USOO844.0627B2

(12) United States Patent (10) Patent No.: US 8.440,627 B2 Kuliopulos et al. (45) Date of Patent: May 14, 2013

(54) GPROTEIN COUPLED RECEPTOR CA 2586344 A1 5, 2006 AGONSTS AND ANTAGONSTS AND ES 101st A2 1392 METHODS OF USE JP 2008519039 T 6, 2008 WO WO 98.00538 1, 1998 (75) Inventors: Athan Kuliopulos, Winchester, MA WO WO 98,34948 8, 1998 (US); Lidija Covic, Lexington, MA WO WO99.437.11 9, 1999 (US); Nicole Kaneider, Innsbruck (AT) WO WO99/62494 12/1999 WO 200181408 A2 11/2001 (73) Assignee: t Medical Center, Inc., Boston, MA W. Wiggs A. 1 558 OTHER PUBLICATIONS (*) Notice: Subject to any disclaimer, the term of this Loetscher et al., Journal of Biological Chemistry, 269:232-237. patent is extended or adjusted under 35 1994.* U.S.C. 154(b) by 0 days. Anand-Srivastava et al., "Cytoplasmic domain of natriuretic peptide receptor-C inhibits adenylyn cyclase'. J. Biol. Chem. (21) Appl. No.: 11/667,042 271 (32):19324-19329 (1996). Andrade-Gordon et al., “Design, Synthesis, and Biological Charac (22) PCT Filed: Nov. 4, 2005 terization of a Peptide-Mimetic Antagonist for a Tethered-Ligand Receptor', Proc. Natl. Acad. Sci. USA, 96(22): 12257-12262 (1999). Aoki et al., “A novel human G-protein-coupled receptor, EDG7, for (86) PCT NO.: PCT/US2005/039959 lysophosphatidic acid with unsaturated fatty-acid moiety'. Annals New York Academy of Sciences. Lysophospholipids and Eicosanoids S371 (c)(1), in Biology and Pathophysiologi, pp. 263-266 (2000). (2), (4) Date: Jan. 15, 2008 Bernatowicz et al., “Development of Potent Thrombin Receptor Antagonist Peptides”, J. Med. Chem., 39:4879-4887 (1996). (87) PCT Pub. No.: WO2O06/052723 Cotecchia et al., “Discrete Amino Acid Sequences of the Ol-Adrenergic Receptor Determine the Selectivity of Coupling to PCT Pub. Date: May 18, 2006 Phosphatidylinositol Hydrolysis”. J. Biol. Chem., 267(3):1633-1639 (1992). (65) Prior Publication Data Coughlin et al., “PARticipation in inflammation”. J. Lin. Invest., US 2008/0214451A1 Sep. 4, 2008 111(1):25-27 (2003). Covic et al., “Intracellular liganding of the PAR1 thrombin receptor by a novel class of cell penetrating peptides'. Blood 96(11): 244a, Related U.S. Application Data Abstract #1050 (2000). Covic et al., “Activation and inhibition of G protein-coupled recep (60) Provisional application No. 60/625,706, filed on Nov. tors by cell-penetrating membrane-tethered peptides'. Proc. Natl. 4, 2004. Acad. Sci. USA, 99(2): 643-648 (2002). Covic et al., “Biphasic Kinetics of Activation and Signaling for PAR1 (51) Int. C. and PAR4 Thrombin Receptors in Platelets”, Biochemistry,39:5458 CI2P 2L/04 (2006.01) 5467 (2000). C07K 4/705 (2006.01) Dalman et al., “Two Peptides from the O-adrenergic receptor alter receptor G protein coupling by distinct mechanisms’. J. Biol. Chem.. A6 IK38/04 (2006.01) 266(17): 11025-11029 (1991). (52) U.S. C. Elliot et al., “Maleimide-Functionalized Lipids that Anchor USPC ...... 514/20.6; 435/69.7:530/402 Polypeptides to Lipid Bilayers and Membranes'. Bioconjugate (58) Field of Classification Search ...... None Chem., 11(6):832-841 (2000). See application file for complete search history. Faruqi et al., “Structure-Function Analysis of Protease-activated Receptor 4 Tethered Ligand Peptides'. J. Biol. Chem. References Cited 275(26): 19728-19734 (2000). (56) George et al., “A Transmembrane Domain-Derived Peptide Inhibits D1 Dopamine Receptor Function without Affecting Receptor U.S. PATENT DOCUMENTS Oligomerization”. J. Biol. Chem., 273(46):30244-30248 (1998). 5,508,384 A * 4/1996 Murphy et al...... 530/324 5,747,267 A 5, 1998 Mulvihill et al. ... 435/721 (Continued) 5,750,370 A * 5/1998 Liet al...... 435/69.1 5,925,549 A 7, 1999 Hsueh et al. .... 435/69.7 Primary Examiner — Marianne P Allen 5,935,936 A * 8/1999 Fasbender et al. 514,44 R 6,096,868 A 8/2000 Halsey et al. ... 530/350 (74) Attorney, Agent, or Firm — McDermott Will & Emery 6,111,075 A 8/2000 Xu et al...... 530/350 LLP 6,111,076 A 8/2000 Fukusumi et al. .. 530/350 6,162,808 A 12/2000 Kindon et al...... 514,269 (57) ABSTRACT 6,548.499 B1 4/2003 Carson 6,864,229 B2 3/2005 Kuliopulos et al...... 514/2 The invention relates generally to G protein coupled receptors 7.304,127 B2 * 12/2007 Saxinger ...... 530/326 (GPCRs) and in particular to GPCRagonists and antagonists, 2003/0148449 A1 8/2003 Kuliopulos et al. . ... 435/69.1 use of these compounds and their pharmaceutical composi 2006,0166274 A1 7/2006 Kuliopulos et al. . ... 435/7.1 2007/0179090 A1 8/2007 Kuliopulos et al. . 514/12 tions, e.g., in the treatment, modulation and/or prevention of 2008/02341.83 A1* 9, 2008 Hallbrink et al...... 514/12 physiological conditions associated with GPCRs, such as in treating conditions in which chemokine receptors play a role, FOREIGN PATENT DOCUMENTS e.g., sepsis, arthritis, inflammation and autoimmune diseases. AU 2005304963 A1 5, 2006 BR PIO5170583. A 9, 2008 37 Claims, 28 Drawing Sheets US 8.440,627 B2 Page 2

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Leger et al., “Protease-Activated Receptors in Cardiovascular Dis eases.” Circulation, 1.14:1070-1077 (2006). * cited by examiner U.S. Patent May 14, 2013 Sheet 1 of 28 US 8.440,627 B2

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FIG. 27 US 8,440,627 B2 1. 2 GPROTEIN COUPLED RECEPTOR development of new GPCR modulators, e.g., agonists, partial AGONSTS AND ANTAGONSTS AND agonists, inverse agonists and antagonists, may have thera METHODS OF USE peutic applications for treating GPCR-related disorders, including sepsis, arthritis, inflammation and autoimmune dis REFERENCE TO RELATED APPLICATIONS 5 CaSCS. This application claims benefit under 35 U.S.C. S371 to SUMMARY OF THE INVENTION PCT Application No. PCT/US2005/039959, filed on Nov. 4, 2005, which claims priority to U.S. Provisional Patent Appli The present invention is based on the discovery of modified cation Ser. No. 60/625,706, filed on Nov. 4, 2004, which is peptides called pepducins which comprise a cell-penetrating incorporated herein by reference. or membrane-tethering moiety to attached to a peptide derived from the first intracellular loop structure of a GPCR. STATEMENT AS TO FEDERALLY SPONSORED Pepducins may be considered chimeric peptides/polypep RESEARCH tides, and are agonists and/or antagonists of receptor-G pro 15 tein signaling. These compositions exhibit selectivity for This invention was made with Government support under their cognate receptor. Grant Numbers R01 HL064701, R01 HL057905 and RO1 Accordingly, the invention provides a pepducin composi CA104406 awarded by the National Institutes of Health. The tion, a chimeric polypeptide including a first domain of a first Government has certain rights in the invention. intracellular loop (i1 loop) or a fragment thereof of a GPCR and a second domain, attached to the first domain. The second FIELD OF THE INVENTION domain is a naturally or non-naturally occurring cell-pen etrating and/or membrane-tethering hydrophobic moiety. The invention relates generally to G protein coupled recep The first domain preferably does not include a native extra tors (GPCRs) and in particular to GPCRagonists and antago cellular portion of the GPCR. The pepducins of the invention nists, use of these compounds and their pharmaceutical com 25 desirably bind to the cognate GPCR from which the first positions, e.g., in the treatment, modulation and/or prevention domain is derived. of physiological conditions associated with GPCRS, such as The first domain (the first intracellular loop (i1 loop) or a in treating conditions in which chemokine receptors play a fragment thereof of a G protein coupled receptor (GPCR)) role, e.g., sepsis, arthritis, inflammation and autoimmune dis includes an amino acid sequence of a luteinizing hormone CaSCS. 30 receptor, a follicle stimulating hormone receptor, a thyroid stimulating hormone receptor, a calcitonin receptor, a gluca BACKGROUND OF THE INVENTION gon receptor; a glucagon-like peptide 1 receptor (GLP-1); a metabotropic glutamate receptor; a parathyroid hormone A variety of hormones, neurotraismitters and biologically receptor, a vasoactive intestinal peptide receptor, a secretin active Substances control, regulate, or adjust the functions of 35 receptor, a growth hormone releasing factor (GRF) receptor; living bodies via specific receptors located in cell mem protease-activated receptors (PARS); cholecystokinin recep branes. Many of these receptors mediate the transmission of tors; somatostatin receptors; melanocortin receptors; ADP intracellular signals by: activating guanine nucleotide-bind receptors; adenosine receptors; thromboxane receptors; ing proteins (G proteins) to which the receptor is coupled. platelet activating factor receptor, adrenergic receptors; 5-HT Such receptors are generically referred to as G protein 40 receptors; chemokine receptors; neuropeptide receptors; coupled receptors (“GPCRs). Binding of a specific signaling opioid receptors; parathyroid hormone (PTH) receptor; or a molecule to the GPCR can cause a conformational change in vasoactive intestinal peptide (VIP) receptor. the receptor, resulting in a form that is able to bind and For example, the first domain (the first intracellular loop (il activate a G protein, thereby triggering a cascade of intracel loop) or a fragment thereof of a G protein coupled receptor lular events that eventually leads to a biological response. 45 (GPCR)) contains an amino acid sequence of a protease Typically, GPCRs interact with G proteins to regulate the activated receptor (PAR) or a chemokine receptor. The pro synthesis of intracellular second messengers such as cyclic tease-activated receptor may be, e.g., PAR1, PAR2, PAR3, or AMP. inositol phosphates, diacylglycerol and calcium ions. PAR4. A chemokine receptor may be a CC or CXC receptor Chemokines are leukocyte attractants and contribute to such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, immune processes that involve leukocyte migration. Leuko 50 CCR8 or CCR9; or CXCR1, CXCR2, CXCR3, CXCR4, cyte trafficking is highly coordinated, and a breakdown of the CXCR5, CXCR6 or CX3CR1; respectively. In another underlying control mechanisms might contribute to exagger embodiment the first domain (the first intracellular loop (i1 ated innate immune activations, such as Systemic inflamma loop) or a fragment thereof of a G protein coupled receptor tory response syndromes or autoimmune diseases. Chemok (GPCR)) may be, e.g., from cholecystokinins A and B ine-induced signaling is mediated by GPCRs, and by 55 (CCKA, CCKB); somatostatin-2 (SSTR2); melanocortin-4 definition their hallmark is leukocyte chemoattraction. In (MC4R); glucagon-like peptide-1 receptor (GLP-1R); P2Y addition, chemokines induce cellular responses that are unre ADP receptor; or from "atypical chemokine receptors such lated to leukocyte migration, like cell Survival, virus-host as NK1, NK2, GRP/bombesin receptors, FPR1, FPRL-1, interactions, tumor growth and metastasis, organogenesis, C3aR or C5aR. In a particular embodiment, pepducins of the and angiogenesis. 60 invention include those for PAR2, CXCR1, CXCR2, CXCR4 GPCRs play a vital role in the signaling processes that and CCR5 chemokine receptors. control cellular metabolism, cell growth and motility, adhe The second domain (the cell-penetrating and/or mem Sion, inflammation, neuronal signaling, and blood coagula brane-tethering hydrophobic moiety) is attached at the N-ter tion. GPCR proteins also have a very important role as targets minal end, the C-terminal end, an amino acid between the for a variety of signaling molecules which control, regulate, 65 C-terminal amino acid and the N-terminal amino acid, or both or adjust the functions of living bodies. GPCRs are involved the N-terminal and C-terminal ends of the first domain. Desir in a wide variety of disorders, as is well-known in the art. The ably, the cell-penetrating and/or membrane-tethering hydro US 8,440,627 B2 3 4 phobic moiety is a lipid such as a straight chain fatty acid, e.g., administered to a subject in need thereof. Suitable inflamma nonanoyl (C); capryl (C); undecanoyl (C); lauroyl (C); tory disorders for treatment may include Chronic Obstructive tridecanoyl (C); myristoyl (C); pentadecanoyl (Cs); Pulmonary Disease (COPD), alkylosing spondylitis, cervical palmitoyl (C); phytanoyl (methyl Substituted Co.); hepta arthritis, fibromyalgia, ischemia reperfusion injury, gut decanoyl (C7); Stearoyl (Cs); nonadecanoyl (Co.); arachi ischemia, juvenile rheumatoid arthritis, lumbosacral arthritis doyl (Co.); heniecosanoyl (C); behenoyl (C); trucisanoyl osteoarthritis, osteoporosis, psoriatic arthritis, rheumatic dis (C); and a lignoceroyl (C) moiety. The cell-penetrating ease, rheumatoid arthritis, eczema, psoriasis, dermatitis, and/or membrane-tethering hydrophobic moiety may be uveitis and conjunctivitis, asthma and bronchitis, ulcers, gin attached to the chimeric polypeptide with, e.g., amide bonds, givitis, Crohn's disease, atrophic gastritis, gastritis varialo Sulfhydryls, amines, alcohols, phenolic groups, or carbon 10 forme, ulcerative colitis, celiac disease, regionalileitis, peptic carbon bonds. Particular embodiments include palmitoyl or ulceration, pyresis, bladder irritation and cystitis, inflamma lithocholic acid (or salts thereof) as the hydrophobic moiety. tory neurological disorders of the central or peripheral ner Other cell-penetrating and/or membrane-tethering hydropho Vous system, multiple Sclerosis, inflammatory neuropathies bic moieties include cholesterol, phospholipids, steroids, and neurological complication of AIDS, autoimmune inflam sphingosine, ceramide, octyl-glycine, 2-cyclohexylalanine, 15 mation, or Surgical trauma. The compositions described benzoylphenylalanine, C or C acyl groups, or C-Cs fatty herein prevent, reverse or reduce the severity of sepsis and acids. associated pathologies such as disseminated intravascular The invention further relates to pharmaceutical composi coagulation (DIC), fibrinolysis, and/or systemic inflamma tions comprising the pepducin compositions of the invention tory responses (SIRS). and a pharmaceutically acceptable carrier, and to kits includ The pepducin compositions of the invention are useful to ing, in one or more containers, these pharmaceutical compo activate or inhibit the activity of a broad range of GPCRs. sitions. Pepducins in accordance with the invention include those that The invention includes methods of treating, reducing the act on chemokine CXC receptors, including CXCR1, severity of, or preventing sepsis, e.g., in a mammalian Subject CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 and CX3CR1; by administering a pepducin including a first domain of a first 25 chemokine CC receptors, including CCR1, CCR2, CCR3, intracellular loop (i1 loop) or a fragment thereof of a G CCR4, CCR5, CCR6, CCR7, CCR8 and CCR9; protease protein coupled receptor (GPCR) and a second domain, activated receptors (PARs), e.g., PAR1, PAR2, PAR4; chole attached to the first domain. The second domain is a naturally cystokinins A and B receptors (CCKA, CCKB), somatosta or non-naturally occurring cell-penetrating, membrane-teth tin-2 (SSTR2) receptor, melanocortin-4 (MC4R) receptor, ering hydrophobic moiety. The Subject has been diagnosed 30 glucagon-like peptide-1 receptor (GLP-1R), Sphingosine with or is at risk of developing sepsis. 1-phosphate (S1P) receptors, e.g., subtypes S1P1 and S1P3, The compositions are also used to treat, reduce the severity EDG receptors, endothelin (ET) receptors, e.g., subtypes of or prevent inflammation and/orangiogenesis. Methods of ET-1, ET-2, ET-3, ETA, ETB, EDG receptors, e.g., subtypes treating or preventing inflammation and/orangiogenesis are EDG-1, EDG-2, EDG-3, EDG-4, EDG-5, EDG-6, and P2Y carried out by administering a chemokine-inhibiting pepdu 35 ADP receptor. Also, pepducins for "atypical chemokine cin including a chimeric polypeptide comprising a first receptors such as NK1, NK2, GRP/bombesin receptors, domain of a first intracellular loop (i1 loop) or a fragment FPR1, FPRL-1, C3aR and C5aR are within the scope of the thereof of a G protein coupled receptor (GPCR) and a second invention. In a particular embodiment, pepducins of the domain, attached to the first domain, in which the second invention include those for PAR2, CXCR1, CXCR2, CXCR4 domain is a naturally or non-naturally occurring cell-pen 40 and CCR5 chemokine receptors. etrating, membrane-tethering hydrophobic moiety. Administration is preferably carried out systemically Such The compositions are also used to treat or reduce the sever as intravenously, e.g., in cases of systemic inflammation, ity of cancer. Methods of treating or reducing the severity of COPD and/or sepsis. Alternatively, the compositions are cancer are carried out by administering a pepducin including delivered Subcutaneously, orally, intranasally (e.g., to treat a chimeric polypeptide comprising a first domain of a first 45 asthma) or locally, e.g., in the form of an adhesive patch, or a intracellular loop (i1 loop) or a fragment thereof of a G cream, foam, ointment (e.g., for the alleviation of symptoms protein coupled receptor (GPCR) and a second domain, of dermatitis, psoriasis or other dermal inflammatory condi attached to the first domain, in which the second domain is a tions. naturally or non-naturally occurring cell-penetrating, mem brane-tethering hydrophobic moiety. 50 BRIEF DESCRIPTION OF THE DRAWINGS The compositions are also used to treat or reduce the sever ity of thrombosis, e.g., coronary, arterial and venous (such as FIG. 1 is a diagram showing GPCR topology. The il loop deep vein or mesenteric) thrombosis. Methods of treating or is illustrated in this figure. preventing thrombosis are carried out by administering a FIG. 2 is a diagram showing CXCR1 and CXCR2 i1 loop pepducin including a chimeric polypeptide comprising a first 55 sequence including transmembrane flanking portions, which domain of a first intracellular loop (i1 loop) or a fragment are aligned to illustrate the similarity in structure. thereof of a G protein coupled receptor (GPCR) and a second FIG.3 is a chart showing chemokine receptors which pep domain, attached to the first domain, in which the second ducins in accordance with the invention may act on, their domain is a naturally or non-naturally occurring cell-pen respective ligands, and the respective cell types in which the etrating, membrane-tethering hydrophobic moiety. 60 receptors may be found. The invention also relates to methods of treating or pre FIG. 4 is a chart showing additional chemokine receptors venting an inflammatory disorder, wherein pepducin com which pepducins inaccordance with the invention may acton, prising a first domain of a first intracellular loop (i1 loop) or their respective ligands, and the respective cell types in which a fragment thereof of a G protein coupled-receptor (GPCR) the receptors may be found. and a second domain, attached to the first domain, wherein the 65 FIG. 5 is a line graph showing the animal response to second domain is a naturally or non-naturally occurring cell pepducintherapy at the time of CLP in an experiment detailed penetrating, membrane-tethering hydrophobic moiety, is in Example 1. US 8,440,627 B2 5 6 FIG. 6 is a line graph showing the results of an experiment addition of 3 nM thrombin (T). The data illustrated in these detailed in Example 2, showing the results of pepducin figures are the results of an experiment as described in detail therapy initiated eight hours after CLP in Example 12. FIGS. 7A-C depict the inhibition of leukocyte infiltration FIG. 25A is a graph illustrating a combination of bivaliru into lung; and TNF-C. production and fibrin deposition in lung 5 din and P4pal-il blocking thrombin-dependent aggregation; after pepducin treatment, as detailed further in Example 3. human platelets were pre-incubated for 2 min with buffer FIGS. 7A and 7B are bar graphs and FIG. 7C are photomi (untreated), bivalirudin (200 nM), RWJ-56110 (1 uM), and/or crographs. PAR1-Ab (74 g/mL) as indicated prior to the addition of 20 FIGS. 8A-D are bar graphs showing the shortening of pM-20 nM thrombin; FIG.25B is a graph illustrating platelets bleeding time in Septic mice, after pepducin treatment. 10 pre-incubated for 2 min with 200 nM bivalirudin plus or FIG. 9 is a graph showing how human neutrophil chemo minus 5 uM P4pal-i1 prior to the addition of the indicated taxis is inhibited by pepducin treatment, as shown in more concentrations of thrombin. The data illustrated in these fig detail in Example 5. ures are the results of an experiment as described in detail in FIG. 10 is a graph showing how leukocyte chemotaxis is 15 Example 13. inhibited by in vivo pepducin treatment, as shown in more FIG. 26 is a bar graph illustrating a combination of biva detail in Example 6. lirudin plus P4pal-il blocking occlusion of carotid arteries in FIG.11 is a line graph showing the results of an experiment guinea pigs. Guinea pigs were treated with bivalirudin, P4pal as described in more detail in Example 7. i1 and/or P1pal-7 (n=3-5 for each treatment group) 5 min FIG. 12 is a graph showing how pepducins of the invention prior to injury of the carotid artery with FeC1 as described in selectively reduce inflammation by demonstrating an inhibi Example 14. P values relative to vehicle-treated are indicated tion in human neutrophil chemotaxis, as shown in more detail at the bottom. in Example 8. FIG.27 is a graph illustrating how il-loop pepducins block FIG. 13 is a graph showing how pepducins of the invention chemotactic migration of breast and ovarian cancer cells. selectively reduce inflammation by demonstrating an inhibi 25 Conditioned media was prepared from NIH3T3. Chemotaxis tion in human monocyte chemotaxis, as shown in more detail assays (20 h) were performed using a 48 blindwell micro in Example 9. chemotaxis chamber (Neuroprobe) equipped with 8 mm pore FIG. 14 is a diagram showing CXCR4 ill-loop sequences nitrocellulose filters for OVCAR-4 human ovarian cancer including transmembrane flanking portions, which may be cells and MDA-MB-231 human breast cancer cells. Data is used in pepducins according to the invention. 30 FIG. 15 is a diagram showing CCR1 ill-loop sequences expressed as chemotaxis index which is the ratio between the including transmembrane flanking portions, which may be distance of migration toward NIH3T3 conditioned media used in pepducins according to the invention. over migration toward RPMI medium alone. FIG. 16 is a diagram showing CCR2 ill-loop sequences DETAILED DESCRIPTION OF THE INVENTION including transmembrane flanking portions, which may be 35 used in pepducins according to the invention. FIG. 17 is a diagram showing CCR4 ill-loop sequences Definitions including transmembrane flanking portions, which may be For convenience, certain terms used in the specification, used in pepducins according to the invention. examples, and appended claims are collected here. FIG. 18 is a diagram showing CCR5 ill-loop sequences 40 “Treating, includes any effect, e.g., lessening, reducing, including transmembrane flanking portions, which may be modulating, or eliminating, that results in the improvement of used in pepducins according to the invention. the condition, disease, disorder, etc. or symptom thereof. FIG. 19 is a diagram showing PAR1 ill-loop sequences “GPCR fragment includes peptides having a portion of including transmembrane flanking portions, which may be the sequence of a GPCR protein which is less than the entire used in pepducins according to the invention. 45 naturally-occurring amino acid sequence of the GPCR. “Iso FIG. 20 is a diagram showing EDG ill-loop sequences lated GPCR fragment includes peptides having a portion of including transmembrane flanking portions, which may be the GPCR protein sequence which is less than the entire used in pepducins according to the invention. sequence, and not containing the naturally-occurring flanking FIG. 21 is a diagram showing NK1-R ill-loop sequences regions. Isolated GPCR fragments lack one or more amino including transmembrane flanking portions, which may be 50 acids which immediately flank the reference fragment in the used in pepducins according to the invention. naturally-occurring molecule. FIG.22 is a bar graph showing the results of an experiment “Isolated intracellular GPCR fragment' includes peptides as described in more detail in Example 10. having an amino acid sequence of the intracellular i1 loop of FIG. 23 is a graph showing the results of an experiment as a GPCR protein, and not containing a sequence from an described in more detail in Example 11. 55 extracellular loop or a transmembrane helix sequence flank FIG. 24A is a bar graph showing HEK cells, transiently ing the intracellular i1 loop. “Isolated extracellular GPCR transfected with PAR1 and/or PAR4, allowed to migrate for fragment includes peptides having an amino acid sequence 24 h toward 0.5 M TFLLRN (SEQID NO. 33) or 500 uM of an extracellular loop of a GPCR protein and not containing AYPGKF (SEQ.IDNO. 34) in the presence or absence of 300 an amino acid of an intracellular loop or transmembrane nMP4pal-il; FIG. 24B is a graph showing human platelets 60 sequence flanking regions of the extracellular loop. pre-incubated for 2 min with 3 uMP4pal-il or buffer (un "Linked' means attached. For example, a peptide and a treated) prior to the addition of 160 uMAYPGKF: FIG.24C cell-penetrating or membrane-tethering moiety are attached is a graph showing human platelets pre-incubated for 2 min to each other in a pepducin via a linkage, i.e., a covalent bond. with 3 uMP4pal-il or buffer (untreated) prior to the addition Preferably, the linkage is a labile bond such as a thiol or ester of 13 uMSFLLRN; and FIG.24Disagraph showing platelets 65 linkage. Pepducin compounds having a labile linkage are pre-incubated for 2 min with 1 uM RWJ-561 10, 5uMP4pal advantageous since accumulation in body tissues is lower i1, or 1 uM RWJ-56110 plus 5 uM P4pal-i1 prior to the compared to compounds with a non-labile linkage. Reduced US 8,440,627 B2 7 8 accumulation in bodily tissues following administration to a contemplated. Supplementary active ingredients can also be subject is associated with decreased adverse side effects in the incorporated into the compositions. Subject. “Small molecules include compositions having a molecu A “GPCR agonist' includes compositions that activate a lar weight of less than about 5 kD and most preferably less GPCR to mimic the action of the endogenous signaling mol than about 4 kD. Small molecules can be nucleic acids, pep ecule specific to that receptor. A “GPCR antagonist' includes tides, polypeptides, peptidomimetics, carbohydrates, lipids compositions that inhibit GPCR activity. GPCR activity is or other organic or inorganic molecules. Libraries of chemi measured by ability to bind to an effector signaling molecule cal and/or biological mixtures, such as fungal, bacterial, or such as G-protein. An “activated GPCR is one which is algal extracts, are known in the art and can be screened with capable of interacting with and activating a G-protein. An 10 any of the assays of the invention. “Target molecules' include molecules with which a GPCR inhibited receptor has a reduced ability to bind extracellular protein binds or interacts in nature, for example, a molecule ligandand/or productively interact with, and activate a G-pro on the surface of a cell which expresses a GPCR interacting tein. protein; a molecule on the Surface of a second cell; a molecule "Cell-penetrating moieties' include compounds or func 15 in the extracellular milieu, a molecule associated with the tional groups which mediate transfer of a Substance from an internal Surface of a cell membrane; or a cytoplasmic mol extracellular space to an intracellular compartment of a cell. ecule. A GPCR target molecule can be a non-GPCR molecule Cell-penetrating moieties shuttle a linked Substance (e.g., a or a GPCR peptide of the invention. In one embodiment, a GPCR peptide or fragment of the invention) into the cyto GPCR target molecule is a component of a signal transduc plasm or to the cytoplasmic space of the cell membrane. For tion pathway that facilitates transduction of an extracellular example, a cell penetrating moiety is a hydrophobic moiety. signal. Such as a signal generated by binding of a compound The hydrophobic moiety is, e.g., a mixed sequence peptide or to a membrane-bound GPCR, through the cell membrane and a homopolymer peptide Such as polyleucine or polyarginine into the cell. The target can be a second intercellular protein at least about 11 amino acids long. The Substance may be a that has catalytic activity or a protein that facilitates the asso peptide such as a GPCR fragment or peptidomimetic of the 25 ciation of downstream signaling molecules with GPCR. invention. The cell penetrating moiety may include at least 10 “Combination therapy' (or “co-therapy') includes the contiguous amino acids, e.g., 1-15 amino acids of a GPCR administration of a pepducin of the invention and at least a transmembrane helix domain. second agent as part of a specific treatment regimen intended “Membrane-tethering moieties' include compounds or to provide the beneficial effect from the co-action of these functional groups which associate with orbind to a cell mem 30 therapeutic agents. The beneficial effect of the combination brane. Thus, the membrane-tethering moiety brings the Sub includes, but is not limited to, pharmacokinetic or pharmaco stance to which the membrane-tethering moiety is attached dynamic co-action resulting from the combination of thera (i.e., the GPCR fragment or peptidomimetic of the invention) peutic agents. Administration of these therapeutic agents in in close proximity to the membrane of a target cell. The cell combination typically is carried out over a defined time membrane is eukaryotic or prokaryotic. The membrane-teth 35 period (usually minutes, hours, days or weeks depending ering moiety is desirably a hydrophobic moiety. The hydro upon the combination selected). “Combination therapy” may, phobic moiety can include a mixed sequence peptide or a but generally is not, intended to encompass the administration homopolymer peptide Such as polyleucine or polyarginine of two or more of these therapeutic agents as part of separate less than 10 amino acids long. The membrane-tethering moi monotherapy regimens that incidentally and arbitrarily result ety can include at least one to seven contiguous amino acids 40 in the combinations of the present invention. “Combination of a GPCR transmembrane helix domain. Preferably, the therapy” is intended to embrace administration of these thera membrane-tethering moiety is at least 10 contiguous amino peutic agents in a sequential manner, that is, wherein each acids (but less than 16 amino acids) of a GPCR transmem therapeutic agent is administered at a different time, as well as brane domain; more preferably, the membrane-tethering moi administration of these therapeutic agents, or at least two of ety is at least 15 contiguous amino acids of a GPCR trans 45 the therapeutic agents, in a substantially simultaneous man membrane domain. Membrane-tethering moieties also ner. Substantially simultaneous administration can be accom include cholesterol, phospholipids, steroids, sphingosine, plished, for example, by administering to the Subject a single ceramide, octyl-glycine, 2-cyclohexylalanine, or ben capsule having a fixed ratio of each therapeutic agent or in Zolylphenylalanine. Other membrane-tethering moieties multiple, single capsules for each of the therapeutic agents. include C or C acyl groups, or a C-Cs fatty acid moiety 50 Sequential or Substantially simultaneous administration of Such as propionoyl (C); butanoyl (C); pentanoyl (Cs); each therapeutic agent can be effected by any appropriate caproyl (C); heptanoyl (C7); and capryloyl (C). The mem route including, but not limited to, oral routes, intravenous brane-tethering moiety may be attached to the C-terminal routes, intramuscular routes, and direct absorption through amino acid, the N-terminal amino acid, or to an amino acid mucous membrane tissues. The therapeutic agents can be between the N-terminal and C-terminal amino acid of the 55 administered by the same route or by different routes. For GPCR fragment in the pepducin. example, a first therapeutic agent of the combination selected “Pharmaceutically or pharmacologically acceptable' may be administered by intravenous injection while the other include molecular entities and compositions that do not pro therapeutic agents of the combination may be administered duce an adverse, allergic or other untoward reaction when orally. Alternatively, for example, all therapeutic agents may administered to an animal, or a human, as appropriate. "Phar 60 be administered orally or all therapeutic agents may be maceutically acceptable carrier includes any and all sol administered by intravenous injection. The sequence in vents, dispersion media, coatings, antibacterial and antifun which the therapeutic agents are administered is not narrowly gal agents, isotonic and absorption delaying agents and the critical. “Combination therapy also can embrace the admin like. The use of Such media and agents for pharmaceutical istration of the therapeutic agents as described above in fur active Substances is well known in the art. Except insofar as 65 ther combination with other biologically active ingredients any conventional media or agent is incompatible with the and non-drug therapies (e.g., Surgery or radiation treatment.) active ingredient, its use in the therapeutic compositions is Where the combination therapy further comprises a non-drug US 8,440,627 B2 9 10 treatment, the non-drug treatment may be conducted at any any other non-naturally-occurring variants that are analogous Suitable time so long as a beneficial effect from the co-action in terms of function, e.g., peptidomimetics. of the combination of the therapeutic agents and non-drug This application is related to copending U.S. patent appli treatment is achieved. For example, in appropriate cases, the cation Ser. No. 10/251,703, entitled “G Protein Coupled beneficial effect is still achieved when the non-drug treatment Receptor Agonists And Antagonists And Methods Of Acti is temporally removed from the administration of the thera vating And Inhibiting G Protein Coupled Receptors. Using peutic agents, perhaps by days or even weeks. The Same the entire contents of this patent application is By “homologous amino acid sequence' is meant an amino incorporated herein by reference. acid sequence, i.e., in a pepducin of the invention, that differs The present invention is based on the discovery of modified 10 peptides called pepducins which comprise a cell-penetrating from a reference amino acid sequence, only by one or more or membrane-tethering moiety to attached to a peptide (e.g., 1, 2, 3, 4 or 5) conservative amino acid Substitutions, or derived from a GPCR i1 loop. Pepducins may be considered by one or more (e.g., 1, 2, 3, 4 or 5) non-conservative amino chimeric peptides/polypeptides, and are agonists and/or acid Substitutions, deletions, or additions located at positions antagonists of receptor-G protein signaling and exhibit selec at which they do not adversely affect the activity of the 15 tivity for their cognate receptor. polypeptide. Preferably, such a sequence is at least 75%, 80%, The pepducins of the invention include a GPCR moiety 85%, 90%, or 95% identical to a reference amino acid derived from the first intracellular loop (i1) of a GPCR, or a Sequence. fragment thereof, and a cell penetrating or membrane-tether Homologous amino acid sequences include peptide ing moiety which partitions the conjugate into and across the sequences that are identical or Substantially identical to a lipid bilayer of target cells; and methods for their use in reference amino acid sequence. By “amino acid sequence treatment of GPCR-mediated conditions. The cell penetrat Substantially identical' is meant a sequence that is at least ing moiety is, e.g., a hydrophobic region of the GPCR frag 90%, preferably 95%, more preferably 97%, and most pref. ment itself. The cell penetrating or membrane-tethering moi erably 99% identical to an amino acid sequence of reference ety anchors the conjugate in the lipid bilayer (or to the cell and that preferably differs from the sequence of reference, if 25 Surface), increasing the effective molarity of the conjugate in at all, by a majority of conservative amino acid Substitutions. the vicinity of the intracellular receptor, e.g., at the receptor-G Conservative amino acid Substitutions typically include protein interface. The exogenous GPCR moiety of the pep Substitutions among amino acids of the same class. These ducin disrupts receptor-G protein interactions and causes sig classes include, for example, (a) amino acids having naling activation and/or inhibition (i.e., agonistic or antago uncharged polar side chains, such as asparagine, glutamine, 30 nistic activity). serine, threonine, and tyrosine; (b) amino acids having basic Pepducins act as receptor-modulating agents by targeting side chains. Such as lysine, arginine, and histidine; (c) amino the intracellular surface of the receptor. For example, pep acids having acidic side chains, such as aspartic acid and ducins of the present invention include PAR1- and PAR4 glutamic acid; and (d) amino acids having nonpolar side based antagonists for anti-hemostatic and anti-thrombotic chains, such as glycine, alanine, Valine, leucine, isoleucine, 35 effects under in vivo conditions. Because thrombin is the proline, phenylalanine, methionine, tryptophan, and cysteine. most potent activator of platelets, PAR1 (Vu et al. Cell 64. Homology is typically measured using sequence analysis 1057 (1991)) and PAR4 (Xu et al. Proc. Natl. Acad. Sci. Software (e.g., Sequence Analysis Software Package of the (USA) 95, 6642 (1998); Covic et al. Biochemistry 39, 5458 Genetics Computer Group, University of Wisconsin Biotech (2000); and Covic et al. Thromb. Haemost. 87, 722 (2002)) nology Center, 1710 University Avenue, Madison, Wis. 40 were chosen as targets. Antagonists of these two receptors are 53705). Similar amino acid sequences are aligned to obtain useful to prevent the thrombotic and proliferative complica the maximum degree of homology (i.e., identity). To this end, tions of acute coronary syndromes, including sepsis. it may be necessary to artificially introduce gaps into the Ischemia reperfusion injury occurs when the blood flow is sequence. Once the optimal alignment has been set up, the restored after an extended period of ischemia. It is a common degree of homology (i.e., identity) is established by recording 45 Source of morbidity and mortality in conditions such as myo all of the positions in which the amino acids of both sequences cardium infarction, stroke, gut ischemia, and cardiopulmo are identical, relative to the total number of positions. nary bypass; for which there is often no specific therapy. Similarity factors include similar size, shape and electrical Pepducins of the invention are administered intravenously to charge. One particularly preferred method of determining provide significant protection against experimental reperfu amino acid similarities is the PAM25O matrix described in 50 sion injury of heart muscle. Dayhoff et al., 5 ATLAS OF PROTEIN SEQUENCE AND STRUCTURE Myocardial infarction prompted by the limited supply of 345-352 (1978& Suppl.), incorporated by reference herein. A cardiac muscle with arterial blood, is a leading cause of heart similarity score is first calculated as the Sum of the aligned failure and death. The emergent restoration of normal circu pairwise amino acid similarity scores. Insertions and dele lation is, therefore, critical for the prevention of the irrevers tions are ignored for the purposes of percent homology and 55 ible damage of cardiac tissues from hypoxia. The re-estab identity. Accordingly, gap penalties are not used in this cal lishment of the normal blood flow can occur naturally or culation. The raw score is then normalized by dividing it by artificially after treatment of myocardial infarction (MI) the geometric mean of the scores of the candidate compound patients with specific drugs. In many cases, however, reper and the reference sequence. The geometric meanis the square fusion of the infarcted area initiates the inflammatory root of the product of these scores. The normalized raw score 60 response that facilitates the damage of cardiac cells and is the percent homology. thereby attenuates the positive effect of restored circulation. It Preferably, a homologous sequence is one that is at least was demonstrated that white blood cells recruited into the 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or hypoxic areas of reperfused tissues significantly contribute to 95% identical to the reference amino acid sequence. Polypep this pathological inflammatory response. tides having a sequence homologous to one of the sequences 65 Organ transplantation is now common with over 22,000 shown in this specification, e.g., Table 3, include naturally procedures performed in the US in 2001; the most commonly occurring allelic variants, as well as mutants and variants or transplanted organs being the kidney, liver and heart. The US 8,440,627 B2 11 12 large numbers of transplants now being conducted is due in The mechanism accommodates the biphasic activation and part to the ability of immunomodulatory agents to control inhibition of the agonists and the inhibition of the antagonists. acute rejection. Delayed graft function, caused by ischemia Pepducins, by virtue of their hydrophobic tether, rapidly reperfusion injury, and host V. graft reaction are the principal transduce the plasma membrane and achieve high effective mechanisms of acute rejection after transplantation. The use molarity at the perimembranous interface. The pepducinago of immunosuppressants has successfully dealt with acute nist first occupies a high-affinity site at the intracellular Sur rejection and the allograft commonly survives for prolonged face of the GPCR. The bound agonist either stabilizes or periods, even though immunosuppressive drug dosages are induces the activated state of the receptor to turn on the reduced to very low levels. associated G protein(s). After this first site becomes saturated, However, immunosuppressants Suppress all immunologic 10 higher concentrations of pepducin begin to occupy a second, reactions, making overwhelming infection the leading cause lower-affinity, inhibitory site that blocks signal transference of death in transplant recipients. Immunosuppressants are to G protein in a dominant manner, perhaps by mimicking the also associated with severe toxicity. The most significant GPCR (e.g., the receptor ill-loop) ground-state interactions complications of drugs used for transplant patients include with the G protein. The inhibition by the pepducinantagonists nephrotoxicity, neurotoxicity, new-onset post-transplant dia 15 is coincident with the inhibitory phase of the agonists, thus betes mellitus, hyperlipidemia, and hypertension. These side the antagonists may also bind at this lower affinity site. Exog effects occur in part because of the ubiquitous expression of enous activation or inhibition of receptors by pepducins could the molecular targets of currently used immunosuppressants. reflect a potential dimerization mode whereby one receptor Therapeutic strategies including pepducins of the present donates its intracellular loops to an adjacent receptor. There invention are able to specifically target ischemia-reperfusion are several examples of receptor dimers that give rise to injury and the Subsequent infiltration of mononuclear cells, a distinct signaling properties (Milligan, Science 288, 65-67 primary cause of acute rejection. (2000)), including the cytokine/GPCRs such as the EPO Severe sepsis is a leading cause of acute hospital admis receptor (Guillard et al., J. Biol. Chem. (2001) 276, 2007 sions and often complicates the clinical course of patients 2013), however, the mechanism(s) of cross-receptor modula treated for other diseases. At the onset of sepsis, the presence 25 tion is unknown. of bacteria and bacterial products Such as endotoxin stimu A naturally-occurring GPCR is a cell surface molecule that lates immune defense mechanisms. Failure to remove the crosses a cell membrane at least once. For example, many invading pathogens initiates hyperactive inflammatory naturally-occurring GPCRs cross a cell membrane seven responses termed systemic inflammatory response syndrome times and contain several intracellular domains. The isolated (SIRS), which is mediated by cytokines and chemokines. 30 GPCR fragment of the invention includes the first intracellu Chemokines are viewed as therapeutic targets in inflamma lar loop domain of a GPCR. The intracellular portion is tion. CXCR1 and CXCR2 are important chemokine receptors selected from the first intracellular loop domain of a one responsible for the activation of neutrophils, endothelium, transmembrane domain G-protein coupled receptor of a epithelium, macrophages and other cells. Mice rendered defi cytokine GPCR, or a fragment thereof, or the first intracellu cient in IL-8 signaling by genetic deletion of their sole IL-8 35 lar loop domain of a multi-polypeptide-GPCR, such as a receptor, CXCR2, protects mice from developing sepsis. GPIb/V/IX receptor or a collagen receptor. In humans, exposure to high systemic IL-8 levels leads to The invention also includes soluble pepducins wherein the endothelial dysfunction and loss of the normal anti-coagulant second domain (cell-penetrating- or membrane-tethering state of endothelium. Disorders of coagulation occur in moiety) optionally includes a naturally occurring contiguous 30-50% of septic patients and the development of overt dis 40 amino acid from a transmembrane domain adjacent to the seminated intravascular coagulation (DIC) is an ominous extracellular or intracellular fragment. For example, the con prognostic sign. Opposing the action of CXCR1/2, CXCR4 struct may contain at least 3, but less than 16 contiguous directs the removal of senescent neutrophils and retains amino acids of a GPCR transmembrane helix domain. The immature leukocytes in the bone marrow. CXCR4 also plays transmembrane domain is not transmembrane domain 1-7 of a prominent role in the homing mechanisms of lymphocytes 45 the CXCR4, transmembrane domain 1-7 of CCKA receptor, and cancer cells, but the function of CXCR4 and its ligand or transmembrane 2 of the CCR5 receptor. In an embodiment, SDF-1C. in acute inflammation remains enigmatic. the second domain includes 1-15 contiguous amino acids of a Apart from administration of antibiotics, the treatment of naturally-occurring transmembrane helix domain immedi sepsis and septic shock is largely limited to Supportive strat ately adjacent to the extracellular or intracellular fragment. egies. Presently no specific therapeutic interventions directed 50 In addition to peptide-based pepducins, the invention against chemokine receptors are available for the treatment of encompasses compositions in which the GPCR fragment sepsis, though Small molecule inhibitors directed against contains a peptidomimetic. For example, the invention CXCR1/2 can protect against reperfusion injury and lung includes pepducin compounds in which one or more peptide damage. Pepducins of the invention can be used to interrupt bonds have been replaced with an alternative type of covalent established systemic inflammation and vascular damage as 55 bond, which is not susceptible to cleavage by peptidases (a well prevent activation of the coagulation cascade without "peptide mimetic' or "peptidomimetic'). Where proteolytic interference with host defense. degradation of peptides following injection into the Subject is The Human PARs include PAR1 (Genbank Accession a problem, replacement of a particularly sensitive peptide Number AF019616); PAR2 (Genbank Accession Number bond with a noncleavable peptide mimetic renders the result XM 003671); PAR3 (Genbank Accession Number 60 ing peptide more stable and thus more useful as a therapeutic. NM 0041101); and PAR4 (Genbank Accession Number Such mimetics, and methods of incorporating them into pep NM 003950.1), the sequences of which are hereby incorpo tides, are well known in the art. Similarly, the replacement of rated by reference. an L-amino acid residue (e.g., with a D-amino acid) renders A two-site mechanism by which pepducins both activate the peptide less sensitive to proteolysis. and inhibit receptor-G protein signaling is described herein, 65 Pepducin compounds of the invention can be synthesized but the inventors do not intend to have their invention be as retro-inverso isomers, which include peptides of reverse limited by this mechanism or the theory on which it is based. sequence and chirality. See, e.g., Jameson et al. Nature 368: US 8,440,627 B2 13 14 744-746 (1994) and Brady et al. Nature 368:692-693 (1994). The GPCR moiety of the pepducins of the present inven The net result of combining D-enantiomers and reverse Syn tion are derived from any cells of a human being or other thesis is that the positions of carbonyl and amino groups in organism (e.g., guinea pig, rat, mouse, chicken, rabbit, pig, each amide bond are exchanged, while the position of the sheep, cattle, monkey, virus, fungi, insects, plants, bacteria, side-chain groups at each alpha carbon is preserved. For 5 etc.), for example, splenic cell, nerve cell, glia cell, beta cell example, if the peptide model is a peptide formed of L-amino of pancreas, marrow cell, mesangial cell, Langerhans cell, acids having the sequence ABC, the retro-inverso peptide epidermic cell, epithelial cell, endothelial cell, fibroblast, analog formed of D-amino acids would have the sequence fibrocyte, muscular cell, fat cell, immunocyte (e.g., macroph CBA. The procedures for synthesizing a chain of D-amino age, T cell, B cell, natural killer cell, mast cell, neutrophil, acids to form the retro-inverso peptides are known in the art. 10 basophil, eosinophilic leukocyte, monocyte, etc.), mega Also useful are amino-terminal blocking groups such as t-butyloxycarbonyl, acetyl, theyl, Succinyl, methoxysuccinyl, karyocyte, synovial cell, chondrocyte, osteocyte, osteoblast, Suberyl, adipyl, aZelayl, dansyl, benzyloxycarbonyl, fluore osteoclast, mammary gland cell, hepatocyte, or interstitial nylmethoxycarbonyl, methoxyaZelayl, methoxyadipyl. cells or precursor cells, stem cells or cancer cells thereof and methoxy Suberyl, and 2,4-dinitrophenyl. Blocking the 15 the like; and any tissues containing Such cells, for example, charged amino- and carboxy-termini of the peptides would brain, various parts of the brain (e.g., olfactory bulb. have the additional benefit of enhancing passage of the pep amygdala, cerebral basal ganglia, hippocampus, thalamus, tide through the hydrophobic cellular membrane and into the hypothalamus, Substhanlamic nucleus, cerebral cortex, cell. medulla, cerebellum, occipital pole, frontal lobe, putamen, An isolated GPCR fragment may be derived from the caudate nucleus, corpus callosum, Substantia nigra), spinal sequence of a Class A GPCR or a Class B GPCR. The isolated cord, pituitary, stomach, pancreas, kidney, liver, genital GPCR fragment is a fragment from any known or unknown organs, thyroid gland, gallbladder, bone marrow, adrenal GPCR, including, but not limited to protease activated recep gland, skin, muscle, lung, digestive tract, blood vessel, heart, tors (PARS, e.g., a thrombin receptor), a luteinizing hormone thymus, spleen, Submandibular gland, peripheral blood, receptor, a follicle stimulating hormone receptor, a thyroid 25 peripheral blood leukocyte, intestinal tract, prostate, testicle, stimulating hormone receptor, a calcitonin receptor, a gluca testis, ovarium, placenta, uterus, bone, joint, Small intestine, gon receptor, a glucagon-like peptide 1 receptor (GLP-1), a large intestine, skeletal muscle and the like, in particular, metabotropic glutamate receptor, a parathyroid hormone brain and various parts of the brain. receptor, a vasoactive intestinal peptide (VIP) receptor, a Cell-penetrating moieties include a lipid, cholesterol, a secretin receptor, a growth hormone releasing factor (GRF) 30 receptor, cholecystokinin receptors, somatostatin receptors, phospholipid, steroid, sphingosine, ceramide, or a fatty acid melanocortin receptors, nucleotide (e.g., ADP receptors), moiety. The fatty acid moiety can be, e.g., any fatty acid adenosine receptors, thromboxane receptors, platelet activat which contains at least eight carbons. For example, the fatty ing factor receptors, adrenergic receptors, 5-hydrox acid can be, e.g., a nonanoyl (Co.); capryl (Co.); undecanoyl ytryptamine (5-HT) receptors, chemokine receptors (e.g., 35 (C); lauroyl (C); tridecanoyl (C); myristoyl (C); pen CXCR4, CCR5), neuropeptide receptors, opioid receptors, tadecanoyl (Cs); palmitoyl (C); phytanoyl (methyl substi erythropoietin receptor, and parathyroid hormone (PTH) tuted C); heptadecanoyl (C7); Stearoyl (Cs); nonade receptor. canoyl (Co.); arachidoyl (Co.); heniecosanoyl (C2); In preferred embodiments, the GPCR is a protease-acti behenoyl (C); trucisanoyl (C); oralignoceroyl (C) moi vated receptor, a peptide receptor, or a nucleotide receptor. In 40 ety. The cell-penetrating moiety can also include multimers particular embodiments, the GPCR is a PAR1, PAR2, PAR3, (e.g., a composition containing more than one unit) of octyl or a PAR4 receptor. In other embodiments, the GPCR is a glycine, 2-cyclohexylalanine, or benzolylphenylalanine. The glucagon-like receptor, a nucleotide receptor, such as a cell-penetrating moiety contains an unsubstituted or a halo P2Y- ADP receptor, a MC4 obesity receptor, a CXCR gen-substituted (e.g., chloro) biphenyl moiety. Substituted receptor (e.g., CXCR4) or CCR5 chemokine receptors, 45 biphenyls are associated with reduced accumulation in body CCKA, or CCKB. tissues, as compared to compounds with a non-substituted An isolated GPCR fragment may include a GPCR frag biphenyl. Reduced accumulation in bodily tissues following ment which is less than 50 contiguous amino acid from the administration to a Subject is associated with decreased GPCR, and does not contain the native extracellular ligand of adverse side effects in the subject. the GPCR. For example, the fragment may contain between 3 50 Preferably, the cell penetrating moiety is a naturally-occur and 30 contiguous amino acids of a GPCR. In preferred ring or non-naturally occurring palmitoyl moiety. Pepducins embodiments, the GPCR fragment comprises a GPCR frag ment which is between 7 and 24 (inclusive) contiguous amino with longer than Cs fatty acids, e.g., palmitoyl, moieties acids. For example, the fragment includes 7, 8, 9, 10, 11, 12, appear to be surprisingly long-lived in Vivo and are Suitable 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 contiguous 55 for Subcutaneous administration. In another embodiment, amino acids of a GPCR. pepducins having shorter than Cs, e.g., myristyl, moieties, Optionally, the amino acid sequence of a GPCR differs appear to be useful for short-term applications, e.g., Surgical from a naturally-occurring amino acid sequence. For applications, and intravenous administration is useful here. example, individual residues from a given domain, e.g., a The cell-penetrating or membrane-tethering moiety may transmembrane helix, extracellular, or intracellular loop, are 60 be attached to the C-terminal amino acid, the N-terminal mutated or substituted with a modified amino acid(s) to amino acid, or to an amino acid between the N-terminal and improve activity of the pepducin. Preferably, the amino acid C-terminal amino acid of the GPCR fragment. sequence of such a GPCR analog differs solely by conserva Also within the invention is a composition which includes tive amino acid Substitutions, i.e., Substitution of one amino a polypeptide having an amino acid sequence of SEQ ID acid for another of the same class, or by non-conservative 65 NOs: 1-14, or portions thereof, linked to a cell penetrating or Substitutions, deletions, or insertions located at positions that membrane-tethering moiety. Particularly Suitable pepducins do not destroy the function of the protein. of the invention include those listed below in Table 1. US 8,440,627 B2 15 16 TABLE 1. i loop pepducins SEQ Lipid ID Receptor Pepducin Sequence Length modification MW NO PAR2 P2i1pal12 FLFRTKKKHPAV 2 palmitate 708 17 CXCR1 X1/2ilpall2 ILYSRVGRSVTD 2 palmitate 6O2 18 CXCR1 X1/2ilmyr10 YSRVGRSVTD O myristate 376 19 CXCR1 x1,21LCA1 O YSRVGRSVTD 0 lithocholate 497 2O CXCR2 x1/2i1pal12 ILYSRVGRSVTD 2 palmitate 6O2 21 CXCR2 X1/2ilmyr10 YSRVGRSVTD O myristate 376 22 CXCR2 x1 21LCA1 O YSRVGRSVTD 0 lithocholate 497 23 CXCR4 X4ilpal10 YQKKLRSMTD O palmitate SO8 24 CXCR4 x4i1pal12 MGYQKKLRSMTD 2 palmitate 697 25 CCR5 CC5i1pal8 KRLKSMTD 8 palmitate 215 26 CCR5 CC5i1pal12 LINCKRLKSMTD 2 palmitate 658 27 PAR1 P1ilpal 11 pal-ILKMKVKKPAV 1 palmitate 491.6 28 PAR4 P411 pal12 pal- 2 palmitate 489.9 29 VLATQAPRLPST PAR4 P411 pal10 pal-ATOAPRLPST O palmitate 277.6 30 PAR4 P411 pal12G pal- 2 palmitate 418.8 31 VLATGAPRLPST PAR4 P411 pal10G pal-ATGAPRLPST O palmitate 206.5 32

The compositions are used to treat, prevent, or ameliorate with Taxotere. The compounds reduce tumor growth migra (reduce the severity of) one or more symptoms associated 25 tion at all stages of tumor development, e.g., early in tumor with diseases and conditions characterized by aberrant GPCR development as well as at later stages of tumor development. activity. Such diseases and conditions include thrombosis, For example, as seen FIG. 27. I1-loop pepducins based on heart attack, stroke, excessive bleeding, asthma, inflamma the CXCR1/2(x1/2pal-il) and CXCR4 (x4pal-il) chemokine tion, pain, inflammatory pain, visceral pain, neurogenic pain, 30 receptors block chemotactic migration of breast and ovarian arthritis, diabetes, HIV infection, anxiety, depression, pulmo CaCCS. nary insufficiency, and various types of cancer. Such methods Certain pepducins of the invention are platelet activation are carried out by contacting a cell, which pathologically inhibitors. The inhibitor contains an isolated fragment of a overexpresses a GPCR with a pepducin GPCR antagonist. protease activated receptor and a cell penetrating moiety For example, the method involves administering to a subject, 35 linked to the GPCR polypeptide. In some embodiments, the e.g., a human patient, in which Such treatment or prevention is protease activated receptor is a thrombin receptor, a trypsin desired a pepducin in an amount Sufficient to reduce the receptor, clotting factor Xa receptor, activated protein C severity of the pathology in the subject. The present invention receptor, tryptase receptor, or a plasmin receptor. The throm also includes pharmaceutical compositions containing any of bin receptor is preferably PAR-4 or PAR-1. the pepducin compositions and a pharmaceutically accept 40 The invention also includes a method of inhibiting platelet able carrier. The invention also includes kits containing the aggregation, by contacting a platelet with a composition of an pharmaceutical compositions. The invention further includes isolated fragment of a protease activated receptor linked to a methods of treating a pathological state in a mammal through cell penetrating moiety as described above. For example, the the administration of any polypeptide of the invention. protease activated receptor is a thrombin receptor, such as a The constructs are also used to inhibit tumor growth and 45 PAR-1 receptor or a PAR-4 receptor. Also within the inven migration. Breast cancer cell invasion is a complex process in tion is a method of inhibiting thrombus formation in a mam which cell migration, proteolytic modifications of tissue mal by administering to the mammal a composition of the basement membranes, and degradation of extracellular matri invention which includes an isolated fragment of a thrombin ces by matrix metalloproteases (MMP) take place. Activation receptor linked to a cell penetrating moiety. of PAR1 by MMP1 plays a critical role in invasion and tum 50 The methods of the invention are carried out by infusing origenesis of breast cancer cells (Boire et al. Cell, 120(3),303 into a vascular lumen, e.g., a jugular vein, peripheral vein or (2005)). A PAR1 i1 loop pepducin antagonist, e.g., the perivascular space, the inhibitory compositions of the Plil pal1 1, is used alone or as an adjuvant cancer treatment, invention. The peripheral vein can be, e.g., a vein located in e.g., when administered with a docetaxel compound Such as the extremities, such as the hand, wrist, or foot. In some Taxotere?R). Preferably, a synergistic antitumor effect is 55 embodiments, the composition is infused into the lungs of the achieved. The effect of intracellular inhibition of PAR1 is mammal, e.g., as an aerosol. In other embodiments, the com demonstrated using the metastatic human breast cancer cell position of the invention is administered by injection. In line MDA-MB-231. Pepducin efficacy alone and in combi various embodiments, the injection can be into the peritoneal nation therapy, e.g., together with Taxotere is analyzed using cavity of the mammal, Subdermally, or Subcutaneously. The the MTT assay and a xenograft mice model. The ICs of the 60 composition of the invention can also be administered trans pepducin P1il pal11 and Taxotere is determined after the dermally. In other embodiments, the composition of the compositions are administered separately or together. The invention is administered vaginally or rectally. The composi Isobologram technique and the combination index (CI) tion can be administered by implanting wound packing mate method is employed to quantify the degree of synergy. rial or a Suppository which is coated or impregnated with the Xenograft data is used to determine whether the tumor growth 65 composition of the invention. rates were suppressed in mice treated with P1il pal11 and Inhibitors of clot formation or platelet aggregation are used Taxotere together compared to the mice that were treated only in medical devices, e.g., as coatings. For example, a vascular US 8,440,627 B2 17 18 endoprosthetic device, e.g., a screen, stent or catheter, invention will be apparent from the description. In the speci includes an inhibitor of thrombus formation which is an iso fication and the appended claims, the singular forms also lated fragment of a thrombin receptor linked to a cell pen include the plural unless the context clearly dictates other etrating moiety. The composition is impregnated in the device wise. Unless defined otherwise, all technical and scientific and diffuses into bodily tissues upon contact with a tissue or terms used herein have the same meaning as commonly implantation of the device; alternatively, the device is coated understood by one of ordinary skill in the art to which this with the pepducin. invention belongs. In the case of conflict, the present Speci Pepducins are also used to inhibit migration and invasion of fication, including definitions, will control. In addition, the a tumor cell by contacting the tumor cell with an isolated materials, methods, and examples are illustrative only and not fragment of a protease activated receptor linked to a cell 10 intended to be limiting. All patents and publications cited in penetrating moiety. The protease activated receptor is a PAR this specification are incorporated herein by reference. 4. PAR-2, or a PAR-1 receptor. Methods of inhibiting G Protein Coupled Receptors metastases of a tumor cell are carried out by contacting the G protein coupled receptors are intrinsic membrane pro tumor cell with an isolated fragment of a protease activated teins which comprise a large Superfamily of receptors. The receptor linked to a cell-penetrating moiety. The tumor cell is 15 family of G protein-coupled receptors (GPCRs) has at least a melanoma cell, a breast cancer cell, a renal cancer cell, a 250 members (Strader et al. FASEB J., 9:745-754, 1995: prostate cancer cell, a lung cancer cell, a colon cancer cell, a Strader et al. Annu. Rev. Biochem. 63:101-32, 1994). It has central nervous system (CNS) cancer cell, a liver cancer cell, been estimated that one percent of human genes may encode a stomach cancer cell, a sarcoma cell, a leukemia cell, or a GPCRs. Many GPCRs share a common molecular architec lymphoma cell. ture and common signaling mechanism. Historically, GPCRs Symptoms of asthma are reduced by administering a have been classified into six families, originally thought to be thrombin or a trypsin/tryptase GPCR based pepducin. unrelated, three of which are found in vertebrates. Recent Accordingly, a method of inhibiting asthma is carried out by work has identified several new GCPR families and suggested administering a composition containing an isolated fragment the possibility of a common evolutionary origin for all of of a thrombin or a trypsin/tryptase GPCR linked to a cell 25 them. penetrating moiety. Preferably, the trypsin/tryptase receptor Many GPCRs share a common structural motif of seven is a PAR-1, PAR-2 or PAR-4 receptor. In various embodi transmembrane helical domains. Some GPCRS, however, do ments, the composition is infused into a vascular lumen, Such not have seven transmembrane helical domains and instead as a peripheral vein, is infused into the lungs of the mammal, can be single-spanning transmembrane receptors. e.g., by inhalation (e.g., as an aerosol), or is administered by 30 Single spanning GPCRs include receptors for cytokines a transdermal route. Such as erythropoietin, EGF, insulin, insulin-like growth fac Inhibitors of platelet activation include an isolated frag tors I and II, and TGF. ment of a nucleotide activated GPCR such as a P2Y recep GPCR families include Class A Rhodopsin like, Class B tor linked to a cell penetrating moiety. Such compositions are Secretin like, Class C Metabotropic glutamate/pheromone, useful in methods of inhibiting platelet aggregation. 35 Class D Fungal pheromone, Class E. cAMP receptors (Dicty In yet another aspect, the invention includes a method of ostelium), and Frizzled/Smoothened family. Putative fami inhibiting thrombus formation in a mammal by administering lies include Ocular albinism proteins, Drosophila odorant a composition including an isolated fragment of a nucleotide receptors, Plant Mlo receptors, Nematode chemoreceptors, activated receptor linked to a cell penetrating moiety to the and Vomeronasal receptors (V1 R & V3R). mammal. In some embodiments, the thrombin receptor is a 40 Class A Rhodopsin like receptors include: Amine recep P2Y receptor. The method can be carried out by infusing tors: Acetylcholine, Alpha Adrenoceptors, Beta Adrenocep into a vascular lumen, e.g., a jugular vein, peripheral vein the tors, Dopamine. Histamine, Serotonin, Octopamine, and inhibitory compositions of the invention. The peripheral vein Trace amine; Peptide receptors: Angiotensin, Bombesin, can be, e.g., a vein located in the extremities. Such as the hand, Bradykinin, C5a anaphylatoxin, Fmet-leu-phe, APJ like, wrist, or foot. In some embodiments, the composition is 45 Interleukin-8, Chemokine receptors (C–C Chemokine, infused into the lungs of the mammal, e.g., as an aerosol. In C X-C Chemokine, BONZO receptors (CXC6R), other embodiments, the composition of the invention is C X3-C Chemokine, and XC Chemokine), CCK receptors, administered by injection. In various embodiments, the injec Endothelin receptors, Melanocortin receptors, Neuropeptide tion can be into the peritoneal cavity of the mammal, Subder Y receptors, Neurotensin receptors, Opioid receptors, Soma mally, or Subcutaneously. In other embodiments, the compo 50 tostatin receptors, Tachykinin receptors, (Substance P(NK1), sition of the invention is administered transdermally. The Substance K (NK2), Neuromedin K (NK3), Tachykinin like composition can be administered by implanting wound pack 1, and Tachykinin like 2), Vasopressin-like receptors (Vaso ing material or a Suppository which is coated or impregnated pressin, Oxytocin, and Conopressin), Galanin like receptors with the composition of the invention. (Galanin, Allatostatin, and GPCR 54), Proteinase-activated In another aspect, the invention includes a vascular 55 like receptors (e.g., Thrombin), Orexin & neuropeptide FF, endoprosthetic device, which includes an inhibitor of throm Urotensin II receptors, Adrenomedulin (G10D) receptors, bus formation which is an isolated fragment of a nucleotide GPR37/endothelin B-like receptors, Chemokine receptor receptor linked to a cell penetrating moiety. In various like receptors, and Neuromedin U receptors; Hormone pro embodiments, the device can be, e.g., a stent or a catheter. In tein receptors: Follicle stimulating hormone, Lutropin-chori Some embodiments, the device is impregnated with or coated 60 ogonadotropic hormone, Thyrotropin, and Gonadotropin; with the inhibitor. (Rhod).opsin receptors; Olfactory receptors; Prostanoid The details of one or more embodiments of the invention receptors: Prostaglandin, Prostacyclin, and Thromboxane: are set forth in the accompanying description below. Nucleotide-like receptors: Adenosine and Purinoceptors: Although any methods and materials similar or equivalent to Cannabis receptors; Platelet activating factor receptors; those described herein can be used in the practice or testing of 65 Gonadotropin-releasing hormone receptors; Thyrotropin-re the present invention, the preferred methods and materials are leasing hormone & Secretagogue receptors: Thyrotropin-re now described. Other features, objects, and advantages of the leasing hormone, Growth hormone secretagogue, and US 8,440,627 B2 19 20 Growth hormone secretagogue like; Melatonin receptors; parison to the prototype GPCR, rhodopsin, using known Viral receptors: Lysosphingolipid & LPA (EDG) receptors: methods, e.g., as described in Palczewski et al., Science 289: Leukotriene B4 receptor: Leukotriene B4 receptor BLT1 and 739 (2000), hereby incorporated by reference. Leukotriene B4 receptor BLT2; and Class A Orphan/other One characteristic feature of most GPCRs is that seven receptors: Platelet ADP & KIO1 receptors, SREB, Mas proto 5 clusters of hydrophobic amino acid residues, or transmem oncogene, RDC1, ORPH, LGR like (hormone receptors), brane regions (TMs, the 7 transmembrane regions are desig GPR, GPR45 like, Cysteinyl leukotriene, Mas-related recep nated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7) are tors (MRGs), and GP40 like receptors. located in the primary structure and pass through (span) the Class B (the secretin-receptor family or family 2) of the cell membrane at each region thereof (FIG. 1). The domains GPCRs is a smaller but structurally and functionally diverse 10 are believed to represent transmembrane alpha-helices con group of proteins that includes receptors for polypeptide hor nected by three intracellular loops (i1, i2, and i3), three extra mones (Calcitonin, Corticotropin releasing factor, Gastric cellular loops (e1, e2, and e3), and amino (N)- and carboxyl inhibitory peptide, Glucagon, Glucagon-like peptide-1.-2, (C)-terminal domains (Palczewski et al., Science 289, 739-45 Growth hormone-releasing hormone, Parathyroid hormone, (2000)). Most GPCRs have single conserved cysteine resi PACAP Secretin, Vasoactive intestinal polypeptide, Diuretic 15 dues in each of the first two extracellular loops which form hormone, EMR1, Latrophilin), molecules thought to mediate disulfide bonds that are believed to stabilize functional pro intercellular interactions at the plasma membrane (Brain tein structure. It is well known that these structures detailed specific angiogenesis inhibitor (BAI)) and a group of Droso above are common among G protein coupled receptor pro phila proteins (Methuselah-like proteins) that regulate stress teins and that the amino acid sequences corresponding to the responses and longevity. area where the protein passes through the membrane (mem Class C Metabotropic glutamate/pheromone receptors brane-spanning region or transmembrane region) and the include Metabotropic glutamate, Metabotropic glutamate amino acid sequences near the membrane-spanning region group I, Metabotropic glutamate group II, Metabotropic are often highly conserved among the receptors. Thus, due to glutamate group III, Metabotropic glutamate other, Extracel the high degree of homology in GPCRS, the identification of lular calcium-sensing, Putative pheromone Receptors, 25 novel GPCRs, as well identification of both the intracellular GABA-B. GABA-B subtype 1, GABA-B subtype 2, and and the extracellular portions of Such novel members, is Orphan GPRC5 receptors. readily accomplished by those of skill in the art. GPCRs can potentially be multi-polypeptide receptors The binding sites for Small ligands of G-protein coupled such as GPIb-V-IX, or the collagen receptor, that exhibit receptors are believed to comprise a hydrophilic socket outside-in-signaling via G proteins. 30 located near the extracellular surface which is formed by Although hundreds of G protein coupled receptor genes or several GPCR transmembrane domains. The hydrophilic cDNAs have been cloned, it is believed that there are still socket is surrounded by hydrophobic residues of the G-pro many uncharacterized G protein coupled receptors which tein coupled receptors. The hydrophilic side of each G-pro have not yet been recognized as GPCRs. tein coupled receptor transmembrane helix is postulated to GPCRs play a vital role in the signaling processes that 35 face inward and form the polar ligand binding site. TM3 has control cellular metabolism, cell growth and motility, adhe been implicated in several GPCRs as having a ligand binding Sion, inflammation, neuronal signaling, and blood coagula site which includes the TM3 aspartate residue. TM5 serines, tion. G protein coupled receptor proteins also have a very a TM6 asparagine and TM6 or TM7 phenylalanines or important role as targets for a variety of signaling molecules tyrosines are also implicated in ligand binding. The ligand which control, regulate, or adjust the functions of living bod 40 binding site for peptide hormones receptors and receptors ies. The signaling species can be endogenous molecules (e.g., with other larger ligands Such as glycoproteins (e.g., lutein neurotransmitters or hormones), exogenous molecules (e.g., izing hormone, follicle stimulating hormone, human chori odorants), or, in the case of visual transduction, light. onic gondaotropin, thyroid-stimulating hormone (Thyrotro For instance, GPCRs include receptors for biogenic pin)), and the Ca"/glutamate/GABA (gamma-aminobutyric amines, e.g., dopamine, epinephrine, histamine, glutamate 45 acid) classes of receptors likely reside in the extracellular (metabotropic effect), acetylcholine (muscarinic effect), and domains and loops. serotonin; receptors for lipid mediators of inflammation Such A key event for the switch from inactive to active receptor as prostaglandins, platelet activating factor, and leukotrienes; is ligand-induced conformational changes of transmembrane receptors for peptide hormones Such as calcitonin, C5a ana helices 3 (TM3) and 6 (TM6) of the GPCRs that have 7 phylatoxin, follicle stimulating hormone, gonadotropin 50 transmembrane spanning helices (Gether and Kolbilka, J. releasing hormone, neurokinin, oxytocin, receptors for pro Biol. Chem. 273, 17979-17982 (1998)). These helical move teases such as thrombin, trypsin, tryptase, activated protein C, ments in turn alter the conformation of the intracellular loops and factor VIIa/Xa; and receptors for sensory signal media of the receptor to promote activation of associated heterotri tors, e.g., retinal photopigments and olfactory stimulatory meric G proteins. Mutagenesis studies (Cotecchia et al., J. molecules. Each molecule is specific to a receptor protein, 55 Biol. Chem. 267: 1633-1639 (1992); Kostenis et al., Bio whereby the specificities of individual physiologically active chemistry 36:1487-1495 (1997); Kjelsberg et al., J. Biol. Substances (including specific target cells and organs), spe Chem. 267:1430-1433 (1992)) demonstrated that the third cific pharmacological actions, specific action strength, action intracellular loop (13) mediates a large part of the coupling time, etc., are decided. Thus, GPCRs are a major target for between receptor and G protein. I3 loops expressed as mini drug action and development. 60 genes have also been shown to directly compete with adren Upon ligand binding, GPCRs regulate intracellular signal ergic receptors for Gd binding (Luttrel et al., Science 259: ing pathways by activating guanine nucleotide-binding pro 1453-1457 (1993)), or can activate G proteins as soluble teins (G proteins). The domain structure of GPCRs are con peptides in cell-free conditions (Okamoto et al. Cell 67, 723 served among members of the GPCR family. Domain 730 (1991)). boundaries of TM helix domains, intracellular loop domains, 65 One particular class of GPCR is the protease activated and extracellular domains of GPCRS are known in the art. receptors (PARs). Protease-activated receptors (PARs) are The structure of unmapped GPCRs is determined by com members of the superfamily of G-protein-coupled receptors US 8,440,627 B2 21 22 that initiate cell signaling by the proteolytic activity of extra schematic representation of transmembrane and loop regions cellular serine proteases. PARs are activated after proteolytic of the PAR1 GPCR is presented in FIG. 1A. cleavage of the amino terminus of the receptor by endogenous One example of a GPCR is the CXCR4 receptor, shown in proteases, including thrombin (PAR-1, -3, and -4) and trypsin/tryptase (PAR-2 and -4). Of these, PAR2 (Nystedt et Table 2 as SEQID NO:15. The seven underlined sequences al., Proc. Natl. Acad. Sci. (USA) 91:9208-9212 (1994)) is a correspond to the seven transmembrane regions of the GPCR. trypsin/tryptase-activated receptor that is important in Thus, the sequence IFLPTIYSIIFLTGIVGNGLVILV (SEQ inflammation and pain, and PAR4 (Xu et al., Proc. Natl. Acad. ID NO:16) corresponds to the first transmembrane region Sci. (USA).95:6642-6646 (1998); Kahn et al., Nature (Lon (TM1). TABLE 2

CXCR4 (SEQ ID NO: 15) MEGISIYTSD NYTEEMGSGD YDSMKEPCFR EENANFNKIF LPTIYSIIFL TGIWGNGLVI

SMTDKYRLHL SWADLLFWIT LPFWAWDAWA NWYFGNFLCK AWHWIYTWNL

YSSWLILAFI SLDRYLAIWH ATNSORPRKL LAEKVVYWGV WIPALLLTIP DFIFANVSEA.

DDRYICDREY FOHIMVGLIL PGIVILSCYC IIISKLSHSK GHOKRKALKT

TWILTLAFFA. CWLPYYIGIS IDSFILLEII KOGCEFENTV HKWISITEAL AFFHCCLNPI

LYAFLGAKFK TSAOHALTSV SRGSSLKILS KGKRGGHSSW STESESSSFH SS. don)394:690-694 (1998)) is a second thrombin receptor that 25 An isolated GPCR fragment is any portion of the GPCR plays a unique role in platelet aggregation (Covic et al., Bio which is less than the full length protein. A peptide containing chemistry 39, 5458-5467 (2000)). an isolated GPCR fragment may contain an amino acid Because both thrombin, trypsin, and tryptase are present in sequence N-terminal and/or C-terminal to the GPCR sequence other than the naturally occurring amino acid inflamed airways, PARs are likely to play a major role in 30 sequence. A peptide containing an isolated transmembrane airway inflammation. Knight et al., J. Allergy Clin. Immunol. sequence of a GPCR may contain only the sequence corre 108:797-803 (2001). sponding to that transmembrane region of the GPCR, or it In addition to its pivotal role in hemostasis, thrombin acti may also contain amino acid sequences N-terminal and/or Vates various cell types such as platelets and vascular Smooth C-terminal to the transmembrane sequence, that are not the muscle cells via proteolytic cleavage of specific cell-surface 35 naturally occurring flanking sequences (i.e., not the loop receptors (PARs), the prototype of which is PAR-1. Thrombin sequences which are adjacent to that region in the naturally receptor activation is likely to play a key role in cardiovascu occurring GPCR sequence). lar disorders such as thrombosis, atherosclerosis and resteno Thus, a peptide containing an isolated transmembrane sis, and as such a thrombin receptor antagonist should have region of the CXCR4 receptor is any peptide that contains any potential utility in the treatment of these disorders. Chackala 40 orall of the contiguous amino acids of an underlined region of mannil, Curr. Opin. Drug Discov. Devel. 4:417-27 (2001). sequence shown in Table 2. Such a peptide does not contain Thrombin is thought to be involved in functional loss after any of the naturally occurring (non-underlined) flanking injury to the mammalian central nervous system (CNS). sequence which corresponds to loop sequences which are Down-regulation of PAR-1 has been shown to increase post adjacent to that TM region in the naturally occurring GPCR traumatic survival of CNS neurons and post-traumatic toxic 45 Sequence. ity of thrombin may be down-regulated by appropriate modu Likewise, a peptide containing an isolated (intracellular or lation of PAR-I receptors. Friedmann et al., Neuroimmunol. extracellular) loop region of the CXCR4 receptor is any pep 121:12-21 (2001). tide that contains any or all contiguous amino acids of a PARS are also involved in a variety of other diseases or non-underlined region of sequence shown in Table 2. Such a indications, including various cancers, cellular proliferation, 50 peptide does not contain any of the naturally occurring trans and pain. membrane sequences, shown as underlined flanking GPCR Domains sequence in Table 2, which are adjacent to that loop region in Most GPCRs are characterized by seven clusters of hydro the naturally occurring GPCR sequence. phobic amino acid residues, or transmembrane regions (TMs, A peptide containing an isolated extracellular domain oran the 7 transmembrane regions are designated as TM1, TM2, 55 isolated intracellular domain can include amino acid TM3, TM4, TM5, TM6, and TM7), that are located in the primary structure and pass through (span) the cell membrane sequences from any (extracellular or intracellular) loop and/ (FIG. 1A). The TM regions are believed to represent trans or the N- or C-terminal domain. Such a peptide does not membrane alpha-helices connected by three intracellular include any sequence from a transmembrane region which is loops (i1, i2, and i3), three extracellular loops (e1, e2, and e3). adjacent to that extracellular domain or intracellular domain GPCRs also contain amino (N)- and carboxyl (C)-terminal 60 in the naturally occurring GPCR sequence. domains (Palczewski et al., Science 289, 739-45 (2000)). The Pharmaceutical Compositions sequences between the transmembrane regions correspond to The pepducins (also referred to herein as “active com GPCR loops, and the location of a loop within a cell deter pounds’) of the invention, and derivatives, fragments, ana mines whether it is an intracellular or an extracellular loop. logs and homologs thereof, can be incorporated into pharma Most GPCRs have single conserved cysteine residues in each 65 ceutical compositions Suitable for administration. Such of the first two extracellular loops which form disulfide bonds compositions typically comprise the pepducin and a pharma that are believed to stabilize functional protein structure. A ceutically acceptable carrier. US 8,440,627 B2 23 24 A pharmaceutical composition of the invention is formu powder of the active ingredient plus any additional desired lated to be compatible with its intended route of administra ingredient from a previously sterile-filtered solution thereof. tion. Examples of routes of administration include parenteral, Oral compositions generally include an inert diluent or an e.g., intravenous (e.g., a peripheral vein, such as found in the edible carrier. They can be enclosed in gelatin capsules or extremities), intraperitoneal, intradermal, Subcutaneous, Sub compressed into tablets. For the purpose of oral therapeutic dermal, oral, intranasal, aerosol (e.g., inhalation), transder administration, the active compound can be incorporated mal (i.e., topical), transmucosal, vaginal, intrauterine, and with excipients and used in the form of tablets, troches, or rectal (e.g., Suppositories) administration. Injectable solu capsules. Oral compositions can also be prepared using a fluid tions containing active compounds of the present invention carrier for use as a mouthwash, wherein the compound in the 10 fluid carrier is applied orally and swished and expectorated or may be administered to the vascular lumen of vessels (e.g., Swallowed. Pharmaceutically compatible binding agents, aorta or jugular vein). Alternatively, active compounds of the and/or adjuvant materials can be included as part of the com present invention may be administered via a device, e.g., stent position. The tablets, pills, capsules, troches and the like can or catheter, impregnated or coated with the active com pounds. contain any of the following ingredients, or compounds of a 15 similar nature: a binder Such as microcrystalline cellulose, Solutions or Suspensions used for administration (e.g., gum tragacanth or gelatin; an excipient such as starch or parenteral) may include the following components: a sterile lactose, a disintegrating agent such as alginic acid, Primogel, diluent such as water for injection, Saline solution, fixed oils, or corn starch; a lubricant such as magnesium Stearate or polyethylene glycols, glycerine, propylene glycol or other Sterotes; a glidant Such as colloidal silicon dioxide; a Sweet synthetic solvents; antibacterial agents such as benzyl alcohol ening agent such as Sucrose or saccharin; or a flavoring agent or methyl parabens; antioxidants such as ascorbic acid or Such as peppermint, methyl salicylate, or orange flavoring. Sodium bisulfite; chelating agents such as ethylenediamine For administration by inhalation or intranasal administra tetraacetic acid (EDTA); buffers such as acetates, citrates or tion, the compounds are delivered in the form of an aerosol phosphates, and agents for the adjustment of tonicity Such as spray from pressured container or dispenser which contains a sodium chloride or dextrose. The pH can be adjusted with 25 Suitable propellant, e.g., a gas such as carbon dioxide, or a acids or bases, such as hydrochloric acid or sodium hydrox nebulizer (e.g., delivery to the lung). ide. A preparation of a pharmaceutical composition of the Systemic administration can also be by transmucosal or present invention can be enclosed in ampoules, disposable transdermal means. For transmucosal or transdermal admin Syringes or multiple dose vials made of glass or plastic. istration, penetrants appropriate to the barrier to be permeated Pharmaceutical compositions suitable for injectable use 30 are used in the formulation. Such penetrants are generally include sterile aqueous solutions (where water soluble) or known in the art, and include, for example, for transmucosal dispersions and sterile powders for the extemporaneous administration, detergents, bile salts, and fusidic acid deriva preparation of sterile injectable solutions or dispersion. For tives. Transmucosal administration can be accomplished intravenous administration, Suitable carriers include physi through the use of nasal sprays or Suppositories. For trans ological saline, bacteriostatic water, Cremophor EL (BASF, 35 dermal administration, the active compounds are formulated Parsippany, N.J.) or phosphate buffered saline (PBS). In all into ointments, salves, gels, or creams as generally known in cases, the composition must be sterile and should be fluid to the art. the extent that easy syringeability exists. It must be stable The active compounds can also be prepared in the form of under the conditions of manufacture and storage and must be Suppositories (e.g., with conventional Suppository bases Such preserved against the contaminating action of microorgan 40 as cocoa butter and other glycerides), a Suppository coating, isms such as bacteria and fungi. The carrier can be a solvent or retention enemas for rectal delivery. The active compounds or dispersion medium containing, for example, water, etha can be similarly prepared for intravaginal or intrauterine nol, polyol (for example, glycerol, propylene glycol, and administration. The active compounds may also be adminis liquid polyethylene glycol, and the like), and Suitable mix tered as impregnated in or as a coating on wound packing tures thereof. The proper fluidity can be maintained, for 45 (e.g., to reduce bleeding). example, by the use of a coating Such as lecithin, by the In one embodiment, the active compounds are prepared maintenance of the required particle size in the case of dis with carriers that will protect the compound against rapid persion and by the use of surfactants. Prevention of the action elimination from the body, such as a controlled release for of microorganisms can be achieved by various antibacterial mulation, including implants and microencapsulated delivery and antifungal agents, for example, parabens, chlorobutanol, 50 systems. Biodegradable, biocompatible polymers can be phenol, ascorbic acid, thimerosal, and the like. In many cases, used. Such as ethylene vinyl acetate, polyanhydrides, polyg it will be preferable to include isotonic agents, for example, lycolic acid, collagen, polyorthoesters, and polylactic acid. Sugars, polyalcohols such as mannitol, Sorbitol, Sodium chlo Methods for preparation of such formulations will be appar ride in the composition. Prolonged absorption of the inject ent to those skilled in the art. The materials can also be able compositions can be brought about by including in the 55 obtained commercially from Alza Corporation and Nova composition an agent which delays absorption, for example, Pharmaceuticals, Inc. Liposomal Suspensions (including aluminum monostearate and gelatin. liposomes targeted to infected cells with monoclonal antibod Sterile injectable solutions can be prepared by incorporat ies to viral antigens) can also be used as pharmaceutically ing the active compound in the required amount in an appro acceptable carriers. These can be prepared according to meth priate solvent with one or a combination of ingredients enu 60 ods known to those skilled in the art, for example, as merated above, as required, followed by filtered sterilization. described in U.S. Pat. No. 4,522,811. Generally, dispersions are prepared by incorporating the It is especially advantageous to formulate oral or parenteral active compound into a sterile vehicle that contains a basic compositions in dosage unit form for ease of administration dispersion medium and the required other ingredients from and uniformity of dosage. Dosage unit form as used herein those enumerated above. In the case of sterile powders for the 65 refers to physically discrete units Suited as unitary dosages for preparation of sterile injectable solutions, methods of prepa the Subject to be treated; each unit containing a predetermined ration are vacuum drying and freeze-drying that yields a quantity of active compound calculated to produce the US 8,440,627 B2 25 26 desired therapeutic effect in association with the required playing a role in releasing the agent to the fluid. The channels pharmaceutical carrier. The specification for the dosage unit can be pre-existing channels or channels left behind by forms of the invention are dictated by and directly dependent released agent or other soluble Substances. on the unique characteristics of the active compound and the Erodible or degradable devices typically may have the particular therapeutic effect to be achieved, and the limita active compounds physically immobilized in the polymer. tions inherent in the art of compounding Such an active com The active compounds can be dissolved and/or dispersed pound for the treatment of individuals. throughout the polymeric material. The polymeric material The pepducins and GPCR peptides can be administered for may be hydrolytically degraded over time through hydrolysis the treatment of various disorders in the form of pharmaceu of labile bonds, allowing the polymer to erode into the fluid, tical compositions. Principles and considerations involved in 10 releasing the active agent into the fluid. Hydrophilic polymers preparing such compositions, as well as guidance in the have a generally faster rate of erosion relative to hydrophobic choice of components are provided, for example, in Reming polymers. Hydrophobic polymers are believed to have almost ton: The Science And Practice OfPharmacy 19th ed. (Alfonso purely surface diffusion of active agent, having erosion from R. Gennaro, et al., editors) Mack Pub.Co., Easton, Pa. 1995; the surface inwards. Hydrophilic polymers are believed to Drug Absorption Enhancement: Concepts, Possibilities, 15 allow water to penetrate the surface of the polymer, allowing Limitations, And Trends, Harwood Academic Publishers, hydrolysis of labile bonds beneath the surface, which can lead Langhorne, Pa., 1994; and Peptide And Protein Drug Deliv to homogeneous or bulk erosion of polymer. ery (Advances. In Parenteral Sciences, Vol. 4), 1991, M. Dek The pharmaceutical compositions can be included in a ker, New York. The formulation herein can also contain more container, pack, or dispenser together with instructions for than one active compound as necessary for the particular administration. indication being treated, preferably those with complemen The pepducin approach according to the present invention tary activities that do not adversely affect each other. Alter allows the rich diversity of intracellular receptor structures to natively, or in addition, the composition can comprise an be exploited both for generation of new therapeutic agents agent that enhances its function, Such as, for example, a and for delineation of the mechanisms of receptor-G protein cytotoxic agent, cytokine, chemotherapeutic agent, or 25 coupling under in vivo conditions. The pepducins discovered growth-inhibitory agent. Such molecules are Suitably present by this strategy may also prove to be more selective to the in combination in amounts that are effective for the purpose extent that the pepducins primarily target the receptor rather intended. The active ingredients can also be entrapped in than the G protein. In addition, many receptors have been microcapsules prepared, for example, by coacervation tech identified by genomic and genetic approaches as being niques or by interfacial polymerization, for example, 30 important in various diseases processes but have no known hydroxymethylcellulose or gelatin-microcapsules and poly ligands—so-called orphan receptors. Pepducin agonists and (methylmethacrylate) microcapsules, respectively, in colloi antagonists can be generated which are tailored to these dal drug delivery systems (for example, liposomes, albumin receptors, and may be useful in determining which signaling microspheres, microemulsions, nano-particles, and nanocap pathways are activated by the orphan receptor in the context Sules) or in macroemulsions. 35 of its native environment. Thus, in the post-genomic era, the The formulations to be used for in vivo administration must pepducin approach may be widely applicable to the targeting be sterile. This is readily accomplished by filtration through of membrane proteins and may open up new experimental sterile filtration membranes. avenues in Systems previously not amenable to traditional Sustained-release preparations can be prepared. Suitable molecular techniques examples of Sustained-release preparations include semiper 40 The compounds of the invention and the other pharmaco meable matrices of Solid hydrophobic polymers containing logically active agent may be administered to a patient simul the antibody, which matrices are in the form of shaped taneously, sequentially or in combination. It will be appreci articles, e.g., films, or microcapsules. Examples of Sustained ated that when using a combination of the invention, the release matrices include polyesters, hydrogels (for example, compound of the invention and the other pharmacologically poly(2-hydroxyethyl-methacrylate), or poly(Vinylalcohol)), 45 active agent may be in the same pharmaceutically acceptable polylactides (U.S. Pat. No. 3,773.919), copolymers of carrier and therefore administered simultaneously. They may L-glutamic acid and ethyl-L-glutamate, non-degradable eth be in separate pharmaceutical carriers such as conventional ylene-vinyl acetate, degradable lactic acid-glycolic acid oral dosage forms which are taken simultaneously. The term copolymers such as the LUPRON DEPOTTM (injectable “combination' further refers to the case where the com microspheres composed of lactic acid-glycolic acid copoly 50 pounds are provided in separate dosage forms and are admin mer and leuprolide acetate), and poly-D-(-)-3-hydroxybu istered sequentially. tyric acid. While polymers such as ethylene-vinyl acetate and The compounds of the invention may be administered to lactic acid-glycolic acid enable release of molecules for over patients (animals and humans) in need of Such treatment in 100 days, certain hydrogels release proteins for shorter time dosages that will provide optimal pharmaceutical efficacy. It periods. 55 will be appreciated that the dose required for use in any Controlled release of active compounds can utilize various particular application will vary from patient to patient, not technologies. Devices, e.g., stents or catheters, are known only with the particular compound or composition selected, having a monolithic layer or a coating incorporating a hetero but also with the route of administration, the nature of the geneous Solution and/or dispersion of an active agent in a condition being treated, the age and condition of the patient, polymeric substance, where the diffusion of a therapeutic 60 concurrent medication or special diets then being followed by agent is rate limiting, as the agent diffuses through the poly the patient, and other factors which those skilled in the art will mer to the polymer-fluid interface and is released into the recognize, with the appropriate dosage ultimately being at the Surrounding fluid. Active compound may be dissolved or discretion of the attendant physician. dispersed in a suitable polymeric material. Such that addi In the treatment of a condition, an appropriate dosage level tional pores or channels are left after the material dissolves. A 65 will generally be about 0.001 to 50 mg per kg patient body matrix device is generally diffusion limited as well, but with weight per day, which may be administered in single or mul the channels or other internal geometry of the device also tiple doses. Preferably, the dosage level will be about 0.01 to US 8,440,627 B2 27 28 about 25 mg/kg per day; more preferably about 0.05 to about ylene glycols, and mixtures thereof and in oils. Under ordi 10 mg/kg per day. For example, a Suitable dosage level is nary conditions of storage and use, these preparations contain about 0.001 to 10 mg/kg per day, preferably about 0.005 to 5 a preservative to prevent the growth of microorganisms. mg/kg per day, and especially about 0.01 to 1 mg/kg per day. Therapeutic or pharmacological compositions of the For example, in patients with acute myocardial infarction, present invention will generally comprise an effective amount a Suitable pepducin (e.g. X1/2pal-il) may be administered i.v. of the component(s) of the combination therapy, dissolved or immediately as abolus dose, followed by additional i.v. injec dispersed in a pharmaceutically acceptable medium. Pharma tions once or twice daily for 2-3 days post MI. The dosage ceutically acceptable media or carriers include any and all may be around 0.1-0.5 mg/kg. Solvents, dispersion media, coatings, antibacterial and anti It will be appreciated that the amount of the compound of 10 fungal agents, isotonic and absorption delaying agents and the invention required for use in any treatment will vary not the like. The use of such media and agents for pharmaceutical only with the particular compounds or composition selected active Substances is well known in the art. Supplementary but also with the route of administration, the nature of the active ingredients can also be incorporated into the therapeu condition being treated, and the age and condition of the tic compositions of the present invention. patient, and will ultimately be at the discretion of the atten 15 The preparation of pharmaceutical or pharmacological dant physician. compositions will be known to those of skill in the art in light The compositions and combination therapies of the inven of the present disclosure. Typically, Such compositions may tion may be administered in combination with a variety of be prepared as injectables, either as liquid solutions or Sus pharmaceutical excipients, including Stabilizing agents, car pensions; Solid forms suitable for Solution in, or Suspension riers and/or encapsulation formulations as described herein. in, liquid prior to injection; as tablets or other solids for oral Aqueous compositions of the present invention comprise administration; as time release capsules; or in any other form an effective amount of the peptides of the invention, dissolved currently used, including cremes, lotions, mouthwashes, or dispersed in a pharmaceutically acceptable carrier or aque inhalants and the like. ous medium. Sterile injectable solutions are prepared by incorporating “Pharmaceutically or pharmacologically acceptable' 25 the active compounds in the required amount in the appropri include molecular entities and compositions that do not pro ate solvent with various of the other ingredients enumerated duce an adverse, allergic or other untoward reaction when above, as required, followed by filtered sterilization. Gener administered to an animal, or a human, as appropriate. "Phar ally, dispersions are prepared by incorporating the various maceutically acceptable carrier includes any and all sol sterilized active ingredients into a sterile vehicle which con vents, dispersion media, coatings, antibacterial and antifun 30 tains the basic dispersion medium and the required other gal agents, isotonic and absorption delaying agents and the ingredients from those enumerated above. In the case of ster like. The use of such media and agents for pharmaceutical ile powders for the preparation of sterile injectable solutions, active Substances is well known in the art. Except insofar as the preferred methods of preparation are vacuum-drying and any conventional media or agent is incompatible with the freeze-drying techniques which yield a powder of the active active ingredient, its use in the therapeutic compositions is 35 ingredient plus any additional desired ingredient from a pre contemplated. Supplementary active ingredients can also be viously sterile-filtered solution thereof. incorporated into the compositions. The preparation of more, or highly, concentrated Solutions For human administration, preparations should meet Ste for intramuscular injection is also contemplated. In this rility, pyrogenicity, general safety and purity standards as regard, the use of DMSO as solvent is preferred as this will required by FDA Office of Biologics standards. 40 result in extremely rapid penetration, delivering high concen The compositions and combination therapies of the inven trations of the active compound(s) or agent(s) to a small area. tion will then generally be formulated for parenteral admin The use of sterile formulations, such as saline-based istration, e.g., formulated for injection via the intravenous, washes, by Surgeons, physicians or health care workers to intramuscular, Subcutaneous, intralesional, or even intraperi cleanse a particular area in the operating field may also be toneal routes. The preparation of an aqueous composition that 45 particularly useful. Therapeutic formulations in accordance contains a composition of the invention or an active compo with the present invention may also be reconstituted in the nent or ingredient will be known to those of skill in the art in form of mouthwashes, or in conjunction with antifingal light of the present disclosure. Typically, such compositions reagents. Inhalant forms are also envisioned. The therapeutic can be prepared as injectables, either as liquid Solutions or formulations of the invention may also be prepared in forms Suspensions; Solid forms Suitable for using to prepare solu 50 Suitable for topical administration, such as in cremes and tions or Suspensions upon the addition of a liquid prior to lotions. injection can also be prepared; and the preparations can also Suitable preservatives for use in such a solution include be emulsified. benzalkonium chloride, benzethonium chloride, chlorobu The pharmaceutical forms suitable for injectable use tanol, thimerosal and the like. Suitable buffers include boric include sterile aqueous solutions or dispersions; formulations 55 acid, Sodium and potassium bicarbonate, sodium and potas including sesame oil, peanut oil or aqueous propylene glycol; sium borates, sodium and potassium carbonate, Sodium and sterile powders for the extemporaneous preparation of acetate, sodium biphosphate and the like, in amounts suffi sterile injectable solutions or dispersions. In all cases the form cient to maintain the pH at between about pH 6 and pH 8, and must be sterile and must be fluid to the extent that easy preferably, between about pH 7 and pH 7.5. Suitable tonicity syringability exists. It must be stable under the conditions of 60 agents are dextran 40, dextran 70, dextrose, glycerin, potas manufacture and storage and must be preserved against the sium chloride, propylene glycol, Sodium chloride, and the contaminating action of microorganisms, such as bacteria and like, such that the sodium chloride equivalent of the oph fungi. thalmic solution is in the range 0.9 plus or minus 0.2%. Solutions of active compounds as free base or pharmaco Suitable antioxidants and stabilizers include sodium bisulfite, logically acceptable salts can be prepared in water Suitably 65 sodium metabisulfite, sodium thiosulfate, thiourea and the mixed with a surfactant, such as hydroxypropylcellulose. like. Suitable wetting and clarifying agents include polysor Dispersions can also be prepared in glycerol, liquid polyeth bate 80, polysorbate 20, poloxamer 282 and tyloxapol. Suit US 8,440,627 B2 29 30 able viscosity-increasing agents include dextran 40, dextran isms can be brought about by various antibacterial and anti 70, gelatin, glycerin, hydroxyethylcellulose, hydroxymethyl fungal agents, for example, parabens, chlorobutanol, phenol, propylcellulose, lanolin, methylcellulose, petrolatum, poly Sorbic acid, thimerosal, and the like. In many cases, it will be ethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, preferable to include isotonic agents, for example, Sugars or carboxymethylcellulose and the like. sodium chloride. Prolonged absorption of the injectable com Upon formulation, therapeutics will be administered in a positions can be brought about by the use in the compositions manner compatible with the dosage formulation, and in Such of agents delaying absorption, for example, aluminum amount as is pharmacologically effective. The formulations monostearate and gelatin. are easily administered in a variety of dosage forms, such as Sterile injectable solutions are prepared by incorporating the type of injectable solutions described above, but drug 10 the active compounds in the required amount in the appropri release capsules and the like can also be employed. ate solvent with various of the other ingredients enumerated In this context, the quantity of active ingredient and Volume above, as required, followed by filtered sterilization. Gener of composition to be administered depends on the host animal ally, dispersions are prepared by incorporating the various to be treated. Precise amounts of active compound required sterilized active ingredients into a sterile vehicle which con for administration depend on the judgment of the practitioner 15 tains the basic dispersion medium and the required other and are peculiar to each individual. ingredients from those enumerated above. In the case of ster A minimal Volume of a composition required to disperse ile powders for the preparation of sterile injectable solutions, the active compounds is typically utilized. Suitable regimes the preferred methods of preparation are vacuum-drying and for administration are also variable, but would be typified by freeze drying techniques which yield a powder of the active initially administering the compound and monitoring the ingredient plus any additional desired ingredient from a pre results and then giving further controlled doses at further viously sterile-filtered solution thereof. intervals. For example, for parenteral administration, a Suit The preparation of more, or highly, concentrated Solutions ably buffered, and if necessary, isotonic aqueous solution for direct injection is also contemplated, where the use of would be prepared and used for intravenous, intramuscular, DMSO as solvent is envisioned to result in extremely rapid Subcutaneous or even intraperitoneal administration. One 25 penetration, delivering high concentrations of the active dosage could be dissolved in 1 ml of isotonic NaCl solution agents to a small area. and either added to 1000 ml of hypodermoclysis fluid or Upon formulation, Solutions will be administered in a man injected at the proposed site of infusion, (see for example, ner compatible with the dosage formulation and in Such Remington's Pharmaceutical Sciences 15th Edition, pages amount as is therapeutically effective. The formulations are 1035-1038 and 1570-1580). 30 easily administered in a variety of dosage forms, such as the In certain embodiments, active compounds may be admin type of injectable solutions described above, but drug release istered orally. This is contemplated for agents which are gen capsules and the like can also be employed. erally resistant, or have been rendered resistant, to proteolysis For parenteral administration in an aqueous solution, for by digestive enzymes. Such compounds are contemplated to examples the solution should be suitably buffered if neces include chemically designed or modified agents; dextrorota 35 sary and the liquid diluent first rendered isotonic with suffi tory peptides; and peptide and liposomal formulations in time cient Saline orglucose. These particular aqueous solutions are release capsules to avoid peptidase and lipase degradation. especially Suitable for intravenous, intramuscular, Subcutane Pharmaceutically acceptable salts include acid addition ous and intraperitoneal administration. In this connection, salts and which are formed with inorganic acids such as, for sterile aqueous media which can be employed will be known example, hydrochloric, hydrobromic, boric, phosphoric, Sul 40 to those of skill in the art in light of the present disclosure. furic acids or phosphoric acids, or Such organic acids as In addition to the compounds formulated for parenteral acetic, oxalic, tartaric, maleic, fumaric, citric, succinic, administration, such as intravenous or intramuscular injec mesylic, mandelic, Succinic, benzoic, ascorbic, methanesul tion, other pharmaceutically acceptable forms include, e.g., phonic, a-keto glutaric, a-glycerophosphoric, glucose-1- tablets or other solids for oral administration; liposomal for phosphoric acids and the like. Salts formed with the free 45 mulations; time-release capsules; and any other form cur carboxyl groups can also be derived from inorganic bases rently used, including cremes. Such as, for example, Sodium, potassium, ammonium, cal Additional formulations suitable for other modes of cium, magnesium, or ferric hydroxides, and Such organic administration include Suppositories. For Suppositories, tra bases as isopropylamine, trimethylamine, histidine, procaine ditional binders and carriers may include, for example, poly and the like. Other examples of pharmaceutically acceptable 50 alkylene glycols or triglycerides; Such suppositories may be salts include quaternary derivatives of the compounds of for formed from mixtures containing the active ingredient in the mula (I) such as the compounds quaternized by compounds range of 0.5% to 10%, preferably 1%-2%. R-T wherein R is C alkyl, phenyl-C alkyl or Cs, Oral formulations include Such normally employed excipi cycloalkyl, and T is a radical corresponding to an anion of an ents as, for example, pharmaceutical grades of mannitol, lac acid. Suitable examples of R include methyl, ethyl and n- and 55 tose, starch, magnesium Stearate, Sodium saccharine, cellu iso-propyl; and benzyl and phenethyl. Suitable examples of T lose, magnesium carbonate and the like. These compositions include halide, e.g., chloride, bromide or iodide. Yet other take the form of Solutions, Suspensions, tablets, pills, cap examples of pharmaceutically acceptable salts also include Sules, Sustained release formulations or powders. internal salts such as N-oxides. In certain defined embodiments, oral pharmaceutical com The carrier can also be a solvent or dispersion medium 60 positions will comprise an inert diluent or assimilable edible containing, for example, water, ethanol, polyol (for example, carrier, or they may be enclosed in hard or soft shell gelatin glycerol, propylene glycol, and liquid polyethylene glycol, capsule, or they may be compressed into tablets, or they may and the like), suitable mixtures thereof, and vegetable oils. be incorporated directly with the food of the diet. For oral The proper fluidity can be maintained, for example, by the use therapeutic administration, the active compounds may be of a coating, Such as lecithin, by the maintenance of the 65 incorporated with excipients and used in the form of ingest required particle size in the case of dispersion and by the use ible tablets, buccal tables, troches, capsules, elixirs, Suspen of Surfactants. The prevention of the action of microorgan sions, syrups, wafers, and the like. Such compositions and US 8,440,627 B2 31 32 preparations should contain at least 0.1% of active com or to be delayed in release. A variety of materials can be used pound. The percentage of the compositions and preparations for Such enteric layers or coatings, such materials including a may, of course, be varied and may conveniently be between number of polymeric acids and mixtures of polymeric acids about 2 to about 75% of the weight of the unit, or preferably with Such materials as shellac, cetyl alcohol and cellulose between 25-60%. The amount of active compounds in such acetate. therapeutically useful compositions is such that a suitable The liquid forms in which the compositions of the inven dosage will be obtained. tion may be incorporated for administration orally or by The tablets, troches, pills, capsules and the like may also injection include aqueous Solution, Suitably flavored syrups, contain the following: a binder, as gum tragacanth, acacia, aqueous or oil suspensions, and emulsions with acceptable cornstarch, or gelatin; excipients, such as dicalcium phos 10 oils such as cottonseed oil, sesame oil, coconut oil or peanut phate; a disintegrating agent, Such as corn starch, potato oil, or with a solubilizing or emulsifying agent Suitable for starch, alginic acid and the like; a lubricant, Such as magne intravenous use, as well as elixirs and similar pharmaceutical sium Stearate; and a Sweetening agent. Such as Sucrose, lac vehicles. Suitable dispersing or Suspending agents for aque tose or saccharin may be added or a flavoring agent, Such as ous Suspensions include synthetic and natural gums such as peppermint, oil of wintergreen, or cherry flavoring. When the 15 tragacanth, acacia, alginate, dextran, Sodium carboxymethyl dosage unit form is a capsule, it may contain, in addition to cellulose, methylcellulose, polyvinylpyrrolidone or gelatin. materials of the above type, a liquid carrier. Various other Compositions for inhalation or insufflation include solu materials may be present as coatings or to otherwise modify tions and Suspensions in pharmaceutically acceptable, aque the physical form of the dosage unit. For instance, tablets, ous or organic solvents, or mixtures thereof, and powders. pills, or capsules may be coated with shellac, Sugar or both. A The liquid or Solid compositions may contain Suitable phar syrup of elixir may contain the active compounds Sucrose as maceutically acceptable excipients as set out above. Prefer a Sweetening agent methyl and propylparabensas preserva ably the compositions are administered by the oral or nasal tives, a dye and flavoring, such as cherry or orange flavor. respiratory route for local or systemic effect. Compositions in The pharmaceutical compositions of this invention may be preferably sterile pharmaceutically acceptable solvents may used in the form of a pharmaceutical preparation, for 25 be nebulized by use of inert gases. Nebulized solutions may example, in Solid, semisolid or liquid form, which contains be breathed directly from the nebulizing device or the nebu one or more of the compound of the invention, as an active lizing device may be attached to a face mask, tent or inter ingredient, in admixture with an organic or inorganic carrier mittent positive pressure breathing machine. Solution, Sus or excipient Suitable for external, enteral or parenteral appli pension or powder compositions may be administered, cations. The active ingredient may be compounded, for 30 preferably orally or nasally, from devices which deliver the example, with the usual non-toxic, pharmaceutically accept formulation in an appropriate manner. able carriers for tablets, pellets, capsules, suppositories, solu For treating clinical conditions and diseases noted above, tions, emulsions, Suspensions, and any otherform Suitable for the compound of this invention may be administered orally, use. The carriers which can be used are water, glucose, lac topically, parenterally, by inhalation spray or rectally in dos tose, gum acacia, gelatin, mannitol, starch paste, magnesium 35 age unit formulations containing conventional non-toxic trisilicate, talc, corn starch, keratin, colloidal silica, potato pharmaceutically acceptable carriers, adjuvants and vehicles. starch, urea and other carriers Suitable for use in manufactur The term parenteral as used herein includes Subcutaneous ing preparations, in Solid, semisolid, or liquid form, and in injections, intravenous, intramuscular, intrasternal injection addition auxiliary, stabilizing, thickening and coloring agents or infusion techniques. and perfumes may be used. The active object compound is 40 Screening and Detection Methods included in the pharmaceutical composition in an amount The composition of the invention can be used to screen sufficient to produce the desired effect upon the process or drugs or compounds that modulate GPCR activity or expres condition of the disease. sion as well as to treat disorders characterized by insufficient For preparing Solid compositions such as tablets, the prin or excessive production of GPCR protein or production of cipal active ingredientis mixed with a pharmaceutical carrier, 45 GPCR protein forms that have decreased or aberrant activity e.g., conventional tableting ingredients such as corn starch, compared to GPCR wild-type protein. lactose, Sucrose, Sorbitol, talc, Stearic acid, magnesium Stear The invention provides a method (also referred to hereinas ate, dicalcium phosphate or gums, and other pharmaceutical a 'screening assay') for identifying modulators, i.e., candi diluents, e.g., water, to form a solid preformulation compo date or test compounds or agents (e.g., peptides, peptidomi sition containing a homogeneous mixture of a compound of 50 metics, small molecules or other drugs) that bind to GPCRs or the invention, or a non-toxic pharmaceutically acceptable salt have a stimulatory or inhibitory effect on, e.g., GPCR protein thereof. When referring to these preformulation compositions expression or GPCR activity. The invention also includes as homogeneous, it is meant that the active ingredient is compounds identified in the screening assays described dispersed evenly throughout the composition so that the com herein. position may be readily subdivided into equally effective unit 55 The invention provides assays for screening candidate or dosage forms such as tablets, pills and capsules. This solid test compounds which bind to or modulate the activity of the preformulation composition is then Subdivided into unit dos membrane-bound form of a pepducin-GPCR complex or bio age forms of the type described above containing from 0.1 to logically-active portion thereof. The test compounds of the about 500 mg of the active ingredient of the invention. The invention can be obtained using any of the numerous tablets or pills of the novel composition can be coated or 60 approaches in combinatorial library methods known in the otherwise compounded to provide a dosage form affording art, including for example, biological libraries; spatially the advantage of prolonged action. For example, the tablet or addressable parallel solid phase or solution phase libraries: pill can comprise an inner dosage and an outer dosage com synthetic library methods requiring deconvolution; the “one ponent, the latter being in the form of an envelope over the bead one-compound library method; and synthetic library former. The two components can be separated by an enteric 65 methods using affinity chromatography selection. The bio layer which serves to resist disintegration in the stomach and logical library approach is limited to peptide libraries, while permits the inner component to pass intact into the duodenum the other four approaches are applicable to peptide, non US 8,440,627 B2 33 34 peptide oligomer or Small molecule libraries of compounds. ing a detectable marker, e.g., luciferase), or detecting a cel See, e.g., Lam, 1997. Anticancer Drug Design 12: 145. lular response, for example, cell survival, cellular differentia Examples of methods for the synthesis of molecular librar tion, or cell proliferation. ies can be found in the art, for example in: DeWitt, et al., 1993. Alternatively, an assay of the invention is a cell-free assay Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. comprising contacting a GPCR peptide or biologically-active Natl. Acad. Sci. U.S.A. 91: 11422, Zuckermann, et al., 1994. portion thereof with a test compound and determining the J.Med. Chem. 37:2678; Cho, et al., 1993. Science 261: 1303: ability of the test compound to bind or modulate (e.g. Stimu Carrell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2059: late or inhibit) the activity of the GPCR protein or biologi Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; cally-active portion thereof. 10 Binding of the test compound to the GPCR can be deter and Gallop, et al., 1994. J. Med. Chem. 37: 1233. mined either directly or indirectly as described above. For Libraries of compounds may be presented in Solution (e.g., example, the assay comprises contacting the pepducin plus Houghten, 1992. Biotechniques 13: 412–421), or on beads the GPCR or biologically-active portion thereof with a known (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. compound which binds GPCR to form an assay mixture, Nature 364: 555-556), bacteria (Ladner, U.S. Pat. No. 5,223, 15 contacting the assay mixture with a test compound, and deter 409), spores (Ladner, U.S. Pat. No. 5.233.409), plasmids mining the ability of the test compound to interact with a (Cull, et al., 1992. Proc. Natl. Acad. Sci. USA89: 1865-1869) GPCR protein, wherein determining the ability of the test or on phage (Scott and Smith, 1990. Science 249: 386-390; compound to interact with a GPCR protein comprises deter Devlin, 1990. Science 249: 404–406; Cwirla, et al., 1990. mining the ability of the test compound to preferentially bind Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382: Felici, 1991. J. to GPCR or biologically-active portion thereofas compared Mol. Biol. 222: 301-310; Ladner, U.S. Pat. No. 5,233,409). to the known compound. An assay is a cell-based assay in which a cell which Determining the ability of the test compound to modulate expresses a membrane-bound form of a GPCR, or a biologi the activity of GPCR can be accomplished, for example, by cally-active portion thereof on the cell Surface, plus a pepdu determining the ability of the GPCR peptide to bind to a cin, is contacted with a test compound and the ability of the 25 GPCR target molecule by one of the methods described above test compound to bind to the GPCR and displace the pepducin for determining direct binding. Alternatively, determining the determined. The test compound could bind at the extracellu ability of the test compound to modulate the activity of GPCR lar Surface, transmembrane domains, or intracellular Surfaces peptide can be accomplished by determining the ability of the of the GPCR target and inhibit or enhance the pepducin GPCR peptide to further modulate a GPCR target molecule. activation of the GPCR. The cell, for example, is of mamma 30 For example, the catalytic/enzymatic activity of the target lian origin or a yeast cell. Determining the ability of the test molecule on an appropriate Substrate can be determined as compound to displace the pepducin from the GPCR protein described above. can be accomplished, for example, by coupling the pepducin The cell-free assay comprises contacting the GPCR pep to a radioisotope or enzymatic label Such that binding of the tide or biologically-active portion thereof with a known com test compound displaces the pepducin from the GPCR or 35 pound which binds the GPCR to form an assay mixture, biologically-active portion thereof. Alternatively, the test contacting the assay mixture with a test compound, and deter compounds can be labeled with 'I, S, ''C, or H, either mining the ability of the test compound to interact with a directly or indirectly, and the pepducin could displace the GPCR, wherein determining the ability of the test compound radio-labeled test compound from the GPCR and the free to interact with a GPCR comprises determining the ability of radio-labeled test compound detected by direct counting of 40 the GPCR peptide to preferentially bind to or modulate the radioemission or by Scintillation counting. Alternatively, test activity of a GPCR target molecule. compounds can be enzymatically-labeled with, for example, The cell-free assays of the invention are amenable to use of horseradish peroxidase, alkaline phosphatase, or luciferase, both the soluble form or the membrane-bound form of GPCR and the enzymatic label detected by increases or decreases in protein. In the case of cell-free assays comprising the mem conversion of an appropriate Substrate to product upon addi 45 brane-bound form of GPCR protein, it may be desirable to tion of pepducin. utilize a solubilizing agent Such that the membrane-bound In another embodiment, an assay is a cell-based assay form of GPCR protein is maintained in solution. Examples of comprising contacting a cell expressing a membrane-bound Such solubilizing agents include non-ionic detergents such as form of GPCR protein, or a biologically-active portion n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, thereof, on the cell surface with a test compound and deter 50 octanoyl-N-methylglucamide, decanoyl-N-methylglucam mining the ability of the test compound to modulate (e.g., ide, Triton RX-100, Triton RX-114, ThesitR, Isotridecypoly stimulate or inhibit) the binding, activity of the pepducin for (ethylene glycol ether), N-dodecyl-N,N-dimethyl-3-ammo the GPCR. nio-1-propane Sulfonate, 3-(3-cholamidopropyl) Determining the ability of the test molecule to interact with dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3- a GPCR target molecule can be accomplished by one of the 55 cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane methods described above for determining direct binding. In sulfonate (CHAPSO). one embodiment, determining the ability of the test molecule It may be desirable to immobilize either GPCR peptide or to inhibit the GPCR peptide interaction with a GPCR target its target molecule to facilitate separation of complexed from molecule can be accomplished by determining the activity of uncomplexed forms of one or both of the proteins, as well as the target GCPR-pepducin complex. For example, the activ 60 to accommodate automation of the assay. Binding of a test ity of the target molecule can be determined by inhibiting compound to GPCR protein, or interaction of GPCR protein GPCR-peptide induction of a cellular second messenger of with a pepducin in the presence and absence of a candidate the GPCR target (i.e. intracellular Ca", diacylglycerol, IP, compound, can be accomplished in any vessel Suitable for etc.), detecting catalytic/enzymatic activity dependent on containing the reactants. Examples of Such vessels include GPCR activation or inhibition, detecting the induction or 65 microtiter plates, test tubes, and micro-centrifuge tubes. In inhibition of a reporter gene (comprising a GPCR-responsive one embodiment, a fusion protein can be provided that adds a regulatory element operatively linked to a nucleic acid encod domain that allows one or both of the proteins to be bound to US 8,440,627 B2 35 36 a matrix. For example, GST-GPCR fusion peptides or GST trapped in the wells by antibody conjugation. Methods for target fusion proteins can be adsorbed onto glutathione detecting Such complexes, in addition to those described Sepharose beads (Sigma Chemical, St. Louis, Mo.) or glu above for the GST-immobilized complexes, include immu nodetection of complexes using antibodies reactive with the tathione derivatized microtiter plates, that are then combined GPCR peptide or target molecule, as well as enzyme-linked with the test compound or the test compound and either the assays that rely on detecting an enzymatic activity associated non-adsorbed target protein or GPCR peptide, and the mix with the GPCR peptide or target molecule. ture is incubated under conditions conducive to complex for Modulators of GPCR protein expression are identified in a mation (e.g., at physiological conditions for salt and pH). method wherein a cell is contacted with a candidate com Following incubation, the beads or microtiter plate wells are pound and the expression of GPCR mRNA or protein in the washed to remove any unbound components, the matrix... 10 cell is determined. The level of expression of GPCR mRNA immobilized in the case of beads, complex determined either or protein in the presence of the candidate compound is com directly or indirectly, for example, as described, vide Supra. pared to the level of expression of GPCR mRNA or protein in Alternatively, the complexes can be dissociated from the the absence of the candidate compound. The candidate com matrix, and the level of GPCR peptide binding or activity is pound can then be identified as a modulator of GPCR mRNA determined using standard techniques. or protein expression based upon this comparison. For Other techniques for immobilizing proteins on matrices are example, when expression of GPCR mRNA or protein is also used in the screening assays of the invention. For greater (i.e., statistically significantly greater) in the presence example, either the GPCR peptide or its target molecule can of the candidate compound than in its absence, the candidate be immobilized utilizing conjugation of biotin and streptavi- compound is identified as a stimulator of GPCR mRNA or din. Biotinylated GPCR peptide or target molecules can be protein expression. Alternatively, when expression of GPCR prepared from biotin-NHS (N-hydroxy-succinimide) using mRNA or protein is less (statistically significantly less) in the techniques well-known within the art (e.g., biotinylation kit, presence of the candidate compound than in its absence, the Pierce Chemicals, Rockford, Ill.), and immobilized in the candidate compound is identified as an inhibitor of GPCR wells of streptavidin-coated 96 well plates (Pierce Chemical). mRNA or protein expression. The level of GPCR mRNA or Alternatively, antibodies reactive with GPCR peptide or tar- protein expression in the cells can be determined by methods get molecules, but which do not interfere with binding of the described herein for detecting GPCR mRNA or protein. GPCR peptide to its cognate GPCR, can be derivatized to the The peptide sequences discussed herein are presented in wells of the plate, and unbound target or GPCR peptide the following Table 3. TABLE 3

SEO ID NO: SEQUENCE Name/Identifier 1 KYVVIIAYALVFLLSLLGNSLVMLVILYSRVGRSVTD CXCR1 HUMAN WYLLNLALADLLFALTLPIWAASK 2 KYVVIITYALVFLLSLLGNSLVMLVILYSRVGRSVTD CXCR1 PANTR WYLLNLALADLLFALTLPIWAASK 3 KYVVIITYALAFLLSLLGNSLVMLVILYSRGGRSVTD CXCR1 GORGO WYLLNLALADLLFALTLPIWAASK 4 KYVVVVIYALVFLLSLLGNSLVMLVILYSRSNRSVTD CXCR1 RABBIT WYLLNLAMADLLFALTMPIWAWSK 5 RQAVVVFYALVFLLSLLGNSLVMLVILYRRRTRSVTD CXCR1 RAT WYWLNLAIADLLFSLTLPFLAWSK 6 KYFVVIIYALVFLLSLLGNSLVMLVILYSRVGRSVTD CXCR2 HUMAN WYLLNLALADLLFALTLPIWAASK 7 SYAVVVIYVLVTLLSLVGNSLVMLVILYNRSTCSVTD CXCR2 MOUSE WYLLNLAIADLFFALTLPWWAASK 8 KYFVVIIYALVFLLSLLGNSLVMLVILYSRVGRSVTD CXCR2 PANTR WYLLNLALADLLFALTLPIWAASK 9 KYFVVIIYALVFLLSLLGNSLVMLVILYSRVGRSVTD CXCR2 MACMU WYLLNLALADLLFALTLPIWAASK 10 KYFVVIIYALVFLLSLLGNSLVILVILYSRVGRSVTD CXCR2 GORGO WYLLNLALADLLFALTLPIWAASK

11 SYVVLITYTLVFLLSLLGNSLVMLVILYSRSTCSVTD CXCR2 RABBIT WYLLNLAIADLLFATTLPTWAASK

12 KYAVVVIYVLVFVLNLLGNSLVIMVVLYSRVSHSVTD CXCR2 CANFA WYLLNLAIADLLFALTLPIWAWSK

13 KYAVVVIDALVFLLSLLGNSLVMLVILYSRIGRSVTD CXCR2 BOVIN WYLLNLAMADLLFAMTLPIWTASK

US 8,440,627 B2 44 TABLE 3 - continued

SEQ ID NO: SEQUENCE Name/Identifier

93. KWASAFIIILCCAIWWENLLWLIAWARNSKFHSAMYL EDG5 RAT FIGNLAASDLLAG

94 GALRGLSWAASCLWWLENLLWLAAITSHMRSRRWWYY EDG6 HUMAN CLWNITLSDLLTG

95 GMLRGPSWAAGCLWWLENAMIWLAAIAIYMRSRRWWYY EDG6 MOUSE CLINITISDLLTG

96 SSLNILFWWICSIIILENLLWLIAWFRNKKFHSAMFF EDG5 BRARE FIGNLAFSDLLAG

97 WIWLCWGTFFCLFIFFSNSLWIAAWIKNRKFHFPFYY EDG7 HUMAN LLANLAAADFFAG

98 WIWLCWGTFFCLFIFFSNSLWIAAWITNRKFHFPFYY EDG7 MOUSE LLANLAAADFFAG

99 WIWLCWGTFFCLFIFFSNSLWIAAWITNRKFHFPFYY EDG7 RAT LLANLAAADFFAG

1 OO WOIVLWAAAYTVIVWTSVVGNVVVMWIILAHKRMRTV NK1R HUMAN TNYFLWNLAFAEAS

101 WQIVLWAAAYTVIVWTSVVGNVVVIWIILAHKRMRTV NK1R MOUSE TNYFLWNLAFAEAC

1 O2 WQIVLWAAAYTVIVWTSVVGNVVVIWIILAHKRMRTV NK1R RAT TNYFLWNLAFAEAC

103 WQIVLWAAAYTVIVWTSVVGNVVVMWIILAHKRMRTV NK1R CAVPO TNYFLWNLAFAEAS

104 WOIALWSWAYSIIVIVSLVGNTIVMWIIIAHKRMRTV NK1R RANCA TNYFLWNLAFAEAS

The peptidic portion of the pepducin, e.g., from the il loop, Nature 401, 808 (1999). Maximum bleeding time allowed may be any suitable length that results in the intended ben was 10 min after which the tail was cauterized. eficial effects, generally 5 to 15, or, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 40 EXAMPLE 1. 29 or 30. Methods for preparing pepducin compounds of the inven To demonstrate how sepsis may be effectively treated by tion are illustrated in the following synthetic schemes and pepducins of the present invention, 6 to 8 week old female example(s). The following schemes, examples and biological CF-1 mice were treated with a 2.5 mg/kg pepducin prepara 45 tion in 20% DMSO, or vehicle (20% DMSO). The time point data are given for the purpose of illustrating the invention, but selected for treatment is at the induction of non-lethal sepsis not for limiting the scope or spirit of the invention. by cecal ligation and puncture (CLP). After an initial dose of Manufacture and Characterization of Pepducin Composi 2.5 mg/kg, 1 mg/kg pepducin (or vehicle) was injected into tions the mice subcutaneously each day for six days. *: Four mice Synthesis by standard Fmoc solid phase synthetic methods 50 out of the vehicle-treated group were sacrificed after six days: and preparation of palmitoylated pepducin peptides was per #: one mouse out of the groups treated with x 1/2i1LCA10 formed as generally described in Covic et al. PNAS 99:643 pepducin was sacrificed on day 6, and all remaining Surviving 648 (2002). Pepducins were purified to >95% purity by Cs or mice were sacrificed at 17 days. FIG. 5 shows the results. In C. reverse phase chromatography and dissolved in DMSO. the left panel, mice were treated with x 1/2pal 16, Bleeding times were performed with 6-8 week-old adult 55 X1/2i1LCA10 or vehicle (20% DMSO) at the time point of male CF-1 mice anaesthetized with an intraperitoneal injec CLP. In the right panel, mice were monitored for body weight tion of Xylazine (10 mg/kg) plus ketamine (50 mg/kg). The every day. Vehicle-treated mice showed severe weight loss, internal jugular vein was cannulated with a 0.28x1.52 mm whereas pepducin-treated mice gained weight after an initial gauge catheter and P1 pal-12 (3 umoles/L), P4pal-10 (3 weight loss. umoles/L) or vehicle alone (DMSO), was infused over 1 min 60 in a total volume of 100 uL. Experiments were performed EXAMPLE 2 blind to injected substance. After 5 min, tails were amputated 2 mm from the tail tip. Tails were immersed in a beaker of There are only few reports where treatment of sepsis is phosphate-buffered saline maintained at 37°C. and bleeding sufficient, even after the onset of systemic inflammatory was visually followed and timed. If bleeding restarted within 65 response syndrome. To demonstrate that sepsis may be effec 5 min, it was recorded as a re-bleed and taken to mark an tively treated by pepducins of the present invention well after unstable hemostasis event as previously described. Law et al., initiating sepsis, mice were treated with a 5.0 mg/kg pepducin US 8,440,627 B2 45 46 preparation in 40% DMSO, or vehicle (40% DMSO). The in mice. Mice were injected with 1 mL of 3% thioglycolate time point selected for treatment is eight hours at the induc i.p. Thioglycollate induces peritoneal macrophages to secret tion of non-lethal sepsis by cecalligation and puncture (CLP). cytokines and leukocytes are then recruited into the peritoneal After an initial dose of 5.0 mg/kg, 2.5 mg/kg pepducin (or cavity. Pepducins were injected i.v. at the indicated doses. vehicle) was injected into the mice Subcutaneously each day After 4 hours, cells were collected by peritoneal lavage, from day 2 to day 3, and 1.0 mg/kg from day 3 to day 6. Eight stained with Giemsa and counted under 40x magnification. hours after CLP, the mice already showed signs of systemic N=60. FIG. 10 shows the results. illness characteristic of sepsis, e.g., ruffled fur, decreased mobility, decreased food and water intake, and closed, EXAMPLE 7 inflamed eyes. FIG. 6 shows the results. In the left panel, *: 10 two mice out the vehicle treated group were sacrificed on day It has been shown that SDF-1C. and CXCR4 are involved in 6. Mice that survived the experiment were sacrificed on day recruiting immature neutrophils from the bone marrow. High 17. In the right panel, mice were monitored for their body levels of CXCR4 expression and activation lead to homing of weight every day. senescent neutrophils to the bone marrow, whereas chemok 15 ines acting on the CXCR2 receptors of neutrophils, i.e., KC, EXAMPLE 3 Gro-C. or IL-8, lead to increased neutrophil mobilization. To demonstrate the utility of pepducin treatment of inflam Blood was drawn 1, 3, 5, 7 and 17 days after CLP, and matory disorders, mice were treated with pepducins of the leukocytes were counted. An initial decrease in leukocytes in invention. FIG. 7 shows the results. Adult respiratory vehicle-treated mice was observed. However, after 3 days of response syndrome (ARDS) results from excessive neutro sepsis, these mice showed high levels of peripheral leuko phil infiltration into lungs and their activation, leading to cytes, which remained at high levels until the end of the fibrin deposition. Sepsis can cause ARDS, which has a high experiment. In X1/2pal16 and X1/2i1LCA10-treated mice, lethality. Panels A and B: Mice were sacrificed 24 hours after leukocyte levels remained in the normal range. In X4il pal12 CLP and BAL was performed. Neutrophils were counted 25 treated mice, leukocytes levels increased immediately, but after staining with Giemsa, TNF was measured by ELISA. decreased 5 days after CLP to very low levels. This observa *p-0.02 vs. vehicle treated group. C Lung histology. Mice tion would match previous reports where either blocking of were sacrificed 48 hours after CLP. CXCR4 or stimulation of CXCR2 lead to neutrophilia in the peripheral blood of mice. The increase of leukocytes in the EXAMPLE 4 30 peripheral blood after X4ilpal 12 treatment shows that CXCR2 blockade prevents peripheral blood neutrophilia. This example demonstrates how pepducin treatment may FIG. 11 shows the results. be used to alleviate another aspect of sepsis. Systemic inflam matory responses, the derangement of coagulation and fibrin EXAMPLE 8 olysis in DIC is mediated by several pro-inflammatory cytok 35 ines. Changes in bleeding time and increases in D-dimer, Chemotaxis assays were performed in modified 48-well which is a product of excessive counter-regulatory fibrinoly micro-chemotaxis chambers. 1 mio neutrophils/mL were sis, is a hallmark of disseminated intravascular coagulation, allowed to migrate toward the chemoattractant in the lower leading to widespread deposition of fibrin in the circulation, wells. Neutrophils were pretreated with either 0.2% DMSO contributing to multi organ failure and death in Septic 40 or the pepducins at the indicate concentrations. N=3. FIG. 12 patients. FIG. 8 shows the results. A. Bleeding time was shows that ill-loop pepducins of the invention have potent and measured 24 h after CLP and immediate onset of treatment. selective anti-inflammatory action. Bleeding time was shortened in vehicle-treated mice, whereas the ill-loop pepducin X1/2i1LCA10- or the i3 loop EXAMPLE 9 pepducin X1/2pal 16-treated mice showed normal bleeding 45 times. B. D-dimer levels were measured 48 hours after CLP. Chemotaxis assays were performed in modified 48-well As a Surrogate marker of the amount of fibrin deposition, micro-chemotaxis chambers. 1 mio neutrophils/mL were D-dimer was increased after CLP to a higher extent in allowed to migrate toward Rantes-ligand for CCR1, CCR3 vehicle-treated mice than in CXCR2 pepducin-treated mice. and CCR5 (20 ng/mL) in the lower wells. Neutrophils were C. Depicts the bleeding time of mice that were treated 8 h after 50 pretreated with either 0.2% DMSO or the pepducins at the CLP. D. Depicts the D-dimer levels of mice treated 8 h after indicate concentrations. N=3. FIG. 13 shows that ill-loop CLP. pepducins of the invention have potent and selective anti inflammatory action. EXAMPLE 5 55 EXAMPLE 10 To demonstrate the utility of pepducin treatment of inflam matory disorders, chemotaxis assays were performed in Since organ failure determines the outcome of patients in modified 48-well microchemotaxis chambers. 1 mio neutro SIRS, liver function was investigated. Mice were sacrificed phils/mL were allowed to migrate toward the chemoattractant 24 hours after CLP and plasma levels of ALT, an enzyme that in the lower wells. Neutrophils were pretreated with either 60 increases with loss of liver function, were measured. In sham 0.2% DMSO or the pepducins at the indicate concentrations. treated mice, i.e., abdominal incision without cecal ligation N=3. FIG. 9 shows the results. and puncture, no elevation of ALT was observed. Vehicle and x4pal12 treated mice showed an increase in ALT, with EXAMPLE 6 x 1/2pal16 and X1/2LCA10 treated mice an increase was 65 observed but it was significantly less compared to vehicle To demonstrate the utility of pepducin treatment of inflam treated mice. FIG. 22 shows the results, that CXCR2 pep matory disorders in vivo, chemotaxis assays were performed ducins can be used to improve liver function. US 8,440,627 B2 47 48 EXAMPLE 11 concentrations, PAR4 is activated by thrombin unless blocked downstream with the PAR4 pepducin. CLP was performed on 6 to 8 week old female CF-1 mice. In the right panel, mice were treated with x4ilpal 12 or vehicle EXAMPLE 1.4 (20% DMSO) at the time point of CLP. After the initial dose of 2.5 mg/kg, animals received 1 mg/kg of pepducin or Arterial thrombosis in guinea pigs is prevented using a vehicle s.c. every day for 6 days. In the left panel, mice were combination of bivalirudin and a PAR4 pepducin. A standard monitored for body weight every day. FIG. 23 shows the carotid artery injury model to assess the efficacy of simulta results. neous administration of bivalirudin and the PAR4 pepducin 10 on arterial thrombosis in guinea pigs was used. Unlike mice EXAMPLE 12 which lack PAR1 on their platelets, guinea pigs share func tional similarity with human platelets and express both PAR1 A more selective PAR4 pepducin based on the first intrac and PAR4. Consistent with earlier results using hirudin, biva ellular loop (i1) of the receptor was developed. Because the il lirudin alone (0.18 mg/kg) prolonged the mean arterial occlu 15 sion time from 13 minto 20 min, though this was not signifi loop is on the opposite side of the receptor relative to the i3 cant (FIG. 26). P4pal-i1 (0.13 mg/kg) prolonged the loop, an ill loop pepducin based on PAR4 exhibits reduced or occlusion time to a similar extent. Co-administration of biva no cross-inhibition of PAR1. PAR4 was expressed on HEK lirudin plus P4 pal-il caused a significant (p=0.0001) protec cells singly or in combination with PAR1. The PAR4 i1 pep tion against acute arterial occlusion. As has been shown with ducin, P4pal-il, completely blocked the chemotactic RWJ-58259, blockade of PAR1 alone with the PAR1 pepdu response of PAR4 on HEK cells and prevented platelet aggre cin, P1 pal-7, caused only partial protection of arterial throm gation to its peptide ligand, AYPGKF (SEQ ID NO. 34), as bosis. In comparison to P4pal-il, supplementation of P1 pal-7 shown in FIGS. 24A-B. P4pal-il was selective for PAR4 and with bivalirudin gave no additional prolongation of the arte did not inhibit the chemotactic response of PAR1 nor did it rial occlusion time. Together, these in vivo data indicate that appreciably inhibit platelet aggregation to the PAR1 peptide 25 PAR4 must also be blocked to achieve significant protection ligand, SFLLRN (SEQID NO. 35), as seen in FIGS. 24A and against thrombosis. C. P4pal-il did not inhibit PAR1 even upon co-expression These experiments support earlier observations with PAR1 with PAR4 indicating that if PAR1 and PAR4 form a complex, and PAR4 pepducinantagonists and blocking antibodies, that the bound il pepducin of PAR4 does not appreciably affect targeting only PAR1 and not PAR4 may have a limited thera signaling from PAR1. Likewise, when used alone, P4pal-il 30 peutic effect. As an alternative therapeutic strategy, prevent had only a minor effect on platelet aggregation to 3 nM ing the interaction of the PAR1 Hir motif with thrombin has thrombin since thrombin also activates PAR1 (FIG. 24D). the dual benefit of directly inhibiting PAR1 and indirectly However, when used in combination with the PAR1 antago inhibiting PAR4. These data demonstrate for the first time that nist, RWJ-561 10, P4pal-il greatly inhibited aggregation to 3 the widely used anti-thrombotic agent, bivalirudin, was effi nM thrombin. Thus, targeting either PAR1 or PAR4 alone has 35 cacious in blocking thrombin activation of both PAR1 and a limited effect on thrombin aggregation, whereas simulta PAR4-dependent platelet aggregation. In combination with a neous inhibition of both PAR1 and PAR4 is effective in block PAR4 pepducin but not a PAR1 pepducin, bivalirudin was ing the response to thrombin, thereby reducing or inhibiting able to prevent acute arterial thrombosis. thrombosis. 40 EXAMPLE 1.5 EXAMPLE 13 Pepducins of the invention are used to interrupt established systemic inflammation and vascular damage as well prevent The effect of bivalirudin on platelet PAR1 and PAR4 activation of the coagulation cascade without interference responses was examined, and the efficacy of inhibiting throm 45 with host defense. il pepducins of the invention, e.g., based bin-PAR1 interactions in combination with PAR4 blockade on CXCR1/2, do not cross-inhibit CXCR4, i1 pepducins on platelet aggregation was assessed. To prevent thrombin completely block the chemotaxis responses of their cognate from binding to the Hir site of PAR1, bivalirudin (aka Hiru receptors on human and mouse neutrophils. X1/2LCA-il is log(R), AngiomaxTM), which binds to thrombin with nanomo selective for the CXCR1/2 IL-8 receptors and does not inhibit lar affinity was used. Despite its widespread use in treating 50 migration of human normouse leukocytes towards SDF-1C. patients with acute coronary syndromes, the effects of biva Likewise, X1/2LCA-il does not inhibit CXCR4 even when lirudin on PAR1 and PAR4-dependent platelet activation have co-expressed with CXCR1 or CXCR2. These data indicate not been determined prior to the invention. Bivalirudin alone that if the CXCR1 and CXCR4 receptors form a complex, the or bivalirudin plus RWJ-56110 gave a similar ECs (10-11 bound il pepducin of CXCR1 does not affect signaling from nM) of thrombin activation of PAR4-dependent aggregation 55 CXCR4. as the PAR1-Ab (FIG. 25A). At concentrations greater than The effects of the CXCR1/2 and CXCR4 pepducins on 11 nM, thrombin regained full activation of PAR4-dependent neutrophil homeostasis under normal versus pro-inflamma aggregation even when RWJ-56110 was supplemented with tory conditions in mice were evaluated. Leukocyte recruit the PAR1-blocking antibody plus bivalirudin. These data ment was first assessed in a thioglycollate peritonitis model. indicate that neither of the Hir-blocking agents completely 60 The X1/2pal-i3 and X1/2LCA-il-pepducins completely inhib prevented interaction of the active site of thrombin with plate ited transmigration of neutrophils into the peritoneal cavity let PAR4. However, addition of the PAR4 pepducin, P4pal-il, with IC50 values of 0.03 mg/kg and 0.15 mg/kg, respectively. to bivalirudin-treated platelets greatly delayed and inhibited Conversely, mice treated with the CXCR4 antagonist, X4 pal the extent of aggregation even at very high thrombin (12-16 i1 (EC50-0.1 mg/kg), showed a substantial increase in peri nM) concentrations (FIG. 25B). These data reveal a mecha 65 toneal neutrophils. nism in which thrombin-docked to the PAR1 Hir-motif The long-term effects of the chemokine pepducins on enhances activation of PAR4, but at high enough thrombin peripheral leukocyte counts were assessed in healthy US 8,440,627 B2 49 50 untreated mice. Pepducins were injected once per day Sub-Q LPS-simulated epithelial and endothelial monolayers. Like (2.5 mg/kg day 1, 1.0 mg/kg days 2-6) and circulating leuko wise, the CXCR1/2 pepducins prevented IL-8 dependent cyte levels measured over a one week period. In the healthy chemotaxis of human macrophages but not monocytes as mice, neither X1/2pal-i3 nor x 1/2LCA-i1 altered the leuko expected. CXCR1/2 pepducins significantly inhibited in vivo cyte count as compared to vehicle-treated mice. In contrast, mouse macrophage transmigration into the peritoneal cavity. X4pal-il caused a leukocytosis in peripheral blood, consistent Together, these data indicate that the pepducins exert their with previous studies. The effects of the chemokine pep therapeutic effects by blocking CXCR1/2 receptors on many ducins on peripheral leukocyte count were quite similar in cell types. septic mice Subjected to cecal ligation and puncture (CLP). Liver failure is a common Sequalae of severe sepsis. Treat Vehicle-treated mice exhibited an initial leukopenia at 24h 10 ment with the CXCR1/2 pepducins right after CLP reduced after CLP, then leukocytosis on day 3. Once per day admin liver damage as evidenced by decreases of 52-87% in liver istration of X1/2pal-i3 or x 1/2LCA-il kept the neutrophil enzyme levels (AST, ALT) whereas the CXCR4 pepducin, count of the CLP-mice within the normal range, whereas x4pal-il, had no effect. When CXCR1/2 pepducin treatment X4pal-il led to an accelerated leukocytosis on day 1 and was initiated 8 h after CLP, the rise in plasma AST and ALT leukopenia by day 7. Together, these data indicate that 15 levels were halted by the 16 h time point and then dropped to CXCR2 and CXCR4 receptors play opposing roles in neu near normal levels. Together, these data indicate that block trophil homeostasis in both normal and pro-inflammatory ade of CXCR1/2 signaling with pepducins reverses several states in mice. criteria of established SIRS in the septic mice, indicating that Chemokine receptor pepducins were tested for the ability this approach is beneficial in the setting of advanced disease. to protector potentially reverse the progression of lethal CLP Spontaneous bleeding and the development of thrombi was peritonitis in mice. In the first set of experiments, pepducins observed in many of the untreated CLP mice, consistent with were given immediately after the CLP procedure at a dose of the development of disseminated intravascular coagulation 2.5 mg/kg. The Subsequent doses were 1 mg/kg until day 6 (DIC). Therefore, systemic platelet activation was observed after which treatment was stopped. None of the mice received by measuring platelet counts and D-dimer levels, a marker of antibiotic therapy. A highly significant decrease was observed 25 the increased fibrin production and subsequent fibrinolysis in sepsis-induced mortality for both the X1/2LCA-il and that occurs during DIC. CXCR1/2 pepducins significantly x 1/2pal-i3 pepducins. Strikingly, over the 17-d observation protected against thrombocytopenia at 24 h even when pep period only 1/34 pepducin-treated mice died, whereas 17/17 ducin treatment was delayed until 8 h after CLP D-dimer of the untreated mice died by day 9. Despite discontinuation levels were found to be highly elevated 48 h after the CLP of CXCR1/2 pepducin therapy at day 6, survival remained 30 procedure in untreated mice. However, X1/2pal-i3 and nearly 100% thereafter. x 1/2LCA-il-treated mice showed significant reductions in The diagnosis of sepsis is often delayed and agents cannot levels of D-dimer. When the CXCR1/2 pepducin treatment be readily administered in a preventative mode, therefore was delayed until 8 h after the CLP procedure, D-dimer levels pepducin treatment was withheld until 8 h after the CLP were still reduced by 60-70%. These data are the first dem procedure. Even with delayed treatment, a highly significant 35 onstration that the coagulopathy that develops in overt sepsis reduction in mortality was seen as compared to untreated is ameliorated by blockade of CXCR2. mice. Overall survival was 26/30 in the delayed CXCR1/2 Recent studies using mice that lack a functional CXCR2 pepducin-treated mice as opposed to 0/20 of the untreated receptor showed increased survival in the CLP sepsis model, mice. Administration of the CXCR4 pepducin, X4 pal-il, had albeit with 3 to 4-fold higher mortality rates than observed in no effect on survival. CXCR1/2-pepducin treated mice 40 our study. The improved outcome in mortality following pep gained weight, were active and maintained normal grooming ducin treatment relative to the effect of deletion of CXCR2 behavior after day 1 even when pepducintherapy was delayed could be due to several factors. CXCR2 (-/-) mice lack by 8 h. CXCR2 during their entire life span and have additional Blockade of CXCR1/2 signaling with pepducins reversed defects in their adaptive immune system making it hard to several criteria of established SIRS in the septic mice. First, 45 distinguish between acute CXCR2-specific effects and com the effect of CXCR1/2 pepducins on systemic KC levels, the pensatory mechanisms arising from abnormal myelopoiesis. mouse IL-8 ortholog was examined. In untreated mice, KC The CF-1 mice used are outbred wild-type animals which levels rose over the 16 h period after CLP and remained might confer a Survival advantage in the context of the severe elevated for at least 48 h. Following administration of the immunogenic challenge from CLP. It is noteworthy that the CXCR1/2 pepducins at the 8 h time point, the systemic KC 50 anti-CXCR1/2 pepducins do not Suppress leukocyte migra levels rapidly dropped and remained low thereafter. Many of tion toward other chemokines such as bacterial fMLP, there the untreated CLP mice became tachypnic and hypoxemic as fore, the pepducin effects may be considered to be immuno early as 24 h and this correlated well with mortality. During modulatory rather than immunosuppressive. Furthermore, sepsis, bacterial endotoxin stimulates lung epithelia to secrete the application of pepducin technology helps validate the IL-8 which recruits and activates leukocytes. The resulting 55 results from genetic knock-out and provides further insight neutrophil margination eventually leads to lung damage. The into the etiology and treatment of complex diseases Such as number of neutrophils in the bronchioalveolar lavage (BAL) sepsis. fluid of untreated mice increased by 100-fold as early as 4h Equivalents after CLP and then rose to 200-300 fold after 8 h. Treatment Those skilled in the art will recognize, or be able to ascer with x 1/2pal-i3 or x 1/2LCA-il at 8 h caused a rapid drop in 60 tain using no more than routine experimentation, numerous BAL neutrophils which stayed at low levels. Histological equivalents to the specific procedures described herein. Such analyses of the lungs harvested at 48 h in the untreated CLP equivalents are considered to be within the scope of the inven mice revealed collapsed alveoli, leukocyte infiltration and tion and are covered by the following claims. Various substi extensive fibrin deposition. x 1/2pal-i3 and X1/2LCA-il gave tutions, alterations, and modifications may be made to the significant protection against fibrin deposition and alveoli 65 invention without departing from the spirit and scope of the appeared histologically normal. The CXCR1/2 pepducins invention as defined by the claims. Other aspects, advantages, also completely blocked transmigration of leukocytes across and modifications are within the scope of the invention. The US 8,440,627 B2 51 52 contents of all references, issued patents, and published nents, processes, and methods of those patents, applications patent applications cited throughout this application are and other documents may be selected for the invention and hereby incorporated by reference. The appropriate compo embodiments thereof.

SEQUENCE LISTING

NUMBER OF SEO ID NOS: 104

SEO ID NO 1. LENGTH: 61 TYPE PRT ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 1.

Lys Tyr Val Val Ile Ile Ala Ala Lell Wall Phe Luell Tell Ser Lell 1. 5 15

Lieu Gly Asn. Ser Lieu Wall Met Lell Wall Ile Luell Tyr Ser Arg Val Gly 2O 25 3 O

Arg Ser Val Thr Asp Wall Tyr Lell Lell Asn Luell Ala Luell Ala Asp Lieu 35 4 O 45

Lieu. Phe Ala Lieu. Thir Lieu. Pro Ile Trp Ala Ala Ser SO 55 60

<21 Os SEQ ID NO 2 LENGTH: 61 TYPE PRT &213s ORGANISM: Pan troglodytes

SEQUENCE: 2

Llys Tyr Val Val Ile Ile Thr Ala Lieu Wall Phe Lieu Luell Ser Lieu. 1. 5 15

Lieu Gly Asn. Ser Lieu Wall Met Lell Wall Ile Luell Tyr Ser Arg Val Gly 2O 25 3 O

Arg Se Wall. Thir Asp Wall Tyr Lell Lell Asn Luell Ala Luell Ala Asp Lieu 35 4 O 45

Lieu. Phe Ala Lieu. Thir Lieu. Pro Ile Trp Ala Ala Ser SO 55 60

<21 Os SEQ ID NO 3 LENGTH: 61 TYPE PRT <213> ORGANISM: Gorilla gorilla

SEQUENCE: 3

Lys Tyr Val Val Ile Ile Thir Tyr Ala Lell Ala Phe Luell Tell Ser Lell 5 15

Lieu Gly Asn. Ser Lieu Wall Met Lell Wall Ile Luell Tyr Ser Arg Gly Gly 2O 25 3 O

Arg Se Wall. Thir Asp Wall Tyr Lell Lell Asn Luell Ala Luell Ala Asp Lieu 35 4 O 45

Lieu. Phe Ala Lieu. Thir Lieu. Pro Ile Trp Ala Ala Ser SO 55 60

SEO ID NO 4. LENGTH: 61 TYPE PRT ORGANISM: Rabbit

< 4 OOs SEQUENCE: 4. Llys Tyr Val Val Val Val Ile Tyr Ala Lieu Val Phe Lieu Lleu Ser Lieu. 1. 5 15 US 8,440,627 B2 53 - Continued Lieu. Gly Asn. Ser Lieu Val Met Lieu Val Ile Lieu. Tyr Ser Arg Ser Asn 2O 25 3O Arg Ser Val Thr Asp Val Tyr Lieu. Lieu. Asn Lieu Ala Met Ala Asp Lieu 35 4 O 45 Lieu. Phe Ala Lieu. Thr Met Pro Ile Trp Ala Val Ser Lys SO 55 6 O

<210s, SEQ ID NO 5 &211s LENGTH: 61 212. TYPE: PRT &213s ORGANISM: Rat

<4 OOs, SEQUENCE: 5 Arg Glin Ala Val Val Val Phe Tyr Ala Lieu Val Phe Lieu. Lieu. Ser Lieu 1. 5 1O 15 Lieu. Gly Asn. Ser Lieu Val Met Lieu Val Ile Lieu. Tyr Arg Arg Arg Thr 2O 25 3O Arg Ser Val Thr Asp Val Tyr Val Lieu. Asn Lieu Ala Ile Ala Asp Lieu 35 4 O 45 Lieu. Phe Ser Lieu. Thir Lieu Pro Phe Leu Ala Val Ser Lys SO 55 6 O

<210s, SEQ ID NO 6 &211s LENGTH: 61 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6 Llys Tyr Phe Val Val Ile Ile Tyr Ala Lieu Val Phe Lieu. Lieu. Ser Lieu. 1. 5 1O 15 Lieu. Gly Asn. Ser Lieu Val Met Lieu Val Ile Lieu. Tyr Ser Arg Val Gly 2O 25 3O Arg Ser Val Thr Asp Val Tyr Lieu. Lieu. Asn Lieu Ala Lieu Ala Asp Lieu 35 4 O 45 Lieu. Phe Ala Lieu. Thir Lieu Pro Ile Trp Ala Ala Ser Lys SO 55 6 O

<210s, SEQ ID NO 7 &211s LENGTH: 61 212. TYPE: PRT &213s ORGANISM: Mouse

<4 OO > SEQUENCE: 7 Ser Tyr Ala Val Val Val Ile Tyr Val Lieu Val Thr Lieu. Leu Ser Lieu. 1. 5 1O 15 Val Gly Asn. Ser Lieu Val Met Lieu Val Ile Lieu. Tyr Asn Arg Ser Thr 2O 25 3O Cys Ser Val Thir Asp Val Tyr Lieu. Lieu. Asn Lieu Ala Ile Ala Asp Lieu. 35 4 O 45 Phe Phe Ala Lieu. Thir Lieu Pro Val Trp Ala Ala Ser Lys SO 55 6 O

<210s, SEQ ID NO 8 &211s LENGTH: 61 212. TYPE: PRT <213> ORGANISM: Pan troglodytes

<4 OOs, SEQUENCE: 8 Llys Tyr Phe Val Val Ile Ile Tyr Ala Lieu Val Phe Lieu. Lieu. Ser Lieu. 1. 5 1O 15 US 8,440,627 B2 55 - Continued Lieu. Gly Asn. Ser Lieu Val Met Lieu Val Ile Lieu. Tyr Ser Arg Val Gly 2O 25 3O Arg Ser Val Thr Asp Val Tyr Lieu. Lieu. Asn Lieu Ala Lieu Ala Asp Lieu 35 4 O 45 Lieu. Phe Ala Lieu. Thir Lieu Pro Ile Trp Ala Ala Ser Lys SO 55 6 O

<210s, SEQ ID NO 9 &211s LENGTH: 61 212. TYPE: PRT <213s ORGANISM: Macaca mulatta

<4 OOs, SEQUENCE: 9 Llys Tyr Phe Val Val Ile Ile Tyr Ala Lieu Val Phe Lieu. Lieu. Ser Lieu. 1. 5 1O 15 Lieu. Gly Asn. Ser Lieu Val Met Lieu Val Ile Lieu. Tyr Ser Arg Val Gly 2O 25 3O Arg Ser Val Thr Asp Val Tyr Lieu. Lieu. Asn Lieu Ala Lieu Ala Asp Lieu 35 4 O 45 Lieu. Phe Ala Lieu. Thir Lieu Pro Ile Trp Ala Ala Ser Lys SO 55 6 O

<210s, SEQ ID NO 10 &211s LENGTH: 61 212. TYPE: PRT <213> ORGANISM: Gorilla gorilla <4 OOs, SEQUENCE: 10 Llys Tyr Phe Val Val Ile Ile Tyr Ala Lieu Val Phe Lieu. Lieu. Ser Lieu. 1. 5 1O 15 Lieu. Gly Asn. Ser Lieu Val Ile Lieu Val Ile Lieu. Tyr Ser Arg Val Gly 2O 25 3O Arg Ser Val Thr Asp Val Tyr Lieu. Lieu. Asn Lieu Ala Lieu Ala Asp Lieu 35 4 O 45 Lieu. Phe Ala Lieu. Thir Lieu Pro Ile Trp Ala Ala Ser Lys SO 55 6 O

<210s, SEQ ID NO 11 &211s LENGTH: 61 212. TYPE: PRT <213> ORGANISM: Rabbit

<4 OOs, SEQUENCE: 11 Ser Tyr Val Val Lieu. Ile Thr Tyr Ile Leu Val Phe Lieu. Leu Ser Lieu. 1. 5 1O 15 Lieu. Gly Asn. Ser Lieu Val Met Lieu Val Ile Lieu. Tyr Ser Arg Ser Thr 2O 25 3O Cys Ser Val Thir Asp Val Tyr Lieu. Lieu. Asn Lieu Ala Ile Ala Asp Lieu. 35 4 O 45 Lieu. Phe Ala Thr Thr Lieu Pro Ile Trp Ala Ala Ser Lys SO 55 6 O

<210s, SEQ ID NO 12 &211s LENGTH: 61 212. TYPE: PRT <213> ORGANISM: Canis familiaris

<4 OOs, SEQUENCE: 12 Llys Tyr Ala Val Val Val Ile Tyr Val Lieu Val Phe Val Lieu. Asn Lieu. 1. 5 1O 15 US 8,440,627 B2 57 58 - Continued Lell Gly Asn. Ser Lieu. Wall Ile Met Val Val Lieu. Tyr Ser Arg Val Ser 2O 25

His Ser Wall Thir Asp Wall Tyr Luell Lieu. Asn Lieu Ala Ile Ala Asp Lieu. 35 4 O 45

Lell Phe Ala Lieu. Thir Lell Pro Ile Trp Ala Val Ser Lys SO 55 6 O

<21Os SEQ ID NO 13 <211 > LENGTH: 61 <212> TYPE PRT &213s ORGANISM: Bovine

< 4 OOs SEQUENCE: 13

Lys Tyr Ala Val Val Wall Ile Asp Ala Luell Wall Phe Lell Luell Ser Luell 1. 1O 15

Lell Gly Asn. Ser Lieu. Wall Met Luell Wall Ile Luell Tyr Ser Arg Ile Gly 25 3O

Arg Ser Val Thr Asp Wall Tyr Luell Luell Asn Luell Ala Met Ala Asp Lieu 35 4 O 45

Lell Phe Ala Met Thir Lell Pro Ile Trp Thir Ala Ser SO 55 6 O

SEQ ID NO 14 LENGTH: 61 TYPE PRT &213s ORGANISM: Rat

SEQUENCE: 14

Arg Tyr Ala Val Val Wall Ile Tyr Wall Luell Wall Thir Lell Luell Ser Luell 1. 5 1O 15

Wall Gly Asn. Ser Lieu. Wall Met Luell Wall Ile Luell Tyr Asn Arg Ser Thr 2O 25 3O

Cys Ser Wall Thir Asp Wall Tyr Luell Luell Asn Luell Ala Ile Ala Asp Lieu 35 4 O 45

Phe Phe Ala Lieu. Thir Lell Pro Wall Trp Ala Ala Ser SO 55 6 O

<210s, SEQ ID NO 15 &211s LENGTH: 352 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 15

Met Glu Gly Ile Ser Ile Thir Ser Asp ASn Tyr Thir Glu Glu Met 1. 5 15

Gly Ser Gly Asp Tyr Asp Ser Met Lys Glu Pro Cys Phe Arg Glu Glu 2O 25 3O

Asn Ala Asn. Phe Asn Ile Phe Luell Pro Thir Ile Tyr Ser Ile Ile 35 4 O 45

Phe Lieu. Thr Gly Ile Wall Gly Asn Gly Luell Wall Ile Lell Val Met Gly SO 55 6 O

Tyr Gln Lys Lell Arg Ser Met Thir Asp Lys Tyr Arg Lieu. His Luell 65 70 8O

Ser Val Ala Asp Lieu Lell Phe Wall Ile Thir Luell Pro Phe Trp Ala Val 85 90 95

Asp Ala Wall Ala Asn Trp Phe Gly Asn Phe Lell Lys Ala Val 105 11 O

His Wall Ile Tyr Thr Wall Asn Luell Tyr Ser Ser Wall Lell Ile Lieu Ala 115 12 O 125 US 8,440,627 B2 59 60 - Continued

Phe Ile Ser Lieu. Asp Arg Tyr Lieu Ala Ile Wall His Ala Thir Asn Ser 13 O 135 14 O

Glin Arg Pro Arg Llys Lieu. Lieu Ala Glu Llys Val Val Tyr Wall Gly Wall 145 150 155 160

Trp Ile Pro Ala Lieu Lleu Lieu. Thir Ile Pro Asp Phe Ile Phe Ala Asn 1.65 17O

Val Ser Glu Ala Asp Asp Arg Tyr Ile Arg Phe Tyr Pro Asn 18O 185 19 O

Asp Leu Trp Val Val Val Phe Glin Phe Gln His Ile Met Wall Gly Luell 195 2OO 2O5

Ile Leu Pro Gly Ile Val Ile Leu Ser Cys Ile Ile Ile Ser 21 O 215 22O

Llys Lieu. Ser His Ser Lys Gly His Glin Lys Ala Luell Thir 225 23 O 235 24 O

Thir Wall Ile Lieu. Ile Leu Ala Phe Phe Ala Cys Trp Lieu. Pro Tyr Tyr 245 250 255

Ile Gly Ile Ser Ile Asp Ser Phe Ile Lieu. Luell Glu Ile Ile Glin 26 O 265 27 O

Gly Cys Glu Phe Glu Asn Thr Val His Ile Ser Ile Thir Glu 27s 28O 285

Ala Lieu Ala Phe Phe His Cys Cys Lieu. Asn. Pro Ile Lieu. Tyr Ala Phe 29 O 295 3 OO

Lieu. Gly Ala Lys Phe Llys Thir Ser Ala Gln His Ala Lieu Thir Ser Wall 3. OS 310 315 32O Ser Arg Gly Ser Ser Lieu Lys Ile Lieu. Ser Lys Gly Lys Arg Gly Gly 3.25 330 335

His Ser Ser Wal Ser Thr Glu Ser Glu Ser Ser Ser Phe His Ser Ser 34 O 345 35. O

<210s, SEQ ID NO 16 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Macaca fascicularis

<4 OOs, SEQUENCE: 16 Ile Phe Leu Pro Thr Ile Tyr Ser Ile Ile Phe Lieu. Thr Gly Ile Val 1. 5 15 Gly Asn Gly Lieu Val Ile Lieu Val 2O

<210s, SEQ ID NO 17 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized <4 OOs, SEQUENCE: 17

Phe Lieu. Phe Arg Thr Llys Llys Llys His Pro Ala Wall 1. 5 1O

<210s, SEQ ID NO 18 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized

<4 OOs, SEQUENCE: 18 US 8,440,627 B2 61 62 - Continued Ile Lieu. Tyr Ser Arg Val Gly Arg Ser Val Thr Asp 1. 5 1O

<210s, SEQ ID NO 19 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized <4 OOs, SEQUENCE: 19 Tyr Ser Arg Val Gly Arg Ser Val Thr Asp 1. 5 1O

<210s, SEQ ID NO 2 O &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized

<4 OOs, SEQUENCE: 2O Tyr Ser Arg Val Gly Arg Ser Val Thr Asp 1. 5 1O

<210s, SEQ ID NO 21 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized

<4 OOs, SEQUENCE: 21 Ile Lieu. Tyr Ser Arg Val Gly Arg Ser Val Thr Asp 1. 5 1O

<210s, SEQ ID NO 22 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized <4 OOs, SEQUENCE: 22 Tyr Ser Arg Val Gly Arg Ser Val Thr Asp 1. 5 1O

<210s, SEQ ID NO 23 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized

<4 OOs, SEQUENCE: 23 Tyr Ser Arg Val Gly Arg Ser Val Thr Asp 1. 5 1O

<210s, SEQ ID NO 24 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized <4 OOs, SEQUENCE: 24 Tyr Glin Llys Llys Lieu. Arg Ser Met Thr Asp 1. 5 1O US 8,440,627 B2 63 64 - Continued

<210s, SEQ ID NO 25 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized

<4 OOs, SEQUENCE: 25 Met Gly Tyr Glin Llys Llys Lieu. Arg Ser Met Thr Asp 1. 5 1O

<210s, SEQ ID NO 26 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized <4 OOs, SEQUENCE: 26 Lys Arg Lieu Lys Ser Met Thr Asp 1. 5

<210s, SEQ ID NO 27 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized

<4 OOs, SEQUENCE: 27 Lieu. Ile Asn. Cys Lys Arg Lieu Lys Ser Met Thr Asp 1. 5 1O

<210s, SEQ ID NO 28 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized

<4 OOs, SEQUENCE: 28 Ile Lieu Lys Met Llys Val Llys Llys Pro Ala Wall 1. 5 1O

<210s, SEQ ID NO 29 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized <4 OOs, SEQUENCE: 29 Val Lieu Ala Thr Glin Ala Pro Arg Lieu. Pro Ser Thir 1. 5 1O

<210s, SEQ ID NO 3 O &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized

<4 OOs, SEQUENCE: 30 Ala Thr Glin Ala Pro Arg Lieu Pro Ser Thir 1. 5 1O US 8,440,627 B2 65 66 - Continued <210s, SEQ ID NO 31 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized

<4 OOs, SEQUENCE: 31 Val Lieu Ala Thr Gly Ala Pro Arg Lieu Pro Ser Thr 1. 5 1O

<210s, SEQ ID NO 32 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized

<4 OOs, SEQUENCE: 32 Ala Thr Gly Ala Pro Arg Lieu Pro Ser Thr 1. 5 1O

<210s, SEQ ID NO 33 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized

<4 OOs, SEQUENCE: 33 Thir Phe Lieu. Lieu. Arg Asn 1. 5

<210s, SEQ ID NO 34 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized

<4 OOs, SEQUENCE: 34 Ala Tyr Pro Gly Llys Phe 1. 5

<210s, SEQ ID NO 35 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: chemically synthesized

<4 OOs, SEQUENCE: 35 Ser Phe Lieu. Lieu. Arg Asn 1. 5

<210s, SEQ ID NO 36 &211s LENGTH: 61 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 36 Asn Lys Ile Phe Leu Pro Thir Ile Tyr Ser Ile Ile Phe Lieu. Thr Gly 1. 5 1O 15 Ile Val Gly Asn Gly Lieu Val Ile Lieu Val Met Gly Tyr Glin Llys Llys 2O 25 3O Lieu. Arg Ser Met Thr Asp Llys Tyr Arg Lieu. His Lieu. Ser Val Ala Asp 35 4 O 45 US 8,440,627 B2 67 68 - Continued

Leu Lleu Phe Wall Ile Thir Lieu. Pro Phe Trp Ala Val Asp SO 55 6 O

SEO ID NO 37 LENGTH: 61 TYPE PRT ORGANISM: Mouse

SEQUENCE: 37

ASn Air g Ile Phe Lieu. Pro Thir Ile Tyr Phe Ile Ile Phe Lieu. Thr Gly 1. 1O 15

Ile Wa l Gly Asn Gly Lieu Wall Ile Leu Val Met Gly Tyr Glin Llys Llys 2O 25 3O

Luell Air g Ser Met Thr Asp Llys Tyr Arg Lieu. His Lieu Ser Wall Ala Asp 35 4 O 45

Lieu. Lue ul Phe Wall Ile Thir Lieu. Pro Phe Trp Ala Val Asp SO 55 6 O

SEQ ID NO 38 LENGTH: 61 TYPE PRT ORGANISM: Macaca fasicularis papillomavirus SEQUENCE: 38

ASn Air g Ile Phe Lieu. Pro Thir Ile Tyr Ser Ile Ile Phe Lieu. Thr Gly 1. 5 1O 15

Ile Wa l Gly Asn Gly Lieu Wall Ile Leu Val Met Gly Tyr Glin Llys Llys 2O 25 3O

Luell Air g Ser Met Thr Asp Llys Tyr Arg Lieu. His Lieu Ser Wall Ala Asp 35 4 O 45

Lieu. Lue ul Phe Wall Ile Thir Lieu. Pro Phe Trp Ala Val Asp SO 55 6 O

SEO ID NO 39 LENGTH: 61 TYPE PRT ORGANISM: Macaca mulatta

< 4 OOs SEQUENCE: 39

ASn Air g Ile Phe Lieu. Pro Thir Ile Tyr Ser Ile Ile Phe Lieu. Thr Gly 1. 5 1O 15

Ile Wa l Gly Asn Gly Lieu Wall Ile Leu Val Met Gly Tyr Glin Llys Llys 2O 25 3O

Luell Air g Ser Met Thr Asp Llys Tyr Arg Lieu. His Lieu Ser Wall Ala Asp 35 4 O 45

Lieu. Lue ul Phe Wall Ile Thir Lieu. Pro Phe Trp Ala Val Asp SO 55 6 O

SEQ ID NO 4 O LENGTH: 61 TYPE PRT ORGANISM: Pan troglodytes

< 4 OOs SEQUENCE: 4 O Asn Lys Ile Phe Lieu. Pro Thir Ile Tyr Ser Ile Ile Phe Lieu. Thr Gly 1. 5 1O 15

Ile Wa l Gly Asn Gly Lieu Wall Ile Leu Val Met Gly Tyr Glin Llys Llys 2O 25 3O

Luell Air g Ser Met Thr Asp Llys Tyr Arg Lieu. His Lieu Ser Wall Ala Asp 35 4 O 45 US 8,440,627 B2 69 70 - Continued

Leu Lleu Phe Wall Ile Thir Lieu. Pro Phe Trp Ala Val Asp SO 55 6 O

<210s, SEQ ID NO 41 &211s LENGTH: 61 212. TYPE: PRT &213s ORGANISM: Felis catus

<4 OOs, SEQUENCE: 41

Asn Arg Ile Phe Lell Pro Thir Wall Ser Ile Ile Phe Lieu. Thr Gly 1. 5 1O 15

Ile Val Gly Asn Gly Lell Wall Ile Luell Wall Met Gly Tyr Gln Lys Llys 2O 25 3O

Lieu. Arg Ser Met Thir Asp Tyr Arg Luell His Lell Ser Val Ala Asp 35 4 O 45

Lieu. Lieu. Phe Wall Lell Thir Lell Pro Phe Trp Ala Wall Asp SO 55 6 O

<210s, SEQ ID NO 42 &211s LENGTH: 61 212. TYPE: PRT &213s ORGANISM: Bovine

<4 OOs, SEQUENCE: 42

Asn Arg Ile Phe Lell Pro Thir Wall Ser Ile Ile Phe Lieu. Thr Gly 1. 1O 15

Ile Val Gly Asn Gly Lell Wall Ile Luell Wall Met Gly Tyr Gln Lys Llys 2O 25 3O

Lieu. Arg Ser Met Thir Asp Tyr Arg Luell His Lell Ser Val Ala Asp 35 4 O 45

Lieu. Lieu. Phe Wall Lell Thir Lell Pro Phe Trp Ala Wall Asp SO 55 6 O

<210s, SEQ ID NO 43 &211s LENGTH: 61 212. TYPE: PRT &213s ORGANISM: Rat

<4 OOs, SEQUENCE: 43

Asn Arg Ile Phe Lell Pro Thir Ile Phe Ile Ile Phe Lieu. Thr Gly 1. 1O 15

Ile Val Gly Asn Gly Lell Wall Ile Luell Wall Met Gly Tyr Gln Lys Llys 2O 25 3O

Lieu. Arg Ser Met Thir Asp Tyr Arg Luell His Lell Ser Val Ala Asp 35 4 O 45

Lieu. Lieu. Phe Wall Ile Thir Lell Pro Phe Trp Ala Wall Asp SO 55 6 O

<210s, SEQ ID NO 44 &211s LENGTH: 50 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 44 Pro Pro Leu Tyr Ser Lieu Val Phe Wall Ile Gly Lieu. Wall Gly Asn. Ile 1. 5 15

Lieu Wal Wall Lieu. Wall Lieu Val Glin Tyr Lys Arg Lieu. Lys Asn Met Thr 25 3O

Ser Ile Tyr Lieu. Lieu. Asn Lieu Ala Ile Ser Asp Lieu. Lell Phe Lieu. Thir 35 4 O 45 US 8,440,627 B2 71 72 - Continued

Lell Pro SO

<210s, SEQ ID NO 45 &211s LENGTH: 51 212. TYPE: PRT &213s ORGANISM: Mouse

<4 OOs, SEQUENCE: 45

Pro Pro Leu Tyr Ser Lieu Val Phe Ile Ile Gly Val Wall Gly Asn. Wall 1. 5 1O 15

Lell Met Ile Leu Wall Leu Met Gln His Arg Arg Lieu. Glin Ser Met Thr 25 3O

Ser Ile Tyr Lieu. Phe Asn Lieu Ala Val Ser Asp Lieu. Wall Phe Lieu. Phe 35 4 O 45

Thir Leul Pro SO

<210s, SEQ ID NO 46 &211s LENGTH: 51 212. TYPE: PRT <213s ORGANISM: Macaca mulatta

<4 OOs, SEQUENCE: 46

Pro Pro Leu Tyr Ser Lieu Val Phe Val Ile Gly Val Wall Gly Asn Lieu. 1. 5 1O 15

Lell Val Val Lieu Val Lieu Val Glin Tyr Lys Arg Lieu. Lys Asn Met Thr 25 3O

Asn Ile Tyr Lieu. Lieu. Asn Lieu Ala Ile Ser Asp Lieu. Lell Phe Lieu. Phe 35 4 O 45

Thir Leul Pro SO

<210s, SEQ ID NO 47 &211s LENGTH: 60 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 47

Pro Pro Leu Tyr Ser Lieu Wall Phe Ile Phe Gly Phe Wall Gly Asn Met 1. 5 1O 15

Lell Wal Wall Lieu. Ile Lieu. Ile Asn. Cys Llys Llys Lieu. Cys Lieu. Thr 2O 25 3O

Asp Ile Tyr Lieu. Lieu. Asn. Luell Ala Ile Ser Asp Lieu. Lell Phe Lieu. Ile 35 4 O 45

Thir Luell Pro Luell Trp Ala His Ser Ala Ala Asn. Glu SO 55 6 O

<210s, SEQ ID NO 48 &211s LENGTH: 60 212. TYPE: PRT &213s ORGANISM: Mouse

<4 OOs, SEQUENCE: 48 Pro Pro Leu Tyr Ser Lieu Val Phe Ile Phe Gly Phe Wall Gly Asn Met 1. 5 1O 15

Lieu Wall Ile Ile Ile Lieu. Ile Gly Cys Llys Llys Lieu. Lys Ser Met Thr 25 3O

Asp Ile Tyr Lieu. Lieu. Asn Lieu Ala Ile Ser Asp Lieu. Lell Phe Lieu. Lieu. 35 4 O 45 US 8,440,627 B2 73 74 - Continued

Thr Lieu Pro Phe Trp Ala His Tyr Ala Ala Asn. Glu SO 55 6 O

<210s, SEQ ID NO 49 &211s LENGTH: 60 212. TYPE: PRT &213s ORGANISM: Rat

<4 OOs, SEQUENCE: 49 Pro Pro Leu Tyr Ser Lieu. Wall Phe Ile Phe Gly Phe Wall Gly Asn Met 1. 5 1O 15

Lieu Wall Ile Ile Ile Lieu. Ile Ser Cys Lys Lell Lys Ser Met Thr 2O 25 3O

Asp Ile Tyr Lieu. Phe Asn Lell Ala Ile Ser Asp Lell Lell Phe Lieu. Lieu. 35 4 O 45

Thr Lieu Pro Phe Trp Ala His Ala Ala ASn Glu SO 55 6 O

<210s, SEQ ID NO 50 &211s LENGTH: 60 212. TYPE: PRT <213s ORGANISM: Macaca mulatta

<4 OOs, SEQUENCE: 50 Pro Pro Leu Tyr Ser Lieu. Wall Phe Ile Phe Gly Phe Wall Gly Asn Met 1. 5 1O 15

Lieu Wal Wall Lieu. Ile Lieu. Ile Asn Cys Lys Lell Ser Lieu. Thir 2O 25 3O

Asp Ile Tyr Lieu. Lieu. Asn Lell Ala Ile Ser Asp Lell Lell Phe Lieu. Ile 35 4 O 45

Thr Lieu Pro Leu Trp Ala His Ser Ala Ala ASn Glu SO 55 6 O

<210s, SEQ ID NO 51 &211s LENGTH: 54 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 51

Pro Pro Leu Tyr Ser Lieu. Wall Phe Wall Phe Gly Lieu. Lell Gly Asn. Ser 1. 5 15

Wal Wal Wall Lieu Wall Lieu. Phe Lys Tyr Lys Arg Lieu. Arg Ser Met Thr 2O 25 3O

Asp Val Tyr Lieu. Lieu. Asn Lell Ala Ile Ser Asp Lieu. Lell Phe Wall Phe 35 4 O 45 Ser Leu Pro Phe Trp Gly SO

<210s, SEQ ID NO 52 &211s LENGTH: 54 212. TYPE: PRT &213s ORGANISM: Mouse

<4 OOs, SEQUENCE: 52

Pro Pro Leu Tyr Ser Lieu Val Phe Luell Lieu. Gly Lieu. Phe Gly Asn. Ser 1. 5 15

Val Val Val Lieu Val Lieu. Phe Lys Tyr Lys Arg Lieu. Lys Ser Met Thr 25 3O

Asp Val Tyr Lieu. Lieu. Asn Lieu Ala Ile Ser Asp Lieu. Lell Phe Wall Lieu. 35 4 O 45 US 8,440,627 B2 75 76 - Continued

Ser Leu Pro Phe Trp Gly SO

<210s, SEQ ID NO 53 &211s LENGTH: 47 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 53 Pro Pro Leu Tyr Ser Lieu Val Phe Ile Phe Gly Phe Wall Gly Asn Met 1. 5 1O 15

Lieu Wall Ile Lieu. Ile Lieu. Ile Asn Cys Lys Arg Lieu. Lys Ser Met Thr 2O 25 3O

Asp Ile Tyr Lieu. Lieu. Asn Lieu Ala Ile Ser Asp Lieu. Phe Phe Lieu. 35 4 O 45

<210s, SEQ ID NO 54 &211s LENGTH: 47 212. TYPE: PRT &213s ORGANISM: Mouse

<4 OOs, SEQUENCE: 54 Pro Pro Leu Tyr Ser Lieu Val Phe Ile Phe Gly Phe Wall Gly Asn Met 1. 5 1O 15

Met Wall Phe Lieu. Ile Lieu. Ile Ser Cys Llys Llys Lieu. Lys Ser Wall Thr 2O 25 3O

Asp Ile Tyr Lieu. Lieu. Asn Lieu Ala Ile Ser Asp Lieu. Lell Phe Lieu. 35 4 O 45

<210s, SEQ ID NO 55 &211s LENGTH: 47 212. TYPE: PRT <213> ORGANISM: Papio hamadryas

<4 OO > SEQUENCE: 55 Pro Pro Leu Tyr Ser Lieu Val Phe Ile Phe Gly Phe Wall Gly Asn. Ile 1. 5 1O 15

Lieu Wal Wall Lieu. Ile Lieu. Ile Asn Cys Lys Arg Lieu. Lys Ser Met Thr 2O 25 3O

Asp Ile Tyr Lieu. Lieu. Asn Lieu Ala Ile Ser Asp Lieu. Lell Phe Lieu. 35 4 O 45

<210s, SEQ ID NO 56 &211s LENGTH: 47 212. TYPE: PRT <213> ORGANISM: Erythrocebus patas

<4 OOs, SEQUENCE: 56 Pro Pro Leu Tyr Ser Lieu Val Phe Ile Phe Gly Phe Wall Gly Asn. Ile 1. 5 1O 15

Lieu Wal Wall Lieu. Ile Lieu. Ile Asn Cys Lys Arg Lieu. Lys Ser Met Thr 2O 25 3O

Asp Ile Tyr Lieu. Lieu. Asn Lieu Ala Ile Ser Asp Lieu. Lell Phe Lieu. 35 4 O 45

<210s, SEQ ID NO 57 &211s LENGTH: 47 212. TYPE: PRT <213s ORGANISM: Macaca mulatta

<4 OO > SEQUENCE: 57 US 8,440,627 B2 77 - Continued Pro Pro Leu Tyr Ser Lieu Val Phe Ile Phe Gly Phe Val Gly Asn Ile 1. 5 1O 15 Lieu Val Val Lieu. Ile Lieu. Ile Asn. Cys Lys Arg Lieu Lys Ser Met Thr 2O 25 3O Asp Ile Tyr Lieu. Lieu. Asn Lieu Ala Ile Ser Asp Lieu. Lieu. Phe Lieu. 35 4 O 45

<210s, SEQ ID NO 58 &211s LENGTH: 47 212. TYPE: PRT <213> ORGANISM: Macaca nemestrina

<4 OOs, SEQUENCE: 58 Pro Pro Leu Tyr Ser Lieu Val Phe Ile Phe Gly Phe Val Gly Asn Ile 1. 5 1O 15 Lieu Val Val Lieu. Ile Lieu. Ile Asn. Cys Lys Arg Lieu Lys Ser Met Thr 2O 25 3O Asp Ile Tyr Lieu. Lieu. Asn Lieu Ala Ile Ser Asp Lieu. Lieu. Phe Lieu. 35 4 O 45

<210s, SEQ ID NO 59 &211s LENGTH: 47 212. TYPE: PRT <213> ORGANISM: Macaca fascicularis

<4 OO > SEQUENCE: 59 Pro Pro Leu Tyr Ser Lieu Val Phe Ile Phe Gly Phe Val Gly Asn Ile 1. 5 1O 15 Lieu Val Val Lieu. Ile Lieu. Ile Asn. Cys Lys Arg Lieu Lys Ser Met Thr 2O 25 3O Asp Ile Tyr Lieu. Lieu. Asn Lieu Ala Ile Ser Asp Lieu. Lieu. Phe Lieu. 35 4 O 45

<210s, SEQ ID NO 60 &211s LENGTH: 47 212. TYPE: PRT <213> ORGANISM: Pan troglodytes

<4 OOs, SEQUENCE: 60 Pro Pro Leu Tyr Ser Lieu Val Phe Ile Phe Gly Phe Val Gly Asn Met 1. 5 1O 15 Lieu Val Ile Lieu. Ile Lieu. Ile Asn. Cys Lys Arg Lieu Lys Ser Met Thr 2O 25 3O Asp Ile Tyr Lieu. Lieu. Asn Lieu Ala Ile Ser Asp Lieu. Phe Phe Lieu. 35 4 O 45

<210s, SEQ ID NO 61 &211s LENGTH: 47 212. TYPE: PRT <213> ORGANISM: Gorilla gorilla <4 OOs, SEQUENCE: 61 Pro Pro Leu Tyr Ser Lieu Val Phe Ile Phe Gly Phe Val Gly Asn Met 1. 5 1O 15 Lieu Val Ile Lieu. Ile Lieu. Ile Asn. Cys Lys Arg Lieu Lys Ser Met Thr 2O 25 3O Asp Ile Tyr Lieu. Lieu. Asn Lieu Ala Ile Ser Asp Lieu. Phe Phe Lieu. 35 4 O 45

<210s, SEQ ID NO 62 &211s LENGTH: 47 US 8,440,627 B2 79 80 - Continued

212. TYPE: PRT &213s ORGANISM: Rat

<4 OOs, SEQUENCE: 62

Pro Pro Leu Tyr Ser Leul Wall Phe Ile Phe Gly Phe Val Gly Asn Met 1. 1O 15

Met Wall Phe Lieu. Ile Lieu. Ile Ser Cys Llys Llys Lieu Lys Ser Met Thr 2O 25 3O

Asp Ile Tyr Lieu Phe Asn Lieu Ala Ile Ser Asp Lieu. Lieu. Phe Lieu. 35 4 O 45

<210s, SEQ ID NO 63 &211s LENGTH: 55 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 63

Phe Wall Pro Ser Wall Tyr Thr Gly Val Phe Wal Wal Ser Luell Pro Luell 1. 5 1O 15

Asn Ile Met Ala Ile Wall Wall Phe Ile Lieu Lys Met Lys Val Lys Llys 2O 25 3O

Pro Ala Val Val Tyr Met Lieu. His Lieu. Ala Thr Ala Asp Wall Lieu. Phe 35 4 O 45

Wall Ser Wall Leu Pro Phe Lys SO 55

<210s, SEQ ID NO 64 &211s LENGTH: 55 212. TYPE: PRT &213s ORGANISM: Mouse

<4 OOs, SEQUENCE: 64

Phe Met Pro Ser Wall Tyr Thr Ile Val Phe Ile Wal Ser Luell Pro Luell 1. 5 1O 15

Asn Wall Lieu Ala Ile Ala Wall Phe Wall Lieu. Arg Met Lys Val Lys Llys 25 3O

Pro Ala Val Val Tyr Met Lieu. His Lieu Ala Met Ala Asp Wall Lieu. Phe 35 4 O 45

Wall Ser Wall Leu Pro Phe Lys SO 55

<210s, SEQ ID NO 65 &211s LENGTH: 55 212. TYPE: PRT &213s ORGANISM: Rat

<4 OOs, SEQUENCE: 65

Phe Ile Pro Ser Wall Tyr Thr Phe Val Phe Ile Wal Ser Luell Pro Luell 1. 5 1O 15

Asn Ile Lieu. Ala Ile Ala Wall Phe Wall Phe Arg Met Lys Val Lys Llys 2O 25 3O Pro Ala Val Val Tyr Met Lieu. His Lieu Ala Met Ala Asp Val Lieu. Phe 35 4 O 45

Wall Ser Wall Leu Pro Phe Lys SO 55

<210s, SEQ ID NO 66 &211s LENGTH: 55 212. TYPE: PRT <213> ORGANISM: Cricetulus longicaudatus US 8,440,627 B2 81 82 - Continued

<4 OOs, SEQUENCE: 66

Phe Ile Pro Ser Val Tyr Thir Phe Wall Phe Wal Wall Ser Luell Pro Luell 1. 5 15

Asn Ile Lieu. Ala Ile Ala Wall Phe Wall Lieu Lys Met Lys Val Lys Llys 25 3O

Pro Ala Wal Wall Tyr Met Lell His Luell Ala Met Ala Asp Wall Lieu. Phe 35 4 O 45

Wall Ser Wall Lieu. Pro Leu Lys SO 55

<210s, SEQ ID NO 67 &211s LENGTH: 55 212. TYPE : PRT <213> ORGANISM: Papio hamadryas

<4 OOs, SEQUENCE: 67

Phe Wall Pro Ser Val Tyr Thir Gly Wall Phe Wal Wall Ser Lieu. Pro Wall 1. 5 1O 15

Asn Ile Met Ala Ile Wall Wall Phe Ile Lieu Lys Met Lys Val Lys Llys 25 3O

Pro Ala Wal Wall Tyr Met Lell His Luell Ala Thir Ala Asp Wall Lieu. Phe 35 4 O 45

Wall Ser Wall Lieu. Pro Phe Lys SO 55

<210s, SEQ ID NO 68 &211s LENGTH: 55 212. TYPE : PRT &213s ORGANISM: Xenopus laevis

<4 OOs, SEQUENCE: 68

Phe Wall Pro Ser Lieu. Tyr Thir Wall Wall Phe Ile Wall Gly Luell Pro Luell 1. 5 1O 15

Asn Lieu. Lieu Ala Ile Ile Ile Phe Luell Phe Llys Met Lys Val Arg Llys 25 3O

Pro Ala Wal Wall Tyr Met Lell Asn Luell Ala Ile Ala Asp Wall Phe Phe 35 4 O 45

Wall Ser Wall Lieu. Pro Phe Lys SO 55

<210s, SEQ ID NO 69 &211s LENGTH: 55 212. TYPE : PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 69

Phe Leu Pro Ile Val Tyr Thir Ile Wall Phe Wal Wall Gly Luel Pro Ser 1. 5 1O 15

Asn Gly Met Ala Lieu. Trp Wall Phe Luell Phe Arg Thr Lys Llys Llys His 25 3O

Pro Ala Val Ile Tyr Met Ala Asn Luell Ala Lieu. Ala Asp Luell Luell Ser 35 4 O 45 Val Ile Trp Phe Pro Leu Lys SO 55

<210s, SEQ ID NO 70 &211s LENGTH: 55 212. TYPE : PRT &213s ORGANISM: Mouse US 8,440,627 B2 83 84 - Continued <4 OO > SEQUENCE: 7 O Phe Leu Pro Val Val Tyr Ile Ile Val Phe Val Ile Gly Lieu Pro Ser 1. 5 15

Asn Gly Met Ala Leu Trp Ile Phe Luell Phe Arg Thr Llys Llys Llys His 2O 25

Pro Ala Val Ile Tyr Met Ala Asn Luell Ala Lieu Ala Asp Lieu. Lieu. Ser 35 4 O 45 Val Ile Trp Phe Pro Leu Lys SO 55

<210s, SEQ ID NO 71 &211s LENGTH: 55 212. TYPE: PRT &213s ORGANISM: Rat

<4 OOs, SEQUENCE: 71 Phe Leu Pro Val Ile Tyr Ile Ile Wall Phe Val Ile Gly Lieu Pro Ser 1. 5 15

Asn Gly Met Ala Leu Trp Val Phe Phe Phe Arg Thr Llys Llys Llys His 2O 25

Pro Ala Val Ile Tyr Met Ala Asn Luell Ala Lieu Ala Asp Lieu. Lieu. Ser 35 4 O 45 Val Ile Trp Phe Pro Leu Lys SO 55

<210s, SEQ ID NO 72 &211s LENGTH: 54 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 72 Lieu. Ile Pro Ala Ile Tyr Lieu. Lieu Wall Phe Val Val Gly Val Pro Ala 1. 5 15

Asn Ala Val Thir Lieu. Trp Met Lieu. Phe Phe Arg Thr Arg Ser Ile Cys 2O 25

Thir Thr Val Phe Tyr Thr Asn Lieu. Ala Ile Ala Asp Phe Lieu. Phe Cys 35 4 O 45 Val Thr Lieu Pro Phe Lys SO

<210s, SEQ ID NO 73 &211s LENGTH: 54 212. TYPE: PRT &213s ORGANISM: Mouse

<4 OO > SEQUENCE: 73

Val Ile Pro Ala Ile Tyr Ile Leu Luell Phe Val Val Gly Val Pro Ser 1. 5 15

Asn. Ile Val Thir Lieu. Trp Llys Lieu. Ser Lieu. Arg Thr Lys Ser Ile Ser 2O 25

Lieu. Wall Ile Phe His Thir Asn Lieu. Ala Ile Ala Asp Lieu. Lieu. Phe Cys 35 4 O 45 Val Thr Lieu Pro Phe Lys SO

<210s, SEQ ID NO 74 &211s LENGTH: 54 212. TYPE: PRT &213s ORGANISM: Rat US 8,440,627 B2 85 86 - Continued

< 4 OOs SEQUENCE: 74

Wall Il e Pro Ala Ile Tyr Ile Luell Wall Phe Wall Ile Gly Wall Pro Ala 1. 1O 15

ASn Il e Wall. Thir Lieu. Trp Lys Luell Ser Ser Arg Thr Lys Ser Ile Cys 25 3O

Luell Wa l Ile Phe His Thir Asn Luell Ala Ile Ala Asp Lell Luell Phe Cys 35 4 O 45

Wall. Thir Lieu Pro Phe SO

SEO ID NO 75 LENGTH: 54 TYPE PRT ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 75

Luell Wa l Pro Ala Lieu. Gly Luell Wall Lieu Wal Wall Gly Luell Pro Ala 1. 5 1O 15

ASn Gl y Lieu Ala Lieu. Trp Wall Luell Ala Thr Glin Ala Pro Arg Leul Pro 2O 25 3O

Ser Thir Met Lieu Lleu. Met Asn Luell Ala Thr Ala Asp Lell Luell Lieu Ala 35 4 O 45

Lieu. All a Luel Pro Pro Arg SO

SEO ID NO 76 LENGTH: 54 TYPE PRT ORGANISM: Mouse

SEQUENCE: 76

Luell Wa l Pro Ala Lieu. Gly Luell Wal Wall Ala Wall Gly Luell Pro Ala 1. 1O 15

ASn Gl y Lieu Ala Lieu. Trp Wall Luell Ala Thr Arg Val Pro Arg Leul Pro 2O 25 3O

Ser Th r Ile Lieu. Lieu. Thir Asn Luell Ala Val Ala Asp Ser Luell Lieu Ala 35 4 O 45

Luell Wa Pro Pro Pro Arg SO

SEO ID NO 77 LENGTH: 54 TYPE PRT ORGANISM: Rat

< 4 OOs SEQUENCE: 77

Luell Wa l Pro Ala Ile Gly Luell Wall Wal Wal Wall Gly Luell Pro Ala 1. 5 1O 15

ASn Gl y Lieu Ala Lieu. Trp Wall Luell Ala Thr Arg Val Pro Arg Leul Pro 2O 25 3O

Ser Th r Ile Lieu. Lieu. Met Asn Luell Ala Val Ala Asp Lell Luell Lieu Ala 35 4 O 45

Luell Wa l Lieu. Pro Pro Arg SO

SEO ID NO 78 LENGTH: SO TYPE PRT <213> ORGANISM: Homo sapiens US 8,440,627 B2 87 88 - Continued <4 OO > SEQUENCE: 78 Lys Lieu. Thir Ser Val Val Phe Ile Lieu. Ile Cys Cys Phe Ile Ile Lieu. 1. 5 1O 15

Glu Asn. Ile Phe Wall Leu Lleu. Thir Ile Trp Llys Thr Lys Llys Phe His 2O 25 3O Arg Pro Met Tyr Tyr Phe Ile Gly Asn Lieu Ala Lieu. Ser Asp Lieu. Lieu. 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 79 &211s LENGTH: 50 212. TYPE: PRT &213s ORGANISM: Mouse

<4 OO > SEQUENCE: 79 Lys Lieu. Thir Ser Val Val Phe Ile Lieu. Ile Cys Cys Phe Ile Ile Lieu. 1. 5 1O 15

Glu Asn. Ile Phe Wall Leu Lleu. Thir Ile Trp Llys Thr Lys Llys Phe His 2O 25 3O Arg Pro Met Tyr Tyr Phe Ile Gly Asn Lieu Ala Lieu. Ser Asp Lieu. Lieu. 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 8O &211s LENGTH: 50 212. TYPE: PRT &213s ORGANISM: Rat

<4 OOs, SEQUENCE: 80 Lys Lieu. Thir Ser Val Val Phe Ile Lieu. Ile Cys Cys Lieu. Ile Ile Lieu. 1. 5 1O 15

Glu Asn. Ile Phe Wall Leu Lleu. Thir Ile Trp Llys Thr Lys Llys Phe His 2O 25 3O Arg Pro Met Tyr Tyr Phe Ile Gly Asn Lieu Ala Lieu. Ser Asp Lieu. Lieu. 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 81 &211s LENGTH: 50 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 81 Llys Lieu Val Met Gly Lieu. Gly Ile Thr Val Cys Ile Phe Ile Met Leu 1. 5 1O 15

Ala Asn Lieu. Lieu Wal Met Wall Ala Ile Tyr Val Asn Arg Arg Phe His 2O 25 3O Phe Pro Ile Tyr Tyr Lieu Met Ala Asn Lieu Ala Ala Ala Asp Phe Phe 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 82 &211s LENGTH: 50 212. TYPE: PRT &213s ORGANISM: Mouse US 8,440,627 B2 90 - Continued <4 OOs, SEQUENCE: 82 Llys Lieu Val Met Gly Lieu. Gly Ile Thr Val Cys Val Phe Ile Met Leu 1. 5 15

Ala Asn Lieu. Lieu Wal Met Wall Ala Ile Tyr Val Asn Arg Arg Phe His 25 Phe Pro Ile Tyr Tyr Lieu Met Ala Asn Lieu Ala Ala Ala Asp Phe Phe 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 83 &211s LENGTH: 50 212. TYPE: PRT <213> ORGANISM: Sheep

<4 OOs, SEQUENCE: 83 Llys Lieu Val Met Gly Lieu. Gly Ile Thr Val Cys Ile Phe Ile Met Leu 1. 5 15

Ala Asn Lieu. Lieu Wal Met Wall Ala Ile Tyr Val Asn Arg Arg Phe His 25 Phe Pro Ile Tyr Tyr Lieu Met Ala Asn Lieu Ala Ala Ala Asp Phe Phe 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 84 &211s LENGTH: 50 212. TYPE: PRT <213> ORGANISM: Bovine

<4 OOs, SEQUENCE: 84 Llys Lieu Val Met Gly Lieu. Gly Ile Thr Val Cys Ile Phe Ile Met Leu 1. 5 15

Ala Asn Lieu. Lieu Wal Met Wall Ala Ile Tyr Val Asn Arg Arg Phe His 25 Phe Pro Ile Tyr Tyr Lieu Met Ala Asn Lieu Ala Ala Ala Asp Phe Phe 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 85 &211s LENGTH: 50 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 85 Thr Lieu. Thir Thr Val Lieu Phe Leu Val Ile Cys Ser Phe Ile Val Lieu. 1. 5 15 Glu Asn Lieu Met Val Lieu. Ile Ala Ile Trp Lys Asn. Asn Llys Phe His 25 Asn Arg Met Tyr Phe Phe Ile Gly Asn Lieu Ala Lieu. Cys Asp Lieu. Lieu 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 86 &211s LENGTH: 50 212. TYPE: PRT &213s ORGANISM: Mouse US 8,440,627 B2 92 - Continued <4 OOs, SEQUENCE: 86 Lieu. Ile Thr Thr Ile Leu Phe Leu Val Thr Cys Ser Phe Ile Wall Lieu. 1. 5 15 Glu Asn Lieu Met Val Lieu. Ile Ala Ile Trp Lys Asn Asn Llys Phe His 25 3O Asn Arg Met Tyr Phe Phe Ile Gly Asn Lieu Ala Lieu. Cys Asp Lieu. Lieu 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 87 &211s LENGTH: 50 212. TYPE: PRT <213> ORGANISM: Fugu rubripes

<4 OO > SEQUENCE: 87 Asp Pro Llys Thr Ile Ala Phe Leu Val Val Cys Ser Phe Ile Ile Lieu. 1. 5 15 Glu Asn Lieu. Thr Val Lieu. Lieu Ala Ile Trp Lys Asn His Arg Phe His 25 3O Asn Arg Met Tyr Phe Phe Ile Gly Asn Lieu Ala Lieu. Cys Asp Lieu. Lieu 35 4 O 45

Ala Ser SO

<210s, SEQ ID NO 88 &211s LENGTH: 50 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 88

Val Val Val Val Ala Lieu. Gly Luell Thir Wal Ser Wall Lieu Wall Lieu. Lieu 1. 5 1O 15

Thir Asn Lieu. Lieu. Wall Ile Ala Ala Ile Ala Ser Asn Arg Arg Phe His 25 3O

Gln Pro Ile Tyr Tyr Lieu. Leu Gly Asn Lieu Ala Ala Ala Asp Lieu. Phe 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 89 &211s LENGTH: 50 212. TYPE: PRT &213s ORGANISM: Mouse

<4 OOs, SEQUENCE: 89

Val Val Val Val Ala Lieu. Gly Luell Thir Wal Ser Wall Lieu Wall Lieu. Lieu 1. 5 1O 15

Thir Asn Lieu. Lieu. Wall Ile Ala Ala Ile Ala Ser Asn Arg Arg Phe His 25 3O

Gln Pro Ile Tyr Tyr Lieu. Leu Gly Asn Lieu Ala Ala Ala Asp Lieu. Phe 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 90 &211s LENGTH: 50 212. TYPE: PRT <213> ORGANISM: Macaca fascicularis US 8,440,627 B2 93 94 - Continued <4 OOs, SEQUENCE: 90 Val Val Val Val Ala Lieu. Gly Lieu. Thr Val Ser Val Lieu Val Lieu. Lieu 1. 5 1O 15 Thir Asn Lieu. Lieu Val Ile Ala Ala Ile Ala Ser Asn Arg Arg Phe His 2O 25 3O Glin Pro Ile Tyr Tyr Lieu. Lieu. Gly Asn Lieu Ala Ala Ala Asp Lieu. Phe 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 91 &211s LENGTH: 50 212. TYPE: PRT <213> ORGANISM: homo sapians

<4 OOs, SEQUENCE: 91 Glin Val Ala Ser Ala Phe Ile Val Ile Lieu. Cys Cys Ala Ile Val Val 1. 5 1O 15 Glu Asn Lieu. Lieu Val Lieu. Ile Ala Val Ala Arg Asn. Ser Llys Phe His 2O 25 3O Ser Ala Met Tyr Lieu. Phe Lieu. Gly Asn Lieu Ala Ala Ser Asp Lieu. Lieu. 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 92 &211s LENGTH: 50 212. TYPE: PRT &213s ORGANISM: Mouse

<4 OOs, SEQUENCE: 92 Llys Val Ala Ser Ala Phe Ile Ile Ile Lieu. Cys Cys Ala Ile Val Val 1. 5 1O 15 Glu Asn Lieu. Lieu Val Lieu. Ile Ala Val Ala Arg Asn. Ser Llys Phe His 2O 25 3O Ser Ala Met Tyr Lieu. Phe Lieu. Gly Asn Lieu Ala Ala Ser Asp Lieu. Lieu. 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 93 &211s LENGTH: 50 212. TYPE: PRT &213s ORGANISM: Rat

<4 OOs, SEQUENCE: 93 Llys Val Ala Ser Ala Phe Ile Ile Ile Lieu. Cys Cys Ala Ile Val Val 1. 5 1O 15 Glu Asn Lieu. Lieu Val Lieu. Ile Ala Val Ala Arg Asn. Ser Llys Phe His 2O 25 3O Ser Ala Met Tyr Lieu. Phe Lieu. Gly Asn Lieu Ala Ala Ser Asp Lieu. Lieu. 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 94 &211s LENGTH: 50 212. TYPE: PRT <213> ORGANISM: homo sapians US 8,440,627 B2 95 96 - Continued <4 OOs, SEQUENCE: 94 Gly Ala Lieu. Arg Gly Lieu. Ser Val Ala Ala Ser Cys Lieu Val Val Lieu. 1. 5 1O 15 Glu Asn Lieu. Lieu Val Lieu Ala Ala Ile Thir Ser His Met Arg Ser Arg 2O 25 3O Arg Trp Val Tyr Tyr Cys Lieu Val Asn. Ile Thr Lieu. Ser Asp Lieu. Lieu 35 4 O 45 Thr Gly SO

<210s, SEQ ID NO 95 &211s LENGTH: 50 212. TYPE: PRT &213s ORGANISM: Mouse

<4 OO > SEQUENCE: 95 Gly Met Lieu. Arg Gly Pro Ser Val Ala Ala Gly Cys Lieu Val Val Lieu. 1. 5 1O 15 Glu Asn Ala Met Val Lieu Ala Ala Ile Ala Ile Tyr Met Arg Ser Arg 2O 25 3O Arg Trp Val Tyr Tyr Cys Lieu. Lieu. Asn. Ile Thr Lieu. Ser Asp Lieu. Lieu 35 4 O 45 Thr Gly SO

<210s, SEQ ID NO 96 &211s LENGTH: 50 212. TYPE: PRT <213> ORGANISM: Brachypodium retusum

<4 OOs, SEQUENCE: 96 Ser Ser Lieu. Asn Ile Leu Phe Val Val Ile Cys Ser Ile Ile Ile Leu 1. 5 1O 15 Glu Asn Lieu. Lieu Val Lieu. Ile Ala Val Phe Arg Asn Llys Llys Phe His 2O 25 3O Ser Ala Met Phe Phe Phe Ile Gly Asn Lieu Ala Phe Ser Asp Leu Lieu. 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 97 &211s LENGTH: 50 212. TYPE: PRT <213> ORGANISM: Homo sapians <4 OO > SEQUENCE: 97 Val Ile Val Lieu. Cys Val Gly Thr Phe Phe Cys Lieu Phe Ile Phe Phe 1. 5 1O 15 Ser Asn. Ser Lieu Val Ile Ala Ala Val Ile Lys Asn Arg Llys Phe His 2O 25 3O Phe Pro Phe Tyr Tyr Lieu. Lieu Ala Asn Lieu Ala Ala Ala Asp Phe Phe 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 98 &211s LENGTH: 50 212. TYPE: PRT &213s ORGANISM: Mouse US 8,440,627 B2 97 98 - Continued <4 OOs, SEQUENCE: 98 Val Ile Val Lieu. Cys Val Gly Thr Phe Phe Cys Lieu Phe Ile Phe Phe 1. 5 15

Ser Asn. Ser Lieu. Wall Ile Ala Ala Wall Ile Thr Asn Arg Llys Phe His 25

Phe Pro Phe Tyr Tyr Lieu. Leu Ala Asn Lieu Ala Ala Ala Asp Phe Phe 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 99 &211s LENGTH: 50 212. TYPE: PRT &213s ORGANISM: Rat

<4 OOs, SEQUENCE: 99 Val Ile Val Lieu. Cys Val Gly Thr Phe Phe Cys Lieu Phe Ile Phe Phe 1. 5 15

Ser Asn. Ser Lieu. Wall Ile Ala Ala Wall Ile Thr Asn Arg Llys Phe His 25

Phe Pro Phe Tyr Tyr Lieu. Leu Ala Asn Lieu Ala Ala Ala Asp Phe Phe 35 4 O 45 Ala Gly SO

<210s, SEQ ID NO 100 &211s LENGTH: 51 212. TYPE: PRT <213> ORGANISM: Homo sapians

<4 OOs, SEQUENCE: 1.OO Trp Glin Ile Val Lieu. Trp Ala Ala Ala Tyr Thr Val Ile Val Val Thr 1. 5 15

Ser Val Val Gly Asn Val Val Val Met Trp Ile Ile Lieu Ala His Lys 25

Arg Met Arg Thr Val Thr Asn Tyr Phe Lieu Wall Asn Lieu Ala Phe Ala 35 4 O 45

Glu Ala Ser SO

<210s, SEQ ID NO 101 &211s LENGTH: 51 212. TYPE: PRT &213s ORGANISM: Mouse

<4 OOs, SEQUENCE: 101 Trp Glin Ile Val Lieu. Trp Ala Ala Ala Tyr Thr Val Ile Val Val Thr 1. 5 15 Ser Val Val Gly Asn Val Val Val Ile Trp Ile Ile Lieu Ala His Lys 25

Arg Met Arg Thr Val Thr Asn Tyr Phe Lieu Wall Asn Lieu Ala Phe Ala 35 4 O 45 Glu Ala Cys SO

<210s, SEQ ID NO 102 &211s LENGTH: 51 212. TYPE: PRT &213s ORGANISM: Rat US 8,440,627 B2

- Continued <4 OOs, SEQUENCE: 102 Trp Glin Ile Val Lieu. Trp Ala Ala Ala Tyr Thr Val Ile Val Val Thr 1. 5 1O 15 Ser Val Val Gly Asn Val Val Val Ile Trp Ile Ile Lieu Ala His Lys 2O 25 3O Arg Met Arg Thr Val Thr Asn Tyr Phe Leu Val Asn Lieu Ala Phe Ala 35 4 O 45 Glu Ala Cys SO

<210s, SEQ ID NO 103 &211s LENGTH: 51 212. TYPE: PRT <213> ORGANISM: Cavia porcellus

<4 OOs, SEQUENCE: 103 Trp Glin Ile Val Lieu. Trp Ala Ala Ala Tyr Thr Val Ile Val Val Thr 1. 5 1O 15 Ser Val Val Gly Asn Val Val Val Met Trp Ile Ile Lieu Ala His Lys 2O 25 3O Arg Met Arg Thr Val Thr Asn Tyr Phe Leu Val Asn Lieu Ala Phe Ala 35 4 O 45

Glu Ala Ser SO

<210s, SEQ ID NO 104 &211s LENGTH: 51 212. TYPE: PRT <213> ORGANISM: Rana catesbeiana

<4 OOs, SEQUENCE: 104 Trp Glin Ile Ala Lieu. Trp Ser Val Ala Tyr Ser Ile Ile Val Ile Val 1. 5 1O 15 Ser Lieu Val Gly Asn. Ile Ile Val Met Trp Ile Ile Ile Ala His Lys 2O 25 3O Arg Met Arg Thr Val Thr Asn Tyr Phe Leu Val Asn Lieu Ala Phe Ala 35 4 O 45

Glu Ala Ser SO

What is claimed is: the group consisting of a: nonanoyl (C); capryl (Co.); unde 1. A chimeric polypeptide comprising: canoyl (C); lauroyl (C); tridecanoyl (C); myristoyl a) a first domain that is an amino acid sequence selected 50 (C); pentadecanoyl (Cs); palmitoyl (C); phytanoyl (me from the group consisting of YOKKLRSMTD (SEQID thyl Substituted Co.); heptadecanoyl (C7); Stearoyl (Cs); NO:24) and MGYQKKLRSMTD (SEQID NO:25) and nonadecanoyl (Co.); arachidoyl (Co.); heniecosanoyl (C); b) a second domain, attached to the first domain, wherein the second domain comprises a cell-penetrating, mem behenoyl (C); trucisanoyl (C); and lignoceroyl (C) moi brane-tethering hydrophobic moiety comprising a lipid, ety. a cholesterol, a phospholipid, a steroid, a sphingosine, a 55 6. The chimeric polypeptide of claim 4, wherein the second ceramide, an octylglycine, a 2-cyclohexylalanine, or a domain comprises a palmitoyl moiety. benzolylphenylalanine, 7. The chimeric polypeptide of claim 4, wherein the second wherein said chimeric polypeptide is an antagonist of a domain comprises a myristoyl (C) or pentadecanoyl (Cs) chemokine receptor. moiety. 2. The chimeric polypeptide of claim 1, wherein the 60 8. The chimeric polypeptide of claim 1, wherein the second chemokine receptor is a CXC chemokine receptor. domain comprises a lithocholic acid or a salt thereof. 3. The chimeric polypeptide of claim 1, wherein the second 9. The chimeric polypeptide of claim 1, wherein the second domain is attached at the N-terminal end of the first domain. domain comprises a hydrophobic moiety that is selected from 4. The chimeric polypeptide of claim 1, wherein the second the group consisting of a cholesterol, a phospholipid, a ste domain comprises a lipid. 65 roid, a sphingosine, a ceramide, an octyl-glycine, a 2-cyclo 5. The chimeric polypeptide of claim 4, wherein the second hexylalanine, a benzolylphenylalanine, and a C-Cs fatty domain comprises a hydrophobic moiety that is selected from acid. US 8,440,627 B2 101 102 10. The chimeric polypeptide of claim 1, wherein the sec selected from the group consisting of a phospholipid, a ste ond domain comprises a steroid. roid, a sphingosine, a ceramide, an octylglycine, a 2-cyclo 11. A pharmaceutical composition comprising the chi hexylalanine, and a benzolylphenylalanine. meric polypeptide of claim 1 and a pharmaceutically accept 25. A pharmaceutical composition comprising the chi able carrier. meric peptide of claim 15 and a pharmaceutically acceptable 12. The chimeric polypeptide of claim 1, wherein the sec carrier. ond domain comprises a hydrophobic moiety that is selected 26. A chimeric polypeptide comprising: from the group consisting of a phospholipid, a steroid, a (a) a first domain that comprises an amino acid sequence sphingosine, a ceramide, an octylglycine, a 2-cyclohexylala that is selected from the group consisting of ILYS nine, and a benzolylphenylalanine. 10 13. The chimeric polypeptide of claim 1, wherein the sec RVGRSVTD (SEQID NO: 18), YSRVGRSVTD (SEQ ond domain is attached to the first domain with an amide ID NO: 19), KRLKSMTD (SEQ ID NO:26), and bond, a Sulfhydryl, an amine, an alcohol, a phenolic group, or LINCKRLKSMTD (SEQID NO:27) and a carbon-carbon bond. (b) a second domain, attached to the first domain, wherein 14. The chimeric polypeptide of claim 1, wherein the sec 15 the second domain comprises a cell-penetrating, mem ond domain is attached at the C-terminal end of the first brane-tethering hydrophobic moiety comprising a lipid, domain. a cholesterol, a phospholipid, a steroid, a sphingosine, a 15. A chimeric polypeptide comprising: ceramide, an octylglycine, a 2-cyclohexylalanine, or a (a) a first domain that comprises an amino acid sequence benzolylphenylalanine; that is selected from the group consisting of ILK wherein said chimeric polypeptide is an antagonist of a MKVKKPAV (SEQ ID NO: 28), VLATOAPRLPST chemokine receptor. (SEQ ID NO: 29), ATOAPRLPST (SEQ ID NO:30, 27. The chimeric polypeptide of claim 26, wherein the VLATGAPRLPST (SEQ ID NO:31), ATGAPRLPST chemokine receptor is a CXC chemokine receptor or a CC (SEQID NO:32), and FLFRTKKKHPAV (SEQID NO: chemokine receptor. 17) and 25 28. The chimeric polypeptide of claim 26, wherein said (b) a second domain, attached to the first domain, wherein second domain comprises a lipid. the second domain comprises a cell-penetrating, mem 29. The chimeric polypeptide of claim 26, wherein said brane-tethering hydrophobic moiety comprising a lipid, second domain comprises a hydrophobic moiety that is a cholesterol, a phospholipid, a steroid, a sphingosine, a selected from the group consisting of a: nonanoyl (C); capryl ceramide, an octylglycine, a 2-cyclohexylalanine, or a 30 (Co.); undecanoyl (C); lauroyl (C); tridecanoyl (C); benzolylphenylalanine; myristoyl (C); pentadecanoyl (Cs); palmitoyl (C); wherein said chimeric polypeptide is an antagonist of a phytanoyl (methyl substituted C); heptadecanoyl (C7); protease-activated receptor (PAR). Stearoyl (Cs); nonadecanoyl (Co.); arachidoyl (Co); 16. The chimeric polypeptide of claim 15, wherein said heniecosanoyl (C); behenoyl (C); trucisanoyl (C); and PAR is PARI, PAR2, or PAR4. 35 lignoceroyl (C) moiety. 17. The chimeric polypeptide of claim 15, wherein the 30. The chimeric polypeptide of claim 26, wherein the second domain comprises a lipid. second domain is attached to the first domain with an amide 18. The chimeric polypeptide of claim 15, wherein the bond, a Sulfhydryl, an amine, an alcohol, a phenolic group, or second domain comprises a hydrophobic moiety that is a carbon-carbon bond. selected from the group consisting of a: nonanoyl (C); capryl 40 (Co.); undecanoyl (C); lauroyl (C); tridecanoyl (C); 31. The chimeric polypeptide of claim 26, wherein the myristoyl (C); pentadecanoyl (Cs); palmitoyl (C); second domain comprises a palmitoyl moiety. phytanoyl (methyl Substituted Co.); heptadecanoyl (C7); 32. The chimeric polypeptide of claim 26, wherein the Stearoyl (Cs); nonadecanoyl (Co.); arachidoyl (Co); second domain comprises a myristoyl (C) or pentadecanoyl heniecosanoyl (C); behenoyl (C); trucisanoyl (C); and 45 (Cs) moiety. lignoceroyl (C4) moiety. 33. The chimeric polypeptide of claim 26, wherein the 19. The chimeric polypeptide of claim 15, wherein the second domain comprises a lithocholic acid or a salt thereof. second domain is attached to the first domain with an amide 34. The chimeric polypeptide of claim 26, wherein the bond, a Sulfhydryl, an amine, an alcohol, a phenolic group, or second domain comprises a hydrophobic moiety that is a carbon-carbon bond. 50 selected from the group consisting of a cholesterol, a phos 20. The chimeric polypeptide of claim 15, wherein the pholipid, a steroid, a sphingosine, a ceramide, an octyl-gly second domain comprises a palmitoyl moiety. cine, a 2-cyclohexylalanine, a benzolylphenylalanine, and a C-Cs fatty acid. 21. The chimeric polypeptide of claim 15, wherein the 35. The chimeric polypeptide of claim 26, wherein the second domain comprises a lithocholic acid or a salt thereof. second domain comprises a steroid. 22. The chimeric polypeptide of claim 15, wherein the 55 36. The chimeric polypeptide of claim 26, wherein the second domain comprises a hydrophobic moiety that is second domain comprises a hydrophobic moiety that is selected from the group consisting of a cholesterol, a phos selected from the group consisting of a phospholipid, a ste pholipid, a steroid, a sphingosine, a ceramide, an octyl-gly roid, a sphingosine, a ceramide, an octylglycine, a 2-cyclo cine, a 2-cyclohexylalanine, a benzolylphenylalanine, and a hexylalanine, and a benzolylphenylalanine. C-Cs fatty acid. 60 23. The chimeric polypeptide of claim 15, wherein the 37. A pharmaceutical composition comprising the chi second domain comprises a steroid. meric peptide of claim 26 and a pharmaceutically acceptable 24. The chimeric polypeptide of claim 15, wherein the carrier. second domain comprises a hydrophobic moiety that is