METHODS OF IDENTIFICATION OF ALLOSTERAMERS AND USES THEREOF

REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

61/172,533, filed April 24, 2009, which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Tumor necrosis factor-alpha (TNFα) and tumor necrosis factor-beta (TNFβ) are homologous multifunctional cytokines and are collectively known as tumor necrosis factor or "TNF." Activities of TNF include: release of other cytokines including IL-I, IL-6, GM-CSF, and IL-10, induction of chemokines, increase in adhesion molecules, growth of blood vessels, release of tissue destructive enzymes and activation of T cells. See, for example, Feldmann et al., 1997, Adv. Immunol., 64:283-350, Nawroth et al, 1986, J. Exp. Med., 163:1363-1375; Moser et al., 1989, J. Clin. Invest., 83:444-455; Shingu et al., 1993, Clin. Exp. Immunol. 94:145-149; MacNaul et al., 1992, Matrix Suppl, 1 :198-199; and Ahmadzadeh et al., 1990, Clin. Exp. Rheumatol. 8:387-391. All of these activities can enhance an inflammatory response.

[0003] TNF initiates its biological effect through its interaction with specific, cell surface receptors. There are two distinct forms of the cell surface tumor necrosis factor receptor (TNFR), designated p75 (also known as TNFRl) and p55 (also known as TNFR2) (Smith et al., 1990, Science 248:1019-1023; Loetscher et al., 1990, Cell 61 :351-359). TNFRl and TNFR2 each bind to both TNFα and TNFβ.

[0004] A number of disorders are associated with deregulation or malfunction of TNF and/or TNF receptors, many of them related to autoimmunity and inflammation. Among such TNF- or TNFR-associated disorders are congestive heart failure, inflammatory bowel diseases (including Crohn's disease), arthritis and asthma. Arthritis is a common crippling condition for which there are no cures and few effective therapies. Approximately one in seven people in the United States are affected by one or more forms of arthritis. Most forms of arthritis are characterized by chronic inflammation of joints resulting from infection, mechanical injury, or immunological disturbance. Rheumatoid arthritis (RA) is a chronic inflammatory disease primarily manifest in the joints by swelling, pain, stiffness, and tissue destruction (Harris, 1990, N. Engl. J. Med, 323:994-996). Although rheumatoid arthritis is not directly and imminently life threatening, recent data suggest that RA results in significantly shorter lifespan, and puts an enormous toll on the both the health system, the overall economy due to lost productivity, as well as quality of life resulting from restricted mobility and activities (Schiff, 1997, Am. J. Med., 1O2(1A):11S-15S).

[0005] At present, over 30-50% of patients with inflammatory bowel disease (IBD), psoriasis, and multiple sclerosis fail to respond to traditional and current disease modifying anti-rheumatic drugs (DMARDs) including new biologies (e.g., anti-TNF antibodies). Therefore, there is a great need for more effective treatment of inflammatory and/or autoimmune diseases.

[0006] Additionally, at present, all agents commercially available that are specific for

TNF or TNFR are administered by injection. There is a need for TNF and TNFR modulators that can be administered orally.

[0007] Finally, at present, design of modulators for a biological target such as a receptor typically requires information about the secondary or tertiary structure of the target. Therefore, there is a need for a simpler and more effective platform for identifying modulators for biological targets for drug discovery purposes.

SUMMARY OF THE INVENTION

[0008] The present invention encompasses the discovery that short peptides based on amino acid sequences of extracellular or transmembrane regions of a TNF receptor are surprisingly effective in modulating (e.g., inhibiting) TNF receptor activities. Thus, the present invention provides, among other things, new and more effective therapeutic compositions and methods for treating inflammatory diseases, disorders or conditions associated with TNF or TNF receptor dysfunction. In addition, the present invention also encompasses the discovery that such effective modulators can be designed simply based on linear amino acid sequences of a biological target without first characterizing any secondary or tertiary structure of the target. Therefore, the present invention further provides a new and effective method to design peptide modulators without involving tertiary or secondary structural modeling of a target. [0009] In one aspect, the present invention provides Allosteramers™ that modulate

TNF receptor activities. As used herein, an Allosteramer™ is a short peptide composed of 5- 25 amino acids derived from body's own proteins that can interact with the protein from which it was derived, or with its associated proteins, without competing with a natural ligand. The terms Allosteramers™ and "peptide modulators" are used interchangeably herein. In some embodiments, the present invention provides a peptide that is a Negative Allosteric Modulator (NAM) of a TNF receptor. In some embodiments, the present invention provides a peptide that is a Positive Allosteric Modulator (PAM) of a TNF receptor.

[0010] In some embodiments, the present invention provides a peptide having a sequence that includes at least 4 (e.g., at least 5, at least 6, at least 7) amino acids from at least 7 (e.g., at least 8, at least 9, at least 10, at least 11, at least 12) contiguous amino acids that appear in an extracellular or transmembrane region of a TNF receptor (e.g., TNFRl, TNFR2), wherein the at least 4 (e.g., at least 5, at least 6, at least 7) amino acids maintain their relative positions and/or spacing as they appear in the TNF receptor. In some embodiments, the present invention provides a peptide having a sequence that includes at least 4 (e.g., at least 5, at least 6, at least 7) amino acids from at least 7 (e.g., at least 8, at least 9, at least 10, at least 11, at least 12) contiguous amino acids that appear in an extracellular or transmembrane region of a TNF receptor (e.g., TNFRl, TNFR2), wherein the at least 4 (e.g., at least 5, at least 6, at least 7) amino acids maintain their relative positions and/or spacing, but in the inverse configuration, as they appear in the TNF receptor.

[0011] In some embodiments, the present invention provides a peptide that is up to 25 amino acids long and comprises a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25) contiguous amino acids that appear in an extracellular or transmembrane region of a TNF receptor. In some embodiments, the present invention provides a peptide that is up to 25 amino acids long and comprises a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25) contiguous amino acids that appear in an extracellular or transmembrane region of a TNF receptor but in the inverse configuration.

[0012] In some embodiments, the present invention provides a peptide that is up to 25 amino acids long and has a sequence identical to at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25) contiguous amino acids that appear in an extracellular or transmembrane region of a TNF receptor. In some embodiments, the present invention provides a peptide that is up to 25 amino acids long and has a sequence identical to at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25) contiguous amino acids that appear in an extracellular or transmembrane region of a TNF receptor but in the inverse configuration.

[0013] In some embodiments, a peptide according to the invention comprises one or more D-amino acid substitutions for corresponding L-amino acids.

[0014] In some embodiments, the TNF receptor suitable for the invention is TNFRl

(SEQ ID NO: 1). In some embodiments, an extracellular or transmembrane region of TNFRl suitable for the invention is selected from the group consisting of

MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQN (SEQ ID NO:3);

HPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRK EMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQN TVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTVLLPLVIFFG LCLLSLLFIGLMYRYQRWKSKLY (SEQ ID NO:4);

VCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHC LSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVH LSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCL (SEQ ID NO:5);

MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSI C CTKCHKGTYLYNDCPGPGQDTDCRECESGSF (SEQ ID NO:6);

TASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFN CSLC (SEQ ID NO:7);

LNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTE DSGTTVLLPLVIFFGLCLLSLLFIGLMYRYQRWKSKLY (SEQ ID NO: 8);

EDSGTTVLLPLVIFFGLCLLSLLFIGLMYRYQRWK (SEQ ID NO:9); and combinations thereof.

[0015] In some embodiments, an extracellular or transmembrane region of TNFRl is selected from the group consisting of MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGK (SEQ ID NO:555);

QNNSICCTKCHKGTYLYN (SEQ ID NO:556); PGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEM (SEQ ID NO:557);

VEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCT CHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTVLLPLVIFFGLCLLS LLFIGLMYRYQRWK (SEQ ID NO:558); and combination thereof.

[0016] In some embodiments, the contiguous amino acids are selected from Tables 1 and 4.

[0017] In some embodiments, a peptide according to the invention is selected from

Table 1 or 2. In some embodiments, a peptide according to the invention is selected from the group consisting of VPHLGDREK (SEQ ID NO: 14) (1-1), TASENHLRHLCS (SEQ ID NO:20) (1-7), CSKCRKEMGQ (SEQ ID NO:21) (1-8), VEISSCTVD (SEQ ID NO:22) (1- 9), SCQEKQNTV (SEQ ID NO:27) (1-14), CTCHAGFF (SEQ ID NO:28) (1-15), LRENECVSC (SEQ ID NO:29) (1-16), CSNCKKSLE (SEQ ID NO:30) (1-17), CTKLCLPQI (SEQ ID NO:31) (1-18), ENVKGTEDS (SEQ ID NO:32) (1-19), GTTVLLPLV (SEQ ID NO:33) (1-20), IFFGLCLLSLL (SEQ ID NO:34) (1-21), SLLFIGLM (SEQ ID NO:35) (1-22), YRYQRWK (SEQ ID NO:36) (1-23) and combination thereof.

[0018] In some embodiments, the TNF receptor suitable for the invention is TNFR2

(SEQ ID NO:2). In some embodiments, an extracellular or transmembrane region of TNFR2 is selected from the group consisting of

MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYYDQT (SEQ ID NO:10);

AQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQV ETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVC KPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQP VSTRSQHTQPTPEPSTAPSTSF (SEQ ID NO: 11); TCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPEC LSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVA RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI (SEQ ID NO: 12);

PGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSF (SEQ ID

NO:13); and combination thereof.

In some embodiments, an extracellular or transmembrane region of TNFR2 is selected from the group consisting of

MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYY (SEQ ID NO:559);

SKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQAC TREQNRICTCRPGW (SEQ ID NO:560);

GCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVA I PGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSF (SEQ ID

NO:561); and combination thereof.

[0019] In some embodiments, the contiguous amino acids are selected from Table 2.

[0020] In some embodiments, a peptide according to the invention is selected from the group consisting of MCCSKCSPG (SEQ ID NO: 85) (2-1), CEDSTYTQL (SEQ ID NO: 86) (2-2), ACTREQNRICT (SEQ ID NO: 87) (2-3) and combination thereof.

[0021] In some embodiments, the present invention provides various derivatives of peptides described herein. In some embodiments, the present invention provides a peptide that is up to 25 amino acids long and has a sequence that includes at least four (e.g., at least five, or at least six) amino acids from any one of the peptides shown in Tables 1, 2, 3 or 4 and wherein the at least four (e.g., at least five, or at least six) amino acids maintain their relative positions as they appear in the corresponding peptide shown in Tables 1, 2, 3 or 4. Alternatively, the present invention provides a peptide that is up to 25 amino acids long and has a sequence that includes at least four (e.g., at least five, or at least six) amino acids from any one of the peptides shown in Tables 1 , 2, 3 or 4 and wherein the at least four (e.g., at least five, or at least six) amino acids maintain their relative positions, but in their inverse configuration, as they appear in the corresponding peptide shown in Tables 1, 2, 3 or 4.

[0022] In some embodiments, the present invention provides a peptide that is up to 25 amino acids long and has a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to any one of the peptides shown in Tables 1, 2, 3 or 4.

[0023] In particular embodiments, the present invention provides a peptide that is up to 25 amino acids long and has a sequence that includes at least four (e.g., at least five, or at least six) amino acids from SCQEKQNTV (SEQ ID NO:27) (1-14) or YRYQRWK (SEQ ID NO:36) (1-23) and wherein the at least four (e.g., at least five or at least six) amino acids maintain their relative positions and/or spacing (or, alternatively, in their inverse configuration) as they appear in SCQEKQNTV (SEQ ID NO:27) (1-14) or YRYQRWK (SEQ ID NO:36) (1-23). In particular embodiments, the present invention provides a peptide that is up to 25 amino acids long and has a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to SCQEKQNTV (SEQ ID NO:27) (1-14) or YRYQRWK (SEQ ID NO:36) (1-23).

[0024] In some embodiments, peptides according to the invention contain one or more L-amino acids, D-amino acids, and/or un-natural amino acids.

[0025] In some embodiments, peptides of the invention contain additional amino acids from a region of the TNF receptor that is separated from the contiguous amino acids.

[0026] In some embodiments, peptides of the invention contain one or more modifications to increase protease resistance, serum stability and/or bioavailability. In some embodiments, suitable modifications are selected from acetylation, glycosylation, biotinylation, substitution with D-amino acid and/or un-natural amino acid, and/or cyclization of the peptide.

[0027] The present invention further provides pharmaceutical compositions containing a peptide described herein and a pharmaceutically acceptable carrier. The present invention also provides methods of inhibiting an activity of a TNF receptor in a mammalian cell by administering into the mammalian cell a peptide or a composition according to the invention. In some embodiments, methods of the invention inhibit an activity of a TNF receptor selected from release of IL-I, IL-6, GM-CSF, and/or IL-10, induction of chemokines, activation of adhesion molecules, growth of blood vessels, release of tissue destructive enzymes and activation of T cells, apoptosis, NF -KB, and/or caspase pathways.

[0028] In some embodiments, the present invention provides methods for treating

TNF-related diseases, disorders or conditions, such as inflammation, by administering to a subject in need of treatment a peptide described herein. In some embodiments, the peptide is administered orally. In some embodiments, the present invention is used to treat inflammation associated with arthritis (e.g., rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, lupus-associated arthritis and/or ankylosing spondylitis); scleroderma; systemic lupus erythematosis; HIV; Sjogren's syndrome; vasculitis; multiple sclerosis; autoimmune thyroiditis; asthma; dermatitis; myasthenia gravis; inflammatory bowel disease (IBD); Crohn's disease; colitis; diabetes mellitus (type I and type II); inflammatory conditions of the skin, cardiovascular system, nervous system, liver, kidney and pancreas; sarcoidosis; scleroderma; cirrhosis; eosinophilic esophagitis; cardiovascular disorders; disorders associated with wound healing; respiratory disorders; acute inflammatory conditions; atopic inflammatory disorders; tumors or cancers; and/or transplant rejection.

[0029] In some embodiments, the present invention provides methods for treating eye diseases or discorders related to TNF (e.g., retinal detachment, retinoschisis, hypertensive retinopathy, diabetic retinopathy, general retinopathy, retinopathy of prematurity, age-related macular degeneration, general macular degeneration, epiretinal membrane, choroidal neovascular membrane, cystoid macular edema, macular hole, retinitis pigmentosa, macular edema, sleritisc, corneal ulcer or abrasion, thygeson's superficial punctate keratopathy, corneal neovascularization, fuchs' dystrophy, keratoconus, keratoconjunctivitis sicca or dry eye, iritis, uveitis, conjunctivitis, pterygium, subconjunctival hemorrhage, glaucoma, keratomycosis , xerophthalmia, cataract and systemic diseases with ocular manifestations including allergies, AIDS and hypertension).

[0030] In another aspect, the present invention provides new methods of designing and identifying peptide modulators (e.g., Allosteramers™) for a biological target (e.g., a receptor) based on a linear amino acid sequence without first characterizing any tertiary or secondary structure of the target.

[0031] In some embodiments, the present invention provides a method of identifying a peptide modulator of a target comprising steps of: (a) providing a plurality of candidate peptides, each of which is up to 25 amino acids long and has a sequence corresponding to at least 5 contiguous amino acids that appear in the target without first characterizing any tertiary or secondary structure of the target; and (b) determining if any of the plurality of candidate peptides inhibit or activate an activity of the target relative to a control. In some embodiments, the plurality of candidate peptides comprises sequences collectively covering the entire length of the target or a portion thereof. In some embodiments, the plurality of peptides has adjacent or overlapping sequences spanning the target or a portion thereof. In some embodiments, the plurality of peptides has overlapping sequences and wherein adjacent overlapping sequences shift by a pre-determined number of amino acids. In some embodiments, the pre-determined number is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0032] In some embodiments, each individual candidate peptide has a sequence at least 70% identical to at least 7 contiguous amino acids that appear in the target, or a sequence at least 70% identical to at least 7 contiguous amino acids that appear in the target but in the inverse configuration. In some embodiments, each individual candidate peptide has a sequence identical to at least 5 contiguous amino acids that appear in the target, or a sequence identical to at least 5 contiguous amino acids that appear in the target but in the inverse configuration. In some embodiments, each individual candidate peptide has a sequence identical to 5, 6, 7, 8, 9, or 10 contiguous amino acids that appear in the target, or a sequence identical to at least 5, 6, 7, 8, 9, or 10 contiguous amino acids that appear in the target but in the inverse configuration.

[0033] In some embodiments, each individual peptide contains all L-, all D- or a mixture of L- and D- amino acids.

[0034] In some embodiments, a target suitable for the present invention is a receptor.

In some embodiments, the receptor suitable for the present invention is a cytokine receptor, a growth factor receptor, a chemokine receptor, a G-protein coupled receptor, a receptor kinase, a guanylyl cyclase receptor, a hormone or neurotransmitter receptor. In some embodiments, the receptor suitable for the invention is a cytokine receptor selected from the group consisting of VEGF receptors, PDGF receptors, IGF-I receptors, FGF receptors, EGF receptors, interleukin receptors, IFNα receptor, IFNβ receptor, TGFβ receptor, NGF/TNF receptors and combinations thereof. In some embodiments, the receptor suitable for the invention is a TNF receptor (e.g., TNFRl or TNFR2). [0035] In some embodiments, the contiguous amino acids appear in an extracellular or transmembrane region of the receptor.

[0036] In some embodiments, a suitable control indicates the activity of the target in the absence of the candidate peptide.

[0037] In some embodiments, step (b) above determining if any of the plurality of candidate peptides inhibit or activate an activity of the target involves performing an in vitro activity assay.

[0038] In some embodiments, a method according to the invention further includes a step of testing the ability of the identified peptide inhibitor or activator to treat a disease, disorder, or condition associated with the target. In some embodiments, the testing step comprises treating an animal model of the disease, disorder, or condition associated with the target.

[0039] In some embodiments, a method according to the invention further includes a step of modifying the identified inhibitor or activator to increase protease resistance, serum stability and/or bioavailability. In some embodiments, the modifying step is selected from acetylation, glycosylation, biotinylation, substitution with D-amino acid or un-natural amino acid, and/or cyclization of the peptide.

[0040] The present invention also encompasses peptide modulators identified using various methods described herein.

[0041] In yet another aspect, the present invention provides a method of identifying a cryptic allosteric site within a target comprising steps of: a) providing a plurality of peptides, wherein each individual peptide is up to 25 (e.g., 5-25, 7-10) amino acids long and the plurality of peptides comprise overlapping or adjacent sequences collectively covering the entire length of the target or a portion thereof; b) assessing the ability of each individual peptide to modulate the target; c) determining an allosteric map by plotting the modulatory effect of the plurality of peptides obtained from step (b) against their corresponding amino acid positions in the target; thereby identifying one or more cryptic allosteric sites within the structure of the target.

[0042] In some embodiments, each individual peptide has a sequence corresponding to at least 5 contiguous amino acids that appear in the target. In some embodiments, each individual peptide has a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, or 12) contiguous amino acids that appear in the target. In some embodiments, each individual peptide has a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, or 12) contiguous amino acids that appear in the target but in the inverse configuration. In some embodiments, each individual peptide has a sequence identical to 5, 6, 7, 8, 9, or 10 contiguous amino acids that appear in the target. In some embodiments, each individual peptide has a sequence identical to 5, 6, 7, 8, 9, or 10 contiguous amino acids that appear in the target but in the inverse configuration. In some embodiments, the plurality of peptides has overlapping sequences and wherein adjacent overlapping sequences shift by a pre-determined number of amino acids. In some embodiments, the pre-determined number of amino acids is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0043] In some embodiments, a method according to the present invention further includes a step of characterizing the identified one or more cryptic allosteric sites by cross- linking or co-crystallization with the corresponding peptides that bind to the cryptic allosteric sites.

[0044] In this application, the use of "or" means "and/or" unless stated otherwise. As used in this application, the term "comprise" and variations of the term, such as "comprising" and "comprises," are not intended to exclude other additives, components, integers or steps. As used in this application, the terms "about" and "approximately" are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0045] Other features, objects, and advantages of the present invention are apparent in the detailed description, drawings and claims that follow. It should be understood, however, that the detailed description, the drawings, and the claims, while indicating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The drawings are provided for illustration purposes only and not for limitation.

[0047] Figure 1. The amino acid sequences of TNFRl (pl9438 TNRlA Human

(SEQ ID NO: I)) and TNFR2 (p2-333 TNRIB Human (SEQ ID NO:2)). The single underlined sequences in TNFRl corresponds to the cysteine rich domain of TNFRl. The double underlined sequences correspond to the potential transmembrane domain. The italic letters (amino acids 1-54) correspond to the pre-ligand assembly domain (PLAD).

[0048] Figure 2 A. Exemplary results illustrating screening of Allosteramers for

TNFR using inhibition of TNFα-induced IL-6 synthesis in human fibroblasts (WI-38) by candidate peptides of the invention. Exemplary peptides 1-1 through 1-23 and 2-1 through 2- 3 are shown here.

[0049] Figure 2B. Additional exemplary screening results using TNFα-induced IL-6 synthesis assays in human fibroblasts (WI-38). Exemplary peptides 1-24 through 1-47 designed using the Module X Walk method, herein defined, are shown here. The sequences of the peptides are shown in Table 1.

[0050] Figure 2C. Further exemplary results illustrating screening of Allosteramers for TNFR using TNFα-induced IL-6 synthesis assay in human fibroblasts (WI-38). Exemplary peptides ALL-I through ALL-24 designed using Module X approach, herein defined, are shown here. The sequences of the peptides are shown in Table 1.

[0051] Figure 2D. Exemplary results illustrating screening of Allosteramers for

TNFR using inhibition of TNFα-induced IL-I production in human umbilical vessels endothelial cells by candidate peptides of the invention. The following peptides were tested: 1-7 (SEQ ID NO:20), 1-8 (SEQ ID NO:21), 1-19 (SEQ ID NO:32), 1-14 (SEQ ID NO:27), 1- 142 (SEQ ID NO:89) (D-peptide version of 1-14), 1-23 (SEQ ID NO:36), 1-232 (SEQ ID NO:94) (D-peptide version of 1-23), 1-21 (SEQ ID NO:34), 1-22 (SEQ ID NO:35), ALL-2 (SEQ ID NO:62), ALL-5 (SEQ ID NO:65), ALL-20 (SEQ ID NO:80). The sequences of the peptides are shown in Table 1, Figures 10 and 11. [0052] Figure 3. Exemplary results illustrating inhibition of TNFα-induced IL-I synthesis in human brain microvascular endothelial cells (HBMED) by exemplary peptide inhibitors. The right panel shows that peptide 1-14 is derived from a loop region in the cysteine rich domain and peptide 1-23 is derived from the potential transmembrane region. According to a 3D structure of TNFRl, peptide 1-23 may be half extra half intra membranous.

[0053] Figure 4. A diagram illustrating that peptides containing sequences from different regions of a TNF receptor demonstrate different TNFR inhibitory activities. For example, peptides derived from the N-terminal region of the receptor are generally less effective in inhibiting TNF-induced IL-6 synthesis than the ones derived from the cysteine rich region or from the last domain in the juxtamembrane regions.

[0054] Figure 5. Exemplary results illustrating the effect of exemplary peptides 1-14 or 1-23 on the dose response of IL-6 synthesis induced by TNFα.

[0055] Figure 6. An exemplary Schild Plot of TNFR 1-23. WI-38 cells were incubated with increasing concentrations of human TNFα and each dose response curve was also generated in the presence of increasing concentrations of peptide TNFRl -23 (e.g., one dose response curve with 10 ^~6 M TNFRl -23, one with 10 ^~7 M TNFRl -23). Twenty four hours later IL-6 measurements were performed with an ELISA kit from R&D Systems according to the manufacturer's instructions. Data was plotted and transformed with Graph Prism software, and exemplary results are shown in Figure 6. Similar analysis was done with peptide TNFR1-14 and exemplary results are shown in Figure 7.

[0056] Figure 7. An exemplary Schild Plot of TNFR 1-14 using similar analysis as described in Figure 6.

[0057] Figure 8. Exemplary dose response of the inhibitory effect of TNFRl -14 and

1-23 on TNFα-induced IL-6 production in human fibroblasts.

[0058] Figure 9. Exemplary results illustrating the in vivo efficacy of exemplary peptides 1-14 and 1-23 using a phorbol myristate acetate (PMA)-induced dermatitis inflammatory model. A peptide that inhibits IL23R (2305) was used as a positive control.

[0059] Figure 10. Exemplary results illustrating the effect of peptide 1-23 on TNF- induced hypotension. Peptide 1-23 was administered orally using gavage. [0060] Figure 11. Exemplary results illustrating exemplary derivatives of peptide 1-

14 and their ability to inhibit TNF -induced IL-6 synthesis.

[0061] Figurel2. Exemplary results illustrating exemplary derivatives of peptide 1-

23 and their ability to inhibit TNF -induced IL-6 synthesis.

[0062] Figure 13. Exemplary illustration of an TNFRl "allosteric map." Short overlapping peptides containing 10 amino acid residues (10-mer peptides) were synthesized along the entire length of TNFRl (each shifted progressively by one amino acid). The effects of these peptides on TNFRl were then assessed using TNFα-induced IL-6 expression as described in Example 1. The percentage of TNF-induced IL-6 production in the presence of candidate peptides were plotted against the corresponding amino acid positions of TNFRl.

DEFINITIONS

[0063] Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. Generally, the procedures of cell cultures, infection, molecular biology methods and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as, for example, Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001; and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 ^rd edition, Cold Spring Harbor Laboratory Press, N.Y., 2001.

[0064] In order for the present invention to be more readily understood, certain terms are first defined. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0065] Allosteramer: As used herein, the term "allosteramer" or "Allosteramer™" refers to a short peptide composed of 5-25 amino acids derived from body's own proteins that can interact with the protein from which it was derived, or with its associated proteins, without competing with a natural ligand. The terms "Allosteramers", "Allosteramers™", "allosteramers", "peptide modulators", and "allosteric modulators" are used interchangeably in this application.

[0066] Allosteric Site: As used herein, the term "allosteric site" refers to any binding site on a target wherein an agent that binds to such site does not compete with a natural ligand binding to the same target.

[0067] Allosteric modulator: As used herein, the term "allosteric modulator" refers to any modulator that can inhibit or activate an activity of a biological target without competing with a natural ligand. In some embodiments, an allosteric modulator binds to an allosteric site. An allosteric modulator can be an allosteric agonist or a Positive Allosteric Modulator (PAM), i.e., a modulator that activates the biological target without competing with a natural ligand. An allosteric modulator can also be an allosteric antagonist or a Negative Allosteric Modulator (NAM), i.e., a modulator that inhibits the biological target without competing with a natural ligand. In some embodiments, an allosteric modulator is both a PAM and a NAM of a biological target. In some embodiments, an allosteric modulator is a Silent Allosteric Modulator (SAM) of a biological target, which binds to an allosteric site, but does not affect the function of a biological target.

[0068] Amino acid: As used herein, the term "amino acid," in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H ₂ N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a synthetic or un-natural amino acid; in some embodiments, an amino acid is a D-amino acid (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, lactic acid); in some embodiments, an amino acid is an L-amino acid. "Standard amino acid" refers to any of the twenty standard amino acids commonly found in naturally occurring peptides including both L- and D- amino acids which are both incorporated in peptides in nature. "Nonstandard" or "unconventional amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. As used herein, "synthetic or un-natural amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxy- and/or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, and/or substitution with other chemical groups that can change the peptide's circulating half- life without adversely affecting its activity. Examples of unconventional or un-natural amino acids include, but are not limited to, citrulline, ornithine, norvaline, 4-(ii)-butenyl-4(i?)-methyl-N- methylthreonine (MeBmt), N-methyl-leucine (MeLeu), aminoisobutyric acid, statine, and N- methyl-alanine (MeAIa). Amino acids may participate in a disulfide bond. The term "amino acid" is used interchangeably with "amino acid residue," and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

[0069] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, at any stage of development. In some embodiments, "animal" refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.

[0070] Approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0071] Autoimmune disorder. As used herein, the term "autoimmune disorder" refers to a disorder resulting from attack of a body's own tissue by its immune system. In some embodiments, autoimmune diseases is diabetes mellitus, multiple sclerosis, premature ovarian failure, scleroderma, Sjogren's disease, lupus, alopecia (baldness), polyglandular failure, Grave's disease, hypothyroidism, polymyosititis, Celiac disease, Crohn's disease, inflammatory bowel disease, ulcerative colitis, autoimmune hepatitis, hypopituitarism, Guillain-Barre syndrome, myocardititis, Addison's disease, autoimmune skin diseases (e.g., psoriasis), uveititis, pernicious anemia, polymyalgia rheumatica, Goodpasture's syndrome, hypoparathyroidism, Hashimoto's thyoriditis, Raynaud's phenomenon, polymyaglia rheumatica, and rheumatoid arthritis.

[0072] Control: As used herein, the term "control" has its art-understood meaning of being a standard against which results are compared. Typically, controls are used to augment integrity in experiments by isolating variables in order to make a conclusion about such variables. In some embodiments, a control is a reaction or assay that is performed simultaneously with a test reaction or assay to provide a comparator. In one experiment, the "test" (i.e., the variable being tested) is applied. In the second experiment, the "control," the variable being tested is not applied. In some embodiments, a control is a historical control (i.e., of a test or assay performed previously, or an amount or result that is previously known). In some embodiments, a control is or comprises a printed or otherwise saved record. A control may be a positive control or a negative control.

[0073] Cryptic Allosteric Site (CAS): As used herein, the term "Cryptic Allosteric

Site (CAS)" refers to any previously unknown binding site outside a natural-ligand binding pocket. As used herein, the terms "Cryptic Allosteric Site (CAS)" and "Cryptic Druggable Site" are used interchangeably. See, the definition of druggable site.

[0074] Dysfunction: As used herein, the term "dysfunction" refers to an abnormal function. Dysfunction of a molecule {e.g., a protein) can be caused by an increase or decrease of an activity associated with such molecule. Dysfunction of a molecule can be caused by defects associated with the molecule itself or other molecules that directly or indirectly interact with or regulate the molecule.

[0075] Druggable site: As used herein, the term "druggable site" refers to any site or region of a biological target that can be used to develop drug candidates. Typically, a druggable site is a binding pocket or site or region that can be used to screen for various drug candidates. In some embodiments, a druggable site is a ligand binding site. In some embodiments, a druggable site is a binding site for an allosteric binding agent. In some embodiments, a druggable site is an Allosteramer™-binding site. As used herein, a druggable site without a natural ligand is also referred to as a cryptic druggable site. In this application, the terms "Cryptic Druggable Site" and "Cryptic Allosteric Site" are used interchangeably.

[0076] Functional: As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.

[0077] Functional derivative: As used herein, the term "functional derivative" denotes, in the context of a functional derivative of an amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence. A functional derivative or equivalent may be a natural derivative or is prepared synthetically. Exemplary functional derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved (e.g., it acts as a non-competitive antagonist of a TNF receptor). The substituting amino acid desirably has chemico-physical properties which are similar to that of the substituted amino acid. Desirable similar chemico-physical properties include similarities in charge, bulkiness, hydrophobicity, hydrophilicity, and the like.

[0078] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism. [0079] In vivo: As used herein, the term "in vivo" refers to events that occur within a multi-cellular organism such as a non-human animal.

[0080] Inflammatory disease, disorder or condition: As used herein, the term an

"inflammatory disease, disorder or condition" refers to any disease, disorder, or condition in which the immune system abnormally activated or suppressed. In some embodiments, an inflammatory disease, disorder, or condition that can be treated according to the invention is inflammation of the upper and lower respiratory tract, for example, bronchial asthma, allergic asthma, non-allergic asthma, lymphomatous tracheobronchitis, allergic hypersensitivity or a hypersecretion condition, such as chronic bronchitis and cystic fibrosis; pulmonary fibrosis of various etiologies (e.g., idiopathic pulmonary fibrosis), chronic obstructive pulmonary disease (COPD), sarcoidosis, allergic and non-allergic rhinitis; allergic or non-allergic urticaria; a skin-related diseases characterized by deregulated inflammation, tissue remodeling, angiogenesis, and neoplasm, a disease of the gastrointestinal tract, such as Crohn's disease, Hirschsprung's disease, diarrhea, malabsorption conditions, and inflammatory conditions; a disorder of the central and peripheral nervous system, such as depression, anxiety states, Parkinson's disease, migraine and other forms of cranial pain, strokes, emesis; a disease of the immune system, such as in the splenic and lymphatic tissues, an autoimmune disease or other immune -related diseases; a disease of the cardiovascular system, such as pulmonary edema, hypertension, atherosclerosis, pre-eclampsia, complex regional pain syndrome type 2, stroke and chronic inflammatory diseases such as arthritis, a bone-related diseases such as rheumatoid arthritis, as well as pain, chronic pain such as fibromyalgia, and other disorders in which the action of neurokinins, tachykinins or other related substances (e.g., hemokinins) are involved in the pathogenesis, pathology, and etiology.

[0081] Additional examples of inflammatory disorders include acne vulgaris; acute respiratory distress syndrome; Addison's disease; allergic intraocular inflammatory diseases, ANCA-associated small-vessel vasculitis; ankylosing spondylitis; atopic dermatitis; autoimmune hemolytic anemia; autoimmune hepatitis; Behcet's disease; Bell's palsy; bullous pemphigoid; cerebral ischaemia; cirrhosis; Cogan's syndrome; contact dermatitis; Cushing's syndrome; dermatomyositis; diabetes mellitus; discoid lupus erythematosus; lupus nephritis; eosinophilic fasciitis; erythema nodosum; exfoliative dermatitis; focal glomerulosclerosis; focal segmental glomerulosclerosis; segmental glomerulosclerosis; giant cell arteritis; gout; gouty arthritis; graft-versus-host disease; hand eczema; Henoch-Schonlein purpura; herpes gestationis; hirsutism; idiopathic cerato- scleritis; idiopathic thrombocytopenic purpura; immune thrombocytopenic purpura inflammatory bowel or gastrointestinal disorders, inflammatory dermatoses; lichen planus; lymphomatous tracheobronchitis; macular edema; multiple sclerosis; myasthenia gravis; myositis; nonspecific fibrosing lung disease; osteoarthritis; pancreatitis; pemphigoid gestationis; pemphigus vulgaris; periodontitis; polyarteritis nodosa; polymyalgia rheumatica; pruritus scroti; pruritis/inflammation, psoriasis; psoriatic arthritis; pulmonary histoplasmosis; relapsing polychondritis; rosacea caused by sarcoidosis; rosacea caused by scleroderma; rosacea caused by Sweet's syndrome; rosacea caused by systemic lupus erythematosus; rosacea caused by urticaria; rosacea caused by zoster-associated pain; sarcoidosis; scleroderma; septic shock syndrome; shoulder tendinitis or bursitis; Sjogren's syndrome; Still's disease; Sweet's disease; systemic lupus erythematosus; systemic sclerosis; Takayasu's arteritis; temporal arteritis; toxic epidermal necrolysis; transplant-rejection and transplant-rejection-related syndromes; tuberculosis; type-1 diabetes; ulcerative colitis; uveitis; vasculitis; and Wegener's granulomatosis. Desirably the autoimmune disorder is inflammatory bowel disease, an inflammatory skin disorder such as psoriasis, or multiple sclerosis.

[0082] Isolated: As used herein, the term "isolated" refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated. In some embodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, the term "isolated cell" refers to a cell not contained in a multi-cellular organism.

[0083] Modulator. As used herein, the term "modulator" refers to a compound that alters or elicits an activity. For example, the presence of a modulator may result in an increase or decrease in the magnitude of a certain activity compared to the magnitude of the activity in the absence of the modulator. In certain embodiments, a modulator is an inhibitor or antagonist, which decreases the magnitude of one or more activities. In certain embodiments, an inhibitor completely prevents one or more biological activities. In certain embodiments, a modulator is an activator or agonist, which increases the magnitude of at least one activity. In certain embodiments the presence of a modulator results in an activity that does not occur in the absence of the modulator. As used herein, the terms "inhibiting," "reducing," "preventing," or "antagonizing," or any variations of these terms as used herein, refer to a measurable decrease of a biological activity. In some embodiments, the decrease is a 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% reduction in the biological activity relative to a control. As used herein, the terms "stimulating," "increasing," or "agonizing," or any variations of these terms as used herein, refer to a measurable increase of a biological activity. In some embodiments, the increase is a 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% increase in the biological activity relative to a control.

[0084] Peptides: As used herein, the term "peptides" refers to macromolecules which comprise a multiplicity of amino or imino acids (or their equivalents) in peptide linkage. In the polypeptide or peptide notation used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention. Peptides may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Peptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, etc. In some embodiments, peptides may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. In some embodiments, peptides may contain up to 25 amino acids.

[0085] Reverse-D peptide: As used herein, the term "reverse-D peptide" refers to peptides containing D-amino acids, arranged in a reverse sequence relative to a peptide containing L-amino acids. For example, the C-terminal residue of an L-amino acid peptide becomes N-terminal for the D-amino acid peptide, and so forth. Reverse D-peptides desirably retain the same tertiary conformation and therefore the same activity, as the L- amino acid peptides, but desirably are more stable to enzymatic degradation in vitro and in vivo, and therefore can have greater therapeutic efficacy than the original peptide (Brady and Dodson, Nature 368:692-693, 1994; and Jameson and McDonnel, Nature 368:744-746, 1994). [0086] Reverse-L peptide: As used herein, the term "reverse-L peptide" refers to peptides containing L-amino acids arranged in a reverse sequence relative to a parent peptide. The C-terminal residue of the parent peptide becomes N-terminal for the reverse-L peptide, and so forth.

[0087] Subject: As used herein, the term "subject" or "patient" refers to any organism to which compositions in accordance with the invention may be administered, e.g. , for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.).

[0088] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0089] Suffering from: An individual who is "suffering from" a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and/or condition.

[0090] Susceptible to: An individual who is "susceptible to" a disease, disorder, and/or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

[0091] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0092] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to any agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect. In some embodiments, a therapeutic agent of the invention refers to a peptide inhibitor or derivatives thereof according to the invention.

[0093] Treating: As used herein, the term "treat," "treatment," or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

DETAILED DESCRIPTION

[0094] The present invention provides, among other things, short peptide modulators such as Allosteramers™ for TNF receptors (e.g., TNFRl or TNFR2) based on amino acid sequences in extracellular or transmembrane regions of the receptor. In some embodiments, Allosteramers according to the invention may be used to bind to the TNF receptor or functionally inhibit or activate the TNF receptors without competing with a natural ligand. Thus, peptide modulators provided herein can be an allosteric agonist or a Positive Allosteric Modulator (PAM) of TNF receptors, i.e., a modulator that activates the TNF receptors without competing with a natural ligand. Peptide modulators provided herein can also be an allosteric antagonist or a Negative Allosteric Modulator (NAM) of TNF receptors, i.e., a modulator that inhibits the TNF receptors without competing with a natural ligand. In some embodiments, peptide modulators provided herein can be both a PAM and a NAM of the TNF receptors. In some embodiments, peptide modulators provided herein can be a Silent Allosteric Modulator (SAM) of the TNF receptors, which binds to a site outside of the ligand binding site, but does not affect the function of the receptor. A SAM of a TNF receptor is particularly useful for detecting the receptor for the purposes of diagnosis or prognosis of a TNF-related disease, disorder or condition.

[0095] The invention provides pharmaceutical compositions and methods for treating

TNF-related diseases, disorders and conditions based on peptide modulators described herein.

[0096] In addition, the invention also provides a new and effective method to design and identify peptide modulators (e.g., Allosteramers) without requiring a priori knowledge of the secondary or tertiary structure of the target or involving tertiary or secondary structural modeling of a target.

[0097] Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of "or" means "and/or" unless stated otherwise. Design of Peptide Modulators by Module X Walk

[0098] Prior to the present invention, design of potential peptide modulators for a biological target typically involves secondary and/or tertiary structural modeling to first identify regions that are involved in ligand binding, conformational change and/or receptor oligomerization. For example, candidate peptide modulators can be designed using the Module X technology, which is described in U.S. Patent No. 7,432,341, and PCT Application No. PCT/IB2006/0022987, both entitled "Cytokine receptor modulators and method of modulating cytokine receptor activity," the entire disclosures of which are hereby incorporated by reference. According to the Module X technology, candidate peptides may be strategically designed to interact with at least one of a flexible region of a receptor that is typically important for the appropriate conformation and activity of the receptor. Such flexible regions include, but are not limited to, juxtamembranous regions, regions containing α helix, β sheet, loops and/or β turns, regions between domains, regions between two β chains, and combinations thereof.

[0099] As described in the Examples section, the inventors of the present application discovered that short peptide modulators (e.g., inhibitors or activators) of a biological target can be successfully designed simply based on the primary linear amino acid sequence of the target without first characterizing any tertiary or secondary structure of the target. This method is referred to as a "walk" or "walking" method. For example, short overlapping or adjacent peptides (e.g., 7-10-mers) can be synthesized along the entire length of a biological target of interest or a portion thereof by progressively walking from the N-terminus to the C- terminus. Inventive methods according to the invention may be used for systematic screening for peptide modulators based on linear amino acid sequence of a target.

[0100] As used herein, a short peptide suitable for the invention includes any peptide that contains up to 25 amino acids (e.g., up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25 amino acids) or equivalents thereof. In some embodiments, a peptide according to the invention contain 5-25 amino acids (e.g., 5-20, 5-15, 5-12, 5-10, 6-25, 6-20, 6-15, 6-12, 6-10, 7-25, 7-20, 7-15, 7-12, or 7-10 amino acids) or equivalents thereof. In some embodiments, peptides according to the invention is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25 amino acids long. [0101] Each individual short peptide may have a sequence corresponding to at least 5

(e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids that appear in the target and a plurality of candidate peptides can be designed to collectively cover the entire length of the target or a portion of interest (e.g., typically a region known to be important structurally or functionally). In some embodiments, such plurality of peptides are designed to have adjacent or overlapping sequences. Typically, two adjacent or overlapping peptide sequences are designed to shift by a pre-determined number of amino acids. Suitable shift can be by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0102] In some embodiments, short peptides suitable for the inventive method are designed to contain a sequence having at least 70% (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to the sequence of at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids that appear in a target of interest. Percentage of amino acid sequence identity can be determined by alignment of amino acid sequences. Alignment of amino acid sequences can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Preferably, the WU-BLAST-2 software is used to determine amino acid sequence identity (Altschul et ah, Methods in Enzymology 266, 460-480 (1996); http://blast.wustl/edu/blast/README.html). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=l, overlap fraction=0.125, word threshold (T)=I 1. HSP score (S) and HSP S2 parameters are dynamic values and are established by the program itself, depending upon the composition of the particular sequence, however, the minimum values may be adjusted and are set as indicated above.

[0103] In some embodiments, short peptides suitable for the inventive method are designed to contain a sequence that includes at least 4 (e.g., at least 5, at least 6, at least 7) amino acids from at least 7 (e.g., at least 8, at least 9, at least 10, at least 11, at least 12) contiguous amino acids that appear in a target of interest, wherein the at least 4 (e.g., at least 5, at least 6, at least 7) amino acids maintain their relative positions and/or spacing as they appear in the target. In some embodiments, short peptides suitable for the inventive method are designed to contain a sequence that includes at least 4 (e.g., at least 5, at least 6, at least 7) amino acids from at least 7 (e.g., at least 8, at least 9, at least 10, at least 11, at least 12) contiguous amino acids that appear in a target of interest, wherein the at least 4 (e.g., at least 5, at least 6, at least 7) amino acids maintain their relative positions and/or spacing, but in the inverse configuration, as they appear in the target.

[0104] In some embodiments, short peptides suitable for the inventive method are designed to contain a sequence that is identical to at least 4 (e.g., at least 5, at least 6, at least 7) contiguous amino acids that appear in a target of interest. In some embodiments, short peptides suitable for the inventive method are designed to contain a sequence that is identical to at least 4 (e.g., at least 5, at least 6, at least 7) contiguous amino acids that appear in a target of interest but in the inverse configuration.

[0105] Suitable short peptides may contain all L-amino acids, all D-amino acids, a mixture of L- and D- amino acids, and/or un-natural amino acids.

[0106] These short peptides (e.g., overlapping or adjacent peptides) are then assessed for their ability to bind to the target or modulate (e.g., inhibit or activate) an activity of the target using various in vitro or in vivo assays known in the art. Such as assays may be performed in e.g., a multi-well format. Thus, inventive methods according to the invention can be developed for low-throughput, high-throughput, or ultra-high throughput screening formats and are amenable to automation.

[0107] Thus, the present invention provides a simplier, efficient and effective method for designing and identifying peptide modulators of a target of interest, which is referred to as the "Module X Walk" technology. It is contemplated that "Module X Walk" methods according to the invention can be used to design and identify candidate peptide modulators of any biological targets including, but not limited to, extracellular targets such as receptors and intracellular proteins such as enzymes, or transcription factors. In particular, the present invention can be used to design and identify candidate peptide modulators of receptors including but not limited to cytokine receptors, growth factor receptors, chemokine receptors, G-protein coupled receptors, receptor kinases, guanylyl cyclase receptors, hormone or neurotransmitter receptors. In some embodiments, the present invention can be used to design and identify candidate peptide modulators of receptors including, but not limited to, VEGF receptors, PDGF receptors, IGF-I receptors, FGF receptors, EGF receptors, interleukin receptors (e.g., IL-IR, IL-2R, IL-3R; IL-4R; IL-5R; IL-6R, IL-7R; IL-8R; IL-9R; IL-10R; IL-I lR; IL- 12R; IL- 13R; IL- 14R; IL- 15R; IL- 16R; IL- 17R; IL-21R; IL-23R, IL- 31R), G protein-coupled receptors, IFNα receptor, IFNβ receptor, TGFβ receptor, NGF receptors, TNF receptors (e.g., TNFRl, TNFR2); RANK; ActRIIB; EpoR; GLPlR; GCGR; ICAM-I; CD2; CD40; CD20; CD22; LRP5/6 receptors; ICOS; Type I interferon receptor 1; IFNGRl (CDl 19 ) & IFNGR2; RAGE receptor; alpha-4-integrin subunit (CD49d) [alpha 4β 1 (VLA-4) and alpha 4β 7 integrins]; EGFR (ErbB-1 /HER-I); BLyS Receptor (BAFF-R); TGF-β receptor I (activin-like kinase-5 ALK5); ActRIIA; CRTH2 receptor; FN14/TWEAKR; Cripto-1; GM-CSF-Receptor; LAMP-2A (lysosome associated membrane protein type 2A); NgR (Nogo receptor); Telomerase; PDGFR-beta receptor; ST2 receptor; DR3 (TRAMP, LARD, WSL-I, TNFRSF25); PDl (PDCDl); TLR2 (CD282); SlPl receptor; CCR2 (CD192) (Note: MCP-I also binds to CCR4); CXCR4 (fusin); P2X7; PTPlB (PTPNl); MMP-9; ADAM-IO (Sheddase); LRPl; Albumin; TAS IRl +3 ("Sweet" taste receptor for amino acids); TAS2R1 - TAS2R50, and TAS2R60 ("Bitter" taste receptors); Tim-3; TSLP receptor; VRl (TRPVl); TPO-R; DR6 (TNFRSF21); PepTl, PepT2, ASBT, OATP-B, D, E or 3, claudin (or other components of the tight junctions); Alpha-9, alpha-10 Nicotinic Acetylcholine receptors; ADORAl (adenosine Al receptor); CD148 (PTPRTJ; DEP-1/PTPη); 0X2R; AdPLA receptor.

[0108] Additional biological targets are described in U.S. Provisional Application entitled "IDENTIFICATION OF CRYPTIC ALLOSTERIC SITES AND USES THEREOF," filed on even date, the disclosures of which are hereby incorporated by reference.

Identification of Cryptic Allosteric Sites

[0109] The inventive Module X Walk method described herein is particularly useful for identification of cryptic allosteric sites within a biological target of interest. As used herein, the term "Cryptic Allosteric Site (CAS)" refers to any previously unknown binding site outside a natural-ligand binding pocket. Such binding sites are useful target sites for drug development, therefore are typically also known as druggable sites. One of the first rationally discovered allosteric sites on an enzyme was the allosteric site in HIV-I reverse transcriptase (RT), which served as a basis for several drug discovery programs for the treatment of AIDS (Kohlstaedt LA et al. Science 1992, 256:1783-1790). Three non-nucleoside RT inhibitors that bind to this allosteric site are now commercially available drugs: efavirenz, nevirapine and delavirdine. [0110] However, prior to the present invention, methods of identifying cryptic allosteric sites or druggable sites on any biological target, mainly involved traditional high- throughput screening followed by X-ray crystallography. See, Oikonomakos et al. "A new allosteric site in glycogen phosphorylase b as a target for drug interactions," Structure Fold Des 2000, 8 :575-584; Yan Y et al. "Inhibition of a mitotic motor protein: where, how, and conformational consequences," JMo/ Biol 2004, 335:547-554; Wright SW et al. "Anilinoquinazoline inhibitors of fructose 1 ,6-bisphosphatase bind at a novel allosteric site: synthesis,in vitro characterization, and X-ray crystallography," J Med Chem 2002, 45:3865- 3877; Wiesmann C, et al. "Allosteric inhibition of protein tyrosine phosphatase IB," Nat Struct MoI Biol 2004, 11 :730-737; Dennis MS et al. "Peptide exosite inhibitors of factor Vila as anticoagulants," Nature 2000; 404:465-470; Hardy JA et al. "Discovery of an allosteric site in the caspases," Proc Natl Acad Sci USA 2004, 101 : 12461-12466; Hardy JA et al. "Discovery of an allosteric site in the caspases," Proc Natl Acad Sci USA 2004, 101 : 12461- 12466.

[0111] The present invention provides methods for identifying cryptic allosteric sites on any biological target simply based on primary amino acid sequences of a biological target without a priori knowledge of the secondary or tertiary structure of the target. For example, short overlapping or adjacent peptides (e.g., 5 to 25 amino acids, and preferably 7 to 10 amino acids) can be synthesized along the entire length of the biological target as described herein. These overlapping or adjacent peptides are then assessed for their ability to bind to the target or modulate (e.g., inhibit or activate) its activity using, e.g., a multi-well format assay. The effect of adjacent or overlapping peptides can be plotted against the corresponding amino acid positions of the target, forming an "allosteric map." Such an "allosteric map" can be used to identify "hot spots," i.e., cryptic allosteric sites, within the structure of the target. As used herein, "hot spots" or "cryptic allosteric sites" of a biological target are defined as regions, the binding of which reduces a measured activitity of the target by at least 20 percent (e.g., at least 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70% 80%, 90% or 100%) or increases a measured activitity of the target by at least 20 percent (at least 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70% 80%, 90%, 100%, 125%, 150%, 175%, 200%, 300%, 400% 500%, 600% 700% 800% 900% or 1,000% ). Alternately, a statistaical test may be used to determine an increase or decrease of activity that is significant or relevant within the contact of a particular assay. An exemplary allosteric map is described in Example 6. [0112] As discussed above, such hot spots or cryptic allosteric sites are particularly effective for Allosteramer design and targeting. In some embodiments, such cryptic allosteric sites can be further characterized by computer modeling, cross-linking and/or co- crytallization with allosteramers that binds to such sites so that improved Allosteramers can be designed. In addition, such cryptic allosteric sites can be used as potential drug targeting sites to screen for small molecules or other drug candidates. See, U.S. Provisional Application entitled "IDENTIFICATION OF CRYPTIC ALLOSTERIC SITES AND USES THEREOF," filed on even date, the disclosures of which are hereby incorporated by reference.

Design of Peptide Modulators for TNF Receptors

[0113] Numerous biological effects of TNF α and TNFβ are mediated by two TNF transmembrane receptors, p75 receptor (referred to as TNF receptor 1 or TNFRl in this application) and p55 receptor (referred to as TNF receptor 2 or TNFR2 in this application). TNFRl is a 75 kDa glycoprotein that has at least been shown to transduce cytotoxic and proliferative signals as well as signals resulting in the secretion of GM-CSF, IL-I and IL-6. TNFR2 is a 55 kDa glycoprotein that has at least been shown to transduce signals resulting in cytotoxic, anti-viral, and proliferative activities of TNFα. See, Smith et al., 1990, Science 248:1019-1023; and Loetscher et al., 1990, Cell 61 :351-359. The amino acid sequences of TNFRl (SEQ ID NO:1) and TNFR2 (SEQ ID NO:2) are shown below and in Figure 1. The pre-ligand assembly domain (PLAD), the cysteine rich domain and potential transmembrane domain are indicated in Figure 1. The extracellular domains (the sequences N-terminal to the potential transmembrane regions shown in Figure 1) of the two receptors have approximately 28% homology.

Amino acid sequence of TNFRl (SEQ ID NO:1)

MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSI C

CTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEIS

SCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHA

GFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTVLLPLVIFFGLCLLSL LFI

GLMYRYQRWKSKLYSIVCGKSTPEKEGELEGTTTKPLAPNPSFSPTPGFTPTLGFSP V

PSSTFTSSSTYTPGDCPNF AAPRREVAPPYQGADPILATALASDPIPNPLQKWEDSAH

KPQSLDTDDPATLYAVVENVPPLRWKEFVRRLGLSDHEIDRLELQNGRCLREAQYS MLATWRRRTPRREATLELLGRVLRDMDLLGCLEDIEEALCGPAALPPAPSLLR (SEQ ID NO:1)

Amino acid sequence of TNFR2 (SEQ ID NO:2)

MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLRE YYDQTAQMCC SKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQAC TREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPG TFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQ

HTQPTPEPSTAPSTSFLLPMGPSPP AEGSTGDF ALPVGLIVGVT ALGLLIIGVVNCVIM

TQVKKKPLCLQREAKVPHLP ADKARGTQGPEQQHLLIT APSSSSSSLESSASALDRRA PTRNQPQ APGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSS QASSTMGDTDSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAG MKPS (SEQ ID NO :2)

[0114] In some embodiments, candidate peptides can be designed using the Module X technology, which is described in U.S. Patent No. 7,432,341, and PCT Application No. PCT/IB2006/0022987, both entitled "Cytokine receptor modulators and method of modulating cytokine receptor activity," the entire disclosures of which are hereby incorporated by reference. According to the Module X technology, peptides suitable for the invention may be strategically designed to interact with at least one of a flexible region of a TNF receptor that is typically important for the appropriate conformation and activity of the receptor. Such flexible regions include, but are not limited to, juxtamembranous regions, regions containing α helix, β sheet, loops and/or β turns, regions between domains, regions between two β chains, and combinations thereof. Various protein analysis tools are available to identify such flexible regions. For example, peptide sequences of TNF receptors can be systematically analyzed to identify regions that reproduce loops, hinge regions and other domains or secondary structures using software such as ProDom, PROSITE, or Predict Protein. In some embodiments, hydrophobic and flexible profiles can be examined using programs such as ProtScale. In some embodiments, flexible or other suitable regions can be identified based on crystallography, molecular modeling, and hydropathy profiles. Thus, in some embodiments, peptides suitable for the invention are designed based on amino acid sequences that appear within such flexible regions. In some embodiments, sequence homology analysis of designed peptides is performed using, e.g., Blast analysis [NCBI] to ascertain that the sequences are unique to the target of interest. [0115] In some embodiments, inventive "Module X Walk" methods described herein are used to design or identify candidate peptide modulators for a TNF receptor. For example, candidate peptides can be systematically designed based on the linear amino acid sequences of TNFRl (SEQ ID NO: 1) or TNFR2 (SEQ ID NO:2) without first characterizing any secondary or tertiary structures or domains of the receptors.

[0116] In some embodiments, candidate peptides may be systematically designed by progressively walking (e.g., shifting progressively by 1, 2, 3, 4, 5 or more amino acids) through the amino acid sequence from the N-terminus to the C-terminus of the entire length of the receptor (e.g., TNFRl or TNFR2). An exemplary 10-mer peptide walk from the N- terminus to C-terminus of TNFRl is provided in Example 6. In some embodiments, candidate peptides may be systematically designed by progressively walking through the amino acid sequence from the N-terminus to the C-terminus of extracellular and transmembrane regions of TNFRl or TNFR2.

[0117] In some embodiments, candidate peptides may be designed based on the linear amino acid sequence in a region that is known to be important functionally or structurally. For example, candidate peptides can be designed based on contiguous amino acids that appear in the N-terminal PLAD domain, the cysteine rich domain, juxtamembranous regions, and/or potential transmembrane domain of the TNF receptors. In some embodiments, candidate peptides may be designed based on amino acid sequence within a pre-determined "hot spot" for allosteric modulation (see, Figure 13).

[0118] In some embodiments, candidate peptides may be designed based on contiguous amino acids that appear in one or more of the following extracellular or transmembrane regions of TNFRl :

MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQN (SEQ ID NO:3);

HPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRK EMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQN TVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTVLLPLVIFFG LCLLSLLFIGLMYRYQRWKSKLY (SEQ ID NO:4); VCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHC LSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVH LSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCL (SEQ ID NO:5);

MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSI C CTKCHKGTYLYNDCPGPGQDTDCRECESGSF (SEQ ID NO:6);

TASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFN CSLC (SEQ ID NO:7);

LNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTE DSGTTVLLPLVIFFGLCLLSLLFIGLMYRYQRWKSKLY (SEQ ID NO: 8);

EDSGTTVLLPLVIFFGLCLLSLLFIGLMYRYQRWK (SEQ ID NO:9).

[0119] In some embodiments, an extracellular or transmembrane region of TNFRl is selected from the group consisting of

MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGK (SEQ ID NO:555);

QNNSICCTKCHKGTYLYN (SEQ ID NO:556); PGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEM (SEQ ID NO:557);

VEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCT CHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTVLLPLVIFFGLCLLS LLFIGLMYRYQRWK (SEQ ID NO:558); and combination thereof.

[0120] In some embodiments, candidate peptides may be designed based on one or more of the following extracellular or transmembrane regions of TNFR2:

MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYYDQT (SEQ ID NO:10);

AQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQV ETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVC KPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQP VSTRSQHTQPTPEPSTAPSTSF (SEQ ID NO: 11); TCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPEC LSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVA RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI (SEQ ID NO: 12);

PGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSF (SEQ ID NO:13).

[0121] In some embodiments, an extracellular or transmembrane region of TNFR2 is selected from the group consisting of

MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYY (SEQ ID NO:559);

SKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQAC TREQNRICTCRPGW (SEQ ID NO:560);

GCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVA I PGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSF (SEQ ID

NO:561); and combination thereof.

[0122] Typically, a suitable peptide according to the invention is a short peptide. As used herein, a short peptide according to the invention includes any peptide that contains up to 25 amino acids (e.g., up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25 amino acids) or equivalents thereof. In some embodiments, peptides according to the invention contain 5-25 amino acids (e.g., 5-20, 5-15, 5-12, 5-10, 6-25, 6-20, 6-15, 6-12, 6-10, 7-25, 7-20, 7-15, 7-12, or 7-10 amino acids) or equivalents thereof. In some embodiments, a peptide according to the invention is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25 amino acids long.

[0123] In some embodiments, candidate peptides are designed to contain a sequence corresponding to at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids that appear in an extracellular or transmembrane region of a receptor, in particular, a TNFR receptor (e.g., TNFRl or TNFR2), described herein.

[0124] In some embodiments, a sequence corresponding to at least 5 (e.g., at least 6,

7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids that appear in an extracellular or transmembrane region of a receptor includes any sequence having at least 70% (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to the sequence of at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids. Percentage of amino acid sequence identity can be determined by alignment of amino acid sequences. Alignment of amino acid sequences can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Preferably, the WU-BLAST-2 software is used to determine amino acid sequence identity (Altschul et al, Methods in Enzymology 266, 460-480 (1996); http://blast.wustl/edu/blast/README.html). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=l, overlap fraction=0.125, word threshold (T)=I 1. HSP score (S) and HSP S2 parameters are dynamic values and are established by the program itself, depending upon the composition of the particular sequence, however, the minimum values may be adjusted and are set as indicated above.

[0125] In some embodiments, a sequence corresponding to at least 5 (e.g., at least 6,

7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids that appear in an extracellular or transmembrane region of a receptor includes any sequence otherwise identical to the sequence of the at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids but incorporating one or more D-amino acid substitutions for corresponding L-amino acids. Peptides containing such a sequence are also known as D- isomers. In some embodiments, a sequence corresponding to at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids that appear in an extracellular or transmembrane region of a receptor includes any sequence that is an inverse of the at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids. Peptides containing such a sequence are also known as reversed L-peptides. In some embodiments, a sequence corresponding to at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids that appear in an extracellular or transmembrane region of a receptor includes any sequence that is otherwise an inverse of the at least 5 (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, or 25) contiguous amino acids but incorporating one or more D-amino acid substitutions for corresponding L-amino acids. Peptides containing such a sequence are also known as reversed D-peptides.

[0126] In some embodiments, a candidate peptide suitable for the invention contains a sequence that includes at least 4 (e.g., at least 5, at least 6, at least 7) amino acids from at least 7 (e.g., at least 8, at least 9, at least 10, at least 11, at least 12) contiguous amino acids that appear in an extracellular region of a TNF receptor (e.g., TNFRl, TNFR2), wherein the at least 4 (e.g., at least 5, at least 6, at least 7) amino acids maintain their relative positions and/or spacing as they appear in the extracellular region of the TNF receptor. In some embodiments, a peptide suitable for the invention contains a sequence that includes at least 4 (e.g., at least 5, at least 6, at least 7) amino acids from at least 7 (e.g., at least 8, at least 9, at least 10, at least 11, at least 12) contiguous amino acids that appear in an extracellular region of a TNF receptor (e.g., TNFRl, TNFR2), wherein the at least 4 (e.g., at least 5, at least 6, at least 7) amino acids maintain their relative positions and/or spacing, but in the inverse configuration, as they appear in the extracellular region of the TNF receptor.

[0127] Exemplary contiguous amino acids that appear in an extracellular or transmembrane region of TNFRl suitable for the invention and exemplary peptides designed based on corresponding amino acids are shown in Table 1. Exemplary contiguous amino acids that appear in an extracellular or transmembrane region of TNFR2 suitable for the invention and exemplary peptides designed based on corresponding amino acids are shown in Table 2. Additional peptides are described in the Examples section (e.g., Tables 3 and 4).

[0128] The present invention also encompass any candidate peptide modulators that contain a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to any one of the amino acid sequence shown in Tables 1, 2, 3 and 4. In addition, the present invention encompasses any exemplary candidate peptide modulators that are D-isomers, reversed L-peptides, or reversed D-peptides of any one of the peptides shown in Tables 1, 2, 3 and 4 as well as forward and reversed peptides with a combination of D- and L- amino acids.

Table 1. Exemplary contiguous amino acids of TNFRl and exemplary peptides

^Differs in amino acids 229-231. **Differs in amino acid 223.

Table 2. Exemplary contiguous amino acids of TNFR2 and exemplary peptides

Peptide Preparation

[0129] Peptides or peptide derivatives of the present invention may be obtained by any method of peptide synthesis known to those skilled in the art, including synthetic (e.g., exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, classical solution synthesis, native-chemical ligation) and recombinant techniques. For example, the peptides or peptides derivatives can be obtained by solid phase peptide synthesis, which in brief, consist of coupling the carboxyl group of the C-terminal amino acid to a resin (e.g., benzhydrylamine resin, chloromethylated resin, hydroxymethyl resin) and successively adding N-alpha protected amino acids. The protecting groups may be any such groups known in the art. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed. Such solid phase synthesis has been described, for example, by Merrifield, J. Am. Chem. Soc. 85: 2149 (1964); Vale et al, Science 213:1394-1397 (1981), in U.S. Patent Numbers 4, 305, 872 and 4,316, 891, Bodonsky et al. Chem. Ind. (London), 38:1597 (1966); and Pietta and Marshall, Chem. Comm. 650 (1970) by techniques reviewed in Lubell et al. "Peptides" Science of Synthesis 21.11, Chemistry of Amides. Thieme, Stuttgart, 713-809 (2005). The coupling of amino acids to appropriate resins is also well known in the art and has been described in U.S. Patent Number 4,244,946. (Reviewed in Houver-Weyl, Methods of Organic Chemistry. VoI E22a. Synthesis of Peptides and Peptidomimetics, Murray Goodman, Editor-in-Chief, Thieme. Stuttgart. New York 2002).

[0130] During any process of the preparation of a peptide of the invention, it may be desirable to protect sensitive reactive groups on any of the molecule concerned. This may be achieved by means of conventional protecting groups such as those described in Protective Groups In Organic Synthesis by T.W. Greene & P.G.M. Wuts, 1991, John Wiley and Sons, New- York; and Peptides: chemistry and Biology by Sewald and Jakubke, 2002, Wiley- VCH, Wheinheim p.142. For example, alpha amino protecting groups include acyl type protecting groups (e.g., trifluoroacetyl, formyl, acetyl), aliphatic urethane protecting groups (e.g., t- butyloxycarbonyl (BOC), cyclohexyloxycarbonyl), aromatic urethane type protecting groups (e.g., fluorenyl-9-methoxy-carbonyl (Fmoc), benzyloxycarbonyl (Cbz), Cbz derivatives) and alkyl type protecting groups (e.g., triphenyl methyl, benzyl). The amino acids side chain protecting groups include benzyl (for Thr and Ser), Cbz (Tyr, Thr, Ser, Arg, Lys), methyl ethyl, cyclohexyl (Asp, His), Boc (Arg, His, Cys) etc. The protecting groups may be removed at a convenient subsequent stage using methods known in the art.

[0131] Further, the peptides of the present invention, including the analogs and other modified variants, may be synthesized according to the FMOC protocol in an organic phase with protective groups. Desirably, the peptides are purified with a yield of 70% with high- pressure liquid chromatography (HPLC) on a C 18 chromatography column and eluted with an acetonitrile gradient of 10-60%. The molecular weight of a peptide can be verified by mass spectrometry (reviewed in Fields, G. B. "Solid-Phase Peptide Synthesis" Methods in Enzymology. Vol. 289, Academic Press, 1997). [0132] Alternatively, peptides of the present invention may be prepared in recombinant systems using, for example, polynucleotide sequences encoding the peptides. It is understood that a peptide may contain more than one of the above-described modifications within the same peptide. Also included in the present invention are pharmaceutically acceptable salt complexes of the peptides of described herein or their derivatives.

[0133] Purification of the synthesized peptides or peptide derivatives may be carried out by standard methods, including chromatography (e.g., ion exchange, size exclusion, and affinity), centrifugation, precipitation or any standard technique for the purification of peptides and peptides derivatives. For example, thin-layered chromatography or reverse phase HPLC may be employed. Other purification techniques well known in the art and suitable for peptide isolation and purification may also be used.

[0134] Where the processes for the preparation of the compounds according to the present invention give rise to mixtures of stereoisomers, these isomers may be separated by conventional techniques such as preparative chromatography. The compounds may be prepared in racemic form, or individual enantiomers may be prepared either by enantiospecific synthesis or by resolution. The compounds may, for example, be resolved into their components enantiomers by standard techniques such as the formation of diastereoisomeric pairs by salt formation with an optically active acid followed by fractional crystallization and regeneration of the free base. The compounds may also be resolved by formation of diastereomeric esters or amides, followed by removal of the chiral auxiliary. Alternatively, the compounds may be resolved using a chiral HPLC column.

Assays to Identify Peptide Modulators

[0135] Screens for peptide modulators (e.g., inhibitors or activators) of a receptor

(e.g., TNFRl or TNFR2) may be based on in vitro or in vivo assays which measure a biological activity of the receptor (e.g., TNFRl or TNFR2).

In vitro assays

[0136] In vitro assays described herein may employ a natural or a recombinant receptor. Cell-based assays can employ cells which express a receptor of interest naturally, or which contain the recombinant receptor of interest. In all cases, the biological activity of the receptor of interest can be directly or indirectly measured. Thus inhibitors or activators of a receptor activity can be identified relative to a control.

[0137] For example, to identify peptides that modulate (e.g., inhibit or activate) an activity of a TNF receptor, a cell-based assay may be used in which a cell expressing the TNF receptor complex or biologically active portion thereof (either natural or recombinant) is contacted with a test peptide to determine the ability of the test peptide to modulate an activity of a TNF receptor. Exemplary cell-based assays include, but are not limited to, proliferation assays, tyrosine phosphorylation assays, migration assays, apoptosis assays, and assays measuring release of IL-I, IL-6, GM-CSF, and/or IL-IO, induction of chemokines, activation of adhesion molecules, growth of blood vessels, release of tissue destructive enzymes and activation of T cells, NF-κB, and/or caspase pathways. For example, candidate peptides may be tested by measuring TNFα induced IL-6 or IL-I synthesis in human fibroblasts, human brain microvascular endothelial cells. Exemplary TNF assays suitable for the invention are described in the Example section. Additional TNF assays are known in the art and can be used for the present invention. It shall be understood that the in vivo experimental model can also be used to carry out an in vitro assay.

[0138] In addition to cell-based assays, cell-free systems can be used to test candidate peptides for their ability to modulate the phosphorylation state of a receptor protein or portion thereof, or an upstream or downstream target protein, using, for example, an in vitro kinase assay. For example, a TNF receptor target molecule (e.g., an immunoprecipitated receptor from a cell line expressing such a molecule), can be incubated with radioactive ATP, e.g., gamma- ³² P-ATP, in a buffer containing MgCl ₂ and MnCl ₂ , e.g., 10 mM MgCl ₂ and 5 mM MnCl ₂ . Following the incubation, the immunoprecipitated receptor target molecule, can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under reducing conditions, transferred to a membrane, e.g., a polyvinylidene difluoride (PVDF) membrane, and autoradiographed. The appearance of detectable bands on the autoradiograph indicates that the receptor substrate has been phosphorylated. Phosphoaminoacid analysis of the phosphorylated substrate can also be performed to determine which residues on the receptor substrate are phosphorylated. Briefly, the radiophosphorylated protein band can be excised from the SDS gel and subjected to partial acid hydrolysis. The products can then be separated by one-dimensional electrophoresis and analyzed on, for example, a phosphoimager and compared to ninhydrin-stained phosphoaminoacid standards. Such assays are described in, for example, Tamaskovic et al. Biol. Chem. 380(5):569-78, 1999. [0139] In some embodiments, in vitro binding assays may be used to identify candidate peptides that can directly or indirectly bind a receptor. In some binding assays, it may be desirable to immobilize the receptor, or an interacting peptide or peptidomimetic of the present invention, to facilitate separation of the complexed form from the uncomplexed form of one or both of the interacting proteins, as well as to accommodate automation of the assay.

[0140] In some embodiments, a fusion protein may be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S- transferase/TNF receptor fusion proteins or glutathione-S-transferase/TNF receptor fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO), or glutathione derivatized microtitre plates, which are then combined with the test peptides, non-adsorbed target protein and/or the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).

[0141] Other techniques for immobilizing proteins on matrices (well-known in the art) can also be used in the screening assays of the invention. For example, either a receptor protein or a molecule that interacts with the receptor can be immobilized by conjugation of biotin and streptavidin. For example, biotinylated TNF receptor protein or TNF receptor interacting molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96-well plates (Pierce Chemical). Alternatively, antibodies reactive with the TNF receptor protein or TNF receptor interacting molecules, but which do not interfere with binding of the TNF receptor protein to its interacting molecule, can be adhered to the wells of the plate to immobilize the TNF receptor.

[0142] In some embodiments, in vitro assays in accordance with the present invention can be carried out on high throughput platforms. High throughput platforms are particularly useful for blind screenings to identify receptor modulators {e.g. , stimulators and/or inhibitors) from a large number of candidate or test peptides. In some embodiments, multiwell plates, e.g., 24-, 48-, 96-, or 384-well plates, may be used for high throughput assays. In some embodiments, high throughput assays can be performed using an automated system so that candidate peptides may be provided and assays may be performed without human intervention. [0143] Peptide modulators may be identified based on a receptor activity assay as compared to a control or reference. In some embodiments, a control or reference indicates a receptor activity detected under otherwise identical conditions but without candidate peptides. In some embodiments, a control or reference indicates the level of the receptor activity of interest detected under otherwise identical conditions in the presence of a known inhibitor or activator. In some embodiments, a control or reference indicates the level of the receptor activity of interest when the receptor is "locked" in a particular activated state.

[0144] In some embodiments, a peptide inhibitor is identified if the peptide causes an at least approximately 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in the receptor activity of interest relative to a control. In some embodiments, a peptide activator is identified if the peptide causes an at least approximately 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% increase in the receptor activity of interest relative to a control.

In vivo Assays

[0145] In some embodiments, the assays described above may be used as initial or primary screens to detect lead peptide inhibitors or activators. Lead peptides can be further assessed in additional in vitro or in vivo assays. For example, TNF inhibitors identified using in vitro assays described herein may be further tested in animal models for clinical efficacy. For example, a peptide inhibitor identified as described herein may be tested in an appropriate animal model such as a rat or a mouse. For example, a peptide can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. In some embodiments, a TNFR peptide inhibitor may be tested in an animal model for its ability to treat diseases, disorders or conditions associated with deregulation or malfunction of TNF or a TNF receptor. Non- limiting animal models which can be used in such assays include: inflammatory bowel disease (IBD), experimental autoimmune encephalitis (EAE), and psoriatic dermatitis models. Such models are standard in the art and exemplary tests are described in the Example sections.

[0146] Additional assays suitable for the invention are described in the Examples section and are known in the art. Peptide Derivatives and Peptidomimetics

[0147] In some embodiments, peptide inhibitors or activators identified according to the methods described herein may be further modified by standard recombinant DNA technology or combinatorial chemistry techniques to provide improved sequence variants or analogs of the originally identified lead peptides.

[0148] Peptide derivatives of the present invention can be made by altering the amino acid sequences by substitution, addition, or deletion or an amino acid residue to provide a functionally equivalent molecule, or functionally enhanced or diminished molecule, as desired. The derivatives of the present invention include, but are not limited to, those containing, as primary amino acid sequence, all or part of the amino acid sequence of the peptides described herein (e.g., peptide inhibitors shown in Tables 1, 2, 3 and 4, in particular, peptides 1-14 and 1-23) including altered sequences containing substitutions of functionally equivalent amino acid residues. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitution for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the positively charged (basic) amino acids include arginine, lysine, and histidine. The nonpolar (hydrophobic) amino acids include leucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophane, and methionine. The uncharged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The negatively charged (acid) amino acids include glutamic acid and aspartic acid. The amino acid glycine may be included in either the nonpolar amino acid family or the uncharged (neutral) polar amino acid family. Substitutions made within a family of amino acids are generally understood to be conservative substitutions. For example, the amino acid sequence of a peptide inhibitor can be modified or substituted.

[0149] In some embodiments, the present invention provides peptide derivatives having a sequence that includes at least four (e.g., at least five, or at least six) amino acids from any peptide shown in Tables 1, 2, 3 or 4 (e.g., SCQEKQNTV (SEQ ID NO:27) (1-14) or YRYQRWK (SEQ ID NO:36) (1-23)) and wherein the at least four (e.g., at least five, or at least six) amino acids maintain their relative positions and/or spacings (or, alternatively, in their inverse configuration) as they appear in the corresponding peptide shown in Tables 1, 2, 3 and 4 (e.g., SCQEKQNTV (SEQ ID NO:27) (1-14) or YRYQRWK (SEQ ID NO:36) (1- 23)). In some embodiments, peptide derivatives according to the invention have a sequence at least 70% (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to any peptide inhibitor shown in Tables 1, 2, 3 or 4 (e.g., SCQEKQNTV (SEQ ID NO:27) (1-14) or YRYQRWK (SEQ ID NO:36) (1-23)). Exemplary peptide derivatives are described in the Examples section (e.g., Figures 11 and 12).

[0150] Longer peptide sequences which result from the addition of additional amino acid residues to the peptides of the invention are also encompassed in the present invention. Such longer peptide sequence would be expected to have the same biological activity (e.g., inhibiting activation of a TNF receptor) as the peptides described above. Truncated versions of peptide inhibitors may be generated and tested for their receptor inhibitory activity. Truncated versions may be generated by deleting amino acids N-terminally, C-terminally and/or internally as long as the truncated peptides retain the same or substantially the same biological activity of the parent peptide.

[0151] Other derivatives included in the present invention are dual peptides consisting of two of the same, or two different peptides of the present invention covalently linked to one another either directly or through a spacer, such as by a short stretch of alanine residues or by a putative site for proteolysis (e.g., by cathepsin, see e.g., U.S. Patent Number 5,126,249 and European Patent Number 495 049). Multimers of the peptides of the present invention consist of polymer of molecules formed from the same or different peptides or derivatives thereof.

[0152] The present invention also encompasses peptide derivatives that are chimeric or fusion proteins containing a peptide described herein, or fragment thereof, linked at its amino- or carboxy-terminal end, or both, to an amino acid sequence of a different protein. Such a chimeric or fusion protein may be produced by recombinant expression of a nucleic acid encoding the protein. For example, a chimeric or fusion protein may contain at least 5 (e.g., at least, 6, 7, 8, 9, 10, 15, 20) amino acids of a peptide of the present invention.

[0153] In addition to peptides consisting only of naturally occurring amino acids, peptidomimetics or peptide analogs are also encompassed by the present invention. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. The non-peptide compounds are termed "peptide mimetics" or peptidomimetics (Fauchere et al., Infect. Immun. 54:283-287 (1986); Evans et al, J. Med. Chem. 30:1229-1239 (1987)). Peptide mimetics that are structurally related to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to the paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity) such as naturally-occurring receptor-binding polypeptides, but have one or more peptide linkages optionally replaced by linkages such as -CH ₂ NH-, -CH ₂ S-, - CH ₂ -CH ₂ -, -CH=CH- (cis and trans), -CH ₂ SO-, -CH(OH)CH ₂ -, -COCH ₂ - etc., by methods well known in the art (Spatola, Peptide Backbone Modifications, Vega Data, 1(3):267 (1983); Spatola et al. Life Sci. 38:1243-1249 (1986); Hudson et al. Int. J. Pept. Res. 14:177-185 (1979); and Weinstein. B., 1983, Chemistry and Biochemistry, of Amino Acids, Peptides and Proteins, Weinstein eds, Marcel Dekker, New- York,). Such peptide mimetics may have significant advantages over naturally-occurring polypeptides including more economical production, greater chemical stability, enhanced pharmacological properties (e.g., half-life, absorption, potency, efficiency, etc), reduced antigenicity and others.

[0154] While peptides may be effective in inhibiting a biological activity of a receptor

(e.g., wild-type TNFRl or TNFR2) in vitro, their effectiveness in vivo might be reduced by the presence of proteases. Serum proteases have specific substrate requirements. The substrate must have both L-amino acids and peptide bonds for cleavage. Furthermore, exopeptidases, which represent the most prominent component of the protease activity in serum, usually act on the first peptide bond of the peptide and require a free N-terminus (Powell et al., Pharm. Res. 10:1268-1273 (1993)). In light of this, it is often advantageous to use modified versions of peptides. The modified peptides retain the structural characteristics of the original L-amino acid peptides that confer biological activity with regard to TNF receptors but are advantageously not readily susceptible to cleavage by protease and/or exopeptidases.

[0155] Systematic substitution of one or more amino acids of a consensus sequence with D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may be used to generate more stable peptides. Thus, a peptide derivative or peptidomimetic of the present invention may be all L, all D or mixed D, L peptide, in either forward or reverse order. The presence of an N-terminal or C-terminal D-amino acid increases the in vivo stability of a peptide since peptidases cannot utilize a D-amino acid as a substrate (Powell et al., Pharm. Res. 10:1268-1273 (1993)). Reverse-D peptides are peptides containing D-amino acids, arranged in a reverse sequence relative to a peptide containing L-amino acids. Thus, the C- terminal residue of an L-amino acid peptide becomes N-terminal for the D-amino acid peptide, and so forth. Reverse D-peptides retain the same secondary conformation and therefore similar activity, as the L-amino acid peptides, but are more stable to enzymatic degradation in vitro and in vivo, and thus have greater therapeutic efficacy than the original peptide (Brady and Dodson, Nature 368:692-693 (1994); Jameson et al, Nature 368:744-746 (1994)). Similarly, a reverse-L peptide may be generated using standard methods where the C-terminus of the parent peptide is now the N-terminus of the reverse-L peptide. It is contemplated that reverse L-peptides of L-amino acid peptides that do not have significant secondary structure (e.g., short peptides) retain the same spacing and conformation of the side chains of the L-amino acid peptide and therefore often have the similar activity as the original L-amino acid peptide. Moreover, a reverse peptide may contain a combination of L- and D- amino acids. The spacing between amino acids and the conformation of the side chains may be retained resulting in similar activity as the original L-amino acid peptide.

[0156] In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods well known in the art (Rizo and Gierasch, Ann. Rev. Biochem. 61 :387-418 (1992)). For example, constrained peptides may be generated by adding cysteine residues capable of forming disulfide bridges and, thereby, resulting in a cyclic peptide. Cyclic peptides have no free N- or C-termini. Accordingly, they are not susceptible to proteolysis by exopeptidases, although they are, of course, susceptible to endopeptidases, which do not cleave at peptide termini. The amino acid sequences of the peptides with N-terminal or C-terminal D-amino acids and of the cyclic peptides are usually identical to the sequences of the peptides to which they correspond, except for the presence of N-terminal or C-terminal D-amino acid residue, or their circular structure, respectively.

[0157] A cyclic derivative containing an intramolecular disulfide bond may be prepared by conventional solid phase synthesis while incorporating suitable S-protected cysteine or homocysteine residues at the positions selected for cyclization such as the amino and carboxy termini (Sah et al., J. Pharm. Pharmacol. 48:197 (1996)). Following completion of the chain assembly, cyclization can be performed either (1) by selective removal of the S- protecting group with a consequent on-support oxidation of the corresponding two free SH- functions, to form a S-S bonds, followed by conventional removal of the product from the support and appropriate purification procedure or (2) by removal of the peptide from the support along with complete side chain de-protection, followed by oxidation of the free SH- functions in highly dilute aqueous solution.

[0158] The cyclic derivative containing an intramolecular amide bond may be prepared by conventional solid phase synthesis while incorporating suitable amino and carboxyl side chain protected amino acid derivatives, at the position selected for cyclization. The cyclic derivatives containing intramolecular -S-alkyl bonds can be prepared by conventional solid phase chemistry while incorporating an amino acid residue with a suitable amino-protected side chain, and a suitable S-protected cysteine or homocysteine residue at the position selected for cyclization.

[0159] Substitution of non-naturally-occurring amino acids for natural amino acids in a subsequence of the peptides can also confer resistance to proteolysis. Such a substitution can, for instance, confer resistance to proteolysis by exopeptidases acting on the N-terminus without affecting biological activity. Examples of non-naturally-occurring amino acids include α,α -disubstituted amino acids, N-alkyl amino acids, lactic acids, C-α-methyl amino acids, β-amino acids, and β-methyl amino acids. Amino acids analogs useful in the present invention may include, but are not limited to, β-alanine, norvaline, norleucine, A- aminobutyric acid, orithine, hydroxyproline, sarcosine, citrulline, cysteic acid, cyclohexylalanine, 2-aminoisobutyric acid, 6-aminohexanoic acid, t-butylglycine, phenylglycine, o-phosphoserine, N-acetyl serine, N-formylmethionine, 3-methylhistidine and other unconventional amino acids. Furthermore, the synthesis of peptides with non-naturally- occurring amino acids is routine in the art.

[0160] Another effective approach to confer resistance to peptidases acting on the N- terminal or C-terminal residues of a peptide is to add chemical groups at the peptide termini, such that the modified peptide is no longer a substrate for the peptidase. One such chemical modification is glycosylation of the peptides at either or both termini. Certain chemical modifications, in particular N-terminal glycosylation, have been shown to increase the stability of peptides in human serum (Powell et al., Pharm. Res. 10:1268-1273 (1993)). Other chemical modifications which enhance serum stability include, but are not limited to, the addition of an N-terminal alkyl group, consisting of a lower alkyl of from one to twenty carbons, such as an acetyl group, and/or the addition of a C-terminal amide or substituted amide group. In particular, the present invention includes modified peptides consisting of peptides bearing an N-terminal acetyl group and/or a C-terminal amide group. [0161] Also included by the present invention are other types of peptide derivatives containing additional chemical moieties not normally part of the peptide, provided that the derivative retains the desired functional activity of the peptide. Examples of such derivatives include (1) N-acyl derivatives of the amino terminal or of another free amino group, wherein the acyl group may be an alkanoyl group (e.g., acetyl, hexanoyl, octanoyl) an aroyl group (e.g., benzoyl) or a blocking group such as F-moc (fluorenylmethyl-O-CO-); (2) esters of the carboxy terminal or of another free carboxy or hydroxyl group; (3) amide of the carboxy- terminal or of another free carboxyl group produced by reaction with ammonia or with a suitable amine; (4) phosphorylated derivatives; (5) derivatives conjugated to an antibody or other biological ligand and other types of derivatives; and (6) derivatives conjugated to a polyethylene glycol (PEG) chain.

Assays to Identify Peptidomimetics

[0162] As described above, non-peptidyl compounds generated to replicate the backbone geometry and pharmacophore display (peptidomimetics) of the peptides identified by the methods of the present invention often possess attributes of greater metabolic stability, higher potency, longer duration of action and better bioavailability.

[0163] The peptidomimetics compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12:145 (1997)). Examples of methods for the synthesis of molecular libraries can be found in the art, for example, in: DeWitt et al. Proc. Natl. Acad. Sci. USA 90:6909 (1993); Erb et al. Proc. Natl. Acad. Sci. USA 91 :11422 (1994); Zuckermann et al. J. Med. Chem. 37:2678 (1994); Cho et al. Science 261 :1303 (1993); Carell et al. Angew. Chem, Int. Ed. Engl. 33:2059 (1994) and ibid 2061; and in Gallop et al. Med. Chem. 37:1233 (1994). Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13:412- 421 (1992)) or on beads (Lam, Nature 354:82-84 (1991)), chips (Fodor, Nature 364:555-556 (1993)), bacteria or spores (U.S. Patent Number 5,223,409), plasmids (Cull et al., Proc. Natl. Acad. ScL USA 89:1865-1869 (1992)) or on phage (Scott and Smith, Science 249:386-390 (1990)), or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[0164] Once a peptide of the present invention is identified, it may be isolated and purified by any number of standard methods including, but not limited to, differential solubility (e.g., precipitation), centrifugation, chromatography (e.g., affinity, ion exchange, size exclusion, and the like) or by any other standard techniques used for the purification of peptides, peptidomimetics or proteins. The functional properties of an identified peptide of interest may be evaluated using any functional assay known in the art. Desirably, assays for evaluating downstream receptor function in intracellular signaling are used (e.g., cell proliferation).

[0165] For example, the peptidomimetics compounds of the present invention may be obtained using the following three-phase process: (1) scanning the peptides of the present invention to identify regions of secondary structure important for recognition and activity toward a TNF receptor; (2) using conformational^ constrained dipeptide surrogates to refine the backbone geometry and provide organic platforms corresponding to these surrogates; and (3) using the best organic platforms to display organic pharmacophores in libraries of candidates designed to mimic the desired activity of the native peptide. In more detail the three phases are as follows. In phase 1, the lead candidate peptides are scanned and their structure abridged to identify the requirements for their activity. A series of peptide analogs of the original are synthesized. In phase 2, the best peptide analogs are investigated using the conformationally constrained dipeptide surrogates. Indolizidin-2-one, indolizidin-9-one, and quinolizidinone amino acids (I ² aa, I ⁹ aa and Qaa respectively) are used as platforms for studying backbone geometry of the best peptide candidates. These and related platforms (reviewed in Halab et al., Biopolymers 55:101-122 (2000); and Hanessian et al. Tetrahedron 53:12789-12854 (1997)) may be introduced at specific regions of the peptide to orient the pharmacophores in different directions. Biological evaluation of these analogs identifies improved lead peptides that mimic the geometric requirements for activity. In phase 3, the platforms from the most active lead peptides are used to display organic surrogates of the pharmacophores responsible for activity of the native peptide. The pharmacophores and scaffolds are combined in a parallel synthesis format. Derivation of peptides and the above phases can be accomplished by other means using methods known in the art. [0166] Structure function relationships determined from the peptides, peptide derivatives, or peptidomimetics of the present invention may be used to refine and prepare analogous molecular structures having similar or better properties. Accordingly, the compounds of the present invention also include molecules that share the structure, polarity, charge characteristics and side chain properties of the peptides described herein. For example, peptidomimetics incorporating one or more lactams or analogues can be prepared according to the present invention. Exemplary methods for preparing peptidomimetics containing lactams or analogues thereof are described in PCT Application NO. PCT/US 10/27969, entitled "INTERLEUKIN-1 RECEPTOR ANTAGONISTS AND USES THEREOF," the disclosure of which is hereby incorporated by reference.

[0167] In summary, based on the disclosure herein, those skilled in the art can develop peptides and peptidomimetics screening assays which are useful for identifying compounds for inhibiting a receptor activity. Compounds so identified may also be shown to activate these receptors. The assays of this invention may be developed for low-throughput, high-throughput, or ultra-high throughput screening formats. Assays of the present invention include assays which are amenable to automation.

Pharmaceutical Compositions

[0168] The peptides, peptide derivatives and peptidomimetics of the present invention are useful in the treatment of diseases, disorders or conditions associated with deregulation or malfunction of TNFα, TNFβ and/or a TNF receptor (e.g., TNFRl or TNFR2).

[0169] TNF related diseases, disorders or conditions include, but are not limited to, the following:

(A) acute and chronic immune and autoimmune pathologies, such as systemic lupus erythematosus (SLE), rheumatoid arthritis, thyroidosis, graft versus host disease, scleroderma, diabetes mellitus, Graves' disease, Beschet's disease, and the like;

(B) infections, including, but not limited to, sepsis syndrome, cachexia, circulatory collapse and shock resulting from acute or chronic bacterial infection, acute and chronic parasitic and/or infectious diseases, bacterial, viral or fungal, such as a HIV, AIDS (including symptoms of cachexia, autoimmune disorders, AIDS dementia complex and infections); (C) inflammatory diseases, such as chronic inflammatory pathologies and vascular inflammatory pathologies, including chronic inflammatory pathologies such as sarcoidosis, chronic inflammatory bowel disease, ulcerative colitis, and Crohn's pathology and vascular inflammatory pathologies, such as, but not limited to, disseminated intravascular coagulation, atherosclerosis, and Kawasaki's pathology:

(D) neurodegenerative diseases, including, but are not limited to, demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders such as lesions of the corticospinal system; disorders of the basal ganglia or cerebellar disorders; hyperkinetic movement disorders such as Huntington's Chorea and senile chorea; drug-induced movement disorders, such as those induced by drugs which block CNS dopamine receptors; hypokinetic movement disorders, such as Parkinson's disease; Progressive supranucleo palsy; Cerebellar and Spinocerebellar Disorders, such as astructural lesions of the cerebellum; spinocerebellar degenerations (spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiple systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and MachadoJoseph)); and systemic disorders (Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, and mitochondrial multi-system disorder); demyelinating core disorders, such as multiple sclerosis, acute transverse myelitis; disorders of the motor unit, such as neurogenic muscular atrophies (anterior horn cell degeneration, such as amyotrophic lateral sclerosis, infantile spinal muscular atrophy and juvenile spinal muscular atrophy); Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy body disease; Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronic alcoholism; Creutzfeldt- Jakob disease; Subacute sclerosing panencephalitis, Hallerrorden- Spatz disease; and Dementia pugilistica, or any subset thereof;

(E) ophthalmic diseases involving TNF such as, but not limited to, retinal detachment, retinoschisis, hypertensive retinopathy, diabetic retinopathy, general retinopathy, retinopathy of prematurity, age-related macular degeneration, general macular degeneration, epiretinal membrane, choroidal neovascular membrane, cystoid macular edema, macular hole, retinitis pigmentosa, macular edema, sleritisc, corneal ulcer or abrasion, thygeson's superficial punctate keratopathy, corneal neovascularization, fuchs' dystrophy, keratoconus, keratoconjunctivitis sicca or dry eye, iritis, uveitis, conjunctivitis, pterygium, subconjunctival hemorrhage, glaucoma, keratomycosis , xerophthalmia , cataract and systemic diseases with ocular manifestations including allergies, AIDS and hypertension. (F) malignant pathologies involving TNF-secreting tumors or other malignancies involving TNF, such as, but not limited to leukemias (acute, chronic myelocytic, chronic lymphocytic and/or myelodyspastic syndrome); lymphomas (Hodgkin's and non-Hodgkin's lymphomas, such as malignant lymphomas (Burkitt's lymphoma or Mycosis fungoides)); carcinomas (such as colon carcinoma) and metastases thereof; cancer-related angiogenesis; infantile haemangiomas;

(G) alcohol-induced hepatitis; and

(H) other diseases related to angiogenesis or VEGF/VPF, such as ocular neovascularization, psoriasis, duodenal ulcers, angiogenesis of the female reproductive tract.

[0170] See, e.g., Berkow et al, eds., The Merck Manual, 16th edition, chapter 11, pp

1380-1529, Merck and Co., Rahway, N. J., 1992, which reference, and references cited therein, are entirely incorporated herein by reference. See also Folkman, Nature Medicine, Volume 1, No. 1 (1995).

[0171] The peptide, peptide derivatives or peptidomimetics according to the invention may also be used to treat other inflammatory or autoimmune diseases, disorders, or conditions described herein.

[0172] The pharmaceutical compositions can be in a variety of forms including oral dosage forms, topic creams, topical patches, ionophoresis forms, suppository, nasal spray and inhaler, eye drops, intraocular injection forms, depot forms, as well as injectable and infusible solutions. Methods for preparing pharmaceutical composition are well known in the art.

[0173] Compositions within the scope of the present invention typically contain the active agent (e.g. peptide, peptide derivative or peptidomimetics) in an amount effective to achieve the desired therapeutic effect while avoiding or minimizing adverse side effects. Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art. For the administration of peptide antagonists and the like, the amount administered desirably is chosen so as to avoid adverse side effects. The amount of the therapeutic or pharmaceutical composition which is effective in the treatment of a particular disease, disorder or condition depends on the nature and severity of the disease, the target site of action, the subject's weight, special diets being followed by the subject, concurrent medications being used, the administration route and other factors that are recognized by those skilled in the art. The dosage can be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the subject. Typically, 0.001 to 100 mg/kg/day is administered to the subject. Effective doses may be extrapolated from dose response curves derived from in vitro or animal model test systems. For example, in order to obtain an effective mg/kg dose for humans based on data generated from rat studies, the effective mg/kg dosage in rat is divided by six.

[0174] Various delivery systems are known and can be used to administer peptides, peptide derivatives or peptidomimetics or a pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention can be administered by any suitable route including, intravenous or intramuscular injection, intraventricular or intrathecal injection (for central nervous system administration), orally, topically, subcutaneously, subconjunctivally, intraocularly, or via intranasal, intradermal, sublingual, vaginal, rectal or epidural routes.

[0175] Other delivery system well known in the art can be used for delivery of the pharmaceutical compositions of the present invention, for example via aqueous solutions, encapsulation in microparticules, or microcapsules.

[0176] The pharmaceutical compositions of the present invention can also be delivered in a controlled release system. For example, a polymeric material can be used (see, e.g., Smolen and Ball, Controlled Drug Bioavailability, Drug product design and performance, 1984, John Wiley & Sons; Ranade and Hollinger, Drug Delivery Systems, pharmacology and toxicology series, 2003, 2 ^nd edition, CRRC Press). Alternatively, a pump may be used (Saudek et al, N. Engl. J. Med. 321 :574 (1989)).

[0177] Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled to a class of biodegradable polymers useful in achieving controlled release of the drug, for example, polylactic acid, polyorthoesters, cross-linked amphipathic block copolymers and hydrogels, polyhydroxy butyric acid, and polydihydropyrans.

[0178] As described above, pharmaceutical compositions of the present invention desirably include a peptide, peptide derivatives or peptidomimetic combined with a pharmaceutically acceptable carrier. The term carrier refers to diluents, adjuvants, excipients or vehicles with which the peptide, peptide derivative or peptidomimetic is administered. Such pharmaceutical carriers include sterile liquids such as water and oils including mineral oil, vegetable oil (e.g., soybean oil or corn oil), animal oil or oil of synthetic origin. Aqueous glycerol and dextrose solutions as well as saline solutions may also be employed as liquid carriers of the pharmaceutical compositions of the present invention. The choice of the carrier depends on factors well recognized in the art, such as the nature of the peptide, peptide derivative or peptidomimetic, its solubility and other physiological properties as well as the target site of delivery and application. For example, carriers that can penetrate the blood brain barrier are used for treatment, prophylaxis or amelioration of symptoms of diseases or conditions (e.g. inflammation or an autoimmune disorder) in the central nervous system. Examples of suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy by Alfonso R. Gennaro, 2003, 21 ^th edition, Mack Publishing Company. Moreover, suitable carriers for oral administration are known in the art and are described, for example, in U.S. Patent Nos. 6,086,918, 6,673,574, 6,960,355, and 7,351,741 and in WO2007/131286, the disclosures of which are hereby incorporated by reference.

[0179] Further pharmaceutically suitable materials that may be incorporated in pharmaceutical preparations of the present invention include absorption enhancers including those intended to increase paracellular absorption, pH regulators and buffers, osmolality adjusters, preservatives, stabilizers, antioxidants, surfactants, thickeners, emollient, dispersing agents, flavoring agents, coloring agents, and wetting agents.

[0180] Examples of suitable pharmaceutical excipients include, water, glucose, sucrose, lactose, glycol, ethanol, glycerol monostearate, gelatin, starch flour (e.g., rice flour), chalk, sodium stearate, malt, sodium chloride, and the like. The pharmaceutical compositions of the present invention can take the form of solutions, capsules, tablets, creams, gels, powders sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides (see Remington: The Science and Practice of Pharmacy by Alfonso R. Gennaro, 2003, 21 ^th edition, Mack Publishing Company). Such compositions contain a therapeutically effective amount of the therapeutic composition, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. The formulations are designed to suit the mode of administration and the target site of action (e.g., a particular organ or cell type).

[0181] The pharmaceutical compositions of the present invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those that form with free amino groups and those that react with free carboxyl groups. Non-toxic alkali metal, alkaline earth metal, and ammonium salts commonly used in the pharmaceutical industry include sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and protamine zinc salts, which are prepared by methods well known in the art. Also included are non-toxic acid addition salts, which are generally prepared by reacting the compounds of the present invention with suitable organic or inorganic acid. Representative salts include the hydrobromide, hydrochloride, valerate, oxalate, oleate, laureate, borate, benzoate, sulfate, bisulfate, acetate, phosphate, tysolate, citrate, maleate, fumarate, tartrate, succinate, napsylate salts, and the like.

[0182] The present invention also provides for modifications of peptides or peptide derivatives such that they are more stable once administered to a subject (i.e., once administered it has a longer half-life or longer period of effectiveness as compared to the unmodified form). Such modifications are well known to those skilled in the art to which this invention pertain (e.g., polyethylene glycol derivatization a.k.a. PEGylation, microencapsulation, etc).

[0183] The TNF receptor antagonists of the present invention may be administered alone or in combination with other active agents (e.g., an anti-inflammatory compound) useful for the treatment, prophylaxis or amelioration of symptoms of a TNF or TNFR associated disease or condition. Thus, the compositions and methods of the present invention can be used in combination with other agents exhibiting the ability to modulate TNF or TNFR activity (e.g., synthesis, release and/or binding to TNFRl or TNFR2) or to reduce the symptoms of the TNF/TNFR associated disease (e.g., an autoimmune or inflammatory disorder).

[0184] The present invention is illustrated in further detail by the following non- limiting examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention.

EXAMPLES Example 1: Identification of TNFR inhibitors using in vitro assays

[0185] TNFα-induced IL-6 or IL-I synthesis assay were used to screen candidate peptides according to the invention. Specifically, in order to characterize the antagonistic effect of a candidate TNFR peptide, we measured TNFα-induced synthesis of cytokines IL-6 and IL-I by human fibroblasts and endothelial cells, respectively, in the presence of 1 μM of different candidate peptides as described above (e.g., exemplary candidate peptides shown in Tables 1 and 2).

TNF a-induced IL-6 production in human fibroblasts (WI-38)

[0186] Cells were cultured in complete DMEM (10 % FBS; 1% penicillin/streptomycin) all from Gibco. At confluence, cells were plated in 24 well plates and cultured until 70 % confluency. Before treatment, cells were starved overnight. Fibroblasts were then pre -incubated with candidate peptides at a concentration of approximately 10 ^"6 M for about 30 minutes and incubated with human recombinant TNFα (10 ng/ml) for 24 hours. IL-6 production was determined in cell culture medium by using an ELISA kit from R&D system according to the manufacturer's instructions.

TNF a-induced IL-I production in human brain microvascular endothelial cells

[0187] HBMVEC (human brain microvascular endothelial cells) cells were cultured in ECM complete medium (10% FBS; 1% penicillin/streptomycin) from Clonetic. At confluence, cells were plated in 24 well plates and cultured until 70 % confluency. Before treatment, cells were starved overnight. The cells were pre-incubated with candidate peptides at a concentration of approximately 10 ^"6 M for about 30 minutes and incubated with human recombinant TNFα (10 ng/ml) for 24 hours. IL-I production was determined in cell culture medium by using an ELISA kit from R&D System according to the manufacturer's instructions.

TNF a-induced IL-I production in human umbilical vessel endothelial cells

[0188] HUVEC (human umbilical vessel endothelial cells) cells were cultured in

EGM-2 (Lonza) complete medium (10 % FBS; 3 mg/ml of gentamicin, 150 ng/ml of amphotericin). At confluence, cells were plated in 24 well plates and cultured until 75 % confluence. Before treatment, cells were starved overnight. Cells were then pre-incubated with candidate peptides at a concentration of approximately 10 ^"6 M for about 30 minutes and incubated with human recombinant TNF α (10 ng/ mL) for 24 hours. IL-I measurements in the culture medium were performed using an ELISA kit from R&D System according to the manufacturer's instructions.

Results

[0189] Candidate peptides 1-1 through 1-23, 2-1, 2-2 and 2-3, were tested using the

IL-6 synthesis assay. Exemplary results of inhibition of IL-6 production are shown in Figure 2A. As shown, most (21 out of 26) of the tested peptides (e.g., peptides 1-1, 1-2, 1-5, 1-6, 1- 7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14, 1-16, 1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 2-1, and 2- 3) inhibited TNFα-induced IL-6 synthesis as compared to the TNF control. In particular, peptides 1-21, 1-22, 1-23, and 2-1 inhibited more than 50% of TNFα-induced IL-6 synthesis. Other peptides such as 1-7, 1-8, 1-11, 1-14, 1-18, 1-19, and 2-3 inhibited more than 20-45% of TNFα-induced IL-6 synthesis. Exemplary peptide inhibitors of TNFRl are summarized in Table 3.

Table 3. Exemplary TNFRl peptide inhibitors

[0190] Additional peptides designed by the Module X Walk method (such as peptides

1-24 through 1-47) and by the Module X approach (such as peptides ALL-I through ALL-24) were tested using the IL-6 synthesis assay described above. Exemplary results are shown in Figure 2B and Figure 2C, respectively. Additional peptides were also screened using TNFα- induced IL-I production in human umbilical vessels endothelial cells described above. Exemplary results are shown in Figure 2D.

[0191] We further verified the TNFR antagonistic activities of those most promising peptides identified using the IL-6 assay in human brain microvascular endothelial cells (HBMEC) using IL-I synthesis assay as described above. Exemplary results are shown in Figure 3. As shown in Figure 3, peptides 1-14 and 1-23 almost completely inhibited TNFα- induced IL-I synthesis in HBMEC. Interestingly, peptides 1-19, 1-21 and 2-22 that were highly effective in inhibiting IL-6 synthesis in fibroblasts failed to inhibit TNFα-induced IL-I synthesis (Figure 3).

[0192] Without wishing to be bound by any theory, it is contemplated that peptides containing sequences from different regions of the TNF receptor may demonstrate different TNFR inhibitory activities. The regions from which certain candidate peptides were derived using Module X methods are shown in Figure 4. For example, peptides derived from the N- terminal region of the receptor are generally less effective than the ones derived from the cysteine rich region in the middle or the last domain in the juxtamembrane regions (e.g., Figure 4). Peptide 1-14 which is derived from the cysteine rich domain and peptide 1-23 which is likely to be half extra half intra membranous exhibited effective inhibition of both TNF-induced IL-6 and IL-I synthesis. However, some TNFR antagonistic peptides exhibit selective inhibitory effects. For example, as shown in Figure 3, peptides 1-19, 1-21 and 1-22 effectively inhibit TNFα-induced IL-6 synthesis but do not inhibit TNFα-induced IL-I synthesis. Therefore, these TNF antagonistic peptides demonstrate functional selectivity, which is characteristic of allosteric modulators.

Example 2. Characterization of modulation capacities of antagonistic peptides

[0193] This example illustrates that the effectiveness and modulation capacities of

TNFR antagonistic peptides can be further characterized using dose response assays. The experiments in this example first show that TNFR antagonistic peptides may have different TNF modulation capacities. For example, peptides 1-14 and 1-23 exhibited different modulation capacities towards TNF-induced IL-6 production in fibroblasts. Specifically, dose response of IL-6 synthesis induced by TNFα were measured in the presence of peptide 1-14 or 1-23 at a constant concentration of approximately 10 ^"6 M. Exemplary results are shown in Figure 5. As shown , peptide 1-14 at a concentration of 10 ^"6 M does not change the EC50 of TNF-induced IL-6 production but causes a 50% decrease of the maximum TNF response which is consistent with the results shown in Figures 2A. On the other hand, peptide 1-23 causes a shift of the affinity curve to the right by approximately 1 log. EC50 of TNF alone is approximately 2 x 10 ^"11 M, while EC50 of TNF in the presence of peptide 1-23 (10 ^"6 M) is approximately 4.1 x 10 ^"10 M. Therefore, both peptides 1-14 and 1-23 demonstrate negative modulation of the receptor's biological response to TNF. However, peptides 1-14 and 1-23 may modulate the receptor's response by different mechanisms.

[0194] As discussed above, peptides 1-14 and 1-23 contain sequences from different regions of the TNFRl receptor (the right panel of Figure 3). Peptide 1-14 was derived from a region close to the TNF binding site and peptide 1-23 was derived from the juxtamembrane region. Without wishing to be bound by any theory, these results further suggest that TNFR peptide inhibitors from different regions demonstrate functional selectivity and are allosteric modulators.

[0195] To further characterize the effects of peptides 1-14 and 1-23, Schild regression analysis was used. Specifically, WI-38 cells were incubated with increasing concentrations of human TNFα and each dose response curve was also generated in the presence of increasing concentrations of peptide TNFR1-23 (e.g., one dose response curve with 10 ^"6 M TNFR1-23, one with 10 ^"7 M TNFR1-23). Twenty four hours later IL-6 measurements were performed with an ELISA kit from R&D Systems according to the manufacturer's instructions. Data was plotted and transformed with Graph Prism software and exemplary results are shown in Figure 6. Similar analysis was done with peptide TNFR1-14 and exemplary results are shown in Figure 7.

[0196] As shown in Figure 6, TNFRl -23 induces a shift to the right characteristic of a

NAM. The effects on the affinity are saturable as illustrated with the plateau of the Schild Plot. The maximal efficacy (Emax) of TNF response is also decreased with the presence of TNFRl -23. The results establish that TNFRl -23 inhibits IL-6 production non-competitive Iy.

[0197] As shown in Figure 7, TNFR1-14 shows a different effect than 1-23 on the

EC50 of IL-6 dose response. 1-14 induces a less pronounced shift to the right and the effect is saturable at lower concentrations than 1-23. The effects of 1-14 on Emax of TNF response is similar to 1-23, i.e., the Emax of TNF efficacy was decreased in the presence of TNFRl- 14. The results indicate that TNFRl -14 induces a saturable effect on affinity decrease and a dose response effect on Emax. Similarly, TNFR1-14 inhibits IL-6 production non- competitively.

[0198] In addition, the effectiveness of TNFR1-14 and 1-23 were characterized using dose response of the inhibitory effect of TNFRl -14 and 1-23 on TNFα-induced IL-6 production in human fibroblasts in the presence of TNF α at a concentration of 10 ng/ml. Specifically, WI-38 cells were pre-incubated with peptides 1-14 and 1-23 at increasing concentrations and incubated 24 hours with human TNFα (10ng/ml). IL-6 measurements were performed as above and exemplary results are shown in Figure 8. As shown in Figure 8, the IC50 for TNFR1-14 is 13OpM and the IC50 for TNFR1-23 is 60 pM.

Example 3: In vivo efficacy of TNFR peptide inhibitors

[0199] This experiment was conducted to show that TNFR peptide inhibitors identified based on in vitro assays can be tested for their ability to treat a disease, disorder or condition associated with TNF or TNFR deregulation or malfunction.

[0200] Peptides 1-14 and 1-23 were chosen for in vivo studies because they showed the most promising efficacy in inhibiting both TNF-induced IL-I and IL-6 synthesis in in vitro assays as described in Example 1. A phorbol myristate acetate (PMA)-induced dermatitis inflammatory model was used to assay the in vivo efficacy of these peptides. PMA-induced dermatitis is an art recognized model of chronic and scaling dermatitis, which is suggestive of psoriatic dermatitis (Petersen et al., Basic & Clinical Pharm Tox 99:104-115 (2006); Schon, J Investig Derm 112:405-410 (1999)). Briefly, the efficacy of anti-TNFR peptides was assessed in CD-I mice. Cutaneous inflammation was induced with PMA in acetone solution applied to the ears of CD-I mice. On day 1, 0.01% PMA was applied to the left ear, while the right ear received acetone vehicle as a control. On day 2, the peptides were administered intraperitoneally twice a day (dosage lmg/kg/day). PMA and acetone control were applied to left and right ears, respectively, between the injections. On day 3, the same procedure as day 2 was repeated. On day 4, the mice received a bolus injection of 1 mg/kg in the morning and sacrificed in the afternoon. Ear thickness was measured with a Mitutoyo Digital caliper. 4 mm ear punches were made and weighed. Exemplary results were shown in Figure 9. As shown in Figure 9, the tested peptides are particularly efficient in inhibiting the increase in ear thickness (about 40% inhibition) (Figure 9, right panel). The peptides also prevent swelling of the ears with an efficacy of approximately 15% (Figure 9, left panel).

Example 4: Oral efficacy of TNFR antagonistic peptide 1-23

[0201] This experiment was conducted to show that the oral efficacy of TNFR antagonistic peptides can be characterized using an animal model. Briefly, the oral biological activity of TNFR antagonistic peptide 1-23 was characterized in a model of TNF-induced hypotension using Sprague-Dawley rats. During the process of inducing inflammation, TNF will cause a decrease in blood pressure via its ability to induce the expression and maturation of cytokines such as IL-I.

[0202] For arterial blood pressure (BP) measurements, Sprague-Dawley rats (300-330 g) were anesthetised with 2% isoflurane. A polypropylene tube (PE-90) was inserted in the jugular vein for injections. Peptides were administrated by gavage at a concentration of approximately 2 mg/kg (500 μl final volume of injection) 30 minutes prior to TNFα intravenous injection (400 ng/200 μl). The left carotid artery was catheterized with a polyethylene PE-10 catheter and BP was recorded using a Statham pressure transducer connected to a Gould recorder. BP measurement variations were recorded for an hour. Exemplary results are shown in Figure 10. As shown in Figure 10, peptide 1-23 orally given inhibited 60% of TNF-induced hypotension after 45 minutes of TNF injection.

Example 5. Derivatives of peptides 1-14 and 1-23

[0203] This experiment was conducted to show that derivatives of TNFR antagonistic peptides can be designed and tested using various in vitro and/or in vivo assays. Further improved TNFR inhibitors can be identified.

[0204] Derivatives of peptide TNFR antagonistic peptides 1-14 and 1-23 were designed and synthesized using methods described herein. The derivatives were tested for their ability to inhibit an activity of a TNF receptor, such as TNF-induced IL-6 synthesis. Exemplary results are shown in Figures 11 and 12. As shown in Figure 11, D-isomer of 1-14 (peptide TNFR 1-142), which contains D-amino acid substitution for each corresponding L- amino acid, shows more efficacy (about 70% inhibition) compare to the initial L-form peptide 1-14 (about 50% inhibition). The inverse L-form of the initial peptide (peptide TNFR 1-141) demonstrated even better efficacy (about 80% inhibition) while the inverse D- form (peptide TNFR 1-143) shows about the same inhibitory effect as the initial L-form peptide 1-14. Scrambled peptides of D and L forms (peptides TNFR 1-144 and 1-145) showed no efficacy (negative controls).

[0205] As shown in Figure 12, L inverse (peptide TNFR 1-231), D isomer (peptide

TNFR 1-232), D inverse (peptide TNFR 1-233) of peptides 1-23 showed comparable inhibitory effect in IL-6 synthesis. Similarly, scrambled L and D forms (peptides TNFR 1- 234 and 1-235) did not show any efficacy and were used as negative controls.

Example 6. Design of Allosteramers for TNFRl by Module X Walk

[0206] Short peptide modulators (e.g., inhibitors or activators) of a receptor can be systematically designed simply based on the primary linear amino acid sequence of the receptor without first characterizing any tertiary or secondary structure of the receptor. This method is also referred to as a "Module X Walk" method. In this example, short overlapping peptides containing 10 amino acid residues (10-mer peptides) were synthesized along the entire length of TNFRl (each shifted progressively by one amino acid). The effects of these peptides on TNFRl were then assessed using TNFα-induced IL-6 expression as described in Example 1. The results were plotted against the amino acid positions, forming an "allosteric map" of the entire protein (Figure 13). According to the allosteric map, two "hot spots" including the following two regions were identified:

Amino acids 51-90: HPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFT (SEQ ID NO:553)

Amino acids 105-140: CRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENL (SEQ ID

NO:554)

The hot spots correspond to regions from which Allosteramers™ were derived based on the Module X process (see Figures 1 and 4). Hence, Module X and Module X Walk process coincide.

[0207] 10-mer L-peptides designed for human TNFRl used in this example are shown in Table 4. Peptides 453 (SEQ ID NO:550), 454 (SEQ ID NO:551), and 455 (SEQ ID

NO:552) were not tested in the assay. Table 4. Exemplary 10-mer L-peptide Walk for Human TNFRl

EQUIVALENTS

[0208] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims. The articles "a", "an", and "the" as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth herein. It should also be understood that any embodiment of the invention, e.g., any embodiment found within the prior art, can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.

[0209] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. Furthermore, where the claims recite a composition, the invention encompasses methods of using the composition and methods of making the composition. Where the claims recite a composition, it should be understood that the invention encompasses methods of using the composition and methods of making the composition.

INCORPORATION OF REFERENCES

[0210] All publications and patent documents cited in this application are incorporated by reference in their entirety to the same extent as if the contents of each individual publication or patent document were incorporated herein.

[0211] What is claimed is: