WO2004020608A2

WO2004020608A2 - Modulators of tnf-alpha and il-1 cell surface receptor activity

Info

Publication number: WO2004020608A2
Application number: PCT/US2003/027381
Authority: WO
Inventors: Lennart Olsson; Tatjana Naranda
Original assignee: Receptron, Inc.
Priority date: 2002-08-29
Filing date: 2003-08-28
Publication date: 2004-03-11
Also published as: WO2004020608A3; AU2003263044A8; CA2496634A1; AU2003263044A1

Abstract

Methods and reagents for modulating the activity of cell surface receptors are provided. Receptor activity modulation occurs when exogenous bioactive compounds are contacted with the receptor activation domain. The modulation can be an increase in receptor activity or a decrease in ligand-induced receptor activity. Also provided are methods for identifying and screening for bioactive compounds.

Description

MODULATORS OF TNF-ALPHA AND IL-1 CELL SURFACE RECEPTOR ACTIVITY

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit of U.S. provisional patent application no. 60/407,396, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION [0002] The present invention relates to methods and reagents for modulating cell surface receptor activity. The invention also relates to assays for identification of agents that modulate receptor activity and compositions containing such agents. The invention finds application in the fields of biology and medicine.

BACKGROUND OF THE INVENTION [0003] Signaling molecules such as neurotransmitters, protein hormones, cytokines and growth factors bind to specific receptors on the surface of the target cells they influence. These signaling molecules (ligands) bind the cell surface receptors, resulting in one or more intracellular signals that alter the behavior of target cells.

[0004] In some cases, receptor signaling results in undesirable effects in the subject. For example, activation of receptors by proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) play key roles in several disease conditions. In such cases, it may be desirable to antagonize the effects of these ligands.

[0005] Thus, methods for modulating the activity of receptors and for identifying receptor- modulating agents will be of significant medical benefit.

SUMMARY OF THE INVENTION [0006] In one aspect, the invention provides a method for modulating the activity of the interleukin 1 (IL-1) receptor. The IL receptor is referred to as "IL-IRI (IL-lR-alpha)." In a related aspect, the invention provides a method for modulating the activity of the TNF-alpha receptor. The TΝF-alpha receptor is referred to as "TΝF-RI (p55)". In an aspect, the invention provides a method for modulating the activity of IL-IRI (IL-lR-alpha) by contacting the receptor with an exogenous compound that binds in the activation domain, wherein the activation domain comprises a sequence identical to, or substantially similar to, SEQ ID ΝO:2. In an embodiment, the IL-IRI (IL-lR-alpha) is human. In an embodiment, receptor activity is decreased. In an embodiment, the contacting takes place in vitro. In an embodiment, the contacting occurs in the presence of IL-1. In an embodiment, the exogenous compound is an oligopeptide. In an embodiment, the oligopeptidehas at least about 8 contiguous residues of a sequence substantially similar to, or identical to, SEQ ID NO:2. [0007] In an aspect, the invention provides a method for screening candidate agents for the ability to modulate activity of a IL-IRI (IL-lR-alpha) by deteπriining the binding of the agent to a receptor activation domain sequence having a sequence of SEQ ID NO:2

[0008] In a related aspect, the invention provides a method for screening candidate agents for the ability to modulate activity of an IL-IRI (IL-lR-alpha) having an activation domain comprising a sequence substantially similar to SEQ ID NO:2 by a) contacting the receptor with a candidate agent and an activation-domain binding oligopeptide comprising a sequence substantially similar to SEQ ID NO:2; and b) determining the binding of the agent or the oligopeptide to the activation domain, wherein the binding of the agent to the activation domain or a reduction in the binding of the oligopeptide to the activation domain identifies a candidate agent for modulating cell-surface receptor activity. In an embodiment, the method includes the step of contacting the receptor with receptor ligand. In an embodiment, the candidate agent or oligopeptide is labeled.

[0009] In a related aspect, the invention provides a method for screening candidate agents for the ability to modulate activity of an IL-IRI (IL-lR-alpha) by identifying a compound that binds in a receptor activation domain of sequence SEQ ID NO:2, and assaying the ability of the compound to modulate activity of the cell surface receptor. In an embodiment, the ability of the agent to reduce ligand-induced receptor activation is assayed. In an embodiment, the ability of the agent to increase, or enhance, ligand-induced receptor activation is assayed.

[0010] In an aspect, the invention provides an isolated oligopeptide that binds the IL-IRI (IL- lR-alpha) activation domain. In an embodiment, the oligopeptide comprises at least 8 amino acids of SEQ ID NO:2. In an embodiment, the oligopeptide antagonizes IL-1 induced activation of a IL-IRI (IL-lR-alpha). The invention also provides a pharmaceutical composition comprising an aforementioned oligopeptide in a sterile form and a pharmaceutically acceptable excipient. [0011] In an aspect, the invention provides a method for treating a condition in a patient characterized by an undesired level of IL-1 receptor activation by administering to a patient an exogenous bioactive compound that binds the IL-1 receptor activation domain and which decreases receptor activity when contacted with the receptor.

[0012] In an aspect, the invention provides a method for treating a condition in a patient characterized by a deficiency of IL-1 by administering to a patient an exogenous bioactive compound that binds in a receptor activation domain and which increases receptor activity. [0013] In an aspect, the invention provides a method for modulating the activity of TNF-RI (p55) by contacting the receptor with an exogenous compound that binds in the activation domain, wherein the activation domain comprises a sequence identical to, or substantially similar to, SEQ ID NO:l. In an embodiment, the TNF-RI (p55) is human. In an embodiment, receptor activity is decreased. In an embodiment, the contacting takes place in vitro. In an embodiment, the contacting occurs in the presence of TNF-alpha. In an embodiment, the exogenous compound is an oligopeptide, e.g., an oligopeptide that comprises at least about 8 residues of a sequence substantially similar to, or identical to, SEQ ID NO: 1.

[0014] In an aspect, the invention provides a method for screening candidate agents for the ability to modulate activity of a TNF-RI (p55) by determining the binding of the agent to a receptor activation domain sequence having a sequence of SEQ ID NO: 1.

[0015] In a related aspect, the invention provides a method for screening candidate agents for the ability to modulate activity of a TNF-RI (p55) having an activation domain comprising a sequence substantially similar to SEQ ID NO:l by a) contacting the receptor with a candidate agent and an activation-domain binding oligopeptide comprising a sequence substantially similar to SEQ ID NO:l; and b) determining the binding of the agent or the oligopeptide to the activation domain, wherein the binding of the agent to the activation domain or a reduction in the binding of the oligopeptide to the activation domain identifies a candidate agent for modulating cell-surface receptor activity. In an embodiment, the method includes the step of contacting the receptor with receptor ligand. In an embodiment, the candidate agent or oligopeptide is labeled.

[0016] In related aspect, the invention provides a method for screening candidate agents for the ability to modulate activity of a TNF-RI (p55) by identifying a compound that binds in a receptor;. activation domain having the sequence SEQ ID NO:l and assaying the ability of the compound to modulate activity of the cell surface receptor. In an embodiment, the ability of the agent to reduce ligand-induced receptor activation is assayed. In an embodiment, the ability of the agent to increase, or enhance, ligand-induced receptor activation is assayed.

[0017] In an aspect, the invention provides an isolated oligopeptide that binds the TNF-RI (p55) activation domain. In an embodiment, the oligopeptide comprises at least 8 amino acids of SEQ ID

NO: 1. In an embodiment, the oligopeptide antagonizes TNF-alpha induced activation of a TNF-RI

(p55).

[0018] In an aspect, the invention provides a pharmaceutical composition comprising an aforementioned oligopeptide in a sterile form and a pharmaceutically acceptable excipient.

[0019] In an aspect, the invention provides a method for treating a condition in a patient characterized by an undesired level of TNF-RI (p55) activation by administering to a patient an exogenous bioactive compound that binds in the TNF-alpha receptor activation domain and which decreases receptor activity when contacted with the receptor.

[0020] In an aspect, the invention provides a method for treating a condition in a patient characterized by an undesired level of TNF-RI (p55) activation by administering to a patient an exogenous bioactive compound that binds in the TNF-RI (p55) activation domain and which increases receptor activity. BRIEF DESCRIPTION OF THE DRAWINGS [0021] Figure 1 is a digital image of a Western Blot showing the antagonistic effect of a peptide having the sequence of SEQ ID NO:l (TNF-al peptide) on TNF-RI (p55) activity, as measured by the inhibition of TNF-α-induced phosphorylation of p38 and ERK kinases in HT-29 cells. [0022] Figure 2 is a digital image of a Western Blot that showing the antagonistic effect of TNF- al peptide on TΝF-RI (p55) activity, as measured by the inhibition of TΝF-α-induced " phosphorylation of ERK kinase in mouse bone marrow macrophages.

[0023] Figure 3 is a digital image of a Western Blot that demonstrates the antagonistic effect of a peptide having the sequence of SEQ ID ΝO:2 (IL-IRI peptide) on IL-IRI (IL-lR-alpha) activity, as measured by dose-responsive inhibition of p38, TRAF6, and IRAK phosphorylation in HepG2 cells. [0024] Figure 4 shows the effect on TNF-α-induced IL-8 production in Normal Human Dermal Fibroblasts (NHDF) cells of a small molecule antagonist and agonist that compete with TNRal peptide for binding to the TNF receptor activation site.

[0025] Figure 5 shows the effect of small molecule compounds on TNFα-induced TNF-RI downstream substrate phosphorylation (p38 protein) in Normal Human Dermal Fibroblasts cells.

DETAILED DESCRIPTION OF THE INVENTION [0026] In one aspect of the invention, methods and compositions for modulating cell surface receptor activity are provided, as well as methods for identification of agents that modulate cell surface receptor activity. As used herein, modulation can be an enliancement or increase in receptor activation (i.e., in the presence of an agent acting as a "receptor agonist") or a decrease in ligand- induced receptor activation (i.e., in the presence of an agent acting as a "receptor antagonist"). The modulatory agents, sometimes referred to herein as "exogenous bioactive compounds" are compounds that bind an extracellular portion of the cell surface receptor termed the "activation domain." [0027] Activation domains are described generally in U.S. patent no. 6,333,031 , which is incorporated herein by reference. The activation domain of a receptor occupies a site distinct from the ligand binding site. Thus, the exogenous bioactive compounds used in the practice of the present invention do not compete with the natural ligand for binding to the receptor. Further, binding to the activation domain of the receptor usually does not substantially change the K_D of the binding of the natural receptor ligand to the ligand binding site.

[0028] Table 1, infra, provides the sequences of activation domains of receptors for TNF-alpha (TNF-RI (p55)) and IL-1 IL-lR-alpha. The activation domain sequences for these receptors were identified generally according to the methods described in U.S. pat. no. 6,333,031 and by cross- species sequence comparisons between human receptors and non-human homologs. Table 1

[0029] The TNF-alpha) and IL-1) receptors are known, and the amino acid sequences, activities and other characteristics of the human receptors and homologs from other animals are well known in the art. Receptor sequences are published in databases, e.g. a human TNF-RI (p55) is described as Swissprot accession number P19438, and a human IL-IRI (IL-lR-alpha) is described as PIR accession number P14778. The receptors used in methods of the invention generally are mammalian, e.g., human, primates (including nonhuman primates), rodents (mice, rats, hamsters, guinea pigs), cows, sheep, pigs, horses, and others. Receptors that may be used include those with the complete sequence of a naturally occurring receptor (including naturally occurring alleles and variants, e.g., naturally occurring mammalian or human alleles) as well as recombinanfly expressed variants, and portions of receptors (e.g., a receptor extracellular domain, or an activation-domain containing fragment comprising at least about 50 or at least about 100 amino acid residues). Suitable receptors for use in the methods of the invention include isolated receptor proteins and activation domain- containing fragments (e.g., for use in binding assays) and receptors expressed on the surface of cells (e.g., often for activity assays). Suitable cells include those that normally expressing a TNF-RI (p55) and/or IL-IRI (IL-lR-alpha) (e.g., without limitation HT-29, HepG2 cells, and NHDF (normal human dermal fibroblasts; Clonetics Inc.) as well as cells in which the receptors are recombinantly expressed. It will be understood that cells that express endogenous receptor can also be engineered to recombinantly express (e.g., overexpress) the same or a different receptor. Methods for recombinant expression of polypeptides are well known in the art. See, e.g., (Sambrook and Russel, 2001, Molecular Cloning: A Laboratory Manual, third edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. The polynucleotide sequences of TNF-RI (ρ55)and IL-IRI (IL-lR-alpha) are also known (e.g., sequences for human receptors) or easily deteirninable. For expression, typically, polynucleotides encoding the receptor are used in expression vectors containing typically include transcriptional and/or translational control signals (e.g., transcriptional regulatory element, promoter, ribosome-binding site, and ATG initiation codon). DNA encoding the receptor or receptor fragment is inserted into DNA constructs capable of introduction into and expression in an in vitro host cell, such as a bacterial (e.g., E. coli, Bacillus subtilus), yeast (e.g., Saccharomyces), insect (e.g., Spodoptera frugiperda), or mammalian cell systems. Numerous examples of mammalian cell culture systems are known (e.g., CHO cells, BaF3 cells). Useful human and nonhuman cell lines are widely available, e.g., from the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108.

[0030] The TNF-RI (p55) and IL-IRI (IL-lR-alpha) act by transducing a signal. Without intending to be bound by a specific mechanism, it is believed that TNF-alpha binding to the extracellular domain of TNF-RI (p55) leads to homotrimerization. Once aggregated, the receptors form a complex with other proteins (e.g., TRADD and TRADD-interacting proteins) to form a complex that activates at least two distinct signaling cascades, apoptosis and NF-kappa B signaling. Without intending to be bound by a specific mechanism, it is believed IL-1 signal transduction pathway is initiated by the binding of IL-1 to (IL-lR-alpha), which then forms a complex with IL- lRAcP, resulting in the recruitment of MyD88 and IRAK. IRAK then dissociates from the receptor complex and interacts with TRAF6, which initiates kinase cascade leading to the activation of JNK and NF-kappaB.

[0031] In one aspect, the invention provides a method for modulating the activity of a TNF-RI

(p55) or IL-1 cell surface receptor by contacting the receptor with an exogenous bioactive compound that binds in the activation domain of the receptor, where the activation domain has a sequence identical or substantially similar to SEQ ID NO:l and 2, and where the binding effects a response in the signaling pathway of said receptor, i.e., modulates receptor activity. As noted, modulation of receptor activity can include an increase in receptor activation in the presence or absence of the natural ligand of the receptor (referred to as agonist activity). Conversely, modulation of receptor activity can include inhibition of receptor activation, or antagonist activity. Thus, by "antagonist" herein it is meant compounds that bind the receptor activation site but do not activate the receptor, and for which ligand-induced receptor activity is inhibited or blocked. The level of inhibition of ligand- induced receptor activity will vary depending on the concentrations of ligand, receptor and exogenous agent, but are often at least about a 5% decrease in receptor activity (e.g., as measured as shown in the Examples, often at least about 25%, and frequently at least about 50% or more. It has been discovered, for example, that binding to the activation sequence of the TNF-RI (p55) by a peptide with the sequence SEQ ID NO:l reduces TNF-RI (p55) activation (e.g., TNFα-induced activation). Similarly, binding to the activation sequence of the IL-IRI (IL-lR-alpha) by a peptide with SEQ ID NO:2 reduces IL-IRI (IL-lR-alpha) activation (e.g., IL-1 induced activation). By "binding in the activation domain" is meant that the exogenous compound binds the receptor through interaction with some, but not necessarily all, amino acid residues in the activation domain. [0032] Also shown in Table 1 are exemplary receptor derived peptide sequences (exemplary RDPSs). As is discussed below, the exemplary RDPSs, their homologs, fragments, and variants, are used to modulate activity of corresponding receptors and to identify still other modulatory agents. The RDPSs shown in Table 1 are "TNF-al peptide" having the sequence set forth as SEQ ID ΝO:l, and "IL-1R peptide " having the sequence set forth as SEQ ID NO:2. [0033] For clarity, each receptor activation sequence and each exemplary receptor derived peptide sequence (RDPS) listed in Table 1 is referred to as "corresponding to," or being the "cognate" of, the receptor from which its sequence is derived. In Table 1, cognate pairs of receptors and activation/peptide sequences presented in the same row. Thus, for example, (i) the TNF-RI (p55) activation sequence (SEQ ID NO:l) and (ii) the TNF-RI (p55) are a cognate pair. Similarly, (i) a receptor derived peptide that modulates the activity of the TNF-alpha receptor and which has sequence similarity to SEQ ID ΝO:l, and (ii) the TNF-alpha receptor are a cognate pair.

Exogenous Bioactive Compounds

[0034] An noted above, receptor modulatory agents are sometimes referred to herein as "exogenous bioactive compounds." Specifically, "exogenous bioactive compound" as used herein, refers to a compound that (1) is not produced endogenously by the cell or organism, i.e., it is artificially introduced to the cell or organism; (2) binds amino acid residues in the activation site of a receptor (e.g., as determined by competition assays); and (3) modulates receptor activity. Exogenous bioactive compounds include oligopeptides described in detail herein, as well as other compounds described herein. Further, exemplary assays for identifying bioactive compounds are described hereinbelow. In an aspect, the invention provides a complex comprising a receptor activation domain and an exogenous bioactive compound (e.g., oliogpeptide) described herein. [0035] A variety of different classes of molecules can act as exogenous bioactive compounds, including oligopeptides, other biomolecules (e.g., saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs) and small organic compounds (e.g., having a molecular weight of more that about 50 and less than about 10,000 daltons, often more than about 100 and less than about 2,500 daltons; most often between about 200 and about 600 daltons). Usually candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an arnine, carbonyl, hydroxyl or carboxyl group, and often at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. In one embodiment, the exogenous bioactive compound is other than a protein or peptide.

[0036] In one embodiment, the exogenous bioactive compound is an oligopeptide. As used herein, "oligopeptide" is used interchangeably with "peptide" and "polypeptide" and refers to a polymer of amino acids. In an embodiment, the oligopeptide comprises a sequence identical or substantially similar to SEQ ID NO:l or SEQ ID NO:2, or to an activation domain-binding subsequence thereof. For example, the oligopeptide compound that binds an activation domain and modulates receptor activity may comprises at least 8 contiguous residues of a sequence in Table 1, and often at least 10, at least 12, at least 15 or at least 20 residues. [0037] As used herein, the term "substantially similar" refers to oligopeptide sequences that may be identical to the receptor activation domain (e.g., SEQ ID NO:l and 2) or that may have a degree of similarity to the receptor activation domain sufficient to allow binding of the oligopeptide to the receptor activation domain resulting in modulation of receptor activity. In some embodiments, the length of the peptide is at least 8 amino acids, usually at least about 12 amino acids, and more usually at least about 18 amino acids. In some embodiments, the length of the peptide is fewer than about 60 amino acids, more usually fewer than about 40 amino acids, more usually fewer than 30 amino acids. [0038] An oligopeptide that is substantially similar to a receptor activation domain and which modulates activity of a receptor may differ from the receptor activation domain by amino acid substitutions, insertions, or deletions as compared to the activation domain. For example, the oligopeptide substantially similar to SEQ. ID. NO:l or 2 may include additional residues, e.g., at the 5' or 3' teirninus of SEQ. ID. NO:l-2 or an activation domain-binding subsequence thereof. In embodiments, the peptide will contain least 8 amino acids, at least about 12 amino acids, at least about 15 amino acids, at least about 18 amino, at least about 21 amino acids, or at least about 24 amino acids identical to a sequence of SEQ ID NO: 1-2 and additional residues that may not be identical to SEQ ID NO:l or 2. In some embodiments, a substantially similar oligopeptide will have an amino acid sequence at least about 60% identical to one of SEQ ID NO: 1-2, at least about 70% identical, often at least about 80% identical, sometimes at least about 90% identical. Sequence identity between two peptide sequences can be easily determined by inspection. Alternatively, algorithms such as the Best Fit sequence program described by Devereux et al, 1984, Nucl. Acid Res. 12:387-95, with default settings preferred.

[0039] As noted, peptides suitable for use as exogenous bioactive compounds of the invention may have amino acid substitutions, insertions, or deletions as compared to sequences of SEQ ID NO: 1-2. In one embodiment, amino acid substitutions are made. In one embodiment the number of changes will not be more than about 30%, sometimes not more than about 20%, sometimes not more than about 10 %, of the number of amino acids in the activation domain, although in some instances higher numbers of alterations may be made. In one embodiment, with reference to the RDPSs shown in Table 1, not more than about five, alternatively not more than about three substitutions or deletions will be made. In general, it is preferable that residues critical for biological activity are either not altered or are conservatively altered (e.g. to conserve charge). Examples of conservative alterations include the substitutions shown in Table 2. Critical residues may be elucidated using known mutagenesis techniques followed by activity or binding assays, e.g., using scanning mutagenesis techniques, wherein single amino acid residues within the oligopeptide are modified by substitution with an aliphatic amino acid, e.g., serine, alanine, glycine, valine, and the like. Table 2

Original Residue Exemplary Substitutions

Ala Ser

Arg Lys

Asn Gin, His

Asp Glu

Cys Ser

Gin Asn

Glu Asp

Gly Pro

His Asn, Gin

He Leu, Val

Leu He, Val

Lys Arg, Gin, Glu

Met Leu, He

Phe Met, Leu, Tyr

Ser Thr

Thr Ser

[0040] Oligopeptide exogenous bioactive compounds can contain naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0041] In one embodiment modifications that are made do not substantially alter the biological activity of the oligopeptide, i.e., the modification does not prevent binding of the oligopeptide to its cognate receptor and does not destroy the modulatory activity.

[0042] Alternatively, variants in which biological function has been modified can be selected for. The substitutions which in general are expected to produce the greatest changes in oligopeptide properties are those in which a nonconservative substitution is made in a critical residue, e.g., (a) a hydrophilic residue, e.g., seryl or fhreonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; ) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.

[0043] Oligopeptides can be made by chemical synthesis, through recombinant means, or any other synthesis method. Usually, the oligopeptides are prepared in accordance with conventional techniques, such as synthesis (for example, use of a Beckman Model 990 peptide synthesizer or other commercial synthesizer). Peptides may be produced directly by recombinant methods (see Sambrook et al. Molecular Cloning: A Laboratory Manual, CSHL Press, Cold Spring Harbor, N.Y., 1989) or as a fusion protein, for example, to a protein that is one of a specific binding pair, allowing purification of the fusion protein by means of affinity reagents, followed by proteolytic cleavage, usually at a site engineered to yield the desired peptide (see for example Driscoll et al. (1993) J. Mol. Bio.232:342- 350).

[0044] In addition to modifications within the peptides, they may also contain additional sequences. For example, the oligopeptides may be extended to: 1) provide convenient linking sites, e.g., cysteine or lysine; 2) to enhance stability; 3) to provide for ease of purification, e.g., epitope or purification (His₆) tags; 4) to alter the physical characteristics, e.g., solubility, charge, etc.; or 5) to stabilize the conformation. The oligopeptides may be joined to non-wild-type flanking regions as fused proteins, joined either by linking groups or covalently linked through cysteine (disulfide) or peptide linkages. The oligopeptide may be linked through a variety of bifunctional agents, such as maleimidobenzoic acid, methyidithioacetic acid, mercaptobenzoic acid, S-pyridyl dithiopropionate, and the like. The oligopeptides may be joined to a single arnino acid at the N- or C-terminus of a chain of arnino acids, or may be internally joined. For example, the subject peptides may be covalently linked to an immunogenic protein, such as keyhole limpet hemocyanin, ovalbumin, and the like, to facilitate antibody production to the subject oligopeptides.

[0045] As noted, the oligopeptides may be shorter than those depicted in Table 1 , i.e., residues from either the N- or C-terminus of the oligopeptide may be deleted with the retention of biological activity, preferably full biological activity. In some cases, internal residues may be removed from the oligopeptide. Generally, this will be done by sequentially removing residues and assaying for the ability to bind to the activation domain of a receptor. Once binding has been established, activation may be evaluated.

[0046] Alternatively, the subject oligopeptides may be expressed in conjunction with other peptides or proteins, so as to be a portion of the chain, either internal, or at the N- or C-terminus. Various post-expression modifications may be achieved. For example, by employing the appropriate coding sequences, one may provide farnesylation or prenylation, such that the subject peptide will be bound to a lipid group at one terminus, and will be able to be inserted into a lipid membrane, such as a liposome. [0047] The subject oligopeptides may also be modified by the addition of chemical moieties or groups. For example, the oligopeptides may be PEGylated, where the polyefhyleneoxy group provides for enhanced lifetime in the blood stream. The subject oligopeptides may also be combined with other proteins, such as the Fc of an IgG isotype to enhance complement binding, or with a toxin, such as ricin, abrin, diphtheria toxin, or the like, particularly the A chain. The oligopeptides may be linked to antibodies for site directed action. For conjugation techniques, see, e.g., U.S. Pat. Nos. 3,817,837; 3,853,914; 3,850,752; 3,905,654; 4,156,081; 4,069,105; and 4,043,989, which are incorporated herein by reference. As outlined herein, the oligopeptides may be labeled as well. [0048] Oligopeptides of the invention can be modified to increase stability, enhance pharmacological properties (half-life, absorption, potency, efficacy) and the like. Exogenous bioactive compounds also include nonpeptide compounds (peptide analogs) structurally similar to the RDPSs shown in Table 1, sometimes referred to known as "peptide mimetics" or "peptidomimetics" (Fauchere, 1986, Adv. Drug Res. 15: 29; Veber and Freidinger, 1985, TINS p.392; and Evans et al., 1987, J. Med. Chem 30: 1229). For example, useful peptidomimetics may be structurally similar to a oligopeptide, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: -CH2NH--, -CH2S-, --CH2 -CH2 -, -CH.dbd.CH-(cis and trans), -COCH2 — , ~CH(OH)CH2 --, and --CH2 SO-, by methods known in the art and further described in the following references: Spatola, A. F. in "Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins," B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general review); Morley, J. S., Trends Pharm Sci (1980) pp.463-468 (general review); Hudson, D. et al., Int J Pept Prot Res (1979) 14:177-185 (-CH2 NH-, CH2 CH2 -); Spatola, A. F. et al., Life Sci (1986) 38:1243-1249 (-CH2 - S); Harm, M. M., J Chem Soc Perkin Trans I (1982) 307-314 (-CH--CH-, cis and trans); Almquist, R. G. et al, J Med Chem (1980) 23:1392-1398 (-OCH2 --); Jennings-White, C. et al., Tetrahedron Lett (1982) 23:2533 (-OCH2 -); Szelke, M. et al., European Appin. EP 45665 (1982) CA: 97:39405 (1982) (~CH(OH)CH2 -); Holladay, M. W. et al., Tetrahedron Lett (1983) 24:4401-4404 (-- C(OH)CH2 --); and Hruby, V. J., Life Sci (1982) 31:189-199 (-CH2 --S-); each of which is incorporated herein by reference. One useful non-peptide linkage is — CH2 NH~. Such peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half- life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.

[0049] In one aspect, the invention provides a complex of an IL-1 receptor activation domain and an exogenous agent, where, when bound to the activation domain of the IL-1 receptor on an IL-1 receptor-expressing cell, the exogenous agent modulates activation of the IL-1 receptor (and where the agent is not an MHC protein or protein comprising the sequence of an MHC protein, or another molecule of the IL-1 receptor). In an embodiment the agent is not a oligopeptide or polypeptide. In an embodiment, the agent is a small molecule with a molecular weight of between about 100 and about 1000 daltons. In one embodiment, the complex is of an IL-1 receptor activation domain on an IL-1 receptor-expressing cell and the exogenous agent.

[0050] In one aspect, the invention provides a complex of a TNFα receptor activation domain and an exogenous agent, where, when bound to the activation domain of the TNFα receptor on an TNFα receptor-expressing cell, the exogenous agent modulates activation of theTNFα receptor (and where the agent is not an MHC protein or protein comprising the sequence of an MHC protein, or another molecule of the TNFα receptor). In an embodiment the agent is not a oligopeptide or polypeptide. In an embodiment, the agent is a small molecule with a molecular weight of between about 100 and about 1000 daltons. In one embodiment, the complex is of an TNFα receptor activation domain on an TNFα receptor- expressing cell and the exogenous agent.

Binding and Activity Assays

[0051] Identification of receptor activation domain sequences, as well as determination of the effect of binding of agents to activation domains (see Examples), permits the design of screening assays for agents that bind the activation domain sequences and modulate receptor activity (exogenous bioactive compounds). Initial screening or validation may be carried out using binding assays, with subsequent determination of the effect of the agent on receptor activity. Alternatively, assays that determine the effect of the agent on receptor activity can be carried out without antecedent binding assays.

[0052] It will be appreciated that assays for activation domain binding and modulatory activity are useful in at least two different, but related, contexts. In one context, screening assays are carried out in which a plurality of assay mixtures are run in parallel with different candidate agents (e.g., high throughput screening assays). Usually candidate agents are assayed at different concentrations to obtain a differential response to the various concentrations. Such assays can be used to identify new exogenous bioactive compounds. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, arnidifϊcation to produce structural analogs.

[0053] In a second, related, context, the assays can be used to assess activity of particular variants of compounds known to bind a receptor activation sequence and modulate receptor activity, such as the receptor derived peptides described in the Examples, infra. For example, the effect of amino acid substitutions, insertions, or deletions in SEQ ID NO:l on the antagonistic activity of the peptide on TNF-RI (p55) activity can be readily assessed. Variants for example, may be selected to develop exogenous bioactive compounds with enhanced half-life or other desirable properties. Useful assays will be apparent to those of skill in the art based on the instant disclosure, and include assays described below as well as those described in U.S. Pat. No. 6,333,031.

Binding Assays

[0054] Agents capable of modulating surface receptor activity can be identified by first screening for the ability to bind an activation sequence of a receptor listed in Table 1. Some embodiments of the various assays described herein utilize human cell surface receptors, although other mammalian receptors may also be used, including receptors from rodents (mice, rats, hamsters, guinea pigs, etc.), farm animals (cows, sheep, pigs, horses, etc.) and primates. Included within the definition of cell surface receptors are proteins having amino acid substitutions, insertions, or deletions of the naturally occurring sequence. Furthermore, included within the definition of cell surface receptors are proteins having portions of cell surface receptors; that is, either the full-length receptor may be used, or functional portions thereof. Thus, in one embodiment, binding to a candidate agent to an oligopeptide having a sequence identical to or substantially similar to SEQ ID NO:l or 2, or a receptor binding fragment or variant thereof, is deteπnined to identify compounds that bind the activation domain and potentially modulate receptor activity.

[0055] In one variation, the assay comprises combining an activation domain of a TNF-RI (p55) or IL-IRI (IL-lR-alpha) cell surface receptor, and a candidate bioactive agent, and determining the binding of the candidate agent to the activation domain. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (e.g., phosphorylation assays), and the like. In one variation, the candidate bioactive agent is labeled, and binding determined directly. In one approach, all or a portion of the cell-surface receptor is attached to a solid support, a labeled candidate agent (for example a fluorescent label) is added, excess and unbound reagent is removed, and the presence of the label is present on the solid support is determined. Various blocking and washing steps may be utilized as is known in the art. Alternatively, the candidate agent can be immobilized.

[0056] Another way to assess binding of an agent to an activation domain uses competitive binding assays to detecting competition between (i) the agent and (ii) a competitor moiety that binds the receptor activation domain, for binding to a TNF-RI (p55) or IL-IRI (IL-lR-alpha) activation domain. Thus, in one variation, the method comprises combining a polypeptide comprising a cell surface receptor activation domain as listed in Table 1, a candidate bioactive agent, and a competitor moiety, and determining the binding of the candidate agent to the activation domain. For example, in one assay the ability of the agent to interfere with the binding of an oligopeptide having a sequence shown in Table 1 and its cognate receptor is determined. Exemplary competitor moieties that bind a receptor activation domain for use in competition assays include oligopeptides having a sequence of SEQ ID NO: 1 or 2, or a receptor binding fragment or variant thereof (e.g., an oligopeptide having a sequence identical or substantially similar to one of SEQ ID NO:l or 2). For use in the assay, examples of polypeptides that comprise a cell surface receptor activation domain as listed in Table 1, as described above, include an oligopeptide having a sequence identical or substantially similar to one of SEQ ID NO:l or 2, a full-length receptor comprising an activation domain as listed in Table 1 (either isolated or expressed by a cell), and a fragment of the receptor a portion of the extracellular portion of the receptor.

[0057] In one variation of the aforementioned assays, the candidate bioactive agent is labeled. Either the candidate bioactive agent, or the competitor moiety (e.g., oligopeptide) is added first to the receptor for a time sufficient to allow binding. Incubations may be performed at any temperature which facilitates optimal activity, typically between 4°C-40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high throughput screening. Typically between 0.1 and 2 hours will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.

[0058] In another variation, the competitor moiety (e.g., oligopeptide) and the candidate bioactive agent are added together. Usually the candidate bioactive agent is added first, and usually in excess. Non-binding of the competitor moiety is an indication that the candidate bioactive agent is binding to the activation domain and thus is capable of modulating receptor activity. Either component can be labeled.

[0059] In an alternative variation, the candidate bioactive agent is added first, with incubation and washing, followed by the competitor moiety (e.g., oligopeptide). The absence of binding by the competitor moiety may indicate that the bioactive agent is bound to the receptor with a higher affinity. Thus, if the candidate bioactive agent is labeled, the presence of the label on the support, coupled with a lack of competitor moiety binding, may indicate that the candidate agent is capable of binding to the activation domain and modulating receptor activity.

[0060] In another variation, the methods comprise combining a cell surface receptor and a competitor moiety (e.g., oligopeptide) as described herein, to form a test mixture. The candidate bioactive agent is added to the test mixture, and the binding of the candidate bioactive agent to the activation domain of the receptor is determined. In this embodiment, either or both of the competitor moiety or the candidate bioactive agent is labeled, with preferred variations utilizing labeled oligopeptides, such that displacement of the label indicates binding by the candidate bioactive agent. [0061] In another variation, the methods comprise differential screening to identity bioactive agents that bind a receptor activation domain. In this embodiment, the methods comprise combining a cell surface receptor and a competitor moiety (e.g., oligopeptide) that binds the activation sequence in a first sample. A second sample comprises a candidate bioactive agent, a cell surface receptor and a competitor moiety. The binding of the competitor moiety is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding the activation domain. That is, if the binding of the competitor moiety is different in the second sample relative to the first sample, the agent is capable of binding the activation domain. [0062] A variety of assay formats will be apparent to those of skill. In one variation of the methods herein, the purified cell surface receptor or candidate agent is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an array, etc.). The insoluble supports may be made of any composition to which peptide or receptor can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. ^■ These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon, or nitrocellulose, Teflon™, and the like. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the peptide or other protein is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the peptide and is nondiffusable. Preferred methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the receptor is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking, and the synthesis of the receptor on the support surface. Following binding of the peptide or receptor, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein. In another variation, cell lines that overexpress the cell surface receptor are used to screen for candidate bioactive agents. [0063] In another variation a homogeneous LANCE assay format, or similar solution phase assay, is used to identify bioactive molecules. In the LANCE type assay, all the reagents are in a solution (in contrast to solid or semi-solid plate assay). Briefly, a receptor of interest (e.g., TNF-RI (p55) or IL-IRI (IL-lR-alpha)) is incubated with biotinylated peptide that specifically binds the modulation domain of the receptor under conditions in which a complex between receptor and the peptide is formed. A Europium-labeled anti-receptor antibody is allowed to bind the complex and streptavidin-APC conjugate is allowed to bind to biotinylated peptide. The formation of the complex is detected by signal emission which occurs as a result of close proximity between Streptavidin-APC conjugate (that binds to biotinylated peptide) and Eu+-labeled anti- receptor antibody. Thus, if the complex between receptor and peptide is formed, strong signal emission occurs. Lack of signal indicates that anti-receptor antibody and streptavidin conjugate are not in a close proximity, generally because the receptor/peptide complex has not been formed. When used as a screening assay, a competition assay between the biotinylated peptide and candidate bioactive molecules (e.g., from a library of compounds) is usually carried out by adding the candidate bioactive molecule before the formation of the peptide-receptor complex. An absence or diminution of signal indicates competition for the modulation site between the biotinyated peptide and the candidate bioactive molecule. [0064] "Labeled," as used herein in the context of binding and activity assays refers to a compound is either directly or indirectly labeled with a compound that provides a detectable signal, e.g., radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin, and the like. For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above. The label can directly or indirectly provide a detectable signal. In some variations, only one of the components is labeled. For example, the oligopeptides may be labeled at tyrosine positions using 125 I, or with fluorophores. Alternatively, more than one component may be labeled with different labels; using ¹²⁵I for the oligopeptides, for example, and a fluorophor for the candidate agents.

[0065] A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g., albumin and detergents, which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, and anti-microbial agents, may be used. The mixture of components may be added in any order that provides for the binding. An oligopeptide that binds a TNF-RI (p55) IL-IRI or (IL-lR-alpha) activation-domain can be referred to as an "activation-domain binding oligopeptide." Examples of such oligopeptides include oligopeptides having the sequence of SEQ ID NO:l or 2 and others identified using the binding assays described herein.

Receptor Activity Assays

[0066] Methods for detecting modulation of activity of a receptor listed in Table 1 are known in the art. As used herein, "receptor activity" has its usual meaning in the art and refers to the biological function associated with binding of natural ligand of the cell surface receptor. Modulation of receptor activity can refer to an increase in the activity (in the presence or absence of the natural ligand) or a decrease in natural-ligand induced activity (generally measured in the presence of activating amounts of natural ligand or ligand analogs that bind the ligand binding site, such as homologs from other species, variants, and the like). [0067] As used herein, the terms "natural ligand," "naturally-occurring ligand," and "endogenous ligand" are used interchangeably, and refer to ligand that is natively produced by the organism expressing the subject receptor, and which binds the ligand binding site of the receptor. For example, tumor necrosis factor alpha is the natural ligand for the TNF-RI (p55) and IL-1 is the natural ligand for the IL-IRI (IL-lR-alpha). Natural ligands are examples of "activating ligands." Other activating ligands are compounds that compete with the natural ligand for binding to the receptor ligand binding site, and which when bound by the receptor cause receptor activation.

[0068] As will be appreciated by those in the art, the specific biological function will vary depending on the identity of the receptor. Generally ligand binding results in a conformational change in the receptor. Thus, in some embodiments, activation of a receptor is detectable as a conformational change either within the receptor, or as a result of monomeric receptors becoming multimeric, which allows the receptor to facilitate signaling. In some cases, the conformational change results in receptor phosphorylation, receptor association with another cell biomolecule (e.g., protein), and/or phosphorylation of another cell protein. Methods for detecting changes in receptor conformation, receptor-biomolecule association, receptor phosphorylation or phosphorylation of other biomolecules in the receptor signaling pathway are well known. Illustrative assays are described in the Examples, infra. Receptor activation can also be detected as a downstream effect mediated by ligand binding to the receptor such as changes in concentration of intracellular biomolecules (e.g., cAMP, see Example 2, infra), metabolic changes (see, e.g., Example 1, infra), cell shape changes, stimulation or inhibition of cell proliferation, activation of substrates in down-stream signaling pathways, and a variety of other measures of receptor activation. Suitable activation assays can be carried out in vitro (e.g., in immortalized cells lines or primary cell cultures) or in vivo (e.g., in non- human animal models, or human subjects). Another assay for receptor activity is measurement of TNF-alpha or IL-1 induced production of IL-8 or IL-6 (see, Bocker et al., 2000, J. Biol. Chem 275:12207-13. See also Suzuki et al, 2000, FEBS Letters 465:23-27). An examplary protocol for measuring receptor activity using this method is provided in Example 3, infra, although many variations will be evident to one of ordinary skill.

Applications

[0069] The exogenous bioactive compounds, e.g., oligopeptides, of the present invention are used in methods of modulating receptor activity. Such modulation finds use in screening assays, studies of the mechanism of action of receptor ligands, therapeutic uses, and other uses that will be apparent to one of ordinary skill in the art.

[0070] In an aspect the invention provides a method for modulating activity of a cell surface receptor by contacting a mammalian cell surface receptor listed in Table 1 and a compound that binds the activation sequence of the receptor (set forth in Table 1 for the human homolog). In one respect, the exogenous compounds may act as receptor antagonists, as seen with, e.g., TNF-al peptide and IL- 1RI peptide (examples of agents that bind the receptor activation sequences of TNF-RI (p55) and IL- IRI (IL-lR-alpha)). Thus, for example, in an aspect the invention provides a method for antagonizing activity a cell surface receptor by contacting a mammalian cell surface receptor and an exogenous compound where the receptor and the exogenous compound are (a) a TNF-RI (p55) and an oligopeptide comprising a sequence substantially similar to SEQ ID NO: 1 ; (b) a TNF-RI (p55) and a compound that competes with an oligopeptide of SEQ ID NO:l for binding the TNF-RI (p55); (c) an IL-IRI (IL-lR-alpha) and an oligopeptide comprising a sequence substantially similar to SEQ ID NO:2; (d) an IL-IRI (IL-lR-alpha) and a compound that competes with an oligopeptide of SEQ ID NO:2 for binding the IL-IRI (IL-lR-alpha).

[0071] It will be recognized by those of ordinary skill that modulation of each of the TNF-RI (p55) and IL-IRI (IL-lR-alpha) has a variety of therapeutic benefits. In general, any of a variety of diseases, symptoms, and conditions mediated, at least in part, by activation of the TNF-RI (p55) or IL-IRI (IL-lR-alpha) can be treated by administering agents that modulate receptor activity. In this context, "treatment" is an approach for obtaining beneficial or desired results, such as alleviation or amelioration of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total). Tumor Necrosis Factor (TNF-alpha) and Interleukin -1 (IL-1) are important effector cytokines for immune responses and inflammation. These cytokines are produced mainly by activated macrophages, and mediate pleiotropic inflammatory and immunoregulatory responses, as well as cytotoxicity, antiviral activity, and stimulation of cell growth.

[0072] In one aspect modulators of the IL-1 and/or TNF-alpha receptors are used as anti- inflammatory agents. Inflammation (e.g., peritonitis, carditis and arthritis) is characterized by edema and tissue injury due to the release of numerous chemotactic pro-inflammatory cytokines including IL-1 and TNF-alpha. Without intending to be bound by any particular mechanism, inflammation causes the induction of the Cox-2 enzyme, leading to the release of prostanoids, which sensitize peripheral nociceptor terminals and produce localized pain hypersensitivity. Induction of Cox-2 results in elevated levels of PGE2 in the cerebrospinal fluid. Major inducers of Cox-2 up-regulation in the CNS are IL-1 and TNF-alpha. Thus, by preventing central prostanoid production by inhibiting the IL-1 or TNF-alpha mediated induction of Cox-2 in neurons or by inhibiting central Cox-2 activity centrally generated inflammatory pain hypersensitivity would be reduced. [0073] In one aspect, modulators of the IL-1 and TNF-alpha receptors are used as anti- inflammatory agents for example in treatment of sepsis, cachexia, rheumatoid arthritis, chronic myelogenous leukemia, asthma, psoriasis, and inflammatory bowel disease (e.g., Crohn's disease, e.g., for sustaining closure of draining fistulas in Crohn's disease patients).

[0074] Depending on the type of disorder, administration of the pharmaceutical composition can serve to enhance the cellular response to endogenous or exogenous ligand, e.g., in ligand resistant states, to replace endogenous ligand, e.g., in ligand deficient states, or to antagonize the effects of ligand (e.g., in cases in which expression of endogenous ligand is detrimental to the subject). Thus, in one embodiment, the exogenous compound contacts the receptor in the presence of the ligand which normally activates the receptor. As above, there may be endogenous ligand present, or exogenous ligand added in addition to the exogenous compound.

Dosages and Formulations of Exogenous Compounds

[0075] An exogenous compound may be formulated as a pharmaceutical composition by combining the compound with a pharmaceutical carrier or diluent and optionally other compounds that enhance therapeutic utility and/or facilitate storage and administration. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Thus, in one aspect the invention provides pharmaceutical compositions that include exogenous compounds of the invention along with pharmaceutically acceptable excipients, carrier or diluent and optionally other compounds that enhance therapeutic utility and/or facilitate storage and administration. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Pharmaceutically acceptable excipients are well known in the art and include sterile water for pharmaceutical use, isotonic solutions such as saline and phosphate buffered saline, physiological saline, PBS, and dextrose solution. In addition, the pharmaceutical composition or formulation can include other carriers, adjuvants, or non-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients and the like. The compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents. Other excipients suitable for administration to a human patient known in the art. See, e.g., Remington: The Science and Practice of Pharmacy (19th edition, 1995, Gennavo, ed.). The pharmaceutical compositions are typically sterile (i.e., manufactured or formulated as a sterile composition) and optionally can be prepared in compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. [0076] The exogenous bioactive compounds of the invention may be administered in a physiologically acceptable carrier to a host. The agents may be administered in a variety of ways, e.g., orally, parenterally (e.g., by intravascular infusion or injection at an epidermal, subcutaneous, intramuscular, or intraperitoneal site), topically, transdermally, or by transmucosal absorption. Depending upon the manner of introduction, the agents may be formulated in a variety of ways. [0077] The formulation of bioactive agent will vary depending upon the purpose of the formulation, the particular mode employed for modulating the receptor activity, the intended treatment, and the like. The concentration of therapeutically active agents in the formulation may vary from about 0.1-100 wt. %. The formulation may involve patches, tablets, capsules, liposomes, time delayed coatings, injectables, or may be formulated in pumps for continuous administration. For example, formulations for injection may comprise a physiologically acceptable medium, such as water, saline, PBS, aqueous ethanol, aqueous ethylene glycols, or the like. Water soluble preservatives which may be employed include sodium bisulfite, sodium thiosulfate, ascorbate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric borate, parabens, benzyl alcohol and phenylefhanol. These agents may be present in individual amounts of from about 0.001% to about 5% by weight and preferably about 0.01% to about 2%. Suitable water soluble buffering agents that may be employed are alkali or alkaline earth carbonates, phosphates, bicarbonates, citrates, borates, acetates, succinates and the like, such as sodium phosphate, citrate, borate, acetate, bicarbonate and carbonate. Additives such as carboxymethylcellulose may be used as a carrier in amounts of from about 0.01% to about 5% by weight. The specific dosage may be determined empirically in accordance with known ways. See, for example, Harrison's Principles of Internal Medicine, 11th ed. Braunwald et al. ed, McGraw Hill Book Co., New York, 1987. [0078] Generally, a therapeutically effective dose of the exogenous bioactive compound will be administered. A therapeutically effective dose is an amount sufficient to modulate receptor activity. This amount is usually in the range of about 0.005-10, more usually from about 0.01-1 mg/kg of host weight, and sometimes from about 0.1 to about 1 mg/kg. Administration may be as often as daily; sometimes not more than once or twice daily, or as infrequent as weekly. The host may be any mammal including domestic animals, pets, laboratory animals and primates, particularly humans. The amount will generally be adjusted depending upon the half life of the compound, where dosages in the lower portion of the range may be employed where the compound has an enhanced half life or is provided as a depot, such as a slow release composition comprising particles, introduced in a matrix which maintains the compound (e.g., peptide) over an extended period of time, e.g., a collagen matrix, use of a pump which continuously infuses the compound over an extended period of time over a substantially continuous rate, or the like. Heller (1987), Biodegradable Polymers in Controlled Drug Delivery, in: CRC Critical Reviews in Therapeutic Drug Carrier Systems, Vol. 1, CRC Press, Boca Raton, Fla., pp 39-90, describes encapsulation for controlled drug delivery, and Di Colo (1992) Biomaterials 13:850-856 describes controlled drug release from hydrophobic polymers.

EXAMPLES [0079] The following examples serve to more fully describe the manner of using the above- described invention. It is understood that these examples in no way serve to limit the scope of this invention, but rather are presented for illustrative purposes. All cells were obtained from the American Type Culture Collection. EXAMPLE 1 BIOLOGICAL ACTIVITY OF TNF-RI (P55 DERIVED PEPTIDE (TNF-A1 PEPTIDE) [0080] Example 1 illustrates that a TNF-RI (p55) derived peptide ("TNR-alp") having the sequence set forth as SEQ ID NO:l in Table 1 is a TNF-o; receptor antagonist. The antagonistic effect of TNR-alp binding to the TNF-α receptor is demonstrated by the inhibitory effect on ligand-induced phosphorylation of ERK1 and p38.

[0081] The biological activity of TNF-al peptide was measured using HT-29 cell and mouse bone marrow macrophage assays. TNF-α activates members of the mitogen-activated protein kinase (MAPK) family whose downstream targets include both c-Jun and c-Fos. The MAPK family comprises three subfamilies: (i) the extracellular signal-regulated kinases of ERK subfamily represented by p42mapk/erk2 and p44mapk/erkl; (ii) the c-Jun NH2-terminal kinases/stress-activated protein kinases or JNK/SAPK subfamily represented by the p46 JNK SAPK; and (iii) the p38mapk subfamily. These assays specifically addressed ERK1 and p38 phosphorylation.

Assay Solutions

[0082] 2x Lysis Buffer is made by combining the following reagents: 50 ml 1M HEPES, pH 7.6;

8.8 g NaCl; 10 ml Triton X-100; 10 ml 0.5M EDTA, pH 8; H₂0 to 500 ml.

[0083] lOOx Protease Inhibitor Mix is made by combining the following reagents: 20 mg

Aprotonin, 2 mg Pepstatin A, 2 mg Leupeptin; 2 mg Cymostatin, 467 mg AEBSF; H₂0 to 20 ml.

[0084] Sample Buffer is 100 μl 50% Glycerol; 0.5% Bromphenyl Blue, 20 μl /3-mercaptoethanol,

40 μl 10% SDS, and 160 μl lx electrophoresis buffer.

[0085] lOx TST is made by combining the following reagents:12.1 g Tris base; 87.7 g NaCl; 7.5 ml Tween-20; 3 g NaN3; H₂0 to 1 liter, pH to 7.4.

[0086] Blotto is 100 ml lOx TST, 5 g Dry Milk; H₂0 to 1 liter.

[0087] lOx TSM is made by combining the following reagents: 121.1 g Tris base; 58.4 g NaCl;

10.2 g MgCl₂x 6H₂0, H₂0 to 1 liter, pH to 9.0.

[0088] BCIP/NBT Substrate: NBT stock: 50 mg/ml in 70% Dimethyl Formamide stored at -

20°C; BCIP stock: 50 mg/ml in 100% Dimethyl Formamide stored at -20°C; Combine 10 ml lx TSM with 66 μl 50 mg/ml NBT and 33 μl 50 mg/ml BCIP just before using.

Cell signaling in HT-29 cells

HT-29 cell growth and starvation [0089] HT-29 (human colonic epithelial) cells were grown and maintained at 37°C and 5% C0₂ in RPMI medium 1640 (ATCC, catalog no. 30-2001) containing 10% fetal bovine serum (FBS, ATCC, catalog no. 30-2020), 100U penicillin/lOOμg/ml streptomycin sulfate (P/S, Applied Scientific, catalog, no. 9366), and 2 ng/ml human granulocyte/macrophage colony-stimulating factor (GM-CSF). The cells were grown to a density no greater than 8 x 10⁵ cells/ml. [0090] When ready to be used in the assay, the cells were starved overnight in RPMI 1640 medium containing 3.5% FBS and P/S in T150 flasks (60 ml/flask) at a density of 8 x 10⁵ cells/ml at environmental conditions of 37°C and 5% C0₂. The cells were then centrifuged at 200 x g for 5 minutes at room temperature. The supernatant was removed, and the cells pooled into one 50 ml conical tube. The cells were washed twice with serum-free medium (RPMI containing P/S) and resuspended to a density of 1.5 x 10⁷ cells/ml in serum-free medium. The cells were then placed in 15 ml conical tubes in 1 ml aliquots, and incubated with the caps loosened for 1 hour at 37°C and 5% C0₂. The lower portion of each tube was tapped several times every 10 minutes to prevent the cells from settling.

TNF-al peptide) modulation of HT-29 cells and immunoprecipitation [0091] TNF-al peptide stock was diluted with ice cold H₂0 (if necessary) to obtain samples having final peptide concentrations of 0.03 μM, 0.3 μM., 3 μM, and 30 μM after addition of 10 μl of each dilution to the 1 ml of HT-29 cell suspension prepared above. The samples were incubated at 37°C and 5% C0₂ for 30 minutes. Fourteen ml of ice cold PBS (Sigma, catalog no. P-3813) was ' added to each sample to stop the assays, and the samples centrifuged at 400 x g for 5 minutes at 4°C. The samples were then placed on ice, the supernatant aspirated, and the cells lysed by adding 800 μl 2x lysis buffer. The cells were incubated on ice for another 30 minutes. Samples were then transferred to 1.5 ml microcentrifuge tubes and spun for 10 minutes at 10,000 x g and 4°C. The supernatant from each sample was then added to anti-phosphotyrosine-coated GammaBind beads, prepared as described below, and incubated overnight at 4°C with end-over-end rotation. [0092] Next, the samples were spun for 15 to 20 seconds and the supernatant discarded. The beads were washed twice with 800 μl lx lysis buffer and once with 800 μl of a 1:1 mix of lysis buffer:125mM Tris, pH 6.8. To each sample was added 45 μl lx sample buffer. The samples were then heated for 5 minutes at 95°C, centrifuged for approximately 20 seconds and examined by SDS- PAGE.

[0093] The GammaBind beads were coated with antibody by washing them 3x with 1 ml ice cold PBS, spinning them for 15 to 20 seconds, and then discarding the supernatant. Ten microliters of anti-phosphotyrosine antibody (PY99, Santa Cruz Biotech, catalog no. sc-7020) was then added per sample to the beads. Next, PBS was added so that the sample contained 50% beads and 50% liquid. The beads were then incubated for 2 hours at room temperature with end-over-end rotation.

Western Blot Analysis [0094] Samples were run on two 8% polyacrylamide gels, transferred to Immobilon-P membranes, blocked with blotto for 1 hour at room temperature, and one membrane incubated with α- p38 antibody and the other with α-ERKl antibody at 4°C overnight. The membranes were then washed 3 times with blotto at room temperature and incubated with α-Rabbit-AP antibody diluted 1 :2000, at room temperature for 2 hours. The membranes were then washed 3 times with blotto, twice with lx TST, once with lx TSM, and developed with BCIP/NBT.

[0095] The Western Blot (digital image) in Figure 1 shows that phosphorylation of both ERK1 and p38 is significantly diminished in the presence of TΝR-alp. Furthermore, TΝR-alp at 0.3 μM was shown to reduce the activation of kinases induced by 3, 10, or 30 ngml of TΝF-α. Thus, the signaling assay using HT-29 cells demonstrated that TΝR-al peptide is a TΝF-α receptor antagonist.

Cell Signaling in Mouse Bone Marrow Macrophages [0096] Mouse bone marrow macrophages were isolated from femoral and tibial bone marrow. Cells were washed twice with PBS and resuspended to a density of 6.5 10⁶ cell/ml in media. Three hundred microliters (6.5 x 10⁵) of cells were mixed with 1.5 ml of methylcellulose media (Stem Cell Technology, catalog no. M3234). At 5-7 days confluent monolayers of macrophages were obtained and stimulated with TΝF-α in the presence or absence of TΝR-alp. After cell lysis, the activation of ERK protein was measured. As shown in Figure 2, (digital image of Western blot with phosphospecific ERK antibody) TΝR-al peptide inhibits TΝF-α induced protein phosphorylation. The effect is observed at max TΝF-α stimulation of 30 ng/ml. Kinetics indicate peptide activity at 15 minutes, with a duration of activity as long as the hormone activity, which is approximately 60 minutes.

[0097] Thus, the cell signaling assay in mouse bone marrow macrophages also demonstrated that TΝR-al peptide is a TΝF-α receptor antagonist.

EXAMPLE 2 ANTAGONIST EFFECT OF IL-IRI QL-IR-ALPHA) DERIVED PEPTIDE QL-1P) [0098] This example demonstrates that IL-IRI (IL-lR-alpha) derived peptide ("IL-lp") having the sequence set forth as SEQ ID NO:2 in Table 1 is an IL-IRI (IL-lR-alpha) antagonist. [0099] The in vitro biological activity of IL-lp was studied using a cell signaling assay in HT-29 and HepG2 cells. TRAF6, IRAKI , and p38 are kinases in the downstream signaling pathway of the IL-IRI (IL-lR-alpha), and IL-1-induced activation of the IL-IRI (IL-lR-alpha) results in phosphorylation of TRAF6, IRAKI, and p38. The effect of IL-lp on IL-1 induced phosphorylation was determined using the techniques described in Example 1, with substitution of appropriate antibodies.

[0100] Figure 3 is a digital image of Western Blots probed with either anti-TRAF6, anti- IRAK1, or anti-p38 showing dose-responsive inhibition of IL-1 induced phosphorylation of TRAF6, IRAKI, and p38 in HepG2 cells. EXAMPLE 3 MEASUREMENT OF TNF-α STIMULATED IL-8 PRODUCTION IN NHDF [0101] This example provides exemplary protocols for measurement of receptor activity. This protocol can be easily modified to measure IL-1 stimulated IL-8 secretion. The assay principle is that Normal Human Dermal Fibroblast (NHDF) cells respond to TNF-α with the secretion of soluble IL-8, which can be measured in a Sandwich ELISA. [0102] Cells:

1. Grow NHDF cells in Clonetics Fibroblast Growth Medium (FGM-2) in a T-75 flask until they are approximately 90% confluent.

2. Seed a 96-well tissue culture plate with 1 x 10⁴ cells/well in FGM-2 medium and ^■ incubate overnight.

3. Wash the cells lx, and starve overnight in FGM-2 containing 0.2% FBS. [0103] Stimulation:

1. Wash the cells lx with FGM-2 containing 0.2% FBS. Aspirate the liquid from the wells.

2. Dilute the exogenous agent (peptide or other compound) in FGM-2 containing 0.2% FBS to 2x the desired final concentration right before it is added to the plate (peptide stock is ImM in H₂0, final concentration in the assay is lOμM). Add lOOμl of each dilution to the appropriate wells, and incubate for 60 minutes at 37°C, 5% C0₂.

3. Dilute hTNF-α to 2x its final concentration in FGM-2 containing 0.2% FBS (i.e. for a final cone, of lOng/ml TNF- α, dilute to 20ng/ml; hTNF- α stock is lOμg/ml in PBS/0.1% BSA). Add lOOμl of the dilution to each well, and incubate at 37°C, 5% C0₂ for 4 hrs.

4. Spin plate at 200xg for 5 rninutes. Remove 150μl of supernatant from each well, and place into the corresponding wells of an ice cold NUNC Minisorp plate. Samples can be stored at - 80°C until they are needed for the IL-8 ELISA, or they can be used in the assay immediately.

5. If the samples were frozen in Step 4, thaw them on ice (this can take a while). Keep the samples on ice until they are used in the ELISA. If the samples are too concentrated, dilute them 1 :4 in FGM-2 containing 0.2% FBS. Use 50μl of each sample in an IL-8 ELISA.

[0104] Signaling in NHDF Cells in Response to TNF-α and Test Compound Cell Growth and Starvation

1. Grow NHDF cells in Fibroblast Growth Medium (FGM-2, Cambrex) in a T-l 50 flask until they are approximately 90% confluent. Grow the cells according to the instructions provided by Cambrex; do not use cells past passage 8.

2. Seed a 6-well tissue culture plate with 1.6 x 10⁵ cells/well in FGM-2 medium and incubate overnight. 3. Wash the cells lx with Starvation Medium (FGM-2 containing growth factors plus

0.2% FBS), and starve overnight in the same medium. [0105] Cell Stimulation

1. Wash the cells lx with Starvation Medium. Aspirate the liquid from the wells.

2. Add 1ml Starvation Medium to each well. Dilute the compounds in DMSO to 333.4x their desired final concentrations. Add 3μl of each diluted compound to the appropriate wells, and 3μl of DMSO to each of the controls; incubate for 45 minutes at 37°C, 5% C0₂.

3. Dilute hTNF- α in Starvation Medium to lOOx its final concentration (i.e. for a final conc.of 6ng/ml TNF- α, dilute to 600ng/ml). Add 10.2μl of the diluted TNF- α to each well, and incubate at 37°C, 5% C0₂ for 15minutes.

4. To stop the reactions, place the 6-well plate on ice, aspirate the medium, and wash the cells with ice cold PBS.

5. Remove as much of the PBS as possible, and lyse the cells with 0.3ml 2x lysis buffer containing 2x protease inhibitors, added just before use. Incubate on ice for 30 minutes.

6. Scrape the cells off of the bottom of each well, and place into clean microcentrifuge tubes.

7. Spin the samples for 10 minutes. For each sample, place 20ul 4x Sample Buffer, and 60ul supernatant into a microcentrifuge tube. Heat the samples for 5 minutes at 95°C, and centrifuge briefly to remove condensation from the top of the tubes. Vortex the samples briefly and examine by SDS-PAGE.

[0106] IL-8 ELISA:

1. On the day before the ELISA is to be performed, coat a NUNC Maxisorp plate with 50μl/well 500ng/ml anti-hIL-8 (R&D, stock 500μg/ml; diluted 1 : 1 ,000 in 25mM NaHC0₃, pH 9.6). Incubate overnight at 4°C while shaking.

2. Block with 200μl/well Blocking Buffer for 1 hr at room temperature. (Blocking Buffer: TBS (20mM Tris, 137mM NaCl, pH 7.6), 0.05% Tween-20, 0.3% Safeway Dry Milk).

3. Wash 3x with 250μl/well Wash Buffer (TBS, 0.05% Tween-20). Do not let the plates sit after washing. Wash, then pipet plate by plate.

4. Prepare the hIL-8 standards in McCoy's medium containing 5% FBS (IL-8 stock is 25μg/ml). Dilute samples to contain the following concentrations of IL-8: 0, 12.5pg/ml, 25pg/ml, 50pg/ml, 75pg/ml, lOOpg/ml, 150pg/ml, and 200pg/ml.

5. Add 50μl of either the diluted IL-8 standards (from step #4 above) or the diluted sample (from step #3 in the previous section) to the appropriate wells of the coated plates; each plate should have a set of standards. Incubate for 3 hrs at room temperature.

6. Wash 3x with 250μl/well Wash Buffer. 7. Dilute biotinylated anti-hIL-8 1 :4,000 in Blocking Buffer (anti-hIL-8-biotin stock is lOOμg/ml), and add 50ul to each well. Incubate over night at 4°C while shaking.

8. Wash 3x with 250μl/well Wash Buffer.

9. Dilute HRP-Streptavidin conjugate 1:100 in Blocking Buffer. Add 50μl to each well. Incubate for 20 minutes at 4°C.

10. Wash 3x with 250μl/well Wash Buffer.

11. Add lOOμl TMB substrate to each well. Incubate for 15 minutes at room temperature.

12. Add 50μl 2M H₂S0₄ to each well. Read on the Wallac at 450nm.

13. Compare effect of added agent on IL-8 production. In the presence of receptor antagonists, IL-8 production is reduced.

EXAMPLE 4 ACΉVΠΎ OF SMALL MOLECULE ANTAGONISTS AND AGONISTS (SYNERGISTS) OF TNF-α [0107] 48,000 compounds from a library of synthetic organic compounds with molecular weight <500 were screened in a peptide displacement assay for binding to the activation domain of the TNF-RI-receptor. Briefly, purified receptor was incubated (in the presence of library compounds) with biotinylated TNRal peptide and complex formation was assayed by (1) capture to a streptavidin coated plate and (2) detection using an anti-TNF-RI (p55) specific antibody in which colorimetrically detectable signal corresponds to formation of complex between TNRal peptide and TNF-RI-receptor. A reduction of signal is indicative of competition between the biotin-TNRal peptide and its binding site on the TNF-RI-receptor has occurred. Competition was detected using non-labeled peptide (control) or certain compounds from the library.

[0108] Molecules that competed with peptide binding were tested for their biological activity in two assays: (i) inhibition of TNF-α induced IL-8 production and (ii) phosphorylation of p38 protein (a substrate specific for the signal transmitted through TNF- α Receptor). Both small molecule antagonist and agonist to TNF-α activity were identified. Total of 67 compounds were identified as antagonists of TNF-α activity (TNF-α-induced IL- 8 production and p38 phosphorylation), and 32 compounds were identified as agonists of TNF-α activity.

[0109] In summary, the primary screen (peptide displacement/binding assay) had a hit rate of 0.47% with 229 compounds showing >70% competition/inhibition.. A first secondary screen (antagonists of TNF-a induced IL-8 production and p38 phosphorylation) had a hit rate of 0.14% (67 compounds). A second secondary screen (enhancers of TNF-a induced IL-8 production and p38 phosphorylation) had a hit rate of 0.07% (32 compounds). [0110] Data representative of compounds with antagonist and agonist activity is presented in Figures 4 and 5.

[0111] Figure 4 shows the effect of two compounds (an antagonist and an agonist) on TNF-α-induced IL-8 production in Normal Human Dermal Fibroblasts (NHDF) cells. Briefly, NHDF cells were seeded to a density of 10⁴ cells/well in a 96-well plate. After starvation overnight in media without serum, cells were washed and compounds of interest were added. After 30 min of incubation, 300 pmols of TNF-α was added and incubation continued for 4 hours at 37°C, 5% CO₂. Plates were spun down and supernatants are transferred to a fresh plate for IL-8 ELISA assay. Cell proliferation solution was added to the pelleted cells in order to determine number of viable cells in each individual well. The final reading of TNF-α induction was adjusted for the number of viable cells. [0112] The amount of TNF-α used for IL-8 induction corresponds to its EC₅₀ value, i.e. 300 pg/ml of TNF-α induces about 550 pg/ml of IL-8 (no TNF-α addition induces about 20 pg/ml of IL-8, maximal induction corresponds to 1200 pg ml of IL-8). The presence of ^■ small molecule agonist or antagonist (in the absence of TNF-α) does not induce production of IL-8. The presence of a small molecule antagonist in the presence of TNF-α (300 pg ml) showed a dose-responsive decrease in IL-8 production; t.e. 5 μM compound reduced IL-8 production by ~ 70%. The presence of small molecule agonist in the presence of TNF-α (300 pg/ml) showed a dose-responsive increase in IL-8 production; i.e. 5 μM compound reduced IL-8 production by -90%.

[0113] Figure 5 shows the effect of various compounds on TNF-RI downstream substrate phosphorylation (p38 protein) in Normal Human Dermal Fibroblasts (NHDF) cells. Briefly, NHDF cells were seeded to a density of 2xl0⁵ cells/well in a 6-well plate. After starvation overnight in media without serum, cells were washed and compounds of interest were added. After 30 min of incubation, TNF-α at 6 ng/ml was added and incubation continued for 30 min at 37 C, 5% CO₂. Media was aspirated and cells were lysed on ice. Lysates were run on SDS-PAGE, transferred to PVDF membranes and incubated with anti-phospho-p38 antibody. In Figure 5, the upper panel shows the antagonistic activity of different small molecules. Compounds B, D, E, F at a 3 μM concentration, and in the presence of 6 ng/ml of TNF-α, inhibited TNF-α-induced substrate phosphorylation. Compound A shows partial antagonistic activity, whereas compound C showed no antagonistic activity (control compound). [0114] The lower panel of Figure 5 demonstrates compounds with agonistic activity to TNF-α. In the presence of 6 ng/ml of TNF-α, compounds H, I, J and K increased TNF-α- induced substrate (p38) phosphorylation. Control compound G showed no change on TNF-α induced substrate phosphorylation.

[0115] All publications, patents, patent applications, and accession numbers (including both polynucleotide and polypeptide sequences and corresponding annotations as of the filing and/or priority application filing dates) cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application were specifically and individually indicated to be so incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

We Claim:

1. A method for modulating the activity of IL-IRI (IL-lR-alpha) by contacting the receptor with an exogenous compound that binds in the activation domain, wherein the activation domain comprises a sequence identical to, or substantially similar to, SEQ ID NO:2.

2. The method of claim 1 wherein receptor activity is decreased.

3. The method of claim 1 wherein the contacting takes place in vitro.

4. The method of claim 1 wherein the IL-IRI (IL-lR-alpha) is human.

5. The method of claim 1 wherein the contacting occurs in the presence of IL-IRI (IL- lR-alpha).

6. The method of claim 1 wherein the exogenous compound is an oligopeptide.

7. The method of claim 6 wherein the oligopeptide comprises at least about 8 residues of a sequence substantially similar to, or identical to, SEQ ID NO:2.

8. A method for screening candidate agents for the ability to modulate activity of a TJ - 1RI (IL-lR-alpha) by determining the binding of the agent to a receptor activation domain sequence having a sequence of SEQ ID NO:2

9. A method for screening candidate agents for the ability to modulate activity of an IL- IRI (IL-lR-alpha) having an activation domain comprising a sequence substantially similar to SEQ ID NO:2 by a) contacting the receptor with a candidate agent and an activation-domain binding oligopeptide comprising a sequence substantially similar to SEQ ID NO:2; and b) determining the binding of the agent or the oligopeptide to the activation domain, wherein the binding of the agent to the activation domain or a reduction in the binding of the oligopeptide to the activation domain identifies a candidate agent for modulating cell- surface receptor activity.

10. The method of claim 9 comprising the step of contacting the receptor with receptor ligand.

11. The method of claim 9 or 10 wherein the candidate agent or oligopeptide is labeled.

12. A method for screening candidate agents for the ability to modulate activity of an IL- IRI (IL-lR-alpha) by identifying a compound that binds in a receptor activation domain of sequence SEQ ID NO:2, and assaying the ability of the compound to modulate activity of the cell surface receptor.

13. The method of claim 12 wherein the ability of the agent to reduce ligand-induced receptor activation is assayed.

14. An isolated oligopeptide that binds the IL-IRI (IL-lR-alpha) activation domain.

15. The oligopeptide of claim 14 wherein the oligopeptide comprises at least 8 arnino acids of SEQ ID NO:2.

16. An oligopeptide of claim 14 that antagonizes IL-1 induced activation of a IL-IRI (IL-lR-alpha).

17. A pharmaceutical composition comprising an oligopeptides of claims 15-16 in a sterile form and a pharmaceutically acceptable excipient.

18. A method for treating a condition in a patient characterized by an undesired level of IL-1 receptor activation by administering to a patient an exogenous bioactive compound that binds the IL-1 receptor activation domain and which decreases receptor activity when contacted with the receptor.

19. A method for treating a condition in a patient characterized by a deficiency of IL-1 by administering to a patient an exogenous bioactive compound that binds in the IL-1 receptor activation domain and which increases receptor activity.

20. A method for modulating the activity of TNF-RI (p55) by contacting the receptor with an exogenous compound that binds in the activation domain, wherein the activation domain comprises a sequence identical to, or substantially similar to, SEQ ID NO:l.

21. The method of claim 20 wherein receptor activity is decreased.

22. The method of claim 20 wherein the contacting takes place in vitro.

23. The method of claim 20 wherein the TNF-RI (p55) is human.

24. The method of claim 21 wherein the contacting occurs in the presence of TNF-alpha.

25. The method of claim 20 wherein the exogenous compound is an oligopeptide.

26. The method of claim 25 wherein the oligopeptide comprises at least about 8 residues of a sequence substantially similar to, or identical to, SEQ ID NO:l.

27. A method for screening candidate agents for the ability to modulate activity of a TNF-RI (p55) by determining the binding of the agent to a receptor activation domain sequence having a sequence of SEQ ID NO:l.

28. A method for screening candidate agents for the ability to modulate activity of a TNF-RI (p55) having an activation domain comprising a sequence substantially similar to SEQ ID NO:l by a) contacting the receptor with a candidate agent and an activation-domain binding oligopeptide comprising a sequence substantially similar to SEQ ID NO:l; and b) determining the binding of the agent or the oligopeptide to the activation domain, wherein the binding of the agent to the activation domain or a reduction in the binding of the oligopeptide to the activation domain identifies a candidate agent for modulating cell- surface receptor activity.

29. The method of claim 28 comprising the step of contacting the receptor with receptor ligand.

30. The method of claim 28 or 29 wherein the candidate agent or oligopeptide is labeled.

31. A method for screening candidate agents for the ability to modulate activity of a TNF-RI (p55) by identifying a compound that binds in a receptor activation domain having the sequence SEQ ID NO:l and assaying the ability of the compound to modulate activity of the cell surface receptor.

32. The method of claim 31 wherein the ability of the agent to reduce ligand-induced receptor activation is assayed.

33. An isolated oligopeptide that binds the TNF-RI (p55) activation domain.

34. The oligopeptide of claim 33 wherein the oligopeptide comprises at least 8 amino acids of SEQ ID NO:l.

35. The oligopeptide of claim 34 that antagonizes TNF-alpha induced activation of a TNF-RI (p55).

36. A pharmaceutical composition comprising an oligopeptides of claims 33 or 34 in a sterile form and a pharmaceutically acceptable excipient. .

37. A method for treating a condition in a patient characterized by an undesired level of TNF-RI (p55) activation by administering to a patient an exogenous bioactive compound that binds in the TΝF-alpha receptor activation domain and which decreases receptor activity when contacted with the receptor.

38. A method for treating a condition in a patient characterized by an undesired level of TΝF-RI (p55) activation by administering to a patient an exogenous bioactive compound that binds in the TΝF-RI (p55) activation domain and which increases receptor activity.