WO2008154008A1 - Methods and compositions for inhibiting toll-like receptor mediated immune responses - Google Patents

Abstract

The present invention provides compounds that are useful in inhibiting signaling activities mediated by Toll-like receptors. The invention also provides therapeutic methods for treating or preventing various diseases or disorders associated with or mediated by aberrant TLR signaling activities. Further provided in the invention are methods of screening for TLR antagonist compounds with improved properties.

Description

Methods and Compositions for Inhibiting Toll-like Receptor Mediated Immune Responses

STATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made in part by U.S. government support by the National Institutes of Health Grant No. GM066119. The U.S. Government therefore has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application Number 60/934,354 (filed June 11, 2007). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.

BACKGROUND OF THE INVENTION

[0003] In the innate immune system, Toll-like receptors (TLRs) function indispensably as sensors of microbial molecules. Upon recognition of microbial components, TLRs quickly engage with their adaptors and initiate innate immune signals by sequentially recruiting downstream signaling mediators. The TLR-mediated innate immune signals generally provide immediate host protection and also stimulate the host's adaptive immune response. At least 11 mammalian TLRs have been identified to date. All TLRs initiate cellular signaling through their cytoplasmic Toll/interleukin-1 receptor (TIR) domain, which triggers signaling pathways by TIR-TIR homophilic interaction with TIR domain containing TLR adaptors. Myeloid differentiation primary response protein 88 (MyD88) is the most common TLR adaptor. Others include MAL/TIRAP, TRAM and TRIF, but all TLRs utilize the MyD88 with the exception of TLR3 which uses only TRIF. TLR signaling consists of MyD88 dependent pathways and MyD88 independent pathways. MyD88 dependent pathways are shared by most TLRs, while MyD88 independent pathways are initiated by TLR3 and TLR4 and require TRIF.

[0004] Despite the fact that TLR-mediated innate immune signals are required to combat invading pathogens, when signaling is not well regulated, uncontrolled activation can result in disruption of host homeostasis leading to chronic inflammation and septic shock. For example, aberrant TLR4 signaling activities are implicated in a number of acute and chronic human diseases. TLR4 is stimulated by LPS, the major pro-inflammatory component of gram negative bacteria. LPS causes much of its morbidity and mortality by activating kinases that control the function of transcription factors (NFKB and AP-I) and ultimately lead to production of proinflammatory cytokines and costimulatory molecules. [0005] There is a need in the art for better means for treating and preventing diseases and medical conditions that are associated with or mediated by aberrant TLR signaling. The present invention is directed to this and other needs.

SUMMARY OF THE INVENTION

[0006] In one aspect, the invention provides methods for inhibiting or suppressing signaling activities of a Toll-like receptor (TLR). The methods involve administering to a subject suffering from undesired signaling activities of a TLR a pharmaceutical composition comprising a therapeutically effective amount of a TLR antagonist compound. The TLR antagonist compound employed in the methods is selected from the group consisting of N-(4- {2-[l-(4-fluorobenzyl)-4-pyridinium]vinyl}phenyl)-N-methylmethanamine iodide, methyl 2- { [ 1 -(4-fluorophenyl)-3-oxo-3-pyridin-3-ylpropyl]thio} benzoate, 4-(5-chloro-2, 1 - benzisoxazol-3-yl)-2-methoxyphenol, N-{4-[2-(l-hexylpyridinium-2-yl)vinyl]phenyl}-N- methylmethanamine iodide, and N-{4-[2-(l-hexylpyridinium-2-yl)vinyl]phenyl}-N- methylmethanamine iodide.

[0007] Some of these methods are directed to inhibiting or suppressing signaling activities of TLR4 and/or TLR2. In some methods, the subjects suffer from sepsis and undesired or aberrant TLR signaling activities associated with sepsis. For example, the subject can suffer from sepsis induced by a Gram-negative bacterial infection or by a Gram-positive bacterial infection. Some other methods are directed to treating subjects suffering from inflammatory disorders. For example, the methods can be directed to inhibiting abnormal TLR signaling activities associated with atherosclerosis. Some other methods of the invention are directed to treating subjects suffering from ischemia-reperfusion injuries, e.g., lung ischemia- reperfusion injury, liver ischemia-reperfusion injury, or brain ischemia-reperfusion injury. Methods are also provided to suppress or ameliorate aberrant TLR signaling associated with myocardial injuries such as myocardial infarction or cardiac ischemia-reperfusion injury. [0008] In a related aspect, the invention provides methods for inhibiting or ameliorating an undesired immune response. The methods entail administering a pharmaceutical composition comprising a therapeutically effective amount of a TLR antagonist compound to a subject suffering from an undesired immune response that is associated with or mediated by a Toll- like receptor. The TLR antagonist compound employed in these methods is selected from the group consisting of N-(4-{2-[l-(4-fluorobenzyl)-4-pyridinium]vinyl}phenyl)-N- methylmethanamine iodide, methyl 2-{[l-(4-fluorophenyl)-3-oxo-3-pyridin-3- ylpropyl]thio}benzoate, 4-(5-chloro-2, l-benzisoxazol-3-yl)-2-methoxyphenol, N-{4-[2-(l - hexylpyridinium-2-yl)vinyl]phenyl}-N-methylmethanarnine iodide, and N-{4-[2-(l- hexylpyridinium-2-yl)vinyl]phenyl}-N-methylmethanamine iodide. In some of these methods, the undesired immune response is mediated by TLR4 and/or TLR2. In some of these methods, the subject to be treated suffers from sepsis. Some other methods are intended to suppress undesired immune responses in subjects suffering from chronic inflammatory disorders such as atherosclerosis. Still some other methods are directed to inhibit undesired immune responses mediated by TLRs in subjects suffering from ischemia- reperfusion injuries or myocardial injuries (e.g., myocardial infarction). [0009] In another aspect, the invention provides methods for identifying TLR antagonist compounds with improved properties. Such methods involve first synthesizing one or more structural analogs of a lead TLR antagonist compound, and then performing a functional assay on the analogs to identify a compound that has an improved biological or pharmaceutical property relative to that of the lead compound. The lead TLR antagonist compound used in these methods is selected from the group consisting of N-(4-{2-[l-(4- fluorobenzyl)-4-pyridinium]vinyl}phenyl)-N-methylmethanamine iodide, methyl 2-{[l-(4- fluorophenyl)-3-oxo-3-pyridin-3-ylpropyl]thio}benzoate, 4-(5-chloro-2,l-benzisoxazol-3-yl)- 2-methoxyphenol, N-{4-[2-(l-hexylpyridinium-2-yl)vinyl]phenyl}-N-methylmethanamine iodide, and N-{4-[2-(l-hexylpyridinium-2-yl)vinyl]phenyl}-N-methylmethanamine iodide. Some of these methods are specifically directed to identify compounds with improved properties for inhibiting signaling activities of TLR4 or TLR2. Some of the methods are directed to identify TLR antagonists that are selective for TLR4 over TLR2. In some preferred embodiments, the methods employ a TLR antagonist compound that is N-{4-[2-(l- hexylpyridinium-2-yl)vinyl]phenyl} -N-methylmethanamine iodide.

[0010] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figures 1A-1C show the structures of five TLR antagonist compounds identified from a TLR4-MyD88 binding screening. The HeLa/CL3-4 cell line-based complementation assay as described in Example 1 below was screened against 16,000 compounds. These five compounds were selected from hits of the primary screening, and their TLR antagonist activities were confirmed in follow-up studies. The compound codes (Maybridge, Cornwall, England) are parenthesized.

[0012] Figures 2A-2B show inhibition of TLR4CD-MyD88 binding by the TLR antagonist compounds. (A) For the coimmunoprecipitation assay, HEK293T cells were transiently transfected with FLAG-TLR4CD and HA-MyD88 vectors (0.5 μg/ml each). After 24 h, cells were treated with the compounds at 10 μM for the indicated length of time (an equivalent amount of DMSO was used as the untreated control). Cells were then lysed and immunoprecipitated with anti-FLAG antibody. Western blotting was performed with both anti-FLAG (TLR4CD detection) and anti-HA (MyD88 detection) antibodies according to standard protocols. Input levels of MyD88 in the crude lysates were also analyzed using anti- HA antibody. The arrows indicated MyD88, TLR4 and IgG light chain (LC). (B) Percent binding of MyD88 to TLR4CD was analyzed using Quantity One software (Bio-Rad). [0013] Figure 3 A-3B show inhibition of LPS-mediated NF-κB induction in RAW264.7 cells. (A) For the gel-shift assay, cells were grown in 12-well tissue culture plates with DMEM/10% FCS for 24 h and pre-treated for 30 min with 0.37, 1.1, 3.3 or 10 μM inhibitory compound as indicated (DMSO was used as the untreated control). Cells were then stimulated with 0.1 μg/ml LPS for 1 h. Nuclear extracts were prepared and analyzed by gel- shift assay using an NF-κB oligonucleotide probe labeled with [γ-³²P]ATP. (B) Percent activation of NF-κB translocation was analyzed using Quantity One Software (Bio-Rad). [0014] Figures 4A-4C show inhibition of LPS-stimulated cell activation by TLR antagonist compounds. (A) Inhibition of LPS-mediated IL-8 promoter activity in HeLa cells. HeLa cells were transiently co-transfected with TLR4/CD14/MD-2 vectors (0.01 μg/ml each) as well as pIL8-promoter-Luc and pSV-β-galactosidase vectors (0.05 μg/ml each). After 24 h, cells were pre-incubated with increasing amounts of compounds for 30 min as indicated and stimulated with 0.1 μg/ml LPS for 6 h. The luciferase activity was then measured and all the luciferase activity was normalized with β-galactosidase activity. Results are shown as the mean ± S.D. *p = 0.0495 versus LPS alone (Mann- Whitney U test). Cont, LPS-negative vehicle control. (B, C) Inhibition of LPS-mediated inflammatory cytokine production in RAW264.7 cells. RAW264.7 cells were pre-incubated with different concentrations of compound for 60 min as indicated. Cells were washed once with DMEM/10% FCS and then stimulated with LPS (0.05 μg/ml) for 16 h. TNF-α (A) or IL-6 (B) in the culture supernatants were measured by ELISA. Results are shown as the mean ± S. D. Cont, LPS negative control. *p = 0.0495 versus LPS alone (Mann- Whitney U test). Note that the compound concentrations used are not the same for all compounds (B and C). Cont, LPS-negative vehicle control.

[0015] Figure 5 shows inhibition of MyD88-dependent NF-κB induction by TLR antagonist compounds. RAW264.7 cells were grown 12-well tissue culture plates with DMEM/10% FCS for 24 h and pre-treated with 10 μM inhibitory compounds 50-F12 (A) and 26-J10 (B) for 30 min. Cells were then stimulated with MALP-2 (50 ng/ml), pIpC (20 μg/ml), LPS (0.1 μg/ml), CpG (20 μg/ml), IL-I β (50 ng/ml), or TNF-α (50 μg/ml) for 1 h. Nuclear extracts were then prepared and gel-shift assay was performed using NF-κB oligonucleotide probe labeled with [γ-³²P]ATP.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

[0016] The present invention is predicated in part on the discovery by the present inventors of several antagonist compounds of Toll-like receptors (TLRs) which can specifically inhibit signaling activities mediated by TLR4 as well as TLR2. As detailed in the Examples below, the inventors developed a β-lactamase fragment complementation based high-throughput screening format to identify compounds which inhibit TLR4 signaling. Specifically, the screening assay employs a stable cell line, HeLa/CL3-4, which expresses MyD88/Bla(a) and TLR4/Bla(b) in which the two β-lactamase fragments complement with each other by virtue of spontaneous MyD88-TLR4 binding via their Toll/IL-IR (TIR) domains. Inhibition of the MyD88-TLR4 binding leads to the disruption of the enzyme complementation and a loss of the lactamase activity. A total of 45 primary hits were identified from screening a library of 16,000 test compounds in a 384-well plate assay format. After re-screening these 45 hits and eliminating compounds that directly inhibited β- lactamase, five TLR antagonist compounds were obtained. These TLR antagonist compounds act as inhibitors of TLR4-MyD88 binding and are effective in inhibiting LPS stimulated cytokine release from RAW264.7 cells. Importantly, none of the compounds showed any cytotoxicity at 20 μM. In addition, some of these compounds were also found to inhibit TLR2-mediated signal transduction.

[0017] In accordance with these discoveries, the present invention provides methods for inhibiting undesired or aberrant TLR signaling activities and for treating diseases and disorders induced by or associated with TLR signaling activities (especially TLR4 and/or TLR2). Examples of such diseases include sepsis, inflammatory or autoimmune disorders such as atherosclerosis and lupus, and ischemia-reperfusion injuries and myocardial dysfunctions. Subjects suitable for treatment with methods of the invention include ones who have or are at risk of developing any of these diseases.

[0018] The invention also provides methods for identifying novel compounds with improved properties in suppressing aberrant TLR signaling activities and treating diseases mediated by abnormal TLR signaling. Typically, these methods entail synthesizing analogs or derivative compounds of one of the TLR antagonist compounds disclosed herein, and then screening for the analog or derivative compounds for improved properties. The following sections provide more detailed guidance for practicing the invention.

II. Definitions

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1^st ed., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3^rd ed., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1^st ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer- Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4^th ed., 2000). In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0020] The term "agent" includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms "agent", "substance", and "compound" are used interchangeably herein.

[0021] The term "analog" or "derivative" is used herein to refer to a molecule that structurally resembles a reference molecule but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screening of analogs to identify variants of known compounds having improved traits (such as higher binding affinity for a target molecule) is an approach that is well known in pharmaceutical chemistry.

[0022] Administration "in conjunction with" one or more other therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. [0023] "Autoimmune disease" refers to a disease caused by an inability of the immune system to distinguish foreign molecules from self molecules, and a loss of immunological tolerance to self antigens, which results in destruction of the self molecules. Examples of autoimmune diseases include but are not limited to systemic lupus erythematosus, Sjogren's syndrome, scleroderma, ulcerative colitis, insulin-dependent diabetes mellitus (IDDM), multiple sclerosis, and rheumatoid arthritis.

[0024] "Autoantigen" refers to a self-antigen normally found within a mammal and normally recognized as self, but due to an auto-immune disease, is erroneously recognized as foreign by the mammal. That is, an autoantigen is not recognized as part of the mammal itself by the lymphocytes or antibodies of that mammal and is erroneously attacked by the immunoregulatory system of the mammal as though such autoantigen were a foreign substance. An autoantigen according to the invention also includes an epitope or a combination of epitopes derived from that autoantigen.

[0025] The term "contacting" has its normal meaning and refers to combining two or more agents (e.g., polypeptides or small molecule compounds) or combining agents and cells. Contacting can occur in vitro, e.g., combining two or more agents or combining an agent and a cell or a cell lysate in a test tube or other container. Contacting can also occur in a cell or in situ, e.g., contacting two polypeptides in a cell by coexpression in the cell of recombinant polynucleotides encoding the two polypeptides, or in a cell lysate. Contacting can also occur inside the body of a subject, e.g., by administering to the subject an agent which then interacts with the intended target (e.g., a tissue or a cell).

[0026] "Inflammation" or "inflammatory response" refers to an innate immune response that occurs when tissues are injured by bacteria, trauma, toxins, heat, or any other cause. The damaged tissue releases compounds including histamine, bradykinin, and serotonin. Inflammation refers to both acute responses (i.e., responses in which the inflammatory processes are active) and chronic responses (i.e., responses marked by slow progression and formation of new connective tissue). Acute and chronic inflammation can be distinguished by the cell types involved. Acute inflammation often involves polymorphonuclear neutrophils; whereas chronic inflammation is normally characterized by a lymphohistiocytic and/or granulomatous response. Inflammation includes reactions of both the specific and non-specific defense systems. A specific defense system reaction is a specific immune system reaction response to an antigen (possibly including an autoantigen). A non-specific defense system reaction is an inflammatory response mediated by leukocytes incapable of immunological memory. Such cells include granulocytes, macrophages, neutrophils and eosinophils.

[0027] "Myocardial injury" means injury to the muscular tissue of the heart. It may be either an acute or nonacute injury in terms of clinical pathology. In any case it involves damage to cardiac tissue and typically results in a structural or compensatory response. Unless otherwise noted, myocardial injury as used herein primarily refers to acute myocardial injury such as acute myocardial infarction (heart attack) and cardiac ischemia/reperfusion. [0028] Acute myocardial infarction (AMI or MI), commonly known as a heart attack, is a disease state that occurs when the blood supply to a part of the heart is interrupted. The resulting ischemia or oxygen shortage causes damage and potential death of heart tissue. [0029] Ischemia is a restriction in blood supply, generally due to factors in the blood vessels, with resultant damage or dysfunction of tissue (e.g., tissues of the heart, lung, and liver). Reperfusion injury refers to damage to tissue caused when blood supply returns to the tissue after a period of ischemia. The absence of oxygen and nutrients from blood creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than restoration of normal function. [0030] Signaling activities of Toll-like receptors (TLRs) or TLR-mediated signaling pathways refer to biological activities and cellular responses induced by the interaction of one or more of TLRs (e.g., TLR4 and/or TLR2) with their cognate ligands (e.g., microbial molecules), and biochemical reactions associated with the cascade of signaling events initiated by the receptor/1 igand interaction. These activities encompass interaction of TLRs with their adaptors, e.g., binding to MyD88 via their cytoplasmic Toll/interleukin-1 receptor (TIR) domains. TLR signaling activities also include activities associated with recruitment of downstream mediators, innate immune response thereafter induced which provides immediate host protection, and stimulation of the host's adaptive immune response (e.g., via activation and maturation of APCs together with production of inflammatory cytokines). For example, TLR signaling activities encompass activation of NFKB and stimulation of costimulatory molecule (CD 14) expression in macrophages. TLR signaling activities are well known and characterized in the art. See, e.g., O'Neill et al., Curr. Opin. Immunol. 18:3-9, 2006; Kawai et al., Cell Death Differ. 13:816-25, 2006; Moynagh, Trends Immunol. 26:469- 76, 2005; and Hoebe et al., Nat. Immunol. 5, 971-974, 2004.

[0031] Stroke is the clinical designation for a rapidly developing loss of brain function due to an interruption in the blood supply to all or part of the brain. Interruption in the blood supply results in depletion of oxygen and glucose in the affected area. This immediately reduces or abolishes neuronal function, and also initiates an ischemic cascade which causes neurons to die or be seriously damaged, further impairing brain function. Stroke can be caused by, e.g., thrombosis, embolism, or hemorrhage.

[0032] The term "subject" for purposes of treatment refers to any animal classified as a mammal, e.g., human and non-human mammals. Examples of non-human animals include dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and etc. Unless otherwise noted, the terms "patient" or "subject" are used herein interchangeably. Preferably, the subject is human.

[0033] TLR mediated immune responses refer to immune responses elicited in a host via recognition by one or more of TLRs (e.g., TLR4 and/or TLR2) of pathogen-associated molecular patterns as well as various endogenous ligands. In addition to innate immunity induced by TLR recognition of a cognate ligand, TLR mediated immune responses also refer to stimulation of adaptive immune reactions as a result of TLR-mediated signaling activities (e.g., cytokine productions and antigen presentation). More detailed description of TLR mediated immune responses is provided in the literature. See, e.g., Trinchieri et al., Nat Rev Immunol. 7:179-90, 2007; and Romagne, Drug Discov. Today 12:80-7, 2007. [0034] The term "treating" or "alleviating" includes the administration of compounds or agents to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease (e.g., an inflammatory disorder or a myocardial dysfunction), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Subjects in need of treatment include those already suffering from the disease or disorder as well as those being at risk of developing the disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease. In the treatment of a disease or disorder associated with or mediated by TLR signaling, a therapeutic agent may directly decrease the pathology of the disease, or render the disease more susceptible to treatment by other therapeutic agents.

III. TLR antagonist compounds

[0035] The invention provides TLR antagonist compounds which can be employed in treating or preventing diseases or medical conditions that are induced by or associated with undesired or abnormal TLR signaling activities or TLR mediated immune responses (e.g., TLR4 or TLR2 signaling pathways). Such diseases or conditions include any abnormal medical conditions or disorders in the development of which TLR signaling activities play a role, e.g., diseases associated with bacterial or viral infections (e.g., sepsis), ischemia- reperfusion injuries (e.g., to the lung, liver and brain), inflammatory disorders including atherosclerosis and autoimmune diseases (e.g., abnormal-immune responses to autoantigens), and myocardial injuries (such as cardiac ischemia-reperfusion injury and myocardial infarction).

[0036] As described in the Examples below, the present inventors identified several compounds which interfere with the binding between MyD88 and TLR4. These compounds are capable of inhibiting TLR4 signaling as measured by cytokine productions in cells stimulated with LPS. In addition, these compounds do not show significant cytotoxicity as cell viability is not affected by the presence of the compounds at a concentration as high as 20 μM. Five exemplary TLR antagonist compounds identified in these studies are N-(4-{2- [l-(4-fluorobenzyl)-4-pyridinium]vinyl}phenyl)-N-methylmethanamine iodide (Compound "50-F12"), methyl 2-{[l-(4-fluorophenyl)-3-oxo-3-pyridin-3-ylpropyl]thio}benzoate (Compound "2-G5"), 4-(5-chloro-2,l-benzisoxazol-3-yl)-2-methoxyphenol (Compound "32-J10"), N-{4-[2-(l-hexylpyridinium-2-yl)vinyl]phenyl}-N-methylmethanamine iodide (Compound "26-J10"), and N-{4-[2-(l-hexylpyridinium-2-yl)vinyl]phenyl}-N- methylmethanamine iodide (Compound "27-N15"). Their structures are shown in Fig. 1. These compounds can be readily obtained from commercial suppliers (e.g., Maybridge, Cornwall, England) or de novo synthesized using routinely practiced methods of organic chemistry.

[0037] Among the five compounds shown in Fig. 1, two compounds, 50-Fl 2 and 26-J10, exhibit the most consistent inhibitory effects in various assays. 50-Fl 2 and 26-J10 showed dose-dependent inhibition of TNF-α and IL-6 production in RAW264.7 cells activated by LPS, among which 26-Jl 0 exhibited stronger inhibition (see Figs. 4B and 4C). These compounds also inhibit TLR2-mediated signal transduction (see Fig. 5). Therefore, the inhibitory activities of these compounds appear to be MyD88-dependent and not TLR4- dependent. MyD88 is a universal adaptor that transmits signals initiated by TLR/IL-1R family members with the exception of TLR3. It has been shown that three different point mutations in MyD88 generated by ethyl nitroso-urea-mediated mouse germ line mutagenesis differentially affected signaling among TLRs, suggesting that the interactions between MyD88 and TLRs are specific to each TLR. The apparent selectivity of the TLR antagonist compounds for TLR2 and TLR4 signaling could be correlated with the adaptor usage of these two receptors. It is known that, a coadaptor, TIRAP/Mal, is known to be essential in the MyD88-dependent signaling pathway shared by TLR2 and TLR4, but not by TLR3, TLR5, TLR7, TLR9, or IL-IR.

IV. Screening for novel TLR antagonist compounds with improved properties [0038] Employing the TLR antagonists described above, the invention provides methods of screening for novel TLR antagonist compounds. These methods are directed to identifying analogs or derivatives of the above-described TLR antagonists with improved properties. An important step in the drug discovery process is the selection of a suitable lead chemical template upon which to base a chemistry analog program. The process of identifying a lead chemical template for a given molecular target typically involves screening a large number of compounds (often more than 100,000) in a functional assay, selecting a subset based on some arbitrary activity threshold for testing in a secondary assay to confirm activity, and then assessing the remaining active compounds for suitability of chemical elaboration. [0039] The TLR antagonists described herein, e.g., the compounds shown in Fig. 1 , provide lead compounds to search for related compounds that have improved biological or pharmaceutical properties. The screening methods of the invention typically involve synthesizing analogs, derivatives or variants of a TLR antagonist (e.g., one shown in Fig. 1). Often, a library of structural analogs of a given TLR antagonist is prepared for the screening. Structures and chemical properties of the TLR antagonists shown in Fig. 1 are all well known and characterized in the art. To synthesize analogs or derivatives based from the chemical backbones of these TLR antagonist compounds, only routinely practiced methods of organic chemistry synthesis are required. For example, combinatorial libraries of chemical analogs of a known compound can be produced by the encoded synthetic libraries (ESL) method as described in WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503 and WO 95/30642. Exemplary methods for synthesizing analogs of various compounds are also described in, e.g., by Overman, Organic Reactions, Volumes 1-62, Wiley-Interscience (2003); Broom et al., Fed Proc. 45: 2779-83, 1986; Ben-Menahem et al., Recent Prog Horm Res. 54:271-88, 1999; Schramm et al., Annu. Rev. Biochem. 67: 693-720, 1998; Bolin et al., Biopolymers 37: 57-66, 1995; Karten et al., Endocr Rev. 7: 44-66, 1986; Ho et al., Tactics of Organic Synthesis, Wiley-Interscience; (1994); and Scheit et al., Nucleotide Analogs: Synthesis and Biological Function, John Wiley & Sons (1980).

[0040] Once a library of candidate structural analogs of a lead TLR antagonist compounds are synthesized, a functional assay is then performed to identify one or of the analogs or derivatives that have an improved biological property relative to that of the TLR antagonist from which the analogs or variants are derived. In some embodiments, a function assay can be performed by contacting a candidate compound with a TLR-expressing cell (e.g., a cell line transiently expressing a TLR as exemplified in the Examples below). This is followed by examining any alteration of a TLR-mediated signaling activity (e.g., NFKB activation) in the cell relative to the signaling activity of the cell in the absence of the candidate compound. The assay can additional include comparing the TLR-mediated signaling activity of the cell in the presence of the candidate compound to the signaling activity of the cell in the presence of a positive or negative control compound. [0041] In some embodiments, relative to the lead compound, structural analog compounds derived from one of these lead TLR antagonist compounds can be screened to identify compounds that have a higher affinity for Myd88 and/or a TLR (e.g., TLR4 or TLR2). The structural analog compounds can also be screened for compounds with stronger ability to inhibit (e.g., lower IC50) the signaling activities of a TLR. For example, structural analogs of a TLR antagonist compound disclosed herein can be screened for enhanced activity in inhibiting TLR4 signaling activities. Examples of TLR4 signaling activities that can be monitored in the screening include, TLR4 binding to MyD88, TLR4 mediated NFKB nuclear translocation, and cytokine production in response to LPS signaling (e.g., IL-6, IL-8 and TNF-α). These activities can be assayed with methods well known in the art, e.g., assays described in the Examples below.

[0042] In some other embodiments, the structural analog compounds can be screened for better selectivity in inhibiting TLR4 signaling relative to any effect on signaling activities mediated by the other Toll-like receptors. As some of the TLR antagonist compounds disclosed herein also inhibit TLR2 mediated signaling activities, candidate compounds can be screened for either mono selectivity (for TLR4 or TLR2 alone) or dual selectivity (for both TLR4 and TLR2). Some compounds identified from the screening can be TLR4 specific antagonists that do not have significant effect on TLR2 signaling at the same concentration. Typically, a selective antagonist compound for a TLR (e.g., TLR4) inhibits signaling activities of the TLR with an IC₅₀ or EC₅₀ that is at least 5 fold, 10 fold, or 50 fold lower than its IC₅₀ for one or more of the other TLRs (e.g., TLR2, TLR3, TLR7 and TLR9). For example, the selective antagonist can inhibit signaling activities of a TLR (e.g., TLR4) with IC₅₀ or EC₅₀ that is lower than 1 μM, preferably lower than 500 nM, 250 nM, 100 nM, and most preferably lower than 50 nM. On the other hand, the compound inhibits signaling activities of one or more of the other TLR receptors (e.g., TLR2, TLR3, TLR7 and TLR9) with an IC₅₀ or EC₅₀ that is at least 5 μM or higher. As demonstrated in the Examples below, selectivity for different TLRs can be assayed by measuring inhibitory activity of a candidate compound on NFKB induction in cells stimulated with cognate ligands for the different TLRs. [0043] In still some other embodiments, the structural analog compounds can be screened for improved pharmacokinetic properties, e.g., in vivo half life. Compounds with such improved properties can be more suitable for various therapeutic applications. Improved pharmaceutical properties of a TLR antagonist analog can be assayed using methods such as those described in, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20^th ed., 2000.

V. Therapeutic applications of TLR antagonist compounds

[0044] TLRs (e.g., TLR4 and TLR2) are expressed on a large number of immune cells as well as epithelial cells and play an essential role in the activation of the innate immune response to microbial pathogens. They impact on adaptive immune reactions and contribute to the initiation and maintenance of the inflammatory response to a multitude of potential microbial pathogens through recognition of pathogen-associated molecular patterns. TLRs also interact with a variety of endogenous human ligands and influence the activity of a wide range of tissues and cell processes. Undesired TLR signaling activities or TLR mediated immune responses are present in various diseases and disorders. Examples include diseases arising from immune response to bacterial or viral infections (e.g., sepsis), ischemia- reperfusion injuries, asthma, acute respiratory distress syndrome, myocardial injuries (e.g., cardiac ischemia), coronary artery diseases (e.g., atherosclerosis), inflammatory disorders (e.g., asthma, inflammatory bowel disease, rheumatoid arthritis, or psoriasis), systemic autoimmune diseases (e.g., systemic lupus erythematosus (SLE), scleroderma and Sjogren's syndrome), ventricular remodeling, vascular collapse, inflammatory bowel disease, acute tubular necrosis, psoriasis, rheumatoid arthritis, pre-term birth, fertility, cancer angiogenesis and transplant rejection.

[0045] The TLR antagonist compounds disclosed in the invention can be employed to inhibit, suppress or ameliorate undesired TLR signaling activities or TLR mediated immune responses in subjects suffering from any of the above noted diseases or disorders. As demonstrated in the Examples below, these compounds are capable of inhibiting NFKB activation through TLR4 or TLR2 signaling pathways. Thus, some embodiments of the invention are directed to inhibiting or suppressing undesired or abnormal signaling activities or immune responses mediated by TLR4 or TLR2. In order to better inhibit or suppress an undesired TLR signaling activity or TLR mediated immune response, these TLR antagonist compounds can be used in conjunction with other known immunosuppressants, e.g., glucocorticoids, azathioprine, cyclosporine, and tacrolimus.

[0046] In some embodiments, the invention provides methods for inhibiting or suppressing undesired TLR signaling activities or immune responses in subjects suffering from acute diseases induced or mediated by infections, e.g., sepsis. Sepsis is a medical condition resulting from the immune response to a severe bacterial infection. Septicaemia is sepsis of the bloodstream caused by bacteremia, which is the presence of bacteria in the bloodstream. The term septicaemia is also used to refer to sepsis in general. Symptoms of sepsis are often related to the underlying infectious process. When the infection crosses into sepsis, the resulting symptoms are tachycardia, tachypnea, fever and/or decreased urination. The immunological response that causes sepsis is a systemic inflammatory response causing widespread activation of inflammation and coagulation pathways. This may progress to dysfunction of the circulatory system and, even under optimal treatment, may result in the multiple organ dysfunction syndrome and eventually death. [0047] The involvement of TLRs and cytokines in sepsis has been well documented. TLRs initiate the inflammatory processes that underlie the clinical response to infections in sepsis and septic shock. In Gram negative bacterial infection, activation of blood monocytes and tissue macrophages occurs through LPS mediated TLR4 receptor signaling (Andonegui et al., J Clin. Invest. 111 : 101 1-1020, 2003; and Tsujimoto et al., Shock 23:39-44, 2005). TLR2 also responds to endotoxin and activates NF-κB (Yang et al., J Immunol 163: 639-643, 1999). It was shown that TLR4 plays a key role for regulating the expression of relevant cytokines within the lung during endotoxic shock (Baumgarten et al., Eur J Anaesthesiol. 23:1041-8, 2006). The TLR4 mediated signaling pathway is known to be critical for the induction of IL- 1, IL-8, and IL-18 that results in the activation of NF-κB and mitogen activated protein (MAP) kinase. Gram positive bacteria may also induce a septic response by a number of cellular components including peptidoglycans, teichoic acids, exotoxins and superantigens. It had been previously thought that Gram positive PAMP molecules were recognized solely by TLR2 and TLR6. However, later studies have revealed that not only is TLR4 a necessary receptor for the innate immune response to LPS, but also critical in the response to staphylococcal enterotoxin B (Calkins et al., J Surg Res 104: 124-130, 2002). Therefore, the TLR antagonist compounds disclosed herein can be employed to inhibit or suppress undesired TLR signaling activities (e.g., TLR4 and/or TLR2 signaling) in subjects with either Gram negative or Gram positive sepsis.

[0048] A subject is considered to have sepsis if infection is highly suspected or proven and two or more of the following systemic inflammatory response syndrome (SIRS) criteria are met: Heart rate > 90 beats per minute; Body temperature < 36 (96.8°F) or > 38°C (100.4⁰F); Hyperventilation (high respiratory rate) > 20 breaths per minute or, on blood gas, a P₃CO₂ less than 32 mm Hg; and White blood cell count < 4000 cells/mm³ or > 12000 cells/mm³ (< 4 x 10⁹ or > 12 x 10⁹ cells/L), or greater than 10% band forms (immature white blood cells). The more critical subsets of sepsis are severe sepsis (sepsis with acute organ dysfunction) and septic shock (sepsis with refractory arterial hypotension). Alternatively, when two or more of the systemic inflammatory response syndrome criteria are met without evidence of infection, patients may be diagnosed simply with "SIRS." Patients with SIRS and acute organ dysfunction may be termed "severe SIRS." Subjects are defined as having "severe sepsis" if they have sepsis plus signs of systemic hypoperfusion; either end organ dysfunction or a serum lactate greater then 4 mmol/dL. Patients are defined as having septic shock if they have sepsis plus hypotension after an appropriate fluid bolus (typically 20 ml/kg of crystaloid). [0049] Conventional treatment of sepsis rests on antibiotics, surgical drainage of infected fluid collections, fluid replacement and appropriate support for organ dysfunction. This may include hemodialysis in kidney failure, mechanical ventilation in pulmonary dysfunction, transfusion of blood products, and drug and fluid therapy for circulatory failure. Ensuring adequate nutrition, if necessary by parenteral nutrition, is important during prolonged illness. The TLR antagonist compounds disclosed herein can be used alone to treat subjects who have been diagnosed to have sepsis as well as subjects who are at the risk of developing sepsis. Alternatively, these compounds can be employed in conjunction with a conventional treatment regimen, e.g., antibiotics.

[0050] In some other embodiments, the invention provides methods for inhibiting or suppressing undesired TLR signaling activities or immune responses that are associated with chronic disorders in which TLR signaling (esp. TLR4 and/or TLR2 signaling) play a role. Examples of such chronic disorders include chronic inflammatory disorders such as atherosclerosis and autoimmune diseases such as lupus. For example, atherosclerosis is a chronic inflammatory disorder affecting the arterial blood vessel. It is a chronic inflammatory response in the walls of arteries, in large part to the deposition of lipoproteins (plasma proteins that carry cholesterol and triglycerides). It is commonly referred to as a "hardening" or "furring" of the arteries. It is caused by the formation of multiple plaques within the arteries. Atherosclerosis causes two main problems. First, the atheromatous plaques, though long compensated for by artery enlargement, eventually lead to plaque ruptures and stenosis (narrowing) of the artery and, therefore, an insufficient blood supply to the organ it feeds. Alternatively, if the compensating artery enlargement process is excessive, then a net aneurysm results. The complications of atherosclerosis are chronic, slowly progressing and cumulative.

[0051] There is abundant evidence of the role played by TLR signaling in the development and progression of atherosclerosis. First, signaling by TLR4 and MyD88 has been implicated in the pathogenesis of atherosclerosis. It was shown that lack of TLR4 or MyD88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein E (Michelsen et al., Proc Natl Acad Sci U S A. 101 : 10679-84, 2004). In addition, modulation of atherosclerosis in mice by TLR2 has also been reported (Mullick et al., J Clin. Invest. 115:3149-56, 2005). It was found that in atherosclerosis-susceptible low-density lipoprotein receptor-deficient (LdIr-/-) mice, complete deficiency of TLR2 led to a reduction in atherosclerosis. [0052] Lupus represents a number of different pathophysiological and clinical syndromes that share autoimmunity against particles of self DNA. Involvement of TLRs, especially TLR3, TLR7, TLR8, and TLR9, in the pathogenesis of autoimmune diseases such as lupus has also been documented. In contrast to other TLRs, which are primarily localized at the outer cell membrane, this subgroup of TLRs is localized intracellularly and recognize nucleic acid motifs or their synthetic analogs (TLR3 and TLR7/8) or hypomethylated bacterial CpG- DNA (TLR9). Macromolecular complexes of RNA or DNA associated with proteins are well known targets of autoimmunity in systemic autoimmune diseases, e.g., mixed connective tissue disease, systemic lupus erythematosus, Sjogren's syndrome, and scleroderma. It was shown that inhibition of TLR7 or TLR7 plus TLR9 attenuates glomerulonephritis and lung injury in experimental lupus (Pawar et al., J. Am. Soc. Nephrol. , 2007). Also, TLR9 activation was shown to have a pathogenetic role in tubulointerstitial inflammation and damage in experimental and human lupus nephritis (Benigni et al., Arthritis Rheum. 56:1569- 78, 2007). These studies provide support that the therapeutic activities of the TLR antagonist compounds can be exploited in inhibiting or suppressing undesired TLR signaling in subjects with chronic inflammatory disorders such as atherosclerosis and systemic autoimmune diseases such as lupus.

[0053] In some other embodiments, the invention provides therapeutic methods for inhibiting or ameliorating aberrant TLR signaling activities or undesired immune responses in subjects suffering from ischemia-reperfusion injuries (e.g., injuries to the heart, lung, liver, brain and other organs), and cardiac dysfunctions such as myocardial infarction and heart failure. The involvement of TLR signaling in ischemia-reperfusion injuries is well established. For example, TLR4 signaling pathway has been shown to be associated with ischemia-reperfusion injuries in the lung (Shimamoto et al., Ann Thorac Surg. 82:2017-23, 2006), ischemia-reperfusion injuries in the liver (Wu et al., Hepatobiliary Pancreat. Dis. Int. 3:250-3, 2004), and ischemia injuries in the brain and stroke (Cao et al., Biochem. Biophys. Res. Commun. 353:509-14, 2007; and Caso et al., Circulation 115:1599-608, 2007). TLR signaling pathways (e.g., TLR4 and TLR2) are also involved in cardiac ischemia-reperfusion injury (Staple et al., Eur J Heart Fail. 8:665-72, 2006; and Sakata et al., Am J Physiol. Heart Circ. Physiol. 292:H503-9, 2007).

[0054] There are additional evidence supporting that the TLR antagonist compounds of the invention are useful in treating ischemia-reperfusion injuries and other cardiac dysfunctions. It was demonstrated that targeting or down-regulating TLR4 or TLR2 can be beneficial in minimizing ischemic-reperfusion-induced tissue damage and organ dysfunction (see, e.g., Li et al., Cardiovasc. Res. 61 :538-47, 2004; and Zhang et al., World J. Gastroenterol. 11 :4423-6, 2005). Reduced myocardial ischemia-reperfusion injury was observed in TLR4-deficient mice (see, e.g., Chong et al., J. Thorac. Cardiovasc. Surg. 128: 170-9, 2004; and Oyama et al., Circulation. 109:784-9, 2004). In rats, modulating TLR4 signaling with beta-D-glucan rapidly induces cardioprotection, e.g., reduced infarct size. It was shown the induced cardioprotection involve decreased association of TLR4 with MyD88, inhibition of I/R induced IRAK and IKKβ activity and decreased NFKB activity (Li et al., Cardiovasc Res. 61 :538-47, 2004). It was also reported that activated TLR4 in monocytes is associated with heart failure after acute myocardial infarction in human patients (see, e.g., Satoh et al., Int. J. Cardiol. 109:226-34, 2006). Inhibition of TLR4 with a specific TLR antagonist, eritoran, has been shown to result in attenuation of myocardial ischemia-reperfusion injury in mice (see, e.g., Shimamoto et al., Circulation. 114:1270-4, 2006). All these evidence indicate that targeting TLR4 and/or TLR2 with the TLR antagonist compounds disclosed herein can be effective in treating or ameliorating inflammatory responses in myocardial injuries such as myocardial infarction and ischemia-reperfusion injuries.

[0055] Subjects having ischemic myocardial injuries such as myocardial infarction or cardiac ischemia/reperfusion can be identified by any of the classical symptoms of acute myocardial infarction. These include chest pain, shortness of breath, nausea, vomiting, palpitations, sweating, and anxiety or a feeling of impending doom. Some myocardial infarctions are silent, without chest pain or other symptoms. These cases can be diagnosed by medical procedures, e.g., electrocardiograms. Medical diagnosis of myocardial infarction can be made by integrating the history of the presenting illness and physical examination with electrocardiogram findings and cardiac markers (blood tests for heart muscle cell damage). A coronary angiogram allows visualization of narrowings or obstructions on the heart vessels, and therapeutic measures can follow immediately. A chest radiograph and routine blood tests may indicate complications or precipitating causes and are often performed on admittance to an emergency department. New regional wall motion abnormalities on an echocardiogram are also suggestive of a myocardial infarction and are sometimes performed in equivocal cases. Technetium and thallium can be used in nuclear medicine to visualize areas of reduced blood flow and tissue viability, respectively.

[0056] Prior to treatment with the TLR antagonist compounds disclosed herein, subjects having or suspected to be suffering from acute myocardial injury usually need to first get emergent care. These include oxygen, aspirin, glyceryl trinitrate and pain relief. The patient will receive a number of diagnostic tests, such as an electrocardiogram (ECG, EKG), a chest X-ray and blood tests to detect elevated creatine kinase or troponin levels (these are chemical markers released by damaged tissues, especially the myocardium). Further treatment may include either medications to break down blood clots that block the blood flow to the heart, or mechanically restoring the flow by dilatation or bypass surgery of the blocked coronary artery. Coronary care unit admission allows rapid and safe treatment of complications such as abnormal heart rhythms.

VI. Pharmaceutical compositions and methods of administration [0057] The TLR antagonist compounds (e.g., compounds shown in Fig. 1) and the other therapeutic agents disclosed herein can be administered directly to subjects in need of treatment. However, these therapeutic compounds are preferable administered to the subjects in pharmaceutical compositions which comprise the TLR antagonist and/or other active agents along with a pharmaceutically acceptable carrier, diluent or excipient in unit dosage form. Accordingly, the invention provides pharmaceutical compositions comprising one or more of the TLR antagonist compounds disclosed herein. The invention also provides a use of these TLR antagonists in the preparation of pharmaceutical compositions or medicaments for treating the above described diseases or medical disorders wherein undesired signaling activities or responses mediated by TLRs (e.g., TLR4 and/or TLR2) are present. [0058] Pharmaceutically acceptable carriers are agents which are not biologically or otherwise undesirable. These agents can be administered to a subject along with a TLR antagonist compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the pharmaceutical composition. The compositions can additionally contain other therapeutic agents that are suitable for treating or preventing aberrant TLR signaling activities or undesired immune response. Pharmaceutically carriers enhance or stabilize the composition or facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The pharmaceutically acceptable carrier employed should be suitable for various routes of administration described herein. Additional guidance for selecting appropriate pharmaceutically acceptable carriers is provided in the art, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20^th ed., 2000. [0059] A pharmaceutical composition containing a TLR antagonist compound described herein and/or other therapeutic agents can be administered by a variety of methods known in the art. The routes and/or modes of administration vary depending upon the desired results. Depending on the route of administration, the active therapeutic agent may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the agent. Conventional pharmaceutical practice may be employed to provide suitable formulations to administer such compositions to subjects. Any appropriate route of administration may be employed, for example, but not limited to, intravenous, parenteral, transcutaneous, subcutaneous, intramuscular, intracranial, intraorbital, intraventricular, intracapsular, intraspinal or oral administration. Depending on the specific conditions of the subject to be treated, either systemic or localized delivery of the therapeutic agents may be used in the treatment.

[0060] In some embodiments, local administration of TLR antagonists is desired in order to achieve the intended therapeutic effect. Many methods of localized delivery of therapeutic agents or formulations can be used in the practice of the invention. For example, local administration of a TLR antagonist to the desired cardiac muscle in a subject can be accomplished by a percutaneous route, by therapeutic cardiac catheterization, by intrapericardial injection or infusion, or by direct intracardiac muscle injection. Suitable methods also include any other routes which allow the therapeutic agent to be applied locally to the heart. For example, the therapeutic agent may be applied from the blood stream, by being placed directly in the heart through the coronary arteries or veins onto the heart surface, or through the ventricular or atrial walls and onto the heart surface. The therapeutic agent may also be applied through direct application during extensive surgical field exposure, or through direct application during minimally invasive exposure, e.g., through a pericardial window or heart port.

[0061] Pharmaceutical compositions of the invention can be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20^th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for molecules of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, e.g., polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

[0062] The TLR antagonists for use in the methods of the invention should be administered to a subject in an amount that is sufficient to achieve the desired therapeutic effect (e.g., eliminating or ameliorating symptoms associated with undesired immune responses) in a subject in need thereof. Typically, a therapeutically effective dose or efficacious dose of the TLR antagonist is employed in the pharmaceutical compositions of the invention. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, and the rate of excretion of the particular compound being employed. It also depends on the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, gender, weight, condition, general health and prior medical history of the subject being treated, and like factors. Methods for determining optimal dosages are described in the art, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20^th ed., 2000. Typically, a pharmaceutically effective dosage would be between about 0.001 and 100 mg/kg body weight of the subject to be treated.

[0063] The TLR antagonist compounds and other therapeutic regimens described herein are usually administered to the subjects on multiple occasions. Intervals between single dosages can be daily, weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the TLR antagonist compounds and the other therapeutic agents used in the subject. In some methods, dosage is adjusted to achieve a plasma compound concentration of 1-1000 μg/ml, and in some methods 25-300 μg/ml or 10-100 μg/ml. Alternatively, the therapeutic agents can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the TLR antagonist compound and the other drugs in the subject. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some subjects may continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the subject can be administered a prophylactic regime.

EXAMPLES

[0064] The following examples are provided to further illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims.

Example 1 Screening for TLR antagonist compounds by protein-protein complementation assay

[0065] A stable cell line expressing chimeras of TLR4 and MyD88 with fragments of β- lactamase was employed for identifying inhibitors of the TIR-TIR interactions by which TLR4 and MyD88 associate. HeLa cells were transfected with plasmids expressing chimeras as noted as well as antibiotic selectable markers. After several rounds of FACS selection for cells expressing the two complementing chimeras, HeLa/CL 3-4 was further characterized. The expression levels of the two proteins were explored by FACS analysis. As anticipated, MyD88/Bla(a) was expressed intracellular^ while TLR4/Bla(b) was expressed on the surface.

[0066] Before attempting to screen for inhibitory compounds, we first tested our HTS assay system using clavulanic acid. The β-lactamase inhibitor clavulanic acid was chosen to use as a model inhibitor during assay development because there was no drug known that inhibits TLR4-MyD88 binding. Assay testing included EC₅₀ value determination and plate uniformity assessment. Preliminary experiments suggested that 3750 cells/well with a 384 well plate gave a good, nearly confluent, mono layer of cells. Clavulanic acid inhibited the β-lactamase activity in HeLa/CL3-4 cells in a dose-dependent manner with an EC₅0 of 0.37 μM. The S/B ratio and the Z¹ value for this dose-response experiment were 3.9 and 0.68, respectively. Since we are not aware of a published value of the EC50 of clavulanic acid for β-lactamase, it is difficult to compare the EC₅₀ value in this study with others. However, the Minimum Inhibitory Concentrations (MICs) of clavulanic acid against various bacteria ranged from 0.1 to 512 mg/L (16). Although the assay systems are different, our EC50 of 0.37 μM (= 0.088 mg/L) is comparable with those MICs.

[0067] The plate uniformity assay was done with three different plate layouts. Each layout resulted in a constant pattern with reliable Z' values ranging from 0.67 to 0.83. Taken together, these data suggested that the proposed assay system could be used for library screening. For library screening, a total of 16,000 compounds in 50 plates were used for the primary screening. Each plate contained a clavulanic acid dose-response control, which was used to determine the hit cutoff for each screening plate. The primary screening yielded a total of 45 hits. These consist of 24 hits selected by each cutoff and additional 21 hits with borderline activities selected by the overall cutoff. These 45 hits were further tested by re- screening. A total of 10 compounds out of 45 were reproducibly positive hits. To detect which compounds were inhibitory because they directly inhibit β-lactamase we utilized a cell line expressing full length β-lactamase. Five of the tested compounds, as well as clavulanic acid, inhibited full length β-lactamase. These compounds were eliminated from further study although they might be useful as lactam antibiotic adjuvants as is clavulanic acid. The remaining 5 compounds were identified and confirmed (see below Examples) as TLR antagonist compounds. They are N-(4-{2-[l-(4-fluorobenzyl)-4-pyridinium]vinyl}phenyl)- N-methylmethanamine iodide (Compound "50-F12"), methyl 2-{[l-(4-fluorophenyl)-3-oxo- 3-pyridin-3-ylpropyl]thio}benzoate (Compound "2-G5"), 4-(5-chloro-2,l-benzisoxazol-3- yl)-2-methoxyphenol (Compound "32-J10"), N-{4-[2-(l-hexylpyridinium-2- yl)vinyl]phenyl}-N-methylmethanamine iodide (Compound "26-J10"), and N-{4-[2-(l- hexylpyridinium-2-yl)vinyl]phenyl}-N-methylmethanamine iodide (Compound "27-N15"). Structures of these 5 TLR antagonist compounds are shown in Fig. 1.

Example 2 Inhibition of MvD88 binding to TLR4 cytoplasmic domain (TLR4CD) by TLR antagonist compounds

[0068] To define whether the selected compounds inhibit TLR4-MyD88 binding, we first studied coimmunoprecipitation of TLR4CD and MyD88. TLR4CD consists of TLR4 transmembrane and cytoplasmic domains. This construct was previously made and shown to co-precipitate with MyD88. HEK293T cells transiently transfected with TLR4CD and MyD88 vectors for 24 h were incubated with the 5 TLR antagonist compounds for different lengths of time. Most compounds directly inhibited the TLR4CD-MyD88 binding, although compound 27-Nl 5 was the least effective. The time course of its effect suggests that it may be unstable, perhaps being metabolized. That the expression levels of TLR4CD and MyD88 (input) did not change before and after addition of the compounds, show that the compounds directly affect the binding between TLR4 and MyD88, but not protein expression.

Example 3 Inhibition of TLR signaling by TLR antagonist compounds [0069] We next investigated whether inhibition of TLR4-MyD88 binding by the compounds is correlated with TLR4 signaling. We first performed NF-κB nuclear translocation experiments using a [γ-³²P]-labeled NF-κB oligonucleotide probe. RAW264.7 cells pretreated with different amounts of the compounds were stimulated with LPS. AU the compounds except 27-Nl 5 inhibited LPS-mediated NF-κB induction in RAW264.7 cells. [0070] We also performed IL-8-promoter reporter gene assay using HeLa cells transiently transfected with TLR4, CD 14 and MD-2 vectors plus the reporter constructs as noted in Figure 4A. LPS-mediated IL-8 promoter activity was inhibited by all the compounds except 27-Nl 5 (Fig. 4A).

[0071] To determine if this transcription factor inhibition really effects production of cytokines, we next performed ELISA to measure TNF-α and IL-6 in RAW264.7 cell cultures. Cells were pre-incubated with different concentrations of the compounds for 60 min, changed with fresh culture media to remove all existing DMSO, and stimulated with LPS for 16 h. As shown in Figures 4B and 4C. IL-6 production was generally more strongly inhibited than TNF-α, even by compound 27-Nl 5. The most inhibitory compound is 26-J10, which is inhibitory of both cytokines at 120 nM.

[0072] We next investigated the inhibitory specificity of the compounds. Using the two most potent compounds (50-F12 and 26-J10), we performed an NF-κB activation assay on RAW 264.7 cells stimulated with a variety of agonists which act through different receptors. In addition to LPS (TLR4 ligand), we used MALP-2 (TLR2 ligand), pIpC (TLR3 ligand), CpG (TLR9 ligand), IL-I β and TNF-α. Interestingly, both compounds inhibited MALP-2 and LPS-induced NF-κB activation while they only slightly inhibited CpG- or IL-I β- mediated NF-κB induction (Fig. 5). However, pIpC and TNF-α mediated NF-κB inductions were not affected. These results suggest that the inhibition is not only MyD88 dependent (Fig. 5), but could also be specific for the particular MyD88 partner.

Example 4 Materials and experimental protocols

[0073] Reagents and antibodies: Potassium clavulanate as a lactamase inhibitor was from Sigma-Aldrich and the lactamase substrate, CCF2AM from Invitrogen (Carlsbad, CA). The 16,000 target compounds used for screening were obtained from Maybridge (Cornwall, England). Lipopolysaccharide (LPS) from Escherichia coli (Ol 1 1 :B4) was purchased from List Biological Laboratories (Campbell, CA) and polyinosinic-polycytidylic acid (pIpC) from Amersham Biosciences (Piscataway, NJ). Anti-FLAG M2-agarose and antibody were obtained from Sigma-Aldrich (St. Louis, MO) and anti-HA.l 1 antibody was obtained from Covance-Berkeley Antibody Company (Richmond, CA). Murine IL-I β and TNFα were purchased from PeproTech (Rocky Hill, NJ). Mouse TNF-α and IL-6 ELISA kits were purchased from BD Biosciences.

[0074] Stable cell line and culture conditions: The stable HeLa line, HeLa/CL3-4, which expresses two β-lactamase fusion proteins, MyD88-Bla(a) and TLR4-Bla(b), was made using two expression constructs, pCDNA3.1/MyD88/Bla(a) and pEF6/TLR4/Bla(b) as previously described in Lee et al., J. Biol. Chem. 279:10564-74, 2004. The stable HeLa/full-Bla line, which expresses a fusion protein of MyD88 with full-length β-lactamase, was made using pCDNA3.1/MyD88/full-length BIa. HeLa/CL3-4 were grown in DMEM with 10% FCS containing 200 μg/mL G418 and 10 μg/mL Blasticidin and HeLa/full-Bla in the same medium containing 200 μg/mL G418 only. Both HeLa/CL3-4 and HeLa/full-Bla were selected using FACSort (BD Biosciences) equipped with excitation at 408 nm and emission at 519/30 nm (CCF, green fluorescence) or 450/40 nm (cleaved CCF2, blue fluorescence). The primary blue-positive cells sorted by FACS were collected, grown in the media described above, and re-sorted. This sequential sorting step was repeated three times. [0075] Assay Optimization: HeLa/CL3-4 cells were plated into regular 384-well plates in a series of different concentrations (Ix 10⁶, 0.5 x 10⁶, 0.25 x 10⁶, 0.125 x 10⁶, and 0.06 x 10⁶ cells per ml) using the FlexDrop Precision Reagent Dispenser (PerkinElmer). Each well contained 30 μl of cells. Cells were incubated at 37⁰C overnight. On the following day, cells were treated with different amounts of clavulanate (10, 3.3, 1.1, 0.37, 0.12, 0.04, 0.014, 0.0046 and 0.0015 μg per mL) for 30 min at 37°C and then loaded with lμM CCF2AM for 2h at room temperature. These two steps were performed using the BioRaptor FRD (Aurora). The plates were then read using the EnVision multi-label reader (PerkinElmer). The data were analyzed with Excel as well as Prism software. The response metric was the ratio of blue fluorescence intensity to green fluorescence intensity.

[0076] To assess assay uniformity across multiple plates HeLa/CL3-4 cells (30 μl of 0.125 x 10⁶ cells/mL) were plated into three 384-well plates with DMEM/10% FCS using the FlexDrop Precision Reagent Dispenser. Cells were incubated at 37⁰C overnight. On the following day, the three plates were treated with 10 μg/mL (High clavulanate), 0.12 μg/mL (Medium clavulanate) or control PBS (Low) with three plate layouts, specifically plate 1 (HML), plate 2 (LHM) and plate 3 (MLH). After 30 min, the plates were loaded with lμM CCF2AM for 2h at room temperature. The plates were then read using the EnVision multi- label reader (PerkinElmer).

[0077] Compound screening and hit selection: HeLa/CL3-4 cells in DMEM/10% FCS (lOμl of 0.22 * 10⁶ cells/ml) were dispensed into low-volume, black clear bottom 384-well plates (Greiner) on the FlexDrop Precision Reagent Dispenser (PerkinElmer). Cells were incubated at 37⁰C overnight. On the following day, cells were treated with lOμM various compounds (10 nl/well) delivered with a pintool (V & P Scientific) on a Beckman FX, except the first two and the last two columns which served as controls. The first two columns were treated with different amounts of clavulanate (10, 3.3, 1.1, 0.37, 0.12, 0.04, 0.014, 0.0046 and 0.0015 μg per mL) while the last two columns were left untreated. The plates were incubated at 37⁰C for 30 min. Thereafter, the plates were read using the EnVision multi-label reader (PerkinElmer).

[0078] Z' was calculated as described in Zhang et al., J. Biomol. Screen. 4:67-73, 1999. Active wells were defined as those with percent inhibition greater than 3 standard deviations from the low control. Percent inhibition was defined as 100*(l -(Well-Median High Control)/(Median Low Control-Median High Control) where High Control was the wells treated with 10 μg/ml clavulanic acid and Low Control was treated with vehicle alone. A total of 24 wells met this definition and 21 more with borderline activity were selected for repeat. Cherry-picked compounds were tested in the primary screening assay in triplicate. [0079] Microscopy. Microscopy was performed as described in Lee et al., J. Biol. Chem. 279:10564-74, 2004. Briefly, cells were cultured in a 12-well plate overnight. Cells were washed twice with a modified physiological saline buffer (10 mM HEPES, 6 mM sucrose, 10 itiM glucose, 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1% probenecid, pH 7.35) and loaded with 1 μM CCF2/AM for 1 h at room temperature. Cells were washed with physiological saline buffer and analyzed by fluorescence microscopy (Zeiss Axiovert IOOTV with a Diagnostics Instruments SPOT cooled CCD camera) using a filter set from Omega Optical XF12-2: 405 nm excitation, 420 nm dichroic mirror, 435 nm long-pass emission. [0080] Electrophoretic mobility shift assay: RAW264.7 cells (0.5 x 10⁶ cells/ml) were grown in 12-well tissue culture plates with DMEM/10% FCS for 24 h and pre-treated with 10 μM inhibitory compounds for 30 min. Cells were then stimulated with LPS (0.1 μg/ml), MALP-2 (50 ng/ml), pIpC (20 μg/ml), CpG (20 μg/ml), IL-I β (50 ng/ml), or TNF-α (50 μg/ml) for 1 h. Cells were lysed with buffer A (10 mM HEPES [pH7.9], 10 mM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 1% NP-40) followed by centrifugation. The pellet was treated with buffer B (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, and 0.1 mM PMSF) to prepare nuclear extracts. EMSA was performed utilizing NF- KB oligonucleotide probe (Promega, Madison, WI) labeled with [γ-³²P]ATP (Amersham Biosciences) using T4 polynucleotide kinase (New England BioLabs) as previously described in Lee et al., Immunity 24, 153-163, 2006.

[0081] Luciferase reporter assay: The luciferase reporter assay was performed as previously described in Lee et al., J. Biol. Chem. 279:10564-74, 2004. Briefly, HeLa cells (0.5 x 10⁶ cells/ml) were grown in 12-well plates with DMEM/10% FCS. On the following day, cells were transiently transfected with 0.01 μg/ml TLR4, CD 14 and MD-2 vectors, along with 0.05 μg/ml pIL8-promoter-Luc vector and pSV-β-galactosidase vector (Promega, Madison, WI). After 24 h, cells were pre-treated with different compounds for 30 and then stimulated with LPS for 6 h. Cell extracts were prepared using the cell culture lysis buffer (Promega) and the luciferase activity was measured using a Luciferase Reporter Assay System (Promega), and β-galactosidase activity was measured using O-nitrophenyl-β-o- galactopyranoside as substrate. Luciferase activity reported in the figures is normalized for transfection efficiency using the β-galactosidase activity.

[0082] Immunoprecipitation and Western blotting: HEK293T cells (0.5 x 10⁶ cells/ml) were transiently transfected with 0.5 μg TLR4 transmembrane-cytoplasmic domain (TLR4CD) (Lee et al., J. Biol. Chem. 279:10564-74, 2004) and MyD88 vectors. Twenty- four hours after transfection, cells were treated with different compounds at the different time points as indicated in the figure legends. Cells were lysed with lysis buffer (150 mM NaCl, 1% Nonidet P40, 5 mM EDTA, 50 mM Tris-HCl, pH 7.5) containing protease inhibitor cocktails (Roche Applied Science). The supernatants were incubated with anti-FLAG M2- agarose (Sigma-Aldrich) and incubated for 3 h at 4⁰C. The mixtures were washed four times with lysis buffer, separated on a 4-12% Bis-Tris NuPAGE gel (Invitrogen), and transferred to nitrocellulose membrane. Western blotting was performed by probing the membrane with both anti-FLAG and anti-HA antibodies according to standard protocols. [0083] Cytokine ELISA: Raw264.7 cells (0.5 x 10⁶ cells/ml) were cultured in a 12-well plate for 24 h and pre-incubated with different concentrations of compounds for 1 h as indicated in the figures. Cells were then washed once with DMEM/10% FCS and stimulated with 0.05 μg/ml LPS for 16 h. TNF-α and IL-6 in the culture supernatants were measured by mouse TNF-α and IL-6 ELISA according to the manufacturer's instructions (BD Biosciences).

[0084] Cell viability test: Raw264.7 cells (0.5 x 10⁶ cells/ml) were cultured in a 12-well plate for 24 h. Cells were washed with PBS once and treated with different compounds at 20 μM or DMSO only, or 70% ethanol for Ih. Cells were then washed with PBS and treated with 2 μM calcein AM and 4 μM EthD-1 solution (Invitrogen) for 30 min. Cells were analyzed by fluorescence microscopy at 495 nm and 528 nm.

***

[0085] 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 one 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.

[0086] All publications, databases, GenBank sequences, patents, and patent applications cited in this specification are herein incorporated by reference as if each was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting or suppressing signaling activities of a Toll-like receptor (TLR), the method comprising administering to a subject suffering from undesired signaling activities of a TLR a pharmaceutical composition comprising a therapeutically effective amount of a TLR antagonist compound, wherein the TLR antagonist compound is selected from the group consisting of N-(4-{2-[l-(4-fluorobenzyl)-4- pyridinium]vinyl}phenyl)-N-rnethylrnethanamine iodide, methyl 2-{[l-(4-fluorophenyl)-3- oxo-3-pyridin-3-ylpropyl]thio}benzoate, 4-(5-chloro-2,l-benzisoxazol-3-yl)-2- methoxyphenol, N-{4-[2-(l-hexylpyridinium-2-yl)vinyl]phenyl}-N-methylrnethanamine iodide, and N-{4-[2-(l-hexylpyridinium-2-yl)vinyl]phenyl}-N-methylmethanamine iodide.

2. The method of claim 1, wherein the Toll-like receptor is TLR4 or TLR2.

3. The method of claim 1 , wherein the subject suffers from sepsis.

4. The method of claim 3, wherein the sepsis is associated with a Gram- negative bacterial infection or a Gram-positive bacterial infection.

5. The method of claim 1 , wherein the subject suffers from a chronic inflammatory disorder.

6. The method of claim 5 , wherein the disorder is atherosclerosis.

7. The method of claim 1, wherein the subject suffers from an ischemia- reperfusion injury.

8. The method of claim 7, wherein the ischemia-reperfusion injury is a lung ischemia-reperfusion injury, a liver ischemia-reperfusion injury, or a brain ischemia- reperfusion injury.

9. The method of claim 1, wherein the subject suffers from a myocardial injury.

10. The method of claim 9, wherein the myocardial injury is cardiac ischemia-reperfusion injury or myocardial infarction.

11. A method of inhibiting or ameliorating an undesired immune response, the method comprising administering to a subject suffering from an undesired immune response mediated by a Toll-like receptor (TLR) a pharmaceutical composition comprising a therapeutically effective amount of a TLR antagonist compound, wherein the TLR antagonist compound is selected from the group consisting of N-(4-{2-[l-(4-fluorobenzyl)-4- pyridinium]vinyl}phenyl)-N-methylrnethanamine iodide, methyl 2-{[l-(4-fluorophenyl)-3- oxo-3-pyridin-3-ylpropyl]thio}benzoate, 4-(5-chloro-2,l-benzisoxazol-3-yl)-2- methoxyphenol, N-{4-[2-(l-hexylpyridinium-2-yl)vinyl]phenyl}-N-methylmethanamine iodide, and N-{4-[2-(l-hexylpyridinium-2-yl)vinyl]phenyl}-N-methylmethanamine iodide.

12. The method of claim 1 1, wherein the undesired immune response is mediated by Toll-like receptor 4 (TLR4) or Toll-like receptor 2 (TLR2).

13. The method of claim 11, wherein the subject suffers from sepsis.

14. The method of claim 11, wherein the subject suffers from a chronic inflammatory disorder.

15. The method of claim 11, wherein the subject suffers from an ischemia- reperfusion injury.

16. A use of a TLR antagonist compound in the manufacture of a medicament for treating or preventing an undesired immune response in a subject, wherein the TLR antagonist compound is selected from the group consisting of N-(4-{2-[l-(4-fluorobenzyl)-4- pyridinium]vinyl}phenyl)-N-methylmethanamine iodide, methyl 2-{[l-(4-fluorophenyl)-3- oxo-3-pyridin-3-ylpropyl]thio}benzoate, 4-(5-chloro-2,l-benzisoxazol-3-yl)-2- methoxyphenol, N- {4-[2-( 1 -hexylpyridinium-2-yl)vinyl]phenyl } -N-methylmethanamine iodide, and N- {4-[2-(l-hexylpyridinium-2-yl)vinyl]phenyl} -N-methylmethanamine iodide.

17. The use of claim 16, wherein the undesired immune response is mediated by Toll-like receptor 4 (TLR4) or Toll-like receptor 2 (TLR2).

18. The use of claim 16, wherein the subject suffers from sepsis.

19. The use of claim 16, wherein the subject suffers from an inflammatory disorder.

20. The use of claim 16, wherein the subject suffers from an ischemia- reperfusion injury.

21. A method for identifying an antagonist of a Toll-like receptor with improved properties, comprising (a) synthesizing one or more structural analogs of a lead TLR antagonist compound; and (b) performing a functional assay on the analogs to identify an analog that has an improved biological or pharmaceutical property relative to that of the lead TLR antagonist compound; thereby identifying a TLR antagonist with improved properties; wherein the lead TLR antagonist compound is selected from the group consisting of N-(4-{2-[l-(4-fluorobenzyl)-4-pyridinium]vinyl}phenyl)-N- methylmethanamine iodide, methyl 2-{[l-(4-fluorophenyl)-3-oxo-3-pyridin-3- ylpropyl]thio}benzoate, 4-(5-chloro-2,l-benzisoxazol-3-yl)-2-methoxyphenol, N-{4-[2-(l- hexylpyridinium-2-yl)vinyl]phenyl}-N-methylmethanarnine iodide, and N-{4-[2-(l- hexylpyridinium-2-yl)vinyl]phenyl}-N-methylmethanamine iodide.

22. The method of claim 21, wherein the improved biological or pharmaceutical property is an enhanced activity in inhibiting signaling activities of TLR4 or TLR2.

23. The method of claim 21, wherein the improved biological or pharmaceutical property is selective inhibition of TLR4 over TLR2.

24. The method of claim 21, wherein the TLR antagonist compound is N-{4- [2-(l-hexylpyridinium-2-yl)vinyl]phenyl}-N-methylmethanamine iodide.