USOO9023787B2

(12) United States Patent (10) Patent No.: US 9,023,787 B2 Yaffe et al. (45) Date of Patent: *May 5, 2015

(54) MAPKAPKINASE-2AS A SPECIFIC TARGET (2013.01); G0IN 2333/4748 (2013.01); G0IN FOR BLOCKING PROLIFERATION OF 2333/9121 (2013.01); G0IN 2500/00 (2013.01); PS3-DEFECTIVE G0IN 2500/02 (2013.01); G06F 19/16 (2013.01); G06F 19/20 (2013.01); G06F 19/22 (71) Applicant: Massachusetts Institute of Technology, (2013.01); A61 K3I/7034 (2013.01); A61 K Cambridge, MA (US) 33/24 (2013.01) (72) Inventors: Michael B. Yaffe, West Roxbury, MA (58) Field of Classification Search (US); Isaac A. Manke, New York, NY None (US); Hans Christian Reinhardt, See application file for complete search history. Cologne (DE) (73) Assignee: Massachusetts Institute of Technology, (56) References Cited Cambridge, MA (US) U.S. PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this 4,244.946 A 1/1981 Rivier patent is extended or adjusted under 35 4,305,872 A 12/1981 Johnston U.S.C. 154(b) by 0 days. 4,316,891 A 2f1982 Guillemin 5,034,506 A 7, 1991 Summerton This patent is Subject to a terminal dis 5,652,122 A 7, 1997 Frankel claimer. 5,670,617 A 9, 1997 Frankel 5,674,980 A 10, 1997 Frankel (21) Appl. No.: 13/893,141 5,747,641 A 5/1998 Frankel 5,804.604 A 9, 1998 Frankel (22) Filed: May 13, 2013 6,683, 172 B1 1/2004 Kotlyarov 8.440,610 B2 * 5/2013 Yaffe et al...... 514/1.1 Prior Publication Data 2004/0101915 A1 5/2004 Deveraux et al...... 435/723 (65) 2004/O127492 A1 7/2004 Vazquez US 2014/OO37755 A1 Feb. 6, 2014 2004/02097.97 A1 10, 2004 Karas 2005.0003387 A1 1/2005 AZa-Blanc 2005, 0079172 A1 4/2005 NaSOff Related U.S. Application Data 2005/0101623 A1 5/2005 Meyers Continuation of application No. 1 1/789.239, filed on 2005/01 19470 A1 6/2005 Manoharan (63) 2005/O137220 A1 6/2005 Anderson Apr. 24, 2007, now Pat. No. 8,440,610, and a 2005.0143371 A1 6/2005 Meyers continuation-in-part of application No. 1 1/273,567, 2005, 0181385 A1 8/2005 Linsley filed on Nov. 14, 2005, now abandoned. 2005, 01968O8 A1 9, 2005 Yaffe (60) Provisional application No. 60/794,451, filed on Apr. 2005/0239731 A1 10/2005 McSwiggen 24, 2006, provisional application No. 60/800,298, 2005.0245475 A1 11/2005 Khvorova filed on May 12, 2006, provisional application No. 2006/0052951 A1 3, 2006 Yaffe 60/873,904, filed on Dec. 8, 2006, provisional FOREIGN PATENT DOCUMENTS application No. 60/627,352, filed on Nov. 12, 2004. WO 2004055O19 T 2004 (51) Int. C. A6 IK3I/00 (2006.01) OTHER PUBLICATIONS A6 IK38/6 (2006.01) A 6LX3/553 (2006.01) Wang et al., J. of the Natl. Canc. Inst., 1996, vol. 88, pp. 956-965.* A6 IK 45/06 (2006.01) (Continued) C07K 7/06 (2006.01) C07K 7/08 (2006.01) CI2N 9/12 (2006.01) CI2O I/48 (2006.01) Primary Examiner — Karlheinz, R Skowronek GOIN 33/573 (2006.01) Assistant Examiner — Roy Teller GOIN33/574 (2006.01) (74) Attorney, Agent, or Firm — Pabst Patent Group LLP A6 IK3I/7034 (2006.01) A6 IK33/24 (2006.01) GO6F 9/16 (2011.01) (57) ABSTRACT GO6F 9/20 (2011.01) GO6F 9/22 (2011.01) The present invention relates to compounds and pharmaceu (52) U.S. C. tical compositions for treating cellular proliferative disorders, CPC ...... A6 IK3I/553 (2013.01); A61 K3I/00 e.g., in patients having one or more p53-deficient cells, (2013.01); A61 K38/16 (2013.01); A61K 45/06 screening assays for identifying Such compounds, and meth (2013.01); C07K 7/06 (2013.01); C07K 7/08 ods for treating Such disorders. (2013.01); C07K 2.299/00 (2013.01): CI2N 9/1205 (2013.01); C12O I/485 (2013.01); G0IN33/573 (2013.01); G0IN33/57407 14 Claims, 17 Drawing Sheets US 9,023,787 B2 Page 2

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(56) References Cited Zhou and Bartek, “Targeting the Checkpoint Kinases: Chemosensitization versus Chernoprotection.” Nat. Rev. Cancer 4:1- OTHER PUBLICATIONS 10 (2004). Zunino and Capranico, “DNA Topoisomerase II as the Primary Tar Zhao and Piwnica-Worms, "ATR-Mediated Checkpoint Pathways Regulate Phosphorylation and Activation of Human Chkl.” Mol. get of Anti-Tumor Tumor .” Anticancer Des. Cell. Bial. 21:4129-4139 (2001). 5:307-317 (1990). Zhao, et al. “Structural Basis for Chkl Inhibition by UCN-01.” J. Blot International Search Report issued in PCT/USO5/41294 (Jun. 20. Chem. 277:46609-46615 (2002b). 2006). Zhou and Elledge, “The DNA Damage Response: Putting Check points in Perspective.” Nature 408:433-439 (2000). * cited by examiner U.S. Patent May 5, 2015 Sheet 1 of 17 US 9,023,787 B2

p38a MAP Kinase Phosphorylation Motif -3 -2 -1 S P +2 M(1.6) P(2.1) M(1.9) P(1.7) SP library F(1.5) M(1.7) L(1.6) V(1.4) Q(1.5) L(1.5) Q(1.5) (1.3) N(1.4) V(1.5) (1.5) -3 P -1 S P +2 G(2.1) Q(1.9) I(1.8) PxSP library (1.5) M(1.8) W(1.7) V(1.5) G(1.7) Y(1.7) Y(1.4) P(1.4) T(1.6) S(1.3) V(1.6) T(1.3) Comparison of p38 MAP Kinase Substrates Substrate -3 2 -1 SIT P +2 3PK1 (T313) W P Q L 3PK1(T201) A L Q C MAPKAP2(T334) V P Q L MAPKAP2(S272) L A G MAPKAP2(T222) S L T C MAPKAP2(T25) Q P P A GADD153(S78) T S Q R GADD153(S81) S P R D MEF2A(T312) P L A W MEF2A(T319) S V T S ATF2(T51) A D Q T ATF2(T53) Q T P T p47phox(S345) G P Q G p47phox(S348) S P G L U.S. Patent May 5, 2015 Sheet 2 of 17 US 9,023,787 B2

G g s

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A/6. A U.S. Patent May 5, 2015 Sheet 3 of 17 US 9,023,787 B2

MAPKAP Kinase-2 Phosphorylation Motif -5 -4 R -2 - 1 S +1 RxxS library L(2,5) Q(1.3), Q(2.6) L(1.6) (1.8) F(1.9) A(1.2) M(1.3) N(1.3) V(1.7) I(1.6) M(1.2) F(1.4) V(1.3) L(1.3) Comparison of MK2 Kinase Substrates Substrate -5 -4 R -2 -1 S +1 HSP-27(82) L S Q L S 5-LO(271) L E Q L L LSP1 (204) D T E L LSP1(252) L A Q A SRF(103) L K T L E GS(7) L N S L V TH(19) F R A V E CDC25B(309) L F S P M CDC25C(216) L Y S P M

A/6. 24 U.S. Patent May 5, 2015 Sheet 4 of 17 US 9,023,787 B2

- 2 OO S. O S 1 OOO s O

SOY 8 OO E 9- 6 Oo C &O 4 OO g 2OO CS H C O O 20O 4OO 6OO 8OO 10OO 12 OO MK2 tidel M A/6. 2A Optimal MK2tide -5 -4 -3 -2 -1 O +1 Motif POSition 1 2 3 4 5 6 7 8 9 10 11 12 Ala His Leu Gin Arg Gin Leu Ser Ile Ala His His Peptide Km M Vmax(pmol/minlug) Vox/Km MK2tide 31 1098 35 L3Atide 29 463 16 Q4Atide 41 1209 29 R5Atide 310 282 <1 Q6Atide 51 1058 21 L7Atide 35 955 27 19Atide 43 627 15 A/6. 26 U.S. Patent May 5, 2015 Sheet 5 Of 17 US 9,023,787 B2

- UV - UV as O E O C S s 2N 4N 2N 4N O A/6. 54 A/6. 36 s

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U.S. Patent May 5, 2015 Sheet 7 Of 17 US 9,023,787 B2

f = Oh t = 12h t = 2h -- UV

with O S E 8 C 9 Z 2N 4N 2N 4N 2N 4N .

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BrdU Labeling Time (Hours) A/6, 4A Brd U (+) gate GFP SiRNA MK2 siRNA

t = Oh (no UV)

y 2N 4N 2N 4 N l t = 20h (no UV)

2N 4N 2N 4N

t = 20h (+ UV)

A/6. 46 U.S. Patent May 5, 2015 Sheet 9 Of 17 US 9,023,787 B2

O GFP siRNA OO 22 MK2 siRNA 8 O

6 O

4. O 2 O | ty O J/m2 5 J/m2 20 J/m25O J/m2 A/6. 4A U.S. Patent May 5, 2015 Sheet 10 of 17 US 9,023,787 B2

sto st mOfif 9. (e. Y. 5.Q p/ 334 N 1 LxRxxS/T () motif

1,

lar MGS7BLKR CDC25A U.S. Patent May 5, 2015 Sheet 11 of 17 US 9,023,787 B2

A/6. 6 COntrol A-T Seckel Fibroblosts Fibroblasts Fibroblosts

Doxorubicin + -

A/G. 76 A/6. 74 cisplot in e 2.5 Confro 2.O initid4 4 dose S|2 oinitial dose 1.5 al re 1.5 E 1.O 1.O O 5 O.5 O5 E 2 O.O O.O 5 O 15 20 25 5 O 15 20 25 -e time days doxorubic in

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P O.O 51o 15 20 25 -o-e time days U.S. Patent May 5, 2015 US 9,023,787 B2

18OO

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Intra-strand crosslinks Topoisomerase inhibition co?t:PA gDDg&DDD t ATR ATR (ATM) UCN-O1 c. . c. . UCN-O1

ct CCCSi 25B

G1/S G2/M checkpoints checkpoint

A/6. 9 U.S. Patent May 5, 2015 Sheet 14 of 17 US 9,023,787 B2

T|Da?auns% OGOCO

Z p53"/ WT E p53 WT/WTMK-2 shRNA p537, luciferase shRNA Op537 , MK 2 ShrNA

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U.S. Patent May 5, 2015 Sheet 16 of 17 US 9,023,787 B2

ZW,

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Normo Cells DD&DD of Ro v / ATM/ATR

M v A/6. W54 p53 Chk 1 y v p21 Cdc25A/B y - v G1/S arrest G2/M arrest Concer Cells

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CdC25A/Bin G1/S arrest G2/M arrest US 9,023,787 B2 1. 2 MAPKAPKNASE-2AS A SPECIFIC TARGET pathway, leading to selective loss of the G checkpoint. These FOR BLOCKING PROLIFERATION OF cells are then entirely dependent on intra-S and G/M check PS3-DEFECTIVE points to maintain their genomic integrity in response to DNA damage. CROSS-REFERENCE TO RELATED In contrast to the DNA damage-specific activation of Chk1 APPLICATIONS and Chk2, the p38 MAPK pathway is a general stress-acti vated kinase pathway that responds to various cellular This application is a continuation-in-part of U.S. applica stimuli, including cytokines, hyperosmolarity, and UV irra tion Ser. No. 1 1/273,567, filed Nov. 14, 2005, which claims diation. Activity of p38 MAPK is important for G/M check benefit of U.S. Provisional Application No. 60/627,352, filed 10 point function in immortalized fibroblasts and HeLa cells Nov. 12, 2004, each of which is hereby incorporated by ref following UV exposure. Furthermore, MAPKAP Kinase-2 CCC. (MK2) is the critical downstream effector kinase of This application also claims benefit of U.S. Provisional p38 MAPK required for UV-induced cell cycle checkpoints in Application Nos. 60/794,451, filed Apr. 24, 2006; 60/800, U2OS cells. 298, filed May 12, 2006; and 60/873,904, filed Dec. 8, 2006, 15 Whether the observed activation of p38 MAPK/MK2 is a each of which is hereby incorporated by reference. direct result of UV-induced DNA lesions, or results instead from other non-genotoxic effects of UV radiation has been STATEMENT AS TO FEDERALLY SPONSORED unclear. Similarly, whether the p38 MAPK/MK2 pathway is RESEARCH an important part of a general cellular response to genotoxic stress has been unclear. There exists a need to better under The present research was Supported by grants from the stand this checkpoint and to develop methods and therapies National Institutes of Health (grant numbers GM60594 and for disease treatment based on this improved understanding. CA112967) and from the National Institute of Environmental Health Sciences (grant number ESO15339). The U.S. govern SUMMARY OF THE INVENTION ment has certain rights to this invention. 25 We now report that MAPKAP kinase-2 is specifically acti REFERENCE TO SEQUENCE LISTING vated in response to DNA damage caused by chemotherapeu tic agents in an ATR and/or ATM-dependent manner, and that The Sequence Listing Submitted Oct. 17, 2013 as a text MAPKAP kinase-2 is critical for the activation of G, S-phase filed named “MIT 12175 CIPCON ST25.txt, created on 30 and G/M checkpoints after exposure to these drugs. Down Jul. 16, 2013, and having a size of 23,787 bytes is hereby regulation of MAPKAP kinase-2 using RNA interference incorporated by reference pursuant to 37 C.F.R. S.1.52(e)(5). profoundly increases the anti-proliferative and cytotoxic effects of cisplatin and doxorubucin on tumor cells in vitro, BACKGROUND OF THE INVENTION and in a murine tumor model in vivo. MAPKAP kinase-2 35 depletion is especially effective in increasing the chemosen The maintenance of genomic integrity is essential for the sitivity of p53-deficient cells, Suggesting that compounds that health of multi-cellular organisms. DNA damage checkpoints target MAPKAP kinase-2 can be used as specific therapeutics constitute a mechanism where cell division is delayed to that can sensitize p53-deficient tumor cells without sensitiz allow repair of damaged DNA, or if the extent of DNA dam ing normal cells. At the systems level, in response to DNA age is beyond repair, induce apoptosis. The three major DNA 40 damage, Chk1 and MAPKAP kinase-2 appear to function in damage-responsive cell cycle checkpoints are the G1/S parallel independent pathways that converge to phosphory checkpoint, intra S-phase checkpoint, and the G/M check late similar molecular targets, such that checkpoint abroga point. tion following MAPKAP kinase-2 depletion can be rescued In response to DNA damage, eukaryotic cells activate a by overexpression of Chk1. complex signaling network to arrest the cell cycle and facili 45 Based on these results, we have invented novel methods of tate DNA repair. This signaling network has traditionally treating cellular proliferative disorders by inhibiting MAP been divided into two major protein kinase pathways, one KAP kinase-2 expression. We have also discovered MAP mediated by Ataxia-Telangiectasia mutated (ATM) through KAP kinase-2 inhibitors, pharmaceutical compounds con Chk2, and the other mediated by Ataxia-Telangiectasia and taining Such inhibitors that are useful for treating cellular Rad-3 related (ATR) through Chk1. Some cross-talk exists 50 proliferative disorders, and screening methods for identifying between the ATM/Chk2 and ATR/Chk1 kinase pathways, additional inhibitors. The methods and compounds of the particularly when signaling through one pathway is partially invention may be used, for example, to treat cancer or to aid or totally deficient. Normally, however the pathways show in the development of other anti-cancer therapies. only partial functional overlap in response to particular forms Accordingly, in one aspect, the invention features a method of DNA damage. The ATM/Chk2 pathway responds prima 55 for treating a cellular proliferative disorder in a patient that rily to DNA double strand breaks (DSBs), while the ATR/ includes the steps of: (a) determining whether the patient has Chk1 pathway is activated by bulky DNA lesions, and fol a p53-deficient cell; and (b) if the patient has a p53-deficient lowing replication fork collapse during S-phase. The tumor cell, administering to the patient a compound, e.g., UCN-01, Suppressor protein p53 is a major downstream effector of that is capable of inhibiting an activity of a MAPKAP these DNA damage kinase pathways. In normal cells, p53 60 kinase-2 polypeptide. Any method can be used to determine dependent signaling results in Garrest, mainly mediated by whether the patient has a p53-deficient cell, e.g., an antibody transcriptional upregulation of p21. In addition, p21 also assay of a cell sample, e.g., from a tumor biopsy. The MAP appears to play a role in Sustaining the G checkpoint after KAP kinase-2 inhibition can be either specific or non-spe Y-irradiation. If the DNA damage is extensive, however, then cific. The activity being inhibited may include, for example, p53-dependent pathways target the damaged cell for apop 65 MAPKAP kinase-2 polypeptide expression or substrate totic cell death through both the intrinsic and extrinsic path binding. The method may also include the step of adminis ways. Most tumor cells show specific disruptions in the p53 tering an additional treatment to the patient, Such as a chemo US 9,023,787 B2 3 4 therapeutic agent or radiation therapy, such that the KAP kinase-2 inhibitor. The chemotherapeutics can be compound and the chemotherapeutic agent or the radiation administered Subsequently or concomitantly to administra therapy are administered in amounts Sufficient to treat the tion of a MAPKAP kinase-2 inhibitor. An exemplary chemo patient’s cellular proliferative disorder. The additional treat therapeutic used in this regimen is UCN-01. Increased ment may be administered simultaneously or nonsimulta chemosensitivity of p53-deficient cells to a MAPKAP neously, e.g., up to twenty-eight days apart, in relation to the kinase-2 treatment may alter a DNA damage-responsive cell administration of the inhibitory compound. Any chemothera cycle checkpoint, in comparison to control p-53 deficient peutic agent or radiation therapy known in the art may be cells in a control patient not receiving the described therapy. useful in the methods of the invention. Exemplary chemo Alteration of a DNA damage-responsive cell cycle check therapeutic agents are antimicrotubule drugs, e.g., nocoda 10 point may occur at G/S phase arrest, where Cdc25a degra Zole; compounds that create double-strand DNA breakage, dation impaired. Alteration of a DNA damage-responsive cell e.g., and ; compounds that induce cycle checkpoint may occurat G2/M phase arrest, where, the single-strand DNA breaks, e.g., camptothecin; and cross interaction between Cdc25b and a 14-3-3 protein is reduced. linking agents, e.g., cisplatin. Exemplary cellular prolifera The above-mentioned alterations of cell cycle checkpoints tive disorders include neoplasms, e.g., any known form of 15 may increase the likelihood of cell death, including cell death cancer. In one embodiment, a solid tumor may be treated by by apoptosis. injecting a MAPKAP kinase-2 inhibitor, alone or in combi The invention further features kits that include: (a) a means nation with an additional therapeutic agent, directly into the of detecting the level of p53 polypeptide expression or activ tumor or by Systemic administration. If given as a mono ity in a cellular sample, e.g., by using an anti-p53 antibody; therapy, the compound is administered in an amount Suffi and (b) a compound that is capable of inhibiting an activity of cient to treat the patient’s cellular proliferative disorder; alter a MAPKAP kinase-2 polypeptide, e.g., UCN-01. In some natively, in the case of combination therapy, the combination instances, the kit can also contain one or more chemothera of compounds is collectively administered in an amount Suf peutic agents. ficient to treat the patient’s cellular proliferative disorder. The invention additionally features a method for treating a An inhibitory compound used in the foregoing method 25 cellular proliferative disorder in a patient including adminis may include a covalently-linked moiety capable of translo tering to the patient a compound that is capable of inhibiting cating across a biological membrane, such as a penetratin or an activity of a MAPKAP kinase-2 polypeptide. In some TAT peptide. Alternatively, such a compound may be admin instances, the cellular proliferative disorder includes the pres istered in the form of a prodrug. Suitable compounds include ence of one or more p53-deficient cells, e.g., tumor cells, in small molecule inhibitors of MAPKAP kinase-2 biological 30 the patient. Administration of a MAPKAP kinase-2 polypep activity, RNA molecules useful in RNA interference therapy, tide together with a chemotherapeutic compound has the RNA molecules useful in antisense therapy, and peptides desired effect of reducing tumor size. Administration of the capable of inhibiting a MAPKAP kinase-2 polypeptide. For described therapy can be, e.g., by direct injection into the example, an RNA molecule useful in the methods of the tumor. invention includes a double-stranded Small interfering 35 The invention further features a method for identifying a nucleic acid (siNA) molecule that is capable of directing compound that may be an inhibitor of Substrate binding to a cleavage of a MAPKAP kinase-2 RNA via RNA interference, MAPKAP kinase-2 polypeptide, the method including the wherein each strand of the siNA molecule is about 18 to 23 steps of contacting the MAPKAP kinase-2 polypeptide and a nucleotides in length, and one strand of the siNA molecule compound capable of binding the MAPKAP kinase-2 includes a nucleotide sequence that is substantially identical 40 polypeptide under conditions allowing the formation of a to the sequence of the MAPKAP kinase-2 RNA. In one complex between the compound and the MAPKAP kinase-2 embodiment, the siNA molecule includes RNA, the sequence polypeptide; contacting the complex with a candidate com of such RNA including, for example, any one of SEQIDNOs: pound; and measuring the displacement of the compound 29-32. Small hairpin nucleic acid (shNA) molecules may also capable of binding the MAPKAP kinase-2 polypeptide from be used in the methods of the invention. Alternatively, anti 45 the MAPKAP kinase-2 polypeptide. The displacement of the sense therapy may be performed by administering a nucleo compound capable of binding identifies the candidate com base oligomer, wherein the sequence of the oligomer is pound as a compound that may be an inhibitor of Substrate complementary to at least 10 consecutive residues of a nucle binding to a MAPKAP kinase-2 polypeptide. In one embodi otide sequence encoding a MAPKAP kinase-2 polypeptide. ment, the compound capable of binding the MAPKAP Therapy may also be performed by utilizing a compound that 50 kinase-2 polypeptide includes a peptide or peptidomimetic, includes a peptide or peptidomimetic, e.g., containing the e.g., containing the amino acid sequence L/F/IXRO/S/T amino acid sequence L/F/IXRIO/S/TLSLIS/THydro LIS/THydrophobic (SEQID NO: 17), wherein the peptide phobic (SEQID NO: 17), wherein X represents any amino or peptidomimetic includes no more than 50 amino acids. For acid and the peptide or peptidomimetic includes no more than example, the peptide or peptidomimetic may include the 50 amino acids. Hydrophobic amino acids are selected from 55 amino acid sequence LQRQLSI (SEQ ID NO: 16). In the the group consisting of alanine, cysteine, isoleucine, leucine, foregoing method, a Substrate-binding fragment of a MAP methionine, phenylalanine, proline, tryptophan, tyrosine, and KAP kinase-2 polypeptide may be utilized in place of a full Valine. In one embodiment, the peptide or peptidomimetic length MAPKAP kinase-2 polypeptide. includes the amino acid sequence LQRQLSI (SEQ ID NO: Variations of the foregoing aspect are also possible in the 16). 60 methods of the invention. The MAPKAP kinase-2 polypep In one aspect of the invention, administering a compound tide, or Substrate-binding fragment thereof, and compound that is capable of inhibiting an activity of a MAPKAP capable of binding the polypeptide may be contacted in the kinase-2 polypeptide for treating a cellular proliferative dis presence of a candidate compound, and any means of mea order in a patient sensitizes p53-deficient cells to chemothera suring the binding of the MAPKAP kinase-2 polypeptide and peutic challenge. This can result in increased likelihood of 65 the compound capable of binding may be used in the methods death of aberrantly proliferating cells in a patient, in compari of the invention. In general, if the amount of binding of the son to p53-deficient cells in a patient not receiving a MAP MAPKAP kinase-2 polypeptide and the compound capable US 9,023,787 B2 5 6 of binding is decreased in the presence of the candidate com retains the biological activity of a corresponding naturally pound in comparison to the amount of binding measured in occurring polypeptide, while having certain biochemical the absence of the candidate compound, then the candidate modifications that enhance the analog's function relative to a compound is determined to be an inhibitor of substrate bind naturally occurring polypeptide. Such biochemical modifica ing using the methods of the invention. tions could increase the analog's protease resistance, mem In another aspect, the invention features a method for iden brane permeability, or half-life, without altering, for example, tifying a compound that may be an inhibitor of Substrate ligand binding. An analog may include an unnatural amino binding to a MAPKAP kinase-2 polypeptide or substrate acid. binding fragment thereof, the method including the steps of By “antisense,” as used herein in reference to nucleic acids, providing a three-dimensional model of the MAPKAP 10 kinase-2 polypeptide having at least one atomic coordinate, is meant a nucleic acid sequence, regardless of length, that is or surrogate thereof, from Table 1 for at least three of the complementary to the coding strand of a gene. residues Ile74, Glu145, Lys188, Glu190, Phe210, Cys224, By "atomic coordinates’ is meant those three-dimensional Tyr225, Thr226, Pro227, Tyr228, Tyr229, and Asp345, or coordinates of the atoms in a crystalline material derived from atomic coordinates that have a root mean Square deviation of 15 mathematical equations related to the patterns obtained on the coordinates of less than 3 A; and producing a structure for diffraction of X-rays by the atoms (X-ray scattering centers) of a candidate compound, the structure defining a molecule the crystalline material. The diffraction data are used to cal having sufficient surface complementary to the MAPKAP culate an electron density map of the unit cell of the crystal. kinase-2 polypeptide to bind the MAPKAP kinase-2 These electron density maps are used to establish the posi polypeptide in an aqueous solution. tions of the individual atoms within the unit cell of the crystal. The invention further features a compound that includes a Atomic coordinates can be transformed, as is known to those peptide or peptidomimetic, e.g., containing the amino acid skilled in the art, to different coordinate systems (i.e., Surro sequence L/F/IXRIQ/S/TLS/THydrophobic (SEQ ID gate systems) without affecting the relative positions of the NO: 17), wherein the peptide or peptidomimetic includes no atOmS. more than 50 amino acids. In one embodiment, the peptide or 25 By “binding to a molecule is meant having a physico peptidomimetic includes the amino acid sequence LQRQLSI chemical affinity for that molecule. Binding may be measured (SEQ ID NO: 16). An inhibitory compound of the invention by any of the methods of the invention, e.g., using an in vitro may include a covalently-linked moiety capable of translo translation binding assay. cating across a biological membrane, such as a penetratin or By “biological activity” of a polypeptide or other com TAT peptide. Alternatively, such a compound may be admin 30 pound is meant having structural, regulatory, or biochemical istered in the form of a prodrug. functions of a naturally occurring molecule. For example, one In another aspect, the invention features a pharmaceutical biological activity of a MAPKAP kinase-2 polypeptide is composition for treating a cellular proliferative disorder in a Substrate binding, e.g., peptide binding, which may be mea patient, the composition including: a compound that is Sured using in Vivo or in vitro binding assays. capable of inhibiting an activity of a MAPKAP kinase-2 35 By "caged compound is meant a biologically active mol polypeptide; and a chemotherapeutic agent, wherein the com ecule coupled to a cleavable moiety Such that the resulting position is formulated in an amount Sufficient to treat the coupled compound lacks biological activity as long as the cellular proliferative disorder. Any chemotherapeutic agent moiety remains attached. Such a moiety prevents bioaction known in the art may be useful in the compositions of the by Sterically shielding one or more chemical groups of the invention. An inhibitory compound useful in the pharmaceu 40 molecule. The moiety may be removed by any means, includ tical composition may include a covalently-linked moiety ing enzymatic, chemical, or photolytic; removal of the moiety capable of translocating across a biological membrane. Such results in restoration of the molecule's biological activity. as a penetratin or TAT peptide. Alternatively, such a com By "candidate compound is meant any nucleic acid mol pound may be administered in the form of a prodrug. Any ecule, polypeptide, or other Small molecule that is assayed for compounds described in any of the foregoing aspects, includ 45 its ability to alter gene or protein expression levels, or the ing Small molecule inhibitors, compounds containing siNA biological activity of a gene or protein by employing one of molecules, antisense RNA molecules, or peptides, may be the assay methods described herein. Candidate compounds useful in the pharmaceutical compositions of the invention. include, for example, peptides, polypeptides, synthesized In any of the foregoing aspects of the invention, it is desir organic molecules, naturally occurring organic molecules, able that the inhibitory compounds be specific inhibitors of 50 nucleic acid molecules, and components thereof. MAPKAP kinase-2, e.g., compounds that inhibit a MAPKAP By “cellular proliferative disorder is meant any pathologi kinase-2 polypeptide without also substantially inhibiting cal condition in which there is an abnormal increase or related kinases such as Chk1, Chk2, and p38 SAPK, although decrease in cell proliferation. Exemplary cellular prolifera compounds that inhibit a MAPKAP kinase-2 polypeptide in a tive disorders include cancer or neoplasms, inflammatory less selective or non-selective manner are also useful in the 55 diseases, or hyperplasias (e.g., some forms of hypertension, methods of the invention. prostatic hyperplasia). As used throughout this specification and the appended By "chemotherapeutic agent' is meant one or more chemi claims, the following terms have the meanings specified. cal agents used in the treatment or control of proliferative By an 'amino acid fragment' is meant an amino acid diseases, including cancer. Chemotherapeutic agents include residue that has been incorporated into a peptide chain via its 60 cytotoxic and cytostatic agents. alpha carboxyl, its alpha nitrogen, or both. A terminal amino By “complex' is meant a chemical association of two or acid is any natural or unnatural amino acid residue at the more molecules. Complexes may include a network of weak amino-terminus or the carboxy-terminus. An internal amino electrostatic bonds that maintain the association of the mol acid is any natural or unnatural amino acid residue that is not ecules. Other types of interactions, such as covalent, ionic, a terminal amino acid. 65 hydrogen bond, hydrophobic, or van der Waals interactions, By “analog is meant a molecule that is not identical but may be present instead of or in addition to electrostatic bonds has analogous features. For example, a polypeptide analog between members of a complex. US 9,023,787 B2 7 8 By "computer modeling' is meant the application of a the nucleic acid sequence of Genbank Accession Nos. computational program to determine one or more of the fol NM 004759 (SEQID NO: 1) or NM 032960 (SEQID NO: lowing: the location and binding proximity of a ligand to a 2), or analog thereof. binding moiety, the occupied space of a bound ligand, the By “MAPKAP kinase-2 polypeptide' and “MK2 are used amount of complementary contact surface between a binding interchangeably herein, and denote a polypeptide Substan moiety and a ligand, the deformation energy of binding of a tially identical to all or a portion of the polypeptide sequence given ligand to a binding moiety, and some estimate of hydro of Genbank Accession Nos. NP 004750 (SEQID NO:3) or genbonding strength, Vander Waals interaction, hydrophobic P49137 (SEQID NO: 4), or analog thereof, and having MAP interaction, and/or electrostatic interaction energies between KAP kinase-2 biological activity. 10 By "neoplasia’’ or "neoplasm' is meant a disease charac ligand and binding moiety. Computer modeling can also pro terized by the pathological proliferation of a cell or tissue and vide comparisons between the features of a model system and its Subsequent migration to or invasion of other tissues or a candidate compound. For example, a computer modeling organs. Neoplasia growth is typically uncontrolled and pro experiment can compare a pharmacophore model of the gressive, and occurs under conditions that would not elicit, or invention with a candidate compound to assess the fit of the 15 would cause cessation of multiplication of normal cells. candidate compound with the model. Examples oftechniques Neoplasias can affect a variety of cell types, tissues, or useful in the above evaluations include: quantum mechanics, organs, including but not limited to an organ selected from the molecular mechanics, molecular dynamics, Monte Carlo group consisting of bladder, bone, brain, breast, cartilage, sampling, systematic searches and distance geometry meth glia, esophagus, fallopian tube, gallbladder, heart, intestines, ods. Further descriptions of computer modeling programs are kidney, liver, lung, lymph node, nervous tissue, ovaries, pan provided elsewhere herein. creas, prostate, skeletal muscle, skin, spinal cord, spleen, By “detectably-labeled' is meant any means for marking stomach, testes, thymus, thyroid, trachea, urogenital tract, and identifying the presence of a molecule, e.g., a peptide or ureter, urethra, uterus, and vagina, or a tissue or cell type a peptidomimetic small molecule that interacts with a MAP thereof. Neoplasias include cancers, such as acoustic neu KAP kinase-2 domain. Methods for detectably-labeling a 25 roma, acute leukemia, acute lymphocytic leukemia, acute molecule are well known in the art and include, without monocytic leukemia, acute myeloblastic leukemia, acute limitation, radionuclides (e.g., with an isotope such as P. myelocytic leukemia, acute myelomonocytic leukemia, acute P, 'I, or S), nonradioactive labeling (e.g., chemilumi promyelocytic leukemia, acute erythroleukemia, adenocarci nescent labeling or fluorescein labeling), and epitope tags. noma, angiosarcoma, astrocytoma, basal cell carcinoma, bile If required, molecules can be differentially labeled using 30 duct carcinoma, bladder carcinoma, brain cancer, breast can markers that can distinguish the presence of multiply distinct cer, bronchogenic carcinoma, cervical cancer, chondrosar molecules. For example, a peptide that interacts with a MAP coma, chordoma, choriocarcinoma, chronic leukemia, KAP kinase-2 domain can be labeled with fluorescein and a chronic lymphocytic leukemia, chronic myelocytic leukemia, MAPKAP kinase-2 domain can be labeled with Texas Red. colon cancer, colon carcinoma, craniopharyngioma, cystad The presence of the peptide can be monitored simultaneously 35 enocarcinoma, embryonal carcinoma, endotheliosarcoma, with the presence of the MAPKAP kinase-2 domain. ependymoma, epithelial carcinoma, Ewing's tumor, glioma, By "fragment' is meant a portion of a polypeptide or heavy chain disease, hemangioblastoma, hepatoma, nucleic acid having a region that is Substantially identical to a Hodgkin’s disease, large cell carcinoma, leiomyosarcoma, portion of a reference protein or nucleic acid and retains at liposarcoma, lung cancer, lung carcinoma, lymphangioen least 50%, 75%, 80%, 90%, 95%, or even 99% of at least one 40 dotheliosarcoma, lymphangiosarcoma, macroglobulinemia, biological activity of the reference protein or nucleic acid. medullary carcinoma, medulloblastoma, melanoma, menin By “hydrophobic” in the context of amino acids is meant gioma, mesothelioma, myxosarcoma, neuroblastoma, non any of the following amino acids: alanine, cysteine, isoleu Hodgkin’s disease, oligodendroglioma, osteogenic sarcoma, cine, leucine, methionine, phenylalanine, proline, tryp ovarian cancer, pancreatic cancer, papillary adenocarcino tophan, tyrosine, or valine. 45 mas, papillary carcinoma, pinealoma, polycythemia Vera, By “inhibit an activity of a MAPKAP kinase-2 polypep prostate cancer, rhabdomyosarcoma, renal cell carcinoma, tide' is meant to reduce one or more biological activities of retinoblastoma, Schwannoma, sebaceous gland carcinoma, MAPKAP kinase-2 polypeptide. Desirably, the inhibition is a seminoma, Small cell lung carcinoma, squamous cell carci decrease of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, noma, Sweat gland carcinoma, synovioma, testicular cancer, 80%, 90% or 95% in biological activity, relative to a control 50 uterine cancer, Waldenstrom's fibrosarcoma, and Wilm's activity, for example the expression or Substrate-binding tumor. capability of a naturally occurring MAPKAP kinase-2 By “nucleic acid' is meant an oligomer or polymer of polypeptide. An example of a compound that inhibits a MAP ribonucleic acid or deoxyribonucleic acid, or analog thereof. KAP kinase-2 polypeptide is UCN-01. This term includes oligomers consisting of naturally occur By “MAPKAP kinase-2 biological activity” is meant any 55 ring bases, Sugars, and interSugar (backbone) linkages as well activity known to be caused in vivo or in vitro by a MAPKAP as oligomers having non-naturally occurring portions which kinase-2 polypeptide. For example, such activity could be function similarly. Such modified or substituted oligonucle caused by at least one of the following: function in a DNA otides are often preferred over native forms because of prop damage response pathway, cell cycle control, transcriptional erties such as, for example, enhanced cellular uptake and regulation, chromatin remodeling, or Substrate binding. In 60 increased stability in the presence of nucleases. one assay for MAPKAP kinase-2 biological activity, the abil Specific examples of Some preferred nucleic acids may ity of MAPKAP kinase-2, or a fragment or mutant thereof contain phosphorothioates, phosphotriesters, methyl phos comprising a substrate-binding domain, to binda Substrate is phonates, short chain alkyl or cycloalkyl interSugar linkages measured. or short chain heteroatomic or heterocyclic interSugar link By “MAPKAP kinase-2 nucleic acid is meant a nucleic 65 ages. Most preferred are those with CH NH-O CH, acid that encodes all or a portion of a MAPKAP kinase-2 CH N(CH)—O CH, CH, O N(CH)—CH, polypeptide or is Substantially identical to all or a portion of CH N(CH)—N(CH)—CH and O N(CH)—CH2— US 9,023,787 B2 10 CH, backbones (where phosphodiester is O—P O—CH). threonine, tyrosine, aspartic acid, histidine amino acid resi Also preferred are oligonucleotides having morpholino back dues, oramino acid analogs. A peptide can be phosphorylated bone structures (Summerton, J. E. and Weller, D.D., U.S. Pat. to the extent of the number of serine, threonine, tyrosine, or No. 5,034.506). In other preferred embodiments, such as the histidine amino acid residues that is present. A phosphopep protein-nucleic acid (PNA) backbone, the phosphodiester tide may be phosphorylated at four independent Ser/Thr/Tyr backbone of the oligonucleotide may be replaced with a residues, at three independent Ser/Thr/Tyr residues, or at two polyamide backbone, the bases being bound directly or indi independent Ser/Thr/Tyr residues. Desirably, a phosphopep rectly to the aza nitrogenatoms of the polyamide backbone (P. tide is phosphorylated at one Ser/Thr/Tyr residue regardless E. Nielsen et al. Science 199: 254, 1997). Other preferred of the presence of multiple Ser. Thr, or Tyr residues. oligonucleotides may contain alkyl and halogen-substituted 10 Typically, a phosphopeptide is produced by expression in a Sugar moieties comprising one of the following at the 2' prokaryotic or eukaryotic cellunder appropriate conditions or position: OH, SH, SCH. F. OCN, O(CH), NH, or in translation extracts where the peptide is Subsequently iso O(CH), CH, where n is from 1 to about 10; C to Co lower lated, and phosphorylated using an appropriate kinase. Alter alkyl, substituted lower alkyl, alkaryl or aralkyl: Cl; Br; CN: natively, a phosphopeptide may be synthesized by standard CF; OCF. O—, S , or N-alkyl: O , S: , or N-alkenyl: 15 chemical methods, for example, using N-C-FMOC-protected SOCH; SOCH: ONO; NO; N. NH; heterocycloalkyl: amino acids (including appropriate phosphoamino acids). In heterocycloalkaryl; aminoalkylamino: polyalkylamino; Sub a desired embodiment, the use of non-hydrolysable phos stituted silyl; an RNA cleaving group; a conjugate; a reporter phate analogs can be incorporated to produce non-hydrolys group; an intercalator, a group for improving the pharmaco able phosphopeptides (Jenkins et al., J. Am. Chem. Soc., kinetic properties of an oligonucleotide; or a group for 124:6584-6593, 2002; herein incorporated by reference). improving the pharmacodynamic properties of an oligonucle Such methods of protein synthesis are commonly used and otide and other Substituents having similar properties. Oligo practiced by standard methods in molecular biology and pro nucleotides may also have Sugar mimetics such as cyclobu tein biochemistry (Ausubel et al., Current Protocols in tyls in place of the pentofuranosyl group. Molecular Biology, John Wiley & Sons, New York, N.Y., Other preferred embodiments may include at least one 25 1994, J. Sambrook and D. Russel, Molecular Cloning: A modified base form. Some specific examples of such modi Laboratory Manual, 3" Edition, Cold Spring Harbor Labo fied bases include 2-(amino)adenine, 2-(methylamino)ad ratory Press, Woodbury N.Y., 2000). In one embodiment, a enine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)ad phosphopeptide is generally not longer than 100 amino acid enine, or other heterosubstituted alkyladenines. residues in length. Shorter phosphopeptides, e.g., less than By “p53-deficient cell' is meant a cell expressing substan 30 50, 25, 20, or 15 residues, are also possible. Phosphopeptides tially less p53 polypeptide, or exhibiting substantially less may be as short as 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid to p53 polypeptide activity, e.g., 10%, 20%, 30%, 40%, 50%, residues long. 60%, 70%, 80%, 90%, 95%, 98%, 99%, or even 100% less By “protein’ or “polypeptide' or “peptide' is meant any p53 expression or activity, than a corresponding wild-type chain of more than two natural or unnatural amino acids, cell. For example, p53-deficient cells, e.g., tumor cells, are 35 regardless of post-translational modification (e.g., glycosyla present in Some neoplastic disorders. p53-deficient cells tion or phosphorylation), constituting all or part of a natu include cells with one or more p53 gene mutations, e.g., point rally-occurring or non-naturally occurring polypeptide or mutations or null mutations, that reduce or eliminate expres peptide, as is described herein. sion or activity. As used herein, a natural amino acid is a natural O-amino By a "peptidomimetic' is meant a compound that is 40 acid having the L-configuration, such as those normally capable of mimicking or antagonizing the biological actions occurring in natural proteins. Unnatural amino acid refers to of a natural parent peptide. A peptidomimetic may include an amino acid, which normally does not occur in proteins, non-peptidic structural elements, unnatural peptides, synthe e.g., an epimer of a natural C-amino acid having the L con sized organic molecules, naturally occurring organic mol figuration, that is to say an amino acid having the unnatural ecules, nucleic acid molecules, and components thereof. 45 D-configuration; or a (D.L)-isomeric mixture thereof, or a Identification of a peptidomimetic can be accomplished by homologue of Such an amino acid, for example, a B-amino screening methods incorporating a binding pair and identify acid, an O.C.-disubstituted amino acid, or an O-amino acid ing compounds that displace the binding pair. Alternatively, a wherein the amino acid side chain has been shortened by one peptidomimetic can be designed in silico, by molecular mod or two methylene groups or lengthened to up to 10 carbon eling of a known protein-protein interaction, for example, the 50 atoms, such as an O-amino alkanoic acid with 5 up to and interaction of a peptide of the invention and a MAPKAP including 10 carbonatoms in a linear chain, an unsubstituted kinase-2 domain. In one embodiment, the peptidomimetic or substituted aromatic (C-aryl or C-aryl lower alkyl), for will displace one member of a binding pair by occupying the example, a Substituted phenylalanine orphenylglycine. Other same binding interface. It is desirable that the peptidomimetic amino acids that may also be incorporated into a polypeptide have a higher binding affinity to the binding interface. 55 include ornithine (O or Orn) and hydroxyproline (Hyp). By “pharmaceutically acceptable excipient' is meant a Polypeptides or derivatives thereof may be fused or carrier that is physiologically acceptable to the Subject to attached to another protein or peptide, for example, as a which it is administered and that preserves the therapeutic Glutathione-S-Transferase (GST) fusion polypeptide. Other properties of the compound with which it is administered. commonly employed fusion polypeptides include, but are not One exemplary pharmaceutically acceptable excipient is 60 limited to, maltose-binding protein, Staphylococcus aureus physiological saline. Other physiologically acceptable protein A, Flag-Tag, HA-tag, green fluorescent proteins (e.g., excipients and their formulations are known to one skilled in eGFP, eYFP, eCFP, GFP, YFP, CFP), red fluorescent protein, the art and described, for example, in “Remington: The Sci polyhistidine (6xHis), and cellulose-binding protein. ence and Practice of Pharmacy.” (20th ed., ed. A. R. Gennaro, By "prodrug is meanta compound that is modified in vivo, 2000, Lippincott Williams & Wilkins). 65 resulting in formation of a biologically active drug com By “phosphopeptide' is meant a peptide in which one or pound, for example by hydrolysis in blood. A thorough dis more phosphate moieties are covalently linked to serine, cussion of prodrug modifications is provided in T. Higuchi US 9,023,787 B2 11 12 and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of ference. An example of a compound that inhibits a MAPKAP the A. C. S. Symposium Series, Edward B. Roche, ed., Bior kinase-2 polypeptide, but does not do so specifically, is UCN eversible Carriers in Drug Design, American Pharmaceutical O1. Association and Pergamon Press, 1987, and Judkins et al., By “substantially identical' is meant a polypeptide or Synthetic Communications 26 (23):4351-4367, 1996, each of nucleic acid exhibiting at least 75%, 85%,90%. 95%, or even which is incorporated herein by reference. 99% identity to a reference amino acid or nucleic acid By “purified’ is meant separated from other components sequence. For polypeptides, the length of comparison that naturally accompany it. Typically, a factor is Substan sequences will generally be at least 35 amino acids, 45 amino tially pure when it is at least 50%, by weight, free from acids, 55 amino acids, or even 70 amino acids. For nucleic proteins, antibodies, and naturally-occurring organic mol 10 acids, the length of comparison sequences will generally beat least 60 nucleotides, 90 nucleotides, or even 120 nucleotides. ecules with which it is naturally associated. The factor may be Sequence identity is typically measured using publicly at least 75%, 90%, or even 99%, by weight, pure. A substan available computer programs. Computer program methods to tially pure factor may be obtained by chemical synthesis, determine identity between two sequences include, but are separation of the factor from natural sources, or production of 15 not limited to, the GCG program package (Devereux et al., the factor in a recombinant host cell that does not naturally Nucleic Acids Research 12:387, 1984), BLASTP. BLASTN, produce the factor. Proteins, vesicles, and organelles may be and FASTA (Altschulet al., J. Mol. Biol. 215:403 (1990). The purified by one skilled in the art using standard techniques well-known Smith Waterman algorithm may also be used to such as those described by Coligan et al. (Current Protocols in determine identity. The BLAST program is publicly available Protein Science, John Wiley & Sons, New York, 2000). The from NCBI and other sources (e.g., BLAST Manual. Altschul factor is desirably at least 2, 5, or 10 times as pure as the et al., NCBI NLM NIH, Bethesda, Md. 20894). These soft starting material, as measured using polyacrylamide gel elec ware programs match similar sequences by assigning degrees trophoresis or column chromatography (including HPLC) of homology to various Substitutions, deletions, and other analysis (Coligan et al., Supra). Exemplary methods of puri modifications. Conservative Substitutions for amino acid fication include (i) salting-out, i.e., (NH4)2SO precipitation; 25 comparisons typically include Substitutions within the fol (ii) conventional chromatography, e.g., ion exchange, size lowing groups: glycine, alanine, Valine, isoleucine, leucine; exclusion, hydrophobic interaction, or reverse-phase; (iii) aspartic acid, glutamic acid, asparagine, glutamine; serine, affinity chromatography, e.g., immunoaffinity, active site threonine; lysine, arginine; and phenylalanine, tyrosine. affinity, dye affinity, or immobilized-metal affinity; and (iv) By “substantially inhibit is meant to reduce one or more preparative electrophoresis, e.g., isoelectric focusing or 30 activities of the molecule being inhibited by at least 50%, 60%, 70%, 80%, 90%, 95%, or even 98% compared to a native PAGE. control activity value. By "RNA interference' (RNAi) is meant a phenomenon By “substrate-binding fragment” in reference to a MAP where double-stranded RNA homologous to a target mRNA KAP kinase-2 polypeptide is meant a portion of the polypep leads to degradation of the targeted mRNA. More broadly 35 tide that is capable of binding a peptide or peptidomimetic defined as degradation of target mRNAS by homologous siR substrate. For example, fragments of MAPKAP kinase-2 NAS. polypeptide that include the region Phe-A6-Asp345 (with ref By “sensitivity” or “sensitivity to an agent' is meant an erence to SEQID NO:3) are substrate-binding fragments. increased likelihood of cell death in response to genotoxic By "surrogate.” in the context of atomic coordinates, is stress. An exemplary means of sensitivity occurs when a 40 meant any modification (e.g., mathematical modification or patient having p53-deficient tumor cell is administered a Scaling) of the coordinates that preserves the relative relation composition including MAPKAP kinase-2 polypeptide ships among the coordinates. inhibitor and a chemotherapeutic agent, resulting in reduction By “three-dimensional model is meant a three-dimen of tumor size. A reduction in tumor size in the described sional representation of a molecule's structure. Computer patient receiving the described therapy is determined in com 45 modeling may be used to generate Such a model in conjunc parison to a control p53-deficient tumor cell in a to control tion with structural data. These data could include X-ray crys patient not receiving the described therapy. Desirably, tumors tallographic data, nuclear magnetic resonance data, electron are reduced is size by 10%, 20%, 30%, 40%, 50%, 60%, 70%, microscopy data, or any other source of experimental or theo 80%, 90% or 95% in comparison to the described control. retical data useful for generating a model of a molecule or By “siNA’ is meant small interfering nucleic acids. One 50 complex of molecules. exemplary siNA is composed of ribonucleic acid (siRNA). By “treating a disease, disorder, or condition is meant siRNAs can be 21-25 nt RNAs derived from processing of preventing or delaying an initial or Subsequent occurrence of linear double-stranded RNA. siRNAs assemble in complexes a disease, disorder, or condition; increasing the disease-free termed RISC (RNA-induced silencing complex) and target Survival time between the disappearance of a condition and its homologous RNA sequences for endonucleolytic cleavage. 55 reoccurrence; stabilizing or reducing an adverse symptom Synthetic siRNAs also recruit RISCs and are capable of associated with a condition; or inhibiting, slowing, or stabi cleaving homologous RNA sequences lizing the progression of a condition. Desirably, at least 20, By “specifically inhibit an activity of a MAPKAP kinase-2 40, 60, 80,90, or 95% of the treated subjects have a complete polypeptide' is meant to reduce one or more biological activi remission in which all evidence of the disease disappears. In ties of MAPKAP kinase-2 polypeptide, without substantially 60 another desirable embodiment, the length of time a patient inhibiting related kinases, e.g., Chk1, Chk2, and p38 SAPK. Survives after being diagnosed with a condition and treated Desirably, the specific inhibition is a decrease of at least 10%, with a compound of the invention is at least 20, 40, 60, 80, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in 100, 200, or even 500% greater than (i) the average amount of biological activity, relative to a control activity, for example time an untreated patient Survives or (ii) the average amount the expression or Substrate-binding capability of a naturally 65 of time a patient treated with another therapy survives. occurring MAPKAP kinase-2 polypeptide. An exemplary By "unnatural amino acid is meant an organic compound means of specific inhibition occurs through use of RNA inter that has a structure similar to a natural amino acid, where it US 9,023,787 B2 13 14 mimics the structure and reactivity of a natural amino acid. KAP kinase-2 siRNA-treated irradiated U2OS cells placed in The unnatural amino acid as defined herein generally 50 ng/ml -containing media for 16 hours. Cells increases or enhances the properties of a peptide (e.g., selec were analyzed for DNA content by PI staining. FIG. 3H is a tivity, stability, binding affinity) when the unnatural amino graph depicting a FACS analysis of MAPKAP kinase-2 acid is either substituted for a natural amino acid or incorpo siRNA-treated irradiated U2OS cells placed in 50 ng/ml rated into a peptide. Unnatural amino acids and peptides nocodazole-containing media for 16 hours. Cells were ana including Such amino acids are described, e.g., in U.S. Pat. lyzed for phospho-histone H3 staining as a marker of mitotic Nos. 6,566,330 and 6,555,522. entry. FIG.3I is a graph depicting the results of an experiment Other features and advantages of the invention will be in which GFP siRNA- or MAPKAP kinase-2-siRNA treated apparent from the following description of the desirable 10 U2OS cells were either mock treated or exposed to 20 J/m of embodiments thereof, and from the claims. UV-C irradiation, and analyzed as described for FIGS. 3A-3H. Representative results of each experiment are shown. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 3.J is a graph depicting the results of an experiment in which GFP siRNA- or MAPKAP kinase-2-siRNA treated FIGS. 1A-1D depict the substrate specificity and kinetic 15 analysis of substrate phosphorylation by p38C. SAPK. FIG. U2OS cells were either mock treated or exposed to 10 Gy of 1A is a table showing p38 substrate specificity determined ionizing radiation, and analyzed as described for FIGS. using oriented peptide library Screening. Residues displaying 3A-3H. Representative results of each experiment are shown. the highest selectivity are shown; those with selection values FIGS. 4A-4D show that MAPKAP kinase-2 is required for >1.7 in bold. Abbreviations: MEF2A, Myocyte Enhancer S-phase arrest and cell survival following DNA damage. In Factor 2: ATF2, Activating Transcription Factor 2: 3PK1, FIG. 4A, GFP siRNA- or MAPKAP kinase-2-siRNA-treated MAP Kinase-activated Protein Kinase-3. FIG. 1B is a graph U2OS cells were mock treated or UV-irradiated and allowed showing the kinetics of in vitrophosphorylation of an optimal to recover for 30 min. BrdU was added and cells were fixed p38 peptide (p38tide) and a peptide from p47phoX (p47 tide) and analyzed by FACS for DNA content and BrdU incorpo by p38C. kinase. FIG. 1C is a graph showing the kinetics of in 25 ration twelve hours later. FIG. 4B is a graph showing the vitro phosphorylation of wild-type GST-p47phox, the percentage of cells in FIG. 4A showing BrdU incorporation at Ser345->Ala mutant, and the Ser348->Ala mutant. Typical two and twelve hours following BrdU addition. In FIG. 4C, data from n=3 experiments is shown. FIG. 1D is a table of GFP siRNA- or MAPKAP kinase-2-siRNA-treated U2OS kinetic parameters for the reactions shown in FIG. 1C. cells were either mock treated or UV-irradiated, allowed to FIGS. 2A-2C depict the substrate specificity and kinetic 30 recover for 30 min, then pulse-labeled with BrdU for 30 analysis of substrate phosphorylation by MAPKAP kinase-2. minutes. At the indicated times after irradiation the distribu FIG. 2A is a table showing MAPKAP kinase-2 substrate tion of DNA content was analyzed in the BrdU-positive popu specificity determined by oriented peptide library Screening. lation. FIG. 4D is a graph showing the results of quantitative Abbreviations: HSP27, Heat Shock protein 27: 5-LO, 5-Li colony forming assays performed by plating cells at a density poxygenase: LSP1, lymphocyte-specific protein; SRF, Serum 35 of ~100 cells per 35 mm dish. Cells were either mock treated Response Factor; GS, Glycogen Synthase: TH, Tyrosine or irradiated at the indicated UV C dose, and assays were Hydroxylase. FIG. 2B is a graph showing the kinetics of in performed in triplicate for each condition. vitrophosphorylation of the optimal MAPKAP kinase-2 pep FIG. 5 is a representation of a unified model of the kinase tide (MK2tide) by MAPKAP kinase-2. FIG. 2C is a table of dependent DNA damage checkpoint. In this model, parallel kinetic parameters for MAPKAP kinase-2 phosphorylation 40 pathways in the DNA damage checkpoint signal transduction of wild-type and mutant MK2tides. network converge on common Substrates by signaling to FIGS. 3A-3J show that MAPKAP kinase-2 is required for downstream kinases with similar phosphorylation motif G/M arrest following DNA damage. FIG. 3A is a graph specificities. (p indicates hydrophobic residues. The dashed depicting a FACS analysis of GFP siRNA-treated non-irradi line from Chk1 to Cdc25B/C indicates that this phosphory ated U2OS cells placed in 50 ng/ml nocodazole-containing 45 lation event remains controversial in response to ionizing media for 16 hours. Cells were analyzed for DNA content by radiation. PI staining. FIG. 3B is a graph depicting a FACS analysis of FIG. 6 is a summary of the requirement for ATM and/or GFP siRNA-treated non-irradiated U2OS cells placed in 50 ATR for the activation of MK2. ng/ml nocodazole-containing media for 16 hours. Cells were FIGS. 7A-7D show that MAPKAP kinase-2 depletion analyzed for phospho-histone H3 staining as a marker of 50 enhances regression of established tumors after DNA dam mitotic entry. FIG.3C is a graph depicting a FACS analysis of aging in a murine model. In FIGS. 7A-7C, GFP siRNA-treated irradiated U2OS cells placed in 50 ng/ml following subcutaneous injection of 10 MAPKAP kinase-2 nocodazole-containing media for an additional 16 hours. siRNA or control (Luciferase) siRNA treated cells into the Cells were analyzed for DNA content by PI staining. FIG. 3D flanks of NCR nude outbred mice, tumor growth was mea is a graph depicting a FACS analysis of GFP siRNA-treated 55 sured every two days. In FIGS. 7A-7C, the arrow indicates the irradiated U2OS cells placed in 50 ng/ml nocodazole-con start of intraperitoneal administration of DMSO, cisplatin, or taining media for an additional 16 hours. Cells were analyzed doxorubicin on day twelve. In the absence of DNA damaging for phospho-histone H3 staining as a marker of mitotic entry. chemotherapy, the MAPKAP kinase-2 depleted tumors were FIG. 3E is a graph depicting a FACS analysis of MAPKAP statistically significantly larger than the control tumors at kinase-2 siRNA-treated non-irradiated U2OS cells placed in 60 each time point beginning on day thirteen (Student's t-test, 50 ng/ml nocodazole-containing media for 16 hours. Cells 2-tailed, p<0.02). In contrast, after cisplatin or doxorubicin were analyzed for DNA content by PI staining. FIG. 3F is a treatment the MAPKAP kinase-2 depleted tumors were sta graph depicting a FACS analysis of MAPKAP kinase-2 tistically smaller than the control tumors beginning on days siRNA-treated non-irradiated U2OS cells placed in 50 ng/ml twenty-one and twenty-three, respectively (p<0.02). Lower nocodazole-containing media for 16 hours. Cells were ana 65 panels are close-up views of the excised tumors. FIG.7D is a lyzed for phospho-histone H3 staining as a marker of mitotic graph showing an analysis of tumor weight at the twenty-six entry. FIG.3G is a graph depicting a FACS analysis of MAP day endpoint. US 9,023,787 B2 15 16 FIG. 8 shows in vitro kinase assays performed in the pres becomes essential for cell survival after DNA damage. Both ence of increasing doses of UCN-01 with Chk1 and MAP pathways are simultaneously inhibited by the indolocarba KAP kinase-2 using the MK-2tide as a substrate. zole drug UCN-01. FIG. 9 is a representation of a model for MAPKAP kinase-2 checkpoint signaling in response to DNA damaging DETAILED DESCRIPTION OF THE INVENTION chemotherapy. Checkpoint function in response to DNA damaging agents normally requires the combined action of The invention features methods and compounds that are both the Chk1 and MAPKAP kinase-2 pathways, and both useful in treating cellular proliferative disorders. The meth pathways are simultaneously inhibited by the indolocarba ods of treatment feature administration of a compound that is 10 capable of inhibiting an activity of a MAPKAP kinase-2 zole drug UCN-01. polypeptide, or a substrate-binding fragment thereof. Such FIGS. 10A-10B are graphs showing the results of a colony compounds include, without limitation, compounds that con survival assay in which mouse embryonic fibroblasts (MEFs) tain peptides, peptidomimetics, or nucleic acid molecules. were treated with increasing doses of doxorubicin or cisplatin The invention further features screening assays for identify for eight hours, rinsed twice with PBS and once with media, 15 ing MAPKAP kinase-2 inhibitors. In addition, the invention and re-plated at an initial density of 5,000 cells/10 cm dish. includes pharmaceutical compositions and compounds, e.g., After eleven days, the number of colonies on the plate was peptides and peptidomimetics, that target the Substrate-bind counted and normalized to the number of colonies formed by ing site of MAPKAP kinase-2, thereby inhibiting it. the same cell type in the absence of treatment with chemo It was recently shown that, in addition to the ATR-Chk1 therapeutic drugs. RNA interference using short hairpin pathway, the p38 SAPK pathway is also required for full RNAs was used with both the p53 wild-type MEFs and the activation of the DNA damage response following UV irra p53 MEF's to knockdown the levels of MAPKAP kinase-2 diation. We now demonstrate that MAPKAP kinase-2, a (MK2 shRNA). Short hairpin RNAs against luciferase (lu direct downstream target of p38 SAPK, is directly respon ciferase shRNA) were used as a control. Loss of MAPKAP sible for phosphorylating Cdc25B and C and maintaining the kinase-2 activity resulted in increased sensitivity to both 25 G, S, and G/M checkpoints in response to UV-induced doxorubicin and cisplatin (i.e. decreased Survival after treat DNA damage. Thus, MAPKAP kinase-2 constitutes a third ment) only in the MEF's that lacked p53. checkpoint kinase, in addition to Chk1 and Chk2, involved in FIG. 11 shows histograms demonstrating MAPKAP coordinating the DNA damage response of higher eukaryotic Kinase 2 mediates a G/Marrest following doxorubicintreat cells. ment. RNAi down-regulation of MK2 ablates the doxorubi 30 A number of important questions regarding this third DNA cin-induced G/M checkpoint (FIG.11). p53-/-MEFs stably damage response pathway have not been previously answered. Is p38 MAPK/MAPKAP kinase-2 activation after expressing control luciferase shRNA (FIG. 11 left panels) or DNA damage dependent on ATR or ATM? Is p38 MAPK/ MK2 shRNA (FIG. 11 right panels) were cultured in the MAPKAP kinase-2 cascade important for DNA damage absence or presence of 10 uM doxorubicin and cell cycle 35 checkpoints in response to other types of genotoxic stress profiles analyzed thirty hours later by FACS using PI for DNA besides UV? How are signals from the Chk1 pathway and the content (blue) and phospho-histone H3 staining as an indica MAPKAP kinase-2 pathway integrated together at the sys tor of mitosis (red). In the lower set of panels, nocodazole tems level? We were particularly interested in investigating (100 ng/ml) was added three hours following doxorubicin whether MAPKAP kinase-2/Chk3 participates in the geno addition. Note that in addition to loss of the prominent G/M 40 toxic stress response of cells exposed to conventional anti checkpoint, the G and S phase components are also elimi cancer chemotherapeutic agents. A demonstration that MAP nated in MK2-depleted cells following doxorubicin-i-nocoda KAP kinase-2 has an important role in preventing cells with Zole treatment. chemotherapy induced DNA damage from progressing FIG. 12 shows histograms demonstrating MK2 controls through the cell cycle would implicate MAPKAP kinase-2 as the S-phase checkpoint in response to cisplatin treatment. 45 a clinically important target for anti-cancer drug design. RNAi down-regulation of MK2 ablates the cisplatin-induced Defining the Optimal Phosphorylation Motif for p38 SAPK S-phase checkpoint (FIG. 12). p53-/- MEF's stably express To identify substrates and targets of the p38 SAPK signal ing control luciferase shRNA (FIG. 12, left panels) or ing pathway involved in DNA damage responses, we deter MK2shRNA (FIG. 12, right panels) were cultured in the mined the optimal substrate phosphorylation motif for p38C. absence or presence of 10 uMcisplatin and cell cycle profiles 50 SAPK using oriented peptide library screening. Efficient pep analyzed thirty hours later by FACS using PI for DNA content tide phosphorylation by p38 SAPK required a fixed Pro resi (blue) and phospho-histone H3 staining as an indicator of due in the Ser+1 position, consistent with the known identi mitosis (red). In the lower set of panels, nocodazole (100 fication of p38 SAPK as a Pro-directed MAP kinase. ng/ml) was added three hours following cisplatin addition. Screening performed with a library containing the degenerate FIGS. 13 A-13B are models for re-wiring of cell cycle 55 sequence XXXXSPXXXX (SEQ ID NO: 8) (X denotes all checkpoint pathways in p53-proficient and deficient cells. amino acids except Cys, Ser, Thr, and Tyr) displayed stron Checkpoint function in p53-proficient cells is mediated pri gest selection for Pro in the Ser-2 position with weaker selec marily through a robust, Sustained p53 response downstream tion for other aliphatic residues (FIG. 1A). Additional selec of ATM, together with Chk1 (FIG. 13A). Although not shown tion was also observed at the Ser-3, Ser-1, and Ser+2 explicitly in the diagram, Chk1 also directly phosphorylates 60 positions. p53 (Shieh et al., 2000). Under these conditions the presence To further refine the optimal phosphorylation motif, a sec of MK2 is not required for cell survival after exposure to DNA ondary screen was performed based on results from the initial damaging agents. In p53-deficient cancer cells (FIG. 13B), screen by using a library with Pro fixed in both the Ser-2 and checkpoint signaling following to exposure to DNA damag Ser+1 positions, and Ser. Thr, and Tyr included in the X ing agents is mediated through the combined action of both 65 positions. This revealed selection for Gln, Met, and Gly in the the Chk1 and the p38 MAPK/MK2 pathways. In this situation Ser-1 position, along with slightly weaker selection for Pro, the p38 MAPK/MK2 branch of checkpoint signaling Ser and Thr (FIG. 1A). Gly was the preferred residue in the US 9,023,787 B2 17 18 Ser-3 position, along with Ile, Val, and Tyr. Hydrophobic Like p47phox, Cdc25B contains a potential p38 SAPK dock residues, particularly aromatic and B-branched amino acids, ing motif, PVONKRRRSV (SEQID NO: 13); however, the were selected at the Ser+2 position. The resulting optimal sequence flanking Ser323, LXRSPSMP (SEQ ID NO: 14), motif for p38C. SAPK determined by oriented peptide library lacks a Pro in the Ser+1 position and does not resemble the screening closely matches the sequence of mapped p38 optimal p38 SAPK motif shown in FIG. 1A. Recombinant MAPK phosphorylation sites on most, though not all, known p38 SAPK readily phosphorylated bacterially produced substrates (FIG. 1A). Cdc25B in vitro. However, this phosphorylation did not A peptide containing the optimal p38 SAPK consensus induce 14-3-3-binding, and a Ser323->Ala mutant form of phosphorylation motif GPQSPI (SEQ ID NO:9), “p38tide.” Cdc25B was phosphorylated by p38 SAPK equivalently to was synthesized for kinetic analysis. This peptide was readily 10 the wild-type Cdc25B protein. These data argue that, while phosphorylated by p38 SAPK in vitro; however, it failed to Cdc25B may be a p38 SAPK substrate, this phosphorylation display saturable Michaelis-Menton-type kinetics (FIG. 1B). event is not responsible for the 14-3-3-binding event that Instead, the initial velocity increased linearly with increasing results in a G/M checkpoint. p38tide concentration up to 1400 uM. This finding suggests Defining the Optimal Phosphorylation Motif for MAPKAP that additional interactions besides an optimal phosphoryla 15 Kinase-2 tion motif are likely to be involved in optimizing p38 SAPK A number of Ser/Thr kinases are activated downstream of Substrate binding. Such as MAP kinase docking sites. p38 SAPK, including MAPKAP Kinases-2 and -3, MNK1 To search for potential p38 SAPK substrates, particularly and MNK2, MSK1 and MSK2, and PRAK. In response to those relevant to DNA damage signaling, the Swiss-Prot data UV-B-induced DNA damage. Sheet al. (Oncogene, 21:1580 base was queried with the p38 SAPK consensus phosphory 1589, 2002) reported that MAPKAPkinase-2 could phospho lation motif using Scansite. Other than GADD153, a known rylate p53 on Ser20, the same site that is phosphorylated by p38 SAPK substrate, we were unable to identify any DNA two well-established checkpoint kinases, Chk1 and Chk2. damage response proteins in the top 250 hits. Database Both Chk1 and Chk2 can also phosphorylate Cdc25 family searching did, however, reveal two tandem near-optimal p38 members to create 14-3-3 binding sites, suggesting that SAPK phosphorylation sites (Ser345 and Ser348) in 25 MAPKAP kinase-2 might share a similar motif. The optimal p47phox, a cytosolic component of the NADPH oxidase substrate phosphorylation motif for MAPKAP kinase-2 was enzyme. A peptide containing this sequence, PGPQSPGSPL therefore investigated using oriented peptide library screen (SEQID NO: 10), “p47 tide was strongly phosphorylated by 1ng. p38 SAPK, but like p38tide, the isolated peptide displayed Efficient peptide phosphorylation by MAPKAP kinase-2 linear non-saturable kinetics (FIG. 1B). 30 was only observed with a library containing a fixed Argin the Wild-type and mutant versions of GST-tagged full-length Ser-3 position (XXXXRXXSXXXX (SEQ ID NO: 15), p47phox protein, rather than isolated peptides, were then where X denotes all amino acids except Cys, Ser. Thr, or Tyr). used as Substrates for in vitro phosphorylation reactions. The A critical step in determining protein kinase phosphorylation wild-type full-length p47phoX protein was rapidly phospho motifs by peptide library screening involves purification of rylated by p38C. SAPK (FIG. 1C). Mutation of Ser345->Ala 35 the phosphorylated peptides from the non-phosphorylated had a more pronounced effect on p47phoX phosphorylation peptide background. In the case of MAPKAP kinase-2, this than mutation of Ser348->Ala, in excellent agreement with was dramatically improved by conversion of all Glu and Asp the observation that the Ser345 site is a better match for the residues to their methyl esters prior to metal-affinity chroma optimal p38 SAPK consensus motif than the Ser348 site. tography and sequencing. This resulting motif revealed clear Simultaneous mutation of both Ser345 and Ser348 to Ala 40 amino acid selection at almost all degenerate positions (FIG. eliminated phosphorylation of p47phox by p38 SAPK alto 2A). MAPKAP kinase-2 displayed strong selection for the gether. Kinetic analysis revealed classical Michaelis-Menton hydrophobic residues Leu, Phe, Ile, and Val in the Ser-5 behavior for p38 SAPK phosphorylation of the wild-type position and the Ser+1 position. Strong selection was also p47phox with a K, of 6.0 M and a V of 36.6 nmol/min/ observed for Gln in the Ser-2 position, and modest selection ug. Mutation of Ser345 to Ala both increased the K and 45 for Leu in the Ser-1 position. The motif determined for MAP reduced the V, while mutation of Ser348 to Ala primarily KAP kinase-2 using oriented peptide library screening is in increased the K. (FIG. 1D). remarkably good agreement with the sequence of mapped These data from isolated peptides and intact proteins argue MAPKAP kinase-2 phosphorylation sites on known sub that efficient substrate phosphorylation by p38 SAPK strates (FIG. 2A, bottom), which primarily contain Leu, Ile or requires sequences with reasonable matches to the optimal 50 Phe in the Ser-5 position; Arg in the Ser-3 position; Gln, Ser, substrate motif determined by oriented peptide library or Thr in the Ser-2 position; Leu, Val or Pro in the Ser-1 screening, and that additional interactions involving MAPK position; and hydrophobic residues along with Glu in the docking sites are likely to be critical for strong kinase-Sub Ser+1 position. The preference for polar residues Ser and Thr strate interactions. Several docking motifs have been identi in addition to Gln in the Ser-2 position in known MAPKAP fied for p38 SAPK, particularly a short cluster of positively 55 kinase-2 substrates would not have been detected by oriented charged amino acid residues often flanked by hydrophobic peptide library screening, since Ser and Thr were not present amino acids. Two sequences corresponding to this type of in the library. docking motif are present near the p38SAPK phosphoryla To verify the peptide library screening results, individual tion sites in p47 phox, IHORSRKRLSQ (SEQ ID NO: 11) peptides (MK2tides) containing the optimal MAPKAP and VRFLQQRRRQA (SEQID NO: 12). Mutation of RRR 60 kinase-2 consensus motif LQRQLSI (SEQ ID NO: 16), or to LLL in the latter motif decreased the rate of p38C. SAPK mutant versions with Ala Substituted at each position in the phosphorylation of p47phox by over 70%. motif, were synthesized and used for kinetic analysis (FIGS. Bulavin et al. (Nature, 411:102-107, 2001) implicated p38 2B and 2C). The optimal MK2tide displayed a K value SAPK in the DNA damage response pathway and reported two-fold lower than the best MAPKAP kinase-2 peptide sub that p38 SAPK was directly responsible for generating a 65 strate known to date, a sequence derived from HSP27. Sub 14-3-3-binding site on Cdc25B (Ser323 in Cdc25B2: Ser309 stitution of Ala at each position in the motif affected K, and in Cdc25B1) in response to UV-C-induced DNA damage. V differently, with some positions showing primarily a US 9,023,787 B2 19 20 K effect (i.e., Arg in the Ser-3 position), while others pho-histone H3 staining (FIGS. 3A and 3B), consistent with revealed a primary effect on V (i.e., Leu in the Ser-5 a nocodazole-mediated M-phase arrest. In response to UV position) (FIG. 2C). The rank order of importance of key irradiation, control cells displayed a prominent G.S, and G residues is Arg-3>Leu-5 Ile--1>Gln-3. The optimal MK2tide distribution, with near-complete loss of phosphohistone H3 had neither the lowest K, northe highest V, but rather had staining, indicating intact G. S., and G checkpoints (FIGS. the highest V/K., ratio, consistent with the fact that the 3C and 3D). peptide library screening approach selects Substrates on the The behavior of the MAPKAP kinase-2 siRNA transfected basis of optimal V/K., rather than low K, or high V. cells was dramatically different. In the absence of UV irra alone. Combining the data from oriented peptide library diation, MAPKAP kinase-2 siRNA transfected cells, like screening, known Substrate sequences, and our kinetic stud 10 control GFP siRNA-transfected cells, accumulate in a 4N ies, the optimal MAPKAP kinase-2 phosphorylation motif is DNA-containing peak with high levels of phospho-histone L/F/IXRIQ/S/TLS/THydrophobic (SEQID NO: 17). H3 staining (FIGS. 3E and 3F). Following UV-induced DNA The optimal MAPKAP kinase-2 substrate motif is an damage, however, the MAPKAP kinase-2 knockdown cells excellent match for the known Ser323 phosphorylation/14 failed to arrest cell cycle progression. Instead, these cells 3-3 binding motif in Cdc25B, as well as the Ser216 phospho 15 proceeded to enter mitosis to the same extent as unirradiated rylation/14-3-3-binding site in Cdc25C (FIG. 2A). Initial cells, as shown by a comparable 4N-DNA peak and similar experiments focused on Cdc25B, since, unlike Cdc25C, levels of phoshohistone H3 staining as those observed in Cdc25B can be produced in modest quantities in bacteria, and un-irradiated cells (FIGS. 3G and 3H). Together with the the Ser323 site in Cdc25B had been previously reported to be Cdc25B/C:14-3-3 results, these FACS data demonstrate that a direct p38 SAPK site. Incubation of recombinant Cdc25B MAPKAP kinase-2 is critical for the UV-induced G/M with purified MAPKAP kinase-2 resulted in significant checkpoint in response to UV-irradiation. In contrast to the Cdc25B phosphorylation and strong binding of the phospho UV response, summarized in FIG. 3I, the G/M checkpoint rylated protein to 14-3-3. Mutation of Ser323->Ala substan response to ionizing radiation in MAPKAP kinase-2 knock tially reduced the ability of MAPKAP kinase-2 to phospho down cells is intact (FIG. 3J). rylate Cdc25B, and completely eliminated the ability of 25 MAPKAP Kinase-2 is Critical for the S-Phase Checkpoint Cdc25B to bind to 14-3-3. These in vitro results strongly and G. Arrest Following UV-Induced DNA Damage suggest that MAPKAP kinase-2 is the critical Cdc25/14-3-3 The MAPKAP kinase-2 knockdown cells in FIGS. 3A-3J checkpoint kinase downstream of DNA damage signals also showed a loss of the G and S-phase checkpoints follow relayed by the p38 SAPK pathway. ing DNA damage, since UV-irradiation of asynchronous cul MAPKAP Kinase-2 is Critical for the G/M Checkpoint Fol 30 tures resulted in accumulation of the cells in a 4N DNA lowing UV-Induced DNA Damage containing peak when the cells were transferred to The importance of MAPKAP kinase-2 in DNA damage nocodazole-containing medium. To investigate the direct role checkpoint function was investigated in U2OS cells. Activa of MAPKAP kinase-2 in S-phase checkpoint function, con tion of MAPKAP kinase-2 in response to UV-C irradiation trol or MAPKAP kinase-2 knockdown U2OS cells were UV induced DNA damage was monitored by its reduced mobility 35 irradiated, allowed to recover for 30 min, and then labeled on SDS-PAGEgels, and by immunoblotting using a phospho with BrdU for various times. In the absence of irradiation, specific antibody against pThr344, a site phosphorylated by 42% of the control siRNA-transfected cells showed substan p38 and required for MAPKAP kinase-2 activation. MAP tial BrdU incorporation after twelve hours, compared with KAP kinase-2 was activated within one hour of irradiation, 53% of the MAPKAP kinase-2-siRNA transfected cells and remained activated for the to eight hour duration of the 40 (FIGS. 4A and 4B). When the cells were irradiated with 20 experiment. The kinetics of MAPKAP kinase-2 activation J/m of UV light prior to BrdU labeling, only 3.5% of the paralleled the ability of Cdc25B from these cells to bind to control siRNA transfected cells showed BrdU incorporation 14-3-3. Based on these data, a two hour time point was chosen at twelve hours. In marked contrast, 48% of the MAPKAP for use in further studies. kinase-2-knockdown cells continued to incorporate Substan RNA interference was used to confirm a direct role for 45 tial amounts of BrdU. A similar difference in BrdU uptake endogenous MAPKAP kinase-2 in the UV-induced DNA between control siRNA-treated cells and MAPKAP kinase damage response. Treatment of U2OS cells with MAPKAP 2-knockdown cells was also seen at shorter times after irra kinase-2-specific siRNA oligonucleotides, but not with con diation (FIG. 4B). trol GFP siRNA oligonucleotides, resulted in a substantial Examination of the FACS profiles twelve hours following reduction of MAPKAP kinase-2 to nearly undetectable levels 50 UV-irradiation revealed a dramatic decrease in the G popu by forty-eight hours after transfection. No reduction in the lation in the MAPKAP kinase-2-knockdown cells compared levels or UV-C-induced activation of p38 SAPK, Chk1 or with the control GFP siRNA-transduced cells (FIG.4A, right Chk2 was observed in these cells. Despite the presence of most upper and lower FACS profiles). This loss of the G these other active kinases, siRNA-mediated knockdown of peak, together with the increased percentage of cells showing MAPKAP kinase-2 caused a loss of both Cdc25B- and 55 BrdU incorporation at twelve hours versus two hours of label Cdc25C-binding to 14-3-3 after UV-C exposure. ing, implies that endogenous MAPKAP kinase-2 plays We studied cell cycle progression in the control GFP and important roles in both the inhibition of DNA synthesis fol MAPKAP kinase-2 knockdown cells following UV-C-irra lowing damage (S-phase checkpoint function), and in the diation using FACS (FIGS. 3A-3J). In these experiments, damage-induced arrest of cells in G prior to S-phase entry cells were irradiated with 20J/m of UV-C radiation, allowed 60 (G/S checkpoint function). Loss of the G1/S and S-phase to recover for two hours, then placed in nocodazole-contain checkpoints in MAPKAP kinase-2 knockdown cells was ing media for an additional sixteen hours to cause any cells associated with higher levels of Cdc25A, decreased levels of progressing through the cell cycle to arrest in mitosis, where p53, and reduced phosphorylation of p53 on Ser20 following they can stained for the mitotic marker phosho-histone H3. UV-irradiation compared with control siRNA-treated cells. Under these conditions, un-irradiated cultures of asynchro 65 The fate of S-phase control or MAPKAP kinase-2 siRNA nous GFP siRNA-transfected cells accumulated in a treated cells in response to UV-C-induced DNA damage was 4N-DNA-containing peak, with prominent levels of phos examined by using FACS. In this experiment, asynchronous US 9,023,787 B2 21 22 cells were mock-treated or irradiated with 20 J/m of UV-C selection for a hydrophobic residue at the Ser+1 position is radiation and then pulse-labeled with BrdU. The cells show explained by a hydrophobic pocket that is conserved at this ing BrdU uptake were Subsequently analyzed ten and twenty position in all three kinases. The pocket is lined by Phe310, hours later (FIG. 4C). In both non-irradiated control and Pro314, Leu317 and Phe359 in Akt/PKB and by Met 167, MAPKAP kinase-2 knockdown cells, the BrdU pulse-labeled Leu171, Val174, Leu178 and Leu179 in Chk1. The corre population showed a late Sand G/M distribution atten hours, sponding Ser+1 pocket in MAPKAP kinase-2 is lined by and a re-appearance of a G peak at twenty hours, indicating Pro223, Pro227, Val234 and Leu235. Within this region, full transit through the cell cycle. In response to UV-C irra Gly312 in Akt/PKB and Gly 169 in Chk1 are replaced by diation, control siRNA-treated cells failed to show significant Tyr225 in MAPKAP kinase-2, which may reduce the depth of BrdU uptake upon which to gate for FACS analysis (FIG. 4C, 10 the MAPKAP kinase-2 hydrophobic pocket and explain lower left panel). In contrast, the large population of MAP selection for branched chain aliphatic residues in this position KAP kinase-2 siRNA treated cells, which had lost the S-phase compared with Phe selection by Akt/PKB and Chk1. checkpoint and incorporated BrdU, went on to display a The marked contrast between Arg selection at the Ser-5 greatly reduced G peak at twenty hours, with many cells position in Akt/PKB with the corresponding selection for showing DNA staining >4N (FIG. 4C, bracket in lower right 15 hydrophobic residues at this position by MAPKAP kinase-2 panel), consistent with mitotic death and exit from the cell and Chk1 is accounted for by the presence of Glu342 in cycle. Akt/PKB at the base of the Ser-5 pocket. This residue is not MAPKAP Kinase-2 Depleted Cells are More Sensitive to conserved in MAPKAP kinase-2 and Chk1, and is instead DNA Damage-Induced Cell Death substituted by Ile255 in MAPKAP kinase-2 and by Ala200 in The experiments in FIGS. 3A-3J and 4A-4C indicate that Chk1. Additional residues, notably Phel47, Pro189, Pro261 MAPKAP kinase-2 is involved in each of the cell cycle and Leu342 in MAPKAP kinase-2, and similarly Phe93, checkpoints triggered by UV-induced DNA damage. To Ile96, Pro98, Pro133 and Leu206 in Chk1, contribute a sig determine the effect of MAPKAP kinase-2 depletion on cell nificant hydrophobic character to this region. survival, we transfected cells with control siRNA or MAP MAPKAP Kinase-2 is Required for the G2/M Checkpoint KAP kinase-2 siRNA for forty-eight hours, trypsinized, 25 Following Doxorubicin Treatment. replated, and analyzed for colony formation in response to Treatment of U2OS cells with doxorubicin generates DNA various doses of UV-C irradiation twelve hours after re-plat double strand breaks, and induced a prominent G/M arrest ing. As shown in FIG. 4D, MAPKAP kinase-2 knockdown between eighteen and thirty hours following treatment. In cells displayed a significant reduction in colony formation addition to this large G2/M population, a minor accumulation when compared to control-treated cells at all doses of UV-C 30 of cells in G and S phase was also observed. To investigate irradiation examined. This difference in survival after UV-C whether MAPKAP kinase-2 activation was involved in the exposure was most pronounced at low to moderate UV doses. checkpoint response, RNA interference was used to generate A Structural Model for MAPKAPKinase-2 Substrate Selec U2OS cells in which MAPKAP kinase-2 protein levels were tivity. stably repressed. Introduction of MAPKAP kinase-2 specific The optimal phosphorylation motif determined for MAP 35 shRNA, but not luciferase shRNA, resulted in a robust knock KAP kinase-2 is strikingly similar to that determined for two down of MAPKAP kinase-2 protein when the entire popula other checkpoint kinases, Chk1 and Chk2. All three of these tion of transfected cells was analyzed. CAMK superfamily members—MAPKAP kinase-2, -Chk1. Asynchronous MAPKAP kinase-2 or luciferase shRNA and Chk2—strongly select for aliphatic residues in the Ser-5 knockdown cells were mock treated or exposed to doxorubi position, Arg in the Ser-3 position, and aromatic/aliphatic 40 cin for thirty hours, and cell cycle progression was monitored residues in the Ser+1 position, along with additional less by FACS. In one set of experiments, the spindle poison stringent selection for particular amino acids in other posi nocodazole was added to the media three hours after addition tions. In contrast, members of the AGC kinase Superfamily, of doxorubicin, to cause any cells progressing through the cell such as Akt/PKB and conventional protein kinase C super cycle to arrest in mitosis. DNA content was monitored by PI family members, preferentially phosphorylate sequences 45 staining; phospho-histone-H3 staining was used as an indi containing Arg residues in both the Ser-5 and Ser-3 positions, cator of mitotic entry. Treatment of control luciferase shRNA and play important roles in anti-apoptotic signaling and other knockdown cells with doxorubicin led to the accumulation of signaling events unique to differentiated cell function, rather cells with 4N DNA content, and a lack of phospho-histone than critical roles in cell cycle control. H3 staining in either the absence or presence of nocodazole. To investigate the structural basis for substrate motif selec 50 The cells expressing the luciferase shRNAs behaved identi tion, we performed molecular modeling studies of activated cally to the untransfected doxorubicin-treated control U2OS MAPKAP kinase-2, using the published MAPKAP kinase cell population, with the prominent 4N DNA component and 2:ADP co-crystal structure (Underwood et al., Structure, the absence of phospho-histone-H3 staining indicative of an 11:627-636, 2003) as a base model. The optimal substrate intact G/M checkpoint. In marked contrast, MAPKAP peptide LQRQLSIA (SEQ ID NO: 6) was modeled into the 55 kinase-2-depleted cells treated with doxorubicin displayed a kinase active site in an extended conformation, and the cell cycle profile essentially identical to that of untreated kinase:Substrate complex compared with the structures of cells. Addition of nocodazole following doxorubicin treat Akt/PKB.AMP-PNP:GSK3-peptide ternary complex (Yang ment to the MAPKAP kinase-2 depleted cells caused them to et al., Nat. Struct. Biol. 9:940-944, 2002) and the Chk1 accumulate in a 4N DNA containing peak, with 36.3% of the crystal structure containing a modeled Cdc25C peptide (Chen 60 cells staining positively for phospho-histone H3, a value simi et al., Cell, 100:681-692, 2000). Strong selection for Arg in lar to that of untreated U2OS cells blocked in mitosis with the Ser-3 position for MAPKAP kinase-2, Akt/PKB and nocodazole (42%). Identical results were obtained using a Chk1 is rationalized by the presence of a conserved glutamate second unrelated RNAi sequence against MAPKAP kinase residue at a similar location in all three kinases (Glu145 in 2, indicating that these results did not arise from RNAi off MAPKAP kinase-2, Glu236 in Akt/PKB and Glu91 in Chk1), 65 target effects. MAPKAP kinase-2 depletion did not alter total to which in Akt/PKB forms a bidentate salt bridge with the Chk1 levels or reduce Chk1 activation following DNA dam Ser-3 arginine guanidino group on GSK3-peptide. Similarly, age. These findings demonstrate that loss of MAPKAP US 9,023,787 B2 23 24 kinase-2 prevents cells from establishing a functional G/M added three hours following cisplatin addition, the MAPKAP checkpoint following doxorubicin-induced DNA damage, kinase-2 depleted cells accumulated in a 4N DNA containing despite the presence of activated Chk1. peak with ~42% of the cells staining strongly for phospho MAPKAPKinase-2 Induces Binding of Cdc25B to 14-3-3 in histone H3. Identical results were obtained in cells treated Response to -Induced DNA Dam with a second unrelated siRNA sequence against MAPKAP age. kinase-2. MAPKAP kinase-2 depletion did not impair acti Two Cdc25 family members, Cdc25B and C, play impor vation of Chk1 after cisplatin exposure. These data imply that tant roles in initiating and maintaining mitotic entry in normal MAPKAP kinase-2 is essential for the cisplatin induced G/S cells, and are prominent targets of the G/M checkpoint. arrest and that loss of MAPKAP kinase-2 enables U2OS cells Cdc25B is believed to function by activating Cdk1/Cyclin B 10 to override the cisplatin-induced G1/S checkpoints, despite at the centrosome in late G2 as an initiator of early mitotic the presence of activated Chikl, and proceed into mitosis. events, while Cdc25C functions to further amplify Cdk1/ MAPKAP kinase-2 is required for Cdc25A degradation in CyclinB activity within a nuclear autoamplification loop once response to cisplatin-induced DNA damage. mitosis has begun. In response to y- or UV-radiation-induced In contrast to the 14-3-3-mediated sequestration of DNA damage, checkpoint kinases phosphorylate Cdc25B 15 Cdc25B and C involved in the G/M checkpoint response, the and C on Ser323 and Ser216, respectively, to induce their G and S phase checkpoints are largely controlled by the binding to 14-3-3 proteins, which, along with a modest reduc phosphorylation-dependent degradation of another Cdc25 tion in their catalytic activity, sequesters them in the cyto isoform, Cdc25A. Based on our observation that depletion of plasm away from their nuclear cyclin/Cdk substrates. Recent MAPKAP kinase-2 resulted in loss of the G/S checkpoint studies suggest that Cdc25B plays a particularly crucial role response, we investigated whether MAPKAP kinase-2 was in initiating and maintaining normal cell cycle G/M check required for the degradation of Cdc25A following cisplatin point responses, since reactivation of Cdc25B is critical for induced DNA damage. Luciferase shRNA control cells and DNA-damaged cells to re-enter the cell cycle. We have MAPKAP kinase-2 depleted cells were treated with cisplatin, shown-above that MAPKAP kinase-2 is capable of directly and cell lysates immunoblotted for Cdc25A at eight and phosphorylating Cdc25B on Ser323 to generate the 14-3-3 25 twelve hours following treatment. Cdc25A levels decreased binding site. We therefore investigated whether MAPKAP dramatically in the control luciferase knockdown cells after kinase-2 signaling was required for association of Cdc25B treatment with cisplatin. In contrast, in the MAPKAP with 14-3-3 in response to DNA damage by chemotherapeu kinase-2 depleted cells, the level of Cdc25A following cispl tic drugs. Control luciferase and MAPKAP kinase-2 knock atin exposure was only minimally reduced, and remained down cells were either mock treated or incubated with cispl 30 comparable to that seen in undamaged cells. These data indi atin, camptothecin, or doxorubicin. Cell lysates were cate that in the absence of MAPKAPkinase-2, U2OS cells are prepared eight hours later and incubated with recombinant defective in targeting Cdc25A for degradation in response to GST-14-3-3B/. Binding of endogenous Cdc25B to 14-3-3 cisplatin induced DNA damage. This inability of MAPKAP was detected by immunoblotting. Both doxorubicin and kinase-2 depleted cells to degrade Cdc25Alikely explains the camptothecin treatment, but not cisplatin exposure, resulted 35 failure of MAPKAP kinase-2 depleted cells to establish a in the generation of stable 14-3-3-binding sites on Cdc25B in Sustained G1/S checkpoint following cisplatin exposure. the luciferase shRNA control cells. No 14-3-3 binding of The degradation of Cdc25A in response to DNA damage Cdc25B, however, was detected in lysates from the MAP involves the direct phosphorylation of Cdc25A by checkpoint KAP kinase-2 depleted cells. This result is in good agreement kinases. In response to UV and Y-irradiation, for example, with the cell cycle studies, which showed loss of the G/M 40 Chk1 phosphorylates Cdc25A at multiple sites facilitating its checkpoint in MAPKAP kinase-2 depleted cells after treat Subsequent ubiquitin-mediated destruction by the proteo ment with the topoisomerase inhibitor doxorubicin. These some. Chk1, however, is activated normally in the MAPKAP data indicate that loss of the chemotherapy-induced G/M kinase-2 depleted cells after cisplatin treatment. Other checkpoint in MAPKAP kinase-2 depleted cells likely arises, kinases besides Chk1, such as Chk2, have been recently at least in part, from loss of Cdc25B binding to 14-3-3 pro 45 reported to be able to phosphorylate Cdc25A on at least some teins. of the same sites as Chk1 under certain conditions. Further MAPKAP Kinase-2 is Required for G/S Checkpoint Arrest more, we have shown that the optimal amino acid sequence Following Cisplatin Treatment. motif on peptides and proteins phosphorylated by MAPKAP In contrast to the G/M checkpoint response observed in kinase-2 is nearly identical to the optimal sequence motif doxorubicin-treated cells, treatment with the DNA intra 50 phosphorylated by Chk1 and Chk2. Wetherefore investigated strand cross-linker cisplatin caused U2OS cells to predomi whether Cdc25A could be a direct MAPKAP kinase-2 Sub nantly accumulate in the G and S phases of the cell cycle over strate. Recombinant Cdc25A was incubated with purified the subsequent thirty hours-. RNA interference was used to MAPKAP kinase-2 or Chk1 in vitro in the presence of 32P investigate the role of MAPKAP kinase-2 in this process. Y-ATP, and phosphorylation monitored by SDS-PAGE/auto Control luciferase knockdown cells showed an identical 55 radiography. MAPKAP kinase-2 phosphorylated Cdc25A in accumulation in G and Safter cisplatin exposure as that seen vitro as efficiently as Chk1. Together, these findings Suggest in U2OS cells lacking shRNA. Addition of nocodazole to the that degradation of Cdc25A in response to cisplatin treatment luciferase knockdown cells three hours following cisplatin either requires direct phosphorylation of Cdc25A by MAP treatment did not reveal the appearance of any mitotic cells KAP kinase-2, or that MAPKAP kinase-2 activity is required over the ensuing twenty-seven hours, as monitored by phos 60 to target Chk1 to Cdc25A in vivo. pho-histone H3 staining, indicating a functionally intact G/S Down-Regulation of MAPKAP Kinase-2 Increases the Sen checkpoint. Depletion of MAPKAP kinase-2 prior to cispl sitivity of Tumor Cells to Chemotherapy. atin exposure resulted in a dramatically different result. Fur The data previously discussed indicate that MAPKAP ther, MAPKAP kinase-2 depleted cells showed a cell cycle kinase-2 is critical for cisplatin- and doxorubicin-triggered profile after cisplatin treatment that was similar to that of 65 G/S and G/Marrest. These checkpoint defects in MAPKAP untreated cells other than a very slight increase in the total kinase-2 depleted cells might render them more sensitive to number of cells in S-phase. Strikingly, when nocodazole was the antiproliferative and cytotoxic effects of chemotherapy. US 9,023,787 B2 25 26 To investigate this, control or MAPKAP kinase-2 knockdown kinase-2. Phosphorylation/activation of MAPKAP kinase-2 U2OS cells were mock treated or incubated with increasing occurred normally after treatment with these DNA damaging doses of cisplatin or doxorubicin for eight hours, washed, agents, regardless of the presence or absence of Chk1. Thus, trypsinized and replated, and assayed for colony formation activation of MAPKAP kinase-2 and Chk1 after genotoxic eight days later. When compared to the control shRNA 5 stress appears to occur independently of each other. treated cells, MAPKAP kinase-2 depleted cells displayed a The MAPKAP Kinase-2 DNA Damage Checkpoint Pheno dramatically increased sensitivity to both cisplatin and doxo type can be Synthetically Rescued by Chk1 Overexpression. rubicin treatment, particularly at relatively low drug doses. The observation that Chk1 and MAPKAP kinase-2 phos For example, luciferase shRNA cells treated with either 10 phorylate the same optimal sequence motif, target a set of uM cisplatin or 1 uM doxorubicinformed ~40% of the num 10 overlapping Substrates, and are activated independently of ber of colonies as those formed by untreated cells, while in each other, prompted us to perform a genetic experiment to MAPKAP kinase-2-depleted cells, these same cisplatin and investigate whether loss of MAPKAP kinase-2 could be res doxorubicin treatments reduced the number of colonies to cued by overexpression of Chk1 in mammalian cells. In these only 4% and 2%, respectively, of those seen in the untreated experiments, luciferase- or MAPKAP kinase-2 shRNA-ex cells. 15 pressing cells were transiently transfected with a mammalian To establish whether the absence of MAPKAP kinase-2 Chk1 expression construct, or with an empty vector control. could also enhance the anti-tumorigenic effect of cisplatin or Cells were exposed to cisplatin, doxorubicin, or UV radiation doxorubicin in vivo, we introduced control or MAPKAP thirty hours following transfection, harvested after an addi kinase-2 siRNAs into H-Ras-V12 transformed p53 MEFs, tional thirty hours, and cell cycle progression analyzed by treated them with either vehicle alone, 1 uM cisplatin or 0.1 FACS. In one set of experiments, nocodazole was added to the uM doxorubicin, and then implanted them into nude mice. media three hours following addition of chemotherapy or UV. Each animal received two injections of MAPKAP kinase-2 to cause any cells progressing through the cell cycle to arrest siRNA-transfected cells (left flank), and two injections of in mitosis. control siRNA transfected cells (right flank), and tumor for Consistent with what we observed previously, luciferase mation was assessed at fifteen days. In the absence of treat 25 shRNA control cells transfected with the empty vector DNA ment with DNA damaging agents, all four injections resulted executed a G/Sarrest following exposure to cisplatin and UV in formation of solid fibrous tumors after fifteen days. In irradiation, and displayed a prominent Garrest in response to general, the size of the tumors resulting from injection of doxorubicin. These cell cycle profiles were unchanged when MAPKAP kinase-2 depleted cells was larger than that from the luciferase shRNA cells were transfected with Chk1. control siRNA-transfected cells. Pre-treatment of the control 30 MAPKAP kinase-2 depleted cells transfected with empty siRNA transfected cells with either cisplatin or doxorubicin vector DNA broke through both checkpoints and accumu prior to implantation did not prevent tumor formation. The lated in mitosis when nocodazole was added to the media. resulting tumors, however, were reduced to ~35% of the size Overexpression of Chk1 in the MAPKAP kinase-2 depleted and weight of the tumors formed by untreated cells. Depletion cells, however, completely restored their ability to establish of MAPKAP kinase-2 prior to treatment with either cisplatin 35 functional checkpoints following genotoxic stress. The cells or doxorubicin completely eliminated the formation of now arrested in G/S in response to cisplatin and UV irradia tumors, indicating that the enhanced sensitivity of these cells tion, and in G following doxorubicin. Addition of nocoda to chemotherapeutic drugs seen in culture was maintained Zole to the growth media of these MAPKAP kinase-2 even when the cells were grown within a normal tissue depleted Chk1 over-expressing cells did not increase the microenvironment. 40 number of phosphohistone H3 positive cells. Thus, overex Taken together with the loss of G/S and G/M checkpoints pression of Chk1 prevented MAPKAP kinase-2 depleted observed by FACS analysis, and the mis-regulation of the cells from progressing through the cell cycle after genotoxic mitotic phosphatases Cdc25A and B, these data provide StreSS. strong evidence that down-regulation of MAPKAP kinase-2 We investigated whether the synthetic rescue of G/S and activity results in enhanced sensitivity of cells to genotoxic 45 G/M checkpoints by Chk1 in MAPKAP kinase-2 depleted stress in vitro and in vivo. These findings have potential cells was also sufficient to reduce their susceptibility to che therapeutic implications, since they suggest that targeting of motherapeutic treatment. Luciferase and MAPKAP kinase-2 MAPKAP kinase-2 with small molecule inhibitors should knockdown cells transfected with Chk1 or vector alone were result in an enhanced sensitivity of tumor cells to conven mock treated or incubated with increasing doses of cisplatin tional chemotherapeutic agents. 50 and doxorubicinforeight hours, or irradiated with 20J/m of MAPKAP Kinase-2 and Chk1 are Activated Independently. UV light. Cells were washed, trypsinized, replated and The activation of MAPKAP kinase-2 by cisplatin, camp assayed for colony formation after eight days as described tothecin, doxorubicin, and UV irradiation that we observed is previously. Further, MAPKAP kinase-2 depleted cells, trans strikingly similar to the activation profile reported for Chk1. fected with the empty control vector, showed enhanced sen Similarly, the impaired G1/S and G/M checkpoints seen after 55 sitivity to the anti-proliferative effects of cisplatin, doxorubi these DNA damaging stimuli in MAPKAP kinase-2 knock cin and UV. Overexpression of Chk1 in these MAPKAP down cells bears some resemblance to what has been reported kinase-2 depleted cells restored their clonogenic survival to for Chk1-deficient cells. These phenotypic similarities levels that were indistinguishable from those seen with con prompted us to further investigate whether the activation of trol cells containing wild-type levels of MAPKAP kinase-2. Chk1 and MAPKAP kinase-2 was interdependent. As previ 60 UCN-01 is a Potent Inhibitor of Both Chk1 and MAPKAP ously discussed, activation of Chk1 in response to cisplatin Kinase-2. and doxorubicin was unimpaired in MAPKAP kinase-2 The staurosporine derivative 7-hydroxystaurosporin/ depleted cells. We therefore investigated the opposite possi UCN-01 inhibits Chk1 with an ICs that is ~1000 fold lower bility—whether the activation of MAPKAP kinase-2 after than that for Chk2, and hence has been used experimentally as DNA damage was dependent on Chk1. U2OS cells were 65 a Chk1-specific inhibitor. Strong circumstantial evidence, depleted of Chk1 using siRNA, exposed to cisplatin and however, suggests that UCN-01 inhibits other kinases doxorubicin, and analyzed for activation of MAPKAP involved in cell cycle control at similar concentrations as US 9,023,787 B2 27 28 those used for Chk1 inhibition studies. For example, Chk1 Since disruption of the MAPKAP kinase-2 signaling path depleted cells maintain phosphorylation of Cdc25C on Ser way enhances chemotherapeutic responses even in the pres 216 both during asynchronous growth and following Y-irra ence of a functional Chk1 response, and since MAPKAP diation. Phosphorylation at this site is lost when cells are kinase-2 knock-out mice are viable, in contrast to Chkl treated with low doses of UCN-01 (-300 nM), indicating that knock-out mice, our results suggest that a MAPKAP kinase-2 UCN-01 inhibitable kinase(s) other than Chk1 participate in specific inhibitor might provide significant clinical benefit Cdc25C phosphorylation. Based on our finding that MAP with fewer undesirable side-effects. In either case, our current KAP kinase-2 is a critical checkpoint regulator, we investi data strongly support the development of clinical MAPKAP gated whether UCN-01 inhibited MAPKAPkinase-2 at doses kinase-2 inhibitors as viable anti-cancer agents. Given the 10 dependence of p53-defective cells on intra-S and G/M typically used in Chk1 inhibition experiments. In vitro kinase checkpoint pathways, targeting MAPKAP kinase-2 may be a assays were performed with Chk1 and MAPKAP kinase-2 particularly efficacious approach to treating these types of using an optimal peptide Substrate with the core consensus human cancers. Thus, therapeutic treatments that interfere sequence LQRQLSI (SEQID NO: 16), similar to the 14-3-3 with MAPKAP kinase-2 function would be expected to pref binding sequence in Cdc25B and C, in the presence of various 15 erentially sensitize p53-deficient cells to treatment with concentrations of UCN-01. As shown in FIG. 8, UCN-01 DNA-damaging chemotherapeutic drugs without similarly potently inhibited both kinases, with an ICso value of -35 nM sensitizing wild-type cells. Disorders, e.g., neoplastic disor for Chk1 and -95 nM for MAPKAP kinase-2. The ICs value ders, that include p53-deficient cells could be treated effec we measured for Chk1 is in good agreement with previously tively and specifically using therapy that combines adminis published data. Importantly, the ICso value we measured for tration of a MAPKAP kinase-2-interfering compound, e.g., MAPKAP kinase-2 is significantly below the concentrations UCN-01, and one or more chemotherapeutic agents, prefer of UCN-01 that are used in “Chk1-specific' checkpoint abro ably at substantially lower levels than would otherwise be gation assays, suggesting that under the conditions used in necessary to treat the disorder, thereby largely sparing normal those studies, both Chk1 and MAPKAP kinase-2 were being cells from the deleterious effects of chemotherapy. inhibited. 25 Model for the Role of MAPKAP Kinase-2 To examine the structural basis for UCN-01 inhibition of Our data show that a crucial role of p38 SAPK in response MAPKAP kinase-2, the structure of the MAPKAP kinase-2: to UV-induced DNA damage is the phosphorylation and acti UCN-01 complex was modeled using coordinates from the vation of MAPKAP kinase-2, leading to MAPKAP kinase published MAPKAP kinase-2:staurosporine structure, and 2-directed phosphorylation of Cdc25 family members to compared the results with the co-crystal structure of Chk1: 30 induce 14-3-3-binding and Subsequent cell cycle arrest. In UCN-01. The 7-hydroxy moiety of UCN-01 can be easily this way, MAPKAP kinase-2 performs similar functions after accommodated into the MAPKAP kinase-2:staurosporine UV-Cinduced DNA damage as those performed by Chk1 and structure, where its closest neighboring residues would be Chk2 after exposure of cells to ionizing radiation. Val118 (2.8 A to Cy2), Leu 141 (3.2 A to C61), and Thr206 MAPKAP kinase-2 undergoes initial activation in the (3.6 A to Cy2). This lack of steric hindrance, and the overall 35 nucleus with Subsequent export of the active kinase to the similarity of the modeled MAPKAP kinase-2:UCN-01 struc cytoplasm. Thus, MAPKAP kinase-2 is well-positioned to ture to the Chk1:UCN-01 structure, provides a structural function as both a nuclear initiator of Cdc25B/C phosphory rationale for the tight binding observed biochemically. lation in response to DNA damage, and as a maintenance To verify that MAPKAP kinase-2 is a direct target of UCN kinase that keeps Cdc25B/C inhibited in the cytoplasm. A 01 in cells, we measured the phosphorylation of the MAP 40 unified model for kinase-dependent DNA damage check KAP kinase-2-specific substrate hsp-27 after heat shock, a points is presented in FIG. 5. In response to ionizing radia stimulus that activates the p38 MAPK/MAPKAP kinase-2 tion, ATM activation of Chk2 and ATR activation of Chk1 pathway. Control luciferase shRNA expressing or MAPKAP leads to phosphorylation of Cdc25 family members on related kinase-2 shRNA expressing U2OS cells were incubated at sequences corresponding to the checkpoint kinase core 42°C. or 37°C. for two hours in the presence or absence of 45 “motif LXRXXIS/THydrophobic (SEQ ID NO: 18). 250 nMUCN-01, and phosphorylation of hsp-27 monitored Similarly, in response to UV-induced DNA damage, ATR by immunoblotting with an antibody against pSer82, a well activates Chk1 and p38 SAPK activates MAPKAP kinase-2, established MAPKAP kinase-2 phosphorylation site. The leading to phosphorylation of the same core motif on Cdc25 hsp-27 was phosphorylated when the control luciferase family members. The major role of Chk1 appears to involve shRNA cells were placed at 42°C. This phosphorylation was 50 phosphorylation of Cdc25A after IR, whereas Chk2 appears completely abrogated by treatment with UCN-01. No phos to phosphorylate all three Cdc25 family members. In the phorylation was observed in MAPKAP kinase-2 knockdown absence of Chk2, Chk1 appears to be able to subsume at least cells placed at 42°C. regardless of the presence or absence of part of this function. Our data now indicate that MAPKAP UCN-01. Likewise, no signal was observed in both the con kinase-2 is the primary effector kinase that targets Cdc25B/C trol and MAPKAPkinase-2 knockdown cells that were main 55 after UV-C exposure. MAPKAP kinase-2 may also be tained at 37°C., with or without UCN-01 treatment. Further involved in Cdc25A phosphorylation, since we observed that more, heat shock was equally effective in promoting the the G and S-phase checkpoints were eliminated in the MAP phosphorylation of hsp-27 on Ser-82, and UCN-01 was KAP kinase-2 knockdown cells. equally effective in blocking Ser-82 phosphorylation in cells The results presented here indicate that the activities of that were depleted of Chk1. Thus, UCN-01 inhibition of 60 both Chk1 and MAPKAP kinase-2 are required for G/S and MAPKAP kinase-2 in vivo is independent of Chk1 function. G/M cell cycle arrest in response to DNA damaging chemo These findings provide strong evidence that UCN-01 is a therapy and UV-irradiation (FIG.9). At a systems level, these direct inhibitor of MAPKAP kinase-2 within cells, and sug observations suggest that the normal DNA damage check gest that the clinical efficacy of UCN-01 in cancer treatment point response involves the unified actions of a dedicated likely arises from the simultaneous inhibition of two parallel 65 DNA damage response pathway (i.e., Chkl) and a potentially but non-redundant checkpoint pathways involving Chk1 and more global stress response pathway (MAPKAP kinase-2). MAPKAP kinase-2. Individual kinase activities emerging from each of these path US 9,023,787 B2 29 30 ways appear to be titered to levels that, in combination, are into kinase buffer. Chk2 expressing cells were lysed in buffer just adequate to arrest the cell cycle after damage, presumably containing 50 mM Tris-HCl, pH 7.5, 250 mM. NaCl, 1 mM facilitating rapid checkpoint release once the DNA damage DTT, 1.0% NP-40, 8 g/mL pepstatin, 8 ug/mL aprotinin, 8 has been repaired. In agreement with this hypothesis, over ug/mL leupeptin, 2 mM NaVO 10 mM NaF. and 1 uM expression of Chk1 rescued both the G/M and G/S cell microcystin, and Chk2 was purified using Ni-NTA agrose cycle checkpoint defects observed in MAPKAP kinase-2 beads. After washing extensively with lysis buffer containing depleted cells. 40 mMimidazole, Chk2 was eluted from the beads with 100 Experimental Procedures mMimidazole in 50 mM Tris-HCl, pH 8.0, and dialyzed into Chemicals, Antibodies, and Drugs. kinase buffer. UCN-01 was the kind gift of R. Schultz, Drug Synthesis 10 Point mutations were generated using the Stratagene Quick and Chemistry Branch, Developmental Therapeutics Pro Change Mutagenesis Kit and confirmed by sequencing the gram, Division of Cancer Treatment and Diagnosis, National entire coding regions. Cancer Institute (Bethesda, Md.). Cisplatin, doxorubicin and Kinase MotifScreening with Oriented Peptide Libraries camptothecin, puromycin, and glutathione beads were pur and In Vitro Kinase Assays. chased from Sigma-Aldrich. Propidium iodide was pur 15 Using the methods of the invention, one skilled in the art chased from Calbiochem. Antibodies against total and phos would be able to utilize a peptide library screen to identify phorylated forms of MAPKAP kinase-2, p38 MAPK, Chk1, peptides that bind to a p38 SAPK polypeptide, to MAPKAP Chk2, ATM/ATR substrate, hsp-27, and p53 (pS20) were kinase-2 polypeptide, or other biologically relevant target. purchased from Cell Signaling Technology (Beverly, Mass.). Peptides identified in Such a screen, or related compounds, Antibodies against B-actin and 5-bromo-2-deoxyuridine would have potential therapeutic benefit due to their ability to (BrdU) were purchased from Sigma-Aldrich; an anti inhibit the biological activity of, e.g., a MAPKAP kinase-2 Cdc25A antibody (MS-640-P1, cocktail) was from NeoMar polypeptide. ker (Fremont, Calif.); an anti-Cdc25B antibody was from Combinatorial peptide library screening was performed Transduction Labs, an anti-GST antibody was from Amer using recombinant purified p38C. SAPK, MK2, Chk1 and sham/GE Healthcare, and an anti-phospho histone H3 anti 25 Chk2 as previously described (Songyang and Cantley, Meth body was from Upstate. Active MAPKAP kinase-2 was pur ods Mol. Biol. 87:87-98, 1998) with minor modifications. chased from Upstate. Propidium Iodide (PI) was purchased Briefly, 5.0 g of recombinant p38C. SAPK, 3.0 ug MK2, 2.0 from Calbiochem, amylose beads were purchased from New Jug Chk1 and 2.0 ug Chk2 were incubated with 1 mg of each England Biolabs, Ni-NTA agarose were purchased from peptide library in 300 ul reaction volumes containing 20 mM QIAGEN, and glutathione beads and BrdU were purchased 30 HEPES, pH 7.5, 10 mM MgCl, 3 mM 2-mercaptoethanol, from Sigma-Aldrich. and 100 uM ATP containing 2.5 uCi of 32P-Y-ATP for 120 Cell Culture. min at 30°C. Under these conditions, approximately 1% of U2OS cells, HeLa cells, U87MG cells and H-Ras-V12 the peptide mixture was phosphorylated. The reaction mix transformed p53 MEFs were cultured in DMEM supple ture was diluted by addition of 300 ul of 30% acetic acid, and mented with 10% FCS and penicillin/streptomycin at 37° C. 35 the phosphorylated peptides separated from unincorporated in a humidified incubator supplied with 5% CO2. GM05849 32P-Y-ATP by DEAE column chromatography (1 ml bed A-T fibroblasts and the corresponding control GMO0637 volume) using isocratic elution with 30% acetic acid. The fibroblasts, and GM18366 ATR-defective Seckel syndrome peptide mixture (both phosphorylated and unphosphorylated, fibroblasts and the corresponding control GMO0023 fibro but free of ATP) eluted within the first 1 ml following the 600 blasts were obtained from the Coriell cell repository and were 40 ul void volume of the column. Samples were dried in a Speed cultured in MEM supplemented with Eagle's salts, 10% FCS, Vac apparatus. and penicillin/streptomycin. For the p38C. SAPK peptide library experiments, the Purification of Recombinant Proteins. sample was resuspended in 200 ul of 50 mM MES, pH 5.5, Constructs encoding GST and MBP-fusion proteins were containing 1 M NaCl. Separation of phosphorylated from transformed into DH5C. or BL21 (DE3) strains of E. coli and 45 non-phosphorylated peptides was achieved by IMAC using recombinant proteins obtained by inducing late log-phase ferric-iminodiacetic acid beads. A 0.5 ml iminodiacetic acid cells with 0.4 mMIPTG at 37°C. for three to five hours. Cells column was charged with 2.5 ml of 20 mM. FeC1 and exten were lysed by sonication in lysis buffer containing 50 mM sively washed with HO, then with 3 ml of 500 mM Tris-HCl, pH 7.5, 250 mM NaCl, 1 mM DTT, 8 ug/mL NHHCO, pH 8.0.3 ml of HO, and 3 ml of 50 mMMES (pH pepstatin, 8 Lug/mL aprotinin, and 8 Lug/mL leupeptin. Fusion 50 5.5)/1 M NaCl. The peptide mixture was applied and the proteins were purified from cell lysates by using amylose or column was developed with 3 ml 50 mM MES, pH 5.5, 1 M glutathione beads. Following extensive washing with PBS NaCl, followed by 4 ml of HO to remove nonphosphorylated containing 0.5% NP-40 and a final wash with PBS, fusion peptides. Phosphorylated peptides were then eluted with 2 ml proteins were eluted from the beads with HEPES, pH 7.2, of 500 mM NHHCO, pH 8.0, and dried in a Speed-Vac containing 40 mM maltose or 20 mM glutathione, followed 55 apparatus, and resuspended in 80 ul H2O. by exchange into PBS using duplicate Sephadex G-25 col Peptide library screens using basophilic kinase-directed umns (NAP-10 columns, Pharmacia). Protein concentrations libraries are complicated by a high background of non-phos were determined using the bicinchoninic acid assay (Pierce) phorylated Asp/Glu-rich peptides that co-purified with the as recommended by the manufacturer, using BSA as the phosphorylated peptides during the immobilized metal affin standard. Full-length Chk1-GST or full-length Chk2-His6 in 60 ity chromatography (IMAC) step prior to peptide sequencing, pFASTBAC was expressed in Sf9 insect cells. Chk1-express greatly complicating the analysis. To overcome this problem, ing cells were lysed in buffer containing 50 mM Tris-HCl, pH we developed a new approach in which peptide libraries are 7.5, 250 mM. NaCl, 1 mM DTT, 1.0% NP-40, 8 ug/mL pep first phosphorylated by the kinase of interest, and then treated , 8 ug/mL aprotinin, 8 ug/mL leupeptin, 2 mMNa3VO4. with methanolic HCl to convert Asp and Glu residues to their 10 mM NaF, and 1 uM microcystin, and Chk1 was purified 65 uncharged methyl esters. Using this approach, the back using glutathione beads. Chk1 was eluted from the beads with ground of nonphosphorylated peptides that adhere to the 10 mM glutathione in 50 mM Tris-HCl, pH 8.0, and dialyzed IMAC column was reduced to insignificant levels. Further US 9,023,787 B2 31 32 more, the Asp and Glu methyl esters were converted back to ten, and twenty minutes. MAPKAP kinase-2 phosphoryla their free acids during the sequencing reaction, allowing tion of MK2tides was performed using peptide concentra selection for these residues, if present in the phosphorylation tions of 5, 10, 20, 40, 80, 160,320, 500, and 1000M, with motif, to be accurately measured. time points taken at three, six, nine, and twelve minutes. From For the MAPKAP kinase-2, Chk1, and Chk2 peptide these enzymatic studies, K. V, and V/K. Values were library experiments, 40 ul of thionyl chloride was added then ascertained. All kinetic experiments were performed a dropwise in a hood to 1 ml of dry methanol. This solution was minimum of three times. For each experimental condition in then used to dissolve each of the dried peptide libraries, the determination of the K, and V values, we verified that followed by stirring at room temperature for one hour. The the reaction rates were linear with respect to time for all peptide library was dried down overnight and resuspended in 10 substrate concentrations and that less than 10% substrate was 100 ul of a 1:1:1 mixture of methanol/acetonitrile/water. A 0.5 phosphorylated. ml iminodiacetic acid column was charged with 2.5 ml of 20 In vitrokinase assays for UCN-01 ICso determination were mM FeCl and extensively washed with HO, then with 3 ml performed in 30 ul reactions containing 20 mM HEPES (pH of 500 mM NHHCO, (pH 8.0), 3 ml of HO, and 3 ml of 50 7.5), 10 mM MgCl2, 3 mM 2-mercaptoethanol, 100 g/ml mM MES, pH 5.5, 1 M NaCl. The peptide mixture was 15 BSA, 50 mM ATP, 10 uCi 32P-Y-ATP, and 50 uM. MK2-tide applied and the column was developed with 4 ml of HO substrate for twenty minutes at 30° C. Chk1 was used at a followed by 3 ml NHHCO, pH 8.0, to remove non-phos concentration of 0.3 M: MAPKAP kinase-2 was used at a phorylated peptides. Phosphorylated peptides were eluted concentration of 0.1 uM. Reactions were terminated by add with 2 ml of 500 mM NHHCO, pH 11.0, dried in a Speed ing an equal Volume of 0.5% phosphoric acid to the reaction Vac apparatus, and resuspended in 40-80 ul H.O. and 5ul was spotted onto P81 paper. Afterwashing 5x in 0.5% Following IMAC purification, libraries (0.5-1.5 nmoles) phosphoric acid, sample were subjected to Scintillation were subjected to automated Edman sequencing using an counting. Cdc25A phosphorylation studies were performed Applied Biosystems model 477A peptide sequencer. Data using GST-Cdc25A immunoprecipitated from HEK293T analysis was performed by normalizing the abundance (mol cells transfected with pCMV GST-Cdc25A, a generous gift %) of each amino acid in the phosphorylated peptide mixture 25 from Dr. W. Harper (Harvard Medical School). In brief, to that present in the starting libraries. The sums of the final HEK293T cells were transfected with pCMV GST-Cdc25A preference ratios were normalized to the total number of construct using the calcium phosphate method described ear amino acids in the degenerate positions within the peptide lier. Cells were harvested thirty-six hours later, lysed in a libraries so that a particular amino acid would have a prefer buffer containing 50 mM Tris-HCl, pH 7.8, 150 mM. NaCl, ence value of 1 in the absence of selectivity at a particular 30 1.0% NP-40, 5 mM EDTA, 2 mM DTT, 8 ug/ml pepstatin, 8 position. The degenerate peptide libraries used for in vitro ug/ml aprotinin, 8 g/ml leupeptin, 2 mM Na3VO4, 10 mM kinase screening with p38 MAP kinase, MK2, Chk1, and NaF. and 1 LM microcystin and cleared by centrifugation. Chk2 consisted of the sequences GAXXXXSPXXXXAKKK Supernatants were precleared with protein G beads for one ISP library (SEQ ID NO: 19), where X denotes all amino hour. GST-Cdc25A was precipitated with 50 ul GSH beads acids except Cys, Ser, Thr, and Tyr; GAXXXXPX 35 (Sigma-Aldrich). Beads were washed five times in kinase SPXXXXXAKKKPxSP library (SEQID NO:20), where X buffer and used in kinase reactions. Kinase reactions were denotes all amino acids except Cys; or GAXXXXRXX performed in 501 of kinase reaction buffer using 0.3 uMChk1 SXXXXAKKK RXXS library (SEQ ID NO: 21), where X and 0.1 uMMAPKAP kinase-2. Reactions were performed at denotes all amino acids except Cys, Ser. Thr and Tyr. In all 30°C. for twenty minutes and terminated by addition of 50 ul libraries, S denotes Ser, P denotes Pro, and R denotes Arg. 40 2x sample buffer. Samples were heated at 95° C. for three Kinase reactions were performed in 30 ul of kinase reac minutes, separated on a 12.5% SDS-PAGE, and visualized tion buffer (20 mM HEPES, pH 7.5, 10 mM MgCl, 3 mM using a phosphor imager (Molecular Dynamics). 2-mercaptoethanol, 100 ug/ml BSA, 50 uM ATP, 10 uCi 14-3-3 Pull-Down Assays, Immunoblotting, and Immun 32P-Y-ATP) containing 2.0 ug of recombinant p47 or Cdc25B ofluorescence. substrate protein or the specified amount of peptide and 0.10 45 U2OS cells were lysed in lysis buffer: 50 mM Tris/HCl, ug of recombinant p38C. SAPK or 0.03 ug of recombinant pH7.8, 150 mM NaCl, 1.0% NP-40, 5 mM EDTA, 2 mM MAPKAP kinase-2 at 30° C. for the indicated time. The DTT, 8 Lug/ml pepstatin, 8 g/ml aprotinin, 8 Lug/ml leupeptin, sequences of the p38 optimal peptide and the p47phoX pep 2 mM NaVO, 10 mM NaF. 1 uM microcystin for twenty tide were KKAZGPQGPQSPIE (SEQID NO: 22) and KKA minutes at 4° C. Clarified lysates (0.5-2 mg protein) were ZGPQSPGSPLE (SEQID NO. 23), respectively. For 14-3-3 50 incubated with 20 uL glutathione beads or amylose beads pulldowns of Cdc25B following in vitro phosphorylation by containing 10-20 lug 14-3-3-GST or 14-3-3-MBP, respec p38 or MAPKAP kinase-2, 2.0 kg of Cdc25B was incubated tively, for 120 minutes at 4°C. Following washing, lysates with 10-fold excess 14-3-3-MBP and analyzed by autorad and bead-bound proteins were analysed by SDS-PAGE, fol iography. For kinetic measurements, the reactions were ter lowed by transfer to PVDF membranes and immunoblotted minated by the addition of an equal volume of 0.5 percent 55 with the indicated antibodies. For immunofluorescence phosphoric acid, and 5ul was spotted onto p81 paper. The p81 experiments, U2OS cells were seeded onto 18 mm cover paper was washed 5x in 0.5 percent phosphoric acid and slips, irradiated or mock-treated, fixed, extracted, and immu added to scintillation fluid for scintillation counting. For in nostained as described previously (Clapperton et al., Nat. vitro phosphorylation reactions, the reactions were termi Struct. Mol. Biol., 11:512-518, 2004). nated by the addition of an equal volume of sample buffer 60 FACS Analysis. followed by heating at 95°C. for 3 min. Samples were ana UV irradiation was performed at 254 nm (UV-C) using a lyzed by SDS-PAGE followed by transfer to nitrocellulose for Stratalinker 2400 (Stratagene). U2OS cells were fixed in 70% autoradiography and immunoblotting. The rate of p38C, phos ethanol overnight at -20°C., permeabilized with PBS con phorylation of isolated peptides and full-length p47phoX pro taining 0.2% Triton X-100 for twenty minutes at 4° C. teins was determined by Scintillation counting using peptide 65 blocked with 2% FBS in PBS, and incubated with 1 lug of concentrations of 100, 400, and 1400M, and protein concen anti-phospho-histone H3 per 10° cells for sixty minutes on trations of 1, 5, 10 and 15 M, with time points taken at five, ice. Following washing, cells were incubated with FITC US 9,023,787 B2 33 34 conjugated goatanti-rabbitantibody (diluted 1:500) for thirty kinase-2 or luciferase. 10° cells were injected into the flanks minutes on ice, washed, and resuspended in PBS containing of nude mice as above, and tumors were allowed to form for 50 ug/ml PI for twenty minutes immediately prior to FACS twelve days. Mice were then treated with either cisplatin (2 analysis. Analysis was performed using a Becton Dickinson mg/kg, intraperitoneal administration 3x per week) or doxo 5 rubicin (4 mg/kg, intraperitoneal administration 3x per FACS machine with CellOuest software. week), monitored for a total of twenty-six days, and then For BrdU incorporation experiments, cells were incubated sacrificed. Tumor diameter was measured periodically during with 30 M BrdU for the indicated times, then fixed and growth and tumors were weighed at the endpoint. Experi permeabilized as above. Cells were denatured in 2N HCl for ments were performed in quadruplicate, and data plotted as twenty minutes at room temperature, neutralized with 0.1M sample means with error bars showing standard deviation. NaBO, (pH 8.5), blocked with 2% FBS in PBS, and incu 10 Structural Modeling. bated with a murine anti-BrdU antibody for sixty minutes on Activated MAPKAP kinase-2 (phosphorylated on Thr ice. Following washing, cells were incubated with FITC 222) to was modeled using the crystal structure of the ADP conjugated goat anti-mouse antibodies and PI as above. complex (Underwood et al., Structure, 11:627-636, 2003) Analysis was performed using a Becton Dickinson FACS with the activation loop (residues 213 to 241) deleted and machine with CellOuest software. 15 rebuilt using the corresponding region (residues 299 to 328) Clonogenic Survival Assay. from the structure of activated Akt/PKB in complex with Cells were either mock-treated or treated with increasing AMP-PNP and GSK3-peptide (Yang et al., Nat. Struct. Biol., doses of doxorubicin or cisplatin. After eight hours of treat 9:940-944, 2002) as a template. A substrate peptide, ment, cells were washed three times with growth media and GRPRTTSFAE (SEQ ID NO. 5), was modeled in the active three times with PBS, trypsinized and replated at a concen site. An optimal peptide, LQRQLSIA (SEQID NO: 6), was tration of 5000 cells/10 cm dish. After eight days, cells were modeled in the active site based on the GSK3-peptide. Coor fixed and stained with 0.1% crystal violet (Sigma-Aldrich). dinates for the activated MAPKAP kinase-2/peptide complex Colonies consisting of >50 cells were counted, and Surviving are listed in Table 1 in standard Protein Data Bank (PDB) fractions were determined by normalization against untreated format (details about the Protein Data Bank and the associ cells. Experiments were performed in triplicate and are plot 25 ated format for coordinates may be found in Berman et al., ted as mean values with standard deviations indicated by the Nuc. Acids Res., 28:235-242, 2000). Table 2 lists pairs of error bars. atoms in the complex that form the closest protein-peptide Murine Tumor Models. contacts and that are useful for designing or identifying addi H-Ras-V 12-transformed p53 MEFs were used for in tional molecules that bind in the active site. A substrate pep vivo tumor formation assays. Cells were transfected with tide, LYRSPSMPL (residues 211-219 of human Cdc25C) siRNA oligonucleotides against GFP or murine MAPKAP (SEQIDNO: 7) in the Chk1 active site was similarly modeled kinase-2 for forty-eight hours, then mock treated or incubated using the GSK3-peptide as a template and manually adjusted with 0.1 uM doxorubicin or 1 uM cisplatin for eight hours, to resemble the published model (Chen et al., Cell, 100:681 washed three times in growth media, three times in PBS, 692, 2000). Structures were superimposed using ALIGN and trypsinized, and resuspended at a concentration of 10 cells/ 35 SUPERIMPOSE. Manual adjustments of the models were ml in PBS. 10° cells were subcutaneously injected into the made using XFIT from the XtalView suite. flanks of nude mice (Ncr nu/nu, Taconic). The structure of MAPKAP kinase-2 bound to UCN-01 was For tumor regression assays, H-Ras-V12 transformed modeled using PyMOL with the structure of MAPKAP p53 MEFs were stably transfected with a lentiviral transfer kinase-2 bound to staurosporine (PDB ID 1NXK) as a base vector encoding for shRNA targeting either MAPKAP model. TABLE 1. Coordinates of the activated MAPKAP kinase-2, peptide complex ATOM 1 N PHE A 46 214.820 109.707 179.069 1.OO 118.35 N ATOM 2 CA PHE A 46 214.336 108.388 178.678 1.OO 109.39 C ATOM 3 C PHE A 46 215.483. 107.382 178. SS6 1.OO 92.52 C ATOM 4 O PHE A 46 216.008 107.116 177483 1.OO 93.11 O ATOM 5 CB PHE A 46 213.617 108.523, 177.33S 1.OO 106.31 C ATOM 6 CG PHE A 46 212.712 107.345 177.122 1.OO 100.53 C ATOM 7 CD1 PHE A 46 213.246 106.138 176.688 1.OO 94.28 C ATOM 8 CD2 PHE A 46 211.347 107.468 177.345 1.OO 81.06 C ATOM 9 CE1 PHE A 46 212.408 105.049 176.48O 1.OO 71.80 C ATOM 10 CE2 PHE A 46 21 O.S1S 106.37O 177.13S 1.OO 83.85 C ATOM 11 CZ PHE A 46 211.041. 105.159 176.704 100 S4.26 C ATOM 12 N HIS A 47 215.896 106.847 179.719 1.OO 99.95 N ATOM 13 CA HIS A 47 216.976 105.867 179.71S 1.OO 110.68 C ATOM 14 C HIS A 47 216.467 104.473 179.342 1.OO 99.74 C ATOM 15 O HIS A 47 215.591 103.903 179.979 1.OO 108.34 O ATOM 16 CB HIS A 47 217.598 105.835 181.111 100 128.34 C ATOM 17 CG HIS A 47 217.973 107.234 181.527 1.OO 172.75 C ATOM 18 ND1 HIS A 47 219.157 107.813 181.211 1.OO 209.78 N ATOM 19 CD2 HIS A 47 217.216 108.147 182.270 100 195.SS C ATOM 2O CE1 HIS A 47 219.113 109.047 181749 1.OO 214.52 C ATOM 21 NE2 HIS A 47 217.963 109.275 182.389 1.OO 216.85 N ATOM 22 N VAL A 48 217.023 103.942 178.238 1.OO 93.29 N ATOM 23 CA VAL. A 48 216.62O 102.613 177.798 1.OO 69.76 C ATOM 24 C VAL A 48 217.795 101.841 177.194 1.OO 72.39 C ATOM 25 O VAL A 48 218.595 102.37O 176.434 1.OO 67.87 O ATOM 26 CB VAL. A 48 21S.S10 102.77O 176.757 1.OO 60.38 C

US 9,023,787 B2 37 38 TABLE 1-continued Coordina es of the activated MAPKAP kinase-2, peptide complex A TOM 05 CA LE 59 229.569 82.753 65.228 OO 54.79 A TOM O6 C LE 59 231.007 82.870 64.670 OO 63.43 A TOM O7 O LE 59 231.766 83.777 65.035 OO 47.27 A TOM O8 CB LE 59 229.605 82,094 66.638 OO 53.52 A TOM 09 CG1 ILE 59 230.284 83.012 67.644 OO SS.42 A TOM 10 CG2 ILE 59 230.376 80.791 66.588 OO 40.15 A TOM 11 CD1 ILE 59 230.311 82.446 69.038 OO 57.36 A TOM 12 N LE 60 231.375 81956 63.777 OO 43.08 A TOM 13 CA LE 60 232.743 81.902 63.274 OO 57.78 A TOM 14 C LE 60 233.086 83.151 62.458 OO 60.65 A TOM 15 O LE 60 234.226 83.590 62.377 OO 72.20 A TOM 16 CB LE 60 232.866 80.659 62.393 OO 38.44 A TOM 17 CG1 ILE 60 231.615 8O.S13 61.518 OO 50.72 A TOM 18 CG2 ILE 60 232.96S 79.403 63.277 OO 49.86 A TOM 19 CD1 ILE 60 231.798 79.479 60.407 OO 46.70 A TOM 2O N ASP 61 232,044 83.704 61811 OO 32.79 A TOM 21 CA. ASP 61 232.254 84863 60.953 OO 58.81 A TOM 22 C ASP 61 232.607 86.117 61.758 OO SO.10 A TOM 23 O ASP 61 233.397 86.956 61.346 OO 78.45 A TOM 24 CB ASP 61 230.972 85.093 60.149 OO 36.9S A TOM 25 CG ASP 61 230.476 83.758 59.608 OO 91.42 A TOM 26 OD1 ASP 61 229.268 83.636 59.400 OO 76.09 A TOM 27 OD2 ASP 61 231.293 82.864 59.405 OO 122.26 A TOM 28 N ASP 62 231.9S4 86.2SO 62.929 OO 62.40 A TOM 29 CA. ASP 62 232.213 87422 63.761 OO 63.46 A TOM 30 C ASP 62 233.328 87.164 64.778 OO 43.90 A TOM 31 O ASP 62 233.994 88.071 65.261 OO 7439 A TOM 32 CB ASP 62 230.918 87.786 64.492 OO 33.21 A TOM 33 CG ASP 62 229.843 88.13S 63.473 OO 76.8O A TOM 34 OD1 ASP 62 230.116 88.979 62.618 OO 99.65 A TOM 35 OD2 ASP 62 228.754 87.573 63S46 OO 84.30 A TOM 36 N TYR 63 233.493 85.877 65.132 OO 4470 A TOM 37 CA TYR 63 234.482 85.539 66.148 OO 62.53 A TOM 38 C TYR 63 235.5O2 84519 65.641 OO 58.43 A TOM 39 O TYR 63 235.388 83.956 64,561 OO 79.42 A TOM 40 CB TYR 63 233.742 84.96S 67.358 OO 50.79 A TOM 41 CG TYR 63 233.100 86.O68 68.120 OO 44.26 A TOM 42 CD1 TYR 63 233.802 86.709 69.136 OO 34.66 A TOM 43 CD2 TYR 63 231.800 86.477 67.81.1 OO 37.87 A TOM CE1 TYR 63 233.220 87.758 69.831 OO 27.94 A TOM 45 CE2 TYR 63 231.215 87.521 68.512 OO 34.56 A TOM 46 CZ TYR 63 231916 88.158 69.519 OO 56.82 A TOM 47 OH TYR 63 231.353 89.224 70.192 OO 41.90 A TOM 48 N LYS 64 236.554 84.325 66.45S OO 69.78 A TOM 49 CA LYS 64 237.542 83.306 66.129 OO 60.63 A TOM 50 C LYS 64 237.831 82.42O 67.340 OO 67.37 A TOM 51 O LYS 64 238.259 82.882 68.389 OO 66.71 A TOM 52 CB LYS 64 238.825 84.004 65.673 OO 85.98 A TOM 53 CG LYS 64 240.084 83.232 66.078 OO 68.10 A TOM S4 CD LYS 64 240.235 81.914 65.315 OO 96.O2 A TOM 55 CE LYS 64 241.289 80.993 65.941 OO 159.06 A TOM 56 NZ LYS 64 241-422 79.778 65.141 OO 151.29 A TOM 57 N VAL 65 237.271 81240 67.130 OO 54.82 A TOM 58 CA VAL 65 237.148 80.288 68.220 OO 65.09 A TOM 59 C VAL 65 238.437 79.492 68.298 OO 65.21 A TOM 60 O VAL 65 238.945 79.029 67.282 OO 79.23 A TOM 61 CB VAL 65 235.960 79.329 68.000 OO 53.30 A TOM 62 CG1 VAL 65 235.948 78.263 69.075 OO 68.94 A TOM 63 CG2 VAL 65 234.659 80.104 68.039 OO SO.OO A TOM 64 N THR 66 238.971 79.338 69.5O2 OO 60.78 A TOM 65 CA. THR 66 240.214 78.602 69.68O OO 73.39 A TOM 66 C THR 66 240.032 77.346 70.515 OO 76.31 A TOM 67 O THR 66 238.969 77.107 71.08O OO 77.40 A TOM 68 CB THR 66 241.267 79.462 70.376 OO 65.28 A TOM 69 OG1 THR 66 241.150 79.289 71798 OO 71.85 A TOM 70 CG2 THR 66 241.067 80.944 70.016 OO 64.31 A TOM 71 N SER 67 241.092 76.554 70.602 OO 86.86 A TOM 72 CA SER 67 241.065 75.318 71.369 OO 86.84 A TOM 73 C SER 67 241.359 75.528 72.838 OO 78.98 A TOM 74 O SER 67 241.140 74.631 73.646 OO 92.44 A TOM 75 CB SER 67 242,082 74.338 70.818 OO 77.74 A TOM 76 OG SER 67 241681 73901 69.540 OO 131.17 A TOM 77 N GLN 68 241.868 76.702 73.185 OO 65.34 A TOM 78 CA GLN 68 242.187 76.981 74.571 OO 79.87 A TOM 79 C GLN 68 240.9SS 76.819 75.448 OO 75.61 A TOM 8O O GLN 68 239.849 77.1.97 75.067 OO 70.47 A TOM 81 CB GLN 68 242.760 78.387 74.7.04 OO 76.67 A TOM 82 CG GLN 68 243.238 78.724 76.100 OO 86.58 US 9,023,787 B2 39 40 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 83 CD GLN 68 244.346 79.757 76.087 OO 151.90 A TOM 84 OE1 GLN 68 244.745 80.271 77.134 OO 147.O3 A TOM 85 NE2 GLN 68 244.860 80.060 74.895 OO 166.06 A TOM 86 N VAL 69 241.15S 76.244 76.625 OO 70.23 A TOM 87 CA VAL 69 240.064 76.O13 77.552 OO 73.71 A TOM 88 C VAL 69 24O157 76.908 78.775 OO 65.57 A TOM 89 VAL 69 241.009 76.704 79.639 OO 83.31 A TOM 90 VAL 69 240.064 74.564 78.023 OO 69.41 A TOM 91 VAL 69 238.896 74.324 78.952 OO 51.08 A TOM 92 VAL 69 240.017 73.648 76.830 OO 57.48 A TOM 93 70 239.276 77.899 78.847 OO 74.02 A TOM 94 CA 70 239.259 78.816 79.977 OO 88.16 A TOM 95 70 238.86S 78.039 81.227 OO 94.93 A TOM 96 70 239.171 78.458 82.343 OO 86.96 A TOM 97 CB 70 238.268 79.956 79.718 OO 80.15 A TOM 98 CG 70 238.660 81.01S 78.68O OO 66.85 A TOM 99 CD1 70 237.42O 81.668 78.097 OO 90.30 A TOM 200 CD2 70 239.553 82,049 79.322 OO 79.20 A TOM 2O1 71 238.194 76.903 81.035 OO 87.34 A TOM 2O2 71 237.781 76.089 82166 OO 87.75 A TOM 2O3 71 236.707 75.077 81.825 OO 83.77 A TOM 204 71 236.158 75.111 80.730 OO 84.32 A TOM 205 72 236.405 74.175 82.757 OO 88.68 A TOM 2O6 72 235.381 73.155 82.534 OO 82.29 A TOM 2O7 72 234.225 73.239 83.509 OO 73.28 A TOM 208 72 234.318 73.871 84.557 OO 95.04 A TOM 209 72 235.976 71.751 82.621 OO 77.46 A TOM 210 72 236.877 71.305 81473 OO 94.19 A TOM 211 CD1 72 237.217 69.818 81.595 OO 139.26 A TOM 212 CD2 72 236.1SO 71.568 80.172 OO 109.03 A TOM 213 73 233.133 72.577 83.158 OO 77.98 A TOM 214 73 231.963 72.592 84.013 OO 71.81 A TOM 215 73 230.762 71853 83.446 OO 99.78 A TOM 216 73 230.843 71171 82.418 OO 93.36 A TOM 217 74 229.63S 72.004 84.136 OO 12.58 A TOM 218 74 228.376 71.365 83.765 OO 14.50 A TOM 219 74 228.103 71.259 82.270 OO 10.24 A TOM 220 74 227.890 70.162 81.750 OO 94.78 A TOM 221 74 227.1.89 72.085 84.439 OO 20.25 A TOM 222 74 227.145 71.707 85.921 OO 27.29 A TOM 223 74 225.889 71.745 83.724 OO 10.29 A TOM 224 74 225.968 72.285 86.673 OO 78.93 A TOM 225 75 228106 72.393 81.58O OO 13.84 A TOM 226 CA 75 227.844 72.395 80.148 OO O8.71 A TOM 227 75 229.044 72.005 79.296 OO OO.S2 A TOM 228 75 228.935 71858 78.079 OO O2.09 A TOM 229 CB 75 227.303 73.756 79.736 OO 95.22 A TOM 230 CG 75 225.898 73.967 80.225 OO 84.98 A TOM 231 OD1 75 224.966 73.315 79.749 OO 88.40 A TOM 232 ND2 75 225.730 74-858 81,199 OO 12.62 A TOM 233 76 230.186 71.820 79.940 OO 99.31 A TOM 234 76 231.360 71.428 79.198 OO 93.20 A TOM 235 76 232489 72428 79.282 OO 96.9S A TOM 236 76 232.666 73.117 80.285 OO O112 A TOM 237 77 233.258 72.506 78.205 OO 82.50 A TOM 238 77 234.386 73.409 78.146 OO 79.18 A TOM 239 77 233.986 74.820 77.757 OO 66.93 A TOM 240 77 233.18.9 75.025 76.852 OO 79.35 A TOM 241 77 23S.410 72.872 77.160 OO 78.70 A TOM 242 78 234.523 75.794 78.470 OO 57.33 A TOM 243 78 234.267 77.179 78.150 OO 49.75 A TOM 244 78 235.512 77.62O 77.410 OO 79.35 A TOM 245 78 236.545 77.888 78.028 OO 64.43 A TOM 246 78 234.138 78.O20 79.386 OO 54.95 A TOM 247 78 234.267 79.482 79.023 OO 45.00 A TOM 248 78 232.810 77.742 80.029 OO S2.90 A TOM 249 79 235414 77.692 76.087 OO 70.72 A TOM 250 CA 79 236.538 78.073 75.245 OO 61.89 A TOM 251 79 236.825 79.563 75.185 OO 64.76 A TOM 252 79 235.930 80394 75.360 OO 63SO A TOM 253 CB 79 236.292 77.599 73.825 OO 51.24 A TOM 254 CG 79 235.907 76.149 73.599 OO 64.90 A TOM 255 CD1 79 235.543 75.983 72.139 OO 49.75 A TOM 256 CD2 79 237.052 75,236 73.978 OO 80.17 A TOM 257 8O 238.090 79.889 74.919 OO 72.17 A TOM 258 CA 8O 238SOS 81.274 74.765 OO 77.62 A TOM 259 8O 238.376 81.604 73.288 OO 66.11 A TOM 260 o 8O 238.775 80.813 72.437 OO 64.63 US 9,023,787 B2 41 42 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 261 CB GLN 8O 239.955 81.486 75.167 OO 52.97 A TOM 262 CG GLN 8O 240.390 82.913 74.863 OO 93.41 A TOM 263 CD GLN 8O 241.715 83.310 75.494 OO 115.14 A TOM 264 OE1 GLN 8O 242.038 84SO2 75.567 OO 116.65 A TOM 26S NE2 GLN 8O 242.490 82.322 75.949 OO 117.09 A TOM 266 N 81 237.792 82.754 72.982 OO 57.33 A TOM 267 CA 81 237.629 83.150 71.596 OO 57.32 A TOM 268 C 81 238.109 84.571 71.400 OO S4.90 A TOM 269 O 81 238.310 85.32O 72.362 OO 61.04 A TOM 270 CB 81 236.160 83.072 71.114 OO 62.17 A TOM 271 CG1 81 235.289 84.OSO 71.897 OO S1.03 A TOM 272 CG2 81 235.628 81.679 71.285 OO 42.99 A TOM 273 CD1 81 233.852 84.088 71.391 OO 59.46 A TOM 274 82 238.287 84.930 70.131 OO 70.25 A TOM 275 CA 82 238.753 86.251 69.763 OO 55.36 A TOM 276 82 237.815 86.911 68.787 OO 62.8O A TOM 277 C 82 237.287 86.260 67.874 OO 65.67 A TOM 278 CB 82 240.145 86.152 69.152 OO 42.74 A TOM 279 CG 82 241.197 85.862 70.151 OO 59.25 A TOM 28O CD1 82 241639 86.857 70.999 OO 44.66 A TOM 281 CD2 82 241.687 84.576 70.314 OO 37.34 A TOM 282 CE1 82 242.SS2 86.576 71.999 OO 57.81 A TOM 283 CE2 82 242.604 84.286 71.315 OO 54.82 A TOM 284 CZ 82 243.032 85.287 72.156 OO 58.83 A TOM 285 83 237.592 88.205 68.997 OO 52.27 A TOM 286 CA 83 236.746 88.9S4 68.104 OO 66.04 A TOM 287 83 237.605 89.492 66.971 OO 53.09 A TOM 288 83 238,453 90.346 67.171 OO 64.85 A TOM 289 CB 83 236.093 90.099 68.830 OO 47.48 A TOM 290 CG 83 23S.214 90.902 67.924 OO 67.70 A TOM 291 OD1 83 235.689 91.744 67.160 OO 72.99 A TOM 292 ND2 83 233.915 90.632 67.977 OO 70.38 A TOM 293 84 237.383 88.974 65.774 OO 63.87 A TOM 294 CA 84 238.133 89.376 64,590 OO 68.2O A TOM 295 84 238.366 90.876 64450 OO 61.51 A TOM 296 84 239.492 91.340 64,537 OO 74.81 A TOM 297 CB 84 237.412 88.859 63.345 OO 55.26 A TOM 298 CG 84 237.312 87.343 63.283 OO 33.38 A TOM 299 CD 84 236.425 86.918 62.154 OO 30.89 A TOM 300 CE 84 236.759 85.521 61.719 OO 46.41 A TOM 301 NZ E. s 84 236.028 85.182 60.476 OO 72.55 A TOM 3O2 85 237.290 91.622 64235 OO 63.12 A TOM 303 CA 85 237.359 93.066 64.043 OO S1.69 A TOM 304 85 237.905 93.905 65.183 OO S8.02 A TOM 305 85 238.197 95.075 64991 OO 86.09 A TOM 306 CB 85 235.978 93.589 63.647 OO 60.23 A TOM 307 CG 85 235.862 95.086 63.509 OO 76.93 A TOM 3O8 CD 85 23S.S16 95.725 64.835 OO 84.92 A TOM 309 NE 85 234.121 96.140 64.907 OO 93.42 A TOM 310 CZ 85 233.541 96.634 65.997 OO 103.30 A TOM 311 NH1 85 234.224 96.777 67.128 OO S4O2 A TOM 312 NH2 85 232.268 96.998 65.949 OO 119.53 A TOM 313 86 238. OSO 93.339 66.370 OO 64.72 A TOM 314 CA 86 238.565 94.123 67.489 OO 54.27 A TOM 315 86 239.717 93.398 68.140 OO 52.40 A TOM 316 86 24O.S2O 93.980 68.856 OO 57.78 A TOM 317 86 237,468 94.392 68.536 OO 58.63 A TOM 3.18 86 236.471 95.246 67.968 OO 54.24 A TOM 319 86 238.045 95.084 69.743 OO 98.48 A TOM 320 N 87 239.772 92.106 67.883 OO 54.70 A TOM 321 CA 87 240.819 91.260 68.397 OO 49.69 A TOM 322 87 240.875 91131 69.927 OO 63.54 A TOM 323 87 241.936 90.881 70.491 OO 78.48 A TOM 324 CB 87 242.148 91.761 67.854 OO 44.04 A TOM 325 CG 87 243.145 90.675 67.561 OO 87.54 A TOM 326 CD 87 242.722 89.811 66.413 OO 70.54 A TOM 327 OE1 87 243.394 88.840 66.083 OO 95.76 A TOM 328 NE2 87 241.601 90.157 65.789 OO 75.13 A TOM 329 88 239.746 91.291 70.608 OO 57.33 A TOM 330 CA 88 239.751 91.135 72.060 OO 69.04 A TOM 331 88 239.352 89.723 72.442 OO 68.23 A TOM 332 88 238.622 89.060 71.702 OO 70.57 A TOM 333 CB 88 238.816 92.138 72.708 OO 73.60 A TOM 334 CG 88 239.297 93.538 72.480 OO 111.93 A TOM 335 CD 88 240.559 93.845 73.228 OO 130.01 A TOM 336 OE1 88 240.728 93.298 74.344 OO 151.01 A TOM 337 U 88 241.376 94.634 72.698 OO 169.07 A TOM 338 LYS 89 239.837 89.261 73.592 OO 80.21 US 9,023,787 B2 43 44 TABLE 1-continued Coordina es of the activated MAPKAP kinase-2, peptide complex A TOM 339 CA LYS 89 239.543 87.904 74.047 OO 67.45 A TOM 340 C LYS 89 238.171 87.815 74.709 OO 67.74 A TOM 341 O LYS 89 237.719 88.752 75.355 OO 71.95 A TOM 342 CB LYS 89 240.632 87.424 75.013 OO 46.26 A TOM 343 N PHE 90 237.504 86.681 74,537 OO 68.69 A TOM 344 CA PHE 90 236.195 86,481 75.128 OO 63.31 A TOM 345 C PHE 90 235.957 85.019 75.477 OO 70.09 A TOM 346 O PHE 90 236.571 84.121 74.892 OO 76.39 A TOM 347 CB PHE 90 235.100 86.935 74.169 OO 52.49 A TOM 348 CG PHE 90 234.975 88.434 74.031 OO 61.34 A TOM 349 CD1 PHE 90 235.523 89.091 72.952 OO 63.36 A TOM 350 CD2 PHE 90 234.238 89.170 74.942 OO 60.92 A TOM 351 CE1 PHE 90 235.330 90.429 72.781 OO 38.41 A TOM 352 CE2 PHE 90 234.048 90.529 74.762 OO 57.58 A TOM 353 CZ PHE 90 234.591 91.155 73.683 OO SO.60 A TOM 3S4 N ALA 91 235.055 84.788 76.431 OO 67.69 A TOM 355 CA ALA 91 234.692 83.444 76.853 OO 44.28 A TOM 356 C ALA 91 233.480 83.OOO 76.052 OO 45.25 A TOM 357 O ALA 91 232.569 83.788 75.795 OO 60.93 A TOM 358 CB ALA 91 234.360 83443 78.302 OO S2.11 A TOM 359 N LEU 92 233.477 81.736 75.653 OO 48.94 A TOM 360 CA LEU 92 232.381 81.179 74.882 OO SO.98 A TOM 361 C LEU 92 231.771 79.995 7S. 610 OO 66.99 A TOM 362 O LEU 92 232.467 79.045 75.939 OO 82.82 A TOM 363 CB LEU 92 232.886 80.710 73.536 OO 39.13 A TOM 364 CG LEU 92 231.838 79.857 72.839 OO 52.58 A TOM 365 CD1 LEU 92 230.636 80.726 72.548 OO 48.33 A TOM 366 CD2 LEU 92 232.404 79.256 71.557 OO 58.60 A TOM 367 N LYS 93 23O469 80.053 75.856 OO 66.30 A TOM 368 CA LYS 93 229.650 79.022 76.486 OO 62.11 A TOM 369 C LYS 93 228.624 78.440 75.508 OO 61.26 A TOM 370 O LYS 93 227.945 79.148 74.774 OO 77.67 A TOM 371 CB LYS 93 228.936 79.645 77.689 OO 79.47 A TOM 372 CG LYS 93 228.640 78.617 78.785 OO 69.18 A TOM 373 CD LYS 93 227.896 79.231 79.974 OO 74.19 A TOM 374 CE LYS 93 227.871 78.303 81.194 OO 107.78 A TOM 375 NZ LYS 93 228.986 78.626 82.083 OO 92.73 A TOM 376 N MET 94 228.SS1 77.095 75.489 OO S3.94 A TOM 377 CA MET 94 227.639 76.436 74.560 OO 63.34 A TOM 378 C MET 94 226.517 75.693 75.290 OO 67.05 A TOM 379 O MET 94 226.740 74.805 76.101 OO 77.93 A TOM 380 CB MET 94 228.447 75451 73.713 OO 54.57 A TOM 381 CG MET 94 229.751 76.056 73.188 OO 66.95 A TOM 382 SD MET 94 230.855 74.810 72.511 OO 128.32 A TOM 383 CE MET 94 231.935 75.912 71.587 OO 110.84 A TOM 384 N LEU 95 225.273 76.1.16 74.999 OO 76.30 A TOM 385 CA LEU 95 224.126 7S.468 75.626 OO 47.37 A TOM 386 C LEU 95 223.284 74.703 74.601 OO 61.07 A TOM 387 O LEU 95 223.143 75.102 73.452 OO 66.44 A TOM 388 CB LEU 95 223.276 76.549 76.295 OO 56.39 A TOM 389 CG LEU 95 224.057 77.342 77.346 OO 78.66 A TOM 390 CD1 LEU 95 223.138 78.105 78.302 OO 71.39 A TOM 391 CD2 LEU 95 224.941 76.453 78.221 OO 68.93 A TOM 392 N GLN 96 222.574 73.621 74.890 OO 60.77 A TOM 393 CA GLN 96 221.662 73.075 73.906 OO 63.29 A TOM 394 C GLN 96 220.493 74.049 73.876 OO 63.17 A TOM 395 O GLN 96 2 9.998 74.481 74.915 OO 63.30 A TOM 396 CB GLN 96 21.193 71.700 74.326 OO 70.27 A TOM 397 N ASP 97 2O.OS6 74.415 72.686 OO 59.87 A TOM 398 CA. ASP 97 8.9SO 75.343 72.589 OO S8.98 A TOM 399 C ASP 97 7.669 74.640 73.022 OO 73.48 A TOM 400 O ASP 97 7.277 73.626 72.448 OO 75.22 A TOM 401 CB ASP 97 8.831 7S.843 71.157 OO 68.14 A TOM 402 CG ASP 97 7.933 77.038 71.045 OO 73.65 A TOM 403 OD1 ASP 97 7.516 77.567 72.096 OO 68.14 A TOM 404 OD2 ASP 97 7.647 77.451 69.906 OO 80.65 A TOM 40S N CYS 98 7.023 75.189 74041 OO 67.24 A TOM 406 CA CYS 98 S.801 74.626 74.586 OO 52.14 A TOM 407 C CYS 98 5.258 75.697 75.510 OO 75.99 A TOM 408 O CYS 98 6.018 76.483 76.068 OO 86.44 A TOM 409 CB CYS 98 6.122 73.415 75.421 OO 52.47 A TOM 410 SG CYS 98 6.922 73906 76.969 OO 68.25 A TOM 411 N PRO 99 3.939 75.722 75.717 OO 81.29 A TOM 412 CA PRO 99 3.283 76.705 76.578 OO 85.87 A TOM 413 C PRO 99 3.996 77.083 77.868 OO 82.76 A TOM 414 O PRO 99 4.153 78.268 78.149 OO 67.85 A TOM 415 CB PRO 99 1926 76.073 76.821 OO 94.07 A TOM 416 CG PRO 99 1642 754.52 75.492 OO 91.19

US 9,023,787 B2 47 48 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 495 C 09 220.952 90.440 80.4O1 OO 49.86 A TOM 496 O 09 221.365 91.583 80.231 OO 77.39 A TOM 497 CB 09 221.OOO 88.905 78.423 OO 58.21 A TOM 498 CG 09 220.711 90.158 77.691 OO 62.11 A TOM 499 CD1 09 219493 90.874 77.686 OO 77.34 A TOM 500 CD2 09 221631 90.902 76.855 OO 77.76 A TOM 5O1 NE1 09 219.557 92.006 76.931 OO 78.01 A TOM 502 CE2 09 220.933 92.041 76.375 OO 87.90 A TOM 503 CE3 09 222.9S4 90.7OO 76.473 OO 105.10 A TOM SO4 CZ2 09 221.572 92.943 75.540 OO 127.04 A TOM 505 CZ3 09 223.594 91.606 75.640 OO 107.99 A TOM SO6 CH2 09 222.895 92.731 75.167 OO 111.87 A TOM 507 N 219.845 90.163 81,113 OO 63.80 A TOM SO8 CA 219.102 91.243 81.753 OO 60.39 A TOM 509 C 219.807 91.738 83.019 OO 56.74 A TOM 510 O G 219416 92.713 83.648 OO 72.39 A TOM 511 CB 217.714 90.704 82.108 OO 55.11 A TOM 512 CG 216.630 91.781 82.038 OO 70.18 A TOM 513 CD 215.241 91.177 81.803 OO 12988 A TOM S14 NE 214.340 91SOO 82.914 OO 98.93 A TOM 515 CZ 214.072 90.515 83.794 OO 97.18 A TOM S16 NH1 213.537 90.802 84.969 OO 106.39 A TOM 517 NH2 214.325 89.245 83.464 OO 84.21 A TOM S18 220.864 90.997 83.409 OO 61.92 A TOM 519 221.578 91.358 84.628 OO 62.44 A TOM 52O 223.023 91.776 84.343 OO 78.96 A TOM 521 223.808 92.067 85.237 OO 57.41 A TOM 522 221.559 90.151 85.567 OO 53.16 A TOM 523 S Elf 223.377 91.758 83.046 OO 76.17 A TOM 524 224.737 92.128 82.670 OO 71.16 A TOM 525 224.979 93.632 82.82S OO 64.82 A TOM 526 226.104 94.113 82.806 OO 63.34 A TOM 527 224.963 91.711 81.216 OO 45.39 A TOM 528 224.992 90.282 81.135 OO 81.43 A TOM 529 223.918 94.403 82.989 OO 73.53 A TOM 530 224.071 95.835 83.163 OO 77.61 A TOM 531 224.644 96.132 84.545 OO 70.17 A TOM 532 224.884 97.28O 84.884 OO 88.91 A TOM 533 222.716 96.537 83.006 OO 79.72 A TOM 534 221.947 96.134 81.754 OO 117.37 A TOM 535 222.616 96.582 80.448 OO 132.38 A TOM 536 OE1 222.305 96.060 79.363 OO 85.14 A TOM 537 NE2 223.522 97.558 80.542 OO 73.13 A TOM 538 224.854 95.098 85.348 OO 77.28 A TOM 539 CA 225.395 95.28O 86.692 OO 65.33 A TOM S4O 226.896 95.029 86.7SO OO 66.81 A TOM 541 227.365 93.910 86. SS3 OO 70.94 A TOM S42 CB 224.699 94.346 87.679 OO 68.99 A TOM 543 SG 225.606 94.158 89.225 OO 70.19 A TOM 544 227.66S 96.073 87.058 OO 57.36 A TOM 545 CA 229.123 96.082 87.171 OO 46.75 A TOM S46 229.770 94.790 87.6SO OO 60.90 A TOM 547 230.735 94.315 87.046 OO 79.32 A TOM S48 CB 229.382 97.226 88.140 OO 46.54 A TOM 549 CG 228.325 98.199 87.764 OO 65.08 A TOM 550 CD 227.093 97.329 87.569 OO 53.52 A TOM 551 229.242 94.217 88.726 OO SO.47 A TOM 552 CA 229.827 93.01S 89.294 OO 62.43 A TOM 553 229.336 91.675 88.787 OO 62.85 A TOM 554 229.654 90.634 89.366 OO 86.03 A TOM 555 CB 229.709 93.085 90.804 OO 66.18 A TOM 556 CG 23 O.S47 94.163 91.405 OO 74.19 A TOM 557 ND1 231.923 94.104 91.422 OO 58.68 A TOM 558 CD2 230.214 95.360 91.945 OO SO.93 A TOM 559 CE1 232.404 95.220 91.945 OO 106.08 A TOM S60 NE2 231.386 95.999 92.269 OO 97.40 A TOM S61 N 228.574 91.693 87.703 OO 58.07 A TOM S62 CA 228.075 90.472 87.097 OO 51.42 A TOM 563 C 228.790 90.375 85.769 OO 57.68 A TOM S64 O 228.912 91.381 85.056 OO 72.95 A TOM 565 CB 226.586 90.572 86.848 OO 6O.OS A TOM 566 CG1 225.867 90.676 88.193 OO 6542 A TOM 567 CG2 226111 89.371 86.OSO OO 56.83 A TOM 568 CD1 22442O 91.064 88.1OO OO 76.8O A TOM 569 N 229.275 89.188 85.427 OO 60.67 A TOM 570 CA 229.979 89.028 84.160 OO 56.50 A TOM 571 C 229.008 89.416 83.044 OO 62.86 A TOM 572 O 227.844 89.008 83.071 OO 618O

US 9,023,787 B2 51 52 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 651 O TYR 28 224.960 70.571 66.956 OO 83.06 A TOM 652 CB TYR 28 226.513 72.996 67.259 OO 73.34 A TOM 653 CG TYR 28 227.849 72.396 67.562 OO 113.80 A TOM 654 CD1 TYR 28 229.OOO 72.939 67.024 OO 118.71 A TOM 655 CD2 TYR 28 227.959 71.253 68.341 OO 128.83 A TOM 656 CE1 TYR 28 230.223 72.372 67.247 OO 148.93 A TOM 657 CE2 TYR 28 229.183 70.667 68.569 OO 160.37 A TOM 658 CZ TYR 28 230.316 71.239 68.018 OO 162.93 A TOM 659 OH TYR 28 231.559 70.688 68.244 OO 162.00 A TOM 660 N ALA 29 226.204 70.240 65.098 OO 112.77 A TOM 661 CA ALA 29 226.016 68.788 65.077 OO 119.60 A TOM 662 C ALA 29 224.556 68.445 64.843 OO 117.04 A TOM 663 O ALA 29 223.981 67.637 65.575 OO 102.48 A TOM 664 CB ALA 29 226.482 68.181 66.398 OO 105.12 A TOM 665 GLY 30 223.957 69.056 63.823 OO 115.24 A TOM 666 GLY 30 222.550 68.810 63.553 OO 115.49 A TOM 667 GLY 30 221.728 69.2SO 64756 OO 107.44 A TOM 668 GLY 30 22O.SO1 69.259 64.722 OO 95.78 A TOM 669 ARG 31 222.425 69.616 65.828 OO 89.82 A TOM 670 ARG 31 221.791 70,067 67.055 OO 92.75 A TOM 671 ARG 31 221.754 71.593 67.105 OO 80.22 A TOM 672 ARG 31 222.756 72.265 66.892 OO 63.19 A TOM 673 ARG 31 222546 69.516 68.271 OO 79.11 A TOM 674 LYS 32 22O.S74 72.121 67.381 OO 68.38 A TOM 675 LYS 32 22O349 73.553 67.487 OO 64.95 A TOM 676 LYS 32 220.944 74.O13 68.828 OO 56.62 A TOM 677 LYS 32 22O449 73.648 69.889 OO 63.01 A TOM 678 LYS 32 218.839 73.782 67.429 OO 50.70 A TOM 679 LYS 32 218.340 75.188 67.516 OO 60.30 A TOM 68O LYS 32 216.821 75.123 67.476 OO 72.13 A TOM 681 LYS 32 216.171 76.486 67.371 OO 106.61 A TOM 682 LYS 32 214692 76.3S4 67.313 OO 65.96 A TOM 683 CYS 33 222O11 74.805 68.787 OO 77.15 A TOM 684 CYS 33 222.648 75.263 70.022 OO 74.16 A TOM 685 CYS 33 222.555 76.745 70.32O OO 65.05 A TOM 686 CYS 33 222.157 77.543 69.471 OO 81:10 A TOM 687 CYS 33 224.114 74.877 70.024 OO 92.23 A TOM 688 33 224.343 73.126 69.850 OO 106.91 A TOM 689 34 222.947 77.099 71.540 OO 67.21 A TOM 690 34 222.921 78.478 71.986 OO 67.45 A TOM 691 34 224.326 78.86S 72.431 OO 63.74 A TOM 692 34 224.804 78.446 73.488 OO 71.31 A TOM 693 34 221.953 78.634 73.15.2 OO S1.96 A TOM 694 34 221.107 79.902 73.18.9 OO 72.36 A TOM 695 CD1 34 220.129 79.885 72.025 OO 66.47

A TOM 696 CD2 34 220.342 79.981 74SO3 OO 63.46 A TOM 697 s 35 224.992 79.663 71.608 OO 69.76 A TOM 698 35 226.338 80.103 71.910 OO 48.46 A TOM 699 35 226.262 81.450 72.608 OO 6O20 A TOM 700 35 225.639 82.381 72.101 OO 49.35 A TOM 701 35 227.130 80.192 70.609 OO 69.61 A TOM 702 35 227.191 78.851 69.873 OO 59.09 A TOM 703 CD1 35 227.585 79.058 68.425 OO 80.07 A TOM 704 CD2 35 228.173 77.943 70.579 OO 92.22 A TOM 705 36 226.896 81.S30 73.775 OO 39.79 A TOM 706 CA 36 226.913 82.744 74.581 OO 48.18 A TOM 707 36 228.321 83.324 74.691 OO 54.20 A TOM 708 36 229.219 82.672 75.204 OO 63.12 A TOM 709 36 226.398 82.453 76.006 OO 57.17 A TOM 710 36 224.997 81.864 75.934 OO 54.12 A TOM 711 36 226.364 83.720 76.829 OO 45.37 A TOM 712 36 224.893 80.475 76.545 OO 94.13 A TOM 713 VAL 37 228.527 84.539 74.200 OO 47.11 A TOM 714 CA VAL 37 229.812 85.185 74.376 OO 51.92 A TOM 715 VAL 37 229.827 86.08.1 75.615 OO 62.30 A TOM 716 VAL 37 228.925 86.871 75.875 OO 65.38 A TOM 717 VAL 37 230.091 85.979 73.099 OO 55.29 A TOM 718 VAL 37 231.461 86.640 73.162 OO 49.20 A TOM 719 VAL 37 230.064 85.035 71.910 OO 33.49 A TOM 720 MET 38 230.893 85.889 76.417 OO 63.64 A TOM 721 CA MET 38 231.026 86.598 77.685 OO 47.48 A TOM 722 MET 38 232.339 87.381 77.769 OO 63.41 A TOM 723 MET 38 233.371 86.975 77.246 OO 60.31 A TOM 724 CB MET 38 231,044 85.548 78.796 OO 76.63 A TOM 725 CG MET 38 229.707 84.836 78.96S OO 72.52 A TOM 726 SD MET 38 229.779 83.570 80.236 OO 81.89 A TOM 727 CE MET 38 228.109 82.937 80.029 OO 180.26 A TOM 728 GLU 39 232.281 88.555 78.427 OO 44.14 US 9,023,787 B2 53 54 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 729 CA GLU 39 233.550 89.154 78.798 OO 46.81 A TOM 730 C GLU 39 234.381 88.099 79.511 OO 61.98 A TOM 731 O GLU 39 233.876 87.285 80.272 OO 68.61 A TOM 732 CB GLU 39 233.291 90.346 79.732 OO 40.2O A TOM 733 CG GLU 39 233.444 89.999 81.216 OO 70.67 A TOM 734 CD GLU 39 233.140 91.231 82.059 OO 76.30 A TOM 735 OE1 GLU 39 231.972 91.5O1 82.306 OO 89.15 A TOM 736 OE2 GLU 39 234.079 91.912 82.459 OO 100.67 A TOM 737 CYS 40 235.664 88.142 79.172 OO 69.16 A TOM 738 CA CYS 40 236.625 87.167 79.666 OO 69.69 A TOM 739 CYS 40 237.322 87.529 80.968 OO 60.14 A TOM 740 CYS 40 238.227 88.349 80.990 OO 82.48 A TOM 741 CB 40 237.676 86.903 78.587 OO 74.24 A TOM 742 SG 40 238.996 85.81.1 79.117 OO 73.10 A TOM 743 41 236.912 86.895 82.OSS OO 71.8O A TOM 744 CA 41 237.524 87.159 83.348 OO 79.OO A TOM 745 41 238.785 86.304 83.509 OO 76.59 A TOM 746 41 238.736 85.081 83.360 OO 77.59 A TOM 747 CB 41 236.523 86.847 84.462 OO 81.97 A TOM 748 CG 41 23S2O3 87.610 84.350 OO 76.77 A TOM 749 CD1 41 234.214 87.100 85.382 OO 96.50 A TOM 750 CD2 41 23S469 89.086 84.53S OO 47.85 A TOM 751 42 239.913 86.940 83.818 OO 90.64 A TOM 752 CA 42 241.158 86.197 83.977 OO 92.77 A TOM 753 42 241.771 86.152 85.366 OO 84.21 A TOM 754 42 242.387 85.153 85.725 OO 120.09 A TOM 755 CB 42 242.2O2 86.698 82.984 OO 92.82 A TOM 756 CG 42 241.951 86.182 81.586 OO 110.94 A TOM 757 OD1 42 241.710 84.961 81.450 OO 82.18 A TOM 758 OD2 42 242.003 86.987 80.632 OO 115.53 A TOM 759 43 241.616 87.220 86.142 OO 76.67 A TOM 760 43 242.178 87.249 87.486 OO 84.99 A TOM 761 43 242.128 85.940 88.269 OO 83.56 A TOM 762 43 242.909 85.744 89.194 OO 94.38 A TOM 763 44 241.216 85.040 87.913 OO 81.49 A TOM 764 44 241.132 83.778 88.621 OO 72.81 A TOM 765 44 240.064 83.784 89.7OO OO 88.08 A TOM 766 LY 44 239.527 84.839 90.045 OO 76.11 A TOM 767 45 239.759 82.603 90.235 OO 72.60 A TOM 768 45 238.746 82.464 91.268 OO 80.92 A TOM 769 45 239. OSO 83.249 92.539 OO 85.24 A TOM 770 45 240.2O6 83446 92.908 OO 76.66 A TOM 771 45 238.543 80.993 91.605 OO 61.62 A TOM 772 45 237.797 80.215 90.539 OO 89.57 A TOM 773 45 238.090 78.727 90.618 OO 126.57 A TOM 774 45 237.257 77.922 90.143 OO 97.67 A TOM 775 45 239.16S 78.367 91.156 OO 103.63 A TOM 776 E. 46 237.988 83.679 93.210 OO 97.07 A TOM 777 46 238.095 84.479 94.419 OO 86.04 A TOM 778 46 239.174 84.06S 95.402 OO 75.32 A TOM 779 46 240.049 84.852 95.732 OO 72.90 A TOM 780 46 236.743 84.S30 95.138 OO 83.71 A TOM 781 46 236.768 85.402 96.395 OO 84.94 A TOM 782 CD1 46 237.338 86.754 96.044 OO 92.90 A TOM 783 CD2 46 235.382 85.551 96.975 OO 116.24 A TOM 784 47 239.127 82.829 95.867 OO 80.55 A TOM 785 CA 47 240.104 82.385 96.847 OO 87.51 A TOM 786 47 241.526 82.244 96.332 OO 92.39 A TOM 787 47 242.478 82546 97.051 OO 104.70 A TOM 788 CB 47 239.611 81.094 97.495 OO 62.88 A TOM 789 CG 47 238.388 81.297 98.330 OO 83.58 A TOM 790 CD1 47 237.514 80.260 98.589 OO 80.71 A TOM 791 CD2 47 238.102 82.557 98.845 OO 82.88 A TOM 792 CE1 47 236.375 80.476 99.342 OO 93.95 A TOM 793 CE2 47 236.971 82.778 99.596 OO SO.19 A TOM 794 CZ 47 236.1OS 81.740 99.844 OO 97.28 A TOM 795 48 241.68O 81.805 95.090 OO 81.24 A TOM 796 CA 48 243.008 81.657 94.525 OO 74.75 A TOM 797 48 243.758 82.970 94.714 OO 83.42 A TOM 798 48 244.679 83.OS3 95.529 OO 88.34 A TOM 799 CB 48 242.902 81301 93.051 OO 76.50 A TOM 800 OG 48 242.158 80.106 92.902 OO 74.94 A TOM 8O1 49 243.347 83.999 93.978 OO 81.76 A TOM 8O2 CA 49 243.974 85.316 94.077 OO 102.63 A TOM 803 ARG 49 244.325 85.650 95.512 OO 107.57 A TOM 804 o ARG 49 245.399 86.169 95.796 OO 115.11 A TOM 805 CB ARG 49 243.038 86.396 93.537 OO 92.17 A TOM 806 CG ARG 49 242.9SO 86.427 92.033 OO 96.27 US 9,023,787 B2 55 56 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 807 CD ARG 49 244.278 86.851 91.433 OO 27.89 A TOM 808 NE ARG 49 244.141 88.079 90.656 OO OS.O3 A TOM 809 CZ ARG 49 243.698 89.227 91.155 OO O6.8O A TOM 810 NH1 ARG 49 243.350 89.307 92.434 OO 91.41 A TOM 811 NH2 ARG 49 243.597 90.293 90.377 OO 81.30 A TOM 812 ILE 50 243.410 85.352 96.423 OO 17.64 A TOM 813 CA ILE 50 243.658 85.636 97.821 OO 11.89 A TOM 814 ILE 50 244.890 84.907 98.32O OO 11.27 A TOM 815 ILE 50 245.841 85.543 98.769 OO 25.08 A TOM 816 ILE 50 242.447 85.262 98.691 OO O2.67 A TOM 817 ILE 50 241.5O1 86.458 OO 81.93 A TOM 818 ILE 50 242.900 84.866 OO 93.62 A TOM 819 ILE 50 241.279 87.159 OO 31.92 A TOM 820 GLN 51 244.898 83.583 OO 93.05 A TOM 821 CA GLN 51 246.045 82.836 OO 12.82 A TOM 822 GLN 51 247.325 83.240 OO 30.96 A TOM 823 GLN 51 248.373 83.378 OO 46.77 A TOM 824 CB GLN 51 245.825 81.329 OO 96.24 A TOM 825 CG GLN 51 246.123 80.751 OO O7.91 A TOM 826 CD GLN 51 246.134 79.231 OO 35.63 A TOM 827 OE1 GLN 51 246.326 78.584 OO 52.64 A TOM 828 NE2 GLN 51 245.929 78.654 OO 63.64 A TOM 829 52 247.241 83452 OO 31.86 A TOM 830 CA 52 248.416 83.833 OO 21.88 A TOM 831 52 248.697 85.331 OO 19.03 A TOM 832 52 249.617 85.765 OO O8.04 A TOM 833 CB 52 248.226 8342O OO 20.82 A TOM 834 CG 52 247.643 82.O15 OO 45.66 A TOM 835 OD1 52 248.152 81.088 OO 61.64 A TOM 836 OD2 52 246.679 81.834 OO 72.99 TER 836 52 A TOM 837 T 59 244.002 91.956 2OS.O11 OO 65.30 A TOM 838 CA t 59 243414 92.956 205.898 OO 99.51 A TOM 839 59 241.893 92.871 205.964 OO 87.22 A TOM 840 59 241.221 92.755 204.944 OO 90.56 A TOM 841 59 243.790 94.404 2O5.473 OO 95.19 A TOM 842 59 243.610 94.560 2O4.061 OO 103.02 A TOM 843 59 245.229 94.718 205.831 OO 121.85 A TOM 844 60 241.360 92.931 2O7.178 OO 90.46 A TOM 845 CA 60 239.921 92.876 2O7.395 OO 95.08 A TOM 846 60 239.243 93.816 2O6.400 OO 97.10 A TOM 847 60 238.191 93.498 205.844 OO 77.77 A TOM 848 60 239.606 93.3O2 208.836 OO 84.OS A TOM 849 60 238.135 93.255 209.218 OO 102.30 A TOM 850 60 237.888 93.636 210.675 OO 107.68 A TOM 851 60 238.512 93.022 211566 OO 102.89 A TOM 852 60 237.063 94.542 210.931 OO 116.03 A TOM 853 61 239.872 94.966 2O6.169 OO 91.94 A TOM 854 CA 61 239.359 95.979 205.252 OO 98.89 A TOM 855 61 239.188 95.480 2O3.826 OO 94.53 A TOM 856 61 238.251 95.874 2O3.129 OO 84.43 A TOM 857 CB 61 240.276 97.204 205.245 OO 94.61 A TOM 858 CG G 61 239.886 98.251 204.206 OO 116.99 A TOM 859 CD 61 240.724 99.506 2O4.347 OO 131.31 A TOM 860 NE 61 240.601 100.069 205.688 OO 142.10 A TOM 861 CZ 61 241.205 101.18O 206.092 OO 132.03 A TOM 862 NH1 61 241.982 101.855 205.257 OO 142.54 A TOM 863 NH2 61 241.026 101.62O 2O7.330 OO 138.86 A TOM 864 62 240.107 94.628 2O3.389 OO 86.92 A TOM 865 CA 62 240,040 94.067 2O2.OSO OO 96.77 A TOM 866 62 238.899 93.063 201.991 OO 93.54 A TOM 867 62 238.101 93.078 201.052 OO 98.52 A TOM 868 CB 62 241.360 93.385 201.699 OO 101.33 A TOM 869 CG 62 242.463 94.360 2O1.317 OO 125.60 A TOM 870 CD 62 243.848 93.755 201448 OO 135.51 A TOM 871 OE1 62 244.823 94.405 201011 OO 144.57 A TOM 872 OE2 62 243.959 92.636 201.999 OO 130.61 A TOM 873 63 238.822 92.199 2O3.002 OO 93.78 A TOM 874 CA 63 237.772 91.187 2O3.071 OO 86.54 A TOM 875 63 236.426 91.889 2O2.976 OO 87.93 A TOM 876 63 235.509 91.418 2O2.297 OO 94.67 A TOM 877 63 237.871 90.419 204.371 OO 72.07 A TOM 878 64 236.319 93.021 2O3.662 OO 75.33 A TOM 879 64 235.098 93.809 2O3.651 OO 81.45 A TOM 880 i 64 234.765 94.222 2O2.223 OO 92.32 A TOM 881 O SER 64 233.708 93.892 2O1.679 OO 89.51 A TOM 882 CB SER 64 23S-281 95.057 2O4.511 OO 61.52 A TOM 883 OG SER 64 234.233 95.977 204.289 OO 85.01 US 9,023,787 B2 57 58 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 884 N GLU 65 235.696 94.949 201622 OO 106.88 A TOM 885 CA GLU 65 235.537 95.436 200.259 OO 107.60 A TOM 886 C GLU 65 23S.138 94.312 99.306 OO 98.22 A TOM 887 O GLU 65 234.309 94.509 OO 80.74 A TOM 888 CB GLU 65 236.834 96.102 OO 117.73 A TOM 889 CG GLU 65 237.296 97.198 OO 121.43 A TOM 890 CD GLU 65 238.640 97.776 OO 129.44 A TOM 891 OE1 GLU 65 239.62O 96.998 OO 132.54 A TOM 892 OE2 GLU 65 238.711 99.007 OO 114.59 A TOM 893 66 235.722 93.134 OO 87.57 A TOM 894 CA 66 235.3S4 92.02O OO 78.81 A TOM 895 66 233.887 91.705 OO 85.13 A TOM 896 66 233.068 91.644 OO 86.94 A TOM 897 66 236.2O7 90.755 OO 76.17 A TOM 898 66 237.628 90.972 OO 76.65 A TOM 899 66 235.567 89.534 OO 77.07 A TOM 900 66 238.532 89.759 OO 56.87 A TOM 901 67 233.546 91.520 OO 80.82 A TOM 902 CA 67 232.166 91.195 OO 77.70 A TOM 903 67 23 228 92.224 OO 73.89 A TOM 904 67 230.152 91.895 OO 72.21 A TOM 905 CB 67 23 978 91.110 OO 67.42 A TOM 906 CG 67 232.544 89.845 2O2.62O OO 65.04 A TOM 907 SD 67 232.087 88.398 201648 OO 75.11 A TOM 908 CE E 67 230.294 88.399 2O1807 OO 53.41 A TOM 909 LYS 68 23 659 93.472 99.962 OO 54.97 A TOM 910 CA 68 230.830 94.534 99.449 OO 60.76 A TOM 911 68 230.456 94.386 97.990 OO 70.78 A TOM 912 68 229.307 94.607 97.629 OO 66.11 A TOM 913 CB 68 23 SO2 95.876 99.659 OO 65.32 A TOM 914 CG 68 230.616 97.027 99.293 OO 49.49 A TOM 915 CD 68 23 208 98.313 99.78O OO 103.77 A TOM 916 CE 68 23 O.191 99.429 99.714 OO 87.12 A TOM 917 NZ 68 230.644 100.674 OO 118.45 A TOM 918 69 23 403 94.023 OO 82.60 A TOM 919 CA 69 23 O61 93.885 OO 89.11 A TOM 920 69 230.176 92.66S OO 81.07 A TOM 921 69 229.262 92.726 OO 75.01 A TOM 922 CB 69 232.327 93.796 OO 73.74 A TOM 923 OG 69 233.058 92.62O OO 77.68 A TOM 924 70 23O448 91S60 OO 75.29 A TOM 925 CA 70 229.630 90.349 OO 74.32 A TOM 926 70 228.232 90.743 OO 63.33 A TOM 927 70 227.255 90.334 OO 57.44 A TOM 928 CB 70 23 O.OS3 89.177 OO 68.61 A TOM 929 70 231.437 88.663 OO 59.95 A TOM 930 70 229.091 88.01S OO 46.17 A TOM 931 70 231.768 87.318 OO 76.19 A TOM 932 71 228.154 91S60 OO 56.09 A TOM 933 71 226.870 92.026 OO 75.47 A TOM 934 71 226110 92.700 OO 69.45 A TOM 935 71 224.947 92.390 OO 76.12 A TOM 936 72 226.792 93.621 OO 71.58 A TOM 937 72 226.212 94.368 OO 73.09 A TOM 938 72 225.715 93.446 OO 69.34 A TOM 939 72 224.645 93.653 OO 74.21 A TOM 940 72 227.243 95.332 OO 62.8O A TOM 941 72 228.028 96.08O OO 91.94 A TOM 942 72 228.717 97.298 OO 112.10 A TOM 943 72 228.051 98.349 OO 116.39 A TOM 944 72 229.919 97.199 OO 121.47 A TOM 945 73 226.487 92.427 OO SO.91 A TOM 946 73 226.039 91.525 OO SS.42 A TOM 947 73 224.666 90.969 OO 69.00 A TOM 948 73 223.779 90.793 OO 68.62 A TOM 949 73 227.036 90.392 OO 59.45 A TOM 950 74 224,491 90.709 OO 59.68 A TOM 951 74 223-240 90.163 OO 55.23 A TOM 952 74 222.137 91.199 OO 66.27 A TOM 953 74 220.991 90.943 OO 59.24 A TOM 954 74 223.439 89.528 OO 72.24 A TOM 955 74 224,492 88.438 OO 44.08 A TOM 956 74 222.132 88.924 OO S2.34 A TOM 957 74 224.090 87.355 OO 105.89 A TOM 958 75 222.473 92.368 OO 46.22 A TOM 959 CA 75 221.477 93.407 OO 60.91 A TOM 960 75 220.804 93.676 OO 48.25 A TOM 961 o 75 219.581 93.742 OO 62.61 US 9,023,787 B2 59 60 TABLE 1-continued Coordinates of the activated MA PKAP kinase-2, peptide complex A TOM 962 GLN 75 22 2.121 94.674 96.144 OO 49.40 A TOM 963 GLN 75 22 .309 95.931 95.912 OO 71.12 A TOM 964 GLN 75 22 661 97.042 96.892 OO 104.15 A TOM 96S GLN 75 22 2.82S 97.446 97.017 OO 76.77 A TOM 966 GLN 75 22 O645 97.544 97.600 OO 11129 A TOM 967 76 22 602 93.798 93.184 OO 72.76 A TOM 968 76 22 O2S 94.078 91.873 OO 74.81 A TOM 969 76 22 O.1OS 92.946 91.416 OO 74.26 A TOM 970 76 21 8.933 93.142 91122 OO 61.26 A TOM 971 76 22 2.168 94.2SS 90.874 OO 79.19 A TOM 972 76 22 628 94.703 89.563 OO 53.89 A TOM 973 76 22 692 96.048 89.209 OO 73.14 A TOM 974 76 22 OS4 93.785 88.683 OO 78.08 A TOM 975 76 22 197 96.471 87.985 OO 58.55 A TOM 976 76 22 0.550 94.209 87.463 OO 60.19 A TOM 977 TY 76 22 O616 95.546 87.115 OO 70.05 A TOM 978 H s 76 22 O.124 95.975 85.898 OO 98.35 A TOM 979 77 22 O626 91.729 91.357 OO 69.60 A TOM 98O 77 21 9.824 90.582 90.986 OO 65.69 A TOM 981 77 21 8.485 90.616 91.705 OO 62.46 A TOM 982 77 21 7.437 90.563 91.057 OO 73.48 A TOM 983 77 22 O.S62 89.297 91.333 OO 59.94 A TOM 984 77 22 1.671 88.9SS 90.357 OO S4O2

A TOM 985 77 22 2.252 87.585 90.679 OO 59.89 A TOM 986 E U 77 22 1.078 88.962 88.964 OO 77.33 A TOM 987 78 2 8.518 90.708 93.035 OO 61.49 A TOM 988 78 7.285 90.746 93.816 OO 57.69 A TOM 989 78 6.419 91.952 93.466 OO 56.16 A TOM 990 78 S.204 91.831 93.368 OO 71.55 A TOM 991 78 7.590 90.741 95.313 OO 58.67 A TOM 992 78 8.154 89.446 95.805 OO 66.35 A TOM 993 ND1 78 8.554 89.259 97.110 OO 49.65 A TOM 994 CD2 78 8437 88.292 95.154 OO 50.67 A TOM 995 CE1 78 9.068 88.049 97.241 OO 77.64 A TOM 996 NE2 78 9.010 87.442 96.069 OO 4453 A TOM 997 79 7.037 93.107 93.259 OO 62.56 A TOM 998 CA SE 79 6.270 94.292 92.906 OO 56.93 A TOM 999 SER 79 5.575 94.094 91.567 OO 44.23 A TOM OOO SER 79 4.83O 94.956 91136 OO 68.45 A TOM OO1 CB SER 79 7.160 95.518 92.8O3 OO 41.33 A TOM OO2 OG SER 79 7.835 9.SSO4 91.554 OO 86.61 A TOM OO3 ILE 8O 5.842 92.997 90.876 OO 60.70 A TOM OO4 CA ILE 8O 5.135 92.778 89.629 OO 66.56 A TOM 005 ILE 8O 4.483 91.409 89.667 OO 76.38 A TOM OO6 ILE 8O 4.223 90.783 88.641 OO 67.28 A TOM OO7 ILE 8O 6.041 92.928 88.368 OO 65.72 A TOM OO8 ILE 8O 7.061 91.804 88.28O OO 98.00 A TOM O09 ILE 8O 6.766 94.248 88.413 OO 65.05 A TOM O10 ILE 8O 7.787 91.783 86.962 OO 42.99 A TOM O11 ASN 81 4.229 90.940 90.88O OO 62.76 A TOM O12 CA ASN 81 3.556 89.672 91.081 OO 6437 A TOM O13 ASN 81 4.257 88.447 90.552 OO 64.72 A TOM O14 ASN 81 3.624 87.605 89.919 OO 76.26 A TOM O15 CB ASN 81 2.156 89.726 90.464 OO 52.97 A TOM O16 CG ASN 81 1.274 90.765 91.118 OO 91.87 A TOM O17 OD1 ASN 81 O607 91.549 90.435 OO 70.51 A TOM O18 ND2 ASN 81 1.261 90.779 92.456 OO 66.84 A TOM O19 ILE 82 5550 88.325 90.800 OO 69.03 A TOM O2O CA ILE 82 6.251 87.140 90.343 OO 58.61 A TOM O21 ILE 82 7.060 86.582 91.510 OO 64.49 A TOM O22 ILE 82 7.652 87.335 92.289 OO 75.24 A TOM O23 ILE 82 7.217 87.452 89.175 OO 67.77 A TOM O24 ILE 82 6.498 88.204 88.058 OO 51.81 A TOM O25 ILE 82 7.765 86.162 88.604 OO 71.OO A TOM O26 ILE 82 7.397 88.520 86.870 OO 51.04 A TOM O27 ALA 83 7.068 85.261 91.639 OO 74.62 A TOM O28 ALA 83 7.907 84.570 92.612 OO 86.67 A TOM O29 ALA 83 8.970 83.710 91.924 OO 88.25 A TOM O3O ALA 83 8.683 82.841 91.110 OO 87.52 A TOM O31 ALA 83 2 7.006 83.691 93.48O OO 82.75 A TOM O32 HIS 84 22 O.241 84.008 92.248 OO 80.72 A TOM O33 HIS 84 22 1.331 83.245 91.654 OO 76.94 A TOM O34 HIS 84 22 1.282 81.775 92.077 OO 80.75 A TOM O35 HIS 84 22 1.411 80.858 91.277 OO 72.26 A TOM O36 HIS 84 22 2.652 83.872 92.102 OO 71.67 A TOM O37 HIS 84 22 3.777 83.319 91.264 OO 61.47 A TOM O38 ND1 HIS 84 22 4.599 84.095 90.515 OO 83.49 A TOM O39 CD2 HIS 84 22 4.158 81983 91.101 OO 84.93 US 9,023,787 B2 61 62 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM CE1 HIS 84 225.456 83.245 89.918 OO 77.60 A TOM NE2 HIS 84 225.215 81974 90.2SO OO 94.64 A TOM N ARG 85 221.128 81570 93.401 OO 76.53 A TOM CA ARG 85 221.070 80.2O7 93.915 OO 77.64 A TOM C ARG 85 222.301 79.398 93.5O1 OO 85.59 A TOM O ARG 85 222.247 78.190 93.308 OO 76.00 A TOM CB ARG 85 219.805 79.546 93.368 OO 6O.OS A TOM CG ARG 85 218.538 80.313 93.747 OO 69.87 A TOM CD ARG 85 217.293 79.421 93.714 OO 88.79 A TOM NE ARG 85 216.984 79.018 92.339 OO 74.62 A TOM CZ ARG 85 216.704 77.720 92.125 OO 102.74 A TOM NH1 ARG 85 216.704 76.86S 93.132 OO 105.16 A TOM NH2 ARG 85 216428 77.299 90.884 OO 124.15 A TOM ASP 86 223.476 8O.OOO 93.397 OO 85.17 A TOM CA ASP 86 224.677 79.238 93.128 OO 65.94 A TOM ASP 86 225.857 80.161 93.327 OO 63.69 A TOM ASP 86 226.821 80.122 92.573 OO 76.8O A TOM CB ASP 86 224.651 78.670 91.712 OO 64.22 A TOM CG ASP 86 225.560 77.461 91.555 OO 69.83 A TOM OD1 ASP 86 225.629 76.654 92.503 OO 114.36 A TOM OD2 ASP 86 226.197 77.304 90.492 OO 103.36 A TOM VAL 87 225.757 80.991 94.364 OO 65.08 A TOM CA VAL 87 226.794 81952 94.717 OO 62.87 A TOM VAL 87 227.923 81.305 95.507 OO 61.46 A TOM VAL 87 228.134 81.605 96.677 OO 76.23 A TOM VAL 87 226.218 83.132 95.539 OO 56.36 A TOM VAL 87 227.311 84.156 95.831 OO 45.04 A TOM VAL 87 225.099 83.791 94.774 OO 49.15 A TOM LYS 88 228.644 80.407 94.852 OO 71.26 A TOM CA LYS 88 229.766 79.731 95.482 OO 71.74 A TOM LYS 88 231.033 80392 94.98S OO 71.11 A TOM LYS 88 231.049 80.975 93.905 OO 76.85 A TOM CB LYS 88 229.770 78.246 95.123 OO 59.58 A TOM CG LYS 88 229.709 77.962 93.639 OO 61.04 A TOM CD LYS 88 228.939 76.679 93.367 OO 96.OO A TOM CE 88 228.669 76.509 91.869 OO 141.45 A TOM NZ 88 227.746 75.371 91.579 OO 122.86 A TOM 89 232.115 80.318 95.776 OO 66.86 A TOM CA 89 233.409 80.911 95.433 OO 82.94 A TOM 89 233.746 80.698 93.967 OO 81:19 A TOM 89 233.989 81.641 93.231 OO 92.51 A TOM CB 89 234.359 80.199 96.381 OO 77.38 A TOM CG 89 233.511 80.063 97.62O OO 79.38 A TOM CD 89 232.197 79.582 97.049 OO 84.38 A TOM 90 233.743 79.447 93.553 OO 79.12 A TOM CA 90 234.019 79.08O 92.178 OO 82.41 A TOM 90 233441 80.092 91.159 OO 81.32 A TOM 90 234.121 80.487 90.213 OO 102.09 A TOM 90 233.432 77.689 91.920 OO 94.71 A TOM 90 233.784 76.609 92.978 OO 121.01 A TOM 90 232.689 76.364 94.043 OO 135.59 A TOM 90 231.485 76.334 93.693 OO 113.86 A TOM 90 233.039 76.185 95.236 OO 133.59 A TOM 91 232.196 80.527 91.3SO OO 80.04 A TOM CA 91 231.584 81.457 90.405 OO 59.50 A TOM 91 231.991 82.905 90.569 OO 72.63 A TOM 91 231.360 83.796 89.998 OO 60.72 A TOM CB 91 230.068 81364 90.471 OO 59.13 A TOM CG 91 229.566 79.975 90.1.89 OO 57.02 A TOM OD1 91 228.797 79.413 90.959 OO 107.48 A TOM ND2 91 23 O.OOS 79.406 89.077 OO 62.85 A TOM 92 233.042 83149 91.342 OO 64.74 A TOM 92 233.524 84.514 91.552 OO 58.75 A TOM 92 234.931 84.6SS 90.987 OO 67.25 A TOM 92 235.906 84.314 91.644 OO 63.30 A TOM 92 233.512 84.856 93.047 OO 64.92 A TOM 92 232.127 84.840 93.709 OO 76.84 A TOM CD1 92 232.285 84.7O6 95.207 OO 63.79 A TOM CD2 92 231.3SO 86.108 93.349 OO 49.09 A TOM 93 23S.O22 85.173 89.767 OO 76.60 A TOM 93 236.299 85.334 89.077 OO 68.12 A TOM 93 236.765 86.779 89.008 OO 67.18 A TOM 93 235.957 87.700 89.001 OO 70.12 A TOM 93 236.16S 84.801 87.657 OO 57.38 A TOM CG 93 235.346 83.517 87.573 OO 67.03 A TOM CD1 93 235.242 83.061 86.137 OO 74.99 A TOM 93 235.985 82.449 88.445 OO 80.12 A TOM TY 94 238.073 86.982 88.9SO OO 66.38 US 9,023,787 B2 63 64 TABLE 1-continued Coordina es of the activated MAPKAP kinase-2, peptide complex A TOM 18 CA TYR 94 238.603 88.333 88.846 OO 70.2O A TOM 19 C TYR 94 238.833 88.697 87.382 OO 65.05 A TOM 20 O TYR 94 239.219 87.856 86.578 OO 55.59 A TOM 21 CB TYR 94 239.903 88.449 89.636 OO 71.53 A TOM 22 CG TYR 94 239.692 88.807 91.085 OO 83.55 A TOM 23 CD1 TYR 94 239.221 90.068 91.446 OO 60.55 A TOM 24 CD2 TYR 94 239.959 87.889 92.095 OO 54.69 A TOM 25 CE1 TYR 94 239.026 90.4O2 92.766 OO 77.54 A TOM 26 CE2 TYR 94 239.764 88.216 93.423 OO 72.23 A TOM 27 CZ TYR 94 239.300 89.472 93.749 OO 80.69 A TOM 28 OH TYR 94 239.118 89.812 95.068 OO 97.12 A TOM 29 N THR 95 238.585 89.952 87.035 OO 56.71 A TOM 30 CA. THR 95 238.754 90.389 85.660 OO 63.48 A TOM 31 C THR 95 240-193 90.2O6 85.176 OO 82.40 A TOM 32 O THR 95 24O436 89.542 84.164 OO 96.10 A TOM 33 CB THR 95 238.334 91.86S 85.494 OO 66.82 A TOM 34 OG1 THR 95 239.028 92.668 86.452 OO 70.80 A TOM 35 CG2 THR 95 236.829 92.022 85.688 OO 54.17 A TOM 36 N SER 96 241.145 90.790 85.897 OO 84.27 A TOM 37 CA. SER 96 242.SS2 90.672 85.532 OO 77.62 A TOM 38 C SER 96 243.388 90.2O2 86.716 OO 88.08 A TOM 39 O SER 96 242.86S 89.980 87.8OS OO 101.07 A TOM 4O CB SER 96 243.089 92.019 85.033 OO 78.67 A TOM 41 OG SER 96 243.063 92.995 86.OSS OO 92.46 A TOM 42 N. LYS 97 244.688 90.038 86.490 OO 106.23 A TOM 43 CA LYS 97 245.601 89.609 87.542 OO 100.72 A TOM 44 C LYS 97 246.OSS 90.885 88.230 OO 93.19 A TOM 45 O LYS 97 246.509 90.870 89.373 OO 91.43 A TOM 46 CB LYS 97 246.792 88.882 86.936 OO 90.18 A TOM 47 N ARG 98 245.911 91.98S 87.500 OO 76.O2 A TOM 48 CA ARG 98 246.275 93.318 87.959 OO 11.08 A TOM 49 C ARG 98 245.815 93.469 89.412 OO OS.33 A TOM 50 O ARG 98 244.993 92.696 89.885 OO O4.59 A TOM S1 CB ARG 98 245.582 94.345 87.048 OO 18.16 A TOM S2 CG ARG 98 246.321 95.666 86.798 OO 44O7 A TOM S3 CD ARG 98 245.911 96.211 85.435 OO S2.31 A TOM S4 NE ARG 98 246.262 97.611 85.210 OO 71.50 A TOM SS CZ. ARG 98 245.92O 98.290 84.115 OO 62.32 A TOM S6 NH1 ARG 98 245.223 97.692 83.152 OO 96.38 A TOM S7 NH2 ARG 98 246.265 99.56S 83.984 OO 46.82 A TOM 58 N PRO 99 246.347 94.461 90.144 OO 15.43 A TOM S9 CA PRO 99 245.943 94.653 91.545 OO 23.33 A TOM 60 C PRO 99 244.581 95.315 91.719 OO 23.60 A TOM 61 O PRO 99 243.991 95.259 92.794 OO 35.30 A TOM 62 CB PRO 99 247.061 95.524 92.105 OO 21.70 A TOM 63 CG PRO 99 247.387 96.397 90.921 OO 33.24 A TOM 64 CD PRO 99 247.411 95.409 89.775 OO 25.16 A TOM 65 N ASN 2OO 244,090 95.943 90.656 OO 22.57 A TOM 66 CA. ASN 2OO 242,810 96.629 90.719 OO O8.91 A TOM 67 C ASN 2OO 241.726 95.826 90.023 OO 95.83 A TOM 68 O ASN 2OO 24O606 96.310 89.835 OO 73.96 A TOM 69 CB ASN 2OO 242.923 98.019 90.090 OO 18.71 A TOM 70 N ALA 2O1 242.059 94.598 89.637 OO 75.18 A TOM 71 CA ALA 2O1 241.093 93.725 88.979 OO 84.90 A TOM 72 C ALA 2O1 239.789 93.860 89.751 OO 91.91 A TOM 73 O ALA 2O1 239.800 94.214 90.929 OO 89.51 A TOM 74 CB ALA 2O1 241.579 92.278 89.022 OO 81.83 A TOM 75 N LE 2O2 238.660 93.6O7 89.097 OO 78.8O A TOM 76 CA LE 2O2 237.378 93.698 89.795 OO 91.70 A TOM 77 C LE 2O2 236.697 92.326 89.886 OO 95.28 A TOM 78 O. LE 2O2 236.837 91.488 88.995 OO 88.21 A TOM 79 CB LE 2O2 236.454 94.707 89.121 OO 76.30 A TOM 80 CG1 ILE 2O2 235.623 94.029 88.059 OO 78.45 A TOM 81 CG2 ILE 2O2 237.278 95.772 88.468 OO 61.72 A TOM 82 CD1 ILE 2O2 234.681 94.991 87.390 OO 169.28 A TOM 83 N LEU 2O3 235.975 92.1O2 90.981 OO 93.25 A TOM 84 CA LEU 2O3 235.308 90.821 91.231 OO 66.28 A TOM 85 C LEU 2O3 233.935 90.746 90.587 OO 67.61 A TOM 86 O LEU 2O3 233.137 91681 90.695 OO 68.70 A TOM 87 CB LEU 2O3 235.195 90.599 92.745 OO 73.90 A TOM 88 CG LEU 2O3 234.947 89.189 93.273 OO S2.16 A TOM 89 CD1 LEU 2O3 236.016 88.244 92.783 OO 61.94 A TOM 90 CD2 LEU 2O3 234.956 89.228 94.779 OO 85.31 A TOM 91 N LYS 204 233.66S 89.628 89.919 OO 58.34 A TOM 92 CA LYS 204 232.392 89.434 89.243 OO 58.89 A TOM 93 C LYS 204 231.788 88.047 89.423 OO 73.88 A TOM 94 O LYS 204 232.490 87.027 89.4O6 OO 67.59 A TOM 95 CB LYS 204 232.540 89.740 87.750 OO 68.02 US 9,023,787 B2 65 66 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 196 CG LYS 204 232.844 91.199 87.461 OO 64.40 A TOM 197 CD LYS 204 232.603 91.552 86.008 OO 62.63 A TOM 198 CE LYS 204 232.786 93.033 85.788 OO 71.16 A TOM 199 NZ LYS 204 232.247 93.468 84.487 OO 94.63 A TOM 2OO N LEU 205 230.472 88.023 89.586 OO 66.OS A TOM 2O1 CA LEU 205 229.750 86.781 89.782 OO 63.28 A TOM 2O2 C LEU 205 229.296 86.241 88.452 OO 53.73 A TOM 2O3 O LEU 205 228.884 87.001 87.583 OO 55.72 A TOM 204 CB LEU 205 228.540 87.024 90.68O OO 6469 A TOM 205 CG LEU 205 227.514 85.905 90.813 OO 47.19 A TOM 2O6 CD1 LEU 205 228.1SO 84.657 91.394 OO 81.96 A TOM 2O7 CD2 LEU 205 226.397 86.387 91.695 OO 6488 A TOM 208 TH 2O6 229.374 84.926 88.300 OO 53.81 A TOM 209 CA TH 2O6 228.976 84.363 87.015 OO 65.26 A TOM 210 TH 2O6 228.239 83.033 87.185 OO 53.82 A TOM 211 TH 2O6 228.098 82.495 88.276 OO 71.37 A TOM 212 TH 2O6 230.236 84.153 86.174 OO 61.13 A TOM 213 TH 2O6 231.023 83.115 86.762 OO 57.86 A TOM 214 TH 2O6 231.064 85,441 86.137 OO 74.17 A TOM 215 AS 2O7 227.721 82.523 86.051 OO 79.42 A TOM 216 CA AS 2O7 227.033 81.239 86.082 OO 59.92 A TOM 217 AS 2O7 225.577 81381 86.532 OO 58.53 A TOM 218 AS 2O7 225.272 81.747 87.660 OO 80.39 A TOM 219 CB AS 2O7 227.785 80.312 87.037 OO 60.66 A TOM 220 CG AS 2O7 227.400 78.869 86.743 OO 80.61 A TOM 221 OD1 AS 2O7 226.540 78.671 85.884 OO 8340 A TOM 222 OD2 AS 2O7 227.955 77.969 87.369 OO 77.29 A TOM 223 PH 208 224.661 81.117 85.582 OO 57.58 A TOM 224 CA PH 208 223-242 81.177 85.911 OO 6542 A TOM 225 PH 208 222.SS4 79.836 85.650 OO 55.77 A TOM 226 PH 208 221.342 79.748 85.504 OO 71.33 A TOM 227 CB PH 208 222.597 82.267 85.055 OO 41.42 A TOM 228 CG PH 208 222.872 83.615 85.655 OO SS. 64 A TOM 229 CD1 PH 208 224.177 84.088 85.707 OO 30.42 A TOM 230 CD2 PH 208 221.825 84-390 86.12S OO 49.87 A TOM 231 CE1 PH 208 224.432 85.349 86.229 OO 68.91 A TOM 232 CE2 PH 208 222.089 85.654 86.645 OO 47.56 A TOM 233 PH 208 223.390 86.139 86.697 OO 56.42 A TOM 234 GLY 209 223.362 78.788 85.594 OO 61.07 A TOM 235 GLY 209 222.825 77.461 85.343 OO 72.68 A TOM 236 GLY 209 221.723 77.08O 86.309 OO 73.62 A TOM 237 GLY 209 220.958 76.161 86.039 OO 77.98 A TOM 238 PHE 2 221.656 77,777 87.441 OO 83.69 A TOM 239 PHE 220.624 77.527 88.439 OO 69.57 A TOM 240 PHE 219.818 78.787 88.732 OO 81.37 A TOM 241 PHE 218.872 78.738 89.507 OO 58.29 A TOM 242 PHE 221.225 77.037 89.753 OO 67.31 A TOM 243 PHE 222.004 75.774 89.633 OO 73.06 A TOM 244 CD1 PHE 221.429 74.645 89.085 OO 114.78 A TOM 245 CD2 PHE 223.306 75.703 90.097 OO 90.32 A TOM 246 CE1 PHE 222.134 73459 89.000 OO 109.98 A TOM 247 CE2 PHE 224,021 74.522 90.017 OO 108.27 A TOM 248 PHE 223.432 73.398 89.467 OO 132.84 A TOM 249 ALA 220.208 79.913 88.137 OO 81.89 A TOM 250 ALA 2 9.490 81.170 88.341 OO 69.02 A TOM 251 ALA 8028 80.898 88.075 OO 58.89 A TOM 252 ALA 7.700 80.102 87.2O6 OO 66.98 A TOM 253 ALA 9.992 82,228 87.381 OO 73.43 A TOM 2S4 LYS 7.147 81.SS2 88.817 OO 66.85 A TOM 255 LYS 5.726 81.329 88.624 OO 74.67 A TOM 2S6 LYS 4.892 82.598 88.785 OO 69.94 A TOM 257 LYS S.106 83.387 89.709 OO 68.45 A TOM 258 LYS 5.242 80.249 89.600 OO 70.15 A TOM 259 LYS 3.744 80.022 89.SS8 OO 104.51 A TOM 260 LYS 3.371 78.669 88.952 OO 15753 A TOM 261 LYS 1877 78.354 89.164 OO 119.37 A TOM 262 LYS 1408 77.209 88.336 OO 93.54 A TOM 263 GLU 4.141 82.794 87.868 OO 1S.OO A TOM 264 GLU 3.459 84,043 87.550 OO 1S.OO A TOM 26S GLU 3.339 84.210 86.033 OO 1S.OO A TOM 266 GLU 2.1.59 83475 85.419 OO 1S.OO A TOM 267 GLU 2.043 83.702 83.925 OO 1S.OO A TOM 268 GLU 1.18O 83.058 83.289 OO 1S.OO A TOM 269 GLU 2.815 84.522 83.385 OO 1S.OO A TOM 270 GLU 2.074 84.087 88.185 OO 1S.OO A TOM 271 O GLU 1548 83.061 88.604 OO 15.36 A TOM 272 N THR 1.491 85.288 88.256 OO 1S.OO A TOM 273 CA THR O. 119 85.515 88.693 OO 1S.OO US 9,023,787 B2 67 68 TABLE 1-continued Coordinates of the activa ed MAPKAP kinase-2, peptide complex A TOM 274 HR 209.149 84.520 88.029 OO A TOM 275 HR 209.187 84.691 86.606 OO A TOM 276 HR 2O7.729 84.751 88.525 OO A TOM 277 HR 209.999 85.380 90.208 OO A TOM 278 O HR 208.821 85.220 90.684 OO A TOM 279 N HR 211.054 85,475 90.986 OO A TOM 28O CA HR 210.986 85.364 92.438 OO A TOM 281 CB HR 212.132 84.493 92.987 OO A TOM 282 OG1 HR 212.031 83.170 92.444 OO A TOM 283 CG2 HR 212.062 84.42O 94.505 OO A TOM 284 HR 211.057 86.739 93.095 OO A TOM 285 HR 212.111 87.316 93.358 OO A TOM 286 ER 209.845 87.327 93.310 OO A TOM 287 CA ER 209.645 88.633 93.871 OO A TOM 288 CB ER 208.582 89.393 93.087 OO A TOM 289 OG ER 2O7.344 88.725 93.OSO OO A TOM 290 ER 209.352 88.649 95.380 OO A TOM 291 ER 209.553 89.753 95.944 OO A TOM 292 HIS 208.923 87.546 95.889 OO A TOM 293 CA HIS 208.587 87.117 97.213 OO A TOM 294 CB HIS 208.248 88.231 98.187 OO A TOM 295 CG HIS 208489 87.994 99.627 OO A TOM 296 CD2 HIS 209.559 88.383 200.390 OO A TOM 297 ND1 HIS 2O7.605 87.415 2OO.SOO OO A TOM 298 CE1 HIS 2O8.130 87.392 201715 OO A TOM 299 NE2 HIS 209.316 87.977 2O1.671 OO A TOM 3OO HIS 207430 86.096 97.115 OO A TOM 301 HIS 2O6.272 86.389 96.895 OO A TOM 3O2 ASN 2O7.933 84.916 97.149 OO A TOM 303 CA ASN 208.098 83.782 96.497 OO A TOM 3O4 CB ASN 2O7.843 83.860 94.969 OO A TOM 305 CG ASN 2O6.412 83.605 94.590 OO A TOM 306 OD1 ASN 205.835 82.541 94.841 OO A TOM 307 ND2 205.794 84.575 93.913 OO A TOM 3O8 209.199 82.856 96.804 OO A TOM 309 S 210.327 83.158 97.160 OO A TOM 310 2O8.738 81.608 96.607 OO A TOM 311 209.612 80.496 96.960 OO A TOM 312 208.946 79.616 98.021 OO A TOM 313 2O7.78O 78.995 97.509 OO A TOM 314 209.953 79.658 95.732 OO A TOM 315 209421 79.706 94.646 OO A TOM 316 211.012 78.785 95.962 OO A TOM 317 : 1564 77.825 95.013 OO A TOM 318 220 2.993 78.219 94.633 OO A TOM 319 220 3.16.1 79.569 93.932 OO A TOM 32O CD1 220 4.636 79.928 93.843 OO A TOM 321 CD2 220 2.537 79.516 92.547 OO A TOM 322 220 1553 76.415 95.594 OO A TOM 323 220 1679 76.279 96.816 OO A TOM 324 221 1.526 75.374 94.729 OO A TOM 325 CA 221 1.397 74.01.1 95.231 OO A TOM 326 CB 221 O.O79 73.365 94.763 OO A TOM 327 OG1 221 O8.970 74.121 95.264 OO A TOM 328 CG2 221 O9.981 71.933 95.268 OO A TOM 329 221 2.562 73.144 94.764 OO A TOM 330 221 2.736 72.026 95.385 OO TER 330 221 A TOM 331 C O 222 3.399 73.479 93.800 OO S.OO A TOM 332 CA 222 4.459 72.638 93.257 OO S.OO A TOM 333 CB 222 5.056 73.250 91.976 OO S.OO A TOM 334 OG1 222 4.OO2 73.557 91.055 OO S.OO A TOM 335 CG2 222 6.025 72.275 91.324 OO S.OO A TOM 336 222 3.808 74.828 90.542 OO 5.8O A TOM 337 222 4.914 75.324 89.669 OO 5.57 A TOM 338 222 3.482 75.613 91.784 OO S.91 A TOM 339 222 2.591 74.609 89.572 OO 5.73 s A TOM 340 222 5.578 72.444 94.276 OO S.OO A TOM 341 C O 222 6.123 73.375 94.857 OO 2.56 A TOM 342 223 5.917 71.098 94.555 OO S.OO A TOM 343 CD 223 S.209 69.889 94.261 OO S.OO A TOM 344 CA 223 7.121 70.849 95.355 OO S.OO A TOM 345 CB R O 223 6.915 69424 95.868 OO S.OO A TOM 346 CG 223 6.042 68.783 94.845 OO S.OO A TOM 347 223 8.396 70.958 94.525 OO S.OO A TOM 348 223 8.639 70.105 93.676 OO 2.12 A TOM 349 CYS 224 9.010 72.137 94.641 OO S.OO A TOM 350 CA CYS 224 22O18O 72.438 93.826 OO S.OO US 9,023,787 B2 69 70 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 351 CB CYS 224 219.754 73079 92.503 OO S.OO A TOM 352 SG CYS 224 219.284 71.899 91.217 OO S.OO A TOM 353 C CYS 224 221.138 73.367 94.56S OO S.OO A TOM 3S4 O CYS 224 220.831 73947 95.595 OO 9.31 A TOM 355 N TY 225 222.314 73.544 93.951 OO S.OO A TOM 356 CA TY 225 223.376 74.375 94.506 OO S.OO A TOM 357 CB TY 225 223.004 74.838 95.916 OO S.OO A TOM 358 CG TY 225 222.663 73.709 96.863 OO S.OO A TOM 359 CD1 TY 225 223.543 72.652 97.060 OO S.OO A TOM 360 CE1 TY 225 223.236 71.62O 97.924 OO S.OO A TOM 361 CD2 TY 225 221.461 73.699 97.556 OO S.OO A TOM 362 CE2 TY 225 221.146 72.667 98.422 OO S.OO A TOM 363 CZ TY 225 222.036 71.631 98.602 OO S.OO A TOM 364 OH TY 225 221.725 70.605 99.464 OO S.OO A TOM 365 TY 225 224.699 73.6.18 94.543 OO S.OO A TOM 366 TY 225 224.872 72.695 93.743 OO 1.69 A TOM 367 TH 226 225.564 74.018 95.383 OO S.OO A TOM 368 CA TH 226 226.833 73.340 95.622 OO S.OO A TOM 369 CB TH 226 228.026 74.279 95.362 OO S.OO A TOM 370 OG1 TH 226 229.137 73.88O 96.175 OO S.OO A TOM 371 CG2 TH 226 227.653 75.716 95.691 OO S.OO A TOM 372 C TH 226 226.916 72.827 97.056 OO S.OO A TOM 373 O TH 226 226.576 73.621 97.967 OO 9.55 A TOM 374 N PRO 227 227.140 71.546 97.345 OO S.OO A TOM 375 CD PRO 227 228.192 71.043 96.515 OO S.OO A TOM 376 CA PRO 227 227.06S 70.837 98.626 OO S.OO A TOM 377 CB PRO 227 227.911 69.587 98.389 OO S.OO A TOM 378 CG PRO 227 228.885 69.992 97.336 OO S.OO A TOM 379 PRO 227 227.621 71.670 99.777 OO S.OO A TOM 380 PRO 227 226.859 71.868 OO O.86 A TOM 381 228 228.838 72.168 OO S.OO A TOM 382 CA 228 229.509 72.872 OO S.OO A TOM 383 CB TY 228 230.999 73.O29 OO S.OO A TOM 384 CG TY 228 23 1.748 71.718 OO S.OO A TOM 385 CD1 228 232.077 71.168 OO S.OO A TOM 386 CE1 TY 228 232.762 69.972 OO S.OO A TOM 387 CD2 TY 228 232.124 71.030 2O1.6O7 OO S.OO A TOM 388 CE2 TY 228 232.808 69.831 201527 OO S.OO A TOM 389 CZ TY 228 233.124 69.307 200.293 OO S.OO A TOM 390 TY 228 233.805 68114 200.210 OO S.OO A TOM 391 TY 228 228.883 74.243 201.095 OO S.OO A TOM 392 TY 228 229.096 74.833 2O2.195 OO O.82 A TOM 393 TY 229 228.075 74.767 2OO.161 OO S.OO A TOM 394 TY 229 227.513 76.1.11 2OO.221 OO S.OO A TOM 395 CB TY 229 227.822 76.871 198.929 OO S.OO A TOM 396 CG TY 229 229.281 77.238 198.768 OO S.OO A TOM 397 CD1 TY 229 229.951 76.991 197.577 OO S.OO A TOM 398 CE1 TY 229 231.283 77.325 197.425 OO S.OO A TOM 399 CD2 TY 229 229.987 77.829 199.807 OO S.OO A TOM 400 CE2 TY 229 231.32O 78.16S 199.661 OO S.OO A TOM 4O1 CZ TY 229 231.962 77.911 198.470 OO S.OO A TOM 4O2 TY 229 233.289 78.245 198.324 OO S.OO A TOM 403 229 226.OOS 76.064 200.443 OO S.OO A TOM 404 229 225.390 77.18O 2OO.S48 OO 0.79 A TOM 40S VA 230 225.363 74.929 2OO.S4O OO S.OO A TOM 4O6 VAL 230 223.918 74-808 200.689 OO S.OO A TOM 407 VAL 230 223.468 73.336 200.596 OO S.OO A TOM 4.08 VAL 230 224.223 72.492 201612 OO S.OO A TOM 409 VAL 230 221.967 73.230 200.819 OO S.OO A TOM 410 VAL 230 223.457 75.378 202.027 OO S.OO A TOM 411 VAL 230 224.048 75.172 2O3.074 OO 9.09 A TOM 412 ALA 231 220.726 77.807 2O2.673 OO S.OO A TOM 413 ALA 231 220.871 75.6O7 2O3.851 OO S.OO A TOM 414 ALA 231 220.417 74.655 2O3.184 OO 949 A TOM 415 ALA 231 222.349 76.103 201.918 OO S.OO A TOM 416 ALA 231 221.6SS 76.68O 2O3.104 OO S.OO A TOM 417 PRO 232 220.755 75.645 2O5.185 OO S.OO A TOM 418 PRO 232 220.885 76.873 205.909 OO S.OO A TOM 419 PRO 232 220.064 74.616 205.968 OO S.OO A TOM 420 PRO 232 22O102 75.171 2O7.392 OO S.OO A TOM 421 PRO 232 220.173 76.648 2O7.213 OO S.OO A TOM 422 PRO 232 218.629 74.4O2 2O5.498 OO S.OO A TOM 423 PRO 232 218.15S 73.251 2OS.S2O OO O.S3 A TOM 424 GLU 233 217.931 75.458 205.055 OO S.OO A TOM 425 GLU 233 216.540 75.343 2O4.633 OO S.OO A TOM 426 GLU 233 215.917 76.732 204,474 OO S.OO A TOM 427 GLU 233 214.503 76.844 2OS.O2O OO S.OO A TOM 428 GLU 233 213.601 77.678 204.133 OO S.OO US 9,023,787 B2 71 72 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 429 GL 233 2.376 77.703 204384 OO A TOM 430 GL 233 4.114 78.309 2O3.185 OO A TOM 431 GL 233 6.428 74.573 2O3.321 OO A TOM 432 GL 233 S392 73.897 2O3.095 OO A TOM 433 VAL 234 7.465 74.585 2O2.462 OO A TOM 434 VAL 234 7.484 73.810 2O1.227 OO A TOM 435 VAL 234 8.603 74.293 200.282 OO A TOM 436 VAL 234 8.713 73.363 199.084 OO A TOM 437 VAL 234 8.331 75.720 199.832 OO A TOM 438 VAL 234 7.690 72.327 2O1.515 OO A TOM 439 VA 234 7.255 71.447 200.787 OO A TOM 440 LE 235 8.26S 72.046 2O2.723 OO A TOM 441 CA LE 235 8.512 70.679 2O3.169 OO A TOM 442 CB LE 235 9.760 70.629 2O4.OS3 OO A TOM 443 CG LE 235 21.077 71.046 2O3.392 OO A TOM 444 CD1 LE 235 22.16S 71.181 2O4.445 OO A TOM 445 CD2 LE 235 21473 70.027 2O2.337 OO A TOM 446 LE 235 7.313 70.129 2O3.934 OO A TOM 447 LE 235 7.361 69.014 2O4SOO OO A TOM 448 GLY 236 6.252 70.887 2O3.946 OO A TOM 449 GLY 236 4.958 70.5O1 2O4.475 OO A TOM 450 GLY 236 3.924 70.292 2O3.386 OO A TOM 451 GLY 236 4.223 70.745 2O2.251 OO A TOM 452 PRO 237 2.800 69.571 2O3.652 OO A TOM 453 PRO 237 2.368 68.973 204.879 OO A TOM 454 CA PRO 237 1.813 69.401 2O2.581 OO A TOM 455 PRO 237 O.885 68.315 2O3.124 OO A TOM 456 PRO 237 O.988 68.445 204605 OO A TOM 457 PRO 237 1.047 70.688 2O2.295 OO A TOM 458 PRO 237 1186 71.681 2O2.987 OO A TOM 459 GLU 238 2 O.242 70.629 201243 OO A TOM 460 GLU 238 2 O9.352 71.651 2OO.704 OO A TOM 461 GLU 238 2 O8.401 72.153 201793 OO A TOM 462 GLU 238 2 O7.415 71.107 2O2.287 OO A TOM 463 GLU 238 2 O6.523 71.625 2O3.397 OO A TOM 464 GLU 238 2 05.595 70.894 2O3.807 OO A TOM 465 GLU 238 2 O6.747 72.764 2O3.860 OO A TOM 466 GLU 238 2 10.146 72.82O 200.131 OO A TOM 467 GLU 238 2 11.191 72.629 OO A TOM 468 LYS 239 2 O9.723 74.116 OO A TOM 469 CA LYS 239 2 10.244 75.246 OO A TOM 470 CB LYS 239 2 O9094 76.047 OO A TOM 471 CG LYS 239 2 O8.369 75.328 OO A TOM 472 CD LYS 239 2 O7.259 76.1.89 OO A TOM 473 CE LYS 239 2 O6.534 7S.469 OO A TOM 474 NZ LYS 239 2 O5.442 76.301 OO A TOM 475 LYS 239 2 1.090 76.155 OO A TOM 476 LYS 239 1196 76.171 OO A TOM 477 e TY 240 1840 77.1.15 OO A TOM 478 TY 240 2.736 78.087 OO A TOM 479 CB TY 240 4.163 77.536 OO A TOM 480 CG TY 240 4.299 76.138 OO A TOM 481 CD1 TY 240 4.813 75.929 OO A TOM 482 CE1 TY 240 4.940 74.655 OO A TOM 483 CD2 TY 240 3.913 75.030 OO A TOM 484 CE2 TY 240 4.038 73.752 OO A TOM 485 CZ TY 240 4.SSO 73.570 OO A TOM 486 TY 240 4.674 72.298 OO A TOM 487 TY 240 2.717 79.406 OO A TOM 488 TY 240 2.436 79.469 OO A TOM 489 AS 241 3.113 80515 OO A TOM 490 AS 241 3.048 81.860 OO A TOM 491 CB AS 241 2.482 82.836 200.725 OO A TOM 492 CG AS 241 1.007 82.608 2OO.994 OO A TOM 493 OD1 AS 241 O.289 82.190 200.062 OO A TOM 494 OD2 AS 241 0.567 82.847 2O2.138 OO A TOM 495 AS 241 4.426 82.330 199.237 OO A TOM 496 AS 241 5.421 81629 199.18O OO A TOM 497 LYS 242 4.597 83.587 198.906 OO A TOM 498 LYS 242 5.786 84.284 198.467 OO A TOM 499 LYS 242 6.900 84.299 199.494 OO A TOM 500 LYS 242 7.948 84.893 199.254 OO A TOM 5O1 LYS 242 5.446 85.726 198.107 OO A TOM 502 LYS 242 4.108 86.236 198.608 OO A TOM 503 LYS 242 3.651 87.417 197.771 OO A TOM SO4 LYS 242 2.555 88.212 198.477 OO A TOM 505 LYS 242 2-18O 89.484 197.778 OO A TOM SO6 SER 243 6.681 83.659 200.637 OO US 9,023,787 B2 73 74 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 507 CA SER 243 217.683 83.627 201.704 OO 84.81 A TOM SO8 C SER 243 218.986 82.926 2O1.333 OO 82.48 A TOM 509 O SER 243 22O.O2S 83.174 2O1948 OO 74.56 A TOM 510 CB SER 243 217.097 82.973 2O2.940 OO 77.67 A TOM 511 OG SER 243 215.9SO 83.691 2O3.344 OO 121.59 A TOM 512 N CYS 244 218.944 82,044 200.343 OO 67.22 A TOM 513 CA CYS 244 220.098 81.217 200.052 OO 78.41 A TOM S1.4 C CYS 244 221.259 82.130 199.674 OO 64.58 A TOM 515 O CYS 244 222.377 81.930 200.139 OO 67.24 A TOM S16 CB CYS 244 219.790 80.254 198.911 OO 64.99 A TOM 517 SG CYS 244 219,163 81.OSO 197.43S OO 91.08 A TOM 518 N ASP 245 220.988 83.129 198.834 OO 49.99 A TOM 519 CA ASP 245 222O16 84.076 198.416 OO 45.00 A TOM 520 C ASP 245 222.720 84.63S 199.651 OO 60.39 A TOM 521 O ASP 245 223.952 84.702 199.688 OO 67.65 A TOM 522 CB ASP 245 221.410 85.236 197.62O OO 69.55 A TOM 523 CG ASP 245 221092 84.874 196.18O OO 60.27 A TOM 524 OD1 ASP 245 221.535 83.802 195.715 OO 73.75 A TOM 525 OD2 ASP 245 220.410 85.679 195.506 OO 70.27 A TOM 526 MET 246 221.937 85.033 200.659 OO 43.54 A TOM 527 CA MET 246 222.494 85.577 2O1898 OO 64.86 A TOM 528 MET 246 223.266 84.526 2O2.684 OO 67.48 A TOM 529 MET 246 224,198 84.855 2O3.425 OO 67.31 A TOM 530 CB MET 246 221.401 86.146 2O2.790 OO S2.62 A TOM 531 CG MET 246 220.63S 87.263 2O2.163 OO 60.79 A TOM 532 SD MET 246 221.708 88.445 2O1.385 OO 66.30 A TOM 533 CE MET 246 222.403 89.309 2O2.804 OO SS.43 A TOM 534 247 222.868 83.26S 2O2.536 OO 54.14 A TOM 535 CA 247 223.551 82.177 2O3.215 OO 62.36 A TOM 536 247 224.934 82.O15 2O2.592 OO 62.90 A TOM 537 247 225.951 82.071 2O3.287 OO 62.42 A TOM 538 CB 247 222.748 80.888 2O3.074 OO 69.04 A TOM 539 247 223.470 79.670 2O3.559 OO 86.2O A TOM S4O CD1 247 224.327 78.896 2O2.844 OO 72.11 A TOM S41 CD2 247 223.430 79.110 204.885 OO 75.65 A TOM S42 NE1 247 224.827 77.885 2O3.636 OO 81.90 A TOM 543 CE2 247 224.293 77.999 204.893 OO 68.57 A TOM 544 CE3 247 222.749 79.446 2O6.064 OO 91.13 A TOM 545 CZ2 247 224.500 77.221 2O6.030 OO 68.02 A TOM S46 CZ3 247 222.952 78.669 2O7.193 OO 82.48 A TOM 547 CH2 247 223.821 77.572 2O7.166 OO 74.62 A TOM S48 248 224.959 81.829 2O1274 OO 77.02 A TOM S49 CA 248 226.2O3 81.676 200.531 OO 73.53 A TOM 550 248 227.156 82.786 200.892 OO 80.75 A TOM 551 248 228.369 82.583 2OO.943 OO 84.06 A TOM 552 CB 248 225.930 81.731 199043 OO 67.92 A TOM 553 OG 248 225.112 80.642 198.668 OO 118.25 A TOM 554 249 226.593 83.967 2O1.130 OO 76.04 A TOM 555 CA 249 227.378 85.132 2O1506 OO 82.09 A TOM 556 249 228.08.1 84.869 2O2.845 OO 84.27 A TOM 557 249 229.298 85.035 202.960 OO 83.90 A TOM 558 CB 249 226.473 86.363 2O1610 OO 85.62 A TOM 559 CG 249 227.049 87.629 2OO.964 OO 77.42 A TOM S60 CD1 249 227.250 87.346 199.493 OO 68.42 A TOM 561 CD2 249 226119 88.832 2O1153 OO 95.64 A TOM S62 250 227.318 84.449 2O3.851 OO 64.22 A TOM 563 250 227.915 84.165 2OS.147 OO 62.72 A TOM S64 250 229.103 83.231 204.998 OO 63.71 A TOM 565 250 230.220 83544 2O5.418 OO 57.98 A TOM 566 AL 251 228.855 82.O76 204387 OO 57.35 A TOM 567 AL 251 229.902 81.091 204.152 OO 72.77 A TOM 568 AL 251 231.130 81.779 2O3.587 OO 65.76 A TOM 569 AL 251 232.239 81624 2O4.093 OO 72.08 A TOM 570 VAL 251 229.474 80.052 2O3.129 OO 72.O2 A TOM 571 VAL 251 230.627 79.119 2O2.852 OO 48.81 A TOM 572 VAL 251 228.2SS 79.312 2O3.617 OO 45.51 A TOM 573 ILE 252 230.927 82.544 2O2.525 OO 65.58 A TOM 574 ILE 252 232.036 83.248 2O1899 OO 68.61 A TOM 575 ILE 252 232.722 84.197 2O2.876 OO 77.12 A TOM 576 ILE 252 233.932 84.098 2O3.088 OO 79.18 A TOM 577 ILE 252 231.564 84.008 2OO.634 OO 65.58 A TOM 578 ILE 252 231.151 82.989 199.570 OO 42.53 A TOM 579 ILE 252 232.679 84-891 200..102 OO 53.46 A TOM S8O ILE 252 230.557 83.587 198.355 OO 59.04 A TOM 581 MET 253 231.956 85091 2O3.498 OO 72.95 A TOM 582 MET 253 232.553 86.O28 204.444 OO 81.03 A TOM 583 MET 253 233.367 85.286 2O5.497 OO 75.68 A TOM S84 MET 253 234.460 85.724 205.883 OO 79.62 US 9,023,787 B2 75 76 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 585 CB MET 253 231.486 86.866 2OS.149 OO 64.17 A TOM S86 CG MET 253 232.083 88.08.1 205.8.19 OO 54.30 A TOM 587 SD MET 253 230.954 88.969 206.885 OO 85.00 A TOM 588 CE MET 253 229.555 89.2SO 205.802 OO 83.83 A TOM 589 N TYR 254 232.829 84.161 205.960 OO 81:14 A TOM 590 CA TYR 254 233.513 83.370 2O6.969 OO 68.03 A TOM 591 C TYR 254 234.879 83.008 2O6.419 OO 74SO A TOM 592 O TYR 254 235.901 83.519 206.879 OO 78.65 A TOM 593 CB TYR 254 232.711 82.107 2O7.279 OO 66.09 A TOM 594 CG TYR 254 233.253 81.291 208.427 OO 68.8O A TOM 595 CD1 TYR 254 234.285 80.388 208.230 OO 93.85 A TOM 596 CD2 TYR 254 232.739 81434 209.713 OO 66.37 A TOM 597 CE1 TYR 254 234.797 79.640 209.281 OO 93.85 A TOM 598 CE2 TYR 254 233.238 80.696 210.775 OO 60.25 A TOM 599 CZ TYR 254 234.270 79.797 210.553 OO 78.16 A TOM 600 OH TYR 254 234.78O 79.043 211.592 OO 83.85 A TOM 6O1 LE 255 234.876 82148 2O5.405 OO 71.34 A TOM 602 CA LE 255 236.099 81690 2O4.774 OO 61.37 A TOM 603 LE 255 237.128 82.782 2O4.SS8 OO 76.55 A TOM 604 LE 255 238.298 82.590 204.870 OO 92.35 A TOM 60S LE 255 235.810 81.046 2O3.443 OO 62.35 A TOM 606 LE 255 234.928 79.819 2O3.653 OO 62.67 A TOM 607 LE 255 237.107 80.661 2O2.790 OO 76.47 A TOM 608 LE 255 234.514 79.131 2O2.387 OO 57.99 A TOM 609 256 236.709 83.925 204024 OO 73.36 A TOM 610 256 237.647 85.022 2O3.809 OO 76.61 A TOM 611 256 238,466 85.295 2O5.06S OO 80.02 A TOM 612 256 239.681 85.116 2O5.074 OO 95.93 A TOM 613 256 236.921 86.316 2O3.430 OO 82.53 A TOM 614 256 236.503 86.623 201.991 OO 67.94 A TOM 615 CD1 256 236.199 88.119 2O1870 OO 83.50 A TOM 616 CD2 256 237.615 86.248 201.038 OO 61.15 A TOM 617 t 257 237.78O 85.718 2O6.124 OO 64.99 A TOM 618 E 257 238.407 86.06O 2O7.399 OO 83.07 A TOM 619 257 239.336 85.046 208.090 OO 92.07 A TOM 62O 257 240.175 85.4SO 208.897 OO 79.63 A TOM 621 257 237.331 86.467 208.402 OO 65.07 A TOM 622 257 236.445 87.661 2O8.054 OO 72.20 A TOM 623 CD1 257 235.475 87.888 209.185 OO 109.20 A TOM 624 CD2 257 237.282 88.906 2O7.843 OO 77.76 A TOM 625 258 239.211 83.751 2O7.805 OO 84.06 A TOM 626 258 240.068 82.782 208.486 OO 65.38 A TOM 627 258 240.811 81822 207.572 OO 79.35 A TOM 628 258 241.865 81.305 2O7.936 OO 89.3S A TOM 629 258 239.2SO 81952 209.465 OO 74-82 A TOM 630 258 238.333 80.661 208.627 OO 88.33 A TOM 631 259 240.259 81.SS2 206.397 OO 77.77 A TOM 632 259 240.927 80.636 2O5.491 OO 86.18 A TOM 633 259 240.211 79.311 205.306 OO 84.64 A TOM 634 259 240.562 78.538 204,415 OO 91.47 A TOM 635 260 239.219 79.030 2O6.145 OO 80.25 A TOM 636 260 238.454 77.788 2O6.024 OO 85.40 A TOM 637 260 236.969 78.064 2O6.208 OO 92.78 A TOM 638 260 236.587 79.064 206.813 OO 109.20 A TOM 639 260 238.936 76.760 2O7.050 OO 90.73 A TOM 640 260 239.030 77.305 208.4S1 OO 78.95 A TOM 641 CD1 260 237.942 77.261 209.312 OO 99.14 A TOM 642 CD2 260 240.204 77.892 208.904 OO 82.37 A TOM 643 CE1 260 238.023 77.785 210.585 OO 88.16 A TOM 644 CE2 260 240.292 78.42O 210.173 OO 97.24 A TOM 645 CZ 260 239.2O1 78.364 211.007 OO 67.63 A TOM 646 OH 260 239.298 78.896 212.269 OO 84.01 A TOM 647 261 236.114 77.183 205.676 OO 89.49 A TOM 648 CA 261 234.656 77.296 205.753 OO 103.35 A TOM 649 261 234.107 77.095 2O7.16.1 OO 96.36 A TOM 6SO 261 234.743 76.459 2O7.995 OO 100.89 A TOM 651 CB 261 234.185 76.209 2O4.791 OO 98.88 A TOM 652 261 235.208 75.152 204.996 OO 95.80 A TOM 653 CD 261 236.493 75.962 2O4.9SO OO 91.52 A TOM 654 262 232.918 77.653 2O7.444 OO 94.45 A TOM 655 CA 262 232.337 77.488 2O8.773 OO 85.15 A TOM 656 262 232.01S 76.028 209.043 OO 68.2O A TOM 657 262 232.519 75.448 209.998 OO 106.94 A TOM 658 CB 262 231.092 78.368 2O8.717 OO 75.57 A TOM 659 CG 262 230.721 78.337 2O7.278 OO 64.63 A TOM 660 CD 262 232.OS3 78.504 206.609 OO 99.03 A TOM 661 263 231.200 7S.419 2O8.191 OO 73.57 A TOM 662 CA 263 230.838 74.021 208.393 OO 79.36 US 9,023,787 B2 77 78 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex ATOM 663 PH 263 23 1864 73.093 207.757 OO O6.15 ATOM 664 O PH 263 231.972 73.009 2O6.530 OO OS.28 ATOM 665 CB PH 263 229.428 73.777 2O7.848 OO 69.85 ATOM 666 CG PH 263 228.42O 74.759 208.374 OO 81.28 ATOM 667 CD1 PH 263 228.128 75.917 2O7.677 OO 13.87 ATOM 668 CD2 PH 263 227.845 74.573 209.613 OO 17.67 ATOM 669 CE1 PH 263 227.285 76.867 208.205 OO 85.69 ATOM 670 CE2 PH 263 227.004 75.520 210.145 OO O8.39 ATOM 671 CZ PH 263 226.729 76.670 209.437 OO 17.73 ATOM 672 N TY 264 232.62O 72.408 208.622 OO 34.25 ATOM 673 CA TY 264 233.681 71.497 2O8.197 OO 47.77 ATOM 674 C TY 264 233.192 70.094 2O7.848 OO 40.09 ATOM 675 O TY 264 232.006 69.787 2O7.979 OO 38.93 ATOM 676 CB TY 264 234.775 71.405 209.284 OO 53.86 ATOM 677 CG TY 264 236.191 71.629 2O8.760 OO 57.09 ATOM 678 CD1 TY 264 237.265 70.886 209.2SO OO 93.79 ATOM 679 CD2 TY 264 236.448 72.574 2O7.768 OO 40.SS ATOM 68O CE1 TY 264 238.553 71.074 2O8.764 OO 220.80 ATOM 681 CE2 TY 264 237.734 72.771 2O7.279 OO 2O1.56 ATOM 682 CZ 264 238.782 72.015 2O7.781 OO 222.82 ATOM 683 264 240069 72.185 2O7.307 OO 22O.S2 ATOM 684 26S 234.118 69.246 2O7.409 OO 141.69 ATOM 685 26S 233.785 67.877 2O7.044 OO 135.35 ATOM 686 26S 234.560 66.896 2O7.915 OO 131.47 ATOM 687 26S 235.792 66.945 2O7.976 OO 126.66 ATOM 688 26S 234.113 67.628 205.566 OO 138.65 ATOM 689 26S 233.591 66.386 2OS.130 OO 107.77 TER 689 26S ATOM 690 274 229.307 63.721 209.431 OO 106.41 ATOM 691 274 228.030 64.307 209.816 OO 119.68 ATOM 692 274 227.987 65.812 209.615 OO 11316 ATOM 693 274 227.553 66.551 210.496 OO 95.55 ATOM 694 275 228.446 66.261 208.449 OO 110.29 ATOM 695 275 228463 67.676 208.112 OO 95.81 ATOM 696 275 227.067 68.162 2O7.746 OO 94.19 ATOM 697 275 226.633 69.2O6 208.220 OO 75.71 ATOM 698 275 229.412 67.938 2O6.943 OO 90.51 ATOM 699 275 229.404 69.380 2O6.472 OO 83.11 ATOM 700 275 230.324 69.611 204.942 OO 99.84 ATOM 701 275 229.028 69.352 2O3.750 OO 79.14 ATOM 702 276 226.362 67.411 2O6.904 OO 80.06 ATOM 703 276 225.018 67.816 2O6. S11 OO 81.42 ATOM 704 276 224.233 68.145 207.770 OO 67.18 ATOM 705 276 223.297 68.931 2O7.738 OO 87.18 ATOM 706 276 224.327 66.7OS 205.731 OO 70.05 ATOM 707 277 224.641 67.552 208.886 OO 90.2O ATOM 708 277 223.982 67.776 2 O.167 OO 86.79 ATOM 709 277 224-402 69.103 O.790 OO 88.87 ATOM 710 277 223.570 69.987 1004 OO 90.25 ATOM 711 277 224.316 66.657 1.170 OO 94.65 ATOM 712 277 223.953 65.387 O.614 OO 12483 ATOM 713 277 223.559 66.871 2.464 OO 101.58 ATOM 71.4 278 225.696 69,224 1.083 OO 83.15 ATOM 715 CA 278 226.273 70.425 1683 OO 85.59 ATOM 716 G 278 225.653 71.666 1.039 OO 80.18 ATOM 717 278 225.537 72.716 1.673 OO 83.67 ATOM 718 CB 278 227.792 70413 1.479 OO 76.56 ATOM 719 CG 278 228.346 68.998 1.313 OO 94.91 ATOM 720 CD 278 229.398 68.6OO 2.352 OO 109.01 ATOM 721 NE 278 230.699 69.199 2.072 OO 121.75 ATOM 722 CZ 278 231.384 69.003 O.944 OO 131.02 ATOM 723 NH1 RG 278 230.892 68.216 O 9.988 OO 101.83 ATOM 724 NH2 278 232.556 69.613 2 O.763 OO 135.84 ATOM 725 279 225.250 71.516 209.778 OO 83.10 ATOM 726 CA 279 224.615 72.579 209.001 OO 82.30 ATOM 727 279 223.167 72.765 209421 OO 76.46 ATOM 728 279 222.778 73.847 209.847 OO 90.2O ATOM 729 279 224.609 72.257 2O7.498 OO 84.92 ATOM 730 279 226.006 72.436 2O6.913 OO 77.12 ATOM 731 279 223.611 73.144 2O6.789 OO 57.01 ATOM 732 279 226.093 72.021 205.455 OO 73.72 ATOM 733 28O 222.371 71.709 209.270 OO 68. SS ATOM 734 28O 220.96S 71757 209.640 OO 66.51 ATOM 735 28O 220.886 72.218 211.090 OO 62.26 ATOM 736 28O 220.039 73.038 211.456 OO 69.34 ATOM 737 28O 220.326 70.383 209.475 OO 49.45 ATOM 738 281 221.787 71.693 211.912 OO 62.31 ATOM 739 281 221846 72.058 213.323 OO 71.30 US 9,023,787 B2 79 80 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 740 C MET 281 222.348 73.489 3.438 OO 78.13 A TOM 741 O MET 281 222.151 74.152 4.450 OO 79.48 A TOM 742 CB MET 281 222.794 71.122 4.075 OO 73.13 A TOM 743 CG MET 281 222.2S3 69.721 4.315 OO 98.38 A TOM 744 SD MET 281 220.8SO 69703 5.434 OO 82.33 A TOM 745 CE MET 281 221.659 70.065 6.963 OO 94.31 A TOM 746 N GLY 282 223.OOO 73.953 2.379 OO 80.51 A TOM 747 CA GLY 282 223.536 75.298 2.354 OO 70.03 A TOM 748 GLY 282 224.577 7S.481 3.435 OO 89.3S A TOM 749 GLY 282 224,566 76.482 4.146 OO 77.94 A TOM 750 e GLN 283 225.479 74.514 3.576 OO 81.75 A TOM 751 GLN 283 226.509 74.624 4.6O2 OO 104.61 A TOM 752 GLN 283 227.939 74.764 4.094 OO 94.71 A TOM 753 GLN 283 228.503 73.843 3.505 OO 88.07 A TOM 754 GLN 283 226.421 73.4SO 5.583 OO 11316 A TOM 755 GLN 283 226.575 72.075 4.982 OO 114.78 A TOM 756 GLN 283 226.574 71.OO2 6.057 OO 133.39 A TOM 757 GLN 283 227.356 71.068 7.005 OO 133.52 A TOM 758 GLN 283 225.691 70.012 S.922 OO 112.55 A TOM 759 TY 284 228.516 75.935 4.347 OO 90.04 A TOM 760 CA TY 284 229.875 76.242 3.934 OO 64.09 A TOM 761 TY 284 230.474 77.137 S.OO8 OO 65.66 A TOM 762 TY 284 229.806 77.468 S.981 OO 88.03 A TOM 763 CB TY 284 229.864 77.002 2.62O OO 82.88 A TOM 764 CG TY 284 229.040 78.259 2.690 OO SO.94 A TOM 765 CD1 TY 284 227.656 78.208 2.619 OO 72.36 A TOM 766 CD2 TY 284 229.645 79.496 2.856 OO 79.70 A TOM 767 CE1 TY 284 226.901 79.355 2.709 OO 69.83 A TOM 768 CE2 TY 284 228.896 80.646 2.951 OO 57.96 A TOM 769 TY 284 22 7.527 80.569 2.877 OO 57.53 A TOM 770 TY 284 226.778 81.716 2.978 OO 94.22 A TOM 771 GL 285 231.725 77.538 4.821 OO 80.73 A TOM 772 GL 285 232.399 78.388 5.785 OO 81.22 A TOM 773 GL 285 233.397 79.305 S.101 OO 90.67 A TOM 774 GL 285 233.713 79.119 3.926 OO 106.16 A TOM 775 GL 285 233.113 77.527 6.807 OO 87.46 A TOM 776 286 233.875 8O303 5.839 OO 86.81 A TOM 777 286 234.88O 81.236 5.334 OO 97.06 A TOM 778 286 236.110 80.887 6.167 OO 100.16 A TOM 779 286 236.527 81.662 7.028 OO 11 O.OS A TOM 78O 286 234.476 82.697 S. 602 OO 78.01 A TOM 781 286 233,214 83.127 4.904 OO 98.13 A TOM 782 CD1 286 232.172 83.698 S. 615 OO 92.96 A TOM 783 CD2 286 233.064 82.959 3.538 OO 102.41 A TOM 784 CE1 286 231.003 84,092 4.98O OO 96.16 A TOM 785 CE2 286 231.892 83.353 2.897 OO 82.36 A TOM 786 CZ 286 230.86S 83.918 3.62O OO 71.90 A TOM 787 287 236.707 79.715 S.909 OO 96.70 A TOM 788 CA 287 237.883 79.170 6.586 OO O3.18 A TOM 789 287 239.086 80.081 6.730 OO 12.32 A TOM 790 287 239.531 80.709 5,770 OO 1413 A TOM 791 CB s 287 238.210 77.940 5.754 OO O5.25 A TOM 792 CG RO 287 237.828 78.359 4.411 OO 92.44 A TOM 793 CD C RO 287 236.474 78.978 4.659 OO OO.S1 A TOM 794 ASN 288 239.608 80.133 7.950 OO 19.25 A TOM 795 CA ASN 288 240.796 80.913 8.239 OO 31.90 A TOM 796 ASN 288 241.953 79.964 7.949 OO 38.51 A TOM 797 ASN 288 241.808 78.742 8.021 OO 43.95 A TOM 798 CB ASN 288 240.826 81347 9.708 OO 33.89 A TOM 799 CG ASN 288 239.631 82.211 20.092 OO S8.92 A TOM 800 OD1 ASN 288 239.383 83.259 9.485 OO 72.68 A TOM 8O1 ND2 ASN 288 238.890 81.779 21.114 OO 39.14 A TOM 802 PRO 289 243.119 80510 7.6O2 OO 45.89 A TOM 803 CA PRO 289 243.399 81.940 7.477 OO 47.78 A TOM 804 PRO 289 242.632 82.643 6.367 OO 40.12 A TOM 805 PRO 289 242.027 83.678 6.608 OO 45.48 A TOM 806 CB PRO 289 244.904 81.967 7.231 OO 60.27 A TOM 807 CG PRO 289 245.107 80.724 6.415 OO 58.40 A TOM 808 CD PRO 289 244.286 79.712 7.185 OO 49.68 A TOM 809 GLU 290 242.665 82.064 S.166 OO 35.89 A TOM 810 CA GLU 290 242.019 82.6O2 3.959 OO 22.33 A TOM 811 GLU 290 241.100 83.810 4.141 OO 11.48 A TOM 812 GLU 290 241.314 84.858 3.531 OO 94.90 A TOM 813 CB GLU 290 241.230 81. SO2 3.236 OO 21.58 A TOM 814 CG GLU 290 241.934 80.15S 3.147 OO 40.70 A TOM 815 CD GLU 290 241.650 79.287 4360 OO 83.86 A TOM 816 GLU 290 241.898 79.762 5.484 OO 93.44 A TOM 817 GLU 290 241.178 78.137 4.193 OO 82.2O US 9,023,787 B2 81 82 TABLE 1-continued Coordina es of the activated MAPKAP kinase-2, peptide complex A TOM 818 N TRP 291 240.08O 83.6SS 4.98O OO O2.18 A TOM 819 CA TRP 291 239.105 84.715 S.221 OO O6.9S A TOM 820 C TRP 291 239.335 85.573 6.457 OO 15.40 A TOM 821 O TRP 291 238.474 86.375 6.824 OO 20.18 A TOM 822 CB TRP 291 237.712 84.106 5.295 OO 10.40 A TOM 823 CG TRP 291 237.364 83.374 4.056 OO O4.11 A TOM 824 CD1 TRP 291 237.782 82.121 3.692 OO 84.92 A TOM 825 CD2 TRP 291 236.556 83.857 2.982 OO O6.19 A TOM 826 NE1 TRP 291 237.282 81.799 2.456 OO O3.99 A TOM 827 CE2 TRP 291 236.529 82.848 1996 OO O2.71 A TOM 828 CE3 TRP 291 235.857 85.048 2.753 OO 70.56 A TOM 829 CZ2 TRP 291 235.827 82.992 O.807 OO 86.03 A TOM 830 CZ3 TRP 291 235.16S 85.189 1578 OO 73.88 A TOM 831 CH2 TRP 291 235.154 84.171 O. 616 OO 14.SS A TOM 832 N SER 292 24O491 85.404 7.091 OO 29.00 A TOM 833 CA. SER 292 240.851 86.158 8.287 OO 24.04 A TOM 834 C SER 292 240.910 87.670 8.051 OO 23.03 A TOM 835 O SER 292 24O661 88.4S5 8.966 OO 13.67 A TOM 836 CB SER 292 242.199 85.658 8.825 OO 25.10 A TOM 837 OG SER 292 243.196 85.669 7.817 OO 35.63 A TOM 838 N GLU 293 241.237 88.08.1 6.829 OO 23.41 A TOM 839 CA GLU 293 241.321 89.508 6.513 OO 27.66 A TOM 840 C GLU 293 240.108 90.026 S. 724 OO 21.92 A TOM 841 O GLU 293 240.091 91.178 5.285 OO 15.91 A TOM 842 CB GLU 293 242.600 89.792 5.737 OO 38.28 A TOM 843 N VAL 294 239.094 89.181 5.555 OO 11.91 A TOM 844 CA VAL 294 237.898 89.568 4.818 OO 95.15 A TOM 845 C VAL 294 236.811 90.136 5.721 OO O3.25 A TOM 846 O VAL 294 236.506 89.573 6.771 OO 98.28 A TOM 847 CB VAL 294 237.322 88.378 4.066 OO 92.93 A TOM 848 CG1 VAL 294 236.182 88.840 3.168 OO 91.77 A TOM 849 CG2 WAL 294 238.418 87.705 3.269 OO 94.47 A TOM 850 N. SER 295 236.218 91.249 S.292 OO 84.68 A TOM 851 CA. SER 295 235.168 91.914 6.056 OO 92.73 A TOM 852 C SER 295 233.919 91.072 6.244 OO 88.74 A TOM 853 O SER 295 233.568 90.2SO 5.399 OO 88.58 A TOM 854 CB SER 295 234.745 93.2O1 5.365 OO 102.34 A TOM 855 OG SER 295 233.464 93.030 4.784 OO 114.40 A TOM 856 N. GLU 296 233.244 91.301 7.362 OO 92.37 A TOM 857 CA GLU 296 232.008 90.600 7.658 OO 104.61 A TOM 858 C GLU 296 230.970 91.166 6.694 OO 112.98 A TOM 859 O GLU 296 229.956 90.523 6.4O6 OO 105.91 A TOM 86O CB GLU 296 231.593 90.857 9.108 OO 99.76 A TOM 861 CG GLU 296 230.316 90.154 9.543 OO 121.36 A TOM 862 CD GLU 296 230.324 88.663 9.236 OO 136.85 A TOM 863 OE1 GLU 296 231.304 87.970 9.609 OO 131.43 A TOM 864 OE2 GLU 296 229.340 88.185 8.619 OO 136.46 A TOM 865 N GLU 297 231.237 92.379 6.2O3 OO 112.50 A TOM 866 CA GLU 297 230.357 93.045 5.247 OO 99.85 A TOM 867 C GLU 297 23O42O 92.241 3.960 OO 92.47 A TOM 868 O GLU 297 229.396 91.854 3.400 OO 93.48 A TOM 869 CB GLU 297 230.838 94.472 4.998 OO 98.76 A TOM 870 CG GLU 297 230.226 95.146 3.787 OO 102.64 A TOM 871 CD GLU 297 23O489 96.642 3.773 OO 129.34 A TOM 872 OE1 GLU 297 229.777 97.379 4.497 OO 134.48 A TOM 873 OE2 GLU 297 231.415 97.081 3.049 OO 104.72 A TOM 874 N VAL 298 231.638 91.993 3.499 OO 80.38 A TOM 875 CA VAL 298 231.843 91.200 2.305 OO 87.49 A TOM 876 C VAL 298 231.282 89.809 2.579 OO 80.88 A TOM 877 O VAL 298 23 O.690 89.176 1.702 OO 71.8O A TOM 878 CB VAL 298 233.333 91.057 1993 OO 64.O2 A TOM 879 CG1 VAL 298 233.542 89.953 O.980 OO 84.65 A TOM 880 CG2 WAL 298 233.873 92.364 1466 OO 112.61 A TOM 881 N LYS 299 231.479 89.331 3.8O1 OO 79.83 A TOM 882 CA LYS 299 230.990 88.01S 4.162 OO 75.16 A TOM 883 C LYS 299 229.478 87.983 4.138 OO 80.77 A TOM 884 O LYS 299 228.872 87.052 3.618 OO 62.14 A TOM 885 CB LYS 299 231517 87.616 5.539 OO 74.57 A TOM 886 CG LYS 299 232.979 87.229 5.509 OO 75.11 A TOM 887 CD LYS 299 233.432 86.563 6.790 OO 92.06 A TOM 888 CE LYS 299 234.841 86.OOS 6.623 OO 125.81 A TOM 889 NZ LYS 299 235.368 85.378 7.867 OO 126.47 A TOM 890 N MET 3OO 228.872 89.021 4.691 OO 78.00 A TOM 891 CA MET 3OO 227.428 89.116 4.735 OO 80.36 A TOM 892 C MET 3OO 226822 89.120 3.327 OO 69.48 A TOM 893 O MET 3OO 225.732 88.587 3.118 OO 75.70 A TOM 894 CB MET 3OO 227.033 90.378 5.5O2 OO 87.97 A TOM 895 CG MET 3OO 226.011 90.131 6.6O1 OO 118.90 US 9,023,787 B2 83 84 TABLE 1-continued Coordina es of the activated MAPKAP kinase-2, peptide complex A TOM 896 SD MET 3OO 2 26.231 88.508 7.393 OO 128.71 A TOM 897 CE MET 3OO 2 24,516 87.872 7.267 OO 113.92 A TOM 898 N LEU 301 2 27.538 89.707 2.369 OO 73.89 A TOM 899 CA LEU 301 2 27.079 89.782 O.972 OO 63.96 A TOM 900 C LEU 301 2 27.048 88.406 O.301 OO 70.75 A TOM 901 O LEU 301 2 26.145 88.086 O9.532 OO 6038 A TOM 902 CB LEU 301 2 27.977 90.735 0.177 OO 63.62 A TOM 903 CG LEU 301 2 27.617 91.019 O8.720 OO S2.63 A TOM 904 CD1 LEU 301 2 26.130 91.284 O8.596 OO 65.57 A TOM 905 CD2 LEU 301 2 28.414 92.212 O8.225 OO 73.24 A TOM 906 N LE 3O2 2 28.046 87.588 O.S92 OO 67.57 A TOM 907 CA LE 3O2 2 28.090 86.2SO O.O37 OO 61.70 A TOM 908 C LE 3O2 2 26.900 85,455 O.S82 OO 75.97 A TOM 909 O LE 3O2 2 26.226 84.741 O9.837 OO 58.38 A TOM 910 CB LE 3O2 2 29.422 85.565 O.402 OO 70.53 A TOM 911 CG1 ILE 3O2 2 30.562 86.271 O9.659 OO 76.98 A TOM 912 CG2 ILE 3O2 2 29.369 84.O86 O.O71 OO 6S.O1 A TOM 913 CD1 ILE 3O2 2 31.930 85.670 O9.869 OO 71.99 A TOM 914 N ARG 303 2 26.63O 85.611 1878 OO 73.53 A TOM 915 CA ARG 303 2 25.526 84.913 2.532 OO 61.85 A TOM 916 C ARG 303 2 24.162 85.2O1 1898 OO 74.OS A TOM 917 O ARG 303 2 23.370 84.286 1.695 OO 79.8O A TOM 918. CB ARG 303 2 25461 85.279 4.009 OO 61.93 A TOM 919 CG ARG 303 2 26.666 84.917 4.852 OO 78.75 A TOM 920 CD ARG 303 2 26.402 85.392 6.284 OO 71.93 A TOM 921 NE ARG 303 2 27.608 85.725 7.038 OO 101.37 A TOM 922 CZ ARG 303 2 28.488 84.827 7.457 OO 93.09 A TOM 923 NH1 ARG 303 2 28.286 83540 7.191 OO 90.11 A TOM 924 NH2 ARG 303 2 29.561 85.211 8.140 OO 76.11 A TOM 925 N ASN 3O4 2 23.865 86.458 1.596 OO 61.15 A TOM 926 CA. ASN 3O4 2 22.575 86.775 O.982 OO 77.76 A TOM 927 C ASN 3O4 2 22.517 86.277 O9.535 OO 74.95 A TOM 928 O ASN 3O4 2 21449 86.208 O8.929 OO 67.98 A TOM 929 CB ASN 3O4 2 22.312 88.291 1.016 OO 83.64 A TOM 930 CG ASN 3O4 2 22.OOO 88.806 2.421 OO 112.22 A TOM 931 OD1 ASN 3O4 2 22.399 89.919 2.802 OO 84.27 A TOM 932 ND2 ASN 3O4 2 21.269 88.004 2 3.197 OO 69.02 A TOM 933 N LEU 305 2 23.671 85.943 208.973 OO 63.99 A TOM 934 CA LEU 305 2 23.711 85.458 2O7.605 OO 54.76 A TOM 935 C LEU 305 2 23.590 83.952 2O7.666 OO 73.10 A TOM 936 O EU 305 2 22.929 83.313 206.841 OO 58.22 A TOM 937 CB 305 2 25.032 85.860 2O6.948 OO 52.05 A TOM 938 CG 305 2 25.127 87.316 2O6.481 OO 58.75 A TOM 939 CD1 U 305 2 26.511 87.621 2O5.932 OO 69.8O A TOM 940 CD2 LEU 305 2 24.075 87.560 2O5.421 OO 47.92

A TOM 941 N EU 306 2 24.240 83.401 208.681 OO 57.81 A TOM 942 CA 306 2 24.256 81975 208.915 OO 59.22 A TOM 943 C 306 2 23087 81.525 209.779 OO 70.17 A TOM 944 O U 306 2 23.205 80.586 21O.S6S OO 86.15 A TOM 945 CB LEU 306 2 25.581 81.582 209.559 OO 54.83 A TOM 946 CG LEU 306 2 26.752 81.718 208.599 OO 56.28 A TOM 947 CD1 LEU 306 2 28.035 81.262 209.237 OO 46.67 A TOM 948 CD2 LEU 306 2 26.445 80.883 2O7.385 OO 50.52 A TOM 949 N LYS 307 2 21.96O 82.212 209.648 OO 70.52 A TOM 9SO CA LYS 307 2 20.772 81.832 210.394 OO 64.84 A TOM 951 C LYS 307 2 2O2S1 80.593 209.676 OO 65.05 A TOM 952 O LYS 307 2 20.153 80.570 208.449 OO 73.22 A TOM 953 CB LYS 307 2 9.720 82.930 210.337 OO 60.26 A TOM 954 CG LYS 307 20.137 84.247 210.952 OO 76.35 A TOM 955 CD LYS 307 9.62O 84.411 212.371 OO 75.87 A TOM 956 CE LYS 307 20.487 83.696 213.389 OO 92.33 A TOM 957 NZ LYS 307 9.963 83.886 214.773 OO 94.73 A TOM 958 N THR 3O8 9.925 79.558 210.439 OO 87.36 A TOM 959 CA. THR 3O8 9.432 78.314 209.864 OO 86.54 A TOM 960 C THR 3O8 8.057 78.489 209.219 OO 80.07 A TOM 961 O THR 3O8 7.722 77.809 208.248 OO 80.48 A TOM 962 CB THR 3O8 9.382 77.198 210.945 OO 80.31 A TOM 963 OG1 THR 3O8 20.615 76.457 210.929 OO 85.50 A TOM 964 CG2 THR 3O8 8.211 76.26S 210.701 OO 108.80 A TOM 965 N GLU 309 7.264 79.407 209.755 OO 86.56 A TOM 966 CA GLU 309 S.934 79.647 209.217 OO 80.34 A TOM 967 C GLU 309 S.964 80.82O 208.2S3 OO 80.14 A TOM 968 O GLU 309 6.353 81.933 208.614 OO 72.43 A TOM 969 CB GLU 309 4.937 79.945 210.345 OO 79.03 A TOM 970 CG GLU 309 3.461 79.716 209.976 OO 107.32 A TOM 971 CD GLU 309 3.028 78.264 210.160 OO 128.86 A TOM 972 OE1 GLU 309 3.354 77.691 211.227 OO 100.32 A TOM 973 OE2 GLU 309 2.364 77.699 209.252 OO 91.75 US 9,023,787 B2 85 86 TABLE 1-continued Coordinates of the activa ed MAPKAP kinase-2, peptide complex A TOM 974 N 5.554 80.585 2O7.002 OO 69.20 A TOM 975 CA 5.536 81.644 2O5.988 OO 75.56 A TOM 976 C 4.955 82.953 2O6.540 OO 73.13 A TOM 977 O 5.697 83.886 206.848 OO 70.2O A TOM 978 CB 4.675 81.041 204.888 OO 58.28 A TOM 979 CG S.049 79.598 2O4.957 OO 61.31 A TOM 98O CD 5.073 79.312 2O6.442 OO 64.66 A TOM 981 N 3.633 83.004 206.683 OO 57.71 A TOM 982 CA 2.952 84.188 2O7.194 OO 63.44 A TOM 983 C 3.671 84899 208.338 OO 65.40 A TOM 984 O 3.424 86.078 208.585 OO 70.08 A TOM 985 CB 1.552 83.839 2O7.666 OO S2.68 A TOM 986 OG1 1.650 82.936 2O8.763 OO 76.00 A TOM 987 CG2 O.781 83.179 2O6.574 OO 44.79 A TOM 988 4.SS1 84.198 209.043 OO 61.23 A TOM 989 CA 5.271 84.831 210.145 OO 58.30 A TOM 990 6.527 85.518 209.629 OO 63.30 A TOM 991 6.995 86.509 210.1.89 OO 65.70 A TOM 992 CB 5.675 83.798 2112O2 OO 78.77 A TOM 993 CG S.2O3 84.12O 212.625 OO 101.21 A TOM 994 CD 5.952 83.321 213.691 OO 105.49 A TOM 995 OE1 7.112 83.612 214.004 OO 110.73 A TOM 996 NE2 S.294 82.305 214.243 OO 82.13 A TOM 997 7.057 84.974 208.545 OO 75.75 A TOM 998 CA 8.278 85.463 2O7.924 OO 58.86 A TOM 999 8.184 86.894 2O7.391 OO 51.81 A TOM 2OOO 7.125 87.341 2O6.952 OO 85.05 A TOM 2001 CB 8.663 84.495 206.807 OO 52.47 A TOM 2002 CG 20.131 84.401 2O6.516 OO 74.86 A TOM 2003 CD 20.355 83.299 2OS.SO6 OO 84.86 A TOM 2004 NE 9.851 82.018 2O5.984 OO 53.52 A TOM 2005 CZ 9.467 81.032 2O5.186 OO 62.95 A TOM 2006 NH1 9.S28 81.192 2O3.874 OO 85.30 A TOM 2007 NH2 9.039 79.888 205.697 OO 49.87 A TOM 2008 9.318 87.590 2O7.430 OO 75.49 A TOM 2009 CA 9.4SO 88.981 206.980 OO 62.90 A TOM 2010 9.115 89.16O 2O5.496 OO 68.2O A TOM 2011 2 9.267 88.229 2O4.694 OO 78.91 A TOM 2012 CB 2 20.888 89.463 2O7.248 OO 79.70 A TOM 2013 CG 2 21145 90.951 2O6.994 OO 7401 A TOM 2014 SD 2 22.873 91.447 2O7.330 OO 84.44 A TOM 2015 CE 2 22.742 2.071 208.957 OO 104.75 A TOM 2016 TH 2 18660 0.357 205.135 OO 63.73 A TOM 2017 CA 2 18.319 O.652 2O3.750 OO 64.98 A TOM 2018 2 19470 347 2O3.030 OO 68.09 A TOM 2019 2 20.317 969 2O3.658 OO 63.59 A TOM 2020 T HR 2 17.075 S45 203.661 OO 62.15 A TOM 2021 2 17.322 2.813 204.286 OO 58.05 A TOM 2022 2 15929 O868 2O4.338 OO 61.97 A TOM 2023 2 19498 236 201707 OO 65.76 A TOM 2O24 CA 2 2O.S66 200.935 OO 66.14 A TOM 2025 2 2O.S64 201.167 OO 71.26 A TOM 2026 2 21.618 201.192 OO 67.36 A TOM 2027 2 20.417 199.426 OO 60.35 A TOM 2028 2 21.714 198.710 OO 52.24 A TOM 2029 ILE 2 9.314 198.854 OO 56.27 A TOM 2O3O 22.942 199.405 OO 55.83 A TOM 2031 9.381 201346 OO 6O.SO A TOM 2032 CA 9.301 2O1.591 OO S8.94 A TOM 2O33 9.858 202.964 OO 61.73 A TOM 2O34 2O.S33 2O3.16.1 OO 69.91 A TOM 2035 CB 7.865 2O1554 OO 68.58 A TOM 2O36 OG1 7.301 200.259 OO 67.56 A TOM 2037 CG2 7.844 201.860 OO 50.05 A TOM 2038 N 9.577 2O3.923 OO 54.23 A TOM 2O39 CA 20.082 205.266 OO 69.88 A TOM 2040 C 21.586 205.348 OO 72.99 A TOM 2041 O 22.262 2O6.221 OO 64.78 A TOM 2042 CB 9.336 2O6.269 OO 82.03 A TOM 2043 CG 7.903 2O6.496 OO 64.99 A TOM 2044 CD 7.152 2O7.486 OO 85.92 A TOM 2045 OE1 6.415 208.315 OO 91.71 A TOM 2046 OE2 2 7.288 2O7.424 OO 88.17 A TOM 2047 N 2 22.103 2O4.421 OO 75.36 A TOM 2048 CA 2 23.519 20438O OO 69.31 A TOM 2049 2 24.279 2O3.800 OO 79.85 A TOM 2OSO 2 2S281 2O4.359 OO 86.43 A TOM 2051 CB 2 23.744 2O3.523 OO 78.91 US 9,023,787 B2 87 88 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 2052 CG PHE 319 225.193 92.144 2O3.2SO OO 82.OS A TOM 2053 CD1 PHE 319 225.978 91.538 204210 OO 6469 A TOM 2O54 CD2 PHE 319 225.757 92.432 202.011 OO 68.6S A TOM 2055 CE1 PHE 319 227.287 91.219 2O3.940 OO 55.30 A TOM 2056 CE2 PHE 319 227.060 92114 201744 OO 75.06 A TOM 2057 CZ PHE 319 227.826 91.506 2O2.711 OO 57.56 A TOM 2O58 N MET 320 223.805 95.404 2O2.679 OO 64.36 A TOM 2059 CA MET 320 224489 96.527 202.066 OO 69.16 A TOM 2060 C MET 320 224.525 97.753 2O2.973 OO 78.25 A TOM 2061 O MET 320 225.304 98.673 2O2.745 OO 86.13 A TOM 2062 CB MET 320 223.848 96.889 200.725 OO 73.27 A TOM 2063 CG MET 320 224.120 95.877 199.63S OO 72.98 A TOM 2O64 SD MET 320 225.832 95.315 199.691 OO 87.32 A TOM 206S CE MET 320 226.586 96.413 198.528 OO 70.2O A TOM 2066 N ASN 321 223.695 97.772 204,006 OO 77.28 A TOM 2O67 CA ASN 321 223.691 98.907 204.912 OO 79.74 A TOM 2O68 C ASN 321 224489 98.692 2O6.174 OO 76.50 A TOM 2069 O ASN 321 224.868 99.651 206.836 OO 95.86 A TOM 2070 CB ASN 321 222.271 99.296 205.267 OO 79.55 A TOM 2O71 CG ASN 321 221.783 100.428 2O4.427 OO 104.33 A TOM 2O72 OD1 ASN 321 222.191 101.578 2O4.622 OO 115.09 A TOM 2O73 ND2 ASN 321 220.926 100.121 2O3.456 OO 119.81 A TOM 2074 HIS 322 224.744 97.439 2O6. S21 OO 82.50 A TOM 2075 CA HIS 322 225.527 97.179 207.707 OO 80.78 A TOM 2O76 HIS 322 226.837 97.948 207.555 OO 84-39 A TOM 207 7 HIS 322 227.483 97.895 2O6.506 OO 83.28 A TOM 2O78 CB HIS 322 225.812 95.691 2O7.851 OO 75.27 A TOM 2O79 CG HIS 322 226.690 95.370 209.013 OO 76.67 A TOM 2080 ND1 HIS 322 227.927 95.950 209.189 OO 84.96 A TOM 2081 CD2 HIS 322 226.504 94.548 210.070 OO 67.42 A TOM 2082 CE1 HIS 322 228.464 95.5O2 210.308 OO 85.58 A TOM 2083 NE2 HIS 322 227.622 94.649 210.862 OO 86.63 A TOM 2O84 323 227.234 98.686 208.603 OO 91.61 A TOM 2085 CA 323 228.447 99.5O1 208.68O OO 84.82 A TOM 2O86 323 229.697 98.867 208.087 OO 79.23 A TOM 2087 323 230.318 99.436 2O7.193 OO 85.59 A TOM 2088 CB 323 228.567 99.772 210.170 OO 74.59 A TOM 2089 CG 323 227.135 99.952 210.553 OO 86.91 A TOM 2090 CD 323 226.484 98.767 209.868 OO 92.14 A TOM 2091 324 230.066 97.692 208.578 OO 81.53 A TOM 2092 CA 324 231.247 97.002 2O8.07.0 OO 80.47 A TOM 2093 324 231.260 97.039 2O6.541 OO 76.75 A TOM 2094 324 232.288 97.293 205.917 OO 63SO A TOM 2095 CB 324 231.225 95.553 208.537 OO 79.62 A TOM 2096 324 232.549 94881 208.546 OO 68.42 A TOM 2097 CD1 324 233.579 95.118 209.408 OO 97.23 A TOM 2098 CD2 324 232.973 93.806 2O7.704 OO 91.77 A TOM 2099 NE1 324 234.614 94.254 209.162 OO 96.32 A TOM 2 OO CE2 324 234.268 93.437 2O8.120 OO 90.15 A TOM O1 CE3 324 232.382 93.118 206.639 OO 85.73 A TOM O2 CZ2 324 234.979 92.411 2O7.515 OO 55.96 A TOM O3 CZ3 324 233.091 92.099 2O6.038 OO 78.54 A TOM O4 CH2 324 234.375 91.755 2O6.475 OO 83.38 A TOM 05 IL 325 230.099 96.785 205.952 OO 61.98 A TOM O6 CA 325 229.956 96.764 2O4S13 OO 63.34 A TOM O7 325 229.832 98.156 2O3.92O OO 81.46 A TOM O8 325 23 O.S18 98.483 2O2.956 OO 101.97 A TOM 09 325 228.723 95.941 204.092 OO 64.13 A TOM 10 325 229.156 94.6.21 2O3.470 OO 57.81 A TOM 11 325 227.909 96.703 2O3.076 OO 79.69 A TOM 12 325 23 O.OOS 93.796 2O4.357 OO 99.33 A TOM 13 326 228.971 98.986 204,497 OO 86.84 A TOM 14 CA 326 228.766 OO.327 2O3.962 OO 97.8O A TOM 15 326 229.941 O1.291 204.108 OO 88.82 A TOM 16 326 230.023 O2.271 2O3.375 OO 96.42 A TOM 17 CB 326 227.509 OO.951 2O4.573 OO 99.22 A TOM 18 CG 326 227.134 O2.287 2O3.957 OO 109.19 A TOM 19 SD 326 225.425 O2.771 2O4.309 OO 131.16 A TOM 2O CE 326 224.679 O2.571 2O2.683 OO 123.49 A TOM 21 LN 327 230.847 O1.016 2O5.042 OO 99.42 A TOM 22 CA L N 327 232.OOS O1880 205.253 OO 100.47 A TOM 23 LN 327 233.330 O1120 205.290 OO 109.31 A TOM 24 LN 327 234.060 O1184 2O6.279 OO 116.79 A TOM 25 CB LN 327 231.856 O2.668 2O6.557 OO 90.31 A TOM 26 CG GLN 327 230.78O O3.743 2O6.538 OO 124.03 A TOM 27 CD GLN 327 230.929 O4.713 205.370 OO 165.27 A TOM 28 GLN 327 232.047 OS.OS1 2O4.958 OO 160.87 A TOM 29 GLN 327 229.795 O5.178 204841 OO 148.79 US 9,023,787 B2 89 90 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 30 N SER 328 233.644 OO.406 2O4.215 OO O9.39 A TOM 31 CA SER 328 234.888 99.643 204.157 OO 17.56 A TOM 32 C SER 328 236.084 OO.S39 2O4.457 OO 26.63 A TOM 33 O SER 328 236.892 OO.239 205.332 OO 22.39 A TOM 34 CB SER 328 235.055 99.008 2O2.777 OO O6.48 A TOM 35 OG SER 328 233.976 98.139 2O2.490 OO O1.32 A TOM 36 N THR 329 236.189 O1642 2O3.725 OO 47.22 A TOM 37 CA THR 329 237.283 O2.582 2O3.919 OO 53.16 A TOM 38 C THR 329 237.668 O2.713 205.393 OO S1.36 A TOM 39 O THR 329 238.849 O2.68O 205.736 OO 65.48 A TOM 40 CB THR 329 236.918 O3.986 2O3.365 OO S9.02 A TOM 41 OG1 THR 329 237.929 O4.930 2O3.748 OO 64.35 A TOM 42 CG2 THR 329 235.553 O4.443 2O3.893 OO 71.23 A TOM 43 LYS 330 236.670 O2.840 2O6.262 OO 38.67 A TOM 44 LYS 330 23691.6 O2994 2O7.691 OO 28.38 A TOM 45 LYS 330 236.937 O1670 208.457 OO 34.38 A TOM 46 LYS 330 236.425 O1586 209.58O OO 38.61 A TOM 47 LYS 330 235.867 O3.931 208.298 OO 34.91 A TOM 48 VAL 331 237.526 OO.637 2O7.860 OO 22.49 A TOM 49 VAL 331 237.608 99.340 208.531 OO 2O.28 A TOM 50 VAL 331 239.066 98.890 208.689 OO 1119 A TOM 51 VAL 331 239.850 98.899 2O7.739 OO 85.41 A TOM 52 VAL 331 236.770 98.239 2O7.785 OO 20.15 A TOM 53 VAL 331 237.342 97.971 206.422 OO 25.69 A TOM S4 VAL 331 236.740 96.946 208.595 OO 24.20 A TOM 55 PRO 332 239.433 98.496 209.916 OO O2.76 A TOM 56 PRO 332 240.737 98.021 210.385 OO O5.52 A TOM 57 PRO 332 241.528 97.215 209.363 OO 12.97 A TOM 58 PRO 332 241.016 96.262 2O8.779 OO 11.93 A TOM 59 CB PRO 332 240.369 97.193 211.609 OO O5.70 A TOM 60 CG PRO 332 239.247 97.970 212.184 OO 22.25 A TOM 61 CD PRO 332 238.418 98.332 210.971 OO OO.61 A TOM 62 LN 333 242.782 97.604 209.154 OO O9.39 A TOM 63 CA LN 333 243.645 96.908 208.206 OO O6.56 A TOM 64 LN 333 244.265 95.714 208.913 OO 99.10 A TOM 65 LN 333 245.347 95.251 208.554 OO 97.72 A TOM 66 CB LN 333 244.744 97.842 2O7.696 OO 17.29 A TOM 67 CG LN 333 244.224 99.042 2O6.922 OO 21.73 A TOM 68 CD LN 333 244.771 99.095 2OS.S10 OO 44.41 A TOM 69 OE1 LN 333 244.442 99.996 2O4.73S OO S3.63 A TOM 70 NE2 333 245.613 98.124 2O5.16S OO 38.94 A TOM 71 334 243.565 95.229 209.931 OO 88.60 A TOM 72 CA 334 244,023 94.081 210.693 OO 12.85 A TOM 73 334 244.310 92.878 209.797 OO 23.95 A TOM 74 334 243.438 92.392 209.086 OO 28.29 A TOM 75 334 242.989 93.668 211.738 OO OS.19 A TOM 76 334 243.01.1 92.243 211.887 OO O2.93 A TOM 77 334 241.607 94.120 211.315 OO O6.97 A TOM 78 335 245.549 92.372 209.832 OO 24.58 A TOM 79 335 245.911 91.224 209.OOS OO 24.62 A TOM 8O 335 245.058 89.999 209.317 OO 19.67 A TOM 81 335 244.513 89.878 210.416 OO O8.06 A TOM 82 335 247.383 91.OO2 209.358 OO 34.66 A TOM 83 335 247.857 92.390 209.673 OO 20.03 A TOM 84 335 246.730 92.881 21O.S47 OO 20.76 A TOM 85 336 244.926 89.110 208.333 OO 10.96 A TOM 86 336 244.175 87.867 208.499 OO 98.45 A TOM 87 336 245087 86.726 2O8.066 OO 87.77

A TOM 88 E 336 245.851 86.877 2O7.117 OO 92.64 A TOM 89 336 242.909 87.852 2O7.632 OO 95.92 A TOM 90 336 241.694 88.725 2O7.963 OO 81.15 A TOM 91 CD1 336 241641 88.972 209.461 OO 94.01 A TOM 92 CD2 E t 336 241.773 90.034 2O7.215 OO 82.79 A TOM 93 337 244.999 85.595 2O8.762 OO 86.OS A TOM 94 337 245.813 84.410 208.469 OO 1OO.OS A TOM 95 337 245.401 83.709 2O7.170 OO 103.25 A TOM 96 337 246.075 82.782 2O6.712 OO 11O.S2 A TOM 97 337 245.687 83.390 209.610 OO 118.04 A TOM 98 337 246.205 83.874 210.930 OO 151.49 A TOM 2 99 ND1 337 246.241 85.210 211.271 OO 169.76 A TOM 2200 CD2 337 246.675 83.198 212.010 OO 16O.S1 A TOM 22O1 CE1 337 246.713 85.337 212. SO2 OO 129.99 A TOM 22O2 NE2 337 246.984 84.132 212.971 OO 150.26 A TOM 22O3 N 338 244.294 84.167 2O6.589 OO 111.23 A TOM 2204 CA 338 243.713 83.590 205.375 OO 105.16 A TOM 2205 338 244.649 82.822 204,440 OO 106.58 A TOM 22O6 338 244.708 81.589 2O4.504 OO 102.89 A TOM 22O7 CB 338 242.957 84.658 2O4.557 OO 100.36 US 9,023,787 B2 91 92 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex A TOM 2208 OG1 THR 338 242.218 85,494 2O5.4S1 OO A TOM 2209 CG2 THR 338 241.961 83.993 2O3.6OS OO A TOM 2210 N SER 339 245.370 83.532 2O3.573 OO A TOM 2211 CA SER 339 246.276 82.877 2O2.622 OO A TOM 2212 C SER 339 246.957 81.641 2O3.2O2 OO A TOM 2213 O SER 339 246.862 80.555 2O2.634 OO A TOM 2214 CB SER 339 247.341 83.8SS 2O2.137 OO A TOM 2215 OG SER 339 246.771 84.88O 201348 OO A TOM 2216 N ARG 340 247.643 81.811 204-329 OO A TOM 2217 CA ARG 340 248.325 80.697 2O4.971 OO A TOM 2218 C ARG 340 247.391 79.508 2O5.121 OO A TOM 2219 O ARG 340 247.SS4 78.480 2O4.454 OO A TOM 2220 CB ARG 340 248.848 81,112 2O6.347 OO A TOM 2221 CG ARG 340 2SO.O41 82.OSS 2O6.292 OO A TOM 2222 CD ARG 340 249.770 83.353 2O7.068 OO A TOM 2223 NE ARG 340 249.6O7 83.107 208.504 OO A TOM 2224 CZ ARG 340 249.378 84.OS3 209.416 OO A TOM 2225 NH1 ARG 340 249.279 85.330 209. OSS OO A TOM 2226 NH2 ARG 340 249.2SS 83.719 21 O.696 OO A TOM 2227 VAL 341 246.412 79.653 2O6.004 OO A TOM 2228 CA VAL 341 245.4S5 78.585 2O6.239 OO A TOM 2229 VAL 341 244.913 78.128 204.891 OO A TOM 2230 VAL 341 244.917 76.939 204566 OO A TOM 2231 VAL 341 244.269 79.077 2O7.078 OO A TOM 2232 VAL 341 243.692 77.928 2O7.866 OO A TOM 2233 VAL 341 244.7OO 80.214 2O7.975 OO A TOM 2234 LEU 342 244.463 79.101 204.107 OO A TOM 2235 CA LEU 342 243.887 78847 2O2.798 OO A TOM 2236 LEU 342 244.771 77.929 201.976 OO A TOM 2237 LEU 342 24428O 77.134 201181 OO A TOM 2238 CB LEU 342 243.666 80.171 202.06S OO A TOM 2239 CG LEU 342 242.513 80.212 201.06S OO A TOM 2240 CD1 LEU 342 241.211 79.86S 201.761 OO A TOM 2241 CD2 LEU 342 242.428 81.595 200.463 OO A TOM 2242 LYS 343 246.078 78.031 2O2.177 OO A TOM 2243 CA LYS 343 246.980 77.154 201.447 OO A TOM 2244 LYS 343 247.211 75.800 2O2.144 OO A TOM 2.245 LYS 343 247.730 74.859 201.559 OO A TOM 2246 CB LYS 343 248.308 77.894 2O1.32O OO A TOM 2247 CG LYS 343 249.233 77.265 200.274 OO A TOM 2248 CD LYS 343 2SO.S28 78.061 200..106 OO A TOM 2249 CE LYS 343 2S1.591 77.288 199.318 OO A TOM 2250 NZ LYS 343 2S2.882 77.968 199.425 OO A TOM 2251 344 246.842 75.714 2O3.442 OO A TOM 2252 CA 344 247.254 74.549 204.240 OO A TOM 2253 344 246.429 73.272 2O3.985 OO A TOM 2254 344 246.915 72.156 204102 OO A TOM 2255 344 247.196 74.935 205.720 OO A TOM 2256 344 248.480 74.589 2O6.485 OO A TOM 2257 344 249.414 75.782 2O6.484 OO 2 A TOM 2258 344 249.252 76.6SO 2O7.336 OO A TOM 2259 344 250.270 75.861 205.6O7 OO 2 A TOM 2260 345 245.127 73.456 2O3.702 OO A TOM 2261 CA 345 244.310 72.298 2O3.321 OO A TOM 2262 345 243.429 72.611 2O2.107 OO A TOM 2263 345 243.779 73.413 2012SO OO A TOM 2264 CB 345 243.435 71.892 2O4.514 OO A TOM 2265 CG 345 242.851 70.495 204291 OO A TOM 2266 OD1 345 243455 69.731 2O3S33 OO A TOM 2267 OD2 345 241.82O 70.182 204.883 OO TER 2267 345 A TOM 242.399 73.184 99.539 OO A TOM CA 240.997 73.382 99.193 OO A TOM CB 24O688 74-874 99.065 OO A TOM CG 239.462 75.245 98.226 OO A TOM CD1 238.302 75.613 99.137 OO A TOM CD2 239.801 76.397 97.295 OO A TOM 240.6SO 72.668 97.890 OO A TOM 241.075 72.982 96.773 OO A TOM 239.765 71.671 97.981 OO A TOM 1 CA 239.268 70.862 96.875 OO A TOM 11 CB 238.690 69.547 97.399 OO A TOM 12 CG 238.232 69.602 98.847 OO A TOM 13 CD 238,424 68.283 OO A TOM 14 OE1 237.494 67.758 OO A TOM 15 NE2 239.634 67.743 OO A TOM 16 238.205 71.615 OO A TOM 17 237.316 72.263 OO US 9,023,787 B2 93 94 TABLE 1-continued Coordinates of the activated MAPKAP kinase-2, peptide complex ATOM 18 N ARG C 6 238.236 71.564 194.739 OO ATOM 19 CA ARG C 6 237.243 72.209 193.873 OO ATOM 2O C ARG C 6 235.863 71.634 194.126 OO ATOM 21 O ARG C 6 235.711 70.424 1943O3 OO ATOM 22 CB ARG C 6 237.578 72.01S 192.398 OO ATOM 23 CG ARG C 6 238.842 72.678 191.953 OO ATOM 24 CD ARG C 6 238.84.O 72.882 190.445 OO ATOM 2S NE ARG C 6 237.880 73.892 189.981 OO ATOM 26 CZ ARG C 6 237.97S 75.204 190.210 OO ATOM 27 NH1 ARG C 6 238.987 75.699 190.913 OO ATOM 28 NH2 ARG C 6 237.069 76.031 189.710 OO ATOM 29 N GLN C 7 234.864 72.520 194186 OO ATOM 3O CA GLN C 7 233.488 72.105 194427 OO ATOM 31. CB GLN C 7 232.904 72.869 195.617 OO ATOM 32 CG GLN C 7 233.852 72.996, 196.798 OO ATOM 33 CD GLN C 7 233.258 73.799 197.940 OO ATOM 34 OE1 GLN C 7 232.067 74.11 6 197.937 OO ATOM 3S NE2 GLN C 7 234.086 74.132 198.923 OO ATOM 36 C GLN C 7 232.623 72.336 193.191 OO ATOM 37 O GLN C 7 232.724 73.384 192.538 OO ATOM 38 N LE C 8 231.805 71.324 192.833 OO ATOM 39 CA LE C 8 230.922 71.415 191.677 OO ATOM 40 CB LE C 8 231.16O 70.233 190.734 OO ATOM 41 CG LE C 8 231.208 70.558. 189.238 OO ATOM 42 CD1 LE C 8 232.592 70.252 188.686 OO ATOM 43 CD2 LE C 8 230.147 69.759 188. SOO OO ATOM 44 C LE C 8 229.459 71.446 192.108 OO ATOM 4S O LE C 8 229.098 70.977 193.190 OO ATOM 46 N SER C 9 228.611 72.OOS 191242 OO ATOM 47 CA. SER C 9 227.183 72.104 191526 OO ATOM 48 C SER C 9 226.402 70.843 191151 OO ATOM 49 O SER C 9 226.93O 69.943 190.490 OO ATOM SO CB SER C 9 226.569 73.323 190.823 OO ATOM S1 OG SE C 9 226.326 73.072 189.444 OO ATOM 52 N ILE C 10 225.174 70.78O 191.613 OO ATOM S3 CA ILE C 10 224.249 69.694 191311 OO ATOM 54 CB ILE C 10 223.837 68.939 192.591 OO ATOM SS CG2 ILE C 10 22S.O2S 68.817 193532 OO ATOM S6 CG1 ILE C 10 222.681 69.664 19328O OO ATOM 57 CD1 ILE C 10 222.397 69.168 194.682 OO ATOM 58 C ILE C 10 222.992 70.220 190.626 OO ATOM 59 O ILE C 10 222.7SS 71.452 190.654 OO ATOM 60 N ALA C 11 222.224 69.342 190.O29 OO ATOM 61 CA. ALA C 11 220.983 69.676 189.342 OO ATOM 62 C ALA C 11 220.122 68.421, 189365 OO ATOM 63 O ALA C 11 220.SS2 67.36O 188.901 OO ATOM 64 CB ALA C 11 221.2S2 70.125 187.902 OO TER 64 ALA C 11 END

45 TABLE 2 Dharmacon Research. Cells were transfected with siRNAs using oligofectamine (Invitrogen) according to the manufac Pairs of contacting atoms in the MAPKAP kinase-2/peptide complex turer's instructions. Cells were typically harvested for further Atom in activated experiments after forty-eight hours. U2OS cells stably MAPKAP kinase-2 Atom in peptide Distance 50 expressing shRNA constructs were generated by lentiviral gene transfer. The RNAi hairpins were cloned into the mul ILE74 CD1 SER9 OG 2.90275 GLU145 OE1 ARG6NH2 194903 tiple cloning site of the lentiviral transfer vector plentiLox LYS188 NZ SER9 CB 2.48O16 3.7puro or -3.7GFP. Amphotropic VSV-G pseudotyped len GLU190 CD GLN7 O 3.33866 tivirus was used for all infections in a BL2+ facility. All PHE210 CE2 SER9 OG 2.78278 55 transfer and packaging constructs were a kind gift from C. P. PHE210 CZ ILE10 O 2.37786 CYS224 SG ALA11 CA 3.36808 Dillon, (MIT). Targeted cells were selected in 8 g/ml puro TYR225 O SER9 CA 3.25655 mycin for four days. Sequences used for RNAi were: TYR225 O ILE1ON 2.88O16 luciferase (shRNA), 5'pTGA CCA GGC ATT CAC AGA THR 226 OG1 GLN 7 OE1 3.42713 AATTCA AGAGATTTC TGT GAA TGC CTG GTC TTT PRO 227 CD LEU 8 O 3.44.686 60 TYR 228 CB GLN 7 OE1 3.02662 TTT C-3' (SEQID NO. 24); hMAPKAP kinase-2 (shRNA), TYR 229 CE1 GLN 7 OE1 3.34283 5'-pTTGACCATCACCGAGTTTATTTCA AGAGAATA ASP 345 O LEU 4N 2.21 OO6 AAC TCG GTG ATG GTCATTTTTTC-3' (SEQID NO: 25); mMAPKAP kinase-2 (shRNA), 5'-pTCG ATG CGT GTTGACTAT GATTCA AGAGAT CAT AGT CAA CAC RNA Interference (RNAi) and Recombinant DNA. 65 GCA TCGTTTTTT C-3' (SEQ ID NO: 26); GFP (siRNA) siRNA duplexes consisting of twenty-one base pairs with a sense 5'-UCC CGG CUA UGUGCAGGA GdTdT-3' (SEQ two-base deoxynucleotide overhang were purchased from ID NO: 27) and antisense strand 5'-CUCCUG CAC AUA US 9,023,787 B2 95 96 GCC GGG AdTdT-3' (SEQ ID NO: 28); mMAPKAP leukemia, chronic lymphocytic leukemia, chronic myelocytic kinase-2 (siRNA), sense 5'-CGA UGC GUG UUG ACU leukemia, colon cancer, colon carcinoma, craniopharyn AUG AdTdT-3' (SEQ ID NO: 29) and antisense strand gioma, cystadenocarcinoma, embryonal carcinoma, endothe 5'-UCAUAGUCA ACACGC AUC GdTdT-3' (SEQID NO: liosarcoma, ependymoma, epithelial carcinoma, Ewings 30); hMAPKAP kinase-2 (siRNA), sense 5'-UGACCAUCA tumor, glioma, heavy chain disease, hemangioblastoma, CCG AGUUUA UdTdT-3' (SEQID NO:31) and anti-sense hepatoma, Hodgkin’s disease, large cell carcinoma, leiomyo strand 5'-AUA AACUCG GUGAUG GUC AdTdT-3' (SEQ sarcoma, liposarcoma, lung cancer, lung carcinoma, lym ID NO:32); Chk1 (siRNA), 5'-UGG CAA CAGUAUUUC phangioendotheliosarcoma, lymphangiosarcoma, macroglo GGU AdTdT-3' (SEQ ID NO: 33) and antisense strand bulinemia, medullary carcinoma, medulloblastoma, 5'-UACCGAAAU ACUGUUGCC AdTdT-3' (SEQID NO: 10 melanoma, meningioma, mesothelioma, myxosarcoma, neu 34). roblastoma, non-Hodgkin’s disease, oligodendroglioma, For overexpression studies, FLAG-6xHis-tagged human osteogenic sarcoma, ovarian cancer, pancreatic cancer, pap Chk1 cDNA was PCR amplified and subcloned into the illary adenocarcinomas, papillary carcinoma, pinealoma, Mlu-1 and Not-1 sites of pHURRA downstream from the polycythemia Vera, prostate cancer, rhabdomyosarcoma, CMV promoter. pHURRA was a kind gift from Dr. H. Paven 15 renal cell carcinoma, retinoblastoma, Schwannoma, Seba stadt (U. of Munster). ceous gland carcinoma, seminoma, Small cell lung carci Therapy noma, squamous cell carcinoma, Sweat gland carcinoma, Therapy according to the invention may be performed synovioma, testicular cancer, uterine cancer, Waldenstrom's alone or in conjunction with another therapy and may be fibrosarcoma, and Wilm's tumor. Any of these diseases or provided at home, the doctors office, a clinic, a hospital’s disorders can include, or be associated with, one or more outpatient department, or a hospital. Treatment generally p53-deficient cells, e.g., tumor cells. begins at a hospital so that the doctor can observe the thera A MAPKAP kinase-2-binding peptide, small molecule, or py's effects closely and make any adjustments that are other compound may be administered within a pharmaceuti needed. The duration of the therapy depends on the age and cally-acceptable diluent, carrier, or excipient, in unit dosage condition of the patient, the stage of the patient’s disease or 25 form. Conventional pharmaceutical practice may be disorder, and how the patient responds to the treatment. Addi employed to provide Suitable formulations or compositions to tionally, a person having a greater risk of developing a disease administer the compounds to patients suffering from a dis or disorder that may be treated by the methods of the inven ease that is caused by excessive cell proliferation. Adminis tion (e.g., a person who is genetically predisposed) may tration may begin before the patient is symptomatic. Any receive prophylactic treatment to inhibit or delay symptoms 30 appropriate route of administration may be employed, for of the disease. Drug administration may be performed at example, administration may be parenteral, intravenous, different intervals (e.g., daily, weekly, or monthly). Therapy intra-arterial, subcutaneous, intramuscular, intracranial, may be given in on-and-off cycles that include rest periods so intraorbital, ophthalmic, intraventricular, intracapsular, that the patient’s body has a chance to build healthy new cells intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, and regain its strength. Therapy may be used to extend the 35 Suppository, or oral administration. For example, therapeutic patient’s lifespan. formulations may be in the form of liquid solutions or Sus For cancer treatment, depending on the type of cancer and pensions; for oral administration, formulations may be in the its stage of development, the therapy can be used to slow the form of tablets or capsules; and for intranasal formulations, in spreading of the cancer, to slow the cancer's growth, to kill or the form of powders, nasal drops, or aerosols. arrest cancer cells that may have spread to other parts of the 40 Combination Therapy body from the original tumor, to relieve symptoms caused by As described above, if desired, treatment with compounds the cancer, or to prevent cancer in the first place. that inhibit MAPKAP kinase-2 polypeptides may be com Administration of Therapeutic Compounds bined with therapies for the treatment of proliferative disease, By selectively disrupting or preventing a compound from Such as radiotherapy, Surgery, or chemotherapy. Chemothera binding to its natural partner(s) through its binding site, one 45 peutic agents that may be administered with compounds (e.g., may inhibit the biological activity or the biological function UCN-01) that interact with a MAPKAP kinase-2 polypeptide of a MAPKAP kinase-2 polypeptide. The methods of the include, but are not limited to, alemtuzumab, , invention feature the use of compounds that inhibit an activity aminoglutethimide, , anastroZole, , of a MAPKAP kinase-2 polypeptide, whether by reducing , bicalutamide, , , carbopl expression of the polypeptide (e.g., RNAi or antisense 50 atin, , , , 2-chlorodeoxyad therapy), or by binding directly to a MAPKAP kinase-2 enosine, cisplatin, , , cytara polypeptide and inhibiting its substrate-binding activity. In bine, cytoxan, , , daunorubicin, some instances, MAPKAP kinase-2 inhibitory compounds , doxorubicin, , , are administered to patients having one or more p53-deficient etodolac, etoposide, exemestane, , , cells, e.g., tumor cells. Exemplary inhibitory compounds will 55 5-, flutamide, formestane, , gentu be described further below. Zumab, goserelin, to hexamethylmelamine, hydroxyurea, Diseases or disorders characterized by inappropriate cell hypericin, , imatinib, interferon, , letro cycle regulation include cellular proliferative disorders, such Zole, leuporelin, , mechlorethamine, melphalen, as neoplasias. Examples of neoplasias include, without limi , 6-mercaptopurine, , mitomy tation, acoustic neuroma, acute leukemia, acute lymphocytic 60 cin, , , nilutamide, nocodazole, pacli leukemia, acute monocytic leukemia, acute myeloblastic leu taxel, , , , rituximab, rofe kemia, acute myelocytic leukemia, acute myelomonocytic coxib, Streptozocin, tamoxifen, , , leukemia, acute promyelocytic leukemia, acute erythroleuke 6-thioguanine, , toremofine, trastuzumab, vinblas mia, adenocarcinoma, angiosarcoma, astrocytoma, basal cell tine, , , and . One or more carcinoma, bile duct carcinoma, bladder carcinoma, brain 65 chemotherapeutic agents may be administered in combina cancer, breast cancer, bronchogenic carcinoma, cervical can tion with one or more compounds that inhibit MAPKAP cer, chondrosarcoma, chordoma, choriocarcinoma, chronic kinase-2 polypeptides. In some instances, combination US 9,023,787 B2 97 98 therapy is administered to patients having one or more p53 RNA insert, and a 4-5-thymidine transcription termination deficient cells, e.g., tumor cells. signal can be employed. The Polymerase III promoters gen In the combination therapies of the invention, the therapy erally have well-defined initiation and stop sites and their components are administered simultaneously, or within transcripts lack poly(A)tails. The termination signal for these twenty-eight days of each other, in amounts sufficient to promoters is defined by the polythymidine tract, and the inhibit the growth of said neoplasm. transcript is typically cleaved after the second uridine. Cleav Depending on the type of cancer and its stage of develop age at this position generates a 3' UU overhang in the ment, the combination therapy can be used to treat cancer, to expressed shRNA, which is similar to the 3' overhangs of slow the spreading of the cancer, to slow the cancer's growth, synthetic siRNAs. Additional methods for expressing the to kill or arrest cancer cells that may have spread to other parts 10 shRNA in mammalian cells are described in the references of the body from the original tumor, to relieve symptoms cited above. caused by the cancer, or to prevent cancer in the first place. Computer programs that employ rational design of oligos Combination therapy can also help people live more comfort are useful in predicting regions of the MAPKAP kinase-2 ably by eliminating cancer cells that cause pain or discomfort. sequence that may be targeted by RNAi. For example, see The administration of a combination of the present inven 15 Reynolds et al., Nat. Biotechnol., 22:326-330, 2004, for a tion allows for the administration of lower doses of each description of the Dharmacon sil)ESIGN tool. Table 3 lists compound, providing similar efficacy and lower toxicity several exemplary nucleotide sequences within MAPKAP compared to administration of either compound alone. Alter kinase-2 that may be targeted for purposes of RNA interfer natively, Such combinations result in improved efficacy in ence. siRNA or shRNA oligos may be made corresponding to treating neoplasms with similar or reduced toxicity. the sequences shown and including an overhang, e.g., a 3' RNA Interference Therapy dTdT overhang and/or a loop. The invention features the novel and therapeutically impor tant discovery that the use of RNA interference (RNAi) to reduce MAPKAP kinase-2 expression renders cells more TABLE 3 Susceptible to chemotherapeutic agents. Accordingly, using 25 MAPKAP kinase-2 RNAi tarcet sequences the methods of the invention, nucleobase oligomers may be employed in double-stranded RNAs for RNAi-mediated Sequence (5' to 3') SEQ ID NO: knockdown of MAPKAP kinase-2 expression. RNAi is a GACCAGGCATTCACAGAAA 35 method for decreasing the cellular expression of specific pro teins of interest (reviewed in Tuschl, Chembiochem 2:239 30 TTGACCACTCCTTGTTATA 36 245, 2001; Sharp, Genes & Devel. 15:485-490, 2000; Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225 GACCACTCCTTGTTATACA 37 232, 2002; and Hannon, Nature 418:244-251, 2002). In TGACCATCACCGAGTTTAT 38 RNAi, gene silencing is typically triggered post-transcrip TCACCGAGTTTATGAACCA 39 tionally by the presence of double-stranded RNA (dsRNA) in 35 a cell. This dsRNA is processed intracellularly into shorter TCAAGAAGAACGCCATCAT 4 O pieces called small interfering RNAs (siRNAs). The intro duction of siRNAs into cells either by transfection of dsRNAs AAGCATCCGAAATCATGAA 41 or through expression of siRNAS using a plasmid-based AGTATCTGCATTCAATCAA 42 expression system is increasingly being used to create loss 40 of-function phenotypes in mammalian cells. CTTTGACCACTCCTTGTTA 43 In one embodiment of the invention, a double-stranded RNA (dsRNA) molecule is made. The dsRNA can be two TTTGACCACTCCTTGT TAT 44 distinct strands of RNA that have duplexed, or a single RNA TACGGATCGTGGATGTGTA 45 strand that has self-duplexed (small hairpin (sh)RNA). Typi 45 cally, dsRNAs are about 21 or 22 base pairs, but may be GGACGGTGGAGAACTCTTT 46 shorter or longer (up to about 29 nucleobases) if desired. dsRNA can be made using standard techniques (e.g., chemi CTTGTTATACACCGTACTA 47 cal synthesis or in vitro transcription). Kits are available, for GACGGTGGAGAACTCTTTA 48 example, from Ambion (Austin, Tex.) and Epicentre (Madi 50 son, Wis.). Methods for expressing dsRNA in mammalian GGAGAACTCTTTAGCCGAA 49 cells are described in Brummelkamp et al. Science 296:550 553, 2002; Paddison et al. Genes & Devel. 16:948-958, 2002. Antisense Therapy Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. As an alternative to RNAi-based approaches, therapeutic Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002: Yu et al. 55 strategies utilizing MAPKAP kinase-2 antisense nucleic Proc. Natl. Acad. Sci. USA99:6047-6052, 2002: Miyagishiet acids may be employed in the methods of the invention. The al. Nature Biotechnol. 20:497-500, 2002; and Lee et al. technique is based on the principle that sequence-specific Nature Biotechnol. 20:500-5052002, each of which is hereby Suppression of gene expression can be achieved by intracel incorporated by reference. lular hybridization between mRNA and a complementary Small hairpin RNAs consist of a stem-loop structure with 60 antisense species. The formation of a hybrid RNA duplex optional 3' UU-overhangs. While there may be variation, may then interfere with the processing/transport/translation stems can range from twenty-one to thirty-one base pairs and/or stability of the target MAPKAP kinase-2 mRNA. Anti (desirably twenty-five to twenty-nine base pairs), and the sense strategies may use a variety of approaches, including loops can range from four to thirty base pairs (desirably four the use to of antisense oligonucleotides and injection of anti to twenty-three base pairs). For expression of shRNAs within 65 sense RNA. An exemplary approach features transfection of cells, plasmid vectors containing, e.g., the polymerase III antisense RNA expression vectors into targeted cells. Anti H1-RNA or U6 promoter, a cloning site for the stem-looped sense effects can be induced by control (sense) sequences; US 9,023,787 B2 99 100 however, the extent of phenotypic changes are highly vari propyl silane. The peptide obtained in Such a manner should able. Phenotypic effects induced by antisense effects are give a single peak by HPLC and is sufficiently pure for car based on changes in criteria Such as protein levels, protein rying on with the assay described below. activity measurement, and target mRNA levels. Peptide Modifications and Unnatural Amino Acids Computer programs such as OLIGO (previously distrib It is understood that modifications can be made to the uted by National Biosciences Inc.) may be used to select amino acid residues of the peptide-containing compounds of candidate nucleobase oligomers for antisense therapy based the invention, to enhance or prolong the therapeutic efficacy on the following criteria: and/or bioavailability of the compound. Accordingly, 1) no more than 75% GC content, and no more than 75% C.-amino acids having the following general formula (I): AT content; 10 2) preferably no nucleobase oligomers with four or more consecutive G residues (due to reported toxic effects, (I) although one was chosen as a toxicity control); R 3) no nucleobase oligomers with the ability to form stable dimers or hairpin structures; and 15 H nN.1 C OH 4) sequences around the translation start site are a preferred | H. region. H O In addition, accessible regions of the target mRNA may be predicted with the help of the RNA secondary structure fold ing program MFOLD (M. Zuker, D. H. Mathews & D. H. where R defines the specific amino acid residue, may undergo Turner, Algorithms and Thermodynamics for RNA Second various modifications. Exemplary modifications of C-amino ary Structure Prediction: A Practical Guide. In: RNA Bio acids, include, but are not limited to, the following formula chemistry and Biotechnology, J. Barciszewski & B. F. C. (II): Clark, eds., NATO ASI Series, Kluwer Academic Publishers, (1999). Sub-optimal folds with a free energy value within 5% 25 of the predicted most stable fold of the mRNA may be pre (II) dicted using a window of 200 bases within which a residue can find a complimentary base to form a base pair bond. Open regions that do not form a base pair may be Summed together with each Suboptimal fold, and areas that consistently are 30 predicted as open may be considered more accessible to the binding to nucleobase oligomers. Additional nucleobase oli gomer that only partially fulfill some of the above selection R. R. R. R. and Rs are independently hydrogen, hydroxy, criteria may also be chosen as possible candidates if they nitro, halo, C.s branched or linear alkyl, Cs alkaryl, het recognize a predicted open region of the target mRNA. 35 eroaryl, and aryl; wherein the alkyl, alkaryl, heteroaryl, and Therapeutically Useful Compounds and Pharmaceutical aryl may be unsubstituted or substituted by one or more Compositions Substituents selected from the group consisting of Cls alkyl, Any compound or pharmaceutical composition that inhib hydroxy, halo, nitro, Cs alkoxy, Cls alkylthio, trihalom its an activity of MAPKAP kinase-2 may be useful in the ethyl, Cs acyl, arylcarbonyl, heteroarylcarbonyl, nitrile, methods of the invention. The model of the activated MAP 40 Cs alkoxycarbonyl, Oxo, arylalkyl (wherein the alkyl group KAP kinase-2/peptide complex described above (Table 1) has from 1 to 5 carbon atoms) and heteroarylalkyl (wherein indicates that peptides, or peptide-like compounds, e.g., pep the alkyl group has from 1 to 5 carbon atoms); alternatively, tidomimetics, may be useful for inhibiting MAPKAP kinase R and R2 are joined to form a Cs cyclic ring, optionally 2. Such compounds achieve this effect by mimicking the including oxygen, sulfur or hydrogen, or Cs alkyl, option natural peptide substrate of MAPKAP kinase-2 and decreas 45 ally substituted by hydroxyl; or R and Rare joined to form ing the extent or rate with which a MAPKAP kinase-2 a C-scyclic ring, optionally substituted by hydroxyl and polypeptide is able to bind to its natural substrates in vivo. optionally including oxygen, Sulfur, C.saminoalkyl, or Cs Accordingly, methods of synthesis or modification of pep alkyl. tides and peptide-like compounds is described below. A compound of the invention that includes a peptide may Peptide Synthesis and Conjugation 50 include, but it is not limited to, an unnatural N-terminal amino A compound of the invention that includes a peptide is acid of the formula (III): prepared as detailed above. Alternatively, peptides can be prepared using standard FMOC chemistry on 2-chlorotrityl chloride resin (Int. J. Pept. Prot. Res. 38, 1991, 555-61). (III) Cleavage from the resin is performed using 20% acetic acid in 55 dichloromehane (DCM), which leaves the side chain still blocked. Free terminal carboxylate peptide is then coupled to 4' (aminomethy)-fluorescein (Molecular Probes, A-1351; Eugene, Oreg.) using excess diisopropylcarbodiimide (DIC) in dimethylformamide (DMF) at room temperature. The to 60 fluorescent N C blocked peptide is purified by silica gel where A' is an amino acid or peptide chain linked via an chromatography (10% methanol in DCM). The N terminal C-amino group; R' and Rare independently hydrogen, Cls FMOC group is then removed using piperidine (20%) in branched or linear Cls alkyl, Cs alkaryl, heteroaryl, and DMF, and the N-free peptide, purified by silica gel chroma aryl, each of which are unsubstituted or substituted with a tography (20% methanol in DCM, 0.5% HOAc). Finally, any 65 substitutent selected from: 1 to 3 of Cs alkyl, 1 to 3 of t-butyl side chain protective groups are removed using 95% halogen, 1 to 2 of OR, N(R)(R), SR, N CONR) trifluoroacetic acid containing 2.5% water and 2.5% triiso NR'R'', methylenedioxy, S(O).R. 1 to 2 of -CF, US 9,023,787 B2 101 102 OCF, nitro, N(R)C(O)(R), C(O)OR, C(O)N The invention also includes modifications of the peptide (R)(R), -1H-tetrazol-5-yl, -SON(R)(R), -N (R)SO, containing compounds of the invention, wherein an unnatural aryl, or - N(R)SOR: R, R and R7 are independently internal amino acid of the formula: selected from hydrogen, C.s linear or branched alkyl, Cs alkaryl, aryl, heteroaryl, and C-7 cycloalkyl, and where two Cs alkyl groups are present on one atom, they optionally are 5 (V) X OR joined to formaCs cyclic ring, optionally including oxygen, nS PC sulfur or NR", where R7 is hydrogen, or Cs alkyl, optionally | YoR6 substituted by hydroxyl; R is hydrogen, F, Cls linear or R O branched alkyl, Cisalkaryl; or R and R' are joined to form 10 a Cs cyclic ring, optionally including oxygen, Sulfur, or 2 NR", where R is hydrogen, or Cs alkyl, optionally substi AS N A tuted by hydroxyl, or R and R are joined to form a Cas R2 cyclic ring, optionally Substituted by hydroxyl and optionally R3 O including oxygen, sulfur or NR", where R is hydrogen, or 15 Cls alkyl; R is hydrogen, F.C.s linear or branched alkyl, is present, where A is an amino acid or peptide chain linked Cisalkaryl; and R is hydrogen, C.s branched or linear Cls via an O-carboxy group: A' is an amino acid or peptide chain alkyl, Cs alkaryl, heteroaryl, and aryl, each of which are linked via an O-amino group; R' and R are independently unsubstituted or substituted with a substitutent selected from: hydrogen, C.s branched or linear Cls alkyl, and Cisalkaryl; 1 to 3 of Cls alkyl, 1 to 3 of halogen, 1 to 2 of —OR, R is hydrogen, F.C.s linear or branched alkyl, Cisalkaryl; N(R)(R), N C(NR)NR'R'', methylenedioxy, S(O).R or R and R' are joined to form a Css cyclic ring, optionally (where m is 0-2), 1 to 2 of CF, OCF, nitro, N(R)C including oxygen, sulfur or NR", where R is hydrogen, or (O)(R), N(R)C(O)(OR), C(O)OR, C(O)N(R) Cs alkyl, optionally substituted by hydroxyl: X is O or S; (R), -1H-tetrazol-5-yl, -SON(R)(R), N(R)SO, aryl, 25 and R and R are independently selected from hydrogen, or - N(R)SO.R. R. Rand Rare independently selected Cs linear or branched alkyl, Cs alkaryl, aryl, heteroaryl, from hydrogen, C.s linear or branched alkyl, Cs alkaryl, and C-7 cycloalkyl, and where two Cls alkyl groups are aryl, heteroaryl, and C-7 cycloalkyl, and where two Cls alkyl present on one atom, they optionally are joined to form a Cs groups are present on one atom, they optionally are joined to cyclic ring, optionally including oxygen, Sulfur or NR". form a C-scyclic ring, optionally including oxygen, Sulfur or 30 where R7 is hydrogen, or Cs alkyl, optionally substituted by NR", where R is hydrogen, or Cs alkyl, optionally substi hydroxyl; or R and Rare joined to form a Css cyclic ring, tuted by hydroxyl. optionally including oxygen, sulfur or NR", where R is A compound of the invention may also include an unnatu hydrogen, or Cs alkyl, optionally substituted by hydroxyl. ral internal amino acid of the formula: A compound of the invention may also include a C-termi 35 nal unnatural internal amino acid of the formula:

(IV) (VI)

40 Q,

where A is an amino acid or peptide chain linked via an t-carboxy group; A' is an amino acid or peptide chain linked 45 where A is an amino acid or peptide chain linked via an via an O-amino group; RandR are independently hydrogen, C-carboxy group; R' and R are independently hydrogen, Cs branched or linear Cs alkyl, Cs alkaryl, heteroaryl, Cs branched or linear Cls alkyl, Cs alkaryl, heteroaryl, and aryl, each of which are unsubstituted or substituted with and aryl, each of which are unsubstituted or substituted with a substitutent selected from: 1 to 3 of Cs alkyl, 1 to 3 of a substitutent selected from: 1 to 3 of Cs alkyl, 1 to 3 of halogen, 1 to 2 of OR, N(R)(R), SR, N CONR) 50 halogen, 1 to 2 of OR, N(R)(R), SR, N CONR) NR'R'', methylenedioxy, S(O).R (m is 1-2), 1 to 2 of NR'R'', methylenedioxy, S(O).R. 1 to 2 of -CF, —CF - OCF, nitro, N(R)C(O)(R), C(O)OR, OCF, nitro, N(R)C(O)(R), C(O)OR, C(O)N —C(O)N(R)(R), -1H-tetrazol-5-yl, -SON(R)(R), (R)(R), -1H-tetrazol-5-yl, -SON(R)(R), N(R)SO. -N(R)SO, aryl, or - N(R)SOR: R. Rand R7 are inde aryl, or - N(R)SOR: R, R and R7 are independently pendently selected from hydrogen, C.s linear or branched 55 selected from hydrogen, C.s linear or branched alkyl, Cs alkyl, Cs alkaryl, aryl, heteroaryl, and C-7 cycloalkyl, and alkaryl, aryl, heteroaryl, and C-7 cycloalkyl, and where two where two Cls alkyl groups are present on one atom, they Cs alkyl groups are present on one atom, they optionally are optionally are joined to form a C-scyclic ring, optionally joined to formaCs cyclic ring, optionally including oxygen, including oxygen, sulfur or NR", where R is hydrogen, or sulfur or NR", where R is hydrogen, or Cs alkyl, optionally Cs alkyl, optionally substituted by hydroxyl; and R is 60 substituted by hydroxyl; R is hydrogen, F, Cls linear or hydrogen, F.C.s linear or branched alkyl, Cisalkaryl; or R branched alkyl, Cs alkaryl; or R and R' are joined to form and R' are joined to form a Css cyclic ring, optionally includ a Css cyclic ring, optionally including oxygen, sulfuror NR". ing oxygen, Sulfur or NR", where R is hydrogen, or Cs where R7 is hydrogen, or Cs alkyl, optionally substituted by alkyl, optionally substituted by hydroxyl, or R and Rare hydroxyl; or R and Rare joined to form a Css cyclic ring, joined to form a C-scyclic ring, optionally Substituted by 65 optionally substituted by hydroxyl and optionally including hydroxyl and optionally including oxygen, Sulfur or NR". oxygen, sulfur or NR", where R7 is hydrogen, or Cs alkyl; where R is hydrogen, or Cls alkyl. R is hydrogen, F.C.s linear or branched alkyl, Cisalkaryl; US 9,023,787 B2 103 104 and Q is OH, OR, or NRR, where R. Rare independently amino terminus and/or carboxyl terminus and/or an internal selected from hydrogen, C.s linear or branched alkyl, Cs site. Such derivatives are highly preferred when targeting alkaryl, aryl, heteroaryl, and C-7 cycloalkyl, and where two intracellular protein-protein interactions, provided they Cs alkyl groups are present on one atom, they optionally are retain the desired functional activity. joined to formaCs cyclic ring, optionally including oxygen, In another example, a peptide derivative binds with sulfur or NR", where R is hydrogen, or Cs alkyl, optionally increased affinity to a ligand (e.g., a MAPKAP kinase-2 substituted by hydroxyl. Methods well known in the art for polypeptide). modifying peptides are found, for example, in “Remington: The peptides or peptide derivatives of the invention are The Science and Practice of Pharmacy.” (20th ed., ed. A. R. obtained by any method of peptide synthesis known to those Gennaro, 2000, Lippincott Williams & Wilkins, Philadel 10 skilled in the art, including synthetic and recombinant tech phia). niques. For example, the peptides or peptide derivatives can Peptidomimetics be obtained by solid phase peptide synthesis which, in brief, Peptide derivatives (e.g., peptidomimetics) include cyclic consists of coupling the carboxyl group of the C-terminal peptides, peptides obtained by Substitution of a natural amino amino acid to a resin and Successively adding N-alpha pro acid residue by the corresponding D-Stereoisomer, or by an 15 tected amino acids. The protecting groups may be any such unnatural amino acid residue, chemical derivatives of the groups known in the art. Before each new amino acid is added peptides, dual peptides, multimers of the peptides, and pep to the growing chain, the protecting group of the previous tides fused to other proteins or carriers. A cyclic derivative of amino acid added to the chain is removed. The coupling of a peptide of the invention is one having two or more addi amino acids to appropriate resins has been described by tional amino acid residues suitable for cyclization. These Rivier et al. (U.S. Pat. No. 4,244.946). Such solid phase residues are often added at the carboxyl terminus and at the syntheses have been described, for example, by Merrifield, J. amino terminus. A peptide derivative may have one or more Am. Chem. Soc. 85:2149, 1964; Vale et al., Science 213: amino acid residues replaced by the corresponding D-amino 1394-1397, 1984; Marki et al., J. Am. Chem. Soc. 10:31.78, acid residue. In one example, a peptide or peptide derivative 1981, and in U.S. Pat. Nos. 4,305,872 and 4,316,891. Desir of the invention is all-L, all-D, or a mixed D.L-peptide. In 25 ably, an automated peptide synthesizer is employed. another example, an amino acid residue is replaced by an Purification of the synthesized peptides or peptide deriva unnatural amino acid residue. Examples of unnatural or tives is carried out by standard methods, including chroma derivatized unnatural amino acids include NC.-methylamino tography (e.g., ion exchange, affinity, and sizing column acids, CC-methylamino acids, and n-methylamino acids. chromatography), centrifugation, differential Solubility, A chemical derivative of a peptide of the invention 30 hydrophobicity, or by any other standard technique for the includes, but is not limited to, a derivative containing addi purification of proteins. In one embodiment, thin layer chro tional chemical moieties not normally a part of the peptide. matography is employed. In another embodiment, reverse Examples of such derivatives include: (a) N-acyl derivatives phase HPLC (high performance liquid chromatography) is of the amino terminal or of another free amino group, where employed. the acyl group may be either an alkanoyl group, e.g., acetyl, 35 Finally, structure-function relationships determined from hexanoyl, octanoyl, an aroyl group, e.g., benzoyl, or a block the peptides, peptide derivatives, and other Small molecules ing group Such as Fmoc (fluorenylmethyl-O CO—), car of the invention may also be used to prepare analogous bobenzoxy (benzyl-O CO—), monomethoxysuccinyl, molecular structures having similar properties. Thus, the naphthyl-NH-CO—, acetylamino-caproyl, adamantyl invention is contemplated to include molecules in addition to NH CO. ; (b) esters of the carboxyl terminal or of another 40 those expressly disclosed that share the structure, hydropho free carboxyl or hydroxy groups; (c) amides of the carboxyl bicity, charge characteristics and side chain properties of the terminal or of another free carboxyl groups produced by specific embodiments exemplified herein. reaction with ammonia or with a suitable amine; (d) glyco In one example, such derivatives or analogs that have the sylated derivatives; (e) phosphorylated derivatives: (f) deriva desired binding activity can be used for binding to a molecule tives conjugated to lipophilic moieties, e.g., caproyl, lauryl, 45 or other target of interest, such as any MAPKAP kinase-2 Stearoyl; and (g) derivatives conjugated to an antibody or polypeptide. Derivatives or analogs that retain, or alterna other biological ligand. Also included among the chemical tively lack or inhibit, a desired property-of-interest (e.g., derivatives are those derivatives obtained by modification of inhibit MAPKAP kinase-2 binding to a natural ligand), can be the peptide bond —CO. NH for example, by: (a) reduc used to inhibit the biological activity of a MAPKAP kinase-2 tion to —CH NH ; (b) alkylation to —CO N(alkyl)—: 50 polypeptide. and (c) inversion to —NH CO . Peptidomimetics may In particular, peptide derivatives are made by altering also comprise phosphonate or Sulfonate moieties. amino acid sequences by Substitutions, additions, or deletions A dual peptide of the invention consists of two of the same, that provide for functionally equivalent molecules, or for or two different, peptides of the invention covalently linked to functionally enhanced or diminished molecules, as desired. one another, either directly or through a spacer. 55 Due to the degeneracy of the genetic code, other nucleic acid Multimers of the invention consist of polymer molecules sequences that encode Substantially the same amino acid formed from a number of the same or different peptides or sequence may be used for the production of recombinant derivatives thereof. peptides. These include, but are not limited to, nucleotide In one example, a peptide derivative is more resistant to sequences comprising all orportions of a peptide of the inven proteolytic degradation than the corresponding non-deriva 60 tion that is altered by the substitution of different codons that tized peptide. For example, a peptide derivative having encode a functionally equivalent amino acid residue within D-amino acid Substitution(s) in place of one or more L-amino the sequence, thus producing a silent change. acid residue(s) resists proteolytic cleavage. The derivatives and analogs of the invention can be pro In another example, the peptide derivative has increased duced by various methods known in the art. The manipula permeability across a cell membrane as compared to the 65 tions that result in their production can occur at the gene or corresponding non-derivatized peptide. For example, a pep protein level. For example, a cloned nucleic acid sequence tide derivative may have a lipophilic moiety coupled at the can be modified by any of numerous strategies known in the

US 9,023,787 B2 111 112 4-bipyrano.3.2-eindole-8-carboxylic acid, 7-methoxy-3,4, (1Z)-6-methoxy-2,3,4,9-tetrahydro-1H-carbazol-1-one 5,10-tetrahydro-1H-2,5-methanoazepino3,4-bindole-1- Oxime, 6-methoxy-3-3-(3-phenylpropyl)aminolpropyl)-2, carboxylic acid, 7-(methylthio)-3,4,5,10-tetrahydro-1H-2.5- 34.9-tetrahydro-1H-beta-carbolin-1-one, methyl 1-oxo-2.3, methanoazepino3,4-bindole-1-carboxylic acid, 4,5-tetrahydro-1H-pyrido4.3-bindole-6-carboxylate, 7-(benzyloxy)-3,4,5,10-tetrahydro-1H-2,5-methanoazepino 5 3-(hydroxymethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta 3,4-bindole-1-carboxylic acid, 7-(methylthio)-3,4,5,10-tet carbolin-1-one, 3-(3-aminopropyl)-6-methoxy-2.3.4.9-tet rahydro-1H-2,5-methanoazepino3,4-bindole-1-carboxylic rahydro-1H-beta-carbolin-1-one, 3-(2-aminoethyl)-6-meth acid, 2.2.2-trifluoroethyl 7-methoxy-3,4,5,10-tetrahydro oxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, ethyl 1H-2,5-methanoazepino3,4-bindole-1-carboxylate, 2,3-di 1-(hydroxyimino)-2.3.4.9-tetrahydro-1H-carbazole-6-car hydroxypropyl 7-methoxy-3,4,5,10-tetrahydro-1H-2,5- 10 boxylate, 2-methoxy-7,8,9,10-tetrahydrocycloheptablin methanoazepino3,4-bindole-1-carboxylate, pyridin-4- dol-6(5H)-one oxime, 3-(hydroxymethyl)-6-methoxy-2,3,4, ylmethyl 7-methoxy-3,4,5,10-tetrahydro-1H-2,5- 9-tetrahydro-1H-beta-carbolin-1-one, 3-(3-aminopropyl)-6- methanoazepino3,4-bindole-1-carboxylate, 2-fluoroethyl methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, 3-(2- 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino.3, aminoethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta 4-bindole-1-carboxylate, allyl 7-methoxy-3,4,5,10-tetrahy 15 carbolin-1-one, ethyl 1-(hydroxyimino)-2,34.9-tetrahydro dro-1H-2,5-methanoazepino 3,4-bindole-1-carboxylate, 1H-carbazole-6-carboxylate, 2-methoxy-7,8,9,10 benzyl 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoaze tetrahydrocycloheptablindol-6(5H)-one oxime, 3-3- pino3,4-bindole-1-carboxylate, 2-(methylthio)ethyl (benzylamino)propyl-6-methoxy-2.3.4.9-tetrahydro-1H 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino.3, beta-carbolin-1-one, 6-methoxy-2,3,4,9-tetrahydro-1H 4-bindole-1-carboxylate, 2-methoxyethyl 7-methoxy-3,4,5, carbazol-1-one oxime, 6-iodo-2.3.4.9-tetrahydro-1H 10-tetrahydro-1H-2,5-methanoazepino3,4-bindole-1-car carbazol-1-one oXime, 6-methoxy-2-methyl-2.3.4.9- boxylatem, 7-methoxy-3,4,5,10-tetrahydro-1H-2,5- tetrahydro-1H-carbazol-1-one oxime, 3-(3-hydroxypropyl)- methanoazepino3,4-bindole-1-carboxylic acid, 7-hydroxy 6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, 3,4,5,10-tetrahydro-1H-2,5-methanoazepino3,4-bindole ethyl 1-oxo-2,3,4,5-tetrahydro-1H-pyrido4,3-bindole-6- 1-carboxylic acid, 2.3,8,10,11,12-hexahydro-1H.7H-9,12 25 carboxylate, 6-methoxy-2.3.4.9-tetrahydro-1H-beta-carbo methanoazepino3,4-bipyrano3.2-eindole-8-carboxylic line-1-thione, methyl 4-oxo-2.3.4.9-tetrahydro-1H-carba acid, 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoaze Zole-8-carboxylate, and 2.3.4.9-tetrahydro-1H-carbazol-1- pino3,4-bindole-1-carboxylic acid, 7-(methylthio)-3,4,5, one oxime. Others are described in U.S. Patent Application 10-tetrahydro-1H-2,5-methanoazepino3,4-bindole-1-car Publication Nos. 2004-0127492, 2005-0101623, 2005 boxylic acid, 7-methoxy-3,4,5,10-tetrahydro-1H-2,5- 30 O137220, and 2005-0143371. methanoazepino3,4-bindole-1-carboxylic acid, Prodrugs and Other Modified Compounds 7-(benzyloxy)-3,4,5,10-tetrahydro-1H-2,5-methanoazepino Interaction of a molecule, e.g., a drug, with a MAPKAP 3,4-bindole-1-carboxylic acid, 7-(methylthio)-3,4,5,10-tet kinase-2 polypeptide can be used to promote enhanced sen rahydro-1H-2,5-methanoazepino3,4-bindole-1-carboxylic sitivity of cells to chemotherapy or radiation treatment. The acid, 6-methoxy-2.3.4.9-tetrahydro-1H-beta-carboline-1- 35 treatment, stabilization, or prevention of a disease or disorder carboxylic acid, 6-(2-oxo-2-phenylethoxy)-2.3.4.9-tetrahy associated with MAPKAP kinase-2 can be mediated by dro-1H-beta-carboline-1-carboxylic acid, 6-methoxy-2-me administering a compound, peptide, or nucleic acid molecule. thyl-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylic In some cases, however, a compound that is effective in vitro acid, 2.2.2-trifluoroethyl 7-methoxy-3,4,5,10-tetrahydro in to disrupting the interaction of a MAPKAP kinase-2 1H-2,5-methanoazepino3,4-bindole-1-carboxylate, 40 polypeptide and a natural Substrate is not an effective thera 6-methoxy-2,3,4,9-tetrahydro-1H-beta-carboline-1-car peutic agent in vivo. For example, this could be due to low boxylic acid, 7-hydroxy-3,4,5,10-tetrahydro-1H-2,5-metha bioavailability of the compound. One way to circumvent this noazepino3.4-bindole-1-carboxylic acid, 6-hydroxy-2-me difficulty is to administer a modified drug, or prodrug, with thyl-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylic improved bioavailability that converts naturally to the origi acid, 7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoaze 45 nal compound following administration. Such prodrugs may pino3,4-bindole-1-carboxylic acid, 6-methoxy-2-methyl-2, undergo transformation before exhibiting their full pharma 34.9-tetrahydro-1H-beta-carboline-1-carboxylic acid, 2,3- cological effects. Prodrugs contain one or more specialized dihydroxypropyl 7-methoxy-3,4,5,10-tetrahydro-1H-2.5- protective groups that are specifically designed to alter or to methanoazepino3,4-bindole-1-carboxylate, 4-ethyl-6- eliminate undesirable properties in the parent molecule. In methoxy-2,3,4,9-tetrahydro-1H-beta-carboline-1- 50 one embodiment, a prodrug masks one or more charged or carboxylic acid, 6-methoxy-4-methyl-2.3.4.9-tetrahydro hydrophobic groups of a parent molecule. Once adminis 1H-beta-carboline-1-carboxylic acid, 8,9,10,11-tetrahydro tered, a prodrug is metabolized in vivo into an active com 7H-pyrido3',4':45pyrrolo2.3-fisoquinolin-7-one pound. trifluoroacetate, 3-(aminomethyl)-6-methoxy-2.3.4.9-tet Prodrugs may be useful for improving one or more of the rahydro-1H-beta-carbolin-1-one trifluoroacetate, 3-(ami 55 following characteristics of a drug: solubility, absorption, nomethyl)-6-methoxy-2.3.4.9-tetrahydro-1H-beta-carbolin distribution, metabolization, excretion, site specificity, Stabil 1-one hydrochloride, 7-methoxy-3,4,5,10-tetrahydroazepino ity, patient acceptability, reduced toxicity, or problems of 3,4-bindol-1(2H)-one, 6-methoxy-2,3,4,9-tetrahydro-1H formulation. For example, an active compound may have beta-carbolin-1-one, 6-methoxy-2,9-dihydro-1H-beta poor oral bioavailability, but by attaching an appropriately carbolin-1-one, 6-hydroxy-2.3.4.9-tetrahydro-1H-beta 60 chosen covalent linkage that may be metabolized in the body, carbolin-1-one, 8,9,10,11-tetrahydro-7H-pyrido3',4':4.5 oral bioavailability may improve sufficiently to enable the pyrrolo-2,3-flisoquinolin-7-one, 3-(aminomethyl)-6- prodrug to be administered orally without adversely affecting methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one, the parent compounds activity within the body. 3-(aminomethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta A prodrug may be carrier-linked, meaning that it contains carbolin-1-one, 6-methoxy-3-3-(2-phenylethyl)amino 65 a group Such as an ester that can be removed enzymatically. propyl-2.3.4.9-tetrahydro-1H-beta-carbolin-1-one, (1E)-6- Optimally, the additional chemical group has little or no phar methoxy-2,3,4,9-tetrahydro-1H-carbazol-1-one oXime, macologic activity, and the bond connecting this group to the US 9,023,787 B2 113 114 parent compound is labile to allow for efficient in vivo acti peptide, 50DuL of GST-MAPKAP kinase-2 polypeptide, or vation. Such a carrier group may be linked directly to the 500DuL of unlabeled polypeptide can be used as a negative parent compound (bipartate), or it may be bonded via a linker control. Once added, all the plates are placed at 4°C. Follow region (tripartate). Common examples of chemical groups ing overnight incubation at 4°C., the fluorescence polariza attached to parent compounds to form prodrugs include tion is measured using a Polarion plate reader (Tecan, esters, methyl esters, Sulfates, Sulfonates, phosphates, alco Research Triangle Park, N.C.). A xenon flash lamp equipped hols, amides, imines, phenyl carbamates, and carbonyls. with an excitation filter of 485 nm and an emission filter of As one example, methylprednisolone is a poorly water 535 nm. The number of flashes is set at 30. Raw data can then soluble drug. In order to be useful for aqueous be converted into a percentage of total interaction(s). All injection or ophthalmic administration, this drug must be 10 further analysis can be performed using SPOTFIRE data converted into a prodrug of enhanced solubility. Methylpred analysis software (SPOTFIRE, Somerville, Mass.) nisolone sodium Succinate ester is much more soluble than Upon selection of active compounds, auto-fluorescence of the parent compound, and it is rapidly and extensively the hits is measured as well as the fluorescein quenching hydrolysed in vivo by cholinesterases to free methylpredniso effect, where a measurement of 2,000 or more units indicates lone. 15 auto-fluorescence, while a measurement of 50 units indicates Caged compounds may also be used as prodrugs. A caged a quenching effect. Confirmed hits can then be analyzed in compound may have, e.g., one or more photolyzable chemi dose-response curves (ICs) for reconfirmation. Best hits in cal groups attached that renders the compound biologically dose-response curves can then be assessed by isothermal inactive. In this example, flash photolysis releases the caging titration calorimetry using a GST-MAPKAP kinase-2 group (and activates the compound) in a spatially or tempo polypeptide fusion. rally controlled manner. Caged compounds may be made or Assays with a candidate compound may be performed in designed by any method known to those of skill in the art. the presence of a compound known to bind MAPKAP kinase For further description of the design and use of prodrugs, 2, and the difference in binding the presence and absence of see Testa and Mayer, Hydrolysis in Drug and Prodrug the compound known to bind may be a useful measure of the Metabolism: Chemistry, Biochemistry and Enzymology, 25 candidate compounds ability to bind to MAPKAP kinase-2. published by Vch. Verlagsgesellschaft Mbh. (2003). Other This assay may be done in any format known to those of skill modified compounds are also possible in the methods of the in the art, e.g., as a displacement assay. invention. For example, a modified compound need not be Alternate Binding and Displacement Assays metabolized to form a parent molecule. Rather, in some Fluorescence polarization assays are but one means to embodiments, a compound may contain a non-removable 30 measure compound-protein interactions in a screening strat moiety that, e.g., increases bioavailability without Substan egy. Alternate methods for measuring compound-protein tially diminishing the activity of the parent molecule. Such a interactions are known to the skilled artisan. Such methods moiety could, for example, be covalently-linked to the parent include, but are not limited to mass spectrometry (Nelson and molecule and could be capable of translocating across a bio Krone, J. Mol. Recognit., 12:77-93, 1999), surface plasmon logical membrane Such as a cell membrane, in order to 35 resonance (Spiga et al., FEBS Lett., 511:33-35, 2002: Rich enhance cellular uptake. Exemplary moieties include pep and Mizka, J. Mol. Recognit., 14:223-8, 2001; Abrantes et al., tides, e.g., penetratin or TAT. An exemplary penetratin-con Anal. Chem., 73:2828-35, 2001), fluorescence resonance taining compound according to the invention is, e.g., a pep energy transfer (FRET) (Bader et al., J. Biomol. Screen, tide comprising the sixteen amino acid sequence from the 6:255-64, 2001: Song et al., Anal. Biochem. 291:133-41, homeodomain of the Antennapedia protein (Derossi et al., J. 40 2001; Brockhoffet al., Cytometry, 44:338-48, 2001), biolu Biol. Chem. 269:10444-10450, 1994), particularly a peptide minescence resonance energy transfer (BRET) (Angers et al., having the amino acid sequence RQIKIWFQNRRMKWKK Proc. Natl. Acad. Sci. USA,97:3684-9, 2000; Xu et al., Proc. (SEQID NO: 50), or including a peptide sequence disclosed Natl. Acad. Sci. USA, 96:151-6, 1999), fluorescence quench by Linet al. (J. Biol. Chem. 270: 14255-14258, 1995). Others ing (Engelborghs, Spectrochim. Acta A. Mol. Biomol. Spec are described in U.S. Patent Application Publication No. 45 trosc. 57:2255-70, 70; Geoghegan et al., Bioconjug. Chem. 2004-0209797 and U.S. Pat. Nos. 5,804,604, 5,747,641, 11:71-7, 2000), fluorescence activated cell scanning/sorting 5,674,980, 5,670,617, and 5,652,122. In addition, a com (Barth et al., J. Mol. Biol. 301:751-7, 2000), ELISA, and pound of the invention could be attached, for example, to a radioimmunoassay (RIA). Solid Support. Pharmaceutical Compositions Screening Assays 50 The pharmaceutical compositions of the present invention Fluorescence polarization assays can be used in displace are prepared in a manner known perse, for example by means ment assays to identify Small molecule peptidomimetics or of conventional dissolving, lyophilizing, mixing, granulating other compounds useful in the methods of the invention. The or confectioning processes. Methods well known in the art for following is an exemplary method for use of fluorescence making compositions and formulations are found, for polarization, and should not be viewed as limiting in any way. 55 example, in “Remington: The Science and Practice of Phar For screening, all reagents are diluted at the appropriate con macy.” (20th ed., ed. A. R. Gennaro, 2000, Lippincott Will centration and the working Solution, kept on ice. The working iams & Wilkins, Philadelphia). stock concentration for GST and GST fusion proteins are ~4 Solutions of the active ingredient, and also suspensions, ng/uL. Fluorescein-labeled peptides can be used at a concen and especially isotonic aqueous solutions or Suspensions, are tration of 1.56 fmol/LL, while cold peptides at 25 pmol/LL. 60 preferably used, it being possible, for example in the case of Samples are incubated at a total volume of 200 uL per well in lyophilized compositions that comprise the active ingredient black flat bottom plates, Biocoat, #359 135 low binding (BD alone or together with a carrier, for example mannitol, for BioSciences; Bedford, Mass.). Assays are started with the Such solutions or Suspensions to be produced prior to use. The successive addition using a Labsystem Multi-Drop 96/384 pharmaceutical compositions may be sterilized and/or may device (Labsystem; Franklin, Mass.) of 50DuL test com 65 comprise excipients, for example preservatives, stabilizers, pounds, diluted in 10% DMSO (average concentration of 28 wetting and/or emulsifying agents, to Solubilizers, salts for uM), 50 uL of 50 mM MES-pH 6.5, 50DuL of Fluorescein regulating the osmotic pressure and/or buffers, and are pre US 9,023,787 B2 115 116 pared in a manner known per se, for example by means of comprise the active ingredient in the form of granules, for conventional dissolving or lyophilizing processes. The said example with fillers, such as lactose, binders, such as Solutions or Suspensions may comprise Viscosity-increasing starches, and/or glidants, such as talc or magnesium Stearate, Substances, such as sodium carboxymethylcellulose, car and if desired with stabilisers. In soft capsules the active boxymethylcellulose, dextran, poly vinylpyrrolidone or gela ingredient is preferably dissolved or suspended in suitable tin. oily excipients. Such as fatty oils, paraffin oil or liquid poly Suspensions in oil comprise as the oil component the Veg ethylene glycols, it being possible also for stabilisers and/or etable, synthetic or semi-synthetic oils customary for injec antibacterial agents to be added. Dyes or pigments may be tion purposes. There may be mentioned as such especially added to the tablets or drage coatings or the capsule casings, liquid fatty acid esters that contain as the acid component a 10 for example for identification purposes or to indicate different long-chained fatty acid having from 8 to 22, especially from doses of active ingredient. 12 to 22, carbon atoms, for example lauric acid, tridecylic The pharmaceutical compositions comprise from approxi acid, myristic acid, pentadecylic acid, palmitic acid, margaric mately 1% to approximately 95%, preferably from approxi acid, Stearic acid, arachidic acid, behenic acid or correspond mately 20% to approximately 90%, active ingredient. Phar ing unsaturated acids, for example oleic acid, elaidic acid, 15 maceutical compositions according to the invention may be, erucic acid, brasidic acid or linoleic acid, if desired with the for example, to in unit dose form, such as in the form of addition of anti oxidants, for example, vitamins E. B-caro ampoules, vials, Suppositories, drages, tablets or capsules. tene, or 3,5-di-tert-butyl-4-hydroxytoluene. The alcohol The formulations can be administered to human patients in component of those fatty acid esters has a maximum of 6 a therapeutically effective amount (e.g., an amount that carbon atoms and is a mono- or poly-hydroxy, for example a decreases, Suppresses, attenuates, diminishes, arrests, or sta mono-, di- or tri-hydroxy, alcohol, for example methanol, bilizes the development or progression of a disease, disorder, ethanol, propanol, butanol or pentanol or the isomers thereof, or infection in a eukaryotic host organism). The preferred but especially glycol and glycerol. The following examples of dosage of therapeutic agent to be administered is likely to fatty acid esters are therefore to be mentioned: ethyl oleate, depend on Such variables as the type and extent of the disor isopropyl myristate, isopropyl palmitate, “Labrafil M 2375' 25 der, the overall health status of the particular patient, the (poly oxyethylene glycerol trioleate, Gattefoss, Paris), “Mig formulation of the compound excipients, and its route of lyol 812 (triglyceride of saturated fatty acids with a chain administration. length of Cs to C, Huls AG, Germany), but especially veg For any of the methods of application described above, a etable oils, such as cottonseed oil, almond oil, olive oil, castor compound that interacts with a MAPKAP kinase-2 polypep oil, Sesame oil, soybean oil and more especially groundnut 30 tide may be applied to the site of the needed therapeutic event oil. (for example, by injection, e.g., direct injection into one or The injection compositions are prepared in customary more tumors), or to tissue in the vicinity of the predicted manner under sterile conditions; the same applies also to therapeutic event or to a blood vessel Supplying the cells introducing the compositions into ampoules or vials and seal predicted to require enhanced therapy. ing the containers. 35 The dosages of compounds that interact with a MAPKAP Pharmaceutical compositions for oral administration can kinase-2 polypeptide depend on a number of factors, includ be obtained by combining the active ingredient with solid ing the size and health of the individual patient, but, generally, carriers, if desired granulating a resulting mixture, and pro between 0.1 mg and 1000 mg inclusive are administered per cessing the mixture, if desired or necessary, after the addition day to an adult in any pharmaceutically acceptable formula of appropriate excipients, into tablets, drage cores or cap 40 tion. In addition, treatment by any of the approaches sules. It is also possible for them to be incorporated into described herein may be combined with more traditional plastics carriers that allow the active ingredients to diffuse or therapies. be released in measured amounts. Suitable carriers are especially fillers, such as Sugars, for EXAMPLES example lactose, Saccharose, mannitol or Sorbitol, cellulose 45 preparations and/or calcium phosphates, for example trical Example 1 cium phosphate or calcium hydrogen phosphate, and binders, Such as starch pastes using for example corn, wheat, rice or The p38 MAPK-MK2 Pathway is Activated by Drugs potato starch, gelatin, tragacanth, methylcellulose, hydrox that Directly Damage DNA ypropylmethylcellulose, Sodium carboxymethylcellulose 50 and/or polyvinyl-pyrrolidone, and/or, if desired, disinte To investigate whether the p38 MAPK/MK2 pathway was grates, such as the above-mentioned Starches, also carboxym involved in the DNA damage response of cells following ethyl starch, crosslinked polyvinylpyrrolidone, agar, alginic exposure to clinically useful chemotherapeutic agents, we acid or a salt thereof. Such as Sodium alginate. Excipients are treated human U2OS osteosarcoma cells with the DNA especially flow conditioners and lubricants, for example 55 crosslinking agent cis-Platinum (cisplatin), the topoi silicic acid, talc, Stearic acid or salts thereof. Such as magne Somerase I inhibitor camptothecin, or the topoisomerase II sium or calcium Stearate, and/or polyethylene glycol. Drage inhibitor doxorubicin. p38 MAPK activation was assessed cores are provided with Suitable, optionally enteric, coatings, using an antibody specific for the Thr-180/Tyr-182 doubly there being used, inter alia, concentrated Sugar Solutions phosphorylated active form of the kinase. Activation of MK2 which may comprise gum arabic, talc, polyvinylpyrrolidone, 60 was monitored by its altered mobility on SDS-PAGE, and by polyethylene glycol and/or titanium dioxide, or coating solu immunoblotting using a phospho-specific antibody for pThr tions in Suitable organic solvents, or, for the preparation of 344, a site in the auto-inhibitory domain whose phosphory enteric coatings, solutions of Suitable cellulose preparations, lation by p38 MAPK results in a dramatic elevation of MK2 such as ethylcellulose phthalate or hydroxypropylmethylcel activity. Prior to exposure of cells to DNA damaging com lulose phthalate. Capsules are dry-filled capsules made of 65 pounds, or in cells treated with DMSO (vehicle) alone, MK2 gelatin and Soft sealed capsules made of gelatin and a plasti ran as a single band that did not cross-react with the anti ciser, Such as glycerol or Sorbitol. The dry-filled capsules may pThr-344 antibody. Within one hour after exposure of the US 9,023,787 B2 117 118 cells to cisplatin and doxorubicin, or within four hour follow examined the response of these otherwise genetically identi ing treatment with camptothecin, MK2 displayed a signifi cal cells to cisplatin and doxorubicin using a colony Survival cant reduction in its electrophoretic mobility. The upshifted assay FIGS. 10A and 10B. Cells were infected with lentivi MK2 band appeared with the same kinetics as both the MK2 ruses delivering shRNAs against luciferase (control) or MK2 pThr-344- and the p38 MAPK pThr-180/pTyr-182 immu and analyzed 6 days later. Luciferase and MK2 knockdown noreactive bands. Activation of MK2 was entirely dependent MEFs were then exposed to increasing doses of cisplatin or on p38 MAPK, since addition of the p38 MAPK selective doxorubicin. As seen in FIGS. 10A and 10B, there was no inhibitor SB203580 to the growth media 30 minutes prior to difference in the number of surviving colonies in the p53 application of the DNA damaging agents completely abol wit/wt MEFs, regardless of the presence or absence of MK2, at ished MK2 activation, while preserving activation of 10 any dose of cisplatin or doxorubicin examined. In contrast, p38 MAPK. Similar results for MK2 activation in response to downregulation of MK2 in p53 cells dramatically reduced cisplatin, camptothecin and doxorubicin were also observed the number of surviving colonies. These results demonstrate in HeLa cervical carcinoma cells, U87MG human glioblas that depletion of MK2 specifically sensitizes p53-deficient toma cells, and primary MEFs (FIG. 10A and data not cells to the anti-proliferative effects of chemotherapy-in shown). The time course of MK2 activation upon treatment 15 with each of these drugs matched the rate of appearance of duced DNA damage. We used Western blot analysis to profile Y—H2AX nuclear foci. These data indicate that treatment of activation of the MK2 pathway and the p53 network follow cells with these genotoxic agents results in MK2 activation, ing cisplatin and doxorubicin treatment in these four cell likely as a direct result of chemotherapy-induced DNA dam lines. The presence or absence of MK2 had no effect on the age. strong induction of p53 and p21 following exposure to cispl atin or doxorubicin in the p53 WT/WT cells. Only minimal Example 2 amounts of the p53 inducer protein p19ARF were detected. Neither p53 or p21 induction was detectable in p53/MEFs in ATM and ATR are Required for p38 MAPK/MK2 the presence or absence of MK2. However, the tumor sup Activation Following Genotoxin-Induced DNA 25 pressor p19ARF was strongly induced in these cells even in Damage but not in Response to UV Irradiation the absence of DNA damaging chemotherapy, likely reflect ing a feedback response due to the inability of these cells to We analyzed the p38 MAPK/MK2 activation profile in induce p53. Thus, we concluded that MK2 is not required for ATM-deficient and ATR-defective fibroblasts (FIG. 6). We the normal p53/p21 induction or stabilization in response to also studied the effect of pharmacological inhibition of these 30 DNA damage in wild-type primary cells, and that MK2 is kinases by addition of caffeine. Activation of the p38 MAPK/ MK2 complex in response to cisplatin, camptothecin or UV unable to induce p21 expression after DNA damage in the exposure occurred normally in ATM deficient fibroblasts, absence of functional p53. while doxorubicin treatment failed to activate either Example 4 p38 MAPK or MK2 in these cells. ATR-defective fibroblasts, 35 on the other hand, failed to activate p38 MAPK or MK2 fol lowing either cisplatin, doxorubicin, or camptothecin expo Down-Regulation of MK2 Leads to Mitotic sure. However, UV-induced p38 MAPK/MK2 activation in Catastrophe after DNA Damage in p537 Cells these cells was unaffected. Similarly, treatment of U2OS cells with 20 mM caffeine, a concentration sufficient to inhibit 40 We speculated that the reduced colony formation observed ATM, ATR and DNA-PK, for thirty minutes prior to exposure in MK2-depleted p53 MEF's after DNA damaging chemo to cisplatin and doxorubicin completely abrogated the therapy might be due to mitotic catastrophe resulting from p38 MAPK/MK2 response, while the activation of these defective cell cycle checkpoints. A hallmark of mitotic catas kinases by UV occurred normally under these conditions. trophe is entry of cells into mitosis despite the presence of Taken together, these data indicate that cisplatin, camptoth 45 damaged DNA, resulting in activation of the apoptotic cell ecin, and doxorubicin require ATR for p38 MAPK/MK2 acti death pathway. To investigate this, luciferase shRNA control vation, that doxorubicin also requires ATM activity, and that and MK2 depleted p53 wild-type or null MEFs were treated UV irradiation is capable of activating the p38 MAPK/MK2 with low doses of doxorubicin or cisplatin for thirty hours and in a manner that is independent of ATM, ATR, or DNA-PK immunostained with antibodies against histone H3 pSer-10 function. 50 as a marker for mitotic entry, histone Y-H2AX as marker for persistent DNA damage, and cleaved caspase-3 as a marker Example 3 for apoptosis. Luciferase shRNA-treated p53 wit/wt and p53/T cells showed robust Y-H2AX foci after exposure to Loss of p53 Renders Cells Dependent on MK2 DNA damaging chemotherapy. No phospho-histone H3 or Signaling for Survival after Chemically-Induced 55 cleaved caspase-3 staining was observed in MK2-containing DNA Damage cells, consistent with an intact DNA damage response regard less of the presence or absence of p53. Similarly, an intact The p53 tumor Suppressor protein plays an important role DNA damage checkpoint response was also observed in MK2 in the cellular response to DNA damage by transcriptionally depleted cells that contained wild-type p53. In sharp contrast, upregulating the Cdk inhibitor p21 to induce a G and G 60 however, in the MK2-deficient p53/T cells, a substantial arrest. Cancer cells frequently show disruptions in the p53 fraction of the Y-H2AX positive cells also stained positively pathway, eliminating this component of the DNA damage for both phospho-histone H3 and cleaved caspase 3. Interest response, and leaving the cells entirely dependent on remain ingly, no caspase 3 staining was observed in Y-H2AX-positive ing checkpoint signaling pathways. To examine whether the cells that did not also contain phospho-histone H3. Thus, in ATR/ATMp38 MAPK-MK2 pathway was required for cell 65 the absence of MK2, p53 null primary cells treated with survival after genotoxin-induced DNA damage in p53 wild cisplatin and doxorubicin lose one or more critical cell cycle type and p53/MEFs, we used RNAi to deplete MK2, and checkpoints and undergo mitotic catastrophe. US 9,023,787 B2 119 120 Example 5 to the MK2 depleted cells caused them to accumulate in a 4N DNA containing peak, with 28.5% of the cells staining posi Down-Regulation of MK2 Causes Regression of tively for phospho-histone H3 (FIG. 11, right lower panels), a Established p53 / Tumors. In Vivo after Low Dose value similar to that of untreated p53/ cells blocked in Treatment with DNA Damaging Agents mitosis with nocodazole. Intriguingly, MK2 depletion did not alter total Chk1 levels or reduce Chk1 activation following We investigated whether the chemo-sensitizing effect of DNA damage. These findings demonstrate that loss of MK2 MK2 depletion in p53 null cells in culture could also be prevents p53-deficient cells from establishing a functional observed when pre-existing p53 deficient tumors were treated G/M checkpoint following doxorubicin-induced DNA dam with DNA damaging drugs in vivo. In these experiments 10 age, despite the presence of activated Chk1. Identical results HRas-V12 transformed p53 / MEFs were stably transfected were obtained using a second unrelated shRNA against to 0 with control shRNA or MK2 shRNA expressed from a murine MK2. Importantly, the checkpoint defect could be fully res U6 promoter, using a lentiviral delivering system. The len cued in the MK2 depleted cells by expressing an shRNA tiviral transfer vector also encoded GFP under the control of resistant form of MK2 at comparable levels to the endog a CMV promoter, allowing for fluorescent detection of 15 enous protein. tumors in situ. Tumors were induced by injection of 10° cells Two Cdc25 family members, Cdc25B and C, play impor into the flanks of nude mice. Twelve days later ~1 cm diam tant roles in initiating and maintaining mitotic entry in normal eter tumors had formed at all injection sites, and treatment cells, and are prominent targets of the G2/M checkpoint. with cisplatin, doxorubicin, or vehicle was begun (FIG. Cdc25B is believed to function by activating Cdk1/Cyclin B 7A-7C). In the absence of treatment with DNA damaging at the centrosome in late G2 as an initiator of early mitotic drugs, the tumors arising from the MK2 depleted cells in the events, while Cdc25C functions to further amplify Cdk1/ right flanks of these animals grew slightly larger than those of CyclinB activity within a nuclear auto-amplification loop the luciferase shRNA control cells in the left flanks (FIGS. once mitosis has begun. In response to Y- or UV-irradiation 7A-7D). Following treatment with cisplatin or doxorubicin, induced DNA damage, checkpoint kinases phosphorylate the control tumors showed either minimal reduction in size, 25 Cdc25B and C on Ser-323 and 216, respectively, to induce or slow continued growth. In contrast, the MK2 depleted their binding to 14-3-3 proteins, sequestering them in the tumors showed a dramatic reduction in weight and diameter. cytoplasm away from their cyclin/Cdk substrates. Cdc25B Tumors depleted of MK2 shrank from 1.3 cm to 0.4 cm over plays a particularly crucial role in initiating and maintaining fourteen days when treated with cisplatin, and from 1.4 to 0.5 normal cell cycle G2/M checkpoint responses, since reactiva cm when treated with doxorubicin. Thus, the sensitizing 30 tion of Cdc25B is critical for DNA-damaged cells to re-enter effect of MK2 depletion on DNA damage induced cell death the cell cycle. We therefore investigated whether MK2 sig in p53-deficient primary cells observed in cell culture was naling was required for association of Cdc25B with 14-3-3 in also maintained in vivo. These results strongly suggest that response to DNA damage by chemotherapeutic drugs. Both MK2 may be a useful target for the design of new cancer doxorubicin and camptothecin treatment, resulted in the gen treatment agents. 35 eration of stable 14-3-3-binding sites on Cdc25B in the luciferase shRNA control cells. No 14-3-3 binding of Example 6 Cdc25B, however, was detected in lysates from the MK2 depleted cells. This result is in good agreement with the cell MK2 is Required for the G/M Checkpoint cycle studies in panel A, which showed loss of the G2/M Following Doxorubicin Treatment in p53-Deficient 40 checkpoint in MK2 depleted cells after treatment with the Cells topoisomerase inhibitor doxorubicin. These data indicate that loss of the G/M checkpoint after DNA lesions in MAPKAP To investigate the molecular mechanisms involved in MK2 Kinase-2-depleted p53-defective cells likely arises, at least in dependent responses to DNA lesions, we examined cell cycle part, from loss of Cdc25B binding to 14-3-3 proteins. profiles of control and MK2 depleted p53 / MEFs. Asyn 45 chronous MK2- or control knock-down p53 / MEF's were Example 7 mock treated or exposed to doxorubicin for thirty hour, and cell cycle distribution monitored by FACS. In one set of MK2 is Required for S-Phase Checkpoint Arrest experiments the spindle poison nocodazole was added to the Following Cisplatin Treatment in p53-Deficient Cells media three hours after addition of doxorubicin, to cause any 50 cells progressing through the cell cycle to arrest in mitosis. Treatment with the DNA intra-strand cross-linker cisplatin DNA content was monitored by PI staining; phospho-his caused p53/T cells to predominantly accumulate in S phase tone-H3 staining was used as an indicator of mitotic entry. As of the cell cycle. RNA interference was used to to investigate shown in the left panels of FIG. 11, treatment of control knock the role of MK2 in this process. p53 / control knock-down down p53 / cells with doxorubicin led to the accumulation 55 cells showed an identical accumulation in S phase after cis of cells with 4N DNA content, and a lack of phospho-histone platin exposure (FIG. 12, left middle panels) as that seen in H3 staining in either the absence or presence of nocodazole, untransfected p53/ cells. Addition of nocodazole to the indicative of an intact G/M checkpoint. These cells express luciferase knock-down cells three hour following cisplatin ing control shRNAs behaved identically to an untransfected treatment did not reveal the appearance of any mitotic cells control p53/ cell population. In marked contrast, MK2 60 over the ensuing twenty seven hours, as monitored by phos depleted p53 / cells treated with doxorubicin displayed a pho-histone H3 staining (FIG. 12, lower left panels), indicat cell cycle profile similar to that of untreated cells (FIG. 11, ing a functionally intact S-phase checkpoint. Depletion of right upper and middle panels), with only a small increase in MK2 prior to cisplatin exposure resulted in a dramatically the 4N peak compared to the doxorubicin-treated luciferase different result. As seen in the right panels of FIG. 12, MK2 shRNA controls, a slightly increased S-phase population, and 65 depleted p53/T cells showed a cell cycle profile after cispl the appearance of a Sub-G population indicative of apopto atin treatment that was similar to that of untreated cells, other sis. Addition of nocodazole following doxorubicin treatment than a slight increase in the total number of cells in S-phase US 9,023,787 B2 121 122 and the appearance of a Sub-G population consistent with ystaurosporin/UCN-01 has been shown to increase the cyto apoptosis. Strikingly, when nocodazole was added three toxicity of chemotherapy and radiation and is currently in hours following cisplatin addition, the MK2 depleted p53/ clinical trials. It has been demonstrated that a major target of cells accumulated in a 4N DNA containing peak with ~25% UCN-01 is the checkpoint kinase Chikl, leading to specula of the cells staining positive for phospho-histone H3. The tion that that the increased chemo- and radiation sensitivity of same cell cycle defects after cisplatin exposure were observed cells treated with UCN-01 is a direct result of Chk1-mediated using a second unrelated shRNA sequence against MK2, and checkpoint abrogation. UCN-01 inhibits Chk1 with an ICso the MK2 shRNA phenotype was completely reversed by that is ~1000 fold lower than that for Chk2, and hence has expression of an RNAi-resistant form of MK2 at physiologi been used experimentally as a Chk1 specific inhibitor. Strong cal levels. Similar to what was observed following doxorubi 10 circumstantial evidence, however, suggests that UCN-01 cin treatment, MK2 depletion did not impair activation of must be inhibiting other kinases involved in cell cycle control Chk1 after cisplatin exposure. These data imply that MK2 is at similar concentrations as those used for Chk1 inhibition essential for the cisplatin induced S-phase arrest in p53-defi studies. For example, Chk1-depleted cells maintain phospho cient cells, and that loss of MK2 enables these cells to over rylation of Ser-216, a well characterized Chk1 target site on ride the cisplatin-induced S-phase checkpoint, despite the 15 Cdc25C, both during asynchronous growth and following presence of activated Chikl, and proceed into mitosis. In Y-irradiation. Phosphorylation at this site is lost, however, contrast to the 14-3-3-mediated sequestration of Cdc25B and when cells are treated with low doses of UCN-01 (-300 nM), C involved in the G2/M checkpoint response, the G and S indicating that UCN-01 inhibitable kinase(s) other than Chk1 phase checkpoints are largely controlled by the phosphoryla participate in Cdc25C Ser-216 phosphorylation. Based on our tion-dependent degradation of another Cdc25 isoform, finding that MK2 depletion results in a dramatically increased Cdc25A. We therefore investigated whether MK2 was chemosensitivity of malignant cells, we asked whether MK2 required for the degradation of Cdc25A following cisplatin might be a UCN-01 target, similar to Chk1. In vitro kinase induced DNA damage. Cdc25A levels were undetectable in assays were performed under identical reaction conditions the control luciferase knock-down cells after treatment with with Chk1 and MK2 using the same optimal peptide substrate cisplatin. In contrast, in the MK2 depleted cells, substantial 25 for both kinases with the core consensus sequence L-Q-R-Q- amounts of Cdc25A remain present in the lysates after cispl L-S-I, in the presence of various concentrations of UCN-01. atin exposure, indicating that in the absence of MAPKAP As shown in FIG. 21A 8, UCN-01 potently inhibited both Kinase-2, p53-/- MEFs cells are defective in targeting kinases, with an ICs value of -35 nM for Chk1 and -95 nM Cdc25A for degradation in response to cisplatin induced for MAPKAP Kinase-2. The ICs value we measured for DNA damage. This impaired ability of MK2 depleted cells to 30 Chk1 is in good agreement with previously published data. degrade Cdc25A likely explains their failure to establish a Importantly, the ICso value we measured for MK2 is signifi Sustained G1/S checkpoint following cisplatin exposure. cantly below the concentrations of UCN-01 that are used in Interestingly, Cdc25A may be a direct target of MK2 in vivo, "Chk1-specific' checkpoint abrogation assays, suggesting since both MK2 and Chk1 phosphorylate Cdc25A equiva that under the conditions used in those studies, both Chk1 and lently in vitro, Chk1 phosphorylation of Cdc25A in vivo has 35 MK2 were being inhibited. To examine the structural basis for been shown to facilitate its ubiquitin-mediated proteolysis in UCN-01 inhibition of MK2, the structure of the MK2:UCN a complex and incompletely understood manner, and MK2 01 complex was modeled using coordinates from the pub and Chk1 phosphorylate the identical optimal sequence lished MK2:staurosporine structure, and compared the motifs when analyzed by oriented peptide library screening. results with the co-crystal structure of Chk1:UCN-01. The 40 7-hydroxy moiety of UCN-01 can be easily accommodated Example 8 into the MK2:staurosporine structure, where its closest neighboring residues would be Val-118 (2.8 A to Cy2), Leu MK2 and Chk1 are Activated Independently by DNA 141 (3.2A to CY1), and Thr-206 (3.6A to Cy2). Lackofsteric Damage, and are Both Potently Inhibited by UCN-01 hindrance, and the overall similarity of the modeled MK2: 45 UCN-01 structure to the Chkl:UCN-01 structure provides a Activation of MK2 by cisplatin, camptothecin, and doxo structural rationale for the tight binding observed biochemi rubicin is strikingly similar to the activation profile reported cally. To verify that MK2 is a direct target of UCN-01 within for Chk1. Similarly, the impaired S-phase and G/M check cells, we measured the phosphorylation of the MK2 substrate points seen after these DNA damaging stimuli in MK2 knock hsp-27 after heat shock, a stimulus that activates the down cells bears some resemblance to what has been previ 50 p38 MAPK/MK2 pathway. Control or MK2 shRNA express ously reported for Chk1-deficient p53-defective cells. These ing U2OS cells were incubated at 42° C. or 37° C. for two phenotypic similarities prompted us to further investigate hours in the presence or absence of 250 nM UCN-01, and whether the activation of Chk1 and MK2 was interdependent. phosphorylation of hsp-27 monitored by immunoblotting. Activation of Chk1 in response to doxorubicin and cisplatin Immunoblotting shows that hsp27 is phosphorylated on Ser was unimpaired in MK2 depleted cells. We therefore inves 55 82 when the control luciferase shRNA cells were placed at tigated the converse—whether the activation of MK2 after 42°C. This phosphorylation was completely abrogated by DNA damage was dependent on Chk1. U2OS cells depleted treatment with UCN-01. No phosphorylation was observed in of Chk1 using siRNA were exposed to cisplatin and doxoru MK2 knock-down cells placed at 42° C. regardless of the bicin, and analyzed for activation of MK2. presence or absence of UCN-01, and no signal was observed Phosphorylation/activation of MK2 occurred normally 60 in both the control and MK2 knock-down cells that were after treatment with these DNA damaging agents, regardless maintained at 37° C., with or without UCN-01 treatment. of the presence or absence of Chk1. Thus, activation of MK2 Heat shock was equally effective in promoting the phospho and Chk1 after drug-induced DNA damage appears to occur rylation of hsp-27 on Ser-82, and UCN-01 was equally effec independently of each other, and both kinases appear to par tive in blocking Ser-82 phosphorylation in cells that were ticipate in parallel DNA damage checkpoint signaling path 65 depleted of Chk1. Thus, UCN-01 inhibits MK2 in vivo, and ways that are necessary for cell survival in the absence of a this effect is independent of Chk1 function. This data dem strong p53 response. The staurosporine derivative 7-hydrox onstrates that UCN-01 is a direct inhibitor of MK2 within US 9,023,787 B2 123 124 cells, and indicates that the clinical efficacy of UCN-01 in becomes essential for cell survival after DNA damage. Both cancer treatment, particularly in p53-defective tumors, likely pathways are simultaneously inhibited by the indolocarba arises from the simultaneous inhibition of both the Chk1 and zole drug UCN-01. MK2 signaling pathways Other Embodiments Example 9 All publications, patents, and patent applications men A Model for Re-Wiring of Cell Cycle Checkpoint tioned in the above specification are hereby incorporated by Pathways in p53-Proficient and Deficient Cells reference. In addition, U.S. Patent Application Publication 10 Nos. US 2005-0196808 and US 2006-0052951 are hereby Checkpoint function in p53-proficient cells is mediated incorporated by reference. Various modifications and varia primarily through a robust, Sustained p53 response down tions of the described method and system of the invention will stream of ATM, together with Chk1 (FIG. 13A). Although not be apparent to those skilled in the art without departing from shown explicitly in the diagram, Chk1 has been shown to also the scope and spirit of the invention. Although the invention directly phosphorylate p53. Under these conditions the pres 15 has been described in connection with specific embodiments, ence of MK2 is not required for cell survival after exposure to it should be understood that the invention as claimed should DNA damaging agents. In p53-deficient cancer cells, check not be unduly limited to such specific embodiments. Indeed, point signaling following exposure to DNA damaging agents various modifications of the described modes for carrying out is mediated through the combined action of both the Chk1 and the invention that are obvious to those skilled in the art are the p38 MAPK/MK2 pathways (FIG. 13B). In this situation intended to be within the scope of the invention. the p38 MAPK/MK2 branch of checkpoint signaling Other embodiments are in the claims.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 5 O

SEO ID NO 1 LENGTH 227 TYPE PRT ORGANISM: Saccharomyces cerevisiae <4 OOs SEQUENCE: 1

Met Ser Ala Ser Thr Thir Ser Luell Glu Glu Tyr Glin Thir Phe Lell 1. 5 15

Glu Lieu. Gly Luell Glu Lys Ala Lieu Arg Phe Gly Ser Phe Lell 2O 25 3 O

Asn Ser Gly Arg Glin Ser Pro Tyr Phe Phe Asn Lieu. Ser Tell Phe Asn 35 4 O 45

Ser Gly Lys Lieu. Luell Ala Asn Lieu. Ala Thir Ala Tyr Ala Thir Ala Ile SO 55 60

Ile Glin Ser Glu Lieu Lys Phe Asp Wall Ile Phe Gly Pro Ala 65 70 7s

Gly Ile Pro Leu Ala Ala Ile Wall Cys Wall Luell Ala Glu Gly 85 90

Gly Thir Phe Gln Gly Ile Glin Tyr Ala Phe Asn Arg Lys Wall 105 110

Asp His Gly Glu Gly Gly Ile Ile Wall Gly Ala Ser Tell Glu Asp 115 12O 125

Arg Val Lieu. Ile Ile Asp Asp Wal Met Thir Ala Gly Thir Ala Ile 13 O 135 14 O

ASn Glu Ala Phe Glu Ile Ile Ser Ile Ala Glin Gly Arg Wall Val Gly 145 15 O 155 16 O

Ile Wall Ala Luell Asp Arg Glin Glu Wall Ile His Glu Ser Asp Pro 1.65 17 O 17s

Glu Arg Thir Ser Ala Thr Glin Ser Wall Ser Arg ASn Wall Pro 18O 185 190

Wall Lell Ser Ile Wall Ser Luell Thir Glin Wall Wall Glin Phe Met Gly Asn 195 2 OO 2O5

Arg Leul Ser Pro Glu Glin Lys Ser Ala Ile Glu Asn Tyr Ala 210 215 22 O US 9,023,787 B2 125 126 - Continued Tyr Gly Ile 225

<210s, SEQ ID NO 2 &211s LENGTH: 294 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 2 Caggtgcagc tigcaggagtic gggcc cagga citggtgaagc Ctt cqgggaC cctgtcc ct c 6 O acctgcgctg. tct Ctggtgg Ct c catcagc agtag taact ggtggagttg ggtcc.gc.ca.g 12 O

CCCC caggga aggggctgga gtggattggg gaaatct atc at agtgggag caccalactac 18O aaccc.gtc.cc ticaagagt cq agt caccata t cagtagaca agt ccaagaa ccagttcticc 24 O Ctgaagctga gct Ctgttgac cqc.cgcggac acggc.cgtgt attactgtgc gaga 294

<210s, SEQ ID NO 3 &211s LENGTH: 571 212. TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae

<4 OOs, SEQUENCE: 3 Met Ser Met Ser Ser Lys Asn. Glu Asn Lys Ile Ser Val Glu Glin Arg 1. 5 1O 15 Ile Ser Thr Asp Ile Gly Glin Ala Tyr Glin Lieu. Glin Gly Lieu. Gly Ser 2O 25 3O Asn Lieu. Arg Ser Ile Arg Ser Lys Thr Gly Ala Gly Glu Val Asn Tyr 35 4 O 45 Ile Asp Ala Ala Lys Ser Val Asn Asp Asn Gln Lieu. Lieu Ala Glu Ile SO 55 6 O Gly Tyr Lys Glin Glu Lieu Lys Arg Glin Phe Ser Thr Lieu. Glin Val Phe 65 70 7s 8O Gly Ile Ala Phe Ser Ile Met Gly Lieu. Leu Pro Ser Ile Ala Ser Val 85 90 95 Met Gly Gly Gly Lieu. Gly Gly Gly Pro Ala Thr Lieu Val Trp Gly Trp 1OO 105 11 O Phe Val Ala Ala Phe Phe Ile Lieu. Leu Val Gly Ile Thr Met Ala Glu 115 12 O 125 His Ala Ser Ser Ile Pro Thr Ala Gly Gly Lieu. Tyr Tyr Trp Thr Tyr 13 O 135 14 O Tyr Tyr Ala Pro Glu Gly Tyr Lys Glu Ile Ile Ser Phe Ile Ile Gly 145 150 155 160 Cys Ser Asn. Ser Lieu Ala Lieu Ala Ala Gly Val Cys Ser Ile Asp Tyr 1.65 17O 17s Gly Lieu Ala Glu Glu Ile Ala Ala Ala Val Thir Lieu. Thir Lys Asp Gly 18O 185 19 O

Asn Phe Glu Val Thr Ser Gly Lys Lieu. Tyr Gly Ile Phe Ala Gly Ala 195 2OO 2O5

Val Val Val Met Cys Ile Cys Thr Cys Val Ala Ser Gly Ala Ile Ala 21 O 215 22O

Arg Lieu. Glin Thr Lieu. Ser Ile Phe Ala Asn Lieu. Phe Ile Ile Val Lieu 225 23 O 235 24 O Lieu. Phe Ile Ala Lieu Pro Ile Gly Thr Lys His Arg Met Gly Gly Phe 245 250 255

Asn Asp Gly Asp Phe Ile Phe Gly Lys Tyr Glu Asn Lieu. Ser Asp Trip 26 O 265 27 O US 9,023,787 B2 127 128 - Continued

Asn Asn Gly Trp Glin Phe Cys Lieu. Ala Gly Phe Met Pro Ala Wall Trp 27s 28O 285

Thir Ile Gly Ser Phe Asp Ser Cys Wall His Glin Ser Glu Glu Ala 29 O 295 3 OO

Asp Ala Ser Wall Pro Ile Gly Ile Ile Ser Ser Ile Ala Wall 3. OS 310 315

Trp Ile Luell Gly Trp Lieu. Ile Ile Ile Lell Met Ala Cys Ile 3.25 330 335

Asn Pro Asp Ile Asp Ser Val Lieu. Asp Ser Gly Phe Luell 34 O 345 35. O

Ala Glin Ile Ile Tyr Asp Ser Lieu. Gly Trp Ala Ile Phe 355 360 365

Met Ser Luell Ile Ala Phe Cys Glin Phe Luell Met Gly Ala Ser Thir 37 O 375 38O

Thir Ala Wall Ser Arg Glin Val Trp Ala Phe Ser Arg Asp Asn Luell 385 390 395 4 OO

Pro Luell Ser Tyr Ile Lys Arg Wall Asp Ser Ser Pro 4 OS

Phe Phe Ala Ile Lieu Ala Ala Cys Wall Gly Ser Lell Ile Luell Luell 425 43 O

Lell Luell Ile Asp Asp Ala Ala Thir Asp Ala Lell Phe Ser Luell Ala 435 44 O 445

Wall Ala Gly Asn Asn Lieu Ala Trp Ser Thir Pro Thir Wall Phe Arg Lel 450 45.5 460

Thir Ser Gly Arg Asp Lieu. Phe Arg Pro Gly Pro Phe Luell Gly Lys 465 470 48O

Ile Trp Ser Pro Ile Val Ala Trp Thir Gly Wall Ala Phe Glin Luell Phe 485 490 495

Ile Ile Ile Luell Wal Met Phe Pro Ser Glin Glin His Gly Ile Thir SOO 505

Ser Thir Met Asn Tyr Ala Cys Val Ile Gly Pro Gly Ile Trp Ile Lel 515 52O 525

Ala Gly Ile Tyr Lys Val Tyr Tyr His Gly 53 O 535 54 O

Ala Thir Asn Luell Ser Asp Asp Asp Thir Glu Ala Wall Gly Ala Asp 5.45 550 555 560

Wall Ile Asp Thir Ile Met Ser Lys Glin Glu Pro 565 st O

<210s, SEQ ID NO 4 &211s LENGTH: 4 OO 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 4.

Met Leu Ser Asn Ser Glin Gly Glin Ser Pro Pro Wall Pro Phe Pro Ala 1. 5 15

Pro Ala Pro Pro Pro Glin Pro Pro Thir Pro Ala Lell Pro His Pro Pro 25

Ala Glin Pro Pro Pro Pro Pro Pro Glin Glin Phe Pro Glin Phe His Wall 35 4 O 45

Ser Gly Luell Glin Ile Llys Llys Asn Ala Ile Ile Asp Asp Lys SO 55 6 O

Wall Thir Ser Glin Val Lieu. Gly Lieu. Gly Ile ASn Gly Wall Luell Glin US 9,023,787 B2 129 130 - Continued

65 70

Ile Phe Asn Thir Glin Glu Phe Ala Lell Met Luell Glin 85 90 95

Asp Pro Lys Ala Arg Arg Glu Wall Glu Luell His Trp Arg Ala Ser 1OO 105 11 O

Glin Pro His Ile Wall Arg Ile Wall Asp Wall Glu Asn Luell 115 12 O 125

Ala Gly Arg Cys Lell Lell Ile Wall Met Glu Cys Lell Asp Gly Gly 13 O 135 14 O

Glu Luell Phe Ser Arg Ile Glin Asp Arg Gly Asp Glin Ala Phe Thir Glu 145 150 155 160

Arg Glu Ala Ser Glu Ile Met Ser Ile Gly Glu Ala Ile Glin 1.65 17O 17s

Lell His Ser Ile Asn Ile Ala His Arg Asp Wall Pro Glu Asn Luell 18O 185 19 O

Lell Thir Ser Lys Arg Pro Asn Ala Ile Luell Lell Thir Asp Phe 195

Gly Phe Ala Glu Thir Thir Ser His Asn Ser Lell Thir Thir Pro 21 O 215 22O

Tyr Thir Pro Tyr Wall Ala Pro Glu Wall Luell Gly Pro Glu Tyr 225 23 O 235 24 O

Asp Ser Asp Met Trp Ser Luell Gly Wall Ile Met Ile Luell 245 250 255

Lell Gly Tyr Pro Pro Phe Ser Asn His Gly Lell Ala Ile Ser 26 O 265 27 O

Pro Gly Met Thir Arg Ile Arg Met Gly Glin Glu Phe Pro Asn 285

Pro Glu Trp Ser Glu Wall Ser Glu Glu Wall Met Lell Ile Arg Asn 29 O 295 3 OO

Lell Luell Thir Glu Pro Thir Glin Arg Met Thir Ile Thir Glu Phe Met 3. OS 310 315

Asn His Pro Trp Ile Met Glin Ser Thir Lys Wall Pro Glin Thir Pro Luell 3.25 330 335

His Thir Ser Arg Wall Lell Glu Asp Glu Arg Trp Glu Asp Wall 34 O 345 35. O

Glu Glu Met Thir Ser Ala Luell Ala Thir Met Arg Wall Asp Glu 355 360 365

Glin Ile Ile Lys Ile Glu Asp Ala Ser Asn Pro Luell Luell Luell 37 O 375

Lys Arg Arg Lys Ala Arg Ala Luell Glu Ala Ala Ala Luell Ala His 385 390 395 4 OO

SEO ID NO 5 LENGTH: TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Substrate Peptide

SEQUENCE: 5 Gly Arg Pro Arg Thr Thr Ser Phe Ala Glu 1. 5

<210s, SEQ ID NO 6 &211s LENGTH: 8 212. TYPE : PRT US 9,023,787 B2 131 132 - Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Substrate Peptide <4 OOs, SEQUENCE: 6 Lieu. Glin Arg Glin Lieu. Ser Ile Ala 1. 5

<210s, SEQ ID NO 7 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Substrate Peptide <4 OO > SEQUENCE: 7 Lieu. Tyr Arg Ser Pro Ser Met Pro Leu 1. 5

<210s, SEQ ID NO 8 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Substrate Peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) ... (4) <223> OTHER INFORMATION: X denotes all amino acids except Cys, Ser, Thr, and Tyr 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222 LOCATION: (7) . . (10 <223> OTHER INFORMATION: X denotes all amino acids except Cys, Ser, Thr, and Tyr

<4 OOs, SEQUENCE: 8

Xaa Xala Xala Xala Ser Pro Xala Xala Xala Xala 1. 5 1O

<210s, SEQ ID NO 9 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Substrate Peptide <4 OOs, SEQUENCE: 9 Gly Pro Glin Ser Pro Ile 1. 5

<210s, SEQ ID NO 10 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Substrate Peptide

<4 OOs, SEQUENCE: 10 Pro Gly Pro Glin Ser Pro Gly Ser Pro Leu 1. 5 1O

<210s, SEQ ID NO 11 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: p38SAPK docking motif US 9,023,787 B2 133 134 - Continued

<4 OOs, SEQUENCE: 11 His Glin Arg Ser Arg Lys Arg Lieu. Ser Glin 1. 5 1O

<210s, SEQ ID NO 12 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: p38SAPK docking motif <4 OOs, SEQUENCE: 12 Val Arg Phe Lieu. Glin Glin Arg Arg Arg Glin Ala 1. 5 1O

<210s, SEQ ID NO 13 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Potential p38 SAPK docking motif <4 OOs, SEQUENCE: 13 Pro Val Glin Asn Lys Arg Arg Arg Ser Val 1. 5 1O

<210s, SEQ ID NO 14 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: Substrate Peptide for Cdc25B 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: X can be any amino acid <4 OOs, SEQUENCE: 14 Leu Xaa Arg Ser Pro Ser Met Pro 1. 5

<210s, SEQ ID NO 15 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Substrate peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) ... (4) <223> OTHER INFORMATION: X denotes all amino acids except Cys, Ser, Thr, or Tyr 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (6) . . (7) <223> OTHER INFORMATION: X denotes all amino acids except Cys, Ser, Thr, or Tyr 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (9) . . (12) <223> OTHER INFORMATION: X denotes all amino acids except Cys, Ser, Thr, or Tyr

<4 OOs, SEQUENCE: 15

Xaa Xala Xala Xala Arg Xaa Xaa Ser Xaa Xala Xaa Xaa 1. 5 1O

<210s, SEQ ID NO 16 &211s LENGTH: 7 US 9,023,787 B2 135 136 - Continued

212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: MAPKAP kinase-2 consensus motif

<4 OOs, SEQUENCE: 16 Lieu. Glin Arg Glin Lieu. Ser Ile 1. 5

<210s, SEQ ID NO 17 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: MAPKAP kinase-2 phosphorylation motif 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: X can be Lleu, Phe, or Ile 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: X can be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: X can be Glin, Ser, or Thr 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (6) . . (6) <223 is OTHER INFORMATION: X can be Ser or Thr

<4 OOs, SEQUENCE: 17 Xaa Xala Arg Xaa Lieu. Xaa 1. 5

<210s, SEQ ID NO 18 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Hydrophobic motif on Calc25 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: X can be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (4) ... (4) <223> OTHER INFORMATION: X can be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: X can be any amino acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (6) . . (6) <223 is OTHER INFORMATION: X can be Ser or Thr

<4 OOs, SEQUENCE: 18 Lieu. Xaa Arg Xaa Xaa Xaa 1. 5

<210s, SEQ ID NO 19 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Substrate Peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (3) . . (6) <223> OTHER INFORMATION: X can be any amino acid except Cys, Ser, Thr, US 9,023,787 B2 137 138 - Continued and Tyr 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (9) . . (12) <223> OTHER INFORMATION: X can be any amino acid except Cys, Ser, Thir, and Tyr

<4 OOs, SEQUENCE: 19 Gly Ala Xala Xala Xaa Xaa Ser Pro Xaa Xala Xaa Xaa Ala Lys Llys Llys 1. 5 1O 15

<210s, SEQ ID NO 2 O &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Substrate Peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (3) . . (6) <223> OTHER INFORMATION: X can be any amino acid except Cys, Ser, Thir, and Tyr 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: X can be any amino acid except Cys, Ser, Thir, and Tyr 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (11) . . (15) <223> OTHER INFORMATION: X can be any amino acid except Cys, Ser, Thir, and Tyr

<4 OOs, SEQUENCE: 2O Gly Ala Xala Xala Xaa Xaa Pro Xaa Ser Pro Xaa Xaa Xaa Xala Xaa Ala 1. 5 1O 15 Llys Llys Llys

<210s, SEQ ID NO 21 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Substrate Peptide 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (3) ... (4) <223> OTHER INFORMATION: X can be any amino acid except Cys, Ser, Thir, and Tyr 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (6) ... (9) <223> OTHER INFORMATION: X can be any amino acid except Cys, Ser, Thir, and Tyr

<4 OOs, SEQUENCE: 21 Gly Ala Xala Xala Ser Xaa Xaa Xala Xaa Ala Lys Llys Llys 1. 5 1O

<210s, SEQ ID NO 22 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Substrate Peptide

<4 OOs, SEQUENCE: 22 Lys Lys Ala Glx Gly Pro Glin Gly Pro Glin Ser Pro Ile Glu 1. 5 1O US 9,023,787 B2 139 140 - Continued

<210s, SEQ ID NO 23 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Substrate Peptide <4 OOs, SEQUENCE: 23 Llys Lys Ala Glix Gly Pro Glin Ser Pro Gly Ser Pro Lieu. Glu 1. 5 1O

<210s, SEQ ID NO 24 &211s LENGTH: 55 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer for RNA.ii.

<4 OOs, SEQUENCE: 24 tgaccaggca ttcacagaaa ttcaa.gagat ttctgtgaat gcc toggt citt tttitc 55

<210s, SEQ ID NO 25 &211s LENGTH: 55 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer for RNA.ii.

<4 OOs, SEQUENCE: 25 ttgaccatca ccgagttt at ttcaa.gagaa taalacticggit gatggtcatt tttitc 55

<210s, SEQ ID NO 26 &211s LENGTH: 55 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer for RNA.ii.

<4 OOs, SEQUENCE: 26 tcqatgcgtg ttgactatga ttcaa.gagat catagt caac acgcatcgtt tttitc 55

<210s, SEQ ID NO 27 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer for RNA.ii. 22 Os. FEATURE: <221 > NAMEAKEY: modified base <222s. LOCATION: (2O) . . (21) <223> OTHER INFORMATION: The last two Ts are deoxythymidines <4 OOs, SEQUENCE: 27 luccc.ggculau glugcaggagt t 21

<210s, SEQ ID NO 28 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer for RNA.ii. 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2O) . . (21) <223> OTHER INFORMATION: The last two Ts denote deoxythymidines

<4 OOs, SEQUENCE: 28

Cucculgcaca ulag.ccgggat t 21 US 9,023,787 B2 141 142 - Continued

SEQ ID NO 29 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer for RNA.ii. FEATURE: NAMEAKEY: misc feature LOCATION: (2O) . . (21) OTHER INFORMATION: The last two Ts denote deoxythymidines SEQUENCE: 29 cgaugogugu ulgaculalugat t 21

SEQ ID NO 3 O LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer for RNA.ii. FEATURE: NAMEAKEY: misc feature LOCATION: (2O) . . (21) OTHER INFORMATION: The last two Ts denote deoxythymidines SEQUENCE: 3 O ulcaulagucaa cacgcaucgt t 21

SEQ ID NO 31 LENGTH: 21 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer for RNA.ii. FEATURE: NAMEAKEY: misc feature LOCATION: (17) . . (17) OTHER INFORMATION: W can be an alanine, or a thymidine/uracil, and denotes weak interactions, 2H-bonds FEATURE: NAMEAKEY: misc feature LOCATION: (2O) . . (21) OTHER INFORMATION: The last two Ts are deoxythymidines

SEQUENCE: 31 lugaccalucac cagulu Walut t 21

SEQ ID NO 32 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer for RNA.ii. FEATURE: NAMEAKEY: misc feature LOCATION: (2O) . . (21) OTHER INFORMATION: The last two Ts are deoxythymidines

SEQUENCE: 32 aluaaacucgg lugalugglucat t 21

SEQ ID NO 33 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer for RNA.ii. FEATURE: NAMEAKEY: misc feature US 9,023,787 B2 143 144 - Continued

<222s. LOCATION: (2O) . . (21) <223> OTHER INFORMATION: The last two Ts are deoxythymidines

<4 OOs, SEQUENCE: 33 luggcaa.cagul aluulu.cggulat t 21

<210s, SEQ ID NO 34 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer for RNA.ii. 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2O) . . (21) <223> OTHER INFORMATION: The last two Ts are deoxythymidines

<4 OOs, SEQUENCE: 34 ulaccgaaaua clugulugcc at t 21

<210s, SEQ ID NO 35 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: MAPKAP kinase-2 RNAi target sequence <4 OOs, SEQUENCE: 35 gacCaggc at t cacagaaa 19

<210 SEQ ID NO 36 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: MAPKAP kinase-2 RNAi target sequence <4 OOs, SEQUENCE: 36 ttgaccactic cittgttata 19

<210s, SEQ ID NO 37 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: MAPKAP kinase-2 RNAi target sequence <4 OO > SEQUENCE: 37 gaccactic ct tdttataca 19

<210s, SEQ ID NO 38 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: MAPKAP kinase-2 RNAi target sequence

<4 OOs, SEQUENCE: 38 tgac catcac cqagttitat 19

<210s, SEQ ID NO 39 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: MAPKAP kinase-2 RNAi target sequence US 9,023,787 B2 145 146 - Continued

<4 OOs, SEQUENCE: 39 t caccgagtt tatgaacca 19

<210s, SEQ ID NO 4 O &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: MAPKAP kinase-2 RNAi target sequence <4 OOs, SEQUENCE: 4 O t caagaagaa cqc cat cat 19

<210s, SEQ ID NO 41 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: MAPKAP kinase-2 RNAi target sequence <4 OOs, SEQUENCE: 41 aagcatc.cga aat catgaa 19

<210s, SEQ ID NO 42 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: MAPKAP kinase-2 RNAi target sequence < 4 OO SEQUENCE: 42 agitatctgcattcaat caa 19

<210s, SEQ ID NO 43 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: MAPKAP kinase-2 RNAi target sequence <4 OOs, SEQUENCE: 43 ctittgaccac tocttgtta 19

<210s, SEQ ID NO 44 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: MAPKAP kinase-2 RNAi target sequence <4 OOs, SEQUENCE: 44 tittgaccact c cttgttat 19

<210s, SEQ ID NO 45 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: MAPKAP kinase-2 RNAi target sequence

<4 OOs, SEQUENCE: 45 tacggat.cgt ggatgtgta 19

<210s, SEQ ID NO 46 &211s LENGTH: 19 US 9,023,787 B2 147 148 - Continued

TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: MAPKAP kinase-2 RNAi target sequence <4 OOs, SEQUENCE: 46 ggacggtgga gaact ctitt 19

SEO ID NO 47 LENGTH 19 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: MAPKAP kinase-2 RNAi target sequence <4 OOs, SEQUENCE: 47 cittgttatac accogtact a 19

SEQ ID NO 48 LENGTH 19 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: MAPKAP kinase-2 RNAi target sequence <4 OOs, SEQUENCE: 48 gacggtggag aact ctitta 19

SEQ ID NO 49 LENGTH 19 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: MAPKAP kinase-2 RNAi target sequence <4 OOs, SEQUENCE: 49 ggagaact ct ttagc.cgaa 19

<210s, SEQ ID NO 50 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Substrate Peptide <4 OOs, SEQUENCE: 50 Arg Glin Ile Lys Ile Trp Phe Glin Asn Arg Arg Met Lys Trp Llys Llys 1. 5 1O 15

The invention claimed is: cin, etoposide, floXuridine, fludarabine, 5-fluorouracil, gem 1. A method of sensitizing cells to chemotherapy compris 50 citabine, hexamethylmelamine, hydroxyurea,-ifosfamid, 1ng irinotecan, letrozole, lomustine, mechlorethamine, mel administering to the cells an effective amount of a MAP phalen, mercaptopurine, 6-mercaptopurine, methotrexate, KAP kinase-2 polypeptide inhibitor to sensitize p53 mitomycin, mitoxantrone, , pentostatin, procarba deficient cells to a DNA damaging chemotherapeutic 55 Zine, raltitrexed, Streptozocin, temozolomide, 6-thioguanine, agent, topotecan, toremofine, trastuzumab, , Vincristine, then administering an effective amount of a chemothera vindesine, and vinorelbine. peutic to the sensitized p53-deficient cells to kill the 3. The method of claim 1, further comprising administer cells wherein the chemotherapeutic agent is adminis ing radiation therapy simultaneously or within twenty-eight tered after and within twenty-eight days of administer 60 days of administering the inhibitor. ing the MAPKAP kinase-2 polypeptide inhibitor. 4. The method of claim 1, wherein the p53-deficient cells 2. The method of claim 1, wherein the chemotherapeutic are cancer cells selected from the group consisting of acoustic agent is selected from the group consisting of altretamine.- neuroma, acute leukemia, acute lymphocytic leukemia, acute amsacrine, azacitidine, bleomycin.-buSulfan, capecitabine, monocytic leukemia, acute myeloblastic leukemia, acute , carmustine, chlorambucil, 2-chlorodeoxyad 65 myelocytic leukemia, acute myelomonocytic leukemia, acute enosine, cisplatin, cyclophosphamide, , Cytoxan, promyelocytic leukemia, acute erythroleukemia, adenocarci dacarbazine, daunorubicin, docetaxel, doxorubicin, epirubi noma, angiosarcoma, astrocytoma, basal cell carcinoma, bile US 9,023,787 B2 149 150 duct carcinoma, bladder carcinoma, brain cancer, breast can 6. The method of claim 5, wherein the DNA damage cer, bronchogenic carcinoma, cervical cancer, chondrosar responsive cell cycle checkpoint is G1/S phase arrest. coma, chordoma, choriocarcinoma, chronic leukemia, 7. The method of claim 6, wherein Cdc25a degradation in chronic lymphocytic leukemia, chronic myelocytic leukemia, the p53-deficient cells is impaired. colon cancer, colon carcinoma, craniopharyngioma, cystad 8. The method of claim 5, wherein the DNA damage enocarcinoma, embryonal carcinoma, endotheliosarcoma, responsive cell cycle checkpoint is G2/M phase arrest. ependymoma, epithelial carcinoma, Ewing's tumor, glioma, 9. The method of claim 8, wherein the interaction between heavy chain disease, hemangioblastoma, hepatoma, Cdc25b and a 14-3-3 protein is reduced in comparison to a Hodgkin’s disease, large cell carcinoma, leiomyosarcoma, p53-deficient control cell. liposarcoma, lung cancer, lung carcinoma, lymphangioen 10 dotheliosarcoma, lymphangiosarcoma, macroglobulinemia, 10. The method of claim 1, wherein cell death is by apop medullary carcinoma, medulloblastoma, melanoma, menin tosis. gioma, mesothelioma, myxosarcoma, neuroblastoma, non 11. The method of claim 10, wherein apoptosis is at least Hodgkin’s disease, oligodendroglioma, osteogenic sarcoma, partially dependent on caspase-3 activation. ovarian cancer, pancreatic cancer, papillary adenocarcino 15 12. The method of claim 1, wherein the MAPKAP kinase mas, papillary carcinoma, pinealoma, polycythemia Vera, 2polypeptide inhibitor is selected from the group consisting prostate cancer, rhabdomyosarcoma, renal cell carcinoma, of a nucleic acid, a peptide and a small molecule. retinoblastoma, Schwannoma, sebaceous gland carcinoma, 13. The method of claim 1, wherein the effective amount of seminoma, Small cell lung carcinoma, squamous cell carci a chemotherapeutic to the cells to kill the cells is lower than noma, Sweat gland carcinoma, synovioma, testicular cancer, the effective amount of the chemotherapeutic to kill cells in uterine cancer, Waldenstrom's fibrosarcoma, and Wilm's the absence of the MAPKAP kinase-2 polypeptide inhibitor. tumor. 14. The method of claim 1, wherein the chemotherapeutic 5. The method of claim 1, wherein the inhibitor alters a agent is administered Subsequent to administering the MAP DNA damage-responsive cell cycle checkpoint of the p53 KAP kinase-2 polypeptide inhibitor. deficient cell. k k k k k