc. Natl. Acad. Sci., USA, 87:8095; and Persson et al., covery Process. Michael J. Sofia, Transcell Technologies Inc. 1991, Proc. Natl. Acad. Sci., USA, 88: 2432; all of which are (Published June/July, 1996); Data Management for Combi incorporated herein by reference) and are of use. These natorial Chemistry. Maryjo Zaborowski, Chiron Corporation expression systems may be used to screen a large number of and Sheila H. DeWitt, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company (Published Novem different members of a library, in the order of about 10 or ber, 1995); and/or The Impact of High Throughput Organic O. Synthesis on R&D in Bio-Based Industries, John P. Devlin 0096. In some embodiments, screening systems rely, for (Published March, 1996); all of which are incorporated herein example, on direct chemical synthesis of library members. by reference. One method involves the synthesis of peptides on a set of pins 0092. Selection protocols for isolating desired members and/or rods, such as described in PCT Publication WO of large libraries are known in the art. In some embodiments, 84/03564 (incorporated herein by reference). A similar selection protocols involve phage display techniques. Such method involving peptide synthesis on beads, which forms a systems, in which diverse peptide sequences are displayed on peptide library in which each bead is an individual library the Surface of filamentous bacteriophage have proven useful member, is described in U.S. Pat. No. 4,631,211 (incorpo for creating libraries of antibody fragments (and the nucle rated herein by reference) and a related method is described in otide sequences that encoding them) for in vitro selection PCT Publication WO92/00091 (incorporated herein by ref and/or amplification of specific antibody fragments that bind erence). A significant improvement of the bead-based meth a target antigen. Nucleotide sequences encoding the V, and/ ods involves tagging each bead with a unique identifier tag, or V regions are linked to gene fragments which encode Such as an oligonucleotide, so as to facilitate identification of leader signals that direct them to the periplasmic space of E. the amino acid sequence of each library member. These coli and, as a result, the resultant antibody fragments are improved bead-based methods are described in PCT Publica displayed on the Surface of the bacteriophage, typically as tion WO93/06121 (incorporated herein by reference). fusions to bacteriophage coat proteins (e.g., pIII and/or pVIII). Alternatively or additionally, antibody fragments are 0097 Another chemical synthesis method involves the displayed externally on lambda phage capsids (phagebodies). synthesis of arrays of peptides (or peptidomimetics) on a An advantage of phage-based display systems is that, because Surface in a manner that places each distinct library member they are biological systems, selected library members may be (e.g., unique peptide sequence) at a discrete, predefined loca amplified simply by growing the phage containing the tion in the array. The identity of each library member is selected library member in bacterial cells. Furthermore, since determined by its spatial location in the array. The locations in the nucleotide sequence that encodes the polypeptide library the array where binding interactions between a predeter member is contained on a phage and/or phagemid vector, mined molecule (e.g., a receptor) and reactive library mem sequencing, expression, and/or Subsequent genetic manipu bers occur is determined, thereby identifying the sequences of lation is relatively straightforward. the reactive library members on the basis of spatial location. 0093 Methods for the construction of bacteriophageanti These methods are described in U.S. Pat. No. 5,143,854; PCT body display libraries and/or lambda phage expression librar Publications WO 90/15070 and WO92/10092; Fodor et al., ies are well known in the art (Kang et al., 1991. Proc. Natl. 1991, Science, 251: 767; and Dower et al., 1991, Ann. Rep. Acad. Sci. USA., 88: 4363; Clackson et al., 1991, Nature, 352: Med. Chem., 26: 271 (all of which are incorporated herein by 624; Lowman et al., 1991, Biochemistry, 30: 10832; Burtonet reference). al., 1991, Proc. Natl. Acad. Sci., USA, 88: 10134; Hoogen 0098. Other systems for generating libraries of polypep boom et al., 1991, Nucleic Acids Res, 19: 4133; Chang et al., tides or nucleotides involve the use of cell-free enzymatic 1991, J. Immunol., 147: 3610; Breitling et al., 1991, Gene, machinery for the invitro synthesis of the library members. In 104: 147; Hawkins et al., 1992, J. Immunol., 22:867; Marks one method, RNA molecules are selected by alternate rounds et al., 1992, J. Biol. Chem., 267: 16007; and Lerner et al., of selection against a target ligand and PCR amplification 1992, Science, 258: 1313; all of which are incorporated herein (Tuerk et al., 1990, Science, 249: 505; and Ellington et al., by reference). Such techniques may be modified if necessary 1990, Nature, 346: 818; both of which are incorporated herein for the expression generally of polypeptide libraries. by reference). A similar technique may be used to identify 0094. One approach has been the use of sclv phage-librar DNA sequences which bind a predetermined human tran ies (Bird et al., 1988, Science, 242: 423: Huston et al., 1988, scription factor (Thiesen et al., 1990, Nucleic Acids Res., 18: Proc. Natl. Acad. Sci., USA, 85:5879; Chaudhary et al., 1990, 3203: Beaudry et al., 1992 Science, 257:635; and PCT Pub Proc. Natl. Acad. Sci, USA, 87: 1066; and Chiswell et al., lications WO92/05258 and WO92/14843; all of which are 1992, Trends Biotech., 10:80; all of which are incorporated incorporated herein by reference). In a similar way, in vitro herein by reference). Various embodiments of scFv libraries translation may be used to synthesize polypeptides as a displayed on bacteriophage coat proteins have been method for generating large libraries. These methods which described. Refinements of phage display approaches are generally comprise stabilized polysome complexes, are known, for example as described in PCT Publications WO described further in PCT Publications WO 88/08453, WO 92/01047, WO 96/06213, and WO 97/08320 (all of which are 90/05785, WO 90/07003, WO 91/02076, WO 91/05058, and incorporated herein by reference). WO92/02536 (all of which are incorporated herein by refer 0095. In certain embodiments, alternative library selec ence). Alternative display systems which are not phage tion technologies include bacteriophage lambda expression based, such as those disclosed in PCT Publications WO US 2010/0247552 A1 Sep. 30, 2010

95/22625 and WO95/11922 (both of which are incorporated In some embodiments, a peptide sequence can be based on the herein by reference), use the polysomes to display polypep sequence of a protein. In some embodiments, a peptide tides for selection. sequence can be a random arrangement of amino acids. Pep 0099. One combinatorial approach in use is based on a tides from panels of peptides comprising random sequences strategy involving the synthesis of libraries containing a dif and/or sequences which have been varied consistently to pro ferent structure on each particle of the solid phase Support, vide a maximally diverse panel of peptides may be used. interaction of the library with a soluble test substance, iden 0105 Peptide inhibitors of protein kinases, such as myo tification of the “bead” which interacts with the test Sub sin light chain kinase (MLCK) and/or CAM kinase, are well stance, and determination of the structure carried by the iden known in the art (Kemp et al., 1991, Methods in Enzymology, tified “bead’ (Lam et al., 1991, Nature, 354:82; incorporated 201:287: Saitoh et al., 1987, J. Biol. Chem., 262:7796; herein by reference). An alternative to this approach is the Nakanishi et al., 1992, J. Biol. Chem., 267:2157; and Strauss sequential release of defined aliquots of the structures from et al., 1992, Am. J. Physiol., 262:C1437; all of which are the Solid Support, with Subsequent determination of activity in incorporated herein by reference). Many protein kinases solution, identification of the particle from which the active regulate themselves through intermolecular autoinhibition structure was released, and elucidation of its structure by (e.g., Johnson et al., 1996, Cell, 85:149; and Kemp et al., direct sequencing (Salmon et al., 1993, Proc. Natl. Acad. Sci., 1994, Trends in Biochem. Sci., 19:440; both of which are USA, 90:11708; incorporated herein by reference) and/or by incorporated herein by reference), in which inhibition is reading its code (Kerr et al., 1993, J. Am. Chem. Soc., 115: achieved via the interaction of amino acid sequences within 2529; Nikolaiev et al., 1993, Pept. Res., 6:161; and Ohlmeyer the kinase which act as pseudosubstrates which block the et al., 1993, Proc. Natl. Acad. Sci., USA, 90:10922; all of catalytic domain of the kinase. The kinase remains inhibited which are incorporated herein by reference). until a specific Ser. Thr, and/or Tyr residue(s) located in the 0100. In some embodiments, soluble random combinato pseudosubstrate is phosphorylated (autophosphorylation), rial libraries may be synthesized using a simple principle for and/or until interaction with an effector eliminates binding of the generation of equimolar mixtures of peptides which was the pseudosubstrate. In the case of PAK, autophosphorylation first described by Furka et al. (1988, Xth International Sym occurs following binding of Cdc42/Rac1 and the Subsequent posium on Medicinal Chemistry, Budapest 1988: Furka et al., conformation changes due to this interaction. Regions around 1988, 14th International Congress of Biochemistry, Prague these phosphorylation sites are regulatory domains that bind 1988; and Furka et al., 1991, Int. J. Peptide Protein Res., at the Substrate binding site, and autophosphorylation fully 37:487: all of which are incorporated herein by reference). opens the catalytic site. Thus, peptide modulators of PAK The construction of soluble libraries for iterative screening may comprise peptides having at least one amino acid has been described (Houghten et al., 1991, Nature, 354:84; sequence corresponding to at least one of these regulatory incorporated herein by reference). Lam et al. disclosed the domains, but with the phosphorylatable residue replaced by novel and unexpectedly powerful technique of using anotheramino acid that is not phosphorylatable. Such that the insoluble random combinatorial libraries. Lam synthesized peptide acts as a "dead-end pseudosubstrate and modulates random combinatorial libraries on Solid phase Supports, so PAK activity Amino acids used to replace the phosphorylat that each Support had a test compound of uniform molecular able residues may be Asp, Glu, and/or Ala. Since PAK cannot structure, and screened the libraries without prior removal of phosphorylate Such peptides, they act as competitive inhibi the test compounds from the Support by solid phase binding tOrS. protocols (Lam et al., 1991, Nature, 354:82; incorporated 0106. In some embodiments, candidate substances may herein by reference). comprise FMRP proteins and/or characteristic portions 0101. In some embodiments, special libraries called thereof. Accordingly, when applied to cells at high concen “diversity files' may be used to assess the specificity, reliabil trations, i.e., in excess of the amount of endogenous FMRP. ity, and/or reproducibility of the new methods. Diversity files the FMRP peptides may act as competitors and bind to endog contain a large number of compounds (e.g., 1000 or more enous PAK, thereby inhibiting binding, and/or phosphoryla Small molecules) representative of many classes of com tion of, endogenous FMRP. The skilled artisan would know pounds that could potentially result in nonspecific detection how much of a competitive peptide inhibitor to add such that in an assay. Diversity files are commercially available and/or the peptide is added to cells and/or cell lysates “in excess.” can be assembled from individual Substances that are com 0107. In some embodiments, peptide modulators of PAK mercially available. are synthesized based on PAK substrates, either exogenous 0102 The present invention provides for screening of (e.g. Myosin light chain kinase (MLCK), regulatory Myosin libraries of candidate substances. Such libraries may be light chain (R-MLC), Myosins I heavy chain, Myosin II exposed to a test Substance (e.g. PAK protein, PAK gene, heavy chain, Myosin VI, Caldesmon, Desmin, Op.18/stath and/or characteristic portion thereof) with or without FMRP. min, Merlin, FilaminA, LIM kinase (LIMK), Ras, Raf, Mek, and the relevant assay(s), described in detail below, carried p47", BAD, caspase 3, estrogen and/or progesterone out. Such libraries may be used in the in vitro, in cyto, and/or receptors, RhoGEF, GEF-H1, NET1, Goz, phosphoglycerate in vivo assay(s) described in detail below. mutase-B, RhoGDI, prolactin, p41', and/or Aurora-A), (0103 Protein and Peptide PAK Modulators and/or intramolecular (e.g., autophosphorylation and/or auto 0104. In some embodiments, PAK modulators may com inhibitory sites). In certain embodiments, peptide modulators prise proteins and/or peptides. In certain embodiments, pep of PAK comprise peptides that compete with PAK's natural tides are proteins having fewer than about 100 amino acids. In binding partners (e.g., FMRP, Cdc42, and/or Rac1) for bind Some embodiments, peptides range from about 5 to about ing to PAK protein. In some embodiments, a peptide com 100, from about 5 to about 50, from about 5 to about 40, from prising the sequence Pro-Pro-Val-Ile-Ala-Pro-Arg-Pro-Glu about 5 to about 35, from about 5 to about 30, from about 5 to His-Thr-Lys-Ser-Val-Tyr-Thr-Arg-Ser (i.e. the Pro-rich PIX about 25, or from about 20 to about 25 amino acids in length. interacting motif of PAK) disrupts the PIX-PAK interaction US 2010/0247552 A1 Sep. 30, 2010 and reduces PAK autophosphorylation (Maruta et al., 2002, Substrates for growing virus include, for example, dog kidney Methods Mol. Biol., 189:75; incorporated herein by refer cells such as MDCK and/or cells from a clone of MDCK, ence). In some embodiments, peptide modulators of PAK MDCK-like cells, monkey kidney cells such as AGMK cells may be non-phosphorylatable analogues and/or pseudoSub including Vero cells, cultured epithelial cells as continuous strates of PAK substrates. cell lines, 293T cells, BK-21 cells, CV-1 cells, and/or any 0108. In some embodiments, protein modulators of PAK other cell type suitable for the production of virus (e.g., cells comprise peptide mimetics. As used herein, the term "peptide available from ATCC, Rockville, Md.). Suitable cell sub mimetics' refers to structures which substitute for peptides in strates include human cells, such as MRC-5 cells. Suitable interactions with natural binding partners, receptors, and/or cell substrates are not limited to cell lines; for example pri enzymes. The mimetic may possess affinity, efficacy, and/or mary cells such as chicken embryo fibroblasts are included. Substrate function. In certain embodiments, a peptide 0113 Alternatively or additionally, protein modulators of mimetic exhibits function(s) of a particular peptide, without PAK in accordance with the present invention may be readily restriction of structure. Peptide mimetics may include amino prepared by Standard, well-established techniques, such as acid residues and/or other chemical moieties which provide solid-phase peptide synthesis (SPPS) as described by Stewart the desired functional characteristics. Peptide mimetics of the etal. (Solid Phase Peptide Synthesis, 2" ed., Pierce Chemical present invention include, but are not limited to, analogs of Company, Rockford, Ill., 1984); and as described by Bodan structural motifs such as the PAK autophosphorylation site, Szky et al. (The Practice of Peptide Synthesis, Springer-Ver the substrate binding site(s) of PAK, the binding site(s) of lag, New York, N.Y., 1984). PAK's natural binding partners, and/or the FMRP binding site 0114. It will be appreciated, of course, that protein modu of PAK. lators of PAK may incorporate amino acid residues which are 0109. In specific embodiments, protein modulators of modified without affecting activity. For example, the termini PAK comprise a FMRP protein, including mutants described may be derivatized to include blocking groups, i.e. chemical herein, a peptide mimetic, a substance that binds to an FMRP substituents suitable to protect and/or stabilize the N- and/or protein, and/or characteristic portions thereof In some C-termini from “undesirable degradation, a term meant to embodiments, a PAK modulator comprises a PAK protein, encompass any type of enzymatic, chemical, and/or bio including mutants described herein, a peptide mimetic, and/ chemical breakdown of the protein at its termini which is or an agent that binds to a PAK protein. Such agents can be likely to affect the function of the protein, i.e. sequential identified by, for example, direct binding assays, such as degradation of the compound at a terminal end thereof two-hybrid assays, GST pulldowns, etc. described in detail 0115 Blocking groups include protecting groups conven below. tionally used in the art of protein chemistry which will not 0110 Inventive protein modulators of PAK (or character adversely affect the in vivo activities of the protein. For istic portions thereof) may be produced, for example, by example, Suitable N-terminal blocking groups may be intro utilizing a host cell system engineered to express an inventive duced by alkylation and/or acylation of the N-terminus PAK modulator-encoding nucleic acid. Examples of Suitable N-terminal blocking groups include 0111. Any system may be used to produce protein modu C-C branched and/or unbranched alkyl groups, acyl groups lators of PAK (and/or characteristic portions thereof), such as Such as formyl and/or acetyl groups, as well as Substituted eggs, baculovirus, plant cells, fungal cells (e.g. yeast cells), forms thereof. Such as the acetamidomethyl (Acm) group. insect cells (e.g. SFM cells, Sf21 cells, Sf9 cells, Schneider Desamino analogs of amino acids are useful N-terminal cells, S2 cells, etc.), avian cells (e.g. chicken embryo fibro blocking groups may be coupled to the N-terminus of the blasts, etc.), mammalian cells (e.g. Chinese hamster ovary protein and/or used in place of the N-terminal reside. Suitable (CHO) cells, HeLa cells, Madin-Darby canine kidney C-terminal blocking groups, in which the carboxyl group of (MDCK) cells, baby hamster kidney (BHK cells), NS0 cells, the C-terminus may or may not be incorporated, include MCF-7 cells, MDA-MB-438 cells, U87 cells, A172 cells, esters, ketones and/or amides. Ester and/or ketone-forming HL60 cells, A549 cells, SP10 cells, DOX cells, DG44 cells, alkyl groups (e.g. lower alkyl groups, such as methyl, ethyl HEK 293 cells, SHSY5Y, Jurkat cells, BCP-1 cells, COS and/or propyl) and/or amide-forming amino groups (e.g. pri cells, Vero cells (African green monkey kidney), GH3 cells, mary amines (—NH), and/or mono- and/or di-alkylamino 9L cells, 3T3 cells, MC3T3 cells, C3H-10T1/2 cells, NIH groups (e.g. methylamino, ethylamino, dimethylamino, 3T3 cells, C6/36 cells, etc.), and/or hybrid cells. Alternatively diethylamino, methylethylamino, etc.) are examples of C-ter or additionally, protein modulators of PAK (and/or character minal blocking groups. Descarboxylated amino acid ana istic portions thereof) may be expressed in cells using recom logues such as agmatine are useful C-terminal blocking binant techniques, such as through the use of an expression groups and may be either coupled to the protein's C-terminal vector (Sambrook et al., Molecular Cloning: A Laboratory residue and/or used in place of it. Further, it will be appreci Manual, 3" ed., Cold Spring Harbor Laboratory Press, Cold ated that the free amino and/or carboxyl groups at the termini Spring Harbor, N.Y., 2001; incorporated herein by reference). may be removed altogether from the protein to yield desa 0112. In some embodiments, protein modulators of PAK mino and/or descarboxylated forms thereof without (and/or characteristic portions thereof) may be produced in adversely affecting protein activity. the context of intact virus, whether otherwise wild type, 0116. Other modifications may be incorporated without attenuated, killed, etc. In some embodiments, protein modu adversely affecting the activity and these include, but are not lators of PAK (and/or characteristic portions thereof) may be limited to, substitution of one or more of the amino acids in produced in the context of virus like particles. For example, the natural L-isomeric form with amino acids in the D-iso virus may be grown in eggs, such as embryonated hen eggs, in meric form. Thus, a protein may include one or more which case the harvested material is typically allantoic fluid. D-amino acid resides, and/or may comprise amino acids Alternatively or additionally, virus may be derived from any which are all in the D-form. Retro-inverso forms of proteins method using tissue culture to grow the virus. Suitable cell in accordance with the present invention are contemplated, US 2010/0247552 A1 Sep. 30, 2010 for example, inverted proteins in which all amino acids are or phosphothreonine). In some embodiments, useful modifi substituted with D-amino acid forms. cations include, e.g., terminal acetylation, amidation, lipida 0117 To ensure that a protein obtained from either chemi tion, phosphorylation, glycosylation, acylation, cal and/or biological synthetic techniques is the desired pro farnesylation, Sulfation, etc. tein, analysis of the protein should be conducted. Such amino I0121 Proteins and/or characteristic portions thereof acid composition analysis may be conducted using high which have been modified using ordinary molecular biologi resolution mass spectrometry to determine the molecular cal techniques so as to improve their resistance to proteolytic weight of the protein. Alternatively or additionally, the amino degradation and/or to optimize solubility properties and/or to acid content of the protein may be confirmed by hydrolyzing render them more Suitable as a therapeutic agent are contem the protein in aqueous acid and separating, identifying, and/or plated. Homologs of Such proteins include those containing quantifying the components of the mixture using HPLC and/ residues other than naturally occurring L-amino acids, e.g., or an amino acid analyzer. Protein sequenators, which D-amino acids and/or non-naturally occurring synthetic sequentially degrade the protein and identify the amino acids amino acids. Proteins of the invention are not limited to in order, may be used to determine definitely the sequence of products of any of the specific exemplary processes listed the protein. Prior to its use, the protein is usually purified to herein. remove contaminants. In this regard, it will be appreciated I0122) Substantially pure protein obtained as described that the protein is often purified so as to meet the standards set herein may be purified by following known procedures for out by the appropriate regulatory agencies. Any one of a protein purification, wherein an immunological, enzymatic, number of a conventional purification procedures may be and/or other assay is used to monitor purification at each stage used to attain the required level of purity including, for in the procedure. Protein purification methods are well known example, reversed-phase high-pressure liquid chromatogra in the art, and are described, for example in Deutscher et al. phy (HPLC) using an alkylated silica column Such as C-, Cs (ed., Guide to Protein Purification, Harcourt Brace Jovanov and/or Cs-silica. A gradient mobile phase of increasing ich, San Diego, Calif., 1990). organic content is generally used to achieve purification, for I0123. In some embodiments, PAK modulators comprise example, acetonitrile in an aqueous buffer, usually containing antibodies (for example, monoclonal and/or polyclonal anti a small amount of trifluoroacetic acid. Ion-exchange chroma bodies, single chain antibodies, chimeric antibodies, and/or tography may be used to separate proteins based on charge. CDR-grafted antibodies) which bind to PAK (i.e., an anti 0118 Acid addition salts of peptides are contemplated in PAKantibody), a PAK modulator, a natural binding partner of accordance with the present invention. Thus, a protein in PAK (e.g. FMRP), and/or an element upstream and/or down accordance with the present invention treated with an inor stream of PAK to inhibit and/or activate an interaction ganic acid (e.g. hydrochloric, hydrobromic, Sulfuric, nitric, between PAK and/or a PAK modulator, natural binding part phosphoric, etc.) and/or an organic acid (e.g. acetic, propi ner, upstream element, and/or downstream element. onic, glycolic, pyruvic, oxalic, malic, malonic, succinic, 0.124 Antibodies generated in accordance with the present maleic, fumaric, tataric, citric, benzoic, cinnamic, mandelic, invention may include, but are not limited to, polyclonal, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicy monoclonal, chimeric (i.e. “humanized”), and/or single chain clic etc.) to provide a water-soluble salt of the protein, is (e.g. recombinant) antibodies. Antibodies of the present suitable for use in the invention. invention may include antibodies with reduced effector func 0119 The present invention provides for sequence vari tions and/or bispecific molecules. Antibodies of the present ants of protein modulators of PAK. Variants may differ from invention may include Fab fragments and/or fragments pro naturally occurring proteins by conservative amino acid duced by a Fab expression library. In the production of anti sequence differences and/or by modifications which do not bodies, screening for the desired antibody may be accom affect sequence. For example, conservative amino acid plished by techniques known in the art, e.g., ELISA (enzyme changes may be made, which although they alter the primary linked immunosorbent assay). sequence of a protein, do not normally alter its function. In 0.125 Antibodies directed against proteins may be gener some embodiments, about 1 about 2, about 3, about 4, about ated using methods that are well known in the art. U.S. Pat. 5, about 10, or more than about 10 conservative amino acid No. 5,436,157 (incorporated herein by reference) discloses changes can be made without any adverse effect on protein methods of raising antibodies to peptides. For the production function. In some embodiments, variants of protein modula of antibodies, various host animals, including but not limited tors of PAK are approximately 60% identical, approximately to rabbits, mice, rats, sheep, goats, chickens, chicken eggs, 70% identical, approximately 80% identical, approximately and/or guinea pigs, can be immunized by injection with a 90% identical, approximately 95% identical, or greater than polypeptide or peptide fragment thereof. To increase the approximately 95% identical to a given protein modulator of immunological response, various adjuvants may be used PAK depending on the host species, including but not limited to 0120 Modifications which do not normally alter primary oil-emulsion and emulsifier-based adjuvants (e.g., complete amino acid sequence include in vivo and/or in vitro chemical Freund's Adjuvant, incomplete Freund's adjuvant, MF59 derivatization of polypeptides, e.g., acetylation, and/or car Novartis, SAF, etc.), gel-type adjuvants (e.g., aluminum boxylation. Modifications of glycosylation, e.g., those made hydroxide, aluminum phosphate, calcium phosphate, etc.), by modifying the glycosylation patterns of a polypeptide microbial adjuvants (e.g., immunomodulatory DNA during its synthesis, processing, and/or in further processing sequences that include CpG motifs; endotoxins such as steps are contemplated, for example, by exposing the monophosphoryl lipid A.; exotoxins such as cholera toxin, E. polypeptide to enzymes which affect glycosylation (e.g., coli heat labile toxin, and pertussis toxin; muramyl dipeptide, mammalian glycosylating and/or deglycosylating enzymes). BCG bacille Calmette-Guerin, corynebacterium, etc.), par Sequences which have phosphorylated amino acid residues ticulate adjuvants (e.g., liposomes, biodegradable micro are contemplated (e.g., phosphotyrosine, phosphoserine, and/ spheres, saponins, etc.), synthetic adjuvants (e.g., nonionic US 2010/0247552 A1 Sep. 30, 2010 block copolymers, muramyl peptide analogues, polyphosp 0.133 Single-chain Fvs (sclvs) are recombinant antibody haZene, synthetic polynucleotides, etc.), polymers (e.g. poly fragments consisting of only the variable light chain (VL) and phosphaZenes, described in U.S. Pat. No. 5,500,161, incor variable heavy chain (VH) covalently connected to one porated herein by reference; pluronic polyols; polyanions; another by a polypeptide linker. Either VL or VH may com etc.), QS21, squalene, tetrachlorodecaoxide, lysolecithin, prise the NH2-terminal domain. The polypeptide linker may peptides, keyhole limpet hemocyanins, dinitrophenol, etc., be of variable length and composition so long as the two and/or combinations thereof. variable domains are bridged without significant steric inter 0126 The generation of polyclonal antibodies is accom ference. Typically, linkers primarily comprise stretches of plished by inoculating the desired animal with the antigen glycine and serine residues with some glutamic acid or lysine and/or isolating antibodies which specifically bind the anti residues interspersed for solubility. gen therefrom. 0.134 Diabodies are dimeric schvs. Diabodies typically 0127. For the preparation of monoclonal antibodies, any have shorter peptide linkers than most scFVs, and they often technique which provides for the production of antibody mol show a preference for associating as dimers. ecules by continuous cell lines in culture may be utilized. For 0.135 An Fv fragment is an antibody fragment which con example, the hybridoma technique (Kohler et al., 1975, sists of one VH and one VL domain held together by nonco Nature, 256:495; incorporated herein by reference), the valent interactions. The term “dsFv as used herein refers to trioma technique, the human B-cell hybridoma technique an Fv with an engineered intermolecular disulfide bond to (Kozbor et al., 1983, Immunology Today, 4:72; incorporated stabilize the VH-VL pair. herein by reference), and/or the EBV-hybridoma technique 0.136 A F(ab')2 fragment is an antibody fragment essen (Cole et al., 1985, in Monoclonal Antibodies and Cancer tially equivalent to that obtained from immunoglobulins by Therapy, Alan R. Liss, Inc., incorporated herein by reference) digestion with an enzyme pepsin at pH 4.0-4.5. The fragment may be employed to produce monoclonal antibodies. In some may be recombinantly produced. embodiments, monoclonal antibodies are produced in germ 0.137 AFab fragment is an antibody fragment essentially free animals utilizing the technology described in PCT Pub equivalent to that obtained by reduction of the disulfide lication WO 90/13678 (incorporated herein by reference). bridge or bridges joining the two heavy chain pieces in the 0128. A nucleic acid encoding a monoclonal antibody F(ab')2 fragment. The Fab' fragment may be recombinantly obtained using the procedures described herein may be produced. cloned and/or sequenced using technology which is available 0.138 A Fab fragment is an antibody fragment essentially in the art (see, e.g., Wright et al., 1992, Critical Rev. Immu equivalent to that obtained by digestion of immunoglobulins mol., 12:125 and references therein; incorporated herein by with an enzyme (e.g. papain). The Fab fragment may be reference). recombinantly produced. The heavy chain segment of the Fab 0129. The term “antibody” refers to any immunoglobulin, fragment is the Fd piece. whether natural or wholly or partially synthetically produced 0.139. An antibody and/or characteristic portion thereof and to derivatives thereof and characteristic portions thereof. produced in a non-human Subject may be recognized to vary An antibody may be monoclonal or polyclonal. An antibody ing degrees as foreign when the antibody is administered to a may be a member of any immunoglobulin class, including human Subject and an immune response against the antibody any of the human classes: IgG, IgM, IgA, Ig), and IgE. may be generated in the Subject. One approach for minimiz 0130. As used herein, an antibody fragment (i.e. charac ing and/or eliminating this problem is to produce chimeric teristic portion of an antibody) refers to any derivative of an and/or humanized antibody derivatives, i.e., antibody mol antibody which is less than full-length. In general, an anti ecules comprising portions which are derived from non-hu body fragment retains at least a significant portion of the man antibodies and/or portions which are derived from full-length antibody's specific binding ability. Examples of human antibodies. Chimeric antibody molecules may antibody fragments include, but are not limited to, Fab, Fab', include, for example, the variable region from an antibody of F(ab')2, scFv, Fv, dsEv diabody, and Fd fragments. a mouse, rat, and/or other species, with human constant 0131) An antibody fragment may be produced by any regions. A variety of approaches for making chimeric anti means. For example, an antibody fragment may be enzymati bodies have been described (Morrison et al., 1985, Proc. Natl. cally or chemically produced by fragmentation of an intact Acad. Sci., USA, 81:6851; Takeda et al., 1985, Nature, 314: antibody and/or it may be recombinantly produced from a 452; U.S. Pat. Nos. 4,816,397 and 4,816,567; European pub gene encoding the partial antibody sequence. Alternatively or lications EP 0173494 and EP 171496; and United Kingdom additionally, an antibody fragment may be wholly or partially patent GB 2177096B; all of which are incorporated herein by synthetically produced. An antibody fragment may option reference). In some embodiments, humanized antibodies ally comprise a single chain antibody fragment. Alternatively have only the hypervariable domains of the variable region of or additionally, an antibody fragment may comprise multiple non-human origin and have other parts of the variable region chains which are linked together, for example, by disulfide of the antibody, especially the conserved framework regions linkages. An antibody fragment may optionally comprise a of the antigen-binding domain, of human origin. Such multimolecular complex. A functional antibody fragment humanized antibodies may be made by any of several tech will typically comprise at least about 50 amino acids and niques known in the art, (e.g., Teng et al., 1983, Proc. Natl. more typically will comprise at least about 200 amino acids. Acad. Sci., USA, 80:7308; Kozbor et al., 1983, Immunology 0.132. In some embodiments, antibodies may include chi Today, 4:727; and Olsson et al., 1982, Meth. Enzymol., 92:3: meric (e.g. “humanized”) and single chain (recombinant) all of which are incorporated herein by reference), and may be antibodies. In some embodiments, antibodies may have made according to the teachings of PCT Publication WO reduced effector functions and/or bispecific molecules. In 92/06193 and/or European Publication EP 02394.00 (both of Some embodiments, antibodies may include fragments pro which are incorporated herein by reference). Humanized duced by a Fab expression library. antibodies may be commercially produced by, for example, US 2010/0247552 A1 Sep. 30, 2010

Scotgen Limited (Twickenham, Great Britain); Antitope molecule. However, the invention should not be construed to (Cambridge, United Kingdom); Fusion Antibodies (Northern be limited solely to the generation of phage encoding Fab Ireland); and Genentech (South San Francisco, Calif.). antibodies. Rather, phage which encode single chain antibod 0140. In some embodiments, techniques described for the ies (scFv/phageantibody libraries) are included in the inven production of single-chain (e.g. recombinant) antibodies tion. Fab molecules comprise the entire Ig light chain, i.e., (U.S. Pat. No. 4,946,778; incorporated herein by reference) they comprise the variable and constant region of the light are adapted to produce protein-specific single-chain antibod chain, but include only the variable region and/or first con ies. In some embodiments, techniques described for the con stant region domain of the heavy chain. Single chain antibody struction of Fab expression libraries (e.g. Huse et al., 1989, molecules comprise a single chain of protein comprising the Science, 246:1275; incorporated herein by reference) are uti Ig Fv fragment. An Ig Fv fragment includes only the variable lized to allow rapid and/or easy identification of monoclonal regions of the heavy and light chains of the antibody, having Fab fragments possessing a desired specificity. no constant region contained therein. Phage libraries com 0141 Antibody fragments which contain the idiotype of prising schv DNA may be generated following the proce the antibody molecule can be generated by known tech dures described in Marks et al. (1991, J. Mol. Biol., 222:581; niques. For example, such fragments include but are not lim incorporated herein by reference). Panning of phage sogen ited to a F(ab')2 fragment which can be produced by pepsin erated for the isolation of a desired antibody is conducted in a digestion of the antibody molecule; Fab' fragments which can manner similar to that described for phage libraries compris be generated by reducing the disulfide bridges of the F(ab') ing Fab DNA. fragment, Fab fragments which can be generated by treating 0146 The invention should be construed to include, for the antibody molecule with papain and/or a reducing agent; example, synthetic phage display libraries in which the heavy and/or FV fragments. and light chain variable regions may be synthesized such that 0142. To generate a phage antibody library, a cDNA they include nearly all possible specificities (Barbas, 1995, library is first obtained from mRNA which is isolated from Nature Med., 1:837; and de Kruifet et al., 1995, J. Mol. Biol., cells (e.g., hybridomas) which express a desired protein (e.g., 248:97; both of which are incorporated herein by reference). a desired antibody) to be expressed on the phage surface. 0147 For use therapeutically, antibody preparations may cDNA copies of the mRNA are produced using reverse tran be unable to fix complement and/or induce other effector Scriptase. cDNA which specifies immunoglobulin fragments functions. Complement fixation may be prevented by dele are obtained by PCR and the resulting DNA is cloned into a tion of the Fc portion of the antibody, by using an antibody Suitable bacteriophage Vector to generate a bacteriophage isotype which is not capable of fixing complement, and/or by DNA library comprising DNA specifying immunoglobulin using a complement fixing antibody in conjunction with a genes. Procedures for making a bacteriophage library com drug which inhibits complement fixation. Alternatively or prising heterologous DNA are well known in the art and are additionally, amino acid residues within the Fc region which described in Sambrook et al. (Molecular Cloning: A Labora are involved in activating complement (see e.g., Tan et al., tory Manual, 3" ed., Cold Spring Harbor Laboratory Press, 1990, Proc. Natl. Acad. Sci., USA, 87:162; and Duncan et al., Cold Spring Harbor, N.Y., 2001; incorporated herein by ref 1988, Nature, 332:738; both of which are incorporated herein erence). by reference) may be mutated to reduce and/or eliminate the 0143 Bacteriophage encoding a desired antibody may be complement-activating ability of an intact antibody. Like engineered such that the antibody is displayed on the Surface wise, amino acids residues within the Fc region which are thereof in such a manner that it is available for binding to its involved in binding of the Fc region to Fc receptors (see e.g., corresponding binding protein (e.g., the antigen against Canfield et al., 1991, J. Exp. Med., 173: 1483; and Lund et al., which the antibody is directed). Thus, when bacteriophage 1991, J. Immunol., 147:2657: both of which are incorporated which express a specific antibody are incubated in the pres herein by reference) may be mutated to reduce and/or elimi ence of a cell which expresses the corresponding antigen, the nate Fc receptor binding if an intact antibody is to be used. bacteriophage will bind to the cell. Bacteriophage which do 0.148. In some embodiments, antibodies may be frag not express the antibody will not bind to the cell. Such pan mented using conventional techniques and the fragments ning techniques are well known in the art. screened for utility in the same manner as for whole antibod 0144 Processes such as those described above, have been ies. For example, F(ab')2 fragments can be generated by treat developed for the production of human antibodies using M13 ing antibody with pepsin. The resulting F(ab')2 fragment can bacteriophage display (Burton et al., 1994, Adv. Immunol., be treated to reduce disulfide bridges to produce Fab' frag 57: 191; incorporated herein by reference). Essentially, a mentS. cDNA library is generated from mRNA obtained from a 0149 Nucleic Acid PAK Modulators population of antibody-producing cells. The mRNA encodes 0150 Nucleic acid modulators of PAK useful in the rearranged immunoglobulin genes and thus, the cDNA present invention include, but are not limited to, RNAi-induc encodes the same. Amplified cDNA is cloned into M13 ing agents, for example, small interfering RNAs (“siRNAs), expression vectors creating a library of phage which express short hairpin RNAs (“shRNAs), etc.; antisense DNAs and/or human Fab fragments on their surface. Phage which display RNAs; ribozymes; DNA for gene therapy; viral fragments, the antibody of interest are selected by antigen binding and including viral DNA and/or RNA; DNA and/or RNA chime are propagated in bacteria to produce soluble human Fab ras; mRNA: plasmids; cosmids; genomic DNA, cDNA; gene immunoglobulin. Thus, in contrast to conventional mono fragments; various structural forms of DNA including single clonal antibody synthesis, this procedure immortalizes DNA stranded DNA, double-stranded DNA, supercoiled DNA and/ encoding human immunoglobulin rather than cells which or triple-helical DNA; Z-DNA; etc. express human immunoglobulin. 0151. In certain embodiments, the present invention pro 0145 The procedures just presented describe the genera vides nucleic acids which encode PAK modulator proteins tion of phage which encode the Fab portion of an antibody and/or characteristic portions thereof In some embodiments, US 2010/0247552 A1 Sep. 30, 2010 the invention provides nucleic acids which are complemen other over the duplex portion. However, as will be appreciated tary to nucleic acids which encode PAK modulator protein by one of ordinary skill in the art, perfect complementarity is and/or characteristic portions thereof not required. Instead, one or more mismatches in the duplex 0152. In some embodiments, PAK modulators comprise formed by the guide strand and the target mRNA is often nucleic acids, including but not limited to PAK-specific tolerated, particularly at certain positions, without reducing RNAi-inducing agents, antisense nucleic acids, and/or the silencing activity below useful levels. For example, there ribozymes. may be 1, 2, 3, or even more mismatches between the target 0153. In some embodiments, the invention provides mRNA and the guide Strand (disregarding the overhangs). nucleic acids which hybridize to nucleic acids encoding PAK Thus, as used herein, two nucleic acid portions such as a guide and/or PAK modulators and/or characteristic portions thereof Strand (disregarding overhangs) and a portion of a target Such nucleic acids may be used, for example, as primers mRNA that are “substantially complementary' may be per and/or as probes. To give but a few examples, such nucleic fectly complementary (i.e., they hybridize to one another to acids may be used as primers in polymerase chain reaction form a duplex in which each nucleotide is a member of a (PCR), as probes for hybridization (including in situ hybrid complementary base pair) or they may have a lesser degree of ization), and/or as primers for reverse transcription-PCR (RT complementarity sufficient for hybridization to occur. One of PCR). ordinary skill in the art will appreciate that the two strands of 0154 RNA interference (RNAi) is an evolutionarily con the siRNA duplex need not be perfectly complementary. In served process in which presence of an at least partly double some embodiments, at least 80%, at least 90%, or more of the stranded RNA molecule in a eukaryotic cell leads to nucleotides in the guide strand of an effective siRNA are sequence-specific inhibition of gene expression. RNAi was complementary to the target mRNA over at least about 19 originally described as a phenomenon in which the introduc contiguous nucleotides. The effect of mismatches on silenc tion of long dsRNA (typically hundreds of nucleotides) into a ing efficacy and the locations at which mismatches may most cell results in degradation of mRNA containing a region readily be tolerated are areas of active study (see, e.g., Rey complementary to one strand of the dsRNA (U.S. Pat. No. nolds et al., 2004, Nat. Biotechnol., 22:326; incorporated 6,506,559; and Fire et al., 1998, Nature, 391:806; both of herein by reference). which are incorporated herein by reference). Subsequent 0157. It will be appreciated that molecules having the studies in D. melanogaster showed that long dsRNAS are appropriate structure and degree of complementarity to a processed by an intracellular RNase III-like enzyme called target gene will exhibit a range of different silencing efficien Dicer into smaller dsRNAs primarily comprised of two cies. A variety of additional design criteria have been devel approximately 21 nucleotide (nt) strands that form a 19 base oped to assist in the selection of effective siRNA sequences. pair duplex with 2 nt 3' overhangs at each end and 5'-phos Numerous Software programs that can be used to choose phate and 3'-hydroxyl groups (see, e.g., PCT Publication WO siRNA sequences that are predicted to be particularly effec 01/75164; U.S. Patent Application Publications 2002/ tive to silence a target gene of choice are available (see, e.g., O086356 and 2003/0108923; Zamore et al., 2000, Cell, 101: Yuan et al., 2004, Nucl. Acids. Res., 32:W130; and Santoyo et 25; and Elbashiret al., 2001, Genes Dev., 15:188; all of which al., 2005, Bioinformatics, 21:1376; both of which are incor are incorporated herein by reference). porated herein by reference). 0155 Short dsRNAs having structures such as this, 0158. As will be appreciated by one of ordinary skill in the referred to as siRNAs, silence expression of genes that art, RNAi may be effectively mediated by RNA molecules include a region that is Substantially complementary to one of having a variety of structures that differ in one or more the two strands. This strand is referred to as the “antisense' or respects from that described above. For example, the length of “guide' strand, with the other strand often being referred to as the duplex can be varied (e.g., from about 17-29 nucleotides); the “sense' strand. The siRNA is incorporated into a ribo the overhangs need not be present and, if present, their length nucleoprotein complex termed the RNA-induced silencing and the identity of the nucleotides in the overhangs can vary complex (RISC) that contains member(s) of the Argonaute (though most commonly symmetric dTdT overhangs are protein family. Following association of the siRNA with employed in synthetic siRNAs). RISC, a helicase activity unwinds the duplex, allowing an 0159. Additional structures, referred to as short hairpin alternative duplex to form the guidestrand and a target mRNA RNAs (shRNAs), are capable of mediating RNA interference. containing a portion Substantially complementary to the An shRNA is a single RNA strand that contains two comple guide Strand. An endonuclease activity associated with the mentary regions that hybridize to one another to form a Argonaute protein(s) present in RISC is responsible for "slic double-stranded “stem, with the two complementary regions ing the target mRNA, which is then further degraded by being connected by a single-stranded loop. shRNAS are pro cellular machinery. cessed intracellularly by Dicer to form an siRNA structure 0156 Considerable progress towards the practical appli containing a guide Strand and an antisense Strand. While cation of RNAi was achieved with the discovery that exog shRNAs can be delivered exogenously to cells, more typi enous introduction of siRNAS into mammalian cells can cally intracellular synthesis of shRNA is achieved by intro effectively reduce the expression of target genes in a ducing a plasmid or vector containing a promoter operably sequence-specific manner via the mechanism described linked to a template for transcription of the shRNA into the above. A typical siRNA structure includes an approximately cell, e.g., to create a stable cell line or transgenic organism. 19 nucleotide double-stranded portion, comprising a guide 0160 While sequence-specific cleavage of target mRNA Strand and an antisense Strand. Each Strand has a 2 nt 3' is currently the most widely used means of achieving gene overhang. Typically the guide strand of the siRNA is perfectly silencing by exogenous delivery of RNAi-inducing entities to complementary to its target gene and mRNA transcript over at cells, additional mechanisms of sequence-specific silencing least 17-19 contiguous nucleotides, and typically the two mediated by short RNA entities are known. For example, strands of the siRNA are perfectly complementary to each post-transcriptional gene silencing mediated by RNAi-induc US 2010/0247552 A1 Sep. 30, 2010

ing entities can occur by mechanisms involving translational DNA may thus be tested and/or triplex formation may be repression. Certain endogenously expressed RNA molecules assayed. The ability to hybridize will depend on the degree of form hairpin structures containing an imperfect duplex por complementarity and/or the length of the antisense nucleic tion in which the duplex is interrupted by one or more mis acids. Generally, the longer the hybridizing nucleic acid, the matches and/or bulges. These hairpinstructures are processed more base mismatches with an RNA it may contain and still intracellularly to yield single-stranded RNA species referred form a stable duplex (or triplex, as the case may be). One to as known as microRNAs (miRNAs), which mediate trans skilled in the art can ascertain a tolerable degree of mismatch lational repression of a target transcript to which they hybrid by use of standard procedures to determine the melting point ize with less than perfect complementarity. siRNA-like mol of the hybridized complex. ecules designed to mimic the structure of miRNA precursors 0164. In some embodiments, oligonucleotides that are have been shown to result in translational repression of target complementary to the 5' end of PAK mRNA (e.g., the 5' genes when administered to mammalian cells. untranslated sequence up to and including the AUG initiation 0161 Thus the exact mechanism by which an RNAi-in codon) may inhibit translation. In some embodiments, oligo ducing entity inhibits gene expression appears to depend, at nucleotides that are complementary to the 3' untranslated least in part, on the structure of the duplex portion of the sequences of PAK mRNA may inhibit translation (Wagner, RNAi-inducing entity and/or the structure of the hybrid 1994, Nature, 372:333; incorporated herein by reference). formed by one strand of the RNAi-inducing entity and a target Therefore, oligonucleotides complementary to the 5' and/or 3' transcript. RNAi mechanisms and the structure of various untranslated, non-coding regions of a PAK gene may be used RNA molecules known to mediate RNAi, e.g., siRNA, in an antisense approach to inhibit translation of endogenous shRNA, miRNA and their precursors, have been extensively PAK mRNA. Oligonucleotides complementary to the 5' reviewed (see, e.g., Dykxhhorn et al., 2003, Nat. Rev. Mol. untranslated region of the mRNA may include the comple Cell Biol. 4:457; Hannon et al., 2004, Nature, 431:3761; and ment of the AUG start codon. Antisense oligonucleotides Meister et al., 2004, Nature, 431:343; all of which are incor complementary to mRNA coding regions are generally less porated herein by reference). It is to be expected that future efficient inhibitors of translation but may be used in accor developments will reveal additional mechanisms by which dance with the invention. Whether designed to hybridize to RNAi may be achieved and will reveal additional effective any of the afore-mentioned regions of a PAK nucleic acid, an short RNAi-inducing entities. Any currently known or sub antisense nucleic acid of the present invention comprises at sequently discovered RNAi-inducing entities are within the least about 6 nucleotides and may comprise about 6 to about scope of the present invention. 50 nucleotides. In certain embodiments, an antisense nucleic 0162 An RNAi-inducing entity that is delivered accord acid comprises least about 10 nucleotides, at least about 17 ing to the methods of the invention and/or is present in a nucleotides, at least about 25 nucleotides, or at least about 50 composition of the invention may be designed to silence any nucleotides. eukaryotic gene. The gene can be a mammalian gene, e.g., a 0.165. In some embodiments, ribozyme molecules human gene. The gene can be a wildtype gene, a mutant gene, designed to catalytically cleave PAK mRNA transcripts may an allele of a polymorphic gene, etc. In certain embodiments, be used to prevent translation of PAK mRNA and/or expres a PAK modulator is an RNAi-inducing entity that targets PAK sion of PAK (see, e.g., PCT Publication WO 90/11364; and expression. The following sequences may be used to design Sarver et al., 1990, Science, 247: 1222; both of which are RNAi-inducing entities that target PAK expression in accor incorporated herein by reference). Ribozymes of the present dance with the guidelines described herein: GenBank Acces invention may cleave mRNA at site specific recognition sion Numbers U24152, U24153, AFO68864, AJO1 1855, sequences in order to destroy PAK mRNAs. Alternatively, AB040812, AF276893; and/or nucleic acids encoding the ribozymes of the present invention may comprise hammer proteins of GenBank Accession Numbers AAA65441, head ribozymes, which cleave mRNAs at locations dictated AAA65442, AAC36097, NP 005875, CAAO9820, by flanking regions that form complementary base pairs with CAC18720, BAA94194, NP 064553, AAF82800, Q9P286, the target mRNA. When hammerhead ribozymes are BAA 11844, AAC47094, AAB95646. In some embodiments, employed, the target mRNA may have the following RNAi-inducing entities can be used to induce isoform-spe sequence of two bases: 5'-UG-3. The construction and/or cific Suppression of PAK expression. In some embodiments, production of hammerhead ribozymes is well known in the art RNAi-inducing entities can be used to induce Suppression of and is described more fully in Haseloff et al. (1988, Nature, all PAK isoforms. 334:585; incorporated herein by reference). There are numer 0163. In specific embodiments, a nucleic acid modulator ous potential hammerhead ribozyme cleavage sites within the of PAK comprises an antisense molecule that binds to a nucleotide sequence of human PAK clNA. In some embodi translational start site, transcriptional start site, and/or splice ments, a ribozyme is engineered so that the cleavage recog junction. Antisense approaches involve the design of oligo nition site is located near the 5' end of the PAK mRNA; i.e., to nucleotides (e.g. DNA and/or RNA) that are complementary increase efficiency and/or minimize the intracellular accumu to PAK mRNA. Antisense oligonucleotides typically bind to lation of non-functional mRNA transcripts. PAK mRNA and/or prevent translation. Alternatively or addi 0166 In certain embodiments, ribozymes may comprise tionally, an antisense oligonucleotide may bind to DNA of a RNA endoribonucleases (hereinafter “Cech-type PAK gene. Such as, for example, a regulatory element. An ribozymes'), such as the one which occurs naturally in Tet antisense oligonucleotide may or may not exhibit absolute rahymena thermophila (known as the IVS, and/or L-19 IVS complementarity. As used herein, a sequence “complemen RNA) and which has been extensively described by Thomas tary to a portion of a nucleic acid refers to a sequence having Cech and collaborators (Zauget al., 1984, Science, 224:574; sufficient complementarity to be able to hybridize with the Zauget al., 1986, Science, 231:470; Zauget al., 1986, Nature, RNA and/or form a stable duplex. In the case of double 324:429; PCT Publication WO 88/04300; and Been et al., Stranded antisense nucleic acids, a single strand of the duplex 1986, Cell, 47:207: all of which are incorporated herein by US 2010/0247552 A1 Sep. 30, 2010 20 reference). The Cech-type ribozymes have an eight base pair modulator is an RNAi-inducing entity that targets FMR1 active site which hybridizes to a target RNA sequence where expression. The following sequences may be used to design after cleavage of the target RNA takes place. RNAi-inducing entities that target FMR1 expression in 0167 Alternatively or additionally, endogenous PAK accordance with the guidelines described herein: GenBank gene expression may be reduced by targeting deoxyribo Accession Numbers AF305881, L29074, U25165, U31501; nucleotide sequences complementary to the regulatory region and/or nucleic acids encoding the proteins of GenBank of the PAK gene (i.e., PAK promoter, enhancers, etc.) to form Accession Numbers AAG.22045, AAB18829, AAC50155, triple helical structures that prevent transcription of the PAK AACSO292. gene (see generally, Helene, 1991, Anticancer Drug Des., 0.172. In certain embodiments, a PAK modulator is an 6:569; Helene et al., 1992, Ann, N.Y. Acad. Sci., 660:27; and RNAi-inducing entity, antisense oligonucleotide, ribozyme, Maher, 1992, Bioassays, 14:807; all of which are incorpo and/or triple-helix inducing agent that targets one or more rated herein by reference). genes that have been shown to positively regulate expression 0168. In certain embodiments, nucleic acid molecules to of PAK and/or FMRP. For example, in certain embodiments, be used in triple helix formation for the inhibition of tran a PAK modulator is an RNAi-inducing entity that targets Scription may be single-stranded and/or composed of deox transcription factors, translation factors, RNA processing fac yribonucleotides. The base composition of these oligonucle tors, protein stabilizing factors, etc. that are involved in posi otides may promote triple helix formation via Hoogsteen base tive regulation of PAK and/or FMRP expression. pairing rules, which generally require sizable stretches of 0173. In some embodiments, a candidate nucleic acid (e.g. purines or pyrimidines to be present on one strandofa duplex. RNAi-inducing agent, antisense oligonucleotide, ribozyme, Nucleotide sequences may be pyrimidine-based, which will and/or triple-helix inducing agent) comprises an O-anomeric result in TAT and/or CGC triplets across the three associated oligonucleotide. An O-anomeric oligonucleotide forms spe strands of the resulting triple helix. Pyrimidine-rich mol cific double-stranded hybrids with complementary RNA in ecules provide base complementarity to a purine-rich region which, contrary to the usual B-units, the strands run parallel to of a single strand of the duplex in a parallel orientation to that each other (Gautier et al., 1987, Nuc. Acids Res., 15:6625; Strand. In some embodiments, nucleic acid molecules may be incorporated herein by reference). The oligonucleotide is a chosen that are purine-rich, for example, containing a stretch 2'-O-methylribonucleotide (Inoue et al., 1987, Nuc. Acids of G residues. These molecules will typically form a triple Res., 15:6131; incorporated herein by reference) and/or a helix with a DNA duplex that is rich in GC pairs, in which the chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS majority of the purine residues are located on a single stand of Lett., 215:327; incorporated herein by reference). the targeted duplex, resulting in CGC triplets across the three 0.174. In some embodiments, in vitro studies are first per Strands in the triplex. formed to quantitate the ability of a candidate nucleic acid 0169. Alternatively or additionally, the potential (e.g. RNAi-inducing agent, antisense oligonucleotide, sequences that can be targeted for triple helix formation may ribozyme, and/or triple-helix inducing agent) to inhibit PAK be increased by creating a so called “switchback” nucleic acid gene expression. These studies may utilize controls that dis molecule. Switchback molecules are synthesized in an alter tinguish between gene inhibition due to the candidate nucleic nating 5'-3', 3'-5' manner, such that they base pair with first acid and nonspecific biological effects of nucleic acids. These one strand of a duplex and then the other, eliminating the studies may compare levels of the target RNA and/or protein necessity for a sizable stretch of either purines or pyrimidines with that of an internal control RNA and/or protein. Results to be present on one strand of a duplex. obtained using the candidate nucleic acid can be compared 0170 As discussed above, nucleic acid modulators of with those obtained using a control nucleic acid. In some PAK can target PAK directly. However, it will be understood embodiments, a control nucleic acid is of approximately the that nucleic acid modulators of PAK may target PAK directly, same length as the candidate nucleic acid, and/or the nucle but instead, might target downstream effectors of PAK, otide sequence of the candidate nucleic acid differs from the upstream activators of PAK, and/or natural binding partners control nucleic acid no more than is necessary to prevent of PAK. For example, in certain embodiments, a PAK modu specific hybridization to the target sequence. lator is an RNAi-inducing entity that targets one or more of 0.175. In some embodiments, a candidate nucleic acid (e.g. the following: Myosin light chain kinase (MLCK), regulatory RNAi-inducing agent, antisense oligonucleotide, ribozyme, Myosin light chain (R-MLC), Myosins I heavy chain, myosin and/or triple-helix inducing agent) may be delivered to cells II heavy chain, Myosin VI, Caldesmon, Desmin, Op.18/stath which express PAK in vivo and/or in vitro. A number of min, Merlin, FilaminA, LIM kinase (LIMK), Ras, Raf, Mek, methods have been developed for delivering nucleic acids to p47P”, BAD, caspase 3, estrogen and/or progesterone cells. For example, nucleic acids may be injected directly into receptors, RhoGEF, GEF-H1, NET1, Gaz, phosphoglycerate the tissue site. Alternatively or additionally, nucleic acids, mutase-B, RhoGDI, prolactin, p41', Aurora-A (Bokochet designed to target desired cells (e.g., linked to peptides and/or al., 2003, Annu. Rev. Biochem., 72:743; and Hofmann et al., antibodies that specifically bind receptors and/or antigens 2004, J. Cell Sci., 117:4343; both of which are incorporated expressed on a target cell Surface), may be administered sys herein by reference), CIB. sphingolipids; G-protein Band/or temically. y subunits; PIX/COOL: GIT/PKL: Nef: Paxillin; NESH: 0176 Since it may be difficult to achieve intracellular SH3-containing proteins (e.g.Nck and/or Grb2); kinases (e.g. concentrations of a candidate nucleic acid (e.g. RNAi-induc Akt, PDK1, PI 3-kinase/p85, Cdk5, Cdc2, Src kinases, Abl, ing agent, antisense oligonucleotide, ribozyme, and/or triple and/or protein kinase A (PKA)); phosphatases (e.g. phos helix inducing agent) sufficient to inhibit gene expression of phatase PP2A, POPX1, and/or POPX2): PDK1, PDK2, PAK, a recombinant DNA construct may be used in which the PI3K, and/or NMDARs. candidate nucleic acid is placed under the control of a strong 0171 In some embodiments, nucleic acid modulators of pol III and/or pol II promoter. The use of such a construct to PAK may target FMRP. In certain embodiments, a PAK transfect, for example, target cells in a patient, may result in US 2010/0247552 A1 Sep. 30, 2010

the transcription of Sufficient amounts of single-stranded other functional characteristics of the nucleic acid are not RNAs that will form complementary base pairs with endog substantially reduced by the substitution. enous PAK transcripts and thereby prevent translation of PAK 0180. It will be appreciated by those of ordinary skill in the mRNA. For example, a vector may be introduced in vivo such art that nucleic acids in accordance with the present invention that it is taken up by a target cell and/or directs the transcrip may comprise nucleotides entirely of the types found in natu tion of a candidate nucleic acid. Such a vector may remain rally occurring nucleic acids, or may instead include one or episomal and/or become chromosomally integrated, as long more nucleotide analogs or have a structure that otherwise as it can be transcribed to produce the desired candidate differs from that of a naturally occurring nucleic acid. U.S. nucleic acid. Such vectors may be constructed by recombi Pat. Nos. 6,403,779; 6,399,754; 6,225,460; 6,127,533; 6,031, nant DNA technology methods standard in the art. Vectors 086; 6,005,087; 5,977,089; and references therein disclose a may be plasmid, viral, and/or others known in the art, used for wide variety of specific nucleotide analogs and modifications replication and/or expression in mammalian cells. Expres that may be used. See Crooke, S. (ed.) Antisense Drug Tech sion of the sequence encoding the candidate nucleic acid may nology. Principles, Strategies, and Applications (1 ed), Marcel Dekker; ISBN: 0824705661: 1st edition (2001) and be by any promoter known in the art to act in mammalian (e.g. references therein (incorporated herein by reference). For human) neuronal cells. Such promoters may be inducible example, 2-modifications include halo, alkoxy and allyloxy and/or constitutive. Such promoters include, but are not lim groups. In some embodiments, the 2'-OH group is replaced by ited to, the SV40 early promoter region (Bernoist et al., 1981, a group selected from H, OR, R, halo, SH, SR, NH, NH, Nature, 290:304; incorporated herein by reference), the pro NR or CN, wherein R is C-C alkyl, alkenyl, or alkynyl, and moter contained in the 31 long terminal repeat of Roussar halo is F, Cl, Br or I. Examples of modified linkages include coma virus (Yamamoto et al., 1980, Cell, 22:787; incorpo phosphorothioate and 5'-N-phosphoramidite linkages. rated herein by reference), the herpes thymidine kinase 0181 Nucleic acids comprising a variety of different promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci., USA, nucleotide analogs, modified backbones, or non-naturally 78:1441; incorporated herein by reference), the promoter occurring internucleoside linkages can be utilized in accor and/or regulatory sequences of FMK1 gene, etc. Any type of dance with the present invention. Nucleic acids of the present plasmid, cosmid, YAC, and/or viral vector may be used to invention may include natural nucleosides (i.e., adenosine, prepare the recombinant DNA construct which can be intro thymidine, guanosine, cytidine, uridine, deoxyadenosine, duced directly into a tissue site. deoxythymidine, deoxyguanosine, and deoxycytidine) or 0177. In some embodiments, a nucleic acid PAK modula modified nucleosides. Examples of modified nucleotides tor may comprise DNA, RNA, chimeric mixtures, deriva include base modified nucleoside (e.g., aracytidine, inosine, tives, characteristic portions, and/or modified versions isoguanosine, nebularine, pseudouridine, 2,6-diaminopurine, thereof. Nucleic acids of the present invention may be single 2-aminopurine, 2-thiothymidine, 3-deaza-5-azacytidine, Stranded and/or double-stranded. A nucleic acid may be 2'-deoxyuridine, 3-nitorpyrrole, 4-methylindole, 4-thiouri modified at the base moiety, Sugar moiety, and/or phosphate dine, 4-thiothymidine, 2-aminoadenosine, 2-thiothymidine, backbone, for example, to improve stability of the molecule, 2-thiouridine, 5-bromocytidine, 5-iodouridine, inosine, hybridization, etc. A nucleic acid may include otherappended 6-azauridine, 6-chloropurine, 7-deaZaadenosine, 7-deaza groups such agents facilitating transport across the cell mem guanosine, 8-azaadenosine, 8-azidoadenosine, benzimida brane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci., Zole, M1-methyladenosine, pyrrolo-pyrimidine, 2-amino-6- USA, 86:6553; Lemaitre et al., 1987, Proc. Natl. Acad. Sci., chloropurine, 3-methyl adenosine, 5-propynylcytidine, USA, 84:648; and PCT Publication WO 88/09810; all of 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-meth which are incorporated herein by reference). ylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoad 0.178 Nucleic acid PAK modulators of the present inven enosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thio tion (including RNAi-inducing agents, antisense oligonucle cytidine), chemically or biologically modified bases (e.g., otides, ribozymes, triple-helix inducing agents, etc.) may be methylated bases), modified Sugars (e.g. 2'-fluororibose, prepared according to any available technique including, but 2'-aminoribose, 2’-azidoribose, 2'-O-methylribose, L-enan not limited to chemical synthesis, enzymatic synthesis, enzy tiomeric nucleosides arabinose, and hexose), modified phos matic or chemical cleavage of a longer precursor, etc. Meth phate groups (e.g., phosphorothioates and 5'-N-phosphora ods of synthesizing RNAS are known in the art (see, e.g., Gait, midite linkages), and combinations thereof. Natural and M. J. (ed.) Oligonucleotide synthesis: a practical approach, modified nucleotide monomers for the chemical synthesis of Oxford Oxfordshire, Washington, D.C.: IRL Press, 1984; nucleic acids are readily available. In some cases, nucleic and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods acids comprising Such modifications display improved prop and applications, Methods in molecular biology, v. 288 (Clif erties relative to nucleic acids consisting only of naturally ton, N.J.) Totowa, N.J.: Humana Press, 2005; both of which occurring nucleotides. In some embodiments, nucleic acid are incorporated herein by reference). modifications described herein are utilized to reduce and/or 0179 Nucleic acid modulators of PAK may comprise prevent digestion by nucleases (e.g. exonucleases, endonu naturally occurring nucleosides, modified nucleosides, natu cleases, etc.). For example, the structure of a nucleic acid may rally occurring nucleosides with hydrocarbon linkers (e.g., an be stabilized by including nucleotide analogs at the 3' end of alkylene) or a polyether linker (e.g., a PEG linker) inserted one or both strands order to reduce digestion. between one or more nucleosides, modified nucleosides with 0182 Modified nucleic acids need not be uniformly modi hydrocarbon or PEG linkers inserted between one or more fied along the entire length of the molecule. Different nucle nucleosides, or a combination of thereof. In some embodi otide modifications and/or backbone structures may exist at ments, nucleotides or modified nucleotides of a nucleic acid various positions in the nucleic acid. One of ordinary skill in can be replaced with a hydrocarbon linker or a polyether the art will appreciate that the nucleotide analogs or other linker provided that the binding affinity, selectivity, and/or modification(s) may be located at any position(s) of a nucleic US 2010/0247552 A1 Sep. 30, 2010 22 acid such that the function of the nucleic acid is not Substan 1%, at least 5%, at least 10%, or at least 20% of the codons tially affected. The modified region may be at the 5'-end have been altered such that the sequence is optimized for and/or the 3'-end of one or both strands. For example, modi expression in the host cell. fied nucleic acids in which approximately 1 to approximately 0187. Nucleic acid variants may be naturally occurring, 5 residues at the 5' and/or 3' end of either of both strands are Such as allelic variants (same locus), homologs (different nucleotide analogs and/or have a backbone modification have locus), and/or orthologs (different organism) and/or may be been employed. The modification may be a 5' or 3' terminal non naturally occurring. Non-naturally occurring variants modification. One or both nucleic acid strands may comprise may be made by mutagenesis techniques, including those at least 50% unmodified nucleotides, at least 80% unmodified applied to polynucleotides, cells, and/or organisms. Variants may contain nucleotide Substitutions, deletions, inversions nucleotides, at least 90% unmodified nucleotides, or 100% and insertions. Variation may occur in the coding and/or unmodified nucleotides. non-coding regions. Variations may produce conservative 0183 Nucleic acids in accordance with the present inven and/or non-conservative amino acid Substitutions (as com tion may, for example, comprise a modification to a Sugar, pared in the encoded product). nucleoside, or internucleoside linkage Such as those 0188 It is not intended that the present invention be lim described in U.S. Patent Publications 2003/0175950, 2004/ ited by the nature of the nucleic acid employed. In some 0192626, 2004/0092470, 2005/0020525, and 2005/0032733 embodiments, nucleic acids in accordance with the present (all of which are incorporated herein by reference). The invention are not synthetic, but are naturally-occurring enti present invention encompasses the use of any nucleic acid ties that have been isolated from their natural environments. A having any one or more of the modification described therein. nucleic acid may be a naturally-occurring and/or non-natu For example, a number of terminal conjugates, e.g., lipids rally-occurring (e.g. chemically-synthesized, artificial, man Such as cholesterol, lithocholic acid, aluric acid, or long alkyl made, etc.) nucleic acid. branched chains have been reported to improve cellular (0189 In some embodiments, nucleic acids are derived uptake. Analogs and modifications may be tested, e.g., using and/or obtained from natural Sources (e.g. viral, fungal, bac any appropriate assay known in the art. In some embodi terial, animal and/or plant sources). Alternatively or addition ments, nucleic acids in accordance with the present invention ally, nucleic acids of the present invention may be prepared by may comprise one or more non-natural nucleoside linkages. any conventional means typically used to prepare nucleic In Some embodiments, one or more internal nucleotides at the acids in large quantity. For example, DNAs and/or RNAs may 3'-end, 5'-end, or both 3'- and 5'-ends of the nucleic acid are be chemically synthesized using commercially available inverted to yield linkages such as a 3'-3' linkage or a 5'-5' reagents and/or synthesizers by methods that are well-known linkage. in the art (see, e.g., Gait, Oligonucleotide Synthesis. A Prac 0184. Nucleic acids comprising modified internucleoside tical Approach, IRL Press, Oxford, England, 1985; incorpo linkages may be synthesized using reagents and/or methods rated herein by reference). RNAs may be produce in high that are well known in the art. For example, methods for yield via in vitro transcription using any of a variety of plas synthesizing nucleic acids containing phosphonate phospho mids known in the art that are suitable for in vitro transcrip rothioate, phosphorodithioate, phosphoramidate methoxy tion, such as pSP72 (Promega Corporation, Madison, Wis.), ethyl phosphoramidate, formacetal, thioformacetal, diisopro pBluescript (Stratagene, La Jolla, Calif.), etc. pylsilyl acetamidate, carbamate, dimethylene-Sulfide 0190. Nucleic acids may be purified by any suitable (—CH2—S CH), dimethylene-sulfoxide ( CH, SO— means, as are well known in the art. For example, the nucleic CH), dimethylene-sulfone ( CH, SO CH). 2'-O- acids may be purified by reverse phase and/or ion exchange alkyl, and/or 2'-deoxy 2'-fluoro phosphorothioate inter HPLC, size exclusion chromatography, and/or gel electro nucleoside linkages are well known in the art (Uhlmann et al., phoresis. Of course, the skilled artisan will recognize that the 1990, Chem. Rev., 90:543; Schneider et al., 1990, Tetrahe method of purification will depend in part on the size of the dron Lett., 31:335; and references therein; all of which are nucleic acid to be purified. incorporated herein by reference). (0191 Small Molecule PAK Modulators 0185. In some embodiments, a nucleic acid, may comprise 0.192 In some embodiments, PAK modulators may com phosphodiester linkages and/or modified linkages such as prise small molecules. For example, U.S. Patent Application phosphotriester, phosphoramidate, siloxane, carbonate, car 2004/007504 (incorporated herein by reference) describes boxymethylester, acetamidate, carbamate, thioether, bridged heterobicyclic pyrazole derivatives that inhibit PAK4 or phosphoramidate, bridged methylene phosphonate, bridged PAK5 and may be used to treat Alzheimer's disease. In some phosphoramidate, bridged phosphoramidate, bridged meth embodiments, heterobicyclic pyrazole derivatives may be ylene phosphonate, phosphorothioate, methylphosphonate, tested for use in treatment of FXS and/or other neurodevel phosphorodithioate, bridged phosphorothioate and/or Sul opmental disorders. fone linkages, and/or combinations of Such linkages. 0193 In some embodiments, Emodin is a small molecule 0186. It will further be understood that, where a heterolo that can be used in the treatment of FXS and/or other neu gous polypeptide is to be expressed in a host cell, it will often rodevelopmental disorders (see, e.g. Example 12). Emodin be desirable to utilize nucleic acid sequences encoding the (also known as 1.3,8-Tri-hydroxy-6-methyl-anthra-quinone; polypeptide that have been adjusted to accommodate codon 6-Methyl-1,3,8-tri-hydroxy-anthra-quinone; Emodol; and preferences of the host cell and/or to link the encoding Frangula-emodin) is a member of a large family of naturally sequences with regulatory elements active in the host cell. occurring anthraquinones and an active ingredient in Chinese Nucleic acids of the present invention may be chosen for herbal medicine. Emodin inhibits HER-2/neutyrosine kinase having codons, which may or may not be preferred for a activity (Zhang et al., 1999, Clin. Cancer Res., 5:343; incor particular expression system. For example, the nucleic acid porated herein by reference) and therefore has anti-cancer may be one in which at least one codon, or in which at least effects in HER-2/neu-overexpressing breast cancer cells (Ja US 2010/0247552 A1 Sep. 30, 2010 yasuriya et al., 1992, J. Nat. Prod., 55:696; incorporated phosphatase PP2A, POPX1, and/or POPX2). Any of these herein by reference). More recently, it has been demonstrated factors may be targeted by PAK modulators in accordance that 40 uMEmodin inhibits human cancer cell migration by with the present invention. interfering with the formation of an active Cdc42/Rac1 and 0200. The present invention encompasses the recognition PAK complex (Huang et al., 2005, Cell. Mol. Life Sci., that PAK modulators may indirectly modulate ERK activity. 62:1167; incorporated herein by reference). For example, considering that PAK functions upstream of (0194 In some embodiments, OSU-03012 (FIGS. 6 and 7) ERK, aberrant activation of PAK (FIG. 9) could cause in the is a small molecule that can be used in the treatment of FXS constitutive activation of ERK pathway kinases. Down-regu and/or other neurodevelopmental disorders (see, e.g. lation of negative regulators of the ERK pathway, Such as Example 12). OSU-03012 (Zhu et al., 2004, Cancer Res., mitogen-activated protein (MAP) kinase phosphatases 64:4309; incorporated herein by reference; also known as (MKPs) and Sprouty proteins, would also result in the con 2-amino-N-(4-5-(2-phenanthrenyl)-3-(trifluoromethyl)- stitutive activation of ERK (see, e.g., Kohno and Pouyssegur, 1H-pyrazol-1-yl-phenyl)acetamide) is derived from cele 2006, Annals Med., 38:200; incorporated by reference coxib. Celecoxib, sold by Pfizer under the brand name CELE herein). BREXR, is a nonsteroidal anti-inflammatory drug that works 0201 AS previously shown, FMR1 KO mice exhibit through the inhibition cyclooxygenase-2 (COX-2). Cele enhanced basal ERK phosphorylation (and presumably activ coxib can be used for the treatment of many conditions ity) (Hou et al., 2006, Neuron, 51:41; incorporated herein by including but not limited to osteoarthritis, rheumatoid arthri reference) while ERK is phosphorylated and activated by tis, analgesia, familial adenomatous polyposis, and cancer. PAK at least in non-neuronal cells (Eblenet al. 2002, Mol Cell Unlike celecoxib, its analog OSU-03012 is not a COX-2 Biol, 22:6023; incorporated herein by reference). ERK is inhibitor, but instead inhibits 3-phosphoinositide-dependent involved in regulation of spine morphology, synaptic plastic protein kinase-1 (PDK-1) and PAK (Porchia et al., 2007, Mol. ity and behaviors (Selcher et al., 2001, Learn Mem, 8:11; and Pharmacol., 72: 1124; incorporated herein by reference). Kelleher et al., 2004, Cell, 116:467; both of which are incor OSU-03012 directly inhibits PAK kinase activity. While not porated herein by reference), therefore, it is possible that PAK wishing to be bound by any one theory, inhibition probably inhibition returns the levels of phospho-ERK in FMR1 KO occurs via competitive inhibition of ATP binding, with IC50 mice to wild-type levels, thereby reversing phenotypes in value of about 500 nM-1 M (Brader and Eccles, 2004, FMR1 KO mice. Tumori, 90:2; incorporated herein by reference). 0202 As shown in FIG. 9, the ERK signaling cascade, OSU-03013 is another derivative of celecoxib that is struc starting from Ras and going downstream, comprises Ras, Raf, turally similar to OSU-03012. Although OSU-03013 has MEK, and ERK. PAK has been shown to phosphorylate and been shown to inhibit PAK activity, it is toxic in vivo and, activate both MEK-1 (Frost et al., 1997, EMBO.J., 16:6426: therefore, not desirable for use as a therapeutic agent (Brader incorporated herein by reference) and Raf-1 (King et al., and Eccles, 2004, Tumori., 90:2; incorporated herein by ref 1998, Nature, 396: 80; and Chaudhary et al., 2000, Curr: erence). Biol., 10:551; both of which are incorporated herein by ref (0195 Activities of PAK Modulators erence). 0196. In some embodiments, PAK modulators target PAK 0203 Thus, the present invention encompasses the recog directly. In some embodiments, PAK modulators target PAK nition that inhibiting any member of the ERK signaling path indirectly. For example, PAK modulators might target down way (e.g. ERK, MEK, Ras, Raf) can be useful for treatment of stream effectors of PAK, upstream effectors of PAK, and/or FXS and/or other neurodevelopmental disorders. The present natural binding partners of PAK. invention provides methods for treating FXS and/or other (0197) PAK Participates in Multiple Signaling Pathways neurodevelopmental disorders comprising administering a 0198 PAK is known to participate in a variety of signaling therapeutically effective amount of an ERK pathway inhibi pathways. To give but one example, FIG. 8 (Klann and Dever, tor (or pharmaceutical composition comprising an ERK path 2004, Nat. Rev. Neurosci., 5:931) shows how PAK fits within way inhibitor) to a patient Susceptible to, Suffering from, the context of the extracellular signal-regulated kinase (ERK) and/or exhibiting one or more symptoms of FXS and/or other and phosphoinositide-3 kinase (PI3K) signaling pathways. neurodevelopmental disorder. The general description of PAK falls downstream of PDK1, PDK2, and PI3K. PAK falls PAK modulators can be applied to ERK pathway inhibitors as upstream of Ras, Raf. MEK, ERK, and Mnk. well. To give but one example, ERK pathway inhibitors may (0199 Although not pictured in FIG.8, PAK falls upstream be proteins, nucleic acids, Small molecules, glycoproteins, of Myosin light chain kinase (MLCK), regulatory Myosin proteoglycans, lipids, and/or carbohydrates, as described light chain (R-MLC), Myosins I heavy chain, myosin II heavy herein. chain, Myosin VI, Caldesmon, Desmin, Op.18/stathmin, Mer 0204 The signaling activity of Ras is dependent upon its lin, FilaminA, LIM kinase (LIMK), p47, BAD, caspase3, association with the inner face of the plasma membrane, and estrogen and/or progesterone receptors, RhoGEF, GEF-H1, this association is dependent upon a post-translational modi NET1, Gaz, phosphoglycerate mutase-B, RhoGDI, prolactin, fication that places afarnesyl group on a cysteine residue near p41', and/or Aurora-A (Bokoch et al., 2003, Annu. Rev. the C-terminus of Ras. This modification is catalyzed by Biochem., 72:743; and Hofmann et al., 2004, J. Cell Sci., farnesyltransferase (FTase). Accordingly, FTase inhibitors 117:4343; both of which are incorporated herein by refer have been developed as anti-Ras compounds. In some ence). The following factors bind to PAK in cells and may fall embodiments, ERK pathway modulators may be FTase downstream of PAK: CIB. sphingolipids; G-protein Band/or inhibitors. Exemplary FTase inhibitors include, but are not y subunits; PIX/COOL: GIT/PKL: Nef: Paxillin; NESH: limited to, FTI-276, FTI-2148, L-739,750, and BZA-2B SH3-containing proteins (e.g.Nck and/or Grb2); kinases (e.g. (Sebti and Der, 2006, Nat. Rev. Cancer, 3:945; Zhu et al., Akd, PDK1, PI 3-kinase/p85, Cdk5, Cdc2, Src kinases, Abl, 2003, Curr: Opin. Investig. Drugs, 4: 1428; both of which are and/or protein kinase A (PKA)); and/or phosphatases (e.g. incorporated herein by reference); AZD-3409 (AstraZeneca; US 2010/0247552 A1 Sep. 30, 2010 24

Lavelle, 1998, Exp. Opin. Invest. Drugs, 7:1015; Williams, phosphorylate substrates (Wan et al., 2004, Cell, 116:855; 1998, Curr. Opin. Ther: Pat., 8:553; Singh and Lingham, incorporated herein by reference). In some embodiments, 2002, Curr: Opin. Drug Discov. Develop., 5:225; Wilson et ureas that can be utilized as ERK pathway modulators may be al., 2004, Eur: J. Cancer. Suppl., 2: Abstract354; Smethurst et urea derivatives (e.g. benzylic ureas, pyridopyrimidinones, al., 2004, Eur: J. Cancer Suppl., 2: Abstract 376; Kelly et al., heteroaryl-substituted diaryl ureas, annelated ureas, etc.). 2005, Proc. Amer: Assoc. Cancer Res., 46: Abstract 5962; all 0209. In some embodiments, ureas in accordance with the of which are incorporated herein by reference); Tipifarnib present invention include urea bioisosteres (see, e.g., Smith et al., 2006, Curr. Topics Med. Chem., 6:1071 and references (R-115777, ZarnestraTM; End et al., 2001, Cancer Res., therein; incorporated herein by reference). In some embodi 61:131: Cunningham et al., 2002, Proc. Amer: Soc. Clin. ments, urea bioisosteres include, but are not limited to, com Oncol., 21: Abstract 502; Lancet et al., 2004, Blood, 104: pounds in which methylene and/or carbonyl moieties have Abstract 874; and Adjei et al., 2001, Proc. Amer: Soc. Clin. been inserted into the urea functionality; glycinamides in Oncol., 20: Abstract 320; all of which are incorporated herein which a methylene moiety has been introduced into the urea; by reference); and/or lonafarnib (Schering-Plough; Sch oxamides having a second carbonyl group added to the urea; 66336, SarasarTM; Yang et al., 2005, Proc. Amer: Soc. Clin. malonamides having both a carbonyl and a methylene moiety Oncol., 24: Abstract 5565; Long et al., 2005, Proc. Amer: inserted into the urea functionality; diacyl hydrazines; Assoc. Cancer Res., 46: Abstract 5968; and Oh et al., 2005, 2-amino-benzimidazole derivatives; and/or pyrrollecarboxa Proc. Amer: Assoc. Cancer Res., 46: Abstract 5967; all of mides. which are incorporated herein by reference). 0210. In some embodiments, ERK pathway modulators in 0205. In some embodiments, ERK pathway modulators accordance with the present invention include bis-aryl imi may be antisense inhibitors of Raf (see, e.g., Smith et al., dazoles and related structures (see, e.g., Smith et al., 2006, 2006, Curr. Topics Med. Chem., 6:1071; and Sridhar et al., Curr. Topics Med. Chem., 6:1071 and references therein; 2005, Mol. Cancer Ther., 4:677; and references therein, both incorporated herein by reference). In some embodiments, of which are incorporated herein by reference). For example, bis-aryl imidazoles include, but are not limited to, the Rafantisense ISIS-5132 (ISIS Pharmaceuticals/Novartis) SB-203580, L-779,450, SB-590885, pyridyl-naphthyl-imi was designed as a 20-mer phosphorothioate oligonucleotide dazoles, and/or derivatives thereof. 0211. In some embodiments, ERK pathway modulators in to inhibit translation of the Raf-1 RNA message into protein accordance with the present invention include benzamides (Lau et al., 1998. Oncogene, 16:1899: Koller et al., 2000, Tr: (see, e.g., Smith et al., 2006, Curr: Topics Med. Chem., 6:1071 Pharm. Sci., 21:142; Monia et al., 1996, Nature Med. 2:668; and references therein; incorporated herein by reference). In and Monia et al., 1996, Proc. Natl. Acad. Sci., USA 93:15481: some embodiments, benzamides include, but are not limited all of which are incorporated herein by reference). To over to, ZM-336372, imatinib, AMN-107, and/or derivatives come degradation and improve intracellular delivery of ISIS thereof. 5132, a liposomal formulation (i.e. LErafAON) is under 0212. In some embodiments, ERK pathway modulators in investigation. accordance with the present invention include OXindoles (see, 0206. In some embodiments, ERK pathway modulators in e.g., Smith et al., 2006, Curr. Topics Med. Chem., 6:1071 and accordance with the present invention include Raf kinase references therein; incorporated herein by reference). In destabilizers (see, e.g., Sridhar et al., 2005, Mol. Cancer Some embodiments, OXindoles include, but are not limited to, Ther., 4:677 and references therein; incorporated herein by GW-5074. reference). In some embodiments, OXindoles include, but are 0213. In some embodiments, ERK pathway modulators in accordance with the present invention include PTK-787, not limited to, geldanamycin and MCP1. thienopyrimidines, styrene (see, e.g., Smith et al., 2006, Curr: 0207. In some embodiments, ERK pathway modulators in Topics Med. Chem., 6:1071 and references therein; incorpo accordance with the present invention include Small molecule rated herein by reference). inhibitors of MEK (see, e.g., Kohno and Pouyssegur, 2006, 0214. Alternatively or additionally, PAK is activated by Annals Med., 38:200; incorporated herein by reference). In PI3K signaling via PDK, and PI3K signaling is involved in some embodiments, small molecule inhibitors of MEK protein synthesis (Hou and Klann, 2004, J. Neurosci., include, but are not limited to, PD98059, UO126, PD184352 24:6352; incorporated herein by reference). Thus, the present (CI-1040), PD0325901, and/or ARRY-14886. invention encompasses the recognition that, a PAK modulator 0208. In some embodiments, ERK pathway modulators may be an inhibitor of the PI3K/PDK pathway. may be ureas (see, e.g., Smith et al., 2006, Curr. Topics Med. 0215 Biological Activities of PAK Modulators Chem., 6:1071 and references therein; incorporated herein by 0216. In some embodiments, substances that are known to reference). Ureas may be derivitized in any way that makes modulate serine/threonine kinase activity may accordingly them suitable as ERK pathway modulators in accordance modulate PAK kinase activity, including but not limited to with the present invention. In some embodiments, ureas may Staurosporin, PD098059, Genistein, tyrphostin B42, be, for example, biaryl ureas, diphenyl ureas, heteroaryl aryl HA1077, K252a, H-7: (1(5-isoquinoline-sulfonyl)-2-meth ureas, quinolinyl ureas, isoquinolinyl ureas, pyridinyl ureas, ylpiperazine), CEP-1347, etc., including analogs, derivatives, etc. Exemplary ureas that may be utilized as ERK pathway and/or mimetics thereof, that retain the ability to modulate modulators include, but are not limited to, 3-thienyl urea 7. PAK activity. isoxazole, pyrazole, and/or BAY 43-9006. BAY 43-9006 0217 list Table 3 Kumar and Table 2 Eswaran (also known as Sorafenib), a novel biary1 urea, inhibits Raf-1 0218. In some embodiments, PAK modulators such as the kinase activity in vitro. The crystal structure of the Raf/BAY ones described in Eswaren et al. (2007, Structure, 15:201; 43-9006 complex revealed that the inhibitor binds in the incorporated herein by reference) and Kumar et al. (2006, adenosine triphosphate (ATP) pocket and interacts with resi Nat. Rev. Cancer, 6:459; incorporated herein by reference) dues of the kinase activation loop of Raf proteins. This inter are used to treat FXS and/or other neurodevelopmental dis action prevents the activation loop and the catalytic residues orders. See, for example, Table 1 (adapted from Eswaren et from adopting a conformation that is competent to bind and al.) and Table 2 (adapted from Kumar et al.): US 2010/0247552 A1 Sep. 30, 2010

TABLE 1.

Exemplary PAK Modulators

Compound Tm Shift ( C.) % Activity at 10 M

Name Chemical Structure PAK4 PAKS PAK6 PAK4 PAKS PAK6

Cokl Inhibitor 7.O- 1.5 7.1 O.3 7.O-O.3 56 12 23

HN C N y

N - N - N NH2 H )

Cdk12 6.5 O.2 5.6 O.3 5.6 O.8 62 7.0 18 Inhibitor III F f -N". HN > F N-N

H- N > NH2

Purvalanol A S.O. O.3 4.5 + 0.2 5.4 0.5 2O 24 48 HN y C No N - N - N

K252a. H 4.5 O.3 5.9 O.3 8.6 1.0 16 22 16 O N US 2010/0247552 A1 Sep. 30, 2010 26

TABLE 1-continued Exemplary PAK Modulators Compound Tm Shift ( C.) % Activity at 10 M

Name Chemical Structure PAK4 PAKS PAK6 PAK4 PAKS PAK6

Staurosporine H 13.1 - 15 12.5 O.3 16.6 OS O O O O N

N N O

O - NHN2

SU11652 6.4 12 53 O2 53 - 0.3 34 43 75

/ \

C / N

TABLE 2 Exemplary PAK1 Inhibitors Inhibitor Mode of Action References hPIP Binds to regulatory domain and blocks Xia et al., 2001, Proc. Natl kinase activity Acad. Sci., USA, 98: 6174 Merlin Binds to PBD and inhibits recruitment to Kissil et al., 2003, Mol. Cell, ocal adhesions 12: 841 Nischarin Binds to kinase domain and inhibits Alahari et al., 2004, kinase activity EMBO J., 23: 2777 P35, CDK5 Phosphorylates Pak and inhibits kinase Nikolic et al., 1998, Nature, activity 395: 194; and Rashid et al., 2001, J. Biol. Chem., 276: 49043 CDC2 Phosphorylates XPAK2 and inhibits Cau et al., 2000, J. Biol. kinase activity Chem., 275: 2367 p 110C Binds to part of kinase domain and Chen et al., 2003, J. Biol. inhibits kinase activity Chem., 278: 20029 POPX1, POPX2 Dephosphorylation of T422 in kinase Koh et al., 2002, Curr. Biol., activation loop 12:317 CRIPak Binds to regulatory domain and blocks Talukder et al., 2006, kinase activity Oncogene, 25: 1311 PAK1 aa 89- Competitive Inhibitor Zhao et al., 1998, Mol. Cell. 143 peptide Biol., 18:2153 CEP-1347 Small molecule - ATPantagonist Nheu et al., 2002, Cancer J., 8:328 GL-2003 ERK inhibitor that blocks Pak activation Hirokawa et al., 2006, Cancer Lett. CDC2, cell division cycle 2: CDK5, cyclin-dependent kinase 5: CRIPak, cysteine-rich inhibitor of PAK1; ERK, extracellular signal regulated kinase; hPIP, human PAKPLC interacting protein 1; PBD, p21-binding domain * The contents of all of which are incorporated herein by reference US 2010/0247552 A1 Sep. 30, 2010 27

0219. In some embodiments, PAK modulators may affect capable of binding to PAK may be used. A PAK modulator the ability of PAK to interact with its natural binding partners, may include a peptide or other Small molecule which is including but not limited to FMRP. In certain embodiments, capable of modulating the binding interaction. such binding blocks the interaction between PAK and its 0224. In some embodiments, PAK modulators are sub natural binding partners (for example, the interaction stances which bind to and/or block the kinase domain of PAK between PAK and FMRP). In some embodiments, such bind and/or the p21-binding domain of PAK, and/or the autophos ing promotes the interaction between PAK and its natural binding partners. However, a PAK modulator need not nec phorylation sites of PAK. In some embodiments, PAK modu essarily bind directly to a catalytic and/or binding site, and lators are short peptides comprising sequences of PAK that may bind, for example, to an adjacent site. Such as an adjacent dominant-negative activity (Kiosses et al., 2002, Circ. Res., site in the PAK polypeptide. A PAK modulator may even bind 90:697; incorporated herein by reference). In some embodi to another Substance (for example, a protein, lipid, carbohy ments, such peptides may not block PAK kinase activity per drate, etc. which is complexed with the enzyme), so long as its se, but may displace PAK from sites of action within the cell, binding modulates PAK activity. which may indirectly modulate PAK activity. 0225. In certain embodiments, PAK modulators may func 0220. The present invention encompasses the discovery tion to alter the ability of PAK to phosphorylate its substrates. that the integrity of the FMRP KH domains facilitates For example, U.S. Pat. No. 6,383,734 (incorporated herein by FMRP's interaction with PAK. In specific embodiments, reference) describes agents that inhibit PAK function by PAK modulators may affect the integrity of one or more KH inhibiting the ability of PAK to phosphorylate Raf. U.S. Pat. domains of FMRP and modulate its ability to bind to PAK. Nos. 6,013,500 and 6,667,168 (both of which are incorpo 0221. In some embodiments, a PAK modulator binds to a rated herein by reference) describe agents that block the ATP natural binding partner of PAK and inhibits and/or promotes binding domain of PAK4 in order to inhibit the kinase func the interaction of PAK with its natural binding partner. In tion of PAK4. another aspect, a PAK modulator binds to PAK and inhibits and/or promotes the interaction of a natural binding partner of 0226. In certain embodiments, PAK modulators may func PAK with PAK. Some modulators that regulate PAK function tion by altering the activity and/or expression of PAK activa in this manner have been described in the art. For example, tors. For example, U.S. Pat. No. 6,046,224 (incorporated U.S. Patent Application 2004/0208880 (incorporated herein herein by reference) describes agents that inhibit PAK func by reference) describes compounds that inhibit PAK1 func tion by blocking 12(S)HETE receptors. 12(S)HETE stimu tion by modulating its interaction with dynein light chain lates PAK activity, so blocking 12(S)HETE function indi 1/protein inhibitors of nitric oxide synthase (DLC1/PIN). rectly inhibits PAK function. U.S. Patent Application 2006/0172360 (incorporated herein 0227. In certain embodiments, inventive PAK modulators by reference) describes compounds that inhibit PAK4 by may comprise phosphatases that inhibit PAK function by modulating its interaction with MKK7. U.S. Patent Applica removing phosphate groups from targets that are phosphory tion 2004/009 1907 (incorporated herein by reference) lated by PAK kinase. Alternatively or additionally, inventive describes compounds that inhibit PAK4 by modulating its PAK modulators may comprise kinases that indirectly acti interaction with GEF/H1 (used to treat cancer). U.S. Patent vate PAK function by phosphorylating targets that are phos Application 2002/0106690 (incorporated herein by refer phorylated by PAK kinase. ence) describes inhibitors that function by altering the inter 0228. In some embodiments, PAK modulators may com action between PAK and the beta subunit of G-protein prise the autoinhibitory domain of PAK, which may block coupled receptors. U.S. Patent Application 2006/0088897 and/or reduce PAK activity. In specific embodiments, the (incorporated herein by reference) describes substances that candidate Substance is an autoinhibitory region of PAK pro modulate PAK by altering the interaction between PAK and tein, such as a peptide comprising at least 25, 50, 75, 100, 125, SH3 domain-containing proteins. U.S. Pat. Nos. 6,013,500 or 150 contiguous amino acids of PAK. In certain embodi and 6,667,168 (both of which are incorporated herein by ments, the protein includes at least 25, 50, 75, 100, 125, 150, reference) describe agents that bind to the GTP-binding 200, or 300 of the N-terminal amino acids of PAK protein. In domain of PAK4 and prevent PAK4 from binding to GTP Some embodiments, PAK modulators may comprise a domi binding proteins and agents that bind the PAK4 cdc42-bind nant-negative PAK protein (e.g., a mutant and/or a character ing domain and prevent PAK4 from interacting with cdc42. istic portion of PAK protein). 0222. In some embodiments, a PAK modulator may bind 0229. In some embodiments, PAK modulators function to to and/or compete for one or more sites on a relevant mol modulate the expression, stability, and/or cellular levels of ecule, for example, a catalytic site and/or a binding site of PAK. For example, U.S. Patent Applications 2004/0102326 PAK. In some embodiments, the PAK modulator interferes (PAK1), 2002/0142325 (PAK2), 2004/0009935 (PAK2), and with and/or inhibits the binding of FMRP to PAK. In certain 2005/0191672 (PAK4) (all of which are incorporated herein embodiments, the PAK modulator competes for an FMRP by reference) describe nucleotide inhibitors of PAK expres binding region of PAK. In some embodiments, the PAK sion that target PAK mRNA for degradation. modulator competes for a PAK-binding region of FMRP. 0230. Alternatively or additionally, inventive PAK modu 0223) Where modulators of an enzyme such as PAK are lators affect PAK levels by increasing and/or decreasing tran concerned, a modulator may include a substrate of PAK scription and/or translation of PAK, PAK substrates, and/or kinase and/or characteristic portion thereof which is capable natural binding partners of PAK. In some embodiments, PAK of binding to PAK. Alternatively or additionally, whole or modulators may affect RNA and/or protein half-life, for portions of a Substrate isolated from a biological Source (e.g. example, by directly affecting mRNA and/or proteinstability. purified from tissues and/or cells) and/or by chemical synthe In certain embodiments, inventive PAK modulators cause the sis may be used to compete with the substrate for binding sites mRNA and/or protein to be more and/or less accessible and/ on the enzyme. Alternatively or additionally, an antibody or Susceptible to nucleases, proteases, and/or the proteasome. US 2010/0247552 A1 Sep. 30, 2010 28

0231. In some embodiments, inventive PAK modulators 0239. As used herein, the term “test substance” refers to affect the processing of mRNAs encoding PAK, PAK sub (1) a PAK protein, a nucleic acid encoding PAK, and/or strates, and/or natural binding partners of PAK. For example, homolog, portion, variant, mutant, and/or derivative thereof. PAK modulators may function at the level of pre-mRNA (2) a natural binding partner of PAK, a nucleic acid encoding splicing, 5' end formation (e.g. capping), 3' end processing a natural binding partner of PAK, and/or a homolog, portion, (e.g. cleavage and/or polyadenylation), nuclear export, and/or variant, mutant, and/or derivative thereof; (3) an FMRP pro association with the translational machinery and/or ribo tein, a nucleic acid encoding FMRP, and/or a homolog, por Somes in the cytoplasm. tion, variant, mutant, and/or derivative thereof, and/or (4) a 0232. In some embodiments, inventive PAK modulators Substrate of PAK kinase, a nucleic acid encoding a substrate affect translational control and/or post-translational modifi of PAK, and/or a homolog, portion, variant, mutant, and/or cation of PAK, PAK substrates, and/or natural binding part derivative thereof. In some embodiments, a test Substance is a ners of PAK. For example, PAK modulators may function at protein and/or characteristic portion thereof comprising a the level of translation initiation, elongation, termination, FMRP-binding portion of PAK. In some embodiments, a test and/or recycling. In some embodiments, PAK modulators Substance is a protein and/or characteristic portion thereof may function at the step of protein folding into secondary, comprising a PAK-binding portion of FMRP. tertiary, and/or quaternary structures. Alternatively or addi 0240. The efficacy of a candidate substance may be tionally, PAK modulators may function at the level of intra assessed by generating dose response curves from data cellular transport (e.g. ER to Golgi transport, intra-Golgi obtained using various concentrations of the candidate Sub transport, Golgi to plasma membrane transport, and/or secre stance. Moreover, a control assay may be performed to pro tion from the cell). In some embodiments, PAK modulators vide a baseline for comparison. In the control assay, the assay may function at the level of post-translational modification is performed in the absence of a candidate Substance. (e.g. cleavage of signal sequences and/or the addition of enti 0241. In some embodiments, inventive PAK modulators ties Such as methyl groups, phosphates, glycan moieties, etc.). inhibit and/or activate PAK activity by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at 0233. In various embodiments, a PAK modulator is a puri least about 50%, at least about 60%, at least about 70%, at fied and/or unpurified synthetic organic molecule and/or least about 80%, at least about 90%, or greater than about naturally occurring organic molecule. 90% as compared with the activity observed under otherwise 0234. In some embodiments, inventive PAK modulators identical conditions lacking a candidate Substance. may cause the level of PAK mRNA and/or protein, an activity 0242. It will, of course, be understood that all screening of PAK protein, the half-life of PAK mRNA and/or protein, methods of the present invention are useful in themselves the binding of PAK mRNA and/or protein to its natural bind notwithstanding the fact that effective PAK modulators may ing partners, and/or the level and/or activity of a Substance not be found. The invention provides methods for screening that phosphorylates a PAK kinase to decrease by at least about for candidate PAK modulators, not solely methods of finding 5%, at least about 10%, at least about 20%, at least about 30%, them. at least about 40%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, at least about 95%, or 0243 Screening substantially 100%. 0244. In some embodiments, screening for PAK modula tors is employed. In some embodiments, high throughput 0235 PAK modulators may be used alone and/or in con screening for PAK modulators is employed. In some embodi junction with other substances which affect PAK activity. ments, such screening identifies Substances that bind to PAK. Additional examples of PAK modulators will be apparent to Typically, large numbers of candidate Substances are immo the skilled person. bilized on a solid substrate. Immobilized candidate sub 0236 Identification and/or Characterization of PAK stances are contacted with PAK and washed. Bound PAK is Modulators then detected by methods well known in the art. 0237. The present invention provides methods of identi 0245 Using high throughput assays, it is possible to fying PAK modulators. In some embodiments, inventive screen up to several thousand candidate Substances in a single methods screen for novel PAK modulators by identifying day. In some embodiments, each well of a microtiter plate can substances that improve and/or treat the symptoms of FXS be used to run a separate assay against a selected candidate and/or other neurodevelopmental disorders. In some embodi Substance, or, if concentration and/or incubation time effects ments, inventive methods identify novel PAK modulators by are to be observed, every 5-10 wells cantest a single candidate identifying substances that affect PAK’s ability to interact Substance. Thus, a single standard microtiter plate can assay with its natural binding partners (e.g. FMRP). In some up to 96 candidate substances. If 1536 well plates are used, embodiments, inventive methods identify novel PAK modu then a single plate can assay up to 1536 candidate Substances. lators by identifying substances that modulate PAK kinase It is possible to assay many plates per day; assay screens for activity. In some embodiments, inventive methods identify up to about 6,000, about 20,000, about 50,000, or more than PAK modulators by identifying substances that modulate about 100,000 different candidate substances are possible PAK expression and/or levels. using high throughput systems in accordance with the present 0238. In some embodiments, inventive methods identify invention. Substances that are known to have a particular function, but 0246 For solid state reactions, a candidate substance may were not previously known to function as PAK modulators. In be bound to the solid state component, directly or indirectly, Some embodiments, inventive methods identify Substances via covalent and/or non covalent linkage e.g., via a tag. A tag that have been identified and/or synthesized, but have not may comprise any of a variety of components. In general, a been attributed any particular function. In some embodi Substance which binds the tag (a tag binder) is fixed to a solid ments, inventive methods identify novel substances that have Support, and the tagged candidate Substance is attached to the never been identified and/or synthesized before. Solid Support by interaction of the tag and/or the tag binder. US 2010/0247552 A1 Sep. 30, 2010 29

0247. A number of tags and/or tag binders may be used, Chemistry, 39(4):718; and Kozal et al., 1996, Nature Medi based upon known molecular interactions well described in cine, 2:753; all describing arrays of biopolymers fixed to solid the literature. For example, where a tag has a natural binder, substrates; all of which are incorporated herein by reference). for example, biotin, protein A, and/or protein G, it may be Non-chemical approaches for fixing tag binders to Substrates used in conjunction with appropriate tag binders (avidin, include other common methods, such as heat, cross-linking streptavidin, neutravidin, the Fc region of an immunoglobu by ultraviolet radiation, and the like. lin, etc.). Antibodies to molecules with natural binders such as (0252) In Vitro Assays biotin and/or appropriate tag binders are widely available 0253) In vitro assays can often be run quickly and/or in (Sigma Immunochemicals, St. Louis, Mo.). large numbers, thereby increasing the amount of information 0248 Similarly, any haptenic and/or antigenic compound obtainable in a short period of time. A variety of vessels may may be used in combination with an appropriate antibody to be used to run the assays, including test tubes, plates, dishes, form a tag/tag binder pair. Thousands of specific antibodies microtiter plates, and/or other Surfaces such as dipsticks and/ are commercially available and many additional antibodies or beads. Some in vitro and in cyto assays have been are described in the literature. For example, in one common described, for example, in PCT Publication WO 06/029337 configuration, the tag is a first antibody and the tag binder is (incorporated herein by reference). a second antibody which recognizes the first antibody. In 0254 The present invention provides in vitro methods for addition to antibody-antigen interactions, receptor-ligand screening for PAK modulators. For example, in some interactions are appropriate as tag and/or tag-binder pairs, embodiments, methods may comprise steps of: (1) providing including but not limited to transferrin, c-kit, viral receptor a test Substance (e.g. PAK protein, PAK gene, and/or charac ligands, cytokine receptors, chemokine receptors, interleukin teristic portion thereof); (2) providing at least one candidate receptors, immunoglobulin receptors and/or antibodies, the Substance; and (3) measuring and/or detecting the influence cadherin family, the integrin family, the selectin family, etc. of the candidate Substance(s) on the test Substance. (see, e.g., Pigott et al., The Adhesion Molecule Facts Book I, 0255. In some embodiments, a test substance (e.g. PAK 1993). Similarly, toxins and/or venoms; viral epitopes; hor protein, PAK gene, and/or characteristic portion thereof) is mones (e.g. opiates, Steroids, etc.); intracellular receptors provided and brought directly and/or indirectly into contact (e.g. which mediate the effects of various Small ligands, with a candidate Substance (e.g. in the form of a library). including Steroids, thyroid hormone, retinoids, vitamin D, Then, the influence of the candidate substance on the test and/or peptides); drugs; lectins; carbohydrates; nucleic acids substance is detected and/or measured. Thereafter, suitable (linear and/or cyclic polymer configurations); proteins; phos PAK modulators may be isolated and/or analyzed. For the pholipids; and/or antibodies may interact with various cell screening of libraries, the use of high-throughput assays are receptors. contemplated and described herein. 0249 Synthetic polymers, such as polyurethanes, polyes 0256 In some embodiments, in vitro assays comprise ters, polycarbonates, polyureas, polyamides, polyethylene binding assays. Binding of a candidate Substance to a test imines, polyarylene Sulfides, polysiloxanes, polyimides, and/ Substance (e.g. PAK protein, PAK gene, and/or characteristic or polyacetates may form appropriate tags and/or tag binders. portion thereof) may, in and of itself, be inhibitory, due to Many other tag/tag binder pairs are useful in assay systems steric, allosteric, and/or charge-charge interactions. The test described herein, as would be apparent to one skilled in the Substance may be free in Solution, fixed to a Support, and/or art expressed in and/or on the surface of a cell. The test substance 0250 Common linkers such as peptides, polyethers, and and/or the candidate substance may be labeled, thereby per the like may serve as tags and may include polypeptide mitting detection of binding. The test Substance is frequently sequences. Such as poly-Gly sequences of between about 5 the labeled species, decreasing the chance that the labeling and 200 amino acids. Such flexible linkers are known to will interfere with and/or enhance binding. Competitive bind persons of skill in the art. For example, poly(ethelyne glycol) ing formats may be performed in which one of the Substances linkers are available from Shearwater Polymers, Inc. (Hunts is labeled, and one may measure the amount of free label ville, Ala.). These linkers optionally have amide linkages, versus bound label to determine the effect on binding. Sulfhydryl linkages, and/or heterofunctional linkages. 0257. In some embodiments, binding assays involve, for 0251 Tagbinders are fixed to solid substrates using any of example, exposing a test Substance to a candidate Substance a variety of methods currently available. Solid substrates are and detecting binding between the test Substance and the commonly derivatized and/or functionalized by exposing all candidate Substance. A binding assay may be conducted in and/or a portion of the Substrate to a chemical reagent which vitro (e.g. in a test tube, comprising Substantially only the fixes a chemical group to the Surface which is reactive with a components mentioned; in cell-free extracts; and/or in Sub portion of the tag binder. For example, groups which are stantially purified components). Alternatively or additionally, Suitable for attachment to a longer chain portion include binding assays may be conducted in cyto and/or in vivo (e.g. amines, hydroxyl, thiol, and/or carboxyl groups. Aminoalkyl within a cell, tissue, organ, and/or organism; described in silanes and/or hydroxyalkylsilanes may be used to function further detail below). alize a variety of Surfaces. Such as glass Surfaces. The con 0258. In certain embodiments, at least one candidate sub struction of Such solid phase biopolymer arrays is well stance is contacted with a test Substance (e.g. PAK protein, described in the literature (see, e.g., Merrifield, 1963, J. Am. PAK gene, and/or characteristic portion thereof) and an effect Chem. Soc., 85:21.49, describing solid phase synthesis of, detected. In some embodiments, for example, a candidate e.g., peptides; Geysen et al., 1987, J. Immun. Meth., 102:259, substance is contacted with PAK protein, and binding to PAK describing synthesis of Solid phase components on pins; protein is tested. In some embodiments, an assay may involve Frank et al., 1988, Tetrahedron, 44:6031, describing synthe contacting a candidate Substance with a characteristic portion sis of various peptide sequences on cellulose disks; Fodor et of PAK protein, including but not limited to a FMRP-binding al., 1991, Science, 251:767; Sheldon et al., 1993, Clinical portion of PAK. Binding of the candidate substance to the US 2010/0247552 A1 Sep. 30, 2010 30

PAK peptide is detected. It will be appreciated that fragments, 0265. In some embodiments, screening methods of the portions, homologs, variants, and/or derivatives of PAK may present invention comprise: (1) obtaining a candidate Sub be employed, provided that they comprise FMRP binding stance; and (2) contacting the candidate Substance with a activity. pre-formed PAK (and/or characteristic portion thereof)- 0259. In some embodiments, assays may involve provid FMRP complex; and (3) determining whether the candidate ing a test Substance (e.g. immobilized on a solid Support), and substance affects the PAK (and/or characteristic portion a non-immobilized candidate substance. The extent to which thereof)-FMRP complex. the test Substance and candidate Substance bind to one 0266. In some embodiments, screening methods of the another is determined Alternatively, the candidate substance present invention comprise (1) obtaining a candidate Sub may be immobilized and the test substance non-immobilized. stance; (2) contacting the candidate Substance with PAK (and/ Such assays may be used to identify candidate Substances or characteristic portion thereof) and a natural binding part capable of binding to PAK and/or fragments, portions, ner; and (3) detecting whether the candidate Substance can compete with the binding interaction between PAK (and/or homologs, variants, and/or derivatives thereof. characteristic portion thereof) and the natural binding partner. 0260. In some embodiments, an antibody that recognizes 0267. In some embodiments, a candidate substance is the test substance (e.g. an O-PAK antibody) is immobilized to determined to be a PAK inhibitor if administering the candi a solid Support (e.g. Protein-A beads). The antibody is con date substance to PAK and the natural binding partner results tacted with the test substance, which binds to the immobilized in decreased binding between PAK and the natural binding antibody. The resulting complex is then brought into contact partner. In some embodiments, a candidate Substance is deter with the candidate substance (purified protein, cellular mined to be a PAK inhibitor if administering the candidate extract, combinatorial library, etc.). If the candidate sub Substance to PAK and the natural binding partner results in an stance interacts with the test Substance, the candidate Sub at least 2-fold decrease in binding between PAK and the stance will become indirectly immobilized to the solid sup natural binding partner. In some embodiments, a candidate port. Presence of the candidate substance on the solid support substance is determined to be a PAK inhibitor if administer can be assayed by any standard technique known in the art ing the candidate Substance to PAK and the natural binding (including, but not limited to, western blotting). This type of partner results in an at least 3-fold, at least 4-fold, at least assay is known in the art as an “immunoprecipitation' assay. 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at 0261. In some embodiments, a test substance (e.g. PAK least 100-fold, at least 200-fold, at least 500-fold, at least protein, PAK gene, and/or characteristic portion thereof) is 1000-fold, at least 10,000-fold, or greater than 10,000-fold immobilized on a Solid Support (e.g. agarose beads). In spe decrease in binding between PAK and the natural binding cific embodiments, the test substance is expressed as a GST partner. In some embodiments, a candidate Substance is deter fusion protein in bacteria, yeast, insect cells, and/or higher mined to be a PAK inhibitor if administering the candidate eukaryotic cell line and/or purified from crude cell extracts Substance to PAK and the natural binding partner results in an using glutathione-agarose beads. As a control, binding of the at least 25%, 50%, 75%, 100%, 200%, 500%, 1000%, or candidate substance, which is not a GST-fusion protein, to the greater than 1000% decrease in binding between PAK and the immobilized PAK protein is determined in the absence of natural binding partner. PAK protein. The binding of the candidate substance to the 0268. In some embodiments, a candidate substance is immobilized PAK protein is then determined This type of determined to be a PAK activator if administering the candi assay is known in the art as a “GST pulldown assay. Alter date substance to PAK and the natural binding partner results natively or additionally, the candidate Substance may be in decreased binding between PAK and the natural binding immobilized and the test substance non-immobilized. partner. In some embodiments, a candidate Substance is deter 0262. It is possible to perform this type of assay using mined to be a PAK activator if administering the candidate different affinity purification systems for immobilizing one of Substance to PAK and the natural binding partner results in an the components, for example Ni-NTA agarose- and/or histi at least 2-fold decrease in binding between PAK and the dine-tagged components. natural binding partner. In some embodiments, a candidate 0263 Binding of a test substance to the candidate sub substance is determined to be a PAK activator if administer stance may be determined by a variety of methods well ing the candidate Substance to PAK and the natural binding known in the art. For example, the non-immobilized compo partner results in an at least 3-fold, at least 4-fold, at least nent may be labeled (with for example, a radioactive label, an 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at epitope tag, and/or an enzyme-antibody conjugate). Alterna least 100-fold, at least 200-fold, at least 500-fold, at least tively or additionally, binding may be determined by immu 1000-fold, at least 10,000-fold, or greater than 10,000-fold nological detection techniques. For example, the reaction decrease in binding between PAK and the natural binding mixture may be subjected to Western blotting and the blot partner. In some embodiments, a candidate Substance is deter probed with an antibody that detects the non-immobilized mined to be a PAK activator if administering the candidate component. Alternatively or additionally, enzyme linked Substance to PAK and the natural binding partner results in an immunosorbent assay (ELISA) may be utilized to assay for at least 25%, 50%, 75%, 100%, 200%, 500%, 1000%, or binding. greater than 1000% decrease in binding between PAK and the 0264. In some embodiments, screening methods of the natural binding partner. present invention comprise: (1) obtaining a candidate Sub 0269. In some embodiments, a candidate substance is stance; (2) contacting the candidate Substance with PAK (and/ determined to be a PAK inhibitor if administering the candi or characteristic portion thereof) and FMRP; and (3) detect date substance to PAK and the natural binding partner results ing inhibition and/or activation of binding between PAK and in increased binding between PAK and the natural binding FMRP in the presence and/or absence of the candidate sub partner. In some embodiments, a candidate Substance is deter Stance. mined to be a PAK inhibitor if administering the candidate US 2010/0247552 A1 Sep. 30, 2010

Substance to PAK and the natural binding partner results in an example, incorporation of phosphate groups can affect physi at least 2-fold increase in binding between PAK and the cochemical properties of the Substrate. Such as electro natural binding partner. In some embodiments, a candidate phoretic mobility, light absorbance, fluorescence and/or substance is determined to be a PAK inhibitor if administer phosphorescence, chromatographic properties, etc. Such ing the candidate Substance to PAK and the natural binding alterations of Substrate physicochemical properties can be partner results in an at least 3-fold, at least 4-fold, at least readily measured by one skilled in the art and used as an 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at indicator of kinase activity. least 100-fold, at least 200-fold, at least 500-fold, at least 0273 Alternatively or additionally, it is well known that 1000-fold, at least 10,000-fold, or greater than 10,000-fold monoclonal or polyclonal antibodies can be generated which increase in binding between PAK and the natural binding selectively recognize phosphorylated forms of the Substrate, partner. In some embodiments, a candidate Substance is deter and thus the degree of binding of such antibodies to substrate mined to be a PAK inhibitor if administering the candidate Subsequent to the kinase reaction may be used as an indirect Substance to PAK and the natural binding partner results in an method of determining kinase activity. Furthermore, it is at least 25%, 50%, 75%, 100%, 200%, 500%, 1000%, or known that many kinases, including PAK kinases, possess the greater than 1000% increase in binding between PAK and the capacity to phosphorylated residues on the same kinase mol natural binding partner. ecule. Such phosphorylation reactions are termed autophos 0270. In some embodiments, a candidate substance is phorylation, and therefore measurement of incorporation of determined to be a PAK activator if administering the candi phosphate into PAK itself catalyzed by the same may be used date substance to PAK and the natural binding partner results to monitor PAK activity. Kinase assays Such as those in increased binding between PAK and the natural binding described above may be performed using purified, partially partner. In some embodiments, a candidate Substance is deter recombinant PAK, and/or PAK which is purified from cells mined to be a PAK activator if administering the candidate that naturally express the protein using purification proce Substance to PAK and the natural binding partner results in an dures such as those described above. at least 2-fold increase in binding between PAK and the 0274 ELISA-based assays may be used to screen for natural binding partner. In some embodiments, a candidate novel PAK substrates. One such assay employs random bioti substance is determined to be a PAK activator if administer nylated peptides that can be phosphorylated by a kinase, Such ing the candidate Substance to PAK and the natural binding as PAK. PAK-specificantibodies are immobilized in the wells partner results in an at least 3-fold, at least 4-fold, at least of a microtiter dish, and samples comprising PAK protein 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at may be diluted into a reaction buffer and subsequently added least 100-fold, at least 200-fold, at least 500-fold, at least to plate wells. Reactions are initiated, for example, by the 1000-fold, at least 10,000-fold, or greater than 10,000-fold addition of the biotinylated peptide substrates to the PAK increase in binding between PAK and the natural binding sample. Reactions are stopped by removing the mixtures, and partner. In some embodiments, a candidate Substance is deter then the plates are washed. Afterwashing, Streptavidin-horse mined to be a PAK activator if administering the candidate radish peroxidase (HRP) is added. Thereafter, unbound Substance to PAK and the natural binding partner results in an streptavidin-HRP is removed, the peroxidase color reaction is at least 25%, 50%, 75%, 100%, 200%, 500%, 1000%, or initiated by addition of the peroxidase substrate, and the greater than 1000% increase in binding between PAK and the optical density is measured in a suitable densitometer. natural binding partner. 0275. In some embodiments, a candidate substance sus (0271 The activity of PAK modulators of the present pected of modulating the binding between FMRP and PAK is invention may be determined by, for example, assaying for a candidate Substance Suspected of modulating the phospho kinase activity of PAK. In such assays PAK and/or a charac rylation of FMRP by PAK. For example, the candidate sub teristic portion thereof produced by recombinant means as stance may be a Substance Suspected of promoting the phos described above is contacted with a substrate in the presence phorylation of FMRP by PAK. In some embodiments, a of a suitable phosphate donor (e.g. ATP) containing radiola candidate Substance Suspected of modulating the phosphory beled phosphate, and PAK-dependent incorporation of radio lation of FMRP by PAK pertains to a candidate substance label into the substrate is measured. By “substrate.” one suspected of inhibiting the phosphorylation of FMRP by means any substance containing a suitable hydroxyl moiety PAK that acts as an acceptor for the Y-phosphate group transferred 0276 Alternatively or additionally, PAK activity may be from a donor molecule such as ATP in a reaction catalyzed by measured by assaying its kinase activity, its ability to interact PAK. A substrate may be an endogenous substrate of PAK, with its natural binding partners, and/or by the detection of i.e. a naturally-occurring Substance that is phosphorylated in events that lead to and/or are consequent of PAK activity in unmodified cells by naturally-occurring PAK and/or any intact cells, in cell lysates, and/or in Systems in which signal other substance that is not normally phosphorylated by PAK ing events are reconstituted in vitro. For example, it is known in a physiological situation, by that may be phosphorylated by that PAK proteins bind to and are activated by GTP-binding PAK in the reaction conditions employed. A substrate may be proteins such as Rac and/or Cdc42. Thus detection of inter a protein or peptide, and the phosphorylation reaction may action of PAK with naturally-occurring activators such as occur on a Substrate serine and/or threonine residue. It is Rac/Cdc42, and/or Substrates may be used as an indicator of well-known to those skilled in the art that non-natural Sub PAK activity. strates can act as Suitable Substrates in kinase assays such as (0277. In some embodiments, a candidate substance is that described above. determined to be a PAK inhibitor if administering the candi 0272. It is well known to those skilled in the art that detec date substance to PAK and a substrate results in decreased tion of kinase-dependent Substrate phosphorylation can be ability of PAK to phosphorylate the substrate. In some effected by a number of means other than measurement of embodiments, a candidate Substance is determined to be a radiolabeled phosphate incorporation into the substrate. For PAK inhibitor if administering the candidate substance to US 2010/0247552 A1 Sep. 30, 2010 32

PAK and the substrate results in an at least 2-fold decrease in (Fields et al., 1994, Trends in Genetics, 10:286; and Colas et the ability of PAK to phosphorylate the substrate. In some al., 1998, TIBTECH, 16:355; both of which are incorporated embodiments, a candidate Substance is determined to be a herein by reference). In this assay, yeast cells express a first PAK inhibitor if administering the candidate substance to fusion protein comprising a test Substance in accordance with PAK and the substrate results in an at least 3-fold, at least the present invention (e.g. PAK protein, PAK gene, and/or a 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least characteristic portion thereof) and a DNA-binding domain of 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, a transcription factor Such as Ga14 and/or Lex A. The cells at least 1000-fold, at least 10,000-fold, or greater than additionally contain a reporter gene whose promoter contains 10,000-fold decrease in the ability of PAK to phosphorylate binding sites for the corresponding DNA-binding domain. By the substrate. transforming the cells with a vector that expresses a second 0278. In some embodiments, a candidate substance is fusion protein comprising a candidate Substance fused to an determined to be a PAK activator if administering the candi activation domain (e.g. from Ga14 and/or herpes simplex date substance to PAK and a substrate results in increased virus VP16) expression of the reporter gene may be increased ability of PAK to phosphorylate the substrate. In some if the candidate substance interacts with the test substance. embodiments, a candidate Substance is determined to be a Consequently this assay may be used for Screening for Sub PAK activator if administering the candidate substance to stances that modulate an interaction between PAK and any PAK and the substrate results in an at least 2-fold increase in number of candidate Substances. In this way, it is possible the ability of PAK to phosphorylate the substrate. In some rapidly to identify novel PAK modulators. embodiments, a candidate Substance is determined to be a 0284. The present invention provides assays involving PAK activator if administering the candidate substance to solid phase-bound PAK proteins and detecting their interac PAK and the substrate results in an at least 3-fold, at least tions with one or more candidate Substances. Thus, a test 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least Substance (e.g. PAK protein and/or a characteristic portion 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, thereof) may contain a detectable marker, such as a radioac at least 1000-fold, at least 10,000-fold, or greater than tive, fluorescent, and/or luminescent label. Furthermore, can 10,000-fold increase in the ability of PAK to phosphorylate didate Substances can be coupled to Substances which permit the substrate. indirect detection (e.g. by means of employing an enzyme (0279. In Cyto Assays which uses a chromogenic Substrate and/or by means of bind 0280. In some embodiments, the present invention pro ing a detectable antibody). Changes in the conformation of vides methods for screening for PAK modulators wherein a PAK as the result of an interaction with a candidate substance candidate substance is contacted with a cell. The cell can then may be detected, for example, by the change in the emission be assayed for various parameters associated with PAK activ of the detectable marker. Alternatively or additionally, the ity. For example, parameters associated with PAK activity Solid phase-bound protein complexes may be analyzed by include, but are not limited to, PAK’s ability to interact with means of mass spectrometry. its natural binding partners (e.g. FMRP) and/or PAK’s ability 0285. The present invention provides assays involving to phosphorylate its Substrates. monitoring activities of downstream effectors of PAK. For 0281. In certain embodiments, cells may be directly example, the invention provides assays involving monitoring assayed for binding between FMRP and PAK Immunohis the activity of ERK pathway members and/or PI3K pathway tochemical techniques, confocal techniques, and/or other members. Kinase activity can be assayed using any method techniques to assess binding are well known to those of skill known in the art. To give but one example, kinase activity can in the art. In some embodiments, a cell is assayed for phos be assayed by using phospho-specific antibodies, as the phos phorylation of FMRP by PAK. Various cell lines may be phorylation state of members of these pathways indicates utilized for Such screening assays, including cells specifically whether the protein is active. For example, a Western blot can engineered for this purpose. Examples of cells used in the be conducted for phosphoERK 1/2, and quantification of screening assays include neuronal cells and/or dendritic cells. changes in pathway activity can be obtained by normalization The cell may be a stimulated cell, such as a cell stimulated to total ERK1/2 levels. Alternatively or additionally, the with a growth factor. One of skill in the art would understand activity of downstream members of the ERK signaling path that the invention disclosed herein contemplates a wide vari way (e.g. translation initiation factors S6, eIF4E, 4EBP1, ety of in cyto assays for measuring parameters that correlate etc.) can also be determined with Western blots probed with with the activity of PAK. phospho-specific antibodies against these proteins (Kelleher 0282. Depending on the assay, cell and/or tissue culture et al., 2004, Cell, 116:467; incorporated herein by reference). may be required. A cell may be examined using any of a In some embodiments, PI3K pathways activity can be moni number of different physiologic assays, as discussed above tored through detection of phosphorylated PI3K or down for binding between FMRP and PAK. Alternatively or addi stream signaling proteins Akt and PDK1. tionally, molecular analysis may be performed, including, but 0286. In some embodiments, screening assays may assay not limited to, western blotting to monitor protein expression PAK activity by monitoring the downstream cellular effects and/or test for protein-protein interactions; northern blotting, of PAK activity. Such effects include, but are not limited to, differential display of RNA, and/or microarray analysis to the formation of peripheral actin microSpikes and/or associ monitor mRNA expression; kinase assays to monitor phos ated loss of stress fibers (Zhao et al., 1998, Mol. Cell Biol., phorylation; mass spectrometry to monitor other chemical 18:2153; incorporated herein by reference) and/or other cel modifications; etc. lular responses. Such as growth, growth arrest, differentiation, 0283. The present invention provides methods for identi and/or apoptosis. In some embodiments, yeast cells are used fying substances that bind to PAK and, therefore, may modu for this type of screening assay. For example, in a PAK yeast late PAK activity. One in cyto method of identifying sub functional assay, when cultured on glucose containing stances that bind to PAK is the two-hybrid system assay medium, PAK yeast cells grow like normal yeast cells. Upon US 2010/0247552 A1 Sep. 30, 2010 exposure to galactose, however, intracellular expression of rate, relieve, delay onset of inhibit progression of reduce PAK is induced, causing yeast cells to die. Substances that severity of and/or reduce incidence of one or more symptoms inhibit PAK activity are identified by their ability to prevent or features of FXS and/or other neurodevelopmental disorder. yeast cells from dying. In some embodiments, a candidate Substance comprises a 0287. In some embodiments, PAK levels are determined PAK modulator. Treatment of these animals with candidate by measuring levels of protein and/or mRNA. Levels of PAK Substances may involve the administration of the Substance, protein and/or characteristic portions thereof are measured in an appropriate form, to the animal Administration is dis using immunoassays such as western blotting and/or ELISA cussed in further detail below, in the section entitled “Admin using antibodies that selectively bind to PAK. For measure istration.” ment of mRNA, amplification (e.g., using polymerase chain reaction PCR, ligase chain reaction LCR), etc.) and/or 0294. Accordingly, in some embodiments, the invention hybridization assays (e.g., northern hybridization, RNAse provides screening systems, including methods and/or com protection, dot blotting, etc.) may be used. In some embodi positions, for determining whether a candidate Substance is ments, levels of protein and/or mRNA can be detected using useful for treating, alleviating, ameliorating, relieving, delay directly- and/or indirectly-labeled detection agents, e.g., fluo ing onset of inhibiting progression of reducing severity of rescently and/or radioactively labeled nucleic acids, radioac and/or reducing incidence of one or more symptoms or fea tively and/or enzymatically labeled antibodies, etc. as tures of a FXS in a mammal In some embodiments, a PAK described herein. modulator is identified that can be used to treat, alleviate, 0288 Alternatively or additionally, PAK expression may ameliorate, relieve, delay onset of inhibit progression of be measured using a reporter gene system. Sucha system may reduce severity of and/or reduce incidence of one or more be devised using a PAK protein promoter operably-linked to symptoms or features of FXS and/or other neurodevelopmen a reporter gene Such as chloramphenicol acetyltransferase, tal disorder. firefly luciferase, bacterial luciferase, O-galactosidase, alka 0295. In some embodiments, screening systems of the line phosphatase, etc. Furthermore, PAK may be used as an present invention may involve the use of the FMR1 knockout indirect reporter via attachment to a second reporter Such as (FMR1 KO) mouse. FMR1 KO mice and FXS patients show red and/or green fluorescent protein (see, e.g., Mistili et al., similar behavioral phenotypes and/or similar abnormalities in 1997, Nature Biotech., 15:961; incorporated herein by refer synaptic morphology in the brain. Their brains have more ence). The reporter construct is typically transfected into a dendritic spines and/or a higher proportion of longer and/or cell. After treatment with a candidate Substance, the amount thinner spines compared to normal individuals. Furthermore, of reporter gene transcription, translation, and/or activity is they display abnormal synaptic function, Such as enhanced measured according to standard techniques known to those of long-term depression (LTD) mediated by metabotropic skill in the art. glutamate receptor in the hippocampus and/or impaired long 0289. In some embodiments, the present invention pro term potentiation (LTP) in the cortex. Therefore, the FMR1 vides methods to determine whether PAK can induce phos KO mouse serves as an accurate model of FXS, and the FMR1 phorylation of FMRP in vivo. In such methods, cells are KO mouse exhibits several quantifiable phenotypes that are transfected with vectors expressing constitutively active useful in the screening methods of the present invention. Such PAK and/or FMRP. Cell extracts are prepared at specified phenotypes include behavior, synaptic morphology, and/or time points after transfection, and the degree of phosphory synaptic function. lation of FMRP is analyzed by western blot analysis. 0296. In specific embodiments, the present invention pro 0290 InVivo Assays vides methods for identifying PAK modulators that may be 0291. In vivo assays involve the use of various animal utilized for treatment of FXS and/or other neurodevelopmen models, including transgenic animals that have been engi tal disorders comprising steps of (1) providing an FMR KO neered to have specific defects and/or carry markers that can mouse exhibiting symptoms of FXS and/or other neurodevel be used to measure the ability of a candidate substance to opmental disorders, (2) administering a candidate Substance reach and/or affect different cells within the organism. Due to to the mouse, and (3) measuring the effect(s) of the candidate their size, ease of handling, and/or information on their physi substance on the symptoms of FXS and/or other neurodevel ology and/or genetic makeup, mice are often used, especially opmental disorder. for transgenics. However, other animals are suitable as well, 0297. In some embodiments, screening methods of the including rats, rabbits, hamsters, guinea pigs, gerbils, wood present invention can measure behavioral symptoms. Such chucks, cats, dogs, sheep, goats, pigs, cows, horses and/or behavioral symptoms can include hyperactivity, Stereotypy, monkeys (including chimps, gibbons and/or baboons). perseverative behavior, anxiety, hypo-anxiety, seizure, Assays for PAK modulators may be conducted using an ani impaired social behavior, and/or cognitive delay. In some mal model derived from any of these species and/or other embodiments, behavioral symptoms can be measured using useful species not listed herein. an open field test. In an open field test, a Subject is allowed to 0292. In such assays, one or more candidate substances are run freely in an open arena (e.g. VersaMax activity monitor administered to an animal, and the ability of the candidate chamber from Accuscan Instruments) and certain behaviors Substance(s) to alter one or more characteristics, as compared are analyzed. These behaviors include, but are not limited to: to a control animal not treated with the candidate Substance (1) hyperactivity, determined by measuring the distance and/ (s), is determined The characteristics may be any of those or length of time traveled by the subject; (2) stereotypy, discussed herein with regard to the symptoms associated with determined by measuring the number of repetitive behaviors FXS (e.g., behavioral symptoms, abnormal synaptic func exhibited by the subject; (3) hypo-anxiety, determined by tion, improperly-formed dendritic spines, etc.). measuring the amount of time the Subject remains in the 0293. The present invention provides methods of screen centerfield relative to the time spent in the corners of the field; ing for candidate Substances that may treat, alleviate, amelio and/or (4) combinations of these. US 2010/0247552 A1 Sep. 30, 2010 34

0298. In some embodiments, behavioral symptoms can be cating impaired social learning, memory, and/or behavior. measured using a trace fear conditioning task. For example, a This test can be conducted in a novel or familiar environment, Subject is placed in a training chamber (Chamber A), where a as significant differences between FMR KO and wild-type tone is sounded, followed by a blank time (also called trace), mice have been observed in Social interaction assays depend and then shock. The sequence is repeated several times to let ing on the degree of familiarity with the environment. Addi the Subject learn the association between tone and shock tional Social behaviors can be monitored in this assay includ across the time gap. To examine whether the Subject remem ing active behaviors (e.g. aggressive attacks, lateral threats, bers this association, at various time point after conditioning, and/or chasing) and passive behaviors (e.g. receiving Sniffing the subject is placed into a new chamber (Chamber B) with a from other mouse and/or not showing signs of Submissive different shape and smell from Chamber A and its response to and/or defensive behavior). the tone is monitored. If the subject learns and remembers that 0303 Prior to an indirect social interaction test mice can tone is associated with shock, it will become immobile (called be housed individually for 4 days. Indirect social interaction “freezing”). Previous studies have shown that attention-dis tasks typically take place in a cage divided in half by a clear tracting stimuli and/or lesion of prefrontal cortex and/or hip perforated partition. The task is run in two different modes: pocampus can interfere with trace fear conditioning. Thus, one involves a familiar environment, by pre-exposure to the the Subject's attention and/or associative memory may be testing chamber, while the other involves a novel environ measured by comparing the degree of freezing both pre- and ment. In some embodiments, test mice are exposed to novel or post-conditioning, often relative to a control Subject. familiar mice. Typically, FMR KO mice behave similarly to 0299. In some embodiments, relevant behavior tests can wild-type mice in a novel environment, but behave signifi include eight-arm maze test and/or sensitization test to cantly differently in a familiar cage. Time spent at the parti amphetamine-induced Stereotypy for Stereotypy and perse tion can be recorded in 2 to 5 minute intervals for a 20 minute Verative behavior. In some embodiments, relevant behavior test. FMR KO mice tend to spend significantly less time tests can include elevated plus maze, light-dark transition, interacting (i.e. at the partition) in the first few minutes and and/or novelty Suppressed feeding test for anxiety, audio take longer to first approach the partition than wild-type mice. genic seizure, Social interaction, and/or social learning for In contrast, FMR KO mice usually spend more time at the social behaviors. In some embodiments, relevant behavior partition during the last time intervals than controls. tests can include Morris water maze (including the reversal 0304. In some experiments, the indirect social interaction version) and/or fear conditioning for learning and memory. test is conducted in a chamber divided into three rooms. The 0300. In some embodiments, behavioral symptoms can be central room which is connected to two rooms independent measured using an audiogenic seizure (AGS) assay. Fragile X from each other, one on the left and one on the right—is humans and mice are susceptible to seizures at early ages. empty. The left room contains an empty cage (i.e. a novel Fragile X mice show a robust phenotype in an AGS assay. object and/or inanimate target), while the right room contains While no 19-21 day old (p 19-21) wildtype mice typically a similar cage enclosing a novel mouse (i.e. a social target). have seizures in this task, the majority of fragile X mice do This task involves a choice between spending time with a have seizures. AGS can be performed essentially as described Social target or an inanimate target, and therefore is called a (Yan et al., 2005, Neuropharmacol., 49:1053; incorporated social preference test. Percent of total time interacting with herein by reference). Briefly, mice are habituated to a behav each object can be recorded. The present invention encom ioral chamber and then exposed to a high intensity siren of passes the recognition that FMR KO mice may spend a sig frequency peak 1800 HZ-6300 Hz at an average sound pres nificantly different amount of time (e.g. more or less) inter sure level above 120 dB at approximately 10 cm for 5 min acting with the Social target than control mice. utes. Behaviors of the mice can be monitored after adminis 0305. In some embodiments, a social dominance tube test tration of sound. Fragile X mice typically (1) run wildly, (2) is conducted in a tube approximately 30 cm long and 3 cm-4 have seizures, and/or (3) die. Wildtype mice typically do not cm in diameter. Mice are placed at opposite ends of the tube exhibit these responses. An AGS phenotype is scored based and released simultaneously. A mouse is pronounced the on the animal's endpoint. “winner when his opponent backs out completely. FMR1 0301 In some embodiments, behavioral symptoms can be KO mice win significantly fewer matches against unfamiliar measured using any of several Social interaction tests. In some wild-type mice than expected by chance. When a FMR1 KO embodiments, home cage behavior and Social interaction are mouse competes against a wild-type non-cagemate, the wild assessed with a variety of tests (Kwon et al., 2006, Neuron, type mouse is the winner in approximately 73% of matches 50:377: Spencer et al., 2005, Genes Brain Behay, 4:420; and (Kwon et al., 2006, Neuron, 50:377; Spencer et al., 2005, Lijam et al., 1997, Cell, 90:895; all of which are incorporated Genes Brain Behay, 4:420; and Lijam et al., 1997, Cell, herein by reference). For example, mice can be observed in 90:895; all of which are incorporated herein by reference). their home cage by Videorecording and scored for various The present invention encompasses the recognition that nonsocial behaviors and/or social behaviors (e.g. grooming, modulation of PAK activity via administration of a PAK mounting, tail pulling, and Sniffing). modulator may ameliorate this phenotype. 0302. In some embodiments, direct social interaction is (0306 Since FMR1 KO mice have been shown to display assessed by exposing mice to a novel conspecific mouse and Some abnormal Social behaviors, the present invention observing approaching and Sniffing behaviors. Percent of encompasses the recognition that home cage social behavior, time spent interacting can be recorded. This is typically including nest building and sleeping behavior, may be altered repeated about 3 days later with the same mice. Control mice in FMR1 KO mice. Nesting patterns are evaluated by placing usually exhibit a decrease in Social interaction the second a cotton nestle into a cage of approximately two, three, or four time, indicating recognition of the familiar mouse and/or mice of the same genotype (e.g. wild-type, FMR1 KO, etc.) normal social learning. FMR KO mice typically do not that receive identical drug treatments. After about 30 minutes exhibit a decrease in Social interaction the second time, indi to 1 hour, the nest can be removed and the height measured. US 2010/0247552 A1 Sep. 30, 2010

Wild-type mice build nests with depths that average 20 mm to 0311. In some embodiments, screening methods of the 50 mm. Mice which display abnormal social behavior, such present invention involve measurement of synaptic morphol as FMR1 KO mice, frequently build shallower nests (e.g. <20 ogy. In some embodiments, the number of dendritic spines is mm) counted. In some embodiments, spine number is examined in Golgi-stained layer II/III pyramidal neurons of the temporal 0307. In some embodiments, sleeping positions of mice in cortex. For example, serial brain sections can be obtained their home cages can be recorded two to four times a day over following the Golgi-Cox technique. Layer II/III pyramidal five consecutive days. Wild-type mice sleep huddled in the neurons in the temporal cortex are visualized by microscopy well-formed, fluffy nests they build. Observations will deter (e.g. under Olympus upright BX61 with motorized XY stage mine whether FMR1 KO mice sleep in scattered, random using Neurolucida/stereology software Microbrightfield). patterns, do not build full nests, or sleep on top of intact nestle On each primary apical dendritic branch, regularly-sized seg material. The present invention encompasses the recognition ments (e.g. ten consecutive 10-um-long dendritic segments) that, if FMK1 KO mice display abnormal phenotypes, ame are analyzed to quantify spine density (e.g. the number of lioration of those phenotypes may occur following modula spines per 10 um long dendritic segment). In some embodi tion of PAK activity. ments, length and/or width of dendritic spines is measured. In 0308. In some embodiments, behavioral symptoms such Some embodiments, spine length is measured from Golgi as hippocampus-dependent spatial learning are assessed stained neurons. using the classical Morris Water Maze test. FMR1 KO mice, 0312. In some embodiments, dendritic spine head size can just like the wild-type controls, learn to find the visible or be examined by electron microscopic analysis of the length of hidden platform with decreasing latency scores over the the postsynaptic density (PSD). In some embodiments, the course of the standard training protocol. However, some dendritic spine heads can be examined by conducting elec groups observe an abnormal phenotype in a reversal trial, a tron microscopic analyses of the proportion of larger, perfo test in which the platform is transferred to the quadrant oppo rated synapses. For example, for electron microscopy, Sub site the initial training quadrant. In particular. FMR1 KO mice jects are anaesthetized and perfused. Blocks of temporal display increased escape latency and path length, Suggesting cortex are embedded, from which sections (e.g. 1 um thick) that they have low response flexibility or high memory inter are cut and stained with toluidine blue (e.g. diluted to 1%) to ference (see, e.g. D'Hooge et al., 1997, Neuroscience, guide the further trimming to isolate layer II/III of temporal 76:367; incorporated herein by reference). cortex. Ultrathin sections (e.g. 90 nm) are then cut and stained 0309. In some embodiments, hippocampal synaptic plas with uracyl acetate and lead citrate. Randomly selected neu ticity is assessed using a standard protocol for mGluR-LTD ropil areas are photographed (e.g. at a 10,000x magnification (see, e.g. Example 5 for a more detailed protocol). Hippoc with a JEOL 1200EX electron microscope) and image nega ampal slices can be prepared from FMRKO mice and control tives are scanned and analyzed. littermates per standard procedures. All experiments are gen 0313. In some embodiments, neurons can be labeled by erally performed blind to genotype. Schaffer collaterals are fluorescent proteins (directly or via indirect staining) in an stimulated and extracellular field potentials measured in Str. animal and/or in culture established from an animal. Fluores radiatum of CA1. mGluR-dependent LTD is elicited both cent microscopy can be used to visualize neurons and mea electrophysiologically (by pairs of stimuli delivered at 1 Hz Sure the number, length, and/or size of spines. In some for 15 minutes to 20 minutes; “PP-LFS) and pharmacologi embodiments, the size of presynaptic terminals is measured. cally (by bath application of 50 uM to 100 uM3,4-dihydrox In some embodiments, the size of presynaptic terminals is yphenylglycine (DHPG) for 5 minutes). The initial slope of examined by conducting electron microscopic analysis of the the field potential is recorded as an indicator of synaptic number of synaptic vesicles and/or docked vesicles. In some strength. Hippocampal slices from FMR1 KO mice show embodiments, presynaptic terminals are labeled by fluores enhanced mCluR-LTD relative to control mice (see, e.g., cent proteins and fluorescent microscopy is used to visualize Huber et al., 2002, Proc. Natl. Acad. Sci., USA, 99.7746; and the presynaptic terminals and measure their size. Nosyreva and Huber, 2006, J. Neurophysiol. 95:3291; both 0314. In certain embodiments, screening methods of the of which are incorporated herein by reference). present invention involve measuring synaptic function. In 0310 Cortical long-term potentiation (LTP) is reduced in Some embodiments, long-term depression (LTD) mediated slices from FMR1 KO mice (see FIG. 14). Coronol brain by metabotropic glutamate receptor (mGluR) can be mea slices containing temporal cortex are prepared from two- to Sured in the hippocampus. For example, hippocampal slices three-month-old male littermates, and left to recover for at are prepared and allowed to recover before recording in oxy least 1 hour before recording in oxygenated (95% O, and 5% genated artificial cerebrospinal fluid (ACSF). Field potentials CO) warm (30°C.) artificial cerebrospinal fluid containing (FPs) in stratum radiatum of area CA1 are evoked by a current 124 mM NaCl, 5 mM KC1, 1.25 mM NaHPO 1 mM pulse to Schaffer collateral axons. Stable baseline responses MgCl, 2 mM CaCl, 26 mM NaHCOs, 10 mM dextrose. are regularly collected by a stimulation intensity (10 LA-30 Field potentials (FPs) in layer II/III evoked by layer IV stimu LA) yielding 50%-60% of the maximal response. mGluR lation are measured as previously described and responses are LTD can be induced by application of mGluRagonist 3.5- quantified as the amplitude of FP in cortex. LTP is induced by dihydroxyphenylglycine (DHPG: e.g. at 100 uM) and/or by TBS, which consisted of eight briefbursts (each with 4 pulses using paired-pulse low-frequency stimulation consisting of at 100 Hz) of stimuli delivered every 200 m.sec. Genetic 900 pairs of stimuli delivered at 1 Hz in the presence of the inhibition of PAK rescues the reduced cortical LTP in the N-methyl-D-aspartate receptor (NMDAR) antagonist D-(-)- FMR1 KO mice, and the present invention encompasses the 2-amino-5-phosphono-pentanoic acid (D-APV; e.g. at 50 recognition that pharmacological inhibition may have the uM). DHPG is a chiral compound, and mGluR-LTD can be same effect (Hayashi et al., 2007, Proc. Natl. Acad. Sci., USA, induced by application of RS-DHPG and S-DHPG, but typi 104:11489; incorporated herein by reference). cally not by application of R-DHPG. US 2010/0247552 A1 Sep. 30, 2010 36

0315. In some embodiments, long-term potentiation lates PAK kinase activity. PAK kinase activity may be mea (LTP) can be measured in the cortex. For example, coronal Sured using standard methods, which are described herein and brain slices containing cortex are prepared and left to recover in Sambrook et al. (Molecular Cloning: A Laboratory before recording in oxygenated ACSF. FPs in layer II/III Manual, 3" ed., Cold Spring Harbor Laboratory Press, Cold evoked by layer IV stimulation are measured and responses Spring Harbor, N.Y., 2001; incorporated herein by reference). are quantified as the amplitude of FP. Cortical LTP can be 0320 In some embodiments, the invention provides induced by theta-burst stimulation (TBS), which consists of screening methods for determining whether a candidate Sub briefbursts of stimuli delivered at regular intervals (e.g. eight stance is useful for treating, alleviating, ameliorating, reliev briefbursts, each with 4 pulses at 100 Hz, of stimuli delivered ing, delaying onset of inhibiting progression of reducing every 200 msec). severity of and/or reducing incidence of one or more symp 0316. In some embodiments, synaptic currents mediated toms or features of a FXS and/or other neurodevelopmental by C.-amino-3-hydroxy-5-methylisoxazole-4-propionic acid disorder in a mammal. This method involves measuring the receptors (AMPARs) and/or NMDARs can be measured in phosphorylation level of FMRP in a cell, tissue, and/or mam the cortex. For example, for measurement of AMPAR-medi mal in the presence and/or absence of the candidate Sub ated miniature excitatory postsynaptic current (mEPSC), tet stance. The candidate Substance is determined to treat, alle rodotoxin, APV, and bicuculline are added in a bath of ACSF. viate, ameliorate, relieve, delay onset of inhibit progression Continuous traces (e.g. 30 msec-60 msec) are collected at of reduce severity of, and/or reduce incidence of one or more regular intervals (e.g. every 8 seconds) and filtered (e.g. at 2 symptoms or features of FXS and/or other neurodevelopmen KHZ). Cells with series resistance above a certain threshold tal disorder if the candidate Substance decreases the phospho level (e.g. 213 mS2) are discarded. The measurement of rylation level of FMRP. NMDAR-EPSC/AMPAR-EPSC ratio is typically done in 0321. In some embodiments, screening methods of the ACSF containing Mg, bicuculline, and glycine. NMDAR present invention involve measuring PAK mRNA and/or pro dependent and AMPAR-dependent responses are discrimi tein levels in a cell, tissue, and/or mammal in the presence nated based on their distinct kinetics and Voltage dependence. and/or absence of the candidate Substance. The candidate Thus, NMDAR-mediated response is valued as the currents Substance is determined to treat, alleviate, ameliorate, relieve, recorded at +40 mV and measured 100 msec after the delay onset of inhibit progression of reduce severity of response onset. The AMPAR-mediated response is taken and/or reduce incidence of one or more symptoms or features from the peak amplitude response recorded at -80 mV. of FXS and/or other neurodevelopmental disorder if the sub 0317. In some embodiments, the present invention pro stance modulates PAK mRNA and/or protein levels. PAK vides methods for identifying PAK modulators that can be mRNA and/or protein levels may be measured using standard utilized to treat FXS and/or other neurodevelopmental disor methods, which are described herein and in Sambrook et al. ders comprising steps of: (1) providing an FMR KO mouse (Molecular Cloning: A Laboratory Manual, 3" ed., Cold exhibiting symptoms of FXS and/or other neurodevelopmen Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., tal disorders, (2) administering a candidate Substance to the 2001; incorporated herein by reference). mouse, and (3) measuring the effect(s) of the candidate Sub 0322. In some embodiments, a strain of C. elegans is uti stance on PAK mRNA and/or protein. lized which is characterized by a phenotype of sterility and/or 0318. In some embodiments, screening methods of the embryonic lethality and/or a defective gonad migration and present invention involve measuring the interaction between which harbors an impaired and/or missing PAK function. The PAK and its natural binding partners. The candidate sub strain of C. elegans utilized by the present invention could be stance is determined to treat, alleviate, ameliorate, relieve, obtainable and/or could be obtained by one or more of the delay onset of inhibit progression of reduce severity of methods for generating a C. elegans having said phenotypes. and/or reduce incidence of one or more symptoms or features Such a model may be used, amongst other things, for charac of FXS and/or other neurodevelopmental disorder if the can terizing the signaling pathways linked to PAK. Such a model didate substance modulates the interaction between PAK and may be used for identifying substances that interfere and/or its natural binding partners. In certain embodiments, the can interact with one or more proteins that are part of a signaling didate Substance is determined to treat, alleviate, ameliorate, pathway linked to PAK. Such a model may be used for iden relieve, delay onset of inhibit progression of reduce severity tification of a PAK modulator using any of the in vivo assays of and/or reduce incidence of one or more symptoms or described herein. features of FXS and/or other neurodevelopmental disorder if 0323. In some embodiments, other animal models for FXS the candidate substance modulates the interaction between and/or other neurodevelopmental disorders can be utilized. PAK and FMRP. The interaction between PAK and its natural For example, a D. melanogaster FXS model could be utilized binding partners may be measured using standard methods, in accordance with the present invention. A Drosophila FXS which are described herein and in Sambrooketal. (Molecular model was established in 2001 (Zhang et al., 2001, Cell, Cloning: A Laboratory Manual, 3" ed., Cold Spring Harbor 107:591; incorporated herein by reference) to provide evolu Laboratory Press, Cold Spring Harbor, N.Y., 2001; incorpo tionary perspective on conserved FMRP functions and to rated herein by reference). allow genetic studies of molecular and cellular functions in 0319. In some embodiments, screening methods of the the context of a relatively simple brain Zhang et al., 2001, present invention involve measuring PAK kinase activity in a Cell, 107:591; Dockendorff et al., 2002, Neuron, 34:973: cell, tissue, and/or mammal in the presence and/or absence of Morales et al., 2002, Neuron, 34:961; Lee et al., 2003, Devel the candidate Substance. A candidate Substance is determined opment, 130:5543; Michelet al., 2004, J. Neurosci., 24:5798: to treat, alleviate, ameliorate, relieve, delay onset of inhibit and Pan et al., 2004, Curr: Biol., 14:1863; all of which are progression of reduce severity of and/or reduce incidence of incorporated herein by reference). In Drosophila, loss of one or more symptoms or features of FXS and/or other neu dFMRP causes significant changes in neuronal structural rodevelopmental disorder if the candidate substance modu morphogenesis and circuit formation (Michel et al., 2004, J. US 2010/0247552 A1 Sep. 30, 2010 37

Neurosci., 24:5798; and Panet al., 2004, Curr. Biol., 14:1863; ized as “improperly-formed when an individual possesses both of which are incorporated herein by reference) and syn abnormally high numbers of dendritic spines. In some aptic differentiation and neurotransmission properties embodiments, dendritic spines are characterized as “improp (Zhanget al., 2001, Cell, 107:591; and Panet al., 2004, Curr: erly-formed when an individual possesses spines that are Biol., 14:1863; both of which are incorporated herein by abnormally long and/or thin. In some embodiments, improp reference) and alterations in behavioral output (Zhang et al., erly-formed dendritic spines are caused by FXS and/or other 2001, Cell, 107:591; Dockendorff et al., 2002, Neuron, neurodevelopmental disorders. 34:973; Morales et al., 2002, Neuron, 34:961; all of which are 0330. In some embodiments, PAK modulators of the incorporated herein by reference) comparable to the human present invention are used in the treatment of abnormal Syn disease. To establish molecular bases for these cellular and aptic function. In certain embodiments, abnormal synaptic behavioral phenotypes, studies were carried out using this function is caused by FXS and/or other neurodevelopmental model to identify proteins that are misregulated in the absence disorders. In specific embodiments, inventive PAK modula of dFMRP. Several such proteins were identified using this tors are used in the treatment of enhanced long-term depres model system (Zhang et al., 2005, Mol. Cell. Proteomics, sion (LTD) mediated by metabotropic glutamate receptor in 4:278; incorporated herein by reference). Such a model may the hippocampus and/or impaired long-term potentiation be used, amongst other things, for characterizing the signal (LTP) in the cortex. In some embodiments, defective ing pathways linked to PAK. Such a model may be used for enhanced long-term depression (LTD) mediated by metabo identifying substances that interfere and/or interact with one tropic glutamate receptor in the hippocampus and/or or more proteins that are part of a signaling pathway linked to impaired long-term potentiation (LTP) in the cortex are PAK. Such a model may be used for identification of a PAK caused by FXS and/or other neurodevelopmental disorders. modulator using any of the in Vivo assays described herein. 0331. In some embodiments, PAK modulators of the 0324. The FXS model systems described above are merely present invention are used in the treatment of one or more of exemplary model systems that can be used in accordance the the following symptoms: hyperactivity, Stereotypy, anxiety, present invention. One of ordinary skill will recognize that the seizure, impaired Social behavior, and/or cognitive delay. In present invention is not limited to these particular in vivo certain embodiments, these symptoms are caused by FXS models, but that any in vivo model in which expression of and/or other neurodevelopmental disorders. FMRP is disrupted may serve as a model system for FXS 0332. In some embodiments, the present invention pro and/or other developmental disorders. In some embodiments, vides a method of treating FXS and/or other neurodevelop any in vivo model in which the organism displays one or more mental disorders comprising steps of (1) providing a patient symptoms of FXS may serve as a model system for FXS exhibiting symptoms of FXS and/or other neurodevelopmen and/or other neurodevelopmental disorders. tal disorders, and (2) administering a therapeutic amount of 0325 Determining the effectiveness of a candidate sub one or more PAK modulators to the patient. In some embodi stance in vivo may involve a variety of different criteria. ments, the present invention provides a method of treating Measuring toxicity and/or dose response may be performed FXS and/or other neurodevelopmental disorders comprising in Subjects in a more meaningful fashion than in in vitro steps of (1) providing a patient suffering from FXS and/or and/or in cyto assays. other neurodevelopmental disorders, and (2) administering a 0326 Methods of Use therapeutic amount of one or more PAK modulators to the 0327. The present invention provides methods of treating patient. In some embodiments, the present invention provides FXS and/or other neurodevelopmental disorders (e.g. POF, a method of treating FXS and/or other neurodevelopmental FXTAS, various forms of mental retardation, and/or autism disorders comprising steps of (1) providing a patient Suscep spectrum disorders (ASD)). In some embodiments, the tible to FXS and/or other neurodevelopmental disorders, and present invention provides methods of treating POF, FXTAS, (2) administering a therapeutic amount of one or more PAK various forms of mental retardation, and/or autism spectrum modulators to the patient. disorders (ASD). In certain embodiments, such methods 0333. In some embodiments, the present invention pro involve modulating PAK activity. In some embodiments, vides methods of treating FXS and/or other neurodevelop PAKactivity is modulated in any manner described herein. In mental disorders comprising steps of (1) providing a patient some embodiments, PAK activity is inhibited. PAK activity exhibiting symptoms of Suffering from, and/or Susceptible to may be inhibited by disrupting its ability to interact with FXS and/or other neurodevelopmental disorders, and (2) FMRP and/or to phosphorylate FMRP. In some embodi administering a substance that modulates the ability of PAK ments, PAK activity is activated. to interact with its natural binding partners. 0328. In some embodiments, the invention provides phar 0334. In some embodiments, the present invention pro maceutical compositions comprising at least one PAK modu vides methods of treating FXS and/or other neurodevelop lator and at least one pharmaceutically acceptable excipient. mental disorders comprising steps of (1) providing a patient In other aspects, a pharmaceutical composition comprising a exhibiting symptoms of Suffering from, and/or Susceptible to candidate Substance is administered to a cell. Such as one in a FXS and/or other neurodevelopmental disorders, and (2) patient suffering from and/or susceptible to FXS and/or other administering a Substance that inhibits and/or disrupts the neurodevelopmental disorders. binding of PAK and FMRP. 0329. In some embodiments, PAK modulators of the 0335. In certain embodiments, the present invention pro present invention are used in the treatment of abnormal Syn vides methods of treating FXS and/or other neurodevelop aptic formation. In certain embodiments, abnormal synaptic mental disorders comprising steps of (1) providing a patient formation is caused by FXS and/or other neurodevelopmental exhibiting symptoms of Suffering from, and/or Susceptible to disorders. In specific embodiments, inventive PAK modula FXS and/or other neurodevelopmental disorders, and (2) tors are used in the treatment of improperly-formed dendritic administering a Substance that modulates the kinase activity spines. In some embodiments, dendritic spines are character of PAK. US 2010/0247552 A1 Sep. 30, 2010

0336. In some embodiments, the present invention pro administering a pharmaceutical composition comprising at vides methods of treating FXS and/or other neurodevelop least one PAK modulator of the present invention to a subject mental disorders comprising steps of (1) providing a patient in need thereof. Inventive PAK modulators may be adminis exhibiting symptoms of Suffering from, and/or Susceptible to tered with other medications used to treat the symptoms of FXS and/or other neurodevelopmental disorders, and (2) FXS. In some embodiments, the compositions are adminis administering a substance that modulates ability of PAK to tered to humans. phosphorylate FMRP. 0337. In some embodiments, the present invention pro 0345 The invention encompasses the preparation and/or vides methods of treating FXS and/or other neurodevelop use of pharmaceutical compositions comprising a PAK mental disorders comprising steps of (1) providing a patient modulator for treatment of FXS and/or other neurodevelop exhibiting symptoms of FXS and/or other neurodevelopmen mental disorders as an active ingredient. Such a pharmaceu tal disorders, and (2) administering a substance that modu tical composition may consist of the active ingredient alone, lates the levels of PAK mRNA and/or protein. in a form suitable for administration to a subject, or the 0338. In some embodiments, inventive PAK modulators pharmaceutical composition may comprise the active ingre may be used to treat diseases, conditions, and/or disorders dient and one or more pharmaceutically acceptable excipi other than FXS. For example, some FMR1 mutations do not ents, one or more additional ingredients, and/or a combina cause FXS, but instead cause other disorders. “Alleles' of the tion of these. The active ingredient may be present in the FMR1 gene are defined by the number of CGG repeats and/or pharmaceutical composition in the form of a physiologically by the methylation status of the FMR1 locus. FMR1 normally acceptable ester and/or salt, Such as in combination with a has between 7-52 CGG repeats (most often 30 repeats) physiologically acceptable cation and/or anion, as is well located in the 5' untranslated region of exon 1. These known in the art. sequences are normally unmethylated, but methylation can 0346 Although the descriptions of pharmaceutical com occur at each C of CG dinucleotide repeats, which silences positions provided herein are principally directed to pharma expression of FMR1. ceutical compositions which are Suitable for administration to 0339. A “full mutation” is characterized by >200 CGG humans, it will be understood by the skilled artisan that such repeats that are methylated. All males and 50% of females compositions are generally suitable for administration to ani possessing the full mutation will develop FXS. In contrast, a mals of all sorts. Modification of pharmaceutical composi “premutation' is characterized by 55-200 CGG repeats that tions Suitable for administration to humans in order to render are not methylated. These individuals are not likely to develop the compositions suitable for administration to various ani FXS, but females exhibit increased twinning and/or an mals is well understood, and the ordinarily skilled veterinary increased occurrence of premature ovarian failure (POF). pharmacologist can design and/or perform such modification 0340 Furthermore, the premutation may lead to fragile with merely ordinary, if any, experimentation. Patients to X-associated tremor ataxia (FXTAS), especially in males. which administration of the pharmaceutical compositions of FXTAS is characterized by intention tremor, ataxia, cognitive the invention is contemplated include, but are not limited to, decline, and/or brain atrophy and is thought to be related to humans and/or other primates; mammals, including commer RNA levels. cially relevant mammals such as cattle, pigs, horses, sheep, 0341 The premutation may lead to cognitive effects. For cats, and/or dogs; and/or birds, including commercially rel example, a premutation carrier's IQ may be within the aver evantbirds Such as chickens, ducks, geese, and/or turkeys. age range, but is likely to be on lower end of the average 0347 Pharmaceutical compositions as described herein range. There is evidence of socio-emotional effects, including may be prepared by any method known or hereafter devel increased shyness and/or social anxiety. oped in the art of pharmaceutics. In general. Such preparatory 0342. Thus, improper expression and/or activity of the methods include the step of bringing the active ingredient into FMR1 gene may lead to diseases, disorders, and/or condi association with one or more excipients and/or one or more tions other than FXS. In some embodiments, inventive PAK other accessory ingredients, and then, if necessary and/or modulators may be used in the treatment of any disease, desirable, shaping and/or packaging the product into a desired condition, and/or disorder caused by improper expression single- or multi-dose unit. and/or function of the FMR1 gene. 0348. A pharmaceutical composition of the invention may be prepared, packaged, and/or sold in bulk, as a single unit Pharmaceutical Compositions dose, and/or as a plurality of single unit doses. As used herein, 0343. The present invention provides PAK modulators a “unit dose' is discrete amount of the pharmaceutical com used in the treatment of FXS and/or other neurodevelopmen position comprising a predetermined amount of the active tal disorders. In some embodiments, the present invention ingredient. The amount of the active ingredient is generally provides pharmaceutical compositions comprising PAK equal to the dosage of the active ingredient which would be modulators and at least one pharmaceutically acceptable administered to a Subject and/or a convenient fraction of Such excipient. The present invention provides pharmaceutical a dosage such as, for example, one-half or one-third of such a compositions comprising a therapeutically effective amount dosage. of inventive PAK modulator(s) appropriately formulated for 0349 The relative amounts of the active ingredient, the administration to a subject Suffering from and/or Susceptible pharmaceutically acceptable excipient(s), and/or any addi to FXS and/or other neurodevelopmental disorders. Such tional ingredients in a pharmaceutical composition of the pharmaceutical compositions may optionally comprise one invention will vary, depending upon the identity, size, and/or or more additional therapeutically-active Substances. condition of the Subject treated and further depending upon 0344. In accordance with some embodiments, methods of the route by which the composition is to be administered. By treating FXS and/or other neurodevelopmental disorders are way of example, the composition may comprise between provided. In some embodiments, inventive methods comprise 0.1% and 100% (w/w) active ingredient. US 2010/0247552 A1 Sep. 30, 2010 39

0350 Pharmaceutical formulations of the present inven fat, cholesterol, wax, and lecithin), colloidal clays (e.g. ben tion may additionally comprise a pharmaceutically accept tonite aluminum silicate and VEEGUM magnesium alu able excipient, which, as used herein, includes any and all minum silicate), long chain amino acid derivatives, high Solvents, dispersion media, diluents, or other liquid vehicles, molecular weight alcohols (e.g. Stearyl alcohol, cetyl alcohol, dispersion or Suspension aids, Surface active agents, isotonic oleyl alcohol, triacetin monostearate, ethylene glycol distear agents, thickening or emulsifying agents, preservatives, Solid ate, glyceryl monoStearate, and propylene glycol monostear binders, lubricants and the like, as suited to the particular ate, polyvinyl alcohol), carbomers (e.g. carboxy polymethyl dosage form desired. Remington's The Science and Practice ene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl of Pharmacy, 21 Edition, A. R. Gennaro, (Lippincott, Will polymer), carrageenan, cellulosic derivatives (e.g. carboxym iams & Wilkins, Baltimore, Md., 2006) discloses various ethylcellulose sodium, powdered cellulose, hydroxymethyl excipients used in formulating pharmaceutical compositions cellulose, hydroxypropyl cellulose, hydroxypropyl methyl and known techniques for the preparation thereof. Except cellulose, methylcellulose), Sorbitan fatty acid esters (e.g. insofar as any conventional excipient is incompatible with a polyoxyethylene sorbitan monolaurate TWEENR 20, poly Substance or its derivatives, such as by producing any unde oxyethylene sorbitan TWEENR 60, polyoxyethylene sor sirable biological effect or otherwise interacting in a delete bitan monooleate TWEENR 80, sorbitan monopalmitate rious manner with any other component(s) of the pharmaceu SPANR 40, sorbitan monostearate SPANR 60, sorbitan tical composition, its use is contemplated to be within the tristearate SPANR) 65, glyceryl monooleate, sorbitan Scope of this invention. monooleate SPANR 80), polyoxyethylene esters (e.g. poly 0351. In some embodiments, the pharmaceutically accept oxyethylene monostearate MYRJR 45), polyoxyethylene able excipient is at least 95%,96%.97%.98%, 99%, or 100% hydrogenated castor oil, polyethoxylated castor oil, poly pure. In some embodiments, the excipient is approved for use oxymethylene stearate, and SOLUTOL), sucrose fatty acid in humans and for veterinary use. In some embodiments, the esters, polyethylene glycol fatty acid esters (e.g. CREMO excipient is approved by United States Food and Drug PHOR), polyoxyethylene ethers, (e.g. polyoxyethylene lau Administration. In some embodiments, the excipient is phar ryl ether BRIJR 30), poly(vinyl-pyrrolidone), diethylene maceutical grade. In some embodiments, the excipient meets glycol monolaurate, triethanolamine oleate, sodium oleate, the standards of the United States Pharmacopoeia (USP), the potassium oleate, ethyl oleate, oleic acid, ethyl laurate, European Pharmacopoeia (EP), the British Pharmacopoeia, sodium lauryl sulfate, PLURONIC(R) F 68, POLOXAMER and/or the International Pharmacopoeia. 188, cetrimonium bromide, cetylpyridinium chloride, benza 0352 Pharmaceutically acceptable excipients used in the Ikonium chloride, docusate Sodium, etc. and/or combinations manufacture of pharmaceutical compositions include, but are thereof. not limited to, inert diluents, dispersing and/or granulating 0356. Exemplary binding agents include, but are not lim agents, Surface active agents and/or emulsifiers, disintegrat ited to, starch (e.g. cornstarch and starch paste); gelatin: Sug ing agents, binding agents, preservatives, buffering agents, ars (e.g. Sucrose, glucose, dextrose, dextrin, molasses, lac lubricating agents, and/or oils. Such excipients may option tose, lactitol, mannitol.); natural and synthetic gums (e.g. ally be included in the inventive formulations. Excipients acacia, Sodium alginate, extract of Irish moss, panwar gum, Such as cocoa butter and Suppository waxes, coloring agents, ghatti gum, mucilage of isapol husks, carboxymethylcellu coating agents, Sweetening, flavoring, and perfuming agents lose, methylcellulose, ethylcellulose, hydroxyethylcellulose, can be present in the composition, according to the judgment hydroxypropyl cellulose, hydroxypropyl methylcellulose, of the formulator. microcrystalline cellulose, cellulose acetate, poly(vinyl-pyr 0353 Exemplary diluents include, but are not limited to, rolidone), magnesium aluminum silicate (VEEGUM), and calcium carbonate, Sodium carbonate, calcium phosphate, larch arabogalactan); alginates; polyethylene oxide; polyeth dicalcium phosphate, calcium Sulfate, calcium hydrogen ylene glycol; inorganic calcium salts; silicic acid; poly phosphate, Sodium phosphate lactose, Sucrose, cellulose, methacrylates; waxes; water, alcohol; etc.; and combinations microcrystalline cellulose, kaolin, mannitol, Sorbitol, inosi thereof. tol, Sodium chloride, dry starch, cornstarch, powdered Sugar, 0357 Exemplary preservatives may include antioxidants, etc., and combinations thereof. chelating agents, antimicrobial preservatives, antifungal pre 0354 Exemplary granulating and/or dispersing agents servatives, alcohol preservatives, acidic preservatives, and include, but are not limited to, potato starch, corn starch, other preservatives. Exemplary antioxidants include, but are tapioca starch, Sodium starch glycolate, clays, alginic acid, not limited to, alpha tocopherol, ascorbic acid, acorbyl palmi guar gum, citrus pulp, agar, bentonite, cellulose and wood tate, butylated hydroxyanisole, butylated hydroxytoluene, products, natural sponge, cation-exchange resins, calcium monothioglycerol, potassium metabisulfite, propionic acid, carbonate, silicates, sodium carbonate, cross-linked polyvi propyl gallate, sodium ascorbate, Sodium bisulfite, sodium nyl-pyrrolidone) (crospovidone), sodium carboxymethyl metabisulfite, and sodium Sulfite. Exemplary chelating agents starch (sodium starch glycolate), carboxymethyl cellulose, include ethylenediaminetetraacetic acid (EDTA), citric acid cross-linked sodium carboxymethyl cellulose (croScarmel monohydrate, disodium edetate, dipotassium edetate, edetic lose), methylcellulose, pregelatinized starch (starch 1500), acid, fumaric acid, malic acid, phosphoric acid, Sodium ede microcrystalline starch, water insoluble starch, calcium car tate, tartaric acid, and trisodium edetate. Exemplary antimi boxymethyl cellulose, magnesium aluminum silicate (VEE crobial preservatives include, but are not limited to, benza GUM), Sodium lauryl Sulfate, quaternary ammonium com lkonium chloride, benzethonium chloride, benzyl alcohol, pounds, etc., and combinations thereof. bronopol, cetrimide, cetylpyridinium chloride, chlorhexi 0355 Exemplary surface active agents and/or emulsifiers dine, chlorobutanol, chlorocresol, chloroxylenol, cresol, include, but are not limited to, natural emulsifiers (e.g. acacia, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phe agar, alginic acid, sodium alginate, tragacanth, chondruX. noxyethanol, phenylethyl alcohol, phenylmercuric nitrate, cholesterol, Xanthan, pectin, gelatin, egg yolk, casein, wool propylene glycol, and thimerosal. Exemplary antifungal pre US 2010/0247552 A1 Sep. 30, 2010 40 servatives include, but are not limited to, butyl paraben, 0361 Liquid dosage forms for oral and parenteral admin methyl paraben, ethyl paraben, propyl paraben, benzoic acid, istration include, but are not limited to, pharmaceutically hydroxybenzoic acid, potassium benzoate, potassium Sor acceptable emulsions, microemulsions, solutions, Suspen bate, Sodium benzoate, Sodium propionate, and Sorbic acid. sions, syrups and elixirs. In addition to the active ingredient Exemplary alcohol preservatives include, but are not limited (S), liquid dosage forms may comprise inert diluents com to, ethanol, polyethylene glycol, phenol, phenolic com monly used in the art such as, for example, water or other pounds, bisphenol, chlorobutanol, hydroxybenzoate, and Solvents, Solubilizing agents and emulsifiers such as ethyl phenylethyl alcohol. Exemplary acidic preservatives include, alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol. 1,3-buty but are not limited to, vitamin A, vitamin C, vitamin E, beta lene glycol, dimethylformamide, oils (in particular, cotton carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic seed, groundnut, corn, germ, olive, castor, and sesame oils), acid, Sorbic acid, and phytic acid. Other preservatives glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols include, but are not limited to, tocopherol, tocopherol acetate, and fatty acid esters of sorbitan, and mixtures thereof. Besides deteroxime mesylate, cetrimide, butylated hydroxyanisol inert diluents, oral compositions can include adjuvants such (BHA), butylated hydroxytoluened (BHT), ethylenediamine, as wetting agents, emulsifying and Suspending agents, Sweet sodium lauryl sulfate (SLS), sodium lauryl ether sulfate ening, flavoring, and perfuming agents. In certain embodi (SLES), sodium bisulfate, sodium metabisulfite, potassium ments for parenteral administration, an active ingredient can sulfite, potassium metabisulfite, GLYDANT PLUSR, PHE be mixed with solubilizing agents such as CREMOPHOR, NONIPR), methylparaben, GERMALL(R) 115, GERMA alcohols, oils, modified oils, glycols, polysorbates, cyclodex BENR II, NEOLONETM, KATHON, and EUXYL. In certain trins, polymers, and combinations thereof. embodiments, the preservative is an anti-oxidant. In other 0362. Injectable preparations, for example, sterile inject embodiments, the preservative is a chelating agent. able aqueous or oleaginous Suspensions may be formulated 0358 Exemplary buffering agents include, but are not lim according to the known art using Suitable dispersing or wet ited to, citrate buffer solutions, acetate buffer solutions, phos ting agents and Suspending agents. A sterile injectable prepa phate buffer Solutions, ammonium chloride, calcium carbon ration may be a sterile injectable solution, Suspension or ate, calcium chloride, calcium citrate, calcium glubionate, emulsion in a nontoxic parenterally acceptable diluent or calcium gluceptate, calcium gluconate, D-gluconic acid, cal Solvent, for example, as a solution in 1,3-butanediol. Among cium glycerophosphate, calcium lactate, propanoic acid, cal the acceptable vehicles and solvents that may be employed cium levulinate, pentanoic acid, dibasic calcium phosphate, are water, Ringer's solution, U.S.P. and isotonic sodium chlo phosphoric acid, tribasic calcium phosphate, calcium hydrox ride solution. In addition, sterile, fixed oils are conventionally ide phosphate, potassium acetate, potassium chloride, potas employed as a solvent or Suspending medium. For this pur sium gluconate, potassium mixtures, dibasic potassium phos pose any bland fixed oil can be employed including synthetic phate, monobasic potassium phosphate, potassium phosphate mono- or di-glycerides. In addition, fatty acids such as oleic mixtures, sodium acetate, sodium bicarbonate, sodium chlo acid are used in the preparation of injectables. ride, Sodium citrate, sodium lactate, dibasic sodium phos 0363. Injectable formulations can be sterilized, for phate, monobasic sodium phosphate, Sodium phosphate mix example, by filtration through a bacterial-retaining filter, or tures, tromethamine, magnesium hydroxide, aluminum by incorporating sterilizing agents in the form of sterile solid hydroxide, alginic acid, pyrogen-free water, isotonic saline, compositions which can be dissolved or dispersed in sterile Ringer's solution, ethyl alcohol, etc., and combinations water or other sterile injectable medium prior to use. thereof. 0364. In order to prolong the effect of a drug, it is often 0359 Exemplary lubricating agents include, but are not desirable to slow the absorption of the drug from subcutane limited to, magnesium Stearate, calcium Stearate, Stearic acid, ous or intramuscular injection. This may be accomplished by silica, talc, malt, glyceryl behanate, hydrogenated vegetable the use of a liquid Suspension of crystalline or amorphous oils, polyethylene glycol, Sodium benzoate, sodium acetate, material with poor water solubility. The rate of absorption of Sodium chloride, leucine, magnesium lauryl Sulfate, sodium the drug then depends upon its rate of dissolution which, in lauryl Sulfate, etc., and combinations thereof. turn, may depend upon crystal size and crystalline form. 0360 Exemplary oils include, but are not limited to, Alternatively, delayed absorption of a parenterally adminis almond, apricot kernel, avocado, babassu, bergamot, black tered drug form is accomplished by dissolving or Suspending current seed, borage, cade, camomile, canola, caraway, car the drug in an oil vehicle. nauba, castor, cinnamon, cocoa butter, coconut, cod liver, 0365 Compositions for rectal or vaginal administration coffee, corn, cotton seed, emu, eucalyptus, evening primrose, are typically suppositories which can be prepared by mixing fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, the active ingredients of this invention with suitable non isopropyl myristate, jojoba, kukui nut, lavandin, lavender, irritating excipients such as cocoa butter, polyethylene gly lemon, litsea cubeba, macademia nut, mallow, mango seed, col, or a Suppository wax which are solid at ambient tempera meadowfoam seed, mink, nutmeg, olive, orange, orange ture but liquid at body temperature and therefore melt in the roughy, palm, palm kernel, peach kernel, peanut, poppy seed, rectum or vaginal cavity and release the active ingredient. pumpkin seed, rapeseed, rice bran, rosemary, Safflower, san 0366 Solid dosage forms for oral administration include dalwood, Sasquana, savoury, sea buckthorn, Sesame, shea capsules, tablets, pills, powders, and granules. In such solid butter, silicone, soybean, Sunflower, tea tree, thistle, tsubaki, dosage forms, the active ingredient is mixed with at least one Vetiver, walnut, and wheat germ oils. Exemplary oils include, inert, pharmaceutically acceptable excipient Such as sodium but are not limited to, butyl Stearate, caprylic triglyceride, citrate or dicalcium phosphate and/or (a) fillers or extenders capric triglyceride, cyclomethicone, diethyl sebacate, dime Such as starches, lactose. Sucrose, glucose, mannitol, and thicone 360, isopropyl myristate, mineral oil, octyldode silicic acid, (b) binders such as, for example, carboxymeth canol, oleyl alcohol, silicone oil, and combinations thereof. ylcellulose, alginates, gelatin, polyvinylpyrrolidinone, US 2010/0247552 A1 Sep. 30, 2010

Sucrose, and acacia, (c) humectants such as glycerol, (d) needle devices such as those described in U.S. Pat. Nos. disintegrating agents such as agar, calcium carbonate, potato 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; or tapioca starch, alginic acid, certain silicates, and sodium 5,015,235; 5,141,496; and 5,417,662. Intradermal composi carbonate, (e) solution retarding agents such as paraffin, (f) tions may be administered by devices which limit the effec absorption accelerators such as quaternary ammonium com tive penetration length of a needle into the skin, Such as those pounds, (g)wetting agents such as, for example, cetyl alcohol described in PCT publication WO 99/34850 and functional and glycerol monoStearate, (h) absorbents such as kaolin and equivalents thereof. Jet injection devices which deliver liquid bentonite clay, and/or (i) lubricants such as talc, calcium vaccines to the dermis via a liquid jet injector and/or via a Stearate, magnesium Stearate, Solid polyethylene glycols, needle which pierces the stratum corneum and produces a jet sodium lauryl sulfate, and mixtures thereof. In the case of which reaches the dermis are suitable. Jet injection devices capsules, tablets and pills, the dosage form may comprise are described, for example, in U.S. Pat. Nos. 5,480,381: buffering agents. 5,599,302; 5,334,144; 5,993,412: 5,649,912; 5,569,189: 0367 Solid compositions of a similar type may be 5,704,911; 5,383,851; 5,893,397; 5,466.220; 5,339,163; employed as fillers in Soft and hard-filled gelatin capsules 5,312,335; 5,503,627; 5,064.413: 5,520,639; 4,596,556; using such excipients as lactose or milk Sugar as well as high 4,790,824; 4.941,880; 4.940,460; and PCT publications WO molecular weight polyethylene glycols and the like. Solid 97/37705 and WO 97/13537. Ballistic powder/particle deliv dosage forms of tablets, dragees, capsules, pills, and granules ery devices which use compressed gas to accelerate vaccine can be prepared with coatings and shells Such as enteric in powder form through the outer layers of the skin to the coatings and other coatings well known in the pharmaceutical dermis are suitable. Alternatively or additionally, conven formulating art. They may optionally comprise opacifying tional syringes may be used in the classical mantoux method agents and can be of a composition that they release the active of intradermal administration. ingredient(s) only, or preferentially, in a certain part of the 0371 Formulations suitable for topical administration intestinal tract, optionally, in a delayed manner. Examples of include, but are not limited to, liquid and/or semiliquid prepa embedding compositions which can be used include poly rations such as liniments, lotions, oil in water and/or water in meric Substances and waxes. Solid compositions of a similar oil emulsions such as creams, ointments and/or pastes, and/or type may be employed as fillers in soft and hard-filled gelatin Solutions and/or Suspensions. Topically-administrable for capsules using Such excipients as lactose or milk Sugar as well mulations may, for example, comprise from about 1.0% to as high molecular weight polethylene glycols and the like. about 10% (w/w) active ingredient, although the concentra 0368 Active ingredients can be in micro-encapsulated tion of the active ingredient may be as high as the solubility form with one or more excipients as noted above. The solid limit of the active ingredient in the solvent. Formulations for dosage forms of tablets, dragees, capsules, pills, and granules topical administration may further comprise one or more of can be prepared with coatings and shells Such as enteric the excipients and/or additional ingredients described herein. coatings, release controlling coatings and other coatings well 0372 A pharmaceutical composition of the invention may known in the pharmaceutical formulating art. In such solid be prepared, packaged, and/or sold in a formulation Suitable dosage forms the active ingredient may be admixed with at for pulmonary administration via the buccal cavity. Such a least one inert diluent such as Sucrose, lactose or starch. Such formulation may comprise dry particles which comprise the dosage forms may comprise, as is normal practice, additional active ingredient and which have a diameter in the range from Substances other than inert diluents, e.g., tableting lubricants about 0.5um to about 7um or from about 1 um to about 6 um. and other tableting aids such a magnesium Stearate and Such compositions are conveniently in the form of dry pow microcrystalline cellulose. In the case of capsules, tablets and ders for administration using a device comprising a dry pow pills, dosage forms may comprise buffering agents. They may der reservoir to which a stream of propellant may be directed optionally comprise opacifying agents and can be of a com to disperse the powder and/or using a self propelling solvent/ position that they release the active ingredient(s) only, or powder dispensing container Such as a device comprising the preferentially, in a certain part of the intestinal tract, option active ingredient dissolved and/or Suspended in a low-boiling ally, in a delayed manner. Examples of embedding composi propellant in a sealed container. Such powders comprise par tions which can be used include polymeric Substances and ticles wherein at least 98% of the particles by weight have a WaxeS. diameter greater than 0.5um and at least 95% of the particles 0369 Dosage forms for topical and/or transdermal admin by number have a diameter less than 7 um. Alternatively, at istration of a composition of this invention may include oint least 95% of the particles by weight have a diameter greater ments, pastes, creams, lotions, gels, powders, Solutions, than 1 Lum and at least 90% of the particles by number have a sprays, inhalants and/or patches. Generally, an active ingre diameter less than 6 Jum. Dry powder compositions may dient is admixed under sterile conditions with a pharmaceu include a solid fine powder diluent such as Sugar and are tically acceptable excipient and/or any needed preservatives conveniently provided in a unit dose form. and/or buffers as may be required. Additionally, the present 0373 Low boiling propellants generally include liquid invention contemplates the use of transdermal patches, which propellants having a boiling point of below 65° F. at atmo often have the added advantage of providing controlled deliv spheric pressure. Generally the propellant may constitute ery of an active ingredient to the body. Such dosage forms 50% to 99.9% (w/w) of the composition, and the active ingre may be prepared, for example, by dissolving and/or dispens dient may constitute 0.1% to 20% (w/w) of the composition. ing the active ingredient in the proper medium. Alternatively The propellant may further comprise additional ingredients or additionally, the rate may be controlled by either providing Such as a liquid non-ionic and/or Solid anionic Surfactant a rate controlling membrane and/or by dispersing the active and/or a solid diluent (which may have a particle size of the ingredient in a polymer matrix and/or gel. same order as particles comprising the active ingredient). 0370 Suitable devices for use in delivering intradermal 0374 Pharmaceutical compositions of the invention for pharmaceutical compositions described herein include short mulated for pulmonary delivery may provide the active ingre US 2010/0247552 A1 Sep. 30, 2010 42 dient in the form of droplets of a solution and/or Suspension. Thus, pharmaceutical compositions containing one or more Such formulations may be prepared, packaged, and/or sold as inventive PAK modulators may be administered to one or aqueous and/or dilute alcoholic Solutions and/or Suspensions, more individuals suffering from, susceptible to, and/or exhib optionally sterile, comprising the active ingredient, and may iting symptoms of FXS. The present invention therefore conveniently be administered using any nebulization and/or encompasses methods of modulating PAK activity, as well as atomization device. Such formulations may further comprise methods of treating FXS and/or other neurodevelopmental one or more additional ingredients including, but not limited disorders in Subjects. to, a flavoring agent such as saccharin Sodium, a Volatile oil, 0380. In some embodiments, a therapeutically effective a buffering agent, a surface active agent, and/or a preservative amount of a pharmaceutical composition comprising a PAK such as methylhydroxybenzoate. Droplets provided by this modulator is delivered to a subject and/or organism prior to, route of administration may have an average diameter in the simultaneously with, and/or after diagnosis with FXS and/or range from about 0.1 um to about 200 um. other neurodevelopmental disorder. In some embodiments, a 0375 Formulations described herein as being useful for therapeutic amount of a pharmaceutical composition is deliv pulmonary delivery are useful for intranasal delivery of a ered to a patient and/or organism prior to, simultaneously pharmaceutical composition of the invention. Anotherformu with, and/or after onset of symptoms of FXS and/or other lation Suitable for intranasal administration is a coarse pow neurodevelopmental disorder. In some embodiments, the der comprising the active ingredient and having an average amount of pharmaceutical composition is sufficient to treat, particle from about 0.2 um to 500 Lum. Such a formulation is alleviate, ameliorate, relieve, delay onset of inhibit progres administered in the manner in which Snuff is taken, i.e. by sion of reduce severity of, and/or reduce incidence of one or rapid inhalation through the nasal passage from a container of more symptoms or features of FXS and/or other neurodevel the powder held close to the nares. opmental disorder. 0376 Formulations suitable for nasal administration may, 0381 Pharmaceutical compositions, according to the for example, comprise from about as little as 0.1% (w/w) and method of the present invention, may be administered using as much as 100% (w/w) of the active ingredient, and may any amount and any route of administration effective for comprise one or more of the excipients and/or additional treatment. The exact amount required will vary from subject ingredients described herein. A pharmaceutical composition to Subject, depending on the species, age, and general condi of the invention may be prepared, packaged, and/or sold in a tion of the subject, the severity of the infection, the particular formulation suitable for buccal administration. Such formu composition, its mode of administration, its mode of activity, lations may, for example, be in the form of tablets and/or and the like. Compositions of the invention are typically lozenges made using conventional methods, and may contain, formulated in dosage unit form for ease of administration and for example, 0.1% to 20% (w/w) active ingredient, the bal uniformity of dosage. It will be understood, however, that the ance comprising an orally dissolvable and/or degradable total daily usage of the compositions of the present invention composition and, optionally, one or more of the excipients will be decided by the attending physician within the scope of and/or additional ingredients described herein. Alternately, Sound medical judgment. The specific therapeutically effec formulations Suitable for buccal administration may com tive dose level for any particular subject or organism will prise a powder and/or an aerosolized and/or atomized solu depend upon a variety of factors including the disorder being tion and/or Suspension comprising the active ingredient. Such treated and the severity of the disorder; the activity of the powdered, aerosolized, and/or aerosolized formulations, specific active ingredient employed; the specific composition when dispersed, may have an average particle and/or droplet employed; the age, body weight, general health, sex and diet size in the range from about 0.1 um to about 200um, and may of the subject; the time of administration, route of adminis further comprise one or more of the excipients and/or addi tration, and rate of excretion of the specific active ingredient tional ingredients described herein. employed; the duration of the treatment; drugs used in com 0377. A pharmaceutical composition of the invention may bination or coincidental with the specific active ingredient be prepared, packaged, and/or sold in a formulation Suitable employed; and like factors well known in the medical arts. for ophthalmic administration. Such formulations may, for 0382 Pharmaceutical compositions of the present inven example, be in the form of eye drops including, for example, tion may be administered by any route. In some embodi a 0.1%/1.0% (w/w) solution and/or suspension of the active ments, pharmaceutical compositions of the present invention ingredient in an aqueous or oily liquid excipient. Such drops are administered by a variety of routes, including oral, intra may further comprise buffering agents, salts, and/or one or venous, intramuscular, intra-arterial, intramedullary, intrath more other of the excipients and/or additional ingredients ecal, parenteral, Subcutaneous, intraventricular, transdermal, described herein. Other opthalmically-administrable formu interdermal, rectal, intravaginal, intraperitoneal, topical (e.g. lations which are useful include those which comprise the by powders, ointments, creams, gels, and/or drops), transder active ingredient in microcrystalline form and/or in a liposo mal, mucosal, nasal, buccal, enteral, Sublingual; by intratra mal preparation. Eardrops and/or eye drops are contemplated cheal instillation, bronchial instillation, and/or inhalation; as being within the scope of this invention. and/or as an oral spray, nasal spray, and/or aerosol. In some 0378 General considerations in the formulation and/or embodiments, inventive compositions are administered intra manufacture of pharmaceutical agents may be found, for venously. In some embodiments, inventive compositions are example, in Remington. The Science and Practice of Phar administered orally. In some embodiments, inventive compo macy 21 ed., Lippincott Williams & Wilkins, 2005 (incor sitions are administered using a continuous IV drip. In some porated herein by reference). embodiments, inventive compositions are administered by retro-orbital injection. Administration 0383. In some embodiments, inventive compositions can 0379 Inventive PAK modulators are useful in the treat be delivered using a pump. In some embodiments, a pump to ment of FXS and/or other neurodevelopmental disorders. be used in accordance with the invention is an external pump. US 2010/0247552 A1 Sep. 30, 2010

In some embodiments, a pump to be used in accordance with currently with another therapeutic agent used to treat the the invention is a pump that is implanted within the body of a same disorder), and/or they may achieve different effects Subject. In some embodiments, a pump to be used in accor (e.g., control of any adverse side effects). In some embodi dance with the invention is a mechanical pump and/or an ments, compositions of the invention are administered with a osmotic pump. second therapeutic agent that is approved by the U.S. Food 0384. In general the most appropriate route of administra and Drug Administration. tion will depend upon a variety of factors including the nature of the composition (e.g., its stability in the environment of the 0389. In will further be appreciated that therapeutically gastrointestinal tract), the condition of the Subject (e.g., active agents utilized in combination may be administered whether the subject is able to tolerate oral administration), together in a single composition or administered separately in etc different compositions. 0385. In certain embodiments, a composition may be 0390. In general, it is expected that agents utilized in com administered in amounts ranging from about 0.001 mg/kg to bination with be utilized at levels that do not exceed the levels about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, at which they are utilized individually. In some embodiments, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 the levels utilized in combination will be lower than those mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 utilized individually. mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from 0391 The pharmaceutical compositions of the present about 1 mg/kg to about 25 mg/kg, of Subject body weight per invention may be administered alone and/or in combination day, one or more times a day, to obtain the desired therapeutic with other agents that are used to treat the symptoms of FXS. effect. The desired dosage may be delivered three times a day, In some embodiments, such agents may treat seizures and/or two times a day, once a day, every other day, every third day, mood instability, including but not limited to as Carbam every week, every two weeks, every three weeks, or every azepine (TEGRETOL(R), Valproic Acid, Divalproex (DEPA four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, KOTE(R), Lithium Carbonate, Gabapentin) (NEURON four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, TINR), Lamotrigine (LAMICTAL(R), Topiramate fourteen, or more administrations). (TOPAMAX(R), Tiagabine (GABITRIL(R), Vigabatrin (SA 0386. It will be appreciated that therapeutic agents and BRIL(R), Phenobarbital, Primidone) (MYSOLINE(R), and/or pharmaceutical compositions of the present invention can be Phenytoin) (DILANTINR). employed in combination therapies. In some embodiments, 0392. In some embodiments, such agents may be central the present invention encompasses “therapeutic cocktails' nervous system stimulants, including but not limited to Meth comprising inventive compositions. The particular combina ylphenidate) (RITALINR), Dextroamphetamine tion of therapies (therapeutics or procedures) to employ in a (DEXEDRINER); ADDERALL(R), Ditropan (CON combination regimen will take into account compatibility of CERTAR), L-acetylcarnitine, Venlafaxine (EFFEXORR), the desired therapeutics and/or procedures and the desired Nefazodone (SERZONE(R), Amantadine (SYMME therapeutic effect to be achieved. It will be appreciated that TREL(R), Buproprion) (WELLBUTRINR). Desipramine, the therapies employed may achieve a desired effect for the Imipramine, and/or Buspirone) (BUSPARR). same purpose. For example, a PAK modulator may be admin istered with another agent that is used to treat symptoms of 0393. In some embodiments, such agents may be antihy FXS and/or other neurodevelopmental disorder. In some pertensive drugs, including but not limited to Clonidine embodiments, the therapies employed may achieve different (CATAPRES(R) and/or Guanfacine (TENEX(R). effects (e.g., control of any adverse side effects). 0394. In certain embodiments, such agents may include 0387 Pharmaceutical compositions of the present inven folic acid. tion may be administered either alone or in combination with 0395. In some embodiments, such agents may be selected one or more other therapeutic agents. By “in combination serotonin reuptake inhibitors, including but not limited to with it is not intended to imply that the agents must be Fluoxetine (PROZACR), Sertraline (ZOLOFTR), and/or administered at the same time and/or formulated for delivery Citalopram (CELEXAR). together, although these methods of delivery are within the 0396. In some embodiments, such agents may be antipsy Scope of the invention. Compositions can be administered chotics, including but not limited to Risperidone) (RIS concurrently with, prior to, or Subsequent to, one or more PERIDAL(R), Olanzepine (ZYPREXAR), and/or Quetiapine other desired therapeutics or medical procedures. In general, (SEROQUEL(R). each agent will be administered at a dose and/or on a time schedule determined for that agent. Additionally, the inven 0397. In certain embodiments, such agents may be used to tion encompasses the delivery of the inventive pharmaceuti treat sleep disturbances, including but not limited to Desyrel) cal compositions in combination with agents that may (TRAZODONE(R) and/or Melatonin. improve their bioavailability, reduce and/or modify their 0398. In will further be appreciated that therapeutically metabolism, inhibit their excretion, and/or modify their dis active agents utilized in combination may be administered tribution within the body. together in a single composition or administered separately in 0388. The particular combination of therapies (therapeu different compositions. tics and/or procedures) to employ in a combination regimen 0399. In some embodiments, inventive PAK modulators will take into account compatibility of the desired therapeu may be administered in combination with one or more other tics and/or procedures and/or the desired therapeutic effect to PAK modulators. be achieved. It will be appreciated that the therapies 0400. One of ordinary skill in the art will understand that employed may achieve a desired effect for the same disorder the examples presented above are not meant to be limiting. (for example, an inventive agent may be administered con The principles presented in the examples above can be gen US 2010/0247552 A1 Sep. 30, 2010 44 erally applied to any combination therapies for treatment of be appreciated that the contents of those cited references are FXS and/or other neurodevelopmental disorders. incorporated herein by reference to help illustrate the state of the art. Kits 0406. The following Examples contain important addi tional information, exemplification and guidance that can be 04.01 The invention provides a variety of kits comprising adapted to the practice of this invention in its various embodi one or more of PAK modulators of the invention. For ments and the equivalents thereof. It will be appreciated, example, the invention provides kits comprising at least one however, that these examples do not limit the invention. PAK modulator and instructions for use. A kit may comprise Variations of the invention, now known and/or further devel multiple different PAK modulators. A kit may comprise any oped, are considered to fall within the scope of the present of a number of additional components or reagents in any invention as described herein and as hereinafter claimed. combination. All of the various combinations are not set forth 04.07 Previous studies have consistently associated men explicitly but each combination is included in the scope of the tal retardation with abnormalities in the number and size of invention. synapses. In FXS patients and in FMR1 KO mice, cortical neurons display more postsynaptic dendritic spines and a 0402. According to certain embodiments of the invention, higher proportion of longer and thinner spines compared to a kit may include, for example, (i) a PAK modulator; (ii) normal individuals. The FMR1 KO mice exhibit impaired instructions for administering the PAK modulator to a subject cortical LTP compared to wild-type mice. Strikingly, the Suffering from, Susceptible to, and/or exhibiting symptoms of abnormalities in cortical synaptic morphology and plasticity FXS and/or other neurodevelopmental disorder. of FMR1 KO mice are opposite to those we observed in 0403. According to certain embodiments of the invention, transgenic (dnPAKTG) mice in which activity of p21-acti a kit for identifying PAK modulators may include, for vated kinase (PAK) is inhibited by its dominant negative form example, (i) an FMR KO mouse; (ii) at least one candidate (dnPAK), specifically in the postnatal forebrain. In the Substance (e.g. Small molecule library, collection of peptides, dnPAK TG mice, we found that cortical neurons display etc.); (iii) a positive control (e.g. known PAK modulator); (iv) fewer dendritic spines and a lower proportion of longer and a negative control (e.g. a substance known not to be a PAK thinner spines compared to wild type mice (Hayashi et al., modulator), (v) pharmaceutically acceptable excipients and 2004, Neuron, 42:773; incorporated herein by reference). equipment necessary to prepare candidate Substances and These transgenic mice exhibit enhanced cortical LTP, in con controls for administration to the mouse; and (vii) instruc trast to the impaired cortical LTP in FMR1 KO mice. One tions for administering the candidate Substance to the mouse hypothesis that may explain these results is that signaling in order to determine whetherit treats, alleviates, ameliorates, pathways mediated by PAK and FMR1 may antagonize each relieves, delays onset of inhibits progression of reduces other to regulate synaptic morphology and function, and this severity of and/or reduces incidence of one or more symp hypothesis was tested in the following examples. toms or features of FXS and/or other neurodevelopmental disorder in the mouse. Example 1 0404 Kits typically include instructions for use of inven PAK Inhibition Rescues Spine Morphological tive PAK modulators. Instructions may, for example, com Abnormalities in FMR1 KO Mice prise protocols and/or describe conditions for identification of PAK modulators, administration of PAK modulators to a Materials and Methods Subject in need thereof, etc. Kits generally include one or 0408 Golgi Analysis more vessels or containers so that some orall of the individual 04.09 Following the Golgi-Cox technique (Ramon-Mo components and reagents may be separately housed. Kits may liner, 1970, Contemporary Research Methods in Neu also include a means for enclosing individual containers in roanatomy, Springer, Berlin, Heidelberg, New York), 120 um relatively close confinement for commercial sale, e.g., a plas thick serial sections were obtained from brains of two-month tic box, in which instructions, packaging materials such as old male littermates. Slides containing these sections were styrofoam, etc., may be enclosed. An identifier, e.g., a bar coded before quantitative analysis, and the code was broken code, radio frequency identification (ID) tag, etc., may be only after the analysis was completed. Layer II/III pyramidal present in or on the kit or in or one or more of the vessels or neurons in the temporal cortex were visualized under Olym containers included in the kit. An identifier can be used, e.g., pus upright BX61 with motorized XY stage using Neurolu to uniquely identify the kit for purposes of quality control, cida/stereology software (Microbrightfield). On each pri inventory control, tracking, movement between worksta mary apical dendritic branch, ten consecutive 10 um-long tions, etc. dendritic segments were analyzed to quantify spine density. To ensure sampling consistency among Golgi analysis and Exemplification electrophysiology experiments, analyses in the temporal cor tex were all carried out in slices or sections corresponding to 04.05 The representative Examples that follow are FIG. 62-67 of the mouse brain atlas (Franklin et al., The intended to help illustrate the invention, and are not intended mouse brain in stereotaxic coordinates, Academic, San to, nor should they be construed to, limit the scope of the Diego, Calif., 1997). invention. Indeed, various modifications of the invention and 0410 Electron Microscopy many further embodiments thereof, in addition to those 0411 Two-month-old male littermates were anaesthetized shown and described herein, will become apparent to those and perfused according to standard procedures. Blocks of skilled in the art from the full contents of this document, temporal cortex were then embedded, from which 1 um thick including the examples which follow and the references to the sections were cut and stained with 1% toluidine blue to guide scientific and patent literature cited herein. It should further the further trimming to isolate layer II/III of temporal cortex. US 2010/0247552 A1 Sep. 30, 2010

90 nm ultrathin sections were then cut and stained with uracyl (95% O, and 5% CO) warm (30°C.) artificial cerebrospinal acetate and lead citrate. Randomly selected neuropil areas fluid containing 124 mM NaCl, 5 mM KCl, 1.25 mM were photographed at a 10,000x magnification with a JEOL NaH2PO 1 mM MgCl2 mM CaCl, 26 mMNaHCO, and 1200EX electron microscope. Image negatives were scanned 10 mM dextrose. Field potentials (FPs) in layer II/III evoked at 1200 dpi and analyzed by OpenLab Program (Improvi by layer IV stimulation were measured as previously sion). Excitatory synapses bearing spines were defined by the described (Hayashi et al., 2004, Neuron 42:773) and presence of a clear post-synaptic density (PSD) facing at least responses were quantified as the amplitude of FP in cortex. three presynaptic vesicles. Micrographs covering 500 um LTP was induced by TBS, which consisted of eight brief 1000 um neuropil regions from each mouse were analyzed bursts (each with 4 pulses at 100 Hz) of stimuli delivered and used for quantitation. PSD length and percentage of every 200 m.sec.V perforated synapses were quantified from the same popula tion of synapses. The measurements were all performed by an Results experimenter blind to the genotype. 0416) Cortical long-term potentiation (LTP) has been Results shown to be reduced in FMR1 KO mice while it is enhanced in dnPAK TG mice (Li et al., 2002, Mol. Cell. Neurosci., 0412 Since spine abnormality is a pathological hallmark 19:138; Zhao et al., 2005, J. Neurosci., 25:7385; and Hayashi in FXS patients and FMR1 KO mice at the cellular level, et al., 2004, Neuron, 42:773; all of which are incorporated dendritic spine morphology was examined by measuring herein by reference). To assess the effect of PAK inhibition on spine density in apical dendrites of Golgi-stained layer II/III the cortical synaptic transmission and plasticity in FMR1 KO pyramidal neurons of the temporal cortex of dMT mice as mice, extracellular field recordings in temporal cortex layer well as their littermates, dnPAKTG, FMR1 KO, and wild II/III synapses were carried out while stimulating layer IV. type mice. The number of spines per 10 um of dendritic Basal synaptic transmission, as measured by field potential segments that run proximal to distal to the neuronal Soma was responses to a range of stimulus intensities, did not differ quantified. In proximal dendritic segments, spine density was between the four genotypes (FIG. 14A). However, as lower indnPAKTG mice compared to wild-type mice while expected, administration of theta-burst stimulation (TBS) at it was higher in FMR1 KO mice compared to wild-type mice 100 Hz produced LTP of a lower magnitude in FMR1 KO (FIGS. 10 and 11). In contrast, spine density indMT mice was mice than in wild-type mice and LTP of a higher magnitude in comparable to that in wild-type controls in all dendritic seg dnPAKTG than in wild-type mice (FIG.14B). In contrast, the ments except segments 7 and 8 (FIG. 11). When averaged magnitude of LTP was indistinguishable between dMT mice over all segments, mean spine density in dMT mice was and wild-type controls at various times following the appli significantly lower than that in FMR1 KO mice and signifi cation of the stimulus (FIG. 14B). This demonstrates that cantly higher than that in dnPAKTG mice (FIG. 12). These PAK inhibition rescues LTP defects in FMR1 KO mice. results indicate that PAK inhibition partially restores the abnormality of spine density in FMR1 KO mice. Example 3 0413. In addition to in increased spine density, cortical neurons from FXS patients and FMR1 KO mice exhibit PAK Inhibition Rescues Multiple Behavioral Defects increased spine length (Hinton et al., 1991, Am. J. Med. in FMR1 KO Mice Genet., 41:289; Comery et al., 1997, Proc. Natl. Acad. Sci., Materials and Methods USA, 94:5401; Irwin et al., 2001, Am. J. Med. Genet., 98:161; and McKinney et al., 2005, Am. J. Med. Genet. B. Neuropsy 0417 Open Field Test chiatr. Genet., 136:98; all of which are incorporated herein by 0418. Two-month-old male littermates were subjected to reference). To investigate whether dnPAK can also restore the open field test according to standard procedures. Each this abnormality, spine length (the radial distance from tip of mouse ran for 10 minutes in a VersaMax activity monitor spine head to dendritic shaft) of Golgi-stained pyramidal chamber (Accuscan Instruments). Open field activity was neurons in the four genotypes was measured. In cumulative detected by photobeam breaks and analyzed by the VersaMax frequency plots, FMR1 KO neurons exhibited a significant software. Stereotypy is recorded when the mouse breaks the shift in the overall spine distribution towards spines of longer same beam (or set of beams) repeatedly. Stereotypy count is length compared to wild-type neurons, while dnPAK TG the number of beam breaks that occur during this period of neurons exhibited the opposite shift to shorter spines (FIG. sterotypic activity. 13). In contrast, spine length distribution of dMT neurons 0419 Trace Fear Conditioning Task overlapped well with that of wild-type neurons (FIG. 13), 0420. Three-month-old male littermates were subjected to indicating that PAK inhibition is sufficient to restore the cor trace fear conditioning as previously described (Zhao et al., tical spine length abnormality in FMR1 KO mice. 2005, J. Neurosci., 25:7385). On day 1, mice were placed in the training chamber (Chamber A, Coulbourn Instruments) Example 2 for 60 seconds before the onset of a 15-second white noise tone (conditioned stimulus or CS). 30 seconds later, mice PAK Inhibition Rescues Reduced Cortical LTP in received a 1-second shock (0.7 mA intensity; unconditioned FMR1 KO Mice stimulus or US). Thus, one trial is composed oftone (CS), 30 Materials and Methods seconds blanktime (also called “trace'), and then shock (US). Seven trials with an intertrial interval (ITI) of 210 seconds 0414 Electrophysiology were performed to let the mice learn the association between 0415. From three-month-old male littermates, coronal tone and shock across a time gap. To examine whether mice brain slices containing temporal cortex were prepared and left remember this association, on day 2, mice were placed into a to recover for at least 1 hour before recording in oxygenated new chamber (Chamber B) with a different shape and smell US 2010/0247552 A1 Sep. 30, 2010 46 from those in Chamber A. After 60 seconds, a 15-second tone memory and/or require a repetition of the recall cue (tone), was repeated seven times with an ITI of 210 seconds. Video but they can eventually (after 5 tone sessions) recall the images were digitized and the percentage of freezing time memory at the level that is not significantly different from the during each ITI was analyzed by Image FZ program (O'Hara wild-type level. & Co). Freezing was defined as the absence of all but respi ratory movement for a 1-second period. Example 4 Results PAK1 and FMRP Physically Interact Materials and Methods 0421) PAK Inhibition Rescues Multiple Behavioral Defects in FMR1 KO Mice 0424 Animal Handling, Experimental Design, and Data 0422 To test if the partial rescue of spine morphology and Analysis the complete rescue of cortical LTP by PAK inhibition could 0425 All strains of mice are of the C57B6 background. ameliorate behavioral deficits present in FMR1 KO mice, the FMR1 KO mice were obtained from Dr. Steven Warren. mice of various genotypes were Subjected to a series of behav dnPAKTG mice were generated previously (Hayashi et al., ioral tasks. In an open field test where mice are placed in a box 2004, Neuron, 42:773). Mouse maintenance and all experi and allowed to run freely for ten minutes. FMR1 KO mice mental procedures were performed in compliance with exhibited three abnormal behaviors compared to wild-type National Institute of Health guidelines. All experiments were mice (Peier et al., 2000, Hum. Mol. Genet., 9:1145). (1) conducted in a blind fashion. Unless specified otherwise, data Hyperactivity: they traveled a longer distance and moved for were analyzed with Statview software (SAS) using one-way a longer period of time (FIG. 15); (2) Stereotypy: they exhib ANOVA test followed by Fisher's protected least significance ited a higher number of repetitive behaviors (FIGS. 15); and difference (PLSD) post hoc test. Values are presented as (3) Hypo-anxiety: they stayed in the center field for a longer mean-SEM. period of time and in the corners of the field for a shorter 0426 Immunoprecipitation and Western Blotting period of time (FIG. 15). In all three behaviors, dMT mice 0427 Mouse brains were homogenized in ice-cold exhibited performance comparable to wild-type controls homogenization buffer (0.32M sucrose; 10 mM Tris-HCl, pH (FIG. 15). This indicated that PAK inhibition in FMR1 KO 7.4.5 mM EDTA; Complete Protease Inhibitor Cocktail Tab mice restores locomotion, repetitive behavior, and anxiety to lets (Roche)) and centrifuged at 1,000xg for 10 minutes at 4 wild-type levels. C. The supernatant was collected and centrifuged at 21,000xg 0423. To further examine whether PAK inhibition can res for 15 minutes at 4° C. The pellet was resuspended in TE cue abnormal cortex-dependent behaviors, trace fear condi buffer (10 mM Tris-HCl pH 7.4.5 mM EDTA) and one-ninth tioning was performed, which is a test that depends on the volume of cold DOC buffer (500 mM Tris-HCl, pH 9.0; 10% integrity of the prefrontal cortex and is sensitive to attention sodium deoxycholate) was added. The mixture was incubated distracting stimuli (McEchron et al., 1998, Hippocampus, in a 37°C. water bath for 30 minutes while shaking and mixed 8:638; and Han et al., 2003, Proc. Natl. Acad. Sci., USA, with one-ninth volume of Buffer T (1% Triton X-100: 1% 100: 13087; both of which are incorporated herein by refer sodium deoxycholate: 500 mM Tris-HCl, pH 9.0). The mem ence). It was previously shown that FMR1 KO mice are brane extract was dialyzed against binding/dialysis buffer (50 impaired in this form of conditioning, which may relate to the mM Tris-HCl, pH 7.4; 0.1% TritonX-100) at 4°C. overnight. attention deficits in FXS patients (Zhao et al., 2005, J. Neu 0428 For immunoprecipitation, the dialyzed membrane rosci., 25:7385; incorporated herein by reference). In this extract was pre-cleared with protein A-Sepharose beads, then task, a conditioning trial was composed of a tone (as the incubated with C.-PAK1 (N-20 from Santa Cruz, Biotech) or conditioned stimulus or CS), then a 30-second time gap (also control rabbit serum (Sigma) in binding/dialysis buffer for 3 called “trace') and finally an electric shock (as the uncondi hours, and then incubated with protein A-Sepharose beads tioned stimulus or US). Seven trials were given to allow the overnight at 4°C. To test binding specificity, C-PAK1 was mice to learn the association between the tone and the shock also incubated with its corresponding blocking peptide (Santa across the 30-second time gap. Mice that learn and remember Cruz, Biotech) prior to incubation with the membrane extract. this association will become immobile (or “freeze') in The beads were washed three times with binding/dialysis response to the tone, even when they are placed into a new buffer. chamber with a different shape and smell compared to the 0429 Proteins that bound to the beads were separated by training chamber. During training, the four genotypes exhib SDS-PAGE, transferred to a nitrocellulose membrane, and ited comparable amounts of freezing in all conditioning trials subjected to western blot analysis. For PAK1 western blots, (FIG. 16), Suggesting normal memory acquisition. However, the membrane was blocked in 10% milk, then incubated with when placed in a new chamber 24 hours after training, both C-PAK1 antibody diluted at 1:1000, then incubated with FMR1 KO mice and dnPAKTG mice exhibited a significant C-rabbit horse radish peroxidase (HRP Sigma) diluted at reduction in tone-induced freezing compared to wild-type 1:1000, and then developed with enhanced chemilumines controls (FIG. 16), indicating an impaired trace fear memory cence (ECL) Renaissance kit (New England Nuclear). For in these two genotypes. dMT mice also showed freezing FMRP western blots, the membrane was processed with the deficits during the first several tone sessions (sessions 1 to 4) Blast blotting amplification system (Perkin Elmer) with compared to wild-type controls (FIG. 16), although the defi C-FMRP antibody (Chemicon) diluted at 1:1000, biotiny cits during these sessions were, on average, less pronounced lated C-mouse diluted at 1:1000, and streptavidin-HRP compared to dnPAKTG or FMR1 KO mice (FIG. 17). How diluted at 1:1000. ever, with additional tone sessions (sessions 5 to 7), freezing 0430 GST Pull-Downs by dMT caught up to that of wild-type while its difference 0431 GEX6p-1 plasmid encoding GST was purchased from FMR1 KO mice almost reached statistical significance from Pharmacia. GST-PAK1 plasmid was obtained from Dr. (p=0.07, FIG. 17). Thus, dMT mice are slow in expressing the Joe Kissil (Kissil et al., 2003, Mol. Cell, 12:841; incorporated US 2010/0247552 A1 Sep. 30, 2010 47 herein by reference). Plasmids encoding FMRP and its and 21). These results show that PAK1 directly binds to mutants were obtained from Dr. Edouard Khandjian FMRP and this interaction requires the integrity of the KH (Mazroui et al., 2003, Hum. Mol. Genet., 12:3087; incorpo domains of FMRP. rated herein by reference). GST and GST-PAK1 proteins were expressed in BL21 E. coli, purified on glutathione Example 5 sepharose 4B (GS4B) beads (Pharmacia), and dialyzed with PBS overnight. FMRP and its mutants were in vitro-trans LTD Mediated by mGluR in the Hippocampus lated with the TNT coupled reticulocyte lysate systems kit 0434 Long-term depression (LTD) mediated by metabo (Promega) and labeled with Transcend tRNA (Promega). tropic glutamate receptor (mGluR) can be measured in the GST or GST-PAK1 was incubated with FMRP or its mutants hippocampus. Hippocampal slices are prepared in ice-cold in binding buffer (50mM Tris-HCl, pH 7.5; 120mMNaCl: 10 dissection buffer (212 mM sucrose, 2.6 mM KCl, 1.25 mM mM MgCl, 5% glycerol: 1% Triton X-100) for 3 hours. NaHPO, 26 mM NaHCO, 5 mM MgCl, 0.5 mM CaCl, GS4B beads were added and incubated for 1 hour. The beads and 10 mM dextrose) and allowed to recover for 1 hours-5 were washed three times with the binding buffer. Proteins that hours before recording in oxygenated (95% O, and 5% CO.) bound to the beads were separated by SDS-PAGE, transferred 30° C. artificial cerebrospinal fluid (ACSF; 124 mM NaC1,5 to a nitrocellulose membrane, and subjected to western blot mMKC1, 1.25 mMNaH2PO 1 mM MgCl2 mM CaCl26 analysis. To detect in vitro-translated FMRP or its mutants, mMNaHCO, and 10 mM dextrose). Field potentials (FPs) in the membrane was blocked, incubated with streptavidin stratum radiatum of area CA1 are evoked by a 200 usec HRP washed, and developed with ECL Renaissance kit. current pulse to Schaffer collateral axons. Stable baseline responses are collected every 30 seconds by a stimulation Results intensity (10 LA-30 LA) yielding 50%-60% of the maximal 0432. The morphological, electrophysiological and response. mGluR-LTD is induced by a 5 minute application behavioral data presented in Examples 1-3 demonstrate that of mGluRagonist 3,5-dihydroxyphenylglycine (DHPG; at PAK inhibition rescues (at least partially) multiple abnor 100 LM) and/or by using paired-pulse low-frequency stimu malities in FMR1 KO mice. To begin to understand the under lation consisting of 900 pairs of stimuli delivered at 1 Hz in lying mechanism, it was determined whether PAK1 and the presence of the N-methyl-D-aspartate receptor (NM FMRP physically interact via immunoprecipitation followed DAR) antagonist D-(-)-2-amino-5-phosphono-pentanoic by Western blot analysis. Since PAK1 and FMRP are both acid (D-APV; at 50 uM). DHPG is a chiral compound, and localized in synapses (Weiler et al., 1997, Proc. Natl. Acad. mGluR-LTD can be induced by application of RS-DHPG and Sci., USA,94:5395; and Hayashi et al., 2004, Neuron, 42:773: S-DHPG, but typically not by application of R-DHPG. The both of which are incorporated herein by reference), synapse initial slope of the field potential is recorded as an indicator of enriched membrane extract was prepared from mouse brain synaptic strength. and Subjected the extract to immunoprecipitation with a PAK1 antibody (C.-PAK1). Proteins that may co-precipitate Example 6 through their direct or indirect interaction with PAK1 were Synaptic Currents Mediated by AMPARs or separated by SDS-PAGE and subjected to Western blot analy NMDARs in the Cortex sis with an FMRP antibody. FMRP immuno-reactivity was observed in PAK1 immunoprecipitates but not in control 0435 Synaptic currents mediated by AMPA receptors serum immunoprecipitates (FIG. 18). This interaction is spe (AMPARs) and/or NMDARs can be measured in the cortex. cific because it did not occur when PAK1 antibody was pre For measurement of AMPAR-mediated miniature excitatory incubated with a blocking peptide, which competes with postsynaptic current (mEPSC), 1 uM tetrodotoxin, 100 uM PAK1 for binding to the PAK1 antibody, prior to immuno APV, and 10 uMbicuculline are added in a bath of ACSF. The precipitation (FIG. 18). This result shows that the endogenous cells are held at -80 mV, and recordings are done at 30° C. PAK1 and FMRP interact, directly or indirectly, in the brain. Continuous 30 msec-60 msec traces are collected at 8 second 0433) To examine whether PAK1 directly interacts with interval and filtered at 2 KHZ. Cells with series resistance FMRP, a glutathione S-transferase (GST)-pull down assay 213 mS2 are discarded. The measurement of NMDAR was performed in which in vitro-translated FMRP was incu EPSC/AMPAR-EPSC ratio is done in ACSF containing 2 bated with either GST or GST-tagged PAK1 (GST-PAK1). mMMg", 10u Mbicuculline, and 50 Mglycine. NMDAR GST-PAK1, but not GST alone, bound to FMRP (FIGS. 19 dependent and AMPAR-dependent responses are discrimi and 20), Suggesting a direct interaction between PAK1 and nated based on their distinct kinetics and Voltage dependence. FMRP. FMRP contains a primary phosphorylation site at Ser Thus, NMDAR-mediated response is valued as the currents 499 and three RNA-binding domains (KH1, KH2 and RGG) recorded at +40 mV and measured 100 msec after the that are conserved among species (FIG. 21; O'Donnell and response onset. The AMPAR-mediated response is taken Warren, 2002, Annu. Rev. Neurosci., 25:315; and Ceman et from the peak amplitude response recorded at -80 mV. al., 2003, Hum. Mol. Genet., 12:3295; both of which are incorporated herein by reference). To map the PAK1-binding Example 7 region on FMRP, a series of deletion or point mutants of Spine Morphology indnPAKTG, FMR1 KO Mice FMRP were used in the GST-pull down assay. An FMRP mutant without the RGG box (ARGG) or phosphorylation 0436 Spine morphology indnPAKTG, FMR1 KO mice domain containing Ser 499 (AS499) was still able to bind to (“dMT mice’) is largely normal compared to wild type mice, PAK1, whereas an FMRP mutant without KH domains indicating that signaling pathways mediated by PAK and (AKH) or with a point mutation in the KH2 domain previ FMRP antagonize each other to regulate spine morphogen ously found in a human with severe FXS (1304N; Feng et al., esis. FMRP is known to bind to mRNA encoding Rac1, the 1997, Mol. Cell, 1:109) was unable to bind to PAK1 (FIGS. 20 upstream activator of PAK, and to antagonize the effect of US 2010/0247552 A1 Sep. 30, 2010 48

Rac1 on dendritic development in Drosophila. If in the donor, Such as ATP, containing radiolabeled phosphate, and mouse, FMRP binds to and subsequently represses the trans PAK-dependent incorporation of radiolabel into FMRP is lation of Rac1 mRNA, FMR1 removal would result in measured. increased levels of Rac1 and enhanced Rac1/PAK-mediated 0441 PAK-dependent FMRP phosphorylation can be signaling. Thus, in dMT mice, Rac1/PAK-mediated signaling measured by monitoring physicochemical properties of would be reversed to wild type level by simultaneous FMR1 FMRP that may occur as a result of incorporation of phos removal and PAK inhibition, leading to normal actin dynam phate groups. Such properties may include electrophoretic ics and spine morphogenesis. mobility, light absorbance, fluorescence and/or phosphores cence, chromatographic properties, etc. Such alterations of Example 8 Substrate physicochemical properties can be readily mea Sured by one skilled in the art and used as an indicator of Analysis of FMRP in Translational Repression of kinase activity. Rac1, PAK1, PAK2, and/or PAK3 mRNA 0442. Alternatively or additionally, monoclonal or poly clonal antibodies may be generated which selectively recog 0437. In addition to an indirect signaling interaction nize phosphorylated forms of FMRP, and thus the degree of between PAK and FMRP possibly via FMRP's capability to binding of such antibodies to FMRP subsequent to the kinase bind to and repress the translation of Rac1 mRNA, FMRP reaction may be used as an indirect method of determining the may directly bind to and repress the translation of mRNAs ability of PAK to phosphorylate FMRP. Kinase assays may be that encode PAKs, such as PAK1, PAK2, and/or PAK3. In this performed using purified, partially recombinant PAK and/or case, FMR removal would result in increased levels of total FMRP, and/or PAK and/or FMRP which is purified from cells PAK, including active PAK. Thus, in dMT mice, the levels of that naturally express the protein using standard purification active PAK would be reversed to wild type level. procedures. Such as those described herein. Example 9 Example 11 Assaying Whether PAK Competes with RNA for PAK Modulators and Autism Binding to FMRP KH Domains 0443) FXS patients constitute ~5% population of patients 0438 PAK may bind to and negatively regulate FMRP's with autism, a complex multigenic disorder characterized by activity. Example 4 shows that the KH RNA binding domains impaired communication, impaired social interactions and of FMRP mediate the binding of FMRP to PAK1. Thus, PAK repetitive interests and behavior. Due to the difficulty in iden may compete with RNAs for binding to the KH domains of tifying Susceptible chromosomal loci and genes, little is FMRP, thus relieving FMRP's activity to repress translation known on the causes and pathogenesis of autism. The asso of these RNAs. To determine whether PAK may compete with ciation between FXS and autism suggests that these two RNAs for binding to the KH domains of FMRP binding disorders may share common genetic factors and underlying assays (such as immunoprecipitation and GST pulldown, patho-physiological mechanisms. PAK modulators, there described herein) may be performed in the presence of a fore, may represent a novel treatment for certain types of substance that may compete with PAK for binding to the KH autism. In support of this idea, the PAK3 gene locates in domains of FMRP. chromosomal locus Xq22, a region linked to autism. It could, 0439. In such assays, FMRP may be free in solution, fixed therefore, be determined whether PAK3 and/or other PAK to a Support, and/or expressed in and/or on the Surface of a genes are mutated inautistic patients. To directly test the link cell. The candidate RNAs and/or PAK may be labeled, between PAK modulation and autism, dnPAKTG mice could thereby permitting detection of binding. Competitive binding be crossed to available autism mouse models (Liam et al., formats may be performed in which one of the substances is 1997, Cell, 90:895; Moretti et al., 2005, Hum. Mol. Genet., labeled, and one may measure the amount of free label versus 14:205; and Kwon et al., 2006, Neuron, 50:377: all of which bound label to determine the effect of the competitor on are incorporated herein by reference) to examine whether binding. For example, PAK may be labeled and the candidate social behavior is reversed to wild type level in the double RNAs may be unlabeled in order to detect the ability of the mutantS. candidate RNAs to compete with PAK for binding to FMRP. Example 12 Example 10 Treatment of FMR KO Mice with Small Molecule Assaying Whether PAK Phosphorylates FMRP PAK Inhibitors 0440 PAK, a serine/threonine kinase, may phosphorylate Preparation of Stock Solutions FMRP. In the brain, FMRP is phosphorylated on Ser 499 and 0444 Emodin (also known as 1.3,8-Tri-hydroxy-6-me its phosphorylation status affects its association with RNA thyl-anthra-quinone; 6-Methyl-1,3,8-tri-hydroxy-anthra and polyribosomes. Additional putative kinase Substrates in quinone; Emodol; Frangula-emodin) is obtained from the mouse brain include Thr 454, Ser 496, Thr501, Ser 503, Sigma-Aldrich (#E7881). Emodin is an orange powder that is Thr517 (Ceman et al., 2003, Hum. Mol. Genet., 12:3295; and soluble in DMSO, ethanol, or 1 N dilute aqueous ammonia. A Mazroui et al., 2003, Hum. Mol. Genet., 12:3087; both of stock solution of 1 mg/ml-50 mg/ml is made and stored at which are incorporated herein by reference). Kinase assays -20° C. may be performed in order to determine whether PAK phos 0445 OSU-03012 (also known as 2-amino-N-(4-5-(2- phorylates FMRP. In such assays PAK and/or a characteristic phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)- portion thereof is contacted with FMRP and/or a characteris phenyl)acetamide) is obtained from Cayman Chemicals tic portion thereof in the presence of a suitable phosphate (#10008005; FIGS. 6 and 7). OSU-03012 is a white solid that US 2010/0247552 A1 Sep. 30, 2010 49 is soluble in organic solvents such as DMSO, ethanol, and three times per week, every other day, one time per day, two DMF. A stock solution of 30 mg/ml-100 mg/ml is made and times per day, three times per day, and/or four times per day. stored at -20°C. 0453 Emodin and/or OSU-03012 can be administered by oral, intravenous, intramuscular, intra-arterial, intramedul Administration of Small Molecule Therapeutics to FMRKO lary, intrathecal, parenteral, Subcutaneous, intraventricular, Mice transdermal, interdermal, rectal, intravaginal, intraperitoneal, 0446 Drug effects are optimized by determining the ideal topical (e.g. by powders, ointments, creams, gels, and/or delivery method, dose, Volume, and dosage schedule. The drops), transdermal, mucosal, nasal, buccal, enteral, and/or level of drug exposure and duration is compared in regions of Sublingual administration; by intratracheal instillation, bron the brain as well as in other organs and the blood following chial instillation, and/or inhalation; and/or as an oral spray, various drug administration methods. nasal spray, and/or aerosol. In some embodiments, Emodin 0447 Small molecule therapeutics, including, but not lim and/or OSU-03012 can be administered by a continuous IV ited to, Emodin and/or OSU-03012, can be administered to drip. In some embodiments, Emodin and/or OSU-03012 can FMRKO mice by injection, and injected mice can be evalu be administered by retro-orbital injection. ated for therapeutic efficacy. Studies are frequently per 0454 Time lapse between last dose and biochemical or formed using male mice, but heterozygous and/or homozy behavioral analyses ranges between 15 minutes and 2 days. gous female mice can also be used in accordance with the For example, time lapse between last dose and biochemical or invention. Studies are frequently performed using adult mice, behavioral analyses can be approximately 15 minutes, but prenatal, infant, and/or juvenile mice can also be utilized approximately 30 minutes, approximately 1 hour, approxi in accordance with the present invention. mately 2 hours, approximately 3 hours, approximately 4 0448 Behavioral phenotypes vary slightly with mouse hours, approximately 5 hours, approximately 6 hours, strain. In some embodiments, a C57BL6 genetic background approximately 9 hours, approximately 12 hours, approxi can be used for small molecule PAK inhibitor studies. In some mately 15 hours, approximately 18 hours, approximately 24 embodiments, a FVB/NJ (FVB) genetic background can be hours, approximately 30 hours, approximately 36 hours, used for small molecule PAK inhibitor studies. In some approximately 42 hours, approximately 48 hours, approxi embodiments, F1 hybrids produced by crossing a male FVB mately 60 hours, or approximately 72 hours. mouse to a female C57BL6 heterozygous FMR1 KO can be 0455 Exemplary Dosage Schedule #1 used for small molecule PAK inhibitor studies. In some 0456 Adult male FMR1 KO mice and littermates are embodiments, F1 hybrids produced by crossing a male administered either placebo or PAK inhibitor (1 mg/kg, 10 C57BL6 mouse to a female FVB heterozygous mouse can be mg/kg, 30 mg/kg, 100 mg/kg, or 300 mg/kg) in a total Volume used for small molecule PAK inhibitor studies. of 200 ul via oral gavage. Mice are monitored for signs of 0449 In general, small molecule PAK modulators are fre toxicity or decline in health. At various time points (1 hour, 4 quently soluble in organic solvents, but not in aqueous solu hours, 12 hours, or 24 hours) following the single drug admin tions. A variety of different solvents are tested to achieve istration, animals are sacrificed. At least two mice are used for maximal therapeutic effects and minimal adverse side effects. each of the 48 conditions. In some experiments, between two For example, Small molecule therapeutics (e.g. Emodin and/ and ten mice are used. In some experiments, more than 10 or OSU-03012) are formulated using water, saline (e.g. phos mice are used. In some experimental runs, only a Subset of phate-buffered saline), 2-hydroxypropyl-beta-cyclodextrin, these conditions is performed. At the time of sacrifice, blood, Tween, methyl cellulose, polyethylene glycol, Cremophor urine, and organs are collected. Brains are dissected into EL (Sigma), oils (e.g. Vegetable oil), and/or Maalox. The Subregions, including the hippocampus, cortex and cerebel Small molecule agent may be formulated as a homogenous lum. Analysis of drug concentration and efficacy in various Solution or colloidal dispersion. fluids and tissues is used to inform future studies by Suggest 0450 A variety of amounts of Emodin and/or OSU-03012 ing optimal drug dosing concentrations and Schedules. are administered to FMRKO mice. For example, dosages of 0457 Exemplary Dosage Schedule #2 about 5ul, about 10 ul, about 20 ul, about 50 ul, about 100 ul, 0458. Adult male FMR1 KO mice and littermates are about 500 ul, about 1000 ul, about 2000 ul, and/or about 3000 treated with PAK inhibitor (e.g. a small molecule such as ul of Emodin and/or OSU-03012 can be administered per Emodin and/or OSU-03012) for 3 days, 1 week, 2 weeks, or dose. Typically, dosages of about 50 ul to about 1.0 ml are 4 weeks prior to behavior analysis. Mice of each genotype utilized. will be randomly divided into three dosing groups with at 0451. Emodin and/or OSU-03012 can be administered at least two mice in each group: Vehicle control and drug treated concentrations of about 0.5 mg/kg, about 1 mg/kg, about 2 (10 mg/kg or 100 mg/kg, or another dose found to be optimal mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about in the previously described pilot experiment). In some experi 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about ments, about five mice are used. In some experiments, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, ten mice are used. In some experiments, more than 10 mice about 14 mg/kg, about 15 mg/kg, about 20 mg/kg, about 50 are used. All drugs are administered once per day in a total mg/kg, about 100 mg/kg, about 250 mg/kg, or about 500 Volume of 2001 by oral gavage. In some experimental runs, mg/kg per dose (mg/kg corresponds to # mg Emodin and/or only a subset of these conditions is performed. Behavior OSU-03012 per kg of animal subject). experiments—including open field, trace fear conditioning, 0452. A variety of dosage schedules for emodin and/or and/or social interaction experiments—are conducted 12-18 OSU-03.012 are utilized. Emodin and/or OSU-03.012 are hours after the last dosing (or at another time point found to be administered one time only and/or are administered multiple optimal in the previous dosing experiment). Mice are sacri times (e.g. at regular intervals). For example, Emodin and/or ficed immediately following final behavior assay and subre OSU-03012 can be administered one time per month, two gions of the brain, as well as other organs and blood, are times per month, one time per week, two times per week, collected and stored. US 2010/0247552 A1 Sep. 30, 2010 50

04.59 Exemplary Dosage Schedule #3 (blood plasma?urine) or per mg basis (sample), based upon 0460 Male FMR1 KO pups and littermates to be used for the amount of sample used for the procedure. audiogenic seizure (AGS) experiments are treated with PAK 0465 For determination of OSU-03012 and metabolite inhibitor (e.g. a small molecule such as Emodin and/or OSU concentrations, samples (e.g. blood plasma, CSF, urine, and 03012) for 1 day, 3 days, 5 days, or 7 days immediately prior methanol extracts from tissues) are subjected to High-Perfor to post natal day 20 (p20), at which time behavioral testing mance Liquid Chromatography (HPLC) using a C18 column occurs. Therefore, PAK inhibitor treatments begin when the as the Solid phase and a linear gradient from acetonitrile/0. pups are p13, p15, p17, or p19. Subcutaneous injections of 025 Mammonium acetate, pH 4.5 (20:80) to acetonitrile/0. placebo or PAK inhibitor (1 mg/kg, 3 mg/kg, or 10 mg/kg) in 025 Mammonium acetate, pH 4.5 (60:40) as the mobile a total Volume of 50 ul/g body weight are given twice per day. phase. OSU-03012 and metabolites are detected by UV For each of the 16 conditions (combinations of days of admin absorbance at 240 nm or by fluorescence with excitation at istration and drug dose), 6-8 FMR1 KO pups and 2 control 240 nm and emission at 380 nm. Samples are spiked with a littermates are used. In some experimental runs, only a Subset known amount of OSU-03012 prior to HPLC as an internal of these conditions is performed. Behavior experiments are standard. The amount of OSU-03012 in the sample is deter conducted 4-8 hours after final injection. mined by comparison to the known amount of internal stan 0461 One of ordinary skill in the art will readily recognize dard, or with samples obtained from untreated mice spiked that these are exemplary, dosage schedules and are not meant with known amounts of OSU-03012. The concentration of to be limiting. A skilled artisan will recognize how the con OSU-03012 is thus determined on a per mL basis (blood ditions of these exemplary dosage schedules may be modified plasma?urine) or per mg basis (sample), based upon the in order to optimize dosage. Such optimizations are contained amount of sample used for the procedure. within the scope of the present invention. 0466 Pharmacokinetic analysis includes plotting mean plasma, CSF, and/or urine concentrations of drug as a func Post-Treatment Biochemical Analyses tion of time, and calculating area under the curve (AUC) for 0462. After treatment, mice are subjected to a number of different times post-injection. Half-life of PAK inhibitors is biochemical analyses in order to evaluate the efficacy of small determined by fitting the concentration-time curve after the molecule treatment. Typically, biological samples to be used peak levels have been reached to a single exponential. Peak in biochemical analyses are obtained post-mortem. However, levels of small molecule PAK inhibitor in collected fluids are biochemical analyses can be performed using biological determined for each drug, dose, and time point combination. samples obtained from living mice. 0467 Similarly, blood and urine can be collected and ana 0463. After the final administration of the compound, lyzed at set intervals throughout the dosage regimen. For mice are subjected to a number of biochemical analyses in example, the peri-orbital sinus can be used as a source of order to evaluate the bioavailability and efficacy of small venous blood, and approximately 0.25 ml of blood can be molecule treatment. Mice are sacrificed at 1 hour, 2 hours, 6 safely collected in a capillary tube at weekly intervals under hours, 12 hours, 24 hours, or 48 hours after final drug admin anesthesia. Similarly, tail vein Venipuncture can be used to istration either by cervical dislocation or overdose of anes collect small blood volumes (typically a few drops) from thesia (e.g. Avertin or Isoflurane), and blood is collected in restrained mice. heparinized tubes immediately thereafter by cardiac punc 0468 Post-mortem, various tissues are collected and ana ture. Blood plasma is separated by centrifugation (e.g. lyzed for drug compounds and associated metabolites (as 20,000xg), frozen, and stored at -80°C. until analysis. Cere described for blood, CSF, and urine samples) and efficacy of brospinal fluid (CSF) is collected from the ventricles, briefly PAK inhibition. Tissues collected include liver, kidney, centrifuged to pellet cell debris, frozen, and stored at -80°C. spleen, lymph node, heart, lung, muscle, skin, spinal cord and until analysis. To determine route and rate of elimination of brain, which can be subdivided into multiple areas including the compound, urine is collected from the bladder, briefly but not limited to cerebral cortex, hippocampus, and cerebel centrifuged to pellet cell debris, and the supernatant frozen lum. Efficacy of drug treatment in the brain is determined by and stored at 80°C. until analysis. Using dissection, tissues homogenization of braintissue, followed by gel electrophore including liver, kidney, spleen, lymph node, heart, lung, sis and Western blotting to monitor activities of PAK and its muscle, skin, spinal cord and brain, which can be subdivided downstream effectors through the use of phospho-specific into multiple areas including but not limited to cerebral cor antibodies. tex, hippocampus, and cerebellum are collected. Emodin, 0469. These Western blots are performed as previously OSU-03012, and their metabolites are extracted from tissues described (Hayashietal., 2004, Neuron, 42:773; and Kelleher using methanol. et al., 2004, Cell, 116:467; both of which are incorporated 0464 For determination of Emodin and metabolite con herein by reference). Briefly, mouse cerebral cortex, hippoc centrations, samples (e.g. blood plasma, CSF, urine, and ampus, and cerebellum are dissected and homogenized in methanol extracts from tissues) are subjected to High-Perfor cold (e.g. 4°C.) RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mance Liquid Chromatography (HPLC) using a C18 column mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0 mM or as the solid phase and either 88%methano 1:12% 0.1%phos 1 mMSDS) with protease (e.g. some or all of Roche Com phoric acid (water) or 40:600.1M phosphoric acid:methanol plete.R., Mini Complete.R., Calbiochem Protease Inhibitor as the mobile phase. Emodin is detected by UV absorbance at Cocktails I, II and III(R), and phosphatase (e.g. Some or all of 220 nm. Samples are spiked with a known amount of emodin Calbiochem Phosphatase Inhibitor Cocktails I, II and III(R) prior to HPLC as an internal standard. The amount of emodin inhibitors. Homogenates are centrifuged to remove debris in the sample is determined by comparison to the known and protein concentrations are determined (e.g. Biorad Brad amount of internal standard, or with samples obtained from ford assay, Biorad Rc-Dc assay, or Molecular Probes untreated mice spiked with known amount of emodin. The NanoCrange protocols as per manufacturer's instructions). In concentration of emodin is thus determined on a per mL basis Some cases, further centrifugation through a Sucrose gradient US 2010/0247552 A1 Sep. 30, 2010

and treatment with detergents is conducted to fractionate the performed on FMRKO mice following PAK inhibitor admin extracts, thereby allowing enrichment of membrane frac istration using the Golgi-Cox technique. Dendritic spine den tions, synaptosome fractions, and post-synaptic density frac sity in areas of the cortex. Such as pyramidal neurons of layer tions. These fractions are subjected to Western blot analysis II/III of temporal cortex, is measured on each primary or using antibodies (e.g. obtainable from Cell Signaling) that secondary apical or basal dendritic branch in 10 um long recognize phospho-specific forms of PAK and downstream dendritic segments. Spine number per 10 um segment and signaling proteins, including p-PAK1 Thr 423 antibody (rec mean spine density averaged over segments are quantified ognizes phosphorylated forms of PAK1, PAK2, and PAK3), and compared (as in FIGS. 11 and 12). FMRKO mice show dually phosphorylated p-ERK1/2, p-S6 (S235/S236), increase spine density with respect to their wild-type litter p-eIF4E (S209), p-4E-BP1 (S65), and p-Akt (S473). Alter mates. This phenotype is rescued or partially rescued by PAK natively or additionally, these fractions are subjected to West inhibition in the FMR KO mice. ern blot analysis using a p-vimentin antibody (S.56; Medical 0477 The Golgi preparation is used to quantify and com & Biological Laboratories). Blots are stripped and reprobed pare dendritic spine length. Increased spine length, and per with anti-sera against total PAK1 (Santa Cruz), PAK3 (Up haps concurrent decreased synapse size, of Golgi stained state), ERK1/2 (Cell Signaling), S6 (Cell Signaling), eIF4E pyramidal neurons is observed in FMRKO mice and may be (Cell Signaling), 4E-BP1 (Cell Signaling), and Akt (Cell rescued by inhibition of PAK activity (as in FIG. 13). Spine Signaling). Results are quantified with software (e.g. NIH length is measured as the radial distance from the tip of the Image J or Li-Cor Odyssey). spine head to the dendritic shaft (see, e.g. Hayashi et al., 2007, 0470 Alternatively or additionally, the extent to which Proc. Natl. Acad. Sci., USA, 104:11489; and Ramon-Moliner, small molecule inhibitors like Emodin and OSU-03012 1970, Contemporary Research Methods in Neuroanatomy, inhibit endogenous PAK catalytic activity in the mouse brain Springer, Berlin; both of which are incorporated herein by is determined using an in vitro kinase assay as described in reference). Hayashi et al., 2004, Neuron, 42:773; and Zenke et al., 1999, 0478 Dendritic spine abnormalities are also observed in J. Biol. Chem., 274:32565; both of which are incorporated green fluorescent protein (GFP) labeled neurons in the cortex, herein by reference). Briefly, catalytic activity of PAK can be such as those in layerV neurons of the barrel cortex or cortical stimulated by GTP-YS-Rac in total forebrain homogenates neurons in culture. Neurons are labeled with GFP through the and measured by the amount of phosphorylated myelin basic use of either a viral vector injected into the cortex or a trans protein (MBP). genic mouse that expresses this fluorescent protein in some 0471. For Western blots and in vitro PAK kinase assays, cortical neurons. Fluorescent neurons are imaged in fixed level of PAK activity (phospho-protein levels for all men brain sections or primary cultures using a two-photon laser tioned proteins above relative to no-drug condition or in vitro scanning microscope. Spine length and density are quantified PAK catalytic activity level relative to no-drug condition) is as described above. FMRKO neurons display increased spine monitored as a function of dose and dosage length. length and increased spine density, as phenotype which may 0472. Measurements of drug toxicity are conducted by be rescued by inhibition of PAK kinase activity (see, e.g., monitoring weight loss, death, motor activity (e.g. open-field Nimchinsky et al., 2001, J. Neurosci., 21:5139; and Livet et test), and/or coordination (e.g. rotorod) for all mentioned al., 2007, Nature, 450:56; both of which are incorporated drug doses and throughout the dosage schedules. herein by reference). 0473. In some embodiments, the optimum concentration of PAK to be used for behavior and electrophysiology experi Post-Treatment Analysis of LTP and LTD ments is approximately the Smaller of the lowest amount of inhibitor required to achieve maximum inhibition of PAK 0479. In some embodiments, hippocampal synaptic plas activity (e.g. from Western Blot and/or kinase assay studies) ticity is assessed using a standard protocol for mGluR-LTD or the lowest amount inhibitor that causes adverse toxic effect (see, e.g. Example 5 for a more detailed protocol). Hippoc (e.g. weight loss, death, motor activity difference, coordina ampal slices are prepared from FMR KO mice and control tion problems, etc.). littermates per standard procedures. All experiments are per formed blind to genotype. Schaffer collaterals are stimulated 0474 Typically, one group of mice is sacrificed and uti and extracellular field potentials measured in Str. radiatum of lized in biochemical experiments (e.g. to determine the level CA1. mGluR-dependent LTD is elicited both electrophysi of PAK kinase activity inhibition in the brain for various ologically (by pairs of stimuli delivered at 1 Hz for 15 minutes dosing schedules, times after final administration, etc.) A to 20 minutes; “PP-LFS) and pharmacologically (by bath separate group of mice are used for behavior analyses. A application of 50 uM to 100 uM3,4-dihydroxyphenylglycine mouse can be used for a single behavioral test or a battery of (DHPG) for 5 minutes). The initial slope of the field potential tests. If a single mouse is used for multiple behavioral tests, is recorded as an indicator of synaptic strength. Hippocampal the tests are typically performed in the following order: open slices from FMR1 KO mice show enhanced mGluR-LTD field, social-interaction, trace fear conditioning, and audio relative to control mice (see, e.g., Huber et al., 2002, Proc. genic seizure analysis. Natl. Acad. Sci., USA, 99.7746; and Nosyreva and Huber, Post-Treatment Morphological Analyses 2006, J. Neurophysiol., 95:3291; both of which are incorpo rated herein by reference). 0475. After treatment, mice are subjected to a number of 0480 Cortical long-term potentiation (LTP) is reduced in histological and/or morphological analyses in order to evalu slices from FMR1 KO mice (see FIG. 14). Coronol brain ate the efficacy of small molecule treatment. A few exemplary slices containing temporal cortex are prepared from two- to histological and/or morphological analyses are described three-month-old male littermates, and left to recover for at below and are explained in further detail in Example 1. least 1 hour before recording in oxygenated (95% O2 and 5% 0476. Since dendritic spine abnormalities are a hallmark CO) warm (30°C.) artificial cerebrospinal fluid containing of loss of FMRP in both humans and mice, Golgi analysis is 124 mM NaC1, 5 mM KC1, 1.25 mM NaH2PO 1 mM US 2010/0247552 A1 Sep. 30, 2010 52

MgCl, 2 mM CaCl, 26 mM NaHCOs, 10 mM dextrose. (-0.7 mA intensity). Thus, one trial is composed of tone, 30 Field potentials (FPs) in layer II/III evoked by layer IV stimu seconds blank time (also called “trace'), and then shock. Five lation are measured as previously described and responses are to ten trials with an intertrial interval (ITI) of 210 seconds are quantified as the amplitude of FP in cortex. LTP is induced by performed. To examine whether mice remember this associa TBS, which consisted of eight briefbursts (each with 4 pulses tion, on day 2 (“tone test'), mice are placed into a new at 100 Hz) of stimuli delivered every 200 m.sec. Genetic chamber with a different shape and smell from the first cham inhibition of PAK rescues the reduced cortical LTP in the ber. After ~60 seconds, a ~15-second tone is repeated for five FMR1 KO mice, and the present invention encompasses the to ten times with an ITI of 210 seconds. Video images are recognition that pharmacological inhibition may have the digitized and the percentage of freezing time during each ITI same effect (Hayashi et al., 2007, Proc. Natl. Acad. Sci., USA, is analyzed (e.g. by Image FZ program). Freezing is defined 104:11489; incorporated herein by reference). as the absence of all but respiratory movement for a 1-second period. See, e.g. Zhao et al. (2005, J. Neurosci., 25:7385; Post-Treatment Behavioral Analyses incorporated herein by reference). 0481 Fragile X mice and littermates treated with PAK 0490 Social Interaction Tests inhibitors or placebo are subjected to various behavioral tasks 0491 Home cage behavior and social interaction are to determine whether pharmacological inhibition of PAK can assessed with a variety of tests (Kwon et al., 2006, Neuron, ameliorate symptoms of Fragile X Syndrome in the mouse 50:377: Spencer et al., 2005, Genes Brain Behav., 4:420; and model. The experimenter is blind to genotype and prior drug Lijam et al., 1997, Cell, 90:895; all of which are incorporated treatment. herein by reference). For example, mice can be observed in 0482. Open Field Test their home cage by Videorecording and scored for various 0483. As described in the specification, the open field test nonsocial behaviors and/or social behaviors (e.g. grooming, records parameters such as horizontal and vertical activity, mounting, tail pulling, and Sniffing). time spent in the center, and stereotopy. Mice are allowed to 0492 Direct social interaction is assessed by exposing run freely in an open arena (e.g. VersaMax activity monitor mice to a novel conspecific mouse and observing approaching chamber from Accuscan Instruments). Mice are allowed to and Sniffing behaviors. Percent of time spent interacting is run about 5 minutes, about 10 minutes, about 15 minutes, recorded. This is repeated about 3 days later with the same about 20 minutes, about 30 minutes, or about 1 hour. After mice. Control mice exhibit a decrease in Social interaction the mice are allowed to run, behaviors are analyzed, including (1) second time, indicating recognition of the familiar mouse hyperactivity, determined by measuring the distance and/or and/or normal social learning. FMRKO mice do not exhibit length of time traveled by the subject; (2) stereotypy, deter a decrease in Social interaction the second time, indicating mined by measuring the number of repetitive behaviors impaired social learning, memory, and/or behavior. This test exhibited by the subject; (3) hypo-anxiety, determined by can be conducted in a novel or familiar environment, as sig measuring the amount of time the Subject remains in the nificant differences between FMR KO and wild-type mice centerfield relative to the time spent in the corners of the field; have been observed in Social interaction assays depending on and (4) combinations of these. the degree of familiarity with the environment. Additional 0484. In certain experiments, open field activity can be Social behaviors can be monitored in this assay including detected by photobeam breaks and analyzed by VersaMax active behaviors (e.g. aggressive attacks, lateral threats, and/ software. Activities measured are the amount of time the or chasing) and passive behaviors (e.g. receiving Sniffing mouse spends in the center of the field, the number of times from other mouse and/or not showing signs of Submissive the mouse exhibits repetitive behaviors (“stereotypy), and and/or defensive behavior). the total distance traveled by the mouse. 0493 Prior to an indirect social interaction test mice are 0485 Audiogenic Seizure (AGS) Assay housed individually for 4 days. Indirect social interaction 0486 Fragile X humans and mice are susceptible to sei tasks typically take place in a cage divided in half by a clear Zures at early ages. Fragile X mice show a robust phenotype perforated partition. The task is run in two different modes: in an audiogenic seizure (AGS) assay. While no 19-21 day old one involves a familiar environment, by pre-exposure to the (p19-21) wildtype mice typically have seizures in this task, testing chamber, while the other involves a novel environ the majority of fragile X mice do have seizures. ment. Test mice are exposed to novel or familiar mice. Typi 0487. AGS is performed essentially as described (Yan et cally, FMRKO mice behave similarly to wild-type mice in a al., 2005, Neuropharmacol., 49:1053; incorporated herein by novel environment, but behave significantly differently in a reference). Briefly, mice are habituated to a behavioral cham familiar cage. Time spent at the partition is recorded in 2 to 5 ber and then exposed to a high intensity siren of frequency minute intervals for a 20 minute test. FMR KO mice spend peak 1800 HZ-6300 Hz at an average sound pressure level significantly less time interacting (i.e. at the partition) in the above 120 dB at approximately 10 cm for 5 minutes. Behav first few minutes and take longer to first approach the partition iors of the mice are monitored after administration of Sound. than wild-type mice. In contrast, FMRKO mice spend more Fragile X mice typically (1) run wildly, (2) have seizures, time at the partition during the last time intervals than con and/or (3) die. Wildtype mice typically do not exhibit these trols. responses. An AGS phenotype is scored based on the animal's 0494. In some experiments, the indirect social interaction endpoint. test is conducted in a chamber divided into three rooms. The 0488 Trace Fear Conditioning central room which is connected to two rooms independent 0489 Mice are subjected to the trace fear conditioning from each other, one on the left and one on the right—is task according to standard procedures. On day 1 ("condition empty. The left room contains an empty cage (i.e. a novel ing'), mice are placed into a training chamber for ~60 sec object and/or inanimate target), while the right room contains onds before the onset of a ~15-second white noise tone. a similar cage enclosing a novel mouse (i.e. a social target). Another ~30 seconds later, mice received a ~1-second shock This task involves a choice between spending time with a US 2010/0247552 A1 Sep. 30, 2010

Social target or an inanimate target, and therefore is called a phase begins in which the previously unbaited arms now have social preference test. Percent of total time interacting with the 4 food pellets and the previously baited arms contain no each object is recorded. FMRKO mice may spend a signifi food reward. Mice with deficits in memory flexibility are cantly different amount of time (e.g. more or less) interacting unable to learn the new task and continue to visit the arms with the Social target than control mice. baited in the training phase. Errors are recorded and com 0495 Social dominance tube test is conducted in a tube pared among FMRKO mice that receive PAK inhibitor, pla approximately 30 cm long and 3 cm-4 cm in diameter. Mice cebo, or no treatment. are placed at opposite ends of the tube and released simulta 0500 Morris Water Maze Test neously. A mouse is pronounced the “winner when his oppo 0501 Hippocampus-dependent spatial learning is nent backs out completely. FMR1 KO mice win significantly assessed using the classical Morris Water Maze test. FMR1 fewer matches against unfamiliar wild-type mice than KO mice, just like the wild-type controls, learn to find the expected by chance. When a FMR1 KO mouse competes visible or hidden platform with decreasing latency scores against a wild-type non-cagemate, the wild-type mouse is the over the course of the standard training protocol. However, winner in approximately 73% of matches (Kwon et al., 2006, Some groups observe an abnormal phenotype in a reversal Neuron, 50:377; Spencer et al., 2005, Genes Brain Behav, trial, a test in which the platform is transferred to the quadrant 4:420; and Lijam et al., 1997, Cell, 90:895; all of which are opposite the initial training quadrant. In particular, FMR1 KO incorporated herein by reference) Inhibition of PAK via mice display increased escape latency and path length, Sug administration of a small molecule inhibitor may ameliorate gesting that they have low response flexibility or high this phenotype. memory interference (see, e.g. D'Hooge et al., 1997, Neuro 0496. Since FMR1 KO mice have been shown to display science, 76:367; incorporated herein by reference). Some abnormal Social behaviors, home cage Social behavior, including nest building and sleeping behavior, may be altered Equivalents and Scope in FMR1 KO mice. Nesting patterns are evaluated by placing 0502. The foregoing has been a description of certain non a cotton nestle into a cage of approximately two, three, or four limiting preferred embodiments of the invention. Those mice of the same genotype (e.g. wild-type, FMR1 KO, etc.) skilled in the art will recognize, or be able to ascertain using that receive identical drug treatments. After about 30 minutes no more than routine experimentation, many equivalents to to 1 hour, the nest can be removed and the height measured. the specific embodiments of the invention described herein. Wild-type mice build nests with depths that average 20 mm to Those of ordinary skill in the art will appreciate that various 50 mm. Mice which display abnormal social behavior, such changes and modifications to this description may be made as FMR1 KO mice, frequently build shallower nests (e.g. <20 without departing from the spirit or scope of the present mm) invention, as defined in the following claims. 0497. In another assay, sleeping positions of mice in their 0503. In the claims articles such as “a”, “an and “the home cages can be recorded two to four times a day over five may mean one or more than one unless indicated to the consecutive days. Wild-type mice sleep huddled in the well contrary or otherwise evident from the context. Claims or formed, fluffy nests they build. Observations will determine descriptions that include “or between one or more members whether FMR1 KO mice sleep in scattered, random patterns, of a group are considered satisfied if one, more than one, or all do not build full nests, or sleep on top of intact nestle material. of the group members are present in, employed in, or other If FMR1 KO mice display abnormal phenotypes, ameliora wise relevant to a given product or process unless indicated to tion of those phenotypes may occur following PAK inhibition the contrary or otherwise evident from the context. The inven using small molecule inhibitors such as Emodin and/or OSU tion includes embodiments in which exactly one member of O3O12. the group is present in, employed in, or otherwise relevant to 0498 Eight-Arm Maze Test a given product or process. The invention also includes 0499 FXS patients have cognitive deficits (e.g. short-term embodiments in which more than one, or all of the group memory impairments) and/or perseverative language and members are presentin, employed in, or otherwise relevant to movements. FXS patients often become anxious with any a given product or process. Furthermore, it is to be understood breach of their normal daily routine. The 8-arm radial maze is that the invention encompasses all variations, combinations, a task used to see if a similar phenotype is also present in the and permutations in which one or more limitations, elements, mouse model of the disease. The maze consists of a central clauses, descriptive terms, etc., from one or more of the arena Surrounded by eight doors which guard the entrances to claims or from relevant portions of the description is intro eight maZe arms. Prominent distal cues Surround the maze. A duced into another claim. For example, any claim that is food pellet is hidden at the distal end of the arms and serves as dependent on another claim can be modified to include one or a reward. In a version of the task specifically designed to test more limitations found in any other claim that is dependent on working memory, all 8 arms are baited with a food pellet. In the same base claim. Furthermore, where the claims recite a each trial, the mouse must visit each of the 8 arms only once composition, it is to be understood that methods of using the to consume the food reward. A second visit to an arm is composition for any of the purposes disclosed herein are considered a working memory error. Mice with poor short included, and methods of making the composition according term memory accrue more errors than wild-type mice. In the to any of the methods of making disclosed herein or other reference memory version of the task, only 4 arms are baited. methods known in the art are included, unless otherwise In this case, the same 4 arms are baited each day during the indicated or unless it would be evident to one of ordinary skill training period (which typically lasts 10-16 days with 1-3 in the art that a contradiction or inconsistency would arise. trials per day). An entry into an unbaited arm is considered a For example, it is to be understood that any of the composi reference memory error. Just as in the previous version of the tions of the invention can be used for inhibiting the formation, task, a second entry into any arm is considered a working progression, and/or recurrence of adhesions at any of the memory error. Once the mice have learned the task, a reversal locations, and/or due to any of the causes discussed herein or US 2010/0247552 A1 Sep. 30, 2010 54 known in the art. It is also to be understood that any of the 0505. Where ranges are given, endpoints are included. compositions made according to the methods for preparing Furthermore, it is to be understood that unless otherwise compositions disclosed herein can be used for inhibiting the indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that formation, progression, and/or recurrence of adhesions at any are expressed as ranges can assume any specific value within of the locations, and/or due to any of the causes discussed the stated ranges in different embodiments of the invention, to herein or known in the art. In addition, the invention encom the tenth of the unit of the lower limit of the range, unless the passes compositions made according to any of the methods context clearly dictates otherwise. It is also to be understood for preparing compositions disclosed herein. that unless otherwise indicated or otherwise evident from the 0504. Where elements are presented as lists, e.g., in context and/or the understanding of one of ordinary skill in Markush group format, it is to be understood that each sub the art, values expressed as ranges can assume any Subrange group of the elements is also disclosed, and any element(s) within the given range, wherein the endpoints of the Subrange are expressed to the same degree of accuracy as the tenth of can be removed from the group. It is also noted that the term the unit of the lower limit of the range. “comprising is intended to be open and permits the inclusion 0506. In addition, it is to be understood that any particular of additional elements or steps. It should be understood that, embodiment of the present invention may be explicitly in general, where the invention, or aspects of the invention, excluded from any one or more of the claims. Any embodi is/are referred to as comprising particular elements, features, ment, element, feature, application, or aspect of the compo steps, etc., certain embodiments of the invention or aspects of sitions and/or methods of the invention (e.g., any PAK modu the invention consist, or consistessentially of Such elements, lator, any biological activity of PAK modulators, any method features, steps, etc. For purposes of simplicity those embodi of identifying PAK modulators, any method of treatment of ments have not been specifically set forth in haec verba FXS and/or other neurodevelopmental disorder, any neurode herein. Thus for each embodiment of the invention that com velopmental disorder, etc.), can be excluded from any one or prises one or more elements, features, steps, etc., the inven more claims. For purposes of brevity, all of the embodiments tion also provides embodiments that consist or consist essen in which one or more elements, features, purposes, or aspects tially of those elements, features, steps, etc. is excluded are not set forth explicitly herein.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 18

SEO ID NO 1 LENGTH: 681 TYPE PRT ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: FMR and FXR amino acid sequences

< 4 OOs SEQUENCE: 1.

Met Glu Asp Lieu. Luell Wall Glu Wall Arg Lell Asp Asn Gly Ala Tyr 1. 5 15

Lys Gly Glin Wall Thir Ala Wall Ala Asp Asp Gly Ile Phe Wall Asp Wall 25 3 O

Asp Gly Wall Pro Glu Ser Met Lys Tyr Pro Phe Wall Asn Wall Arg Lell 35 4 O 45

Pro Pro Glu Glu Thir Wall Glu Wall Ala Ala Pro Ile Phe Glu Glu Gly SO 55 60

Met Glu Wall Glu Wall Phe Thir Arg Thir Asn Asp Arg Glu Thir Gly 65 70

Trp Trp Wall Gly Ile Ile Met Arg Lys Ala Glu Ile Ala Wall 85 90 95

Ala Ile Gly Phe Glu Thir Ser Tyr Thir Glu Ile Glu Lell Gly 1OO 105 110

Arg Lell Arg Ala Asn Ser Asn Pro Pro Ile Thir Ala Thir Phe 115 12O 125

Glin Phe Thir Luell Pro Wall Pro Glu Glu Luell Arg Glu Glu Ala Glin 13 O 135 14 O

Lys Asp Gly Ile His Lys Glu Phe Glin Arg Thir Ile Asp Ala Gly Wall 145 15 O 155 16 O US 2010/0247552 A1 Sep. 30, 2010 55

- Continued

Cys Asn Tyr Ser Arg Asp Lieu. Asp Ala Lieu. Ile Val Ile Ser Llys Phe 1.65 17O 17s Glu. His Thr Glin Lys Arg Ala Ser Met Lieu Lys Asp Met His Phe Arg 18O 185 19 O Asn Lieu. Ser Glin Llys Wal Met Lieu. Lieu Lys Arg Thr Glu Glu Ala Ala 195 2OO 2O5 Arg Glin Lieu. Glu Thir Thr Llys Lieu Met Ser Arg Gly Asn Tyr Val Glu 21 O 215 22O Glu Phe Arg Val Arg Asp Asp Lieu Met Gly Lieu Ala Ile Gly Ser His 225 23 O 235 24 O Gly Ser Asn. Ile Glin Ala Ala Arg Thr Val Asp Gly Val Thr Asn. Ile 245 250 255 Glu Lieu. Glu Glu Lys Ser Cys Thr Phe Lys Ile Ser Gly Glu. Thr Glu 26 O 265 27 O Glu Ser Val Glin Arg Ala Arg Ala Met Lieu. Glu Tyr Ala Glu Glu Phe 27s 28O 285 Phe Glin Val Pro Arg Glu Lieu Val Gly Llys Val Ile Gly Lys Asn Gly 29 O 295 3 OO Arg Ile Ile Glin Glu Ile Val Asp Llys Ser Gly Val Phe Arg Ile Llys 3. OS 310 315 32O Ile Ala Gly Asp Asp Glu Glin Asp Glin Asn. Ile Pro Arg Glu Lieu Ala 3.25 330 335 His Val Pro Leu Val Phe Ile Gly Thr Val Glu Ser Ile Ala Asn Ala 34 O 345 35. O Llys Val Lieu. Lieu. Lieu. Tyr His Lieu. Ser His Lieu Lys Glu Val Glu Glin 355 360 365 Lieu. Arg Glin Glu Lys Met Glu Ile Asp Glin Gln Lieu. Arg Ala Ile Glin 37 O 375 38O Glu Ser Ser Met Gly Ser Thr Glin Ser Phe Pro Val Thr Arg Arg Ser 385 390 395 4 OO Glu Arg Gly Tyr Ser Ser Asp Ile Glu Ser Val Arg Ser Met Arg Gly 4 OS 41O 415 Gly Gly Gly Gly Glin Arg Gly Arg Val Arg Gly Arg Gly Gly Gly Gly 42O 425 43 O Pro Gly Gly Gly Asn Gly Lieu. Asn Glin Arg Tyr His Asn. Asn Arg Arg 435 44 O 445 Asp Glu Asp Asp Tyr Asn. Ser Arg Gly Asp His Glin Arg Asp Glin Glin 450 45.5 460 Arg Gly Tyr Asn Asp Arg Gly Gly Gly Asp Asn Thr Gly Ser Tyr Arg 465 470 47s 48O Gly Gly Gly Gly Gly Ala Gly Gly Pro Gly Asn. Asn Arg Arg Gly Gly 485 490 495 Ile Asin Arg Arg Pro Pro Arg Asn Asp Glin Glin Asn Gly Arg Asp Tyr SOO 505 51O Gln His His Asn His Thr Thr Glu Glu Val Arg Glu Thr Arg Glu Met 515 52O 525 Ser Ser Val Glu Arg Ala Asp Ser Asn. Ser Ser Tyr Glu Gly Ser Ser 53 O 535 54 O Arg Arg Arg Arg Arg Gln Lys Asn. Asn. Asn Gly Pro Ser Asn. Thir Asn 5.45 550 555 560 US 2010/0247552 A1 Sep. 30, 2010 56

- Continued Gly Ala Val Ala Asn. Asn. Asn. Asn Llys Pro Glin Ser Ala Glin Glin Pro 565 st O sts Glin Glin Glin Glin Pro Pro Ala Pro Gly Asn Lys Ala Ala Lieu. Asn Ala 58O 585 59 O Ser Asp Ala Ser Lys Glin Asn. Ser Gly Asn Ala Asn Ala Ala Gly Gly 595 6OO 605 Ala Ser Llys Pro Lys Asp Ala Ser Arg Asin Gly Asp Llys Glin Glin Ala 610 615 62O Gly Thr Glin Glin Glin Glin Pro Ser Glin Val Glin Glin Glin Glin Ala Ala 625 630 635 64 O Glin Glin Glin Glin Pro Llys Pro Arg Arg Asn Lys Asn Arg Ser Asn. Asn 645 650 655 His Thr Asp Glin Pro Ser Glin Glin Glin Glin Lieu Ala Glu Asn. Wall Lys 660 665 67 O Lys Glu Gly Lieu Val Asn Gly. Thir Ser 675 68O

<210s, SEQ ID NO 2 &211s LENGTH: 632 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: FMR and FXR amino acid sequences < 4 OO > SEQUENCE: 2 Met Glu Glu Lieu Val Val Glu Val Arg Gly Ser Asn Phe Ala Phe Tyr 1. 5 1O 15 Lys Ala Phe Wall Lys Asp Wal His Glu Asp Ser Ile Thr Val Ala Phe 2O 25 3O Glu Asn. Asn Trp Glin Pro Asp Arg Glin Ile Pro Phe His Asp Val Arg 35 4 O 45 Phe Pro Pro Pro Val Gly Tyr Asn Lys Gly Ile Asn Glu Ser Asp Glu SO 55 6 O Val Glu Val Tyr Ser Arg Ala Asn. Glu Lys Glu Pro Cys Cys Trp Trip 65 70 7s 8O Lieu Ala Lys Val Arg Met Ile Lys Gly Glu Phe Tyr Val Ile Glu Tyr 85 90 95 Ala Ala Cys Asp Ala Thr Tyr Asn. Glu Ile Val Thir Ile Glu Arg Lieu 1OO 105 11 O Arg Ser Val Asn Pro Asn Llys Pro Ala Thir Lys Asp Thr Glu Phe His 115 12 O 125 Lys Ile Llys Lieu. Asp Val Pro Glu Asp Lieu. Arg Glin Met Cys Ala Lys 13 O 135 14 O Glu Ala Ala His Lys Asp Phe Llys Lys Ala Val Gly Ala Phe Ser Val 145 150 155 160 Thr Tyr Asp Pro Glu Asn Tyr Gln Leu Val Ile Leu Ser Ile Asin Glu 1.65 17O 17s Val Thr Ser Lys Arg Ala His Met Lieu. Ile Asp Met His Phe Arg Ser 18O 185 19 O Lieu. Arg Thr Lys Lieu. Ser Lieu. Ile Met Arg Asn. Glu Glu Ala Ser Lys 195 2OO 2O5 Glin Lieu. Glu Ser Ser Arg Glin Lieu Ala Ser Arg Phe His Glu Glin Phe 21 O 215 22O US 2010/0247552 A1 Sep. 30, 2010 57

- Continued Ile Val Arg Glu Asp Lieu Met Gly Lieu Ala Ile Gly Thr His Gly Ala 225 23 O 235 24 O Asn. Ile Glin Glin Ala Arg Llys Val Pro Gly Val Thr Ala Ile Lieu. Asp 245 250 255 Glu Asp Thr Cys Thr Phe His Ile Tyr Gly Glu Asp Glin Asp Ala Val 26 O 265 27 O Llys Lys Ala Arg Ser Phe Lieu. Glu Phe Ala Glu Asp Tyr Ile Glin Val 27s 28O 285 Pro Arg Asn Lieu Val Gly Llys Val Ile Gly Lys Asn Gly Lieu Lys Ile 29 O 295 3 OO Glin Glu Ile Val Asp Llys Ser Gly Val Val Arg Val Arg Ile Glu Ala 3. OS 310 315 32O Glu Asin Glu Lys Asn Val Pro Glin Glu Glu Glu Ile Met Pro Pro Asn 3.25 330 335 Ser Lieu Pro Ser Asn. Asn. Ser Arg Val Gly Pro Asn Ala Pro Glu Glu 34 O 345 35. O Llys Llys His Lieu. Asp Ile Lys Glu Asn. Ser Thr His Phe Ser Glin Pro 355 360 365 Asn Ser Thir Lys Val Glin Arg Val Lieu Val Ala Ser Ser Val Val Ala 37 O 375 38O Gly Glu Ser Glin Llys Pro Glu Lieu Lys Ala Trp Gln Gly Met Val Pro 385 390 395 4 OO Phe Val Phe Val Gly Thr Lys Asp Ser Ile Ala Asn Ala Thr Val Lieu. 4 OS 41O 415 Lieu. Asp Tyr His Lieu. Asn Tyr Lieu Lys Glu Val Asp Gln Lieu. Arg Lieu. 42O 425 43 O Glu Arg Lieu. Glin Ile Asp Glu Glin Lieu. Arg Glin Ile Gly Ala Ser Ser 435 44 O 445 Arg Pro Pro Pro Asn Arg Thr Asp Llys Glu Lys Ser Tyr Val Thir Asp 450 45.5 460 Asp Gly Glin Gly Met Gly Arg Gly Ser Arg Pro Tyr Arg Asn Arg Gly 465 470 47s 48O His Gly Arg Arg Gly Pro Gly Tyr Thr Ser Gly Thr Asn Ser Glu Ala 485 490 495 Ser Asn Ala Ser Glu Thr Glu Ser Asp His Arg Asp Glu Lieu. Ser Asp SOO 505 51O Trp Ser Lieu Ala Pro Thr Glu Glu Glu Arg Glu Ser Phe Lieu. Arg Arg 515 52O 525 Gly Asp Gly Arg Arg Arg Gly Gly Gly Gly Arg Gly Glin Gly Gly Arg 53 O 535 54 O Gly Arg Gly Gly Gly Phe Lys Gly Asn Asp Asp His Ser Arg Thr Asp 5.45 550 555 560 Asn Arg Pro Arg ASn Pro Arg Glu Ala Lys Gly Arg Thir Thr Asp Gly 565 st O sts Ser Lieu. Glin Ile Arg Val Asp Cys Asn. Asn. Glu Arg Ser Val His Thr 58O 585 59 O Llys Thr Lieu. Glin Asn. Thir Ser Ser Glu Gly Ser Arg Lieu. Arg Thr Gly 595 6OO 605 Lys Asp Arg Asn. Glin Llys Lys Glu Lys Pro Asp Ser Val Asp Gly Glin 610 615 62O Gln Pro Leu Val Asn Gly Val Pro US 2010/0247552 A1 Sep. 30, 2010 58

- Continued

625 630

SEQ ID NO 3 LENGTH: 61.2 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: FMR and FXR amino acid sequences

<4 OOs, SEQUENCE: 3

Met Ala Asp Wall Thir Wall Glu Wall Arg Gly Ser Asn Gly Ala Phe Tyr 1. 5 15

Lys Gly Phe Ile Lys Asp Wall His Glu Asp Ser Lell Thir Wall Wall Phe 2O 25

Glu Asn Asn Trp Glin Pro Glu Arg Glin Wall Pro Phe Asn Glu Wall Arg 35 4 O 45

Lell Pro Pro Pro Pro Asp Ile Glu Ile Ser Glu Gly Asp Glu SO 55 6 O

Wall Glu Wall Ser Arg Ala Asn Asp Glin Glu Pro Gly Trp Trp 65 70

Lell Ala Wall Arg Met Met Gly Glu Phe Wall Ile Glu Tyr 85 90 95

Ala Ala Asp Ala Thir Asn Glu Ile Wall Thir Phe Glu Arg Luell 1OO 105 11 O

Arg Pro Wall Asn Glin Asn Thir Wall Lys Asn Thir Phe Phe 115 12 O 125

Thir Wall Asp Wall Pro Glu Asp Luell Arg Glu Ala Ala Asn Glu 13 O 135 14 O

Asn Ala His Asp Phe Ala Wall Gly Ala Arg Ile Phe 145 150 155 160

His Pro Glu Thir Thir Glin Luell Met Ile Luell Ser Ala Ser Glu Ala 1.65 17O 17s

Thir Wall Arg Wall Asn Ile Luell Ser Asp Met His Lell Arg Ser Ile 18O 185 19 O

Arg Thir Lys Luell Met Lell Met Ser Arg Asn Glu Glu Ala Thir His 195

Lell Glu Thir Glin Lell Ala Ala Ala Phe His Glu Glu Phe Wall 21 O 215 22O

Wall Arg Glu Asp Lell Met Gly Luell Ala Ile Gly Thir His Gly Ser Asn 225 23 O 235 24 O

Ile Glin Glin Ala Arg Wall Pro Gly Wall Thir Ala Ile Glu Luell Asp 245 250 255

Glu Asp Thir Gly Thir Phe Arg Ile Tyr Gly Glu Ser Ala Asp Ala Wall 26 O 265 27 O

Ala Arg Gly Phe Lell Glu Phe Wall Glu Asp Phe Ile Glin Wall 28O 285

Pro Arg Glu Luell Wall Gly Lys Wall Ile Gly Lys Asn Gly Arg Wall Ile 29 O 295 3 OO

Glin Glu Ile Wall Asp Lys Ser Gly Wall Wall Arg Ile Arg Ile Glu Gly 3. OS 310 315

Asp Asn Glu Asn Lys Lell Pro Arg Glu Asp Gly Met Wall Pro Phe Wall 3.25 330 335

Phe Wall Gly Thir Glu Ser Ile Gly Asn Wall Glin Wall Luell Luell Glu US 2010/0247552 A1 Sep. 30, 2010 59

- Continued

34 O 345 35. O Tyr His Ile Ala Tyr Lieu Lys Glu Val Glu Gln Lieu. Arg Met Glu Arg 355 360 365 Lieu. Glin Ile Asp Glu Glin Lieu. Arg Glin Ile Gly Ser Arg Ser Tyr Ser 37 O 375 38O Gly Arg Gly Arg Gly Arg Arg Gly Pro Asn Tyr Thr Ser Gly Tyr Gly 385 390 395 4 OO Thr Asn Ser Glu Lieu. Ser Asn Pro Ser Glu Thr Glu Ser Glu Arg Lys 4 OS 41O 415 Asp Glu Lieu. Ser Asp Trp Ser Lieu Ala Gly Glu Asp Asn Arg Asp Ser 42O 425 43 O Arg His Glin Arg Asp Ser Arg Arg Arg Pro Gly Gly Arg Gly Arg Ser 435 44 O 445 Val Ser Gly Gly Arg Gly Arg Gly Gly Pro Arg Gly Arg Llys Ser Ser 450 45.5 460 Ile Ser Ser Val Lieu Lys Asp Pro Asp Ser Asn Pro Tyr Ser Lieu. Lieu. 465 470 47s 48O Asp Asn Thr Glu Ser Asp Glin Thr Ala Asp Thr Asp Ala Ser Glu Ser 485 490 495 His His Ser Thr Asn Arg Arg Arg Arg Ser Arg Arg Arg Arg Thr Asp SOO 505 51O Glu Asp Ala Val Lieu Met Asp Gly Met Thr Glu Ser Asp Thr Ala Ser 515 52O 525 Val Asn. Glu Asn Gly Lieu Val Thr Val Ala Asp Tyr Ile Ser Arg Ala 53 O 535 54 O Glu Ser Glin Ser Arg Glin Arg Asn Lieu Pro Arg Glu Thir Lieu Ala Lys 5.45 550 555 560 Asn Llys Lys Glu Met Ala Lys Asp Val Ile Glu Lieu. His Gly Pro Ser 565 st O sts Glu Lys Ala Ile Asn Gly Pro Thir Ser Ala Ser Gly Asp Asp Ile Ser 58O 585 59 O Llys Lieu. Glin Arg Thr Pro Gly Glu Glu Lys Ile Asn. Thir Lieu Lys Glu 595 6OO 605

Glu Asn. Thir Glin 610

<210s, SEQ ID NO 4 &211s LENGTH: 664 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: FMR and FXR amino acid sequence <4 OOs, SEQUENCE: 4 Met Gly Gly Lieu Ala Ser Gly Gly Asp Val Glu Pro Gly Lieu Pro Val 1. 5 1O 15 Glu Val Arg Gly Ser Asn Gly Ala Phe Tyr Lys Gly Phe Wall Lys Asp 2O 25 3O Val His Glu Asp Ser Val Thir Ile Phe Phe Glu Asn Asn Trp Glin Ser 35 4 O 45 Glu Arg Glin Ile Pro Phe Gly Asp Val Arg Lieu Pro Pro Pro Ala Asp SO 55 6 O Tyr Asn Lys Glu Ile Thr Glu Gly Asp Glu Val Glu Val Tyr Ser Arg US 2010/0247552 A1 Sep. 30, 2010 60

- Continued

Ala Asn. Glu Glin Glu Pro Cys Gly Trp Trp Lieu Ala Arg Val Arg Met 85 90 95 Met Lys Gly Asp Phe Tyr Val Ile Glu Tyr Ala Ala Cys Asp Ala Thr 1OO 105 11 O Tyr Asn. Glu Ile Val Thir Lieu. Glu Arg Lieu. Arg Pro Val Asn Pro Asn 115 12 O 125 Pro Leu Ala Thr Lys Gly Ser Phe Phe Llys Val Thr Met Ala Val Pro 13 O 135 14 O Glu Asp Lieu. Arg Glu Ala Cys Ser Asn. Glu Asn Val His Lys Glu Phe 145 150 155 160 Llys Lys Ala Lieu. Gly Ala Asn. Cys Ile Phe Lieu. Asn. Ile Thir Asn. Ser 1.65 17O 17s Glu Lieu. Phe Ile Lieu. Ser Thir Thr Glu Ala Pro Val Lys Arg Ala Ser 18O 185 19 O Lieu. Lieu. Gly Asp Met His Phe Arg Ser Lieu. Arg Thr Llys Lieu. Lieu. Lieu. 195 2OO 2O5 Met Ser Arg Asn. Glu Glu Ala Thir Lys His Lieu. Glu Thir Ser Lys Glin 21 O 215 22O Lieu Ala Ala Ala Phe Glin Glu Glu Phe Thr Val Arg Glu Asp Lieu Met 225 23 O 235 24 O Gly Lieu. Ala Ile Gly Thr His Gly Ala ASn Ile Glin Glin Ala Arg Lys 245 250 255 Val Pro Gly Val Thr Ala Ile Glu Lieu. Gly Glu Glu Thr Cys Thr Phe 26 O 265 27 O Arg Ile Tyr Gly Glu Thr Pro Glu Ala Cys Arg Glin Ala Arg Ser Tyr 27s 28O 285 Lieu. Glu Phe Ser Glu Asp Ser Val Glin Val Pro Arg Glu Lieu Val Gly 29 O 295 3 OO Llys Val Ile Gly Lys Asn Gly Arg Val Ile Glin Glu Ile Val Asp Llys 3. OS 310 315 32O Ser Gly Val Val Arg Val Arg Val Glu Gly Asp Asn Asp Llys Lys Asn 3.25 330 335 Pro Arg Glu Glu Gly Met Val Pro Phe Ile Phe Val Gly Thr Arg Glu 34 O 345 35. O Asn. Ile Ser Asn Ala Glin Ala Lieu. Lieu. Lieu. Tyr His Lieu. Ser Tyr Lieu 355 360 365 Glin Glu Val Glu Glin Lieu. Arg Lieu. Glu Arg Lieu. Glin Ile Asp Glu Glin 37 O 375 38O Lieu. Arg Glin Ile Glin Lieu. Gly Phe Arg Pro Pro Gly Ser Gly Arg Gly 385 390 395 4 OO Ser Gly Gly Ser Asp Lys Ala Gly Tyr Ser Thr Asp Glu Ser Ser Ser 4 OS 41O 415 Ser Ser Lieu. His Ala Thr Arg Thr Tyr Gly Gly Ser Tyr Gly Gly Arg 42O 425 43 O Gly Arg Gly Arg Arg Thr Gly Gly Pro Ala Tyr Gly Pro Ser Ser Asp 435 44 O 445 Val Ser Thir Ala Ser Glu Thr Glu Ser Glu Lys Arg Glu Lieu Pro Asn 450 45.5 460 Arg Ala Gly Pro Gly Asp Arg Asp Pro Pro Thr Arg Gly Glu Glu Ser 465 470 47s 48O US 2010/0247552 A1 Sep. 30, 2010 61

- Continued

Arg Arg Arg Pro Thr Gly Gly Arg Gly Arg Gly Pro Pro Pro Ala Pro 485 490 495 Arg Pro Thir Ser Arg Tyr Asn Ser Ser Ser Ile Ser Ser Val Lieu Lys SOO 505 51O Asp Pro Asp Ser ASn Pro Tyr Ser Lieu. Lieu. Asp Thir Ser Glu Pro Glu 515 52O 525 Pro Pro Val Asp Ser Glu Pro Gly Glu Pro Pro Pro Ala Ser Ala Arg 53 O 535 54 O Arg Arg Arg Ser Arg Arg Arg Arg Thr Asp Glu Asp Arg Thr Val Met 5.45 550 555 560 Asp Gly Gly Lieu. Glu Ser Asp Gly Pro Asn Met Thr Glu Asn Gly Lieu 565 st O sts Glu Asp Glu Ser Arg Pro Glin Arg Arg Asn Arg Ser Arg Arg Arg Arg 58O 585 59 O Asn Arg Gly Asn Arg Thr Asp Gly Ser Ile Ser Gly Asp Arg Glin Pro 595 6OO 605 Val Thr Val Ala Asp Tyr Ile Ser Arg Ala Glu Ser Glin Ser Arg Glin 610 615 62O Ser Ala Pro Lieu. Glu Arg Thr Llys Pro Ser Lieu. Asp Ser Lieu. Ser Gly 625 630 635 64 O Glin Lys Gly Asp Ser Val Ser Lys Lieu Pro Lys Gly Pro Ser Glu Asn 645 650 655 Gly Glu Lieu. Ser Ala Pro Lieu. Glu 660

<210s, SEQ ID NO 5 &211s LENGTH: 614 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence for mouse FMR1 <4 OOs, SEQUENCE: 5 Met Glu Glu Lieu Val Val Glu Val Arg Gly Ser Asn Gly Ala Phe Tyr 1. 5 1O 15 Lys Ala Phe Wall Lys Asp Wal His Glu Asp Ser Ile Thr Val Ala Phe 2O 25 3O Glu Asn. Asn Trp Glin Pro Glu Arg Glin Ile Pro Phe His Asp Val Arg 35 4 O 45 Phe Pro Pro Pro Val Gly Tyr Asn Lys Asp Ile Asn Glu Ser Asp Glu SO 55 6 O Val Glu Val Tyr Ser Arg Ala Asn. Glu Lys Glu Pro Cys Cys Trp Trip 65 70 7s 8O Lieu Ala Lys Val Arg Met Ile Lys Gly Glu Phe Tyr Val Ile Glu Tyr 85 90 95 Ala Ala Cys Asp Ala Thr Tyr Asn. Glu Ile Val Thir Ile Glu Arg Lieu 1OO 105 11 O Arg Ser Val Asn Pro Asn Llys Pro Ala Thir Lys Asp Thr Phe His Lys 115 12 O 125 Ile Llys Lieu. Glu Val Pro Glu Asp Lieu. Arg Gln Met Cys Ala Lys Glu 13 O 135 14 O Ser Ala His Lys Asp Phe Llys Lys Ala Val Gly Ala Phe Ser Val Thr 145 150 155 160 US 2010/0247552 A1 Sep. 30, 2010 62

- Continued

Tyr Asp Pro Glu Asn Tyr Glin Lieu Val Ile Lieu. Ser Ile Asin Glu Val 1.65 17O 17s Thir Ser Lys Arg Ala His Met Lieu. Ile Asp Met His Phe Arg Ser Lieu. 18O 185 19 O Arg Thr Lys Lieu. Ser Lieu. Ile Lieu. Arg Asn. Glu Glu Ala Ser Lys Glin 195 2OO 2O5 Lieu. Glu Ser Ser Arg Glin Lieu Ala Ser Arg Phe His Glu Glin Phe Ile 21 O 215 22O Val Arg Glu Asp Lieu Met Gly Lieu Ala Ile Gly Thr His Gly Ala Asn 225 23 O 235 24 O Ile Glin Glin Ala Arg Llys Val Pro Gly Val Thr Ala Ile Asp Lieu. Asp 245 250 255 Glu Asp Thr Cys Thr Phe His Ile Tyr Gly Glu Asp Glin Asp Ala Val 26 O 265 27 O Llys Lys Ala Arg Ser Phe Lieu. Glu Phe Ala Glu Asp Val Ile Glin Val 27s 28O 285 Pro Arg Asn Lieu Val Gly Llys Val Ile Gly Lys Asn Gly Lys Lieu. Ile 29 O 295 3 OO Glin Glu Ile Val Asp Llys Ser Gly Val Val Arg Val Arg Ile Glu Ala 3. OS 310 315 32O Glu Asin Glu Lys Ser Val Pro Glin Glu Glu Glu Ile Met Pro Pro Ser 3.25 330 335 Ser Lieu Pro Ser Asn. Asn. Ser Arg Val Gly Pro Asn. Ser Ser Glu Glu 34 O 345 35. O Llys Llys His Lieu. Asp Thir Lys Glu Asn. Thir His Phe Ser Glin Pro Asn 355 360 365 Ser Thr Llys Val Glin Arg Val Lieu Val Val Ser Ser Ile Val Ala Gly 37 O 375 38O Gly Pro Gln Llys Pro Glu Pro Lys Ala Trp Gln Gly Met Val Pro Phe 385 390 395 4 OO Val Phe Val Gly. Thir Lys Asp Ser Ile Ala Asn Ala Thr Val Lieu. Lieu 4 OS 41O 415 Asp Tyr His Lieu. Asn Tyr Lieu Lys Glu Val Asp Gln Lieu. Arg Lieu. Glu 42O 425 43 O Arg Lieu. Glin Ile Asp Glu Glin Lieu. Arg Glin Ile Gly Ala Ser Ser Arg 435 44 O 445 Pro Pro Pro Asn Arg Thr Asp Llys Glu Lys Gly Tyr Val Thir Asp Asp 450 45.5 460 Gly Glin Gly Met Gly Arg Gly Ser Arg Pro Tyr Arg Asn Arg Gly. His 465 470 47s 48O Gly Arg Arg Gly Pro Gly Tyr Thr Ser Gly Thr Asn Ser Glu Ala Ser 485 490 495 Asn Ala Ser Glu Thr Glu Ser Asp His Arg Asp Glu Lieu. Ser Asp Trip SOO 505 51O Ser Lieu Ala Pro Thr Glu Glu Glu Arg Glu Ser Phe Lieu. Arg Arg Gly 515 52O 525 Asp Gly Arg Arg Arg Gly Gly Gly Gly Arg Gly Glin Gly Gly Arg Gly 53 O 535 54 O Arg Gly Gly Gly Phe Lys Gly Asn Asp Asp His Ser Arg Thr Asp Asn 5.45 550 555 560 US 2010/0247552 A1 Sep. 30, 2010 63

- Continued Arg Pro Arg Asn Pro Arg Glu Ala Lys Gly Arg Thr Ala Asp Gly Ser 565 st O sts

Lell Glin Ser Ala Ser Ser Glu Gly Ser Arg Lieu. Arg Thr Gly Lys Asp 58O 585 59 O Arg Asin Gln Lys Lys Glu Lys Pro Asp Ser Val Asp Gly Lieu. Glin Pro 595 6OO 605

Lell Val Asn Gly Val Pro 610

<210s, SEQ ID NO 6 &211s LENGTH: 545 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Human PAK1, PAK2 and PAK3 amino acid sequences

<4 OOs, SEQUENCE: 6

Met Ser Asn. Asn Gly Lieu. Asp Ile Glin Asp Lys Pro Pro Ala Pro Pro 1. 5 15

Met Arg Asn. Thir Ser Thr Met Ile Gly Wall Gly Ser Asp Ala Gly 2O 25

Thir Lieu. Asn His Gly Ser Llys Pro Lieu Pro Pro Asn Pro Arg Arg 35 4 O 45

Llys Lys Asp Arg Phe Tyr Arg Ser Ile Luell Pro Gly Asp Thir SO 55 6 O

Asn Llys Llys Llys His Lys Glu Arg Pro Arg Ile Ser Lell Pro Ser Asp 65 70

Phe Glu. His Thir Ile His Val Gly Phe Asp Ala Wall Thir Gly Glu Phe 85 90 95

Thir Gly Met Pro Glu Gln Met Ala Arg Luell Luell Glin Thir Ser Asn Ile 1OO 105 11 O

Thir Llys Ser Glu Gln Llys Lys Asn Pro Glin Ala Wall Lell Asp Wall Luell 115 12 O 125

Glu Phe Tyr Asn Ser Lys Llys Thr Ser Asn Ser Glin Met Ser 13 O 135 14 O

Phe Thir Asp Llys Ser Ala Glu Asp Tyr Asn Ser Ser Asn Ala Luell Asn 145 150 155 160

Wall Lys Ala Val Ser Glu Thr Pro Ala Wall Pro Pro Wall Ser Arg Asp 1.65 17O

Glu Asp Asp Asp Asp Asp Asp Ala Thr Pro Pro Pro Wall Ile Ala Pro 18O 185 19 O

Arg Pro Glu. His Thr Lys Ser Val Tyr Thir Arg Ser Wall Ile Arg Pro 195 2OO

Lell Pro Val Thr Pro Thr Arg Asp Val Ala Thir Ser Pro Ile Ser Pro 21 O 215 22O

Thir Glu Asn Asn Thr Thr Pro Pro Asp Ala Luell Thir Arg Asn Thir Glu 225 23 O 235 24 O

Gln Lys Llys Llys Pro Llys Met Ser Asp Glu Glu Ile Luell Arg 245 250 255

Lell Arg Ser Ile Val Ser Val Gly Asp Pro Lys Tyr Thir Arg 26 O 265 27 O

Phe Glu Lys Asp Gly Glin Gly Ala Ser Gly Thir Wall Tyr Thir Ala Met 27s 28O 285 US 2010/0247552 A1 Sep. 30, 2010 64

- Continued Asp Wall Ala Thr Gly Glin Glu Val Ala Ile Lys Gln Met Asn Lieu. Glin 29 O 295 3 OO Glin Glin Pro Llys Lys Glu Lieu. Ile Ile Asin Arg Ile Lieu Val Met Arg 3. OS 310 315 32O Glu Asn Lys Asn Pro Asn. Ile Val Asn Tyr Lieu. Asp Ser Tyr Lieu Val 3.25 330 335 Gly Asp Glu Lieu Met Val Val Met Glu Tyr Lieu Ala Gly Gly Ser Lieu. 34 O 345 35. O Thr Asp Val Val Thr Glu Thr Cys Met Asp Glu Gly Glin Ile Ala Ala 355 360 365 Val Cys Arg Glu. Cys Lieu. Glin Ala Lieu. Glu Phe Lieu. His Ser Asn Glin 37 O 375 38O Val Ile His Arg Asp Ile Llys Ser Asp Asn. Ile Lieu. Lieu. Gly Met Asp 385 390 395 4 OO Gly Ser Val Lys Lieu. Thr Asp Phe Gly Phe Cys Ala Glin Ile Thr Pro 4 OS 41O 415 Glu Glin Ser Lys Arg Ser Thr Met Val Gly Thr Pro Tyr Met Met Ala 42O 425 43 O Pro Glu Val Val Thr Arg Lys Ala Tyr Gly Pro Llys Val Asp Ile Met 435 44 O 445 Ser Leu Gly Ile Met Ala Ile Glu Met Ile Glu Gly Glu Pro Pro Tyr 450 45.5 460 Lieu. Asn. Glu Asn. Pro Lieu. Arg Ala Lieu. Tyr Lieu. Ile Ala Thir Asn Gly 465 470 47s 48O Thr Pro Glu Lieu. Glin Asn Pro Glu Lys Lieu. Ser Ala Ile Phe Arg Asp 485 490 495 Phe Lieu. Asn Arg Cys Lieu. Asp Met Asp Val Glu Lys Arg Gly Ser Ala SOO 505 51O Lys Glu Lieu. Lieu. Glin His Glin Phe Lieu Lys Ile Ala Llys Pro Lieu. Ser 515 52O 525 Ser Lieu. Thr Pro Lieu. Ile Ala Ala Ala Lys Glu Ala Thr Lys Asn. Asn 53 O 535 54 O

His 5.45

<210s, SEQ ID NO 7 &211s LENGTH: 525 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Human PAK1, PAK2 and PAK3 amino acid sequences <4 OO > SEQUENCE: 7 Met Ser Asp Asin Gly Glu Lieu. Arg Asp Llys Pro Pro Ala Pro Pro Val 1. 5 1O 15 Arg Met Ser Ser Thr Ile Phe Ser Thr Gly Gly Lys Asp Pro Leu Ser 2O 25 3O Ala Asn His Ser Lieu Lys Pro Lieu Pro Ser Val Pro Arg Arg Llys Llys 35 4 O 45 Pro Arg His Lys Ile Ile Ser Ile Phe Ser Gly Thr Glu Lys Gly Ser SO 55 6 O Llys Llys Lys Glu Lys Glu Arg Pro Arg Ile Ser Pro Pro Ser Asp Phe 65 70 7s 8O US 2010/0247552 A1 Sep. 30, 2010 65

- Continued Glu. His Thir Ile His Val Gly Phe Asp Ala Val Thr Gly Glu Phe Thr 85 90 95 Gly Met Pro Glu Gln Met Ala Arg Lieu. Leu Gln Thr Ser Asn Ile Thr 1OO 105 11 O Llys Lieu. Glu Gln Llys Lys Asn Pro Glin Ala Val Lieu. Asp Val Lieu Lys 115 12 O 125 Phe Tyr Asp Ser Asn Thr Val Lys Gln Lys Tyr Lieu Ser Phe Thr Pro 13 O 135 14 O Pro Glu Lys Asp Gly Lieu Pro Ser Gly Thr Pro Ala Lieu. Asn Ala Lys 145 150 155 160 Gly Thr Glu Ala Pro Ala Val Val Thr Phe Glu Glu Asp Asp Asp Glu 1.65 17O 17s Glu Thir Ala Pro Pro Val Ile Ala Pro Arg Pro Asp His Thr Lys Ser 18O 185 19 O Ile Tyr Thr Arg Ser Val Ile Asp Pro Val Pro Ala Pro Val Gly Asp 195 2OO 2O5 Ser His Val Asp Cys Ala Ala Lys Ser Lieu. Asp Llys Glin Llys Llys Llys 21 O 215 22O Pro Llys Met Thir Asp Glu Glu Ile Met Arg Llys Lieu. Arg Thir Ile Val 225 23 O 235 24 O Ser Ile Gly Asn. Pro Llys Llys Llys Tyr Thr Arg Tyr Glu Lys Asp Gly 245 250 255 Gln Gly Ala Ser Gly Thr Val Phe Thr Ala Thr Asp Val Ala Leu Gly 26 O 265 27 O Glin Glu Val Ala Ile Llys Glin Ile Asn Lieu. Glin Lys Glin Pro Llys Llys 27s 28O 285 Glu Lieu. Ile Ile Asn Arg Ile Lieu Val Met Lys Glu Lieu Lys Asn Pro 29 O 295 3 OO Asn. Ile Val Asn. Phe Lieu. Asp Ser Tyr Lieu Val Gly Asp Glu Lieu. Phe 3. OS 310 315 32O Val Val Met Glu Tyr Lieu Ala Gly Gly Ser Lieu. Thr Asp Val Val Thr 3.25 330 335 Glu Thir Ala Cys Met Asp Glu Ala Glin Ile Ala Ala Val Cys Arg Glu 34 O 345 35. O Cys Lieu. Glin Ala Lieu. Glu Phe Lieu. His Ala Asn. Glin Val Ile His Arg 355 360 365 Asp Ile Llys Ser Asp Asn Val Lieu. Lieu. Gly Met Glu Gly Ser Val Lys 37 O 375 38O Lieu. Thir Asp Phe Gly Phe Cys Ala Glin Ile Thr Pro Glu Glin Ser Lys 385 390 395 4 OO Arg Ser Thr Met Val Gly Thr Pro Tyr Met Met Ala Pro Glu Val Val 4 OS 41O 415 Thir Arg Lys Ala Tyr Gly Pro Llys Val Asp Ile Met Ser Lieu. Gly Ile 42O 425 43 O Met Ala Ile Glu Met Val Glu Gly Glu Pro Pro Tyr Lieu. Asn Glu Asn 435 44 O 445 Pro Lieu. Arg Ala Lieu. Tyr Lieu. Ile Ala Thr Asn Gly Thr Pro Glu Lieu. 450 45.5 460 Glin Asn Pro Glu Lys Lieu. Ser Pro Ile Phe Arg Asp Phe Lieu. Asn Arg 465 470 47s 48O Cys Lieu. Glu Met Asp Val Glu Lys Arg Gly Ser Ala Lys Glu Lieu. Lieu. US 2010/0247552 A1 Sep. 30, 2010 66

- Continued

485 490 495 Gln His Pro Phe Leu Lys Lieu Ala Lys Pro Leu Ser Ser Lieu. Thr Pro SOO 505 Lieu. Ile Met Ala Ala Lys Glu Ala Met Lys Ser Asn Arg 515 525

SEQ ID NO 8 LENGTH: 544 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Human PAK1, PAK2 and PAK3 amino acid sequences

<4 OOs, SEQUENCE: 8

Met Ser Asp Gly Lell Asp Asn Glu Glu Lys Pro Pro Ala Pro Pro Luell 1. 5 1O 15

Arg Met Asn Ser Asn Asn Arg Asp Ser Ser Ala Lell Asn His Ser Ser 25 3O

Pro Luell Pro Met Ala Pro Ser Glu ASn Lys Ala Arg Luell 35 4 O 45

Arg Ser Ile Phe Pro Gly Gly Gly Asp Thir Asn Glu SO 55 6 O

Lys Glu Arg Pro Arg Ile Ser Luell Pro Ser Asp Phe Glu His Thir Ile 65 70

His Wall Gly Phe Asp Ala Wall Thir Gly Glu Phe Thir Gly Ile Pro Glu 85 90 95

Glin Met Ala Arg Lell Lell Glin Thir Ser Asn Ile Thir Luell Glu Glin 105 11 O

Asn Pro Glin Ala Wall Luell Asp Wall Luell Phe Asp Ser 115 12 O 125

Glu Thir Wall Asn Asn Glin Met Ser Phe Thir Ser Gly Asp 13 O 135 14 O

Lys Ser Ala His Gly Tyr Ile Ala Ala His Pro Ser Ser Thir Thir 145 150 155 160

Ala Ser Glu Pro Pro Lell Ala Pro Pro Wall Ser Arg Arg Arg Asp Arg 1.65 17O 17s

Arg Arg Arg Arg Arg Arg Asp Arg Asn Glu Pro Pro Pro Wall Ile Ala 18O 185 19 O

Pro Arg Pro Glu His Thir Ser Ile Tyr Thir Arg Ser Wall Wall Glu 195

Ser Ile Ala Ser Pro Ala Wall Pro Asn Glu Wall Thir Pro Pro Ser 21 O 215 22O

Ala Glu Asn Ala Asn Ser Ser Thir Luell Tyr Arg Asn Thir Asp Arg Glin 225 23 O 235 24 O

Arg Ser Lys Asn Thir Asp Glu Glu Ile Lell Arg Luell Arg 245 250 255

Ser Ile Wall Ser Wall Gly Asp Pro Lys Lys Thir Arg Glu Arg 26 O 265 27 O

Asp Gly Glin Gly Ala Ser Gly Thir Wall Tyr Thir Ala Luell Asp Ile 27s 285

Ala Thir Gly Glin Glu Wall Ala Ile Glin Met Asn Lell Glin Glin Glin 29 O 295 3 OO

Pro Glu Lell Ile Ile Asn Arg Ile Luell Wall Met Arg Glu Asn US 2010/0247552 A1 Sep. 30, 2010 67

- Continued

3. OS 310 315 32O Lys Asn Pro Asn. Ile Val Asn Tyr Lieu. Asp Ser Tyr Lieu Val Gly Asp 3.25 330 335 Glu Lieu Met Val Val Met Glu Tyr Lieu Ala Gly Gly Ser Lieu. Thir Asp 34 O 345 35. O Val Val Thr Glu Thr Cys Met Asp Glu Gly Glin Ile Ala Ala Val Cys 355 360 365 Arg Glu. Cys Lieu. Glin Ala Lieu. Asp Phe Lieu. His Ser Asn. Glin Val Ile 37 O 375 38O His Arg Asp Ile Llys Ser Asp Asn. Ile Lieu. Lieu. Gly Met Asp Gly Ser 385 390 395 4 OO Val Lys Lieu. Thir Asp Phe Gly Phe Cys Ala Glin Ile Thr Pro Glu Gln 4 OS 41O 415 Ser Lys Arg Ser Thr Met Val Gly Thr Pro Tyr Met Met Ala Pro Glu 42O 425 43 O Val Val Thir Arg Lys Ala Tyr Gly Pro Llys Val Asp Ile Met Ser Lieu 435 44 O 445 Gly Ile Met Ala Ile Glu Met Val Glu Gly Glu Pro Pro Tyr Lieu. Asn 450 45.5 460 Glu Asn Pro Lieu. Arg Ala Lieu. Tyr Lieu. Ile Ala Thr Asn Gly Thr Pro 465 470 47s 48O Glu Lieu. Glin ASn Pro Glu Arg Lieu. Ser Ala Val Pro Arg Asp Phe Lieu. 485 490 495 Asn Arg Cys Lieu. Glu Met Asp Val Glu Arg Arg Gly Ser Ala Lys Glu SOO 505 51O Lieu. Lieu Gln His Phe Pro Lieu Lys Lieu Ala Lys Pro Lieu. Ser Ser Lieu. 515 52O 525 Thr Pro Lieu. Ile Ile Ala Ala Lys Glu Ala Ile Lys Asn. Ser Ser Arg 53 O 535 54 O

<210s, SEQ ID NO 9 &211s LENGTH: 591 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Human PAK4, PAK5 PAK6 and PAK7 amino acid sequences

<4 OOs, SEQUENCE: 9 Met Phe Gly Lys Arg Llys Lys Arg Val Glu Ile Ser Ala Pro Ser Asn 1. 5 1O 15 Phe Glu. His Arg Val His Thr Gly Phe Asp Gln His Glu Glin Llys Phe 2O 25 3O Thr Gly Lieu Pro Arg Glin Trp Glin Ser Lieu. Ile Glu Glu Ser Ala Arg 35 4 O 45 Arg Pro Llys Pro Leu Val Asp Pro Ala Cys Ile Thr Ser Ile Glin Pro SO 55 6 O Gly Ala Pro Llys Thir Ile Val Arg Gly Ser Lys Gly Ala Lys Asp Gly 65 70 7s 8O Ala Lieu. Thir Lieu Lleu Lieu. Asp Glu Phe Glu Asn Met Ser Val Thr Arg 85 90 95 Ser Asn. Ser Lieu. Arg Arg Asp Ser Pro Pro Pro Pro Ala Arg Ala Arg 1OO 105 11 O US 2010/0247552 A1 Sep. 30, 2010 68

- Continued Gln Glu Asn Gly Met Pro Glu Glu Pro Ala Thir Thr Ala Arg Gly Gly 115 12 O 125 Pro Gly Lys Ala Gly Ser Arg Gly Arg Phe Ala Gly His Ser Glu Ala 13 O 135 14 O Gly Gly Gly Ser Gly Asp Arg Arg Arg Ala Gly Pro Glu Lys Arg Pro 145 150 155 160 Llys Ser Ser Arg Glu Gly Ser Gly Gly Pro Glin Glu Ser Ser Arg Asp 1.65 17O 17s Lys Arg Pro Leu Ser Gly Pro Asp Val Gly Thr Pro Gln Pro Ala Gly 18O 185 19 O Lieu Ala Ser Gly Ala Lys Lieu Ala Ala Gly Arg Pro Phe Asn. Thir Tyr 195 2OO 2O5 Pro Arg Ala Asp Thr Asp His Pro Ser Arg Gly Ala Glin Gly Glu Pro 21 O 215 22O His Asp Wall Ala Pro Asn Gly Pro Ser Ala Gly Gly Lieu Ala Ile Pro 225 23 O 235 24 O Gln Ser Ser Ser Ser Ser Ser Arg Pro Pro Thr Arg Ala Arg Gly Ala 245 250 255 Pro Ser Pro Gly Val Lieu. Gly Pro His Ala Ser Glu Pro Glin Leu Ala 26 O 265 27 O Pro Pro Ala Cys Thr Pro Ala Ala Pro Ala Val Pro Gly Pro Pro Gly 27s 28O 285 Pro Arg Ser Pro Glin Arg Glu Pro Glin Arg Val Ser His Glu Glin Phe 29 O 295 3 OO Arg Ala Ala Lieu Gln Lieu Val Val Asp Pro Gly Asp Pro Arg Ser Tyr 3. OS 310 315 32O Lieu. Asp Asn. Phe Ile Lys Ile Gly Glu Gly Ser Thr Gly Ile Val Cys 3.25 330 335 Ile Ala Thr Val Arg Ser Ser Gly Lys Lieu Val Ala Val Lys Llys Met 34 O 345 35. O Asp Lieu. Arg Lys Glin Glin Arg Arg Glu Lieu. Lieu. Phe Asn. Glu Val Val 355 360 365 Ile Met Arg Asp Tyr Gln His Glu Asn Val Val Glu Met Tyr Asn Ser 37 O 375 38O Tyr Lieu Val Gly Asp Glu Lieu. Trp Val Val Met Glu Phe Lieu. Glu Gly 385 390 395 4 OO Gly Ala Lieu. Thir Asp Ile Val Thr His Thr Arg Met Asn Glu Glu Gln 4 OS 41O 415 Ile Ala Ala Val Cys Lieu Ala Val Lieu. Glin Ala Lieu. Ser Val Lieu. His 42O 425 43 O Ala Glin Gly Val Ile His Arg Asp Ile Llys Ser Asp Ser Ile Lieu. Lieu 435 44 O 445 Thir His Asp Gly Arg Val Llys Lieu. Ser Asp Phe Gly Phe Cys Ala Glin 450 45.5 460 Val Ser Lys Glu Val Pro Arg Arg Llys Ser Leu Val Gly Thr Pro Tyr 465 470 47s 48O Trp Met Ala Pro Glu Lieu. Ile Ser Arg Lieu Pro Tyr Gly Pro Glu Val 485 490 495 Asp Ile Trp Ser Lieu. Gly Ile Met Val Ile Glu Trp Val Asp Gly Glu SOO 505 51O Pro Pro Tyr Phe Asn Glu Pro Pro Leu Lys Ala Met Lys Met Ile Arg US 2010/0247552 A1 Sep. 30, 2010 69

- Continued

515 52O 525 Asp Asn Lieu Pro Pro Arg Lieu Lys Asn Lieu. His Llys Val Ser Pro Ser 53 O 535 54 O Lieu Lys Gly Phe Lieu. Asp Arg Lieu. Lieu Val Arg Asp Pro Ala Glin Arg 5.45 550 555 560 Ala Thr Ala Ala Glu Lieu Lleu Lys His Pro Phe Lieu Ala Lys Ala Gly 565 st O sts Pro Pro Ala Ser Ile Val Pro Leu Met Arg Glin Asn Arg Thr Arg 58O 585 59 O

<210s, SEQ ID NO 10 &211s LENGTH: 718 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Human PAK4, PAK5 PAK6 and PAK7 amino acid sequences

<4 OOs, SEQUENCE: 10 Met Phe Gly Lys Llys Llys Llys Lys Ile Glu Ile Ser Gly Pro Ser Asn 1. 5 1O 15 Phe Glu. His Arg Val His Thr Gly Phe Asp Pro Glin Glu Glin Llys Phe 2O 25 3O Thr Gly Lieu Pro Glin Glin Trp His Ser Lieu. Lieu Ala Asp Thir Ala Asn 35 4 O 45 Arg Pro Llys Pro Met Val Asp Pro Ser Cys Ile Thr Pro Ile Glin Leu SO 55 6 O Ala Pro Met Lys Thr Ile Val Arg Gly Asn Llys Pro Cys Lys Glu Thr 65 70 7s 8O Ser Ile Asin Gly Lieu. Lieu. Glu Asp Phe Asp Asn. Ile Ser Val Thr Arg 85 90 95 Ser Asn. Ser Lieu. Arg Lys Glu Ser Pro Pro Thr Pro Asp Glin Gly Ala 1OO 105 11 O Ser Ser His Gly Pro Gly His Ala Glu Glu Asn Gly Phe Ile Thr Phe 115 12 O 125 Ser Glin Tyr Ser Ser Glu Ser Asp Thir Thr Ala Asp Tyr Thr Thr Glu 13 O 135 14 O Llys Tyr Arg Glu Lys Ser Lieu. Tyr Gly Asp Asp Lieu. Asp Pro Tyr Tyr 145 150 155 160 Arg Gly Ser His Ala Ala Lys Glin Asn Gly His Wal Met Lys Met Lys 1.65 17O 17s His Gly Glu Ala Tyr Tyr Ser Glu Val Llys Pro Lieu Lys Ser Asp Phe 18O 185 19 O Ala Arg Phe Ser Ala Asp Tyr His Ser His Lieu. Asp Ser Lieu. Ser Lys 195 2OO 2O5 Pro Ser Glu Tyr Ser Asp Lieu Lys Trp Glu Tyr Glin Arg Ala Ser Ser 21 O 215 22O Ser Ser Pro Leu Asp Tyr Ser Phe Glin Phe Thr Pro Ser Arg Thr Ala 225 23 O 235 24 O Gly. Thir Ser Gly Cys Ser Lys Glu Ser Leu Ala Tyr Ser Glu Ser Glu 245 250 255 Trp Gly Pro Ser Lieu. Asp Asp Tyr Asp Arg Arg Pro Llys Ser Ser Tyr 26 O 265 27 O US 2010/0247552 A1 Sep. 30, 2010 70

- Continued Lieu. Asn Gln Thr Ser Pro Gln Pro Thr Met Arg Glin Arg Ser Arg Ser 27s 28O 285 Gly Ser Gly Lieu Gln Glu Pro Met Met Pro Phe Gly Ala Ser Ala Phe 29 O 295 3 OO Lys Thr His Pro Glin Gly His Ser Tyr Asn Ser Tyr Thr Tyr Pro Arg 3. OS 310 315 32O Lieu. Ser Glu Pro Thr Met Cys Ile Pro Llys Val Asp Tyr Asp Arg Ala 3.25 330 335 Gln Met Val Lieu Ser Pro Pro Leu Ser Gly Ser Asp Thr Tyr Pro Arg 34 O 345 35. O Gly Pro Ala Lys Lieu Pro Glin Ser Glin Ser Lys Ser Gly Tyr Ser Ser 355 360 365 Ser Ser His Glin Tyr Pro Ser Gly Tyr His Lys Ala Thr Lieu. Tyr His 37 O 375 38O His Pro Ser Leu Gln Ser Ser Ser Glin Tyr Ile Ser Thr Ala Ser Tyr 385 390 395 4 OO Lieu. Ser Ser Leu Ser Leu Ser Ser Ser Thr Tyr Pro Pro Pro Ser Trp 4 OS 41O 415 Gly Ser Ser Ser Asp Glin Gln Pro Ser Arg Val Ser His Glu Glin Phe 42O 425 43 O Arg Ala Ala Lieu Gln Lieu Val Val Ser Pro Gly Asp Pro Arg Glu Tyr 435 44 O 445 Lieu Ala Asn. Phe Ile Lys Ile Gly Glu Gly Ser Thr Gly Ile Val Cys 450 45.5 460 Ile Ala Thr Glu Lys His Thr Gly Lys Glin Val Ala Val Lys Llys Met 465 470 47s 48O Asp Lieu. Arg Glin Glin Arg Arg Glu Lieu. Lieu. Phe Asn. Glu Val Val Ile 485 490 495 Met Arg Asp Tyr His His Asp Asn Val Val Asp Met Tyr Ser Ser Tyr SOO 505 51O Lieu Val Gly Asp Glu Lieu. Trp Val Val Met Glu Phe Lieu. Glu Gly Gly 515 52O 525 Ala Lieu. Thir Asp Ile Val Thr His Thr Arg Met Asn Glu Glu Glin Ile 53 O 535 54 O Ala Thr Val Cys Lieu. Ser Val Lieu. Arg Ala Lieu. Ser Tyr Lieu. His Asn 5.45 550 555 560 Glin Gly Val Ile His Arg Asp Ile Llys Ser Asp Ser Ile Lieu. Lieu. Thir 565 st O sts Ser Asp Gly Arg Ile Llys Lieu. Ser Asp Phe Gly Phe Cys Ala Glin Val 58O 585 59 O Ser Lys Glu Val Pro Lys Arg Llys Ser Leu Val Gly Thr Pro Tyr Trp 595 6OO 605 Met Ala Pro Glu Val Ile Ser Arg Lieu Pro Tyr Gly Thr Glu Val Asp 610 615 62O Ile Trp Ser Leu Gly Ile Met Val Ile Glu Met Ile Asp Gly Glu Pro 625 630 635 64 O Pro Tyr Phe Asn. Glu Pro Pro Lieu. Glin Ala Met Arg Arg Ile Arg Asp 645 650 655 Ser Lieu Pro Pro Arg Val Lys Asp Lieu. His Llys Val Ser Ser Val Lieu. 660 665 67 O Arg Gly Phe Lieu. Asp Lieu Met Lieu Val Arg Glu Pro Ser Glin Arg Ala US 2010/0247552 A1 Sep. 30, 2010 71

- Continued

675 68O 685 Thir Ala Glin Glu Lieu. Lieu. Gly His Pro Phe Lieu Lys Lieu Ala Gly Pro 69 O. 695 7 OO Pro Ser Cys Ile Val Pro Leu Met Arg Glin Tyr Arg His His 7 Os 71O 71s

<210s, SEQ ID NO 11 &211s LENGTH: 681 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Human PAK4, PAK5 PAK6 and PAK7 amino acid sequences

<4 OOs, SEQUENCE: 11 Met Phe Arg Llys Llys Llys Llys Lys Arg Pro Glu Ile Ser Ala Pro Glin 1. 5 1O 15 Asn Phe Gln His Arg Val His Thr Ser Phe Asp Pro Lys Glu Gly Lys 2O 25 3O Phe Val Gly Lieu Pro Pro Gln Trp Glin Asn. Ile Lieu. Asp Thir Lieu. Arg 35 4 O 45 Arg Pro Llys Pro Val Val Asp Pro Ser Arg Ile Thr Arg Val Glin Lieu SO 55 6 O Gln Pro Met Lys Thr Val Val Arg Gly Ser Ala Met Pro Val Asp Gly 65 70 7s 8O Tyr Ile Ser Gly Lieu. Lieu. Asn Asp Ile Glin Llys Lieu. Ser Val Ile Ser 85 90 95 Ser Asn. Thir Lieu. Arg Gly Arg Ser Pro Thir Ser Arg Arg Arg Ala Glin 1OO 105 11 O Ser Lieu. Gly Lieu. Lieu. Gly Asp Glu. His Trp Ala Thr Asp Pro Asp Met 115 12 O 125 Tyr Lieu. Glin Ser Pro Glin Ser Glu Arg Thr Asp Pro His Gly Lieu. Tyr 13 O 135 14 O Lieu. Ser Cys Asn Gly Gly Thr Pro Ala Gly His Lys Gln Met Pro Glin 145 150 155 160 Pro Glu Pro Glin Ser Pro Arg Val Lieu Pro Asn Gly Lieu Ala Ala Lys 1.65 17O 17s Glin Ser Lieu. Gly Pro Ala Glu Phe Glin Gly Ala Ser Glin Arg Cys Lieu. 18O 185 19 O Gln Leu Gly Ala Cys Lieu. Glin Ser Ser Pro Pro Gly Ala Ser Pro Pro 195 2OO 2O5 Thr Gly Thr Asn Arg His Gly Met Lys Ala Ala Lys His Gly Ser Glu 21 O 215 22O Glu Ala Arg Pro Glin Ser Cys Lieu Val Gly Ser Ala Thr Gly Arg Pro 225 23 O 235 24 O Gly Gly Glu Gly Ser Pro Ser Pro Llys Thr Arg Glu Ser Ser Leu Lys 245 250 255 Arg Arg Lieu. Phe Arg Ser Met Phe Lieu. Ser Thr Ala Ala Thr Ala Pro 26 O 265 27 O Pro Ser Ser Ser Lys Pro Gly Pro Pro Pro Glin Ser Lys Pro Asn Ser 27s 28O 285 Ser Phe Arg Pro Pro Glin Lys Asp Asn Pro Pro Ser Lieu Val Ala Lys 29 O 295 3 OO US 2010/0247552 A1 Sep. 30, 2010 72

- Continued Ala Glin Ser Leu Pro Ser Asp Gln Pro Val Gly Thr Phe Ser Pro Leu 3. OS 310 315 32O Thir Thr Ser Asp Thr Ser Ser Pro Gln Lys Ser Lieu. Arg Thr Ala Pro 3.25 330 335 Ala Thr Gly Glin Leu Pro Gly Arg Ser Ser Pro Ala Gly Ser Pro Arg 34 O 345 35. O Thir Trp His Ala Glin Ile Ser Thr Ser Asn Lieu. Tyr Lieu Pro Glin Asp 355 360 365 Pro Thr Val Ala Lys Gly Ala Lieu Ala Gly Glu Asp Thr Gly Val Val 37 O 375 38O Thir His Glu Glin Phe Lys Ala Ala Lieu. Arg Met Val Val Asp Glin Gly 385 390 395 4 OO Asp Pro Arg Lieu Lleu Lieu. Asp Ser Tyr Val Lys Ile Gly Glu Gly Ser 4 OS 41O 415 Thr Gly Ile Val Cys Lieu Ala Arg Glu Lys His Ser Gly Arg Glin Val 42O 425 43 O Ala Wall Lys Met Met Asp Lieu. Arg Lys Glin Glin Arg Arg Glu Lieu. Lieu 435 44 O 445 Phe Asin Glu Val Val Ile Met Arg Asp Tyr Gln His Phe Asin Val Val 450 45.5 460 Glu Met Tyr Lys Ser Tyr Lieu Val Gly Glu Glu Lieu. Trp Val Leu Met 465 470 47s 48O Glu Phe Lieu. Glin Gly Gly Ala Lieu. Thir Asp Ile Val Ser Glin Val Arg 485 490 495 Lieu. Asn. Glu Glu Glin Ile Ala Thr Val Cys Glu Ala Val Lieu. Glin Ala SOO 505 51O Lieu Ala Tyr Lieu. His Ala Glin Gly Val Ile His Arg Asp Ile Llys Ser 515 52O 525 Asp Ser Ile Lieu. Lieu. Thir Lieu. Asp Gly Arg Val Llys Lieu. Ser Asp Phe 53 O 535 54 O Gly Phe Cys Ala Glin Ile Ser Lys Asp Val Pro Lys Arg Llys Ser Lieu. 5.45 550 555 560 Val Gly Thr Pro Tyr Trp Trp Met Ala Pro Glu Val Ile Ser Arg Ser 565 st O sts Lieu. Tyr Ala Thr Glu Val Asp Ile Trp Ser Leu Gly Ile Met Val Ile 58O 585 59 O Glu Met Val Asp Gly Glu Pro Pro Tyr Phe Ser Asp Ser Pro Val Glin 595 6OO 605 Ala Met Lys Arg Lieu. Arg Asp Ser Pro Pro Pro Llys Lieu Lys Asn. Ser 610 615 62O His Llys Val Ser Pro Val Lieu. Arg Asp Phe Lieu. Glu Arg Met Lieu Val 625 630 635 64 O Arg Asp Pro Glin Glu Arg Ala Thr Ala Glin Glu Lieu. Lieu. Asp His Pro 645 650 655 Phe Lieu. Leu Gln Thr Gly Lieu Pro Glu. Cys Lieu Val Pro Leu. Ile Gly 660 665 67 O Lieu. Tyr Arg Lys Glin Thir Ser Thr Cys 675 68O

<210s, SEQ ID NO 12 &211s LENGTH: 681 212. TYPE: PRT US 2010/0247552 A1 Sep. 30, 2010 73

- Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Human PAK4, PAK5 PAK6 and PAK7 amino acid sequences

<4 OOs, SEQUENCE: 12 Met Phe Arg Llys Llys Llys Llys Lys Arg Pro Glu Ile Ser Ala Pro Glin 1. 5 1O 15 Asn Phe Gln His Arg Val His Thr Ser Phe Asp Pro Lys Glu Gly Lys 2O 25 3O Phe Val Gly Lieu Pro Pro Gln Trp Glin Asn. Ile Lieu. Asp Thir Lieu. Arg 35 4 O 45 Arg Pro Llys Pro Val Val Asp Pro Ser Arg Ile Thr Arg Val Glin Lieu SO 55 6 O Gln Pro Met Lys Thr Val Val Arg Gly Ser Ala Met Pro Val Asp Gly 65 70 7s 8O Tyr Ile Ser Gly Lieu. Lieu. Asn Asp Ile Glin Llys Lieu. Ser Val Ile Ser 85 90 95 Ser Asn. Thir Lieu. Arg Gly Arg Ser Pro Thir Ser Arg Arg Arg Ala Glin 1OO 105 11 O Ser Lieu. Gly Lieu. Lieu. Gly Asp Glu. His Trp Ala Thr Asp Pro Asp Met 115 12 O 125 Tyr Lieu. Glin Ser Pro Glin Ser Glu Arg Thr Asp Pro His Gly Lieu. Tyr 13 O 135 14 O Lieu. Ser Cys Asn Gly Gly Thr Pro Ala Gly His Lys Gln Met Pro Trp 145 150 155 160 Pro Glu Pro Glin Ser Pro Arg Val Lieu Pro Asn Gly Lieu Ala Ala Lys 1.65 17O 17s Ala Glin Ser Lieu. Gly Pro Ala Glu Phe Glin Gly Ala Ser Glin Arg Cys 18O 185 19 O Lieu Gln Leu Gly Ala Cys Lieu. Glin Ser Ser Pro Pro Gly Ala Ser Pro 195 2OO 2O5 Pro Thr Gly Thr Asn Arg His Gly Met Lys Ala Ala Lys His Gly Ser 21 O 215 22O Glu Glu Ala Arg Pro Glin Ser Cys Lieu Val Gly Ser Ala Thr Gly Arg 225 23 O 235 24 O Pro Gly Gly Glu Gly Ser Pro Ser Pro Llys Thr Arg Glu Ser Ser Lieu. 245 250 255 Lys Arg Arg Lieu. Phe Arg Ser Met Phe Lieu. Ser Thr Ala Ala Thr Ala 26 O 265 27 O Pro Pro Ser Ser Ser Lys Pro Gly Pro Pro Pro Glin Ser Llys Pro Asn 27s 28O 285 Ser Ser Phe Arg Pro Pro Gln Lys Asp Asin Pro Pro Ser Leu Val Ala 29 O 295 3 OO Lys Ala Glin Ser Lieu Pro Ser Asp Gln Pro Val Gly Thr Phe Ser Pro 3. OS 310 315 32O Lieu. Thir Thr Ser Asp Thir Ser Ser Pro Glin Lys Ser Lieu. Arg Thr Ala 3.25 330 335 Pro Ala Thr Gly Glin Leu Pro Gly Arg Ser Ser Pro Ala Gly Ser Pro 34 O 345 35. O Arg Thr Trp His Ala Glin Ile Ser Thr Ser Asn Lieu. Tyr Lieu Pro Glin 355 360 365 US 2010/0247552 A1 Sep. 30, 2010 74

- Continued Asp Pro Thr Val Ala Lys Gly Ala Lieu Ala Gly Glu Asp Thr Gly Val 37 O 375 38O Val Thr His Glu Glin Phe Lys Ala Ala Lieu. Arg Met Val Val Asp Glin 385 390 395 4 OO Gly Asp Pro Arg Lieu Lleu Lieu. Asp Ser Tyr Val Lys Ile Gly Glu Gly 4 OS 41O 415 Ser Thr Gly Ile Val Cys Lieu Ala Arg Glu Lys His Ser Gly Arg Glin 42O 425 43 O Val Ala Wall Lys Met Met Asp Lieu. Arg Lys Glin Glin Arg Arg Glu Lieu 435 44 O 445 Lieu. Phe Asin Glu Val Val Ile Met Arg Asp Tyr Gln His Phe Asin Val 450 45.5 460 Val Glu Met Tyr Lys Ser Tyr Lieu Val Gly Glu Glu Lieu. Trp Val Lieu. 465 470 47s 48O Met Glu Phe Leu Gln Gly Gly Ala Lieu. Thir Asp Ile Val Ser Glin Val 485 490 495 Arg Lieu. Asn. Glu Glu Glin Ile Ala Thr Val Cys Glu Ala Val Lieu. Glin SOO 505 51O Ala Lieu Ala Tyr Lieu. His Ala Glin Gly Val Ile His Arg Asp Ile Llys 515 52O 525 Asp Ser Asp Ile Lieu. Lieu. Thir Lieu. Asp Gly Arg Val Llys Lieu. Ser Asp 53 O 535 54 O Phe Gly Phe Cys Ala Glin Ile Ser Lys Asp Val Pro Lys Arg Llys Ser 5.45 550 555 560 Lieu Val Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Ile Ser Arg Ser 565 st O sts Lieu. Tyr Ala Thr Glu Val Asp Ile Trp Ser Leu Gly Ile Met Val Ile 58O 585 59 O Glu Met Val Asp Gly Glu Pro Pro Tyr Phe Ser Asp Ser Pro Val Glin 595 6OO 605 Ala Met Lys Arg Lieu. Arg Asp Ser Pro Pro Pro Llys Lieu Lys Asn. Ser 610 615 62O His Llys Val Ser Pro Val Lieu. Arg Asp Phe Lieu. Glu Arg Met Lieu Val 625 630 635 64 O Arg Asp Pro Glin Glu Arg Ala Thr Ala Glin Glu Lieu. Lieu. Asp His Pro 645 650 655 Phe Lieu. Leu Gln Thr Gly Lieu Pro Glu. Cys Lieu Val Pro Leu. Ile Glin 660 665 67 O Lieu. Tyr Arg Lys Glin Thir Ser Thr Cys 675 68O

<210s, SEQ ID NO 13 &211s LENGTH: 719 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Human PAK4, PAK5, and PAK6 amino acid sequences <4 OOs, SEQUENCE: 13 Met Phe Gly Lys Llys Llys Llys Lys Ile Glu Ile Ser Gly Pro Ser Asn 1. 5 1O 15 Phe Glu. His Arg Val His Thr Gly Phe Asp Pro Glin Glu Glin Llys Phe 2O 25 3O US 2010/0247552 A1 Sep. 30, 2010 75

- Continued Thr Gly Lieu Pro Glin Glin Trp His Ser Lieu. Lieu Ala Asp Thir Ala Asn 35 4 O 45 Arg Pro Llys Pro Met Val Asp Pro Ser Cys Ile Thr Pro Ile Glin Leu SO 55 6 O Ala Pro Met Lys Thr Ile Val Arg Gly Asn Llys Pro Cys Lys Glu Thr 65 70 7s 8O Ser Ile Asin Gly Lieu. Lieu. Glu Asp Phe Asp Asn. Ile Ser Val Thr Arg 85 90 95 Ser Asn. Ser Lieu. Arg Lys Glu Ser Pro Pro Thr Pro Asp Glin Gly Ala 1OO 105 11 O Ser Ser His Gly Pro Gly His Ala Glu Glu Asn Gly Phe Ile Thr Phe 115 12 O 125 Ser Glin Tyr Ser Ser Glu Ser Asp Thir Thr Ala Asp Tyr Thr Thr Glu 13 O 135 14 O Llys Tyr Arg Glu Lys Ser Lieu. Tyr Gly Asp Asp Lieu. Asp Pro Tyr Tyr 145 150 155 160 Arg Gly Ser His Ala Ala Lys Glin Asn Gly His Wal Met Lys Met Lys 1.65 17O 17s His Gly Glu Ala Tyr Tyr Ser Glu Val Llys Pro Lieu Lys Ser Asp Phe 18O 185 19 O Ala Arg Phe Ser Ala Asp Tyr His Ser His Lieu. Asp Ser Lieu. Ser Lys 195 2OO 2O5 Pro Ser Glu Tyr Ser Asp Lieu Lys Trp Glu Tyr Glin Arg Ala Ser Ser 21 O 215 22O Ser Ser Pro Leu Asp Tyr Ser Phe Glin Phe Thr Pro Ser Arg Thr Ala 225 23 O 235 24 O Gly. Thir Ser Gly Cys Ser Lys Glu Ser Leu Ala Tyr Ser Glu Ser Glu 245 250 255 Trp Gly Pro Ser Lieu. Asp Asp Tyr Asp Arg Arg Pro Llys Ser Ser Tyr 26 O 265 27 O Lieu. Asn Gln Thr Ser Pro Gln Pro Thr Met Arg Glin Arg Ser Arg Ser 27s 28O 285 Gly Ser Gly Lieu Gln Glu Pro Met Met Pro Phe Gly Ala Ser Ala Phe 29 O 295 3 OO Lys Thr His Pro Glin Gly His Ser Tyr Asn Ser Tyr Thr Tyr Pro Arg 3. OS 310 315 32O Lieu. Ser Glu Pro Thr Met Cys Ile Pro Llys Val Asp Tyr Asp Arg Ala 3.25 330 335 Gln Met Val Lieu Ser Pro Pro Leu Ser Gly Ser Asp Thr Tyr Pro Arg 34 O 345 35. O Gly Pro Ala Lys Lieu Pro Glin Ser Glin Ser Lys Ser Gly Tyr Ser Ser 355 360 365 Ser Ser His Glin Tyr Pro Ser Gly Tyr His Lys Ala Thr Lieu. Tyr His 37 O 375 38O His Pro Ser Leu Gln Ser Ser Ser Glin Tyr Ile Ser Thr Ala Ser Tyr 385 390 395 4 OO Lieu. Ser Ser Leu Ser Leu Ser Ser Ser Thr Tyr Pro Pro Pro Ser Trp 4 OS 41O 415 Gly Ser Ser Ser Asp Glin Gln Pro Ser Arg Val Ser His Glu Glin Phe 42O 425 43 O Arg Ala Ala Lieu Gln Lieu Val Val Ser Pro Gly Asp Pro Arg Glu Tyr US 2010/0247552 A1 Sep. 30, 2010 76

- Continued

435 44 O 445 Lieu Ala Asn. Phe Ile Lys Ile Gly Glu Gly Ser Thr Gly Ile Val Cys 450 45.5 460 Ile Ala Thr Glu Lys His Thr Gly Lys Glin Val Ala Val Lys Llys Met 465 470 47s 48O Asp Lieu. Arg Lys Glin Glin Arg Arg Glu Lieu. Lieu. Phe Asn. Glu Val Val 485 490 495 Ile Met Arg Asp Tyr His His Asp Asn Val Val Asp Met Tyr Ser Ser SOO 505 51O Tyr Lieu Val Gly Asp Glu Lieu. Trp Val Val Met Glu Phe Lieu. Glu Gly 515 52O 525 Gly Ala Lieu. Thir Asp Ile Val Thr His Thr Arg Met Asn Glu Glu Gln 53 O 535 54 O Ile Ala Thr Val Cys Lieu. Ser Val Lieu. Arg Ala Lieu. Ser Tyr Lieu. His 5.45 550 555 560 Asn Glin Gly Val Ile His Arg Asp Ile Llys Ser Asp Ser Ile Lieu. Lieu 565 st O sts Thir Ser Asp Gly Arg Ile Llys Lieu. Ser Asp Glu Gly Phe Cys Ala Glin 58O 585 59 O Val Ser Lys Glu Val Pro Lys Arg Llys Ser Leu Val Gly Thr Pro Tyr 595 6OO 605 Trp Met Ala Pro Glu Val Ile Ser Arg Lieu Pro Tyr Gly Thr Glu Val 610 615 62O Asp Ile Trp Ser Lieu. Gly Ile Met Val Ile Glu Met Ile Asp Gly Glu 625 630 635 64 O Pro Pro Tyr Phe Asn Glu Pro Pro Leu Glin Ala Met Arg Arg Ile Arg 645 650 655 Asp Ser Lieu Pro Pro Arg Val Lys Asp Lieu. His Llys Val Ser Ser Val 660 665 67 O Lieu. Arg Gly Phe Lieu. Asp Lieu Met Lieu Val Arg Glu Pro Ser Glin Arg 675 68O 685 Ala Thr Ala Glin Glu Lieu. Lieu. Gly. His Pro Phe Lieu Lys Lieu Ala Gly 69 O. 695 7 OO Pro Pro Ser Cys Ile Val Pro Leu Met Arg Glin Tyr Arg His His 7 Os 71O 71s

<210s, SEQ ID NO 14 &211s LENGTH: 591 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Human PAK4, PAK5 and PAK6 amino acid sequences <4 OOs, SEQUENCE: 14 Met Phe Gly Lys Arg Llys Lys Arg Val Glu Ile Ser Ala Pro Ser Asn 1. 5 1O 15 Phe Glu. His Arg Val His Thr Gly Phe Asp Gln His Glu Glin Llys Phe 2O 25 3O Thr Gly Lieu Pro Arg Glin Trp Glin Ser Lieu. Ile Glu Glu Ser Ala Arg 35 4 O 45 Arg Pro Llys Pro Leu Val Asp Pro Ala Cys Ile Thr Ser Ile Glin Pro SO 55 6 O Gly Ala Pro Llys Thir Ile Val Arg Gly Ser Lys Gly Ala Lys Asp Gly US 2010/0247552 A1 Sep. 30, 2010 77

- Continued

Ala Lieu. Thir Lieu Lleu Lieu. Asp Glu Phe Glu Asn Met Ser Val Thr Arg 85 90 95 Ser Asn. Ser Lieu. Arg Arg Asp Ser Pro Pro Pro Pro Ala Arg Ala Arg 1OO 105 11 O Gln Glu Asn Gly Met Pro Glu Glu Pro Ala Thir Thr Ala Arg Gly Gly 115 12 O 125 Pro Gly Lys Ala Gly Ser Arg Gly Arg Phe Ala Gly His Ser Glu Ala 13 O 135 14 O Gly Gly Gly Ser Gly Asp Arg Arg Arg Ala Gly Pro Glu Lys Arg Pro 145 150 155 160 Llys Ser Ser Arg Glu Gly Ser Gly Gly Pro Glin Glu Ser Ser Arg Asp 1.65 17O 17s Lys Arg Pro Leu Ser Gly Pro Asp Val Gly Thr Pro Gln Pro Ala Gly 18O 185 19 O Lieu Ala Ser Gly Ala Lys Lieu Ala Ala Gly Arg Pro Phe Asn. Thir Tyr 195 2OO 2O5 Pro Arg Ala Asp Thr Asp His Pro Ser Arg Gly Ala Glin Gly Glu Pro 21 O 215 22O His Asp Wall Ala Pro Asn Gly Pro Ser Ala Gly Gly Lieu Ala Ile Pro 225 23 O 235 24 O Gln Ser Ser Ser Ser Ser Ser Arg Pro Pro Thr Arg Ala Arg Gly Ala 245 250 255 Pro Ser Pro Gly Val Lieu. Gly Pro His Ala Ser Glu Pro Glin Leu Ala 26 O 265 27 O Pro Pro Ala Cys Thr Pro Ala Ala Pro Ala Val Pro Gly Pro Pro Gly 27s 28O 285 Pro Arg Ser Pro Glin Arg Glu Pro Glin Arg Val Ser His Glu Glin Phe 29 O 295 3 OO Arg Ala Ala Lieu Gln Lieu Val Val Asp Pro Gly Asp Pro Arg Ser Tyr 3. OS 310 315 32O Lieu. Asp Asn. Phe Ile Lys Ile Gly Glu Gly Ser Thr Gly Ile Val Cys 3.25 330 335 Ile Ala Thr Val Arg Ser Ser Gly Lys Lieu Val Ala Val Lys Llys Met 34 O 345 35. O Asp Lieu. Arg Lys Glin Glin Arg Arg Glu Lieu. Lieu. Phe Asn. Glu Val Val 355 360 365 Ile Met Arg Asp Tyr Gln His Glu Asn Val Val Glu Met Tyr Asn Ser 37 O 375 38O Tyr Lieu Val Gly Asp Glu Lieu. Trp Val Val Met Glu Phe Lieu. Glu Gly 385 390 395 4 OO Gly Ala Lieu. Thir Asp Ile Val Thr His Thr Arg Met Asn Glu Glu Gln 4 OS 41O 415 Ile Ala Ala Val Cys Lieu Ala Val Lieu. Glin Ala Lieu. Ser Val Lieu. His 42O 425 43 O Ala Glin Gly Val Ile His Arg Asp Ile Llys Ser Asp Ser Ile Lieu. Lieu 435 44 O 445 Thir His Asp Gly Arg Val Llys Lieu. Ser Asp Phe Gly Phe Cys Ala Glin 450 45.5 460 Val Ser Lys Glu Val Pro Arg Arg Llys Ser Leu Val Gly Thr Pro Tyr 465 470 47s 48O