WO2008101109A1 - Withanolides, probes and binding targets and methods of use thereof - Google Patents

Abstract

Novel withanolide chemical genetic probes identify the in vivo binding target of withaferin A, which is the type-III intermediate filament protein vimentin. In addition, a withanolide-based small molecule screening method screens drug candidates that target type-III intermediate filament proteins. The method includes introducing a tagged linker covalently bonded to the withanolide molecule to form a withanolide probe. Better or alternative small molecule compounds as potential drug candidates can be generated based on their likely affinity for the determined binding site in vimentin. The affinity labeled withanolide can also be used to find intermediate filament-associated proteins using chemical proteomics by extracting proteins from cells that were exposed to withanolide-biotin analog. The withanolide probes can be used to monitor expression of vimentin, in tumor samples or other diseased tissues. Withaferin A and its analogs can be used as a treatment of diseases associated with aberrant type-III intermediate filament protein production, including diseases of vimentin-associated disorders, such as cancers, angiofibrotic diseases, and chronic inflammation, GFAP-associated disorders such as gliopathies, motor neuron degeneration, scar formation, traumatic brain injury, stroke, epilepsy and mood disorders and desmin-associated disorders relating to cardiomyopathies and musculoskeletal disorders.

Description

WITHANOLIDES, PROBES AND BINDING TARGETS AND METHODS OF USE THEREOF

FIELD OF THE INVENTION

[0001] The present invention relates to compounds for targeting human or animal disease states characterized by aberrant expression of the type-Ill intermediate filament proteins, methods and compounds for detecting type-Ill intermediate filament proteins, compounds for use in screening small molecules that target intermediate filament proteins, and methods for treating diseases using small molecules which target the type- Ill intermediate filament proteins.

BACKGROUND OF THE INVENTION

[0002] Natural products that demonstrate pharmacological efficacy are very important chemical scaffolds for drug design. While the vast majority of small molecule natural products will eventually be screened for biological activity, only a small fraction of these drug-like agents will move forward in the drug development pipeline. Currently, a considerable amount of effort is being directed through high throughput screening to select lead candidates based on their ability to bind to validated disease-associated protein targets.

[0003] A new approach has emerged that employs biologically active small molecules as cell permeable probes to identify novel binding targets and to study their function. Since pharmacological activity of a small molecule resides principally through binding interaction with its hypothetical biological target(s) (e.g., protein receptor), it is the discovery of such a protein receptor is of critical importance in drug development. Furthermore, the identification of the specific amino acid of the protein target to which the small molecule binds offers molecular insight into the specificity of such binding interactions. Consequently, the use of the binding site information allows one to look for similar binding sites in other classes of proteins, molecular information that helps expand the target cell types/organisms and clinical indications for the small molecule drug lead. The discovery of VIAGRA^® for male sexual dysfunction is one such example where research on phoshodiesterase inhibitors for cardiovascular dysfunction led to the serendipitous identification of a novel therapeutic area for this class of drugs. [0004] In small molecule protein target discovery research, the most critical element is the innovative chemical design to convert a drug-like chemical compound into a useful biologically active chemical probe that affords the ability to use the reagent for identification of its in vivo protein binding target. Careful consideration is paid to not alter any of the small molecule's biological activities when the synthetic analog is generated. In addition, it is also important that a level of specificity for protein binding is demonstrated. Chemical radioisotope tagging is commonly used to generate a radiolabeled analog of the small molecule to help in protein target detection. However, a major drawback with this approach is that this method does not afford a direct means to isolate the binding target of this agent.

[0005] One recent screening method uses affinity tagging small molecules with biotin to form a small molecule analog, which seemingly provides the potential for isolating a small molecule binding target. Unfortunately, current techniques of biotin affinity tagging have not realized the full potential of isolating the binding target for small molecule agents due, in part, to the presence of the biotin attached to the small molecule, which may interfere with the natural binding of the small molecule analog with its target.

[0006] One small molecule is the natural product Withaferin A (hereinafter "WFA"), which is a potent angiogenesis inhibitor that targets the ubiquitin-proteasome pathway in vascular endothelial cells. WFA is an important prototype of the withanolide class of natural products and is a highly oxygenated steroidal lactone that is found in the medicinal plant Withania somnifera and its related solanaceas species. The withanolides are known to exert very potent and diverse cytotoxic, anti-stress, cardioactive, central nervous system, and immunomodulatory activities. Since the early discovery of WFA during the 1960s, the major interest has been on its anti-tumor cytotoxic activities. The mesenchymal type-Ill intermediate filament ("IF") protein vimentin plays a critical role in wound healing, angiogenesis and cancer growth.

[0007] The role of vimentin in human and animal disorders, including those disorders associated with aberrant or altered expression of vimentin, and diseases associated with angiogenesis are known in the prior art, e.g. by the following references, U.S. Patent Nos. 5,716,787; 5,932,545; 6,846,841 ; 7,132,245; 7,175,844; U.S. Patent application publication no. 2006/0014225; Hertig, A., Verine, J., Mougenot, B., Jouanneau, C, Ouali, N., Sebe, P., Glotz, D., Ancel, P.Y., Rondeau, E. and Xu-Dubois Y.C.; "Risk factors for early epithelial to mesenchymal transition in renal grafts," Am J Transplant; 6(12):2937-46 (2006 Dec) (hereinafter "Hertig et al."); Kenyon, B.M., et al., "Effects of thalidomide and related metabolites in a mouse corneal model of neovascularization," Exp. Eye Res., 64:971-978 (1997) (hereinafter "Kenyon et al."); Cole, C. H., et al., "Thalidomide in the management of chronic graft-versus-host disease in children following bone marrow transplantation," Bone Marrow Transplantation, 14:937-942 (1994) (hereinafter "Cole et al."); Russell, M. E., et al., "Chronic cardiac rejection in the LEW to F344 rat model," J. Clin. Invest, 97(3):833-838 (1996) (hereinafter "Russell et al."); Kwon, Y.S. and Kim, J. C, "Inhibition of corneal neovascularization by rapamycin," Exp MoI Med. ;38(2):173-9 (2006 Apr 30) (hereinafter "Kwon et al."); Zogakis, T.G. and Libutti, S. K., "General aspects of anti-angiogenesis and cancer therapy," Expert Opin Biol Ther, 1 (2):253-75, (2001 Mar), Review; Kirsch, M., Santahus, T., Black, P.M. and Schackert, G., "Therapeutic anti-angiogenesis for malignant brain tumors," Onkologie, 24(5):423-30 (2001 Oct) Review (hereinafter "Zogakis et al."); Manzano, R., Peyman, G., Khan, P., Carvounis, P., Kivilcim, M., Ren, M., Lake, J. and Chevez-Barhos, P., "Inhibition of experimental corneal neovascularization by Bevacizumab(AVASTIN)," Br J Ophthalmol. (2006 Dec 19) (hereinafter "Manzano et al."); Yeh, J. R., Mohan, R. and Crews, CM., "The antiangiogenic agent TNP-470 requires p53 and p21 CIP/WAF for endothelial cell growth arrest," Proc Natl Acad Sci U S A., 97(23): 12782-7 (2000 Nov 7) (hereinafter "Yeh et al."); Yu, Y., Moulton, K.S., Khan, M. K., Vineberg, S., Boye, E., Davis, V.M., O'Donnell, P. E., Bischoff, J. and Milstone, D. S., "E-selectin is required for the antiangiogenic activity of endostatin," Proc Natl Acad Sci U S A., 101 (21 ):8005-10 (2004 May 25) (hereinafter "Yu et al."); Murthy, R.C., McFarland, T.J., Yoken, J., Chen, S., Barone, C, Burke, D., Zhang, Y., Appukuttan, B. and Stout, J. T., "Corneal transduction to inhibit angiogenesis and graft failure," Invest Ophthalmol Vis Sci. 44(5): 1837-42 (2003 May) (hereinafter Murthy et al."); Rogers, M.S., Rohan, R.M., Birsner, A.E. and D'Amato, R.J., "Genetic loci that control the angiogenic response to basic fibroblast growth factor," FASEB J., 18(10):1050-9 (2004 JuI) (hereinafter "Rogers et al."); Zhang, M., Volpert, O., Shi, Y.H. and Bouck, N., "Maspin is an angiogenesis inhibitor," Nat Med, 6(2):196-9 (2000 Feb) (hereinafter "Zhang et al."); and Mor-Vaknin, N., Puntuheh, A., Sitwala, K., Markovitz, D. M., "Vimentin is secreted by activated macrophages," Nat Cell Biol, 5(1 ):59-63 (2003 Jan) (hereinafter "Mor-Vaknin et al."); all herein incorporated by reference. [0008] Although the role of vimentin, and diseases associated with angiogenesis are known in the prior art, the non-cytotoxic anti-inflammatory and immunomodulatory mechanisms of WFA have thus far remained rather poorly characterized. These latter disease-altering activities are highly pertinent to the practice of ayurveda, a traditional form of Indian medicine, which has borne out many effective formulations from W. somnifera, especially for the treatment of chronic human diseases such as arthritis and female bleeding disorders.

[0009] Other type-Ill intermediate filament proteins include desmin, glial fibrillary acid protein ("GFAP") and pehphehn. Production of desmin is linked with various desmin-related myopathies and musculoskeletal disorders characterized by aberrant levels or altered forms of intermediate filament desmin, which results in protein aggregation and tissue and organ dysfunction. Aberrant levels or altered forms of type-Ill intermediate filament GFAP is associated with neurological diseases, spinal and neuromuscular injuries that are characterized by inflammatory reactive astrocytes and glial cells expressing the aforementioned GFAP. In addition, gliosis, motor-neuron degeneration, scar formation, age-related macular degeneration, macular edema, retinoblastoma, glioblastoma, gliomas, retinopathy retinal detachment, glaucoma, retinopathy of prematurity, diabetic retinopathy, proliferative diabetic retinopathy, proliferative vitreoretinopathy, retinitis pigmentosa, uveitis, glioma, glioblastoma, astroglioma, glial tumors, retinoblastoma, optic nerve damage, retinal ischemia, bomb blast injuries, chemical injury, thermal burns, viral infections, Alexander disease, Alzheimer's disease, depression, anxiety, migraine, schizophrenia, bipolar disorders, addiction, Parkinson's disease, Huntington's disease, traumatic brain and spinal cord injury, developmental disorders, depression and mood disorder, stroke and epilepsy are associated with aberrant or altered forms of GFAP. Although the correlation of the presence of aberrant levels or altered forms of the type-Ill intermediate filament proteins to the aforementioned diseases are known, a mechanism for blocking the intermediate filament production remains poorly characterized.

[0010] There are many desmin-related cardiomyopathies where mutation in the desmin gene produced dramatic effects in the desmin polymer formation. Studies have shown a link between type-Ill IF proteins and associated disorders. Mutations in the human desmin gene cause familial or sporadic forms of skeletal myopathy frequently associated with cardiac abnormalities, lntrasarcoplasmic amyloidosis and increased ubiquitinated proteins ("UPS") are observed in human failing hearts. Thus, UPS impairment is linked to important pathogenic mechanism underlying cardiac disorders with abnormal protein aggregation, in accordance with "Primary desminopathies," Schroder, R., Vrabie, A., Goebel, H. H., J Cell MoI Med, 1 1 (3):416-26 (2007 May-Jun)

(hereainfter "Schroder"); "The biology of desmin filaments: how do mutations affect their structure, assembly, and organisation?," Bar, H., Strelkov, S.V., Sjoberg, G., Aebi, U. and Herrmann, H., J Struct Biol, 148(2):137-52 (2004 Nov) (hereinafter "Bar"); and "Intermediate filament-related myopathies," Banwell, B. L., Pediatr Neurol, 24(4):257-63 (2001 Apr) (hereinafter "Banwell").

[0011] Aberrant protein aggregation is essential for a mutant desmin to impair the proteolytic function of the ubiquitin-proteasome system in cardiomyocytes, as shown by Liu, J., Tang, M., Mestril, R. and Wang, X, J MoI Cell Cardiol, 40(4):451-4 (2006 Apr) (hereinafter "Liu April 2006"); and Liu, J., Chen, Q., Huang, W., Horak, K.M., Zheng, H., Mestril, R. and Wang, X., "Impairment of the ubiquitin-proteasome system in desminopathy mouse hearts," FASEB J., 20(2):362-4 (2006 Feb) (hereinafter "Liu Feb. 2006"). For instance, a heterogenous R350P desmin missense mutation which resides in the evolutionary highly conserved α-helical coiled-coil desmin rod domain has pathogenic effects on the assembly of desmin IF in cultured cells and in vitro. This mutation also causes endogenous vimentin cytoskeleton in 3T3 fibroblasts cells to be disrupted displaying cytoplasmic aggregates reminiscent of desmin-positive protein deposits seen in the immunohistochemical and ultrastructural analysis of skeletal muscle derived from the index patient of affected family.

[0012] The developmental disorder known as Alexander Disease is caused by mutations in the astrocyte's IF, GFAP, which results in abnormal aggregation of IF and cell dysfunction. After neurotrauma, ischemia, or neurodegenerative disease, astrocytes upregulate their expression of the intermediate filament proteins GFAP, vimentin, and nestin. Astrocytes become activated ("reactive") in response to changes in the extracellular environment in these conditions. The hallmark of activated astrocytes is hypertrophy of the cellular processes and upregulation of the IF's, GFAP, vimentin and re-expression of nestin, leading to prominent cytoskeleton in the soma and processes of the astrocytes, as shown by Pekny, M. and Pekna, M., "Astrocyte intermediate filaments in CNS pathologies and regeneration," J Pathol 204, 428-37 (2004) (hereinafter "Pekny"). [0013] In acquired inflammatory and traumatic disorders, such as Multiple

Sclerosis and spinal cord injuries, activation of the astrocytes in response to the insult is associated with dys-regulation of IFs, GFAP and vimentin, and formation of gliosis and a glial scar impairs neuro-regeneration and contributes to disability. It has been demonstrated that the absence of vimentin and GFAP in mice can increase neurogenesis and survival of grafts, as reported in Widestrand, A., Faijerson, J., Wilhelmsson, U., Smith, P. L., Li, L., Sihlbom, C, Eriksson, P. S. and Pekny, M., "Increased neurogenesis and astrogenesis from neural progenitor cells grafted in the hippocampus of GFAP-/- Vim-/- mice," Stem Cells, 25(10):2619-27 (2007 Oct) (hereinafter "Faijerson"). Also, the absence of GFAP and vimentin attenuates retinal detachment-induced reactive gliosis and, subsequently, limits photoreceptor degeneration, further implicating the importance of GFAP and vimentin overexpression in ocular diseases such as retinal degeneration and glaucoma, shown in Nakazawa, T., Takeda, M., Lewis, G. P., Cho, K.S., Jiao, J., Wilhelmsson, U., Fisher, S. K., Pekny, M., Chen, D. F. and Miller, J.W., "Attenuated glial reactions and photoreceptor degeneration after retinal detachment in mice deficient in glial fibrillary acidic protein and vimentin," Invest Ophthalmol Vis Sci, 48(6):2760-8 (2007 Jun) (hereinafter "Nakazawa"); "Novel mutations in exon 6 of the GFAP gene affect a highly conserved IF motif in the rod domain 2B and are associated with early onset infantile Alexander disease," as shown by Hartmann, H., Herchenbach, J., Stephani, U., Ledaal, P., Donnerstag, F., Lucke, T., Das, A.M., Christen, H.J., Hagedorn, M. and Meins, M, Neuropediatrics, 38(3):143-7 (2007 Jun) (hereinafter "Hartmann"); Eng, L. F., Ghirnikar, R. S. and Lee, Y. L., "Glial fibrillary acidic protein: GFAP-thirty-one years (1969-2000)," Neurochem Res 25, 1439-51 (2000) (hereinafter "Eng"); and Brenner, M., et al., "Mutations in GFAP, encoding glial fibrillary acidic protein, are associated with Alexander disease," Nat Genet 27, 1 17-20 (2001 ) (hereinafter "Brenner"). [0014] In addition, prior studies have shown a link between interfering with type-Ill

IF proteins and treatment of disorders. Epigallocatechin-3-gallate (EGCG) is the major active polyphenol of green tea and is widely recognized for its anti-angiogenic activity and tumor targeting activities, as shown by Fassina, G., Vene, R., Morini, M., Minghelli, S., Benelli, R., Noonan, D. M., and Albini, A., "Mechanisms of inhibition of tumor angiogenesis and vascular tumor growth by epigallocatechin-3-gallate," Clin Cancer Res 10, 4865-4873, (2004) (hereinafter "Fassina"). EGCG binding to the N-terminal end of vimentin to prevent its phosphorylation is believed to explain further the anti-tumor and anti-angiogenic activities of this inhibitor, as shown by EGCG binds to vimentin and prevents its phosphorylation and thereby inhibits cell proliferation, as shown by Ermakova, S., Choi, B.Y., Choi, H. S., Kang, B. S., Bode, A.M. and Dong, Z. J. Biol. Chem., 280: 16882 - 16890 (Apr 2005) (hereinafter "Ermakova"). [0015] The mechanism of prostaglandin 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) in treatment of renal disease is thought to be mediated by vimentin binding, as shown by "Identification of novel protein targets for modification by 15-deoxy-Delta12,14- prostaglandin J2 in mesangial cells reveals multiple interactions with the cytoskeleton," Stamatakis, K., Sanchez-Gomez, F. J., and Perez-Sala, D., J Am Soc Nephrol. 17(1 ):89-9 (2006 Jan) (hereinafter "Stamatakis"). Furthermore, these investigators have proposed that this class of prostaglandin may exert its anti-tumor and anti-inflammatory activities by direct modification of the cysteine 328 residue of vimentin, as shown in "Study of protein targets for covalent modification by the antitumoral and anti-inflammatory prostaglandin PGA1 : focus on vimentin," Gharbi, S., Garzόn, B., Gayarre, J., Timms, J. and Perez-Sala, D., J Mass Spectrom, 42(1 1 ): 1474-84 (2007 Nov) (hereinafter "Gharbi").

[0016] Targeting endothelial vimentin in a mouse tumor model significantly inhibited tumor growth and reduced microvessel density, as disclosed in "Gene expression of tumor angiogenesis dissected: specific targeting of colon cancer angiogenic vasculature," van Beijnum, J. R., Dings, R. P., van der Linden, E., Zwaans, B.M., Ramaekers, F.C., Mayo, K.H., Griffioen, A.W., Blood, 108(7):2339-48 (2006 Oct 1 ) (hereinafter "Van Beijnum").

SUMMARY OF THE INVENTION

[0017] The present invention relates to compounds for targeting functions of type-Ill intermediate filament proteins, such as vimentin, desmin, pehphehn and glial fibrillary acid protein ("GFAP"), thus treating disorders associated with aberrant levels or altered forms of type-Ill intermediate filament proteins. These compounds include small molecules, such as WFA and its analogs, collectively referred to as withanolides. The ability of certain withanolides to bind vimentin is positively correlated to the anti-angiogenic and anti-inflammatory activity of this compound class. The ability of certain withanolides to bind desmin, periphehn and GFAP is correlated to controlling heart disease, especially fibrosis, and the treatment of neurological diseases, spinal and neuromuscular injuries characterized by inflammatory reactive astrocytes. Glial cells expressing aberrant levels or altered forms of the type-Ill intermediate filament, GFAP, are associated with gliosis, motor neuron degeneration, scar formation, neural cell and stem cell transplant failure, early and late forms of age-related macular degeneration, macular edema, retinal detachment, proliferative diabetic retinopathy, retinopathy of prematurity, glaucoma, proliferative vitreoretinopathy, retinitis pigmentosa, uveitis, retinoblastoma, gliomas, glioblastoma, glial tumors, optic nerve damage , retinal ischemia, bomb blast injury, chemical injury, thermal burns, viral infections, Alexander disease, Alzheimer's disease, anxiety, migraine, schizophrenia, bipolar disorders, addiction, Parkinson's disease, Huntington's disease, traumatic brain injury, development disorders, depression and mood disorders, stroke and epilepsy. In another aspect, the pharmacological control of enteric neurogliopathies, such as Crohn's disease and necrotizing enterocolitis would be disease of the enteric nervous system (ENS) where the dose of drug is altered to result in desired level of GFAP expression. [0018] The present invention also relates to compounds and methods for detecting type-Ill intermediate filament proteins, such as vimentin, desmin, GFAP and periphehn. For example, withanolide derivatives with chemical or radioactive tags can be used as detection probes and/or assist with the isolation of withanolide binding proteins and target-associated co-isolated proteins.

[0019] The present invention also relates to a method for screening small molecules as potential drug candidates that interfere with binding of WFA to its target protein binding site by use of a tagged withanolide analog, as demonstrated with biotinylated WFA. For example, a withanolide analog can be used to screen drugs that target intermediate filament proteins. In one form, the affinity tag biotin, which is covalently bonded to a hydrocarbon linker having a chain of C1-C20 (of structures shown) covalently bonded to the withanolide.

[0020] Affinity tagging with biotin allows for both the detection of the target protein of the small molecule and the target sites by isolation of the small molecule-bound protein target using affinity chromatography. The isolation of the target bound to the small molecule analog allows for the determination of the binding site. From knowing the target binding site, one can generate and develop tailored new classes of small molecule compounds, which may be even better drug candidates than the parental molecule. [0021] Two key aspects, which allow for the isolation of the small molecule analog bound to its target, are that the placement of the biotin moiety should not hinder the small molecule's pharmacophores and that the small molecule biotin adduct must be separated by a chemical linker long enough that it allows the small molecule to enter into living cells and bind to its target in vivo. [0022] A highly validated approach was used to test the effectiveness of the present screening method using small molecule analogs labeled with an affinity tag by designing a WFA-biotin affinity reagent as a test small molecule analog. The WFA-biotin affinity reagent utilized previous known structure-activity relationship studies to determine the pharmacophores of WFA. This information was used to generate a novel cell- permeable affinity analog of WFA that retains biological activity and identifies its covalent binding protein, as disclosed by Yokota, et al., "Development of Withafehn A.," Bioorg Med Chem Lett. (2006), herein incorporated by reference.

[0023] A small molecule screening method comprises generating an affinity labeled withanolide analog by binding withanolide compound to an affinity tag via a linker group; introducing the affinity labeled withanolide analog to a cell culture that has been exposed to small molecule drug candidates; and contacting the affinity labeled withanolide analog with one of: (i) a purified protein that has been exposed to one or more small molecule drug candidates; (ii) a cell extract that has been exposed to one or more small molecule drug candidates; and (iii) a protein mixture that has been exposed to one or more small molecule drug candidates.

[0024] Using the present method, a small molecule can be selected as a potential drug based on its binding to the target. In addition, the present method can be used to determine a target binding site for the small molecule and/or the withanolide compound with the target.

[0025] In addition, a second small molecule can be generated based on its likely affinity for the determined target binding site, leading to other potential drug candidates. [0026] The present invention, in another form thereof, relates to a method of producing a small molecule probe comprising a withanolide compound that has been conjugated to an affinity tag via a linker group. The affinity tag may be a biotin moiety and the linker group may be a C1-C20 long hydrocarbon chain linker. [0027] The present invention, in another form thereof, relates to an affinity labeled screening compound analog comprising a withanolide covalently bonded to a linker molecule, which is itself covalently bonded to an affinity moiety tag. [0028] Without being bound to any particular theory, the present invention in another form, concerns a method for treating diverse human or animal disorders characterized by aberrant or altered levels of one or more type-Ill intermediate filament proteins selected from the group consisting of vimentin, desmin, GAP and pehphehn. The method comprises administering an effective amount of WFA or a withanolide analog compound to an individual or animal in need of treatment therefrom to bind to the one or more type-Ill intermediate filament proteins including, but not limited to, vimentin, desmin, GFAP, and pehpherin, thereby treating the disease associated with altered levels of the type-Ill intermediate filament protein(s). The disorders include, but are not limited to, angiofibroic diseases such as tumors, macular edema, proliferative diabetic retinopathy, macular degeneration, neovascular glaucoma, corneal neovascularization, and endometriosis and diseases with scar tissue formation such as scleroderma, keloids, kidney fibrosis, pulmonary fibrosis, cardiac fibrosis, chemotherapy/radiation induced lung fibrosis, pancreatitis, inflammatory bowel disease, Crohn's disease, necrotizing enterocolitis, hypertrophic scar, nodular fasciitis, eosinophilic fasciitis, Dupuytren's contracture, general fibrosis syndrome, characterized by replacement of normal muscle tissue by fibrous tissue in varying degrees, retroperitoneal fibrosis, liver fibrosis, and acute fibrosis, chronic inflammation such as Crohn's disease, ulcerative colitis, psoriasis, sarcoidosis, and rheumatoid arthritis, and organ transplant failure. With regard to GFAP, the disorders treatable include gliosis, motor neuron degeneration and scar formation, and also including, but not limited to, early and late forms of age-related macular degeneration, macular edema, Alexander disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, traumatic brain injury, developmental disorders, depression and mood disorders, stroke, and epilepsy. The treatment with WFA and its analogs includes treatment of glaucoma. BRIEF DESCRIPTION OF THE FIGURES

[0029] Figure 1 a shows the chemical structures of WFA and 12-D WS, a WFA analog,

[0030] Figure 1 b is a Coomassie blue stained gel depicting affinity isolation of

WFA-B binding proteins,

[0031] Figure 1c is a protein blot depicting WFA-B binding to a 56 kDa protein in

HUVECs,

[0032] Figure 1 d is a protein blot depicting WFA-B binding to vimentin in HUVECs, and

[0033] Figure 1 e is a protein blot depicting tetramehc soluble hamster vimentin incubated with WFA-B in the presence of different doses of WFA or inactive congener

12-D WS;

[0034] Figure 2a is a snapshot of a MD-simulated solvent accessible surface area binding structure showing WFA binding in the cleft between the A and A' α-helices of the vimentin tetramer,

[0035] Figure 2b is an enlargement of a portion of Figure 2a, showing the A-ring twist-boat and B-ring half-chair conformation of WFA accommodated deep within the binding cleft of the vimentin tetramer,

[0036] Figure 2c is a ribbon model showing hydrogen bonding between Gln324 of the vimentin A-helix and the C1 position oxygen atom, and Asp331 of the vimentin

A'-helix and the C4 hydroxyl group, and

[0037] Figure 2d depicts the alpha orientations of the C5 (OH) and C6-C7

(epoxide) of the inactive withanolide congener 12-D WS appose Cys328 of vimentin;

[0038] Figure 3a depicts tetrameric soluble hamster vimentin polymerized in the presence of 170 mM NaCI,

[0039] Figure 3b depicts polymerization of tetrameric vimentin in the presence of

25 μM WFA, [0040] Figure 3c depicts irregular fragmented aggregated structures,

[0041] Figure 3d shows that 12-D WS does not disrupt vimentin polymerization,

[0042] Figures 3e-3j are micrographs in which the BAECs are treated with DMSO

(Figures 3e, 3g and 3i) or 3 μM WFA (Figures 3f, 3h and 3j) for 18 hours, stained for vimentin using a monocolonal anti-vimentin antibody (Figures 3e and 3f), and co-stained with phalloidin-rhodamine (Figures 3g and 3h),

[0043] Figure 3k is a western blot analysis of HUVECs showing dose-responsive increases in vimentin cleavage products produced by WFA treatment, and

[0044] Figure 3i is a western blot depicting higher concentrations and longer periods of exposure to WFA cause reduction in levels of the 56 kDa protein;

[0045] Figure 4a comprises an upper panel blot depicting MCF-7 cells which lack endogenous IF proteins which were transfected with human vimentin cDNA or vector control and after 24 hour cells treated with either vehicle or 2 μM WFA,

[0046] Figure 4b is a plot showing 2OS proteasome preparation incubated with vehicle, Epoxomicin or WFA in the presence of LLVY-AMC substrate,

[0047] Figure 4c is a graph of embryonic fibroblast cell lines derived from vimentin-deficient mice and wild-type littermates treated with vehicle or 5 μM WFA,

[0048] Figure 4d is a micrograph showing BAECs transduced with WFA-modified vimentin and cells stained for vimentin,

[0049] Figure 4e is a micrograph depicting BAECs transduced with WFA-modified vimentin and stained for actin,

[0050] Figure 4f is a micrograph depicting BAECs transduced with vehicle-treated vimentin having well distributed orchestration of vimentin filaments, and

[0051] Figure 4g depicts BAECs transduced with vehicle-treated vimentin having well distributed orchestration of actin cytoskeleton; [0052] Figure 5a comprises four panels of various micrographs in which wild-type mice and vimentin-deficient mice were subjected to corneal chemical injury and treated with vehicle or WFA, and

[0053] Figure 5b are plots quantifying neovascularization from each group of mice from Figure 5a;

[0054] Figure 6a is a comparison of human type-Ill intermediate filament protein sequences vimentin, SEQ ID NO:15; desmin, SEQ ID NO:16; and GFAP, SEQ ID

NO: 17; where bold letters contain the WFA-binding unique cysteine amino acid that is present in the 2B rod domain conserved motif of these proteins, and

[0055] Figure 6b comprises the WFA-binding motif in mouse vimentin, desmin and

GFAP proteins shown as being conserved;

[0056] Figures 7a-7e are micrographs of stained mouse heart tissue, where

Figure 7a shows WFA-binding patterns, Figure 7b shows desmin expression and

Figure 7c shows extensive overlap of WFA and desmin, all in cardiac muscles of heart tissue; Figures 7d-7f are enlarged images of Figures 7a-7c, respectively;

[0057] Figure 8 is a western blot showing the effect of WFA in targeting of injury induced expression of polyubiquitinated proteins in wild type and vimentin-deficient mice

[0058] Figure 9a is a blot probed with antibody against GFAP, in accordance with the present invention, and

[0059] Figure 9b is a blot probed with anti-vimentin antibody, in accordance with the present invention;

[0060] Figures 10a-1 Od are micrographs of astrocytes stained with anti-GFAP antibody, where Figure 10a is vehicle treated, Figure 10b is 200 nM WFA, Figure 10c is

1 μm WFA and Figure 10d is 5 μm WFA;

[0061] Figures 1 1 a-1 1 d are astrocytes stained with anti-vimentin antibody, where

Figure 1 1 a is vehicle treated, Figure 1 1 b is 200 nM WFA, Figure 1 1 c is 1 μM WFA and

Figure 1 1 d is 5 μm WFA; [0062] Figures 12a-12d are micrographs depicting angiofibrotic expression of heme oxygenase-1 (HO-1 ) abrogated by WFA treatment, where Figure 12a shows no expression of HO-1 in uninjured cornea, Figure 12b shows that no staining occurs in injured corneas of vehicle-treated mice in the absence of primary antibody (Veh-Ab),

Figure 12c shows strong staining in corneal stroma in vehicle-treated mice in presence of primary antibody (Veh); and Figure 12d shows the absence of staining in WFA-treated mice in presence of HO-1 primary antibody (WA);

[0063] Figures 13a and 13b are micrographs showing frozen sections of corneal tissue stained with CD31 -FITC conjugated antibody, where Figure 13b shows that the presence of corneal vessels in stroma of vehicle-treated sample (Veh) is abundant compared to that in WFA-treated sample (WA) shown in Figure 13a;

[0064] Figures 14a, 14b and 14c are micrographs showing that corneal transketolase (TKT) expression is retained in injured corneas with WFA-treament, where

Figure 14a shows abundant expression of TKT in uninjured corneas, Figure 14b shows retention of TKT antigenicity in WFA-treated injured corneas, and Figure 14c shows TKT antigen loss in corneas of vehicle-treated mice;

[0065] Figure 15a is a protein blot showing biotin label incorporated in the 56 kDa and 50 kDa protein in a WFA-competitive manner, where the presence of endogenous

70 kDa biotinylated band is unaffected by WFA, as indicated by the arrowhead;

[0066] Figure 15b is a western blot probed with antibody to vimentin;

[0067] Figure 15c is the western blot of Figure 15a subsequently probed with

GFAP (rabbit polyclonal), which demonstrates that WFA-B binding identifies both vimentin and GFAP targets in a WFA-competitive manner;

[0068] Figure 16 is a western blot showing that WFA modulates TNF-α-induced ubiquitination in astrocytes;

[0069] Figures 17a-17d comprise a series of stained retinal tissue in which

Figure 17a is a stain of non-injured wild-type mice showing basal GFA expression localized to ganglion cell layer, Figure 17b is a stain of injured wild-type-Veh, Figure 17c is a stain of injured wild-type mice treated with WFA, and Figure 17d is a stain of injured Vim KO-Veh;

[0070] Figures 18a-18b are stains of retinal tissue which show that WFA downregulates vimentin expression in glial cells of alkali injured mice eyes during retinal gliosis, where Figure 18a is a stain for GFAP in injured WT-WFA, and Figure 18b is a stain for GFAP in injured Vim KO-WFA;

[0071] Figure 19 comprises panels A-D, corresponding to retinal tissue stains from injured WT-Veh probed with anti-GFAP (panel A) or anti-Vim (panel C), and cell stains from injured WT-WFA probed with anti-GFAP (panel B) and anti-Vim (panel D); [0072] Figure 20 comprises panels A and B, in which panel A corresponds to non-injured wild-type retinal tissue stained with CD31 , and panel B corresponds to injured WT-WFA retinal tissue stained with CD31 ; and

[0073] Figure 21 comprises panels A-D, in which panel A corresponds to non-injured wild-type retinal tissue stained for vimentin and GFAP, panel B corresponds to injured wild-type-Veh retinal tissue stained for vimentin and GFAP, panel C corresponds to injured wild-type-WFA retinal tissue, and panel D corresponds to injured Vim KO-Veh retinal tissue stained for vimentin and GFAP.

DETAILED DESCRIPTION

[0074] A novel withanolide chemical genetic probe is used to identify, in vivo, the binding target of withafehn A, which includes type-Ill intermediate filament proteins. For example, a withanolide-based small molecule is used in a screening method to screen drug candidates that target type-Ill intermediate filament proteins. The method includes introducing a tagged linker covalently bonded to the withanolide molecule to form a withanolide analog. The linker may be a C₁-C₂0 long hydrocarbon chain linker and the affinity tag can be a biotin moiety. [0075] As a result, the linker spaces the affinity group sufficiently from the small molecule moiety so as to prevent the affinity tag from interfering with the normal binding of the small molecule with its target. Accordingly, the present invention allows for a more accurate identification of a small molecule's binding site on a target and identification of the target binding sites without the affinity group interfering with the binding of the small molecule with the target.

[0076] The affinity tag and linker are selected so as to not interfere with the uptake of the small molecule analog by a target cell. The exact composition or form of the linker group is not particularly relevant so long as the linker group sufficiently spaces the affinity group from the small molecule and the linker group does not adversely effect the binding of the small molecule with its target. The affinity tag can be biotin or any other appropriate affinity tag which can be covalently bound to the linker. [0077] Better or alternative potential small molecule compounds as potential drug candidates can be generated based on their likely affinity for the determined binding site. [0078] The affinity labeled withanolide can also be used to find intermediate filament-associated proteins via chemical proteomics by extracting proteins from cells that were exposed to withanolide-biotin analog. The withanolide probes can be used to monitor expression of the intermediate filament-associated proteins, such as vimentin, desmin, GFAP and pehphehn.

[0079] Withaferin A and its analogs can be used as a treatment for intermediate filament associated protein disorders by binding to target binding sites of type-Ill filament proteins. The binding of withaferin A and its analogs to type-Ill filament proteins can treat disorders associated with the type-Ill filament proteins. These disorders include vimentin-associated cancers, such as epithelial-to-mesenchymal transition found in epithelial cancers, such as breast cancer associated with vimentin. The withaferin A analogs also can be used as a treatment for other vimentin associated disorders, including but not limited to a broad range of angiofibrotic diseases with scar tissue formation, chronic inflammation, and organ transplant failure. In addition, the withafehn A analogs can be used to treat other type-Ill intermediate filament-associated protein disorders associated with desmin, including cardiac diseases, cardiomyopathies and musculoskeletal disorders characterized by aberrant levels or altered forms of desmin, which result in protein aggregation and tissue and organ dysfunction. [0080] In addition, WFA and withanolide analogs may be used for the treatment of neurological diseases, spinal and neuromuscular injuries that are characterized by inflammatory reactive astrocytes, and disorders associated with glial cells expressing aberrant levels or altered forms of GFAP, which is associated with gliosis, motor neuron degeneration, scar formation, early and late forms of age-related macular degeneration, macular edema, Alexander disease, Alzheimer's disease, Parkinson's disease, traumatic brain injury, developmental disorders, depression and mood disorders, stroke, and epilepsy. [0081] Examples of preferred withanolide analogs include the following structures:

where:

R' is a methyl group of a phenyl group;

R" is a methyl group, ethyl group or a propyl group;

R'" is an amino acid; and

(a) Ra, Rb & Rc are -OH; or

(b) Ra, Rb and Rc are independently -O-Rd-Re,

Rd is a straight or branched alkyl with up to 12 carbons or aralkyl, Re is -OH₁-NH₂, -Cl, Br, -I, -F, CF₃, or biotin, digoxigenin, BODIFY

(δ-chloromethyl^^-difluoro-I ^^J-tetramethyl-^bora-Sa^a-diaza-s-indacene) succinate, or radioactive ligand; or

(c) Ra, Rb & Rc are independently -O-C(=O)-Rd-Re,

Rd is a straight or branched alkyl with up to 12 carbons or aralkyl, (i) Re is -OH₁-NH₂, -Cl, Br, -I, -F, CF₃ or biotin, digoxigenin, BODIFY (δ-chloromethyW^-difluoro-i ^δJ-tetramethyW-bora-Sa^a-diaza-s-indacene) succinate, or radioactive ligand -O-C(=O)-R-Rd, where R is mono- di- tri- ehyleneglycol; or

(ii) Re is OH, -NH₂, -Cl, Br, -I, -F, CF₃ or biotin, digoxigenin, BODIFY (δ-chloromethyW^-difluoro-i ^δJ-tetramethyW-bora-Sa^a-diaza-s-indacene) succinate, or radioactive ligand; or

(d) Ra, Rb & Rc are independently -O-C(=O)-X-NH-Re where X is a straight or branched alkyl with up to 12 carbons or mono, di- tri- ehyleneglycol,

Re is -OH₁-NH₂, -Cl, Br, -I, -F, CF₃ or biotin, digoxigenin, BODIFY (8-chloromethyl- 4,4-difluoro-1 ,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) succinate, or radioactive ligand or Rd is -(C=O)-Re,

Re is Cy5.5 acetate, Fluorescein acetate, 2-Naphthoxy acetate, Benzoyl, Benzoyl benzyl acetate, phloro-acetophenone acetate, 4-methoxy-2-hydroxy-benzoate, Alexa succinate, Coumarin acetate, 1-naphthyl, 1 -, or 1 ,3- or, 1 ,3,5-methoxy-benzyl, 1 to 5 fluoro-benzyl or piperazynyl; or

(e) Ra, Rb & Rc are independently -O-C(=O)-X-Y-Z, where X is a straight or branched alkyl with up to 12 carbons or mono- di- tri- ehyleneglycol

(i) Y is penta, hexa, hepta and octapeptides comprising any combination of amino acids selected from the group consisting of Leu, Ala, Pro Tyr, lie, hydroxy proline, and Cys; and Z is (argine)8, Z = -OH, -O-benzyl, -NH₂; or (ii) Y is (argine)8, and Z = penta, hexa, hepta and octapeptides comprising any combination of amino acids selected from the group consisting of Leu, Ala, Pro Tyr, lie, hydroxy proline, and Cys.

[0082] The preferred dose for administration of a withanolide compound composition in accordance with the present invention is that amount which will be effective in preventing or treating a type-Ill filament protein associated disease such as, but not limited to, cancers, a broad range of angiofibrotic diseases with scar tissue formation, chronic inflammation and organ transplant failure. One skilled in the art would readily recognize that this amount will vary greatly depending on the nature of the disorder and the condition of a patient. An "effective amount" of the withanolide compound or pharmaceutical agent to be used in accordance with the invention is intended to mean a nontoxic but sufficient amount of the agent, such that the desired prophylactic or therapeutic effect is produced. Thus, the exact amount of the withanolide compound or a particular agent that is required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular carrier or adjuvant being used and its mode of administration, and the like. Similarly, the dosing regimen should also be adjusted to suit the individual to whom the composition is administered and will once again vary with age, weight, metabolism, etc. of the individual. Accordingly, the "effective amount" of any particular withanolide compound or composition will vary based on the particular circumstances, and an appropriate effective amount may be determined in each case of application by one of ordinary skill in the art using only routine experimentation. [0083] It will be clear to one of ordinary skill in the art which type-Ill intermediate filament protein associated conditions or diseases will benefit from the present treatment, based on the role of the protein in such conditions or diseases and the effect the binding of a withanolide compound or a particular agent to the protein has on the role of protein. For example, it will be known to one of ordinary skill in the art, which angiogenesis diseases, including cancers, associate with vimentin, which will benefit from the present treatment with a withanolide compound or a particular agent in accordance with the present treatment method. Similarly, it will be known to one of ordinary skill in the art which diseases and disorders associated with desmin and GFAP production will benefit from the present treatment.

[0084] The present small molecule screening method in which small molecule analogs comprising small molecules covalently bonded to an affinity tag via a linker was established using various experiments with WFA binding proteins. These experiments confirm that affinity tagged small molecules can be used to determine the specific binding sites on target molecules, such as proteins. From knowing the target binding sites, new or additional small molecules can be developed as potential drug candidates, which have affinity for the target binding site. Thus, the present experiments demonstrate the effectiveness of a screening method which can be used to determine new potential drug candidates which bind to those target binding sites. [0085] For example, as shown by Eckes, B. et al., "Impaired wound healing in embryonic and adult mice lacking vimentin," J. Cell Sci. 1 13, 2455-2462 (2000); and van Beijnum, J. R. et al., "Gene expression of tumor angiogenesis dissected: specific targeting of colon cancer angiogenic vasculature," Blood 108, 2339-2348 (2006), both herein incorporated by reference, the small molecule angiogenesis inhibitor WFA binds to tetrameric vimentin by covalently modifying the cysteine residue in its conserved α-helical coiled coil 2B domain. Building on what is know in the art with regard to the role of vimentin with regard to disease conditions such as fibrosis and inflammation as discussed in the above background section, WFA binding to tetrameric vimentin induces filamentous aggregation in vitro, which manifests in vivo as punctuate cytoplasmic aggregates that co-localize with vimentin and actin. WFA's potent dominant-negative effect on F-actin requires vimentin expression and induces apoptosis. Finally, WFA inhibits capillary growth in a mouse model of corneal neovascularization, but this drug- induced inhibition is compromised in vimentin deficient mice. Thus, WFA is useful for incorporation as a novel chemical genetic probe of vimentin functions, and illuminates a potential new molecular target for withanolide-based therapeutics for treating angioproliferative and malignant diseases, and as a model for IF protein-related human dystrophies.

[0086] The prototypic withanolide WFA (Figure 1 a), which is abundant in the

Indian medicinal plant Withania somnifera, is a potent inhibitor of angiogenesis and tumor growth. To understand the mode of action of this natural product, a chemical genetic approach was exploited that affords isolation of small molecule binding target(s) in accordance with Crews, CM. and Splittgerber, U., "Chemical genetics: exploring and controlling cellular processes with chemical probes," Trends Biochem. Sci. 24, 317-320 (1999); Schreiber, S. L., "Chemical genetics resulting from a passion for synthetic organic chemistry," Bioorg. Med. Chem. 6, 1 127-1 152 (1998), both herein incorporated by reference. Towards this end, a novel WFA-biotin analog, WFA-B, was synthesized, as shown below.

Withaferin A Glycine

WFA-Gly-LC-LC-biotin (WFA-B)

[0087] To generate the WFA-biotinylated analog, the C27 hydroxyl group of WFA was first dehvatized with glycine, which introduced a free amine functional group for subsequent biotin coupling. The amine group was coupled with a biotinylated 12-hydrocarbon chain linker to produce WFA-Gly-LC-LC-biotin (hereinafter "WFA-B"), as taught by Yokota, Y., Bargagna-Mohan, P., Ravindranath, P.P., Kim, K. B. and Mohan, R., "Development of Withaferin A analogs as probes of angiogenesis," Bioorg. Med. Chem. Lett. 16, 2603-2607 (2006), herein incorporated by reference. [0088] WFA-B binds to a 56 kDa protein that is irreversibly targeted by WFA in vivo in human umbilical vein endothelial cells (HUVECs). To isolate this WFA-target, bovine aortic endothelial cells (BAECs) were treated with WFA-B, and biotinylated proteins were affinity-purified over NEUTRAVIDIN^® columns and fractionated by gel electrophoresis. Gels stained with Coomassie blue dye confirmed the isolation of this 56 kDa protein (Figure 1 b). [0089] Affinity isolation of WFA-B-binding proteins was conducted as follows.

BAECs were preincubated with DMSO (Veh) or with 5 μM WFA for 30 minutes and subsequently with 5 μM WFA-B for 2 hours. Cell lysates prepared in 1 % Triton X-100 buffer were purified over NEUTRAVIDIN^® affinity columns and subjected to SDS-PAGE. The gel was stained with Coomassie blue. The arrow points to the 56 kDa protein band and asterisks mark the co-eluted 51 kDa and 43 kDa proteins.

[0090] LC-MS/MS characterization of this protein identified the 56 kDa protein as vimentin (27% protein coverage), an IF protein that is abundant in mesenchymal cells. To further support that vimentin is bound by WFA-B in vivo, HUVECs were treated with WFA-B in the presence and absence of unconjugated WFA, and total cellular lysates were gel-fractionated and protein blots probed with streptavidin-HRP. Referring to Figure 1 c, the blot confirms that WFA-B binds to the 56 kDa protein in HUVECs. This experiment was conducted using cells preincubated with DMSO or WFA for 30 minutes and subsequently with WFA-B for 2 hours. Soluble proteins extracted in 1 % Triton X-100 were fractionated by SDS-PAGE and blotted. This blot, as well as those of Figures 1 d and 1 e, were developed with Streptavidin-HRP. Biotin label is incorporated in this 56 kDa protein in a WFA-competitive manner.

[0091] As shown in the blot of Figure 1 d, WFA-B binds vimentin in HUVECs. The blot of Figure 1 d was produced using cell cultures preincubated with DMSO (Veh) or with 5 μM WFA or 1 μM (WFA^*) for 30 minutes and subsequently with 5 μM WFA-B for 2 hours. Cell lysates prepared in 1 % Triton X-100 buffer were purified over NEUTRAVIDIN^® affinity columns and subjected to SDS-PAGE and western blotted with antivimentin V9 antibody.

[0092] Referring now to Figure 1 e, in vitro ligand binding assays were performed which show that vimentin is bound by WFA-B in a WFA-competitive manner. The assays were performed using soluble hamster vimentin incubated with different doses of WFA or inactive congener 12-D WS for 1 hour and subsequently with either 0.3 or 1 μM WFA-B for 1 hour. The proteins were fractionated by SDS-PAGE, blotted and biotinylated adduct was detected by streptaviding-HRP with chemiluminescence. [0093] The WFA-B-affinity chromatographic approach (Figure 1 b) also led to co-isolation of 51 kDa β-tubulin and 43 kDa β-actin. Since vimentin has been reported to interact with actin and it is a cargo for microtubule-dependent transport, it is not unexpected that actin and tubulin would co-isolate with WFA-B-modified vimentin. [0094] Next, to identify the amino acid residue(s) of vimentin covalently modified by WFA, purified hamster vimentin was incubated with WFA and the protein-ligand complexes subjected to tryptic digestion and LC-MS/MS analysis. A search for the position of adduct formation (a molecular mass shift of 470) in the tryptic fragments of vimentin revealed that the sole cysteine residue at position 327 (position 328 in human vimentin) in the α-helical coil coiled 2B rod domain of vimentin is uniquely modified by WFA (data now shown). A three-dimensional model of the WFA-vimentin complex was developed using x-ray crystal structures of vimentin and WFA. Molecular modeling studies revealed a stable binding mode for WFA in the surface binding pocket of tetramehc vimentin between the pair of head-to-tail α-helical dimers (Figure 2a). In this simulated model, the C3 and C6 carbons of WFA lie in close proximity to the cysteine residue in the vimentin A-helix (Figure 2b), permitting a nucleophilic attack by this thiol group on the electrophilic carbon centers (Figure 2c). Remarkably, the amino acid residues of vimentin (Gln324, Cys328 and Asp 331 ) that make contact with WFA (Figure 2c) are identical from fish to mammals, as shown in the table below.

Species Sequence Sequence Listing Identifier

Human: RQAKQESTEYRRQVQALTCEVDSLKGTNESLERQMREMEEN SEQ ID NO: 1

Chimpanzee: RQAKQESTEYRRQVQAPTCEVDSLKGTNESLERQMREMEEN SEQ ID NO: 2

Macaque: RQAKQESNEYRRQVQALTCEVDSLKGTNESLERQMREMEEN SEQ ID NO: 3

A.Green monkey: RQAKQESNEYRRQVQALTCEVDSLKGTNESLERQMREMEEN SEQ ID NO: 4

Rhesus monkey: RQAKQESNEYRRQVQALTCEVDSLKGTNESLERQMREMEEN SEQ ID NO: 5

Pig: RQAKQESNEYRRQVQALTCEVDSLKGTNESLERQMREMEEN SEQ ID NO: 6 Species Sequence Sequence Listing Identifier

^~D5gi RQAKQESNEYRRQVQALTCEVDSLKGTNESLEHQMREMEEN SEQ ID NO: 7

Bovine: RQAKQESNEYRRQVQALTCEVDSLKGTNESLERQMREMEEN SEQ ID NO: 8

Hamster: RQAKQESNEYRRQVQALTCEVDSLKGTNESLERQMREMEEN SEQ ID NO: 9

Mouse: RQAKQESNEYRRQVQALTCEVDSLKGTNESLERQMREMEEN SEQ ID NO: 10

Rat: RQAKQESNEYRRQVQALTCEVDSLKGTNESLERQMREMEEN SEQ ID NO: 11

Chicken: RQAKQEANEYRRQIQALTCEVDSLKGSNESLERQMREMEEN SEQ ID NO: 12

Frog: RQAKQETSDFRRQIQALTCEVDSLKGSNESYERQMREMEEN SEQ ID NO: 13

Fish (Trout): RQAKQEANEYRRQVQALTCEVDSLKGTNESMERQMRELEES SEQ ID NO: 14

[0095] As shown from the table, the binding site of WFA in vimentin is evolutionary conserved. Amino acid sequences from within the 2B domain of vimentins of different vertebrate species reveal the overall high level of sequence conservation. The specific amino acids that make contact with WFA (bold), as determined from our molecular modeling studies, are evolutionarily identical.

[0096] Another feature illustrated by this model is that the orientation of C27 hydroxyl group places it outside the binding cleft; thus, biotinylated WFA is able to retain the flexibility to bind immobilized NEUTRAVIDIN^® after modifying tetramehc vimentin. On the other hand, the simulated model of vimentin/the inactive congener 12-D WS (Figure 2d) reveals that steric hindrances from the C5 alpha-hydroxyl and the C6-C7 epoxide of WFA are likely to prevent such a nucleophilic attack by the reactive thiol group on 12-D WS. This distinction between these two withanolides in their binding modes with vimentin is further corroborated by comparing the molecular docking- simulated internuclear distances between WFA and 12-D WS with vimentin, and this data is consistent with lack of in vitro binding activity of 12-D WS to vimentin (Figure 1 d) and the absence of its endothelial cell targeting activity.

[0097] The clinical importance of the cysteine residue in vimentin lies in its propensity for being preferentially oxidized in vimentin compared to other cytoskeletal proteins from rheumatoid arthritis patients. Because this unique cysteine residue under oxidizing conditions can participate in disulfide cross-linking between a pair of vimentin dimers leading to disruption to the filament structure in vitro, how chemical modification of cysteine by WFA affects the vimentin IF structure was investigated. Employing soluble tetrameric vimentin in filament polymerization assays in vitro, although WFA at high doses does not block filament assembly per se, the drug induces formation of filamentous aggregates, many of which display amorphous condensed structures as revealed in negatively stained transmission electron micrographs (Figures 3a-3j). [0098] Figure 3a depicts tetrameric soluble hamster vimentin polymerized in the presence of 170 mM NaCI by incubation at 37°C for 1 hour. The protein was fixed with 0.5% glutaraldehyde, stained with uranyl acetate and observed by transmission electron microscopy. The presence of vehicle solvent does not interfere with filament formation. [0099] Figure 3b depicts polymerization of tetrameric vimentin in the presence of

25 μM WFA, which produces extensive filamentous aggregates, and Figure 3c depicts many irregular fragmented aggregated structures. As shown in Figure 3d, 12-D WS (25 μM) does not disrupt vimentin polymerization.

[00100] BAECs treated with DMSO (Figures 3e, 3g, 3i) or 3 μM WFA (Figures 3f, 3h, 3j) for 18 hours were stained for vimentin using a monoclonal anti-vimentin antibody (green) and co-stained with phalloidin-rhodamine (red). The vimentin (Figures 3e, 3f) and phalloidin-stained images (Figures 3g, 3h), in fluorescence overlap, reveal the presence of numerous cytoplasmic particulate granules that co-stain for vimentin and disrupted F-actin in WFA-treated cells compared to controls (Figures 3i, 3j). Figure 3k is a western blot analysis of HUVECs which shows dose-responsive increases in vimentin cleavage products (arrows) with WFA treatment after 2 hours, as detected with the monoclonal anti-vimentin V9 antibody. Figure 3I depicts higher concentrations and longer periods of exposure to WFA (4 hours) cause reduction in levels of the 56 kDa protein (asterisk) and increased abundance of cleavage products of vimentin (arrows). [00101] The amorphous condensed structure phenotype is not observed with lower doses of WFA or equivalent high dose of the inactive congener 12-D WS. To further corroborate that vimentin aggregation by WFA treatment is associated with perturbation of the cytoskeleton structure, the drug effects in BAECs were investigated by immunostaining. Cells treated with 3 μM WFA showed condensation of vimentin filaments around the perinuclear region and the presence of numerous, vimentin-positive staining particles in the cytoplasm (compare Figure 3e with Figure 3f). Importantly, these vimentin particulates strongly co-stain for actin (compare Figure 3h with Figure 3j), exemplifying the role of the WFA-binding domain in control of cytoskeletal structure through the actin-binding activity of vimentin 2B domain. [00102] As it became apparent that vimentin targeting by WFA may initiate signaling events leading to cellular apoptosis, the effect of WFA on soluble tetramehc vimentin was investigated. Using western blot analysis of soluble proteins from HUVECs treated with WFA, dose-dependent (above 2 μM) increases in vimentin cleavage products became detectable by 2 hours after drug treatment (Figures 3k and 3I). Additionally, two-dimensional-western blot analysis reveals that WFA-B also causes several full-length (-53-56 kDa) isoforms of vimentin to disappear in a manner similar to WFA-induced effects in HUVECs (data not shown), data that further supports the in vivo drug-mimetic effect of this WFA analog.

[00103] Since IF protein aggregation has been shown to negatively impact the ubiquitin proteasome pathway (UPP) resulting in proteasome inhibition, an investigation as to whether the UPP-targeting activity of WFA is also regulated in a vimentin- dependent manner was undertaken. Employing the widely used MCF-7 vimentin- deficient model of Bar, H. et al., "Forced expression of desmin and desmin mutants in cultured cells: impact of myopathic missense mutations in the central coiled-coil domain on network formation," Exp. Cell. Res. 312, 1554-1565 (2006), herein incorporated by reference, it was determined that co-treatment with WFA increases levels of polyubiquitinated proteins in vimentin transfected MCF-7 cells, but not in the vector controls (Figure 4a). MCF-7 cells that lack endogenous IF proteins were transfected with human vimentin cDNA or a vector control and after 24 hours cells were treated with either vehicle or 2 μM WFA for 1 hour. Cell lysates were prepared and equal amounts of protein subjected to SDS-PAGE and protein blots probed with anti-ubiquitin antibody. Blots were re-probed with anti-GAPDH antibody.

[00104] Referring to Figure 4b, 2OS proteasome (bovine) preparation was incubated with vehicle (Con), 0.5 μM, 1 .0 μM and 10 μM WFA or with 10 nM Epoxomicin (Epx) in the presence of LLVY-AMC substrate at room temperature. Fluorescence readings from triplicate samples were obtained at different time intervals from the 96-well plate using excitation at 355 nm and emission at 430 nm. The release of product was plotted against time for each concentration of inhibitor.

[00105] Referring to Figure 4c, embryonic fibroblast cell lines derived from vimentin-deficient mice and wild-type littermates were treated with vehicle or 5 μM WFA for 24 hours. Cells were harvested and stained with annexin V-FITC and propidium iodide and extent of apoptosis assessed by flow cytometry. The fold-apoptotis for drug over vehicle-treated samples for each cell line was plotted (n = 2 experiments). [00106] Referring to Figure 4d, BAECs were transduced with WFA-treated vimentin and cells were stained after 18 hours. Cells transduced with WFA-modified vimentin show condensed vimentin filaments localized largely in and around the nucleus and the presence of vimentin-staining particulates (Figure 4e). Cells transduced with vehicle- treated vimentin show well distributed orchestration of vimentin IFs (Figure 4f) and actin cytoskeleton (Figure 4g). Results representative of n = 3 experiments are shown. [00107] Referring back to Figure 4a, to corroborate that vimentin-targeting and not the possible direct inhibition of the 2OS proteasome by WFA mediates its UPP-targeting function, tests were conducted on WFA in proteasome kinetic assays. Whereas epoxomicin, a highly selective proteasome inhibitor, significantly inhibits the 2OS proteasome's major catalytic function at 10 nM, WFA, even at cytotoxic concentrations of 10 μM, minimally inhibits this catalytic activity (Figure 4b). The Kobs/[l] (IVT¹S^"1), a measure of the efficiency of inactivation, was calculated to be 340 ± 80 (0.5-10 μM) for WFA, while that of epoxomicin is 44,510 ± 7,000 (10-75 nM). Contrary to a recent report of Yang, H., Shi, G. & Dou, Q. P., "The tumor proteasome is a primary target for the natural anticancer compound Withafehn A isolated from 'Indian Winter Cherry'," MoI. Pharmacol. Nov 8 online (2006), herein incorporated by reference, the presently acquired data provides evidence that WFA-modified vimentin may mediate sequestration of ubiqitinated proteins and result in proteasome inhibition in vivo. Thus, employing cell lines derived from vimentin-deficient and isogenic wild-type mouse strains confers enhanced resistance to drug-induced apoptosis with wild-type cells having a 7- fold greater rate of apoptosis (Figure 4c). Furthermore, exploiting a protein transduction methodology to introduce the WFA-modified, dominant-negative, vimentin tetramers into cells, immunostaining demonstrates that endogenous vimentin IFs and F-actin are found to aggregate, whereas cells transduced with vehicle-treated vimentin do not produce this phenotype (compare Figure 4d with Figure 4f). Collectively, these findings demonstrate that vimentin-targeting by WFA can affect the severe alterations in the cytoskeleton architecture (Figures 4e and 4g).

[00108] Since WFA exerts potent angiogenesis inhibitory activity in vivo, drug activity on de novo capillary growth in vimentin deficiency was investigated. The mouse model of injury-induced corneal neovascularization shows that WFA markedly suppresses neovascularization in wild-type mice (73% inhibition, n = 8; P = 0.002), whereas it only marginally attenuates neovascularization in vimentin-null mice (29% inhibition; n = 10; P = 0.005) (Figure 5), revealing that inhibition of capillary growth by WFA is mediated predominantly by vimentin. Interestingly, the vascularization response of vimentin-deficient mice is not as extensive as in wild-type mice, which is consistent with previous reports on impaired angiogenesis in vimentin deficiency. [00109] WFA has been recently shown to bind annexin Il in cancer cell lines, as shown by Falsey, R. R. et al., "Actin microfilament aggregation induced by Withaferin A is mediated by annexin II," Nat. Chem. Biol. 2, 33-38 (2006), herein incorporated by reference. However, this 36 kDa protein is not detected in in vivo WFA-affinity purified proteins from human or bovine endothelial cells (Figure 1 b), nor was it detected by western blotting of N EUTRAVI Dl N^®-puhfied proteins in our studies (data not shown). Thus, differences in the construction of the biotinylated WFA analogs (e.g., inclusion of a long, linear hydrocarbon linker between the natural product and biotin), or their applications for target isolation (in vitro versus in vivo labeling), could account for different targets being bound to WFA. It is also possible that WFA has context-related, binding specificities for different targets in vivo, a speculation that will need to be rigorously tested.

[00110] Vimentin is the primary target of WFA in vivo, and this small molecule can perturb vimentin function. Use of a small molecule to inhibit vimentin function can serve as a complementary approach to classical genetic studies of disorders of IFs, shown in Bar, H., Strelkov, S. V., Sjoberg, G., Aebi, U. and Herrmann, H., "The biology of desmin filaments: how do mutations affect their structure, assembly, and organisation?," J. Struct. Biol. 148, 137-152 (2004), herein incorporated by reference. Thus, WFA and derivative steroidal-lactones represent a useful chemical genetic tool for studies of the type-Ill IF proteins. As vimentin modulates the immune response and is overexpressed in prostate and other cancers, WFA holds great promise as a potential lead for the development of small molecule therapeutics.

[00111] Based on the foregoing experiments and test results, there are many potential applications for development of small molecule based probes and analogs. For example, the binding of the WFA probe can be used to modulate vimentin protein function and, thus, the binding pocket can serve as a novel target for the development of small molecule agonists or antagonists of vimentin function. In another use, one can monitor intracellular transport and localization of vimentin protein to different subcellular locations in vivo by conjugating a detection label, such as a fluorescent tag, radiolabel or biotin, to WFA. Since these probes bind selectively and covalently to vimentin, they allow one to follow this marker in a whole range of known tissues from a variety of proliferative diseases, such as cancer, arthritis, diabetes, etc., serving essentially as a sensitive diagnostic tool.

[00112] In another use, WFA-based analogs can be employed to more potently modify vimentin function and thereby generate new classes of pharmacological drugs for treatment of human diseases. In addition, the WFA-vimentin binding site information could be employed to probe similar binding sites in other IF proteins, for which the cysteine group is known to be present in a similar coiled coil domain of the IFs. [00113] The following experimental conditions provide additional background and details regarding the aforementioned experiments, which support the present screening method as an effective method for identifying potential small molecule drugs and their respective binding sites.

[00114] All cell culture supplies were purchased from Gibco unless otherwise specified. Antibodies were from SantaCruz Biotechnology unless otherwise specified. WFA and 12-D WS were obtained from Chromadex and stock solutions were freshly prepared in DMSO. All cell reagents were purchased from Invitrogen. [00115] HUVECs and BAECs cells were obtained from Cascade Biologicals and cultured according to vendor's protocols. MCF-7 breast cancer cell line was obtained from ATCC and cultured in RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS). MFT-16 cells from embryo fibroblasts of vimentin homozygous-deficient mice (Vim^"'^") and MFT-6 cells from embryo fibroblasts of wild-type (Vim^+/+) mice were obtained from Robert Evans (University of Colorado, Denver) and cultured in F12:DMEM (1 :1 ) medium supplemented with 5% FBS. All cells were cultured in humidified incubators at

37°C-5% CO₂ conditions. Isolation of WFA-B-binding proteins by affinity chromatography. [00116] The synthesis and chemical characterization of WFA-B, demonstration of its biological activity in endothelial cells, and use to identify biotinylated proteins from HUVECs was previously reported (Yokota 2006). For scale-up studies, BAECs were pre-incubated with DMSO or 5μM WFA for 1 hour and subsequently treated with 5 μM WFA-B for 2 hours. Cells were washed in ice-cold phosphate buffered saline (PBS) and cytoplasmic extracts were prepared in Buffer A (5 mM Tris, pH 7.6, 50 mM NaF, 1 % Triton X-100, 5 mM EGTA), supplemented with a proteinase inhibitor cocktail (Roche). After centhfugation, equal amounts of protein were pre-cleared on agarose beads (Sigma) to remove non-specific agarose-binding proteins. The beads were centhfuged and pre-cleared cell lysates were repeatedly loaded three times on columns containing NEUTRAVIDIN^®-agarose beads (Pierce) to maximize immobilization of biotinylated proteins. After extensive washing with ice-cold Buffer A, bound biotinylated proteins were eluted in Laemeli gel loading buffer containing β-mercaptoethanol and fractioned by SDS-PAGE on 15 x 15 cm gels. Gels were stained with Coomassie blue dye and bands corresponding to 56 kDa, 51 kDa and 43 kDa protein were excised for mass spectrometry analysis.

Identification of affinity purified proteins by LC-MS/MS.

[00117] All mass spectra reported in this disclosure were acquired by the University of Kentucky Mass Spectrometry Facility. Gel pieces were digested with trypsin, and LC/MS/MS spectra were acquired on a ThermoFinnigan LCQ "Classic" quadrupole ion trap mass spectrometer (Finnigan Co., San Jose, CA). Separations were performed with an HP 1 100 high performance liquid chromatograph modified with a custom splitter to deliver 4 μl/min to a custom C18 capillary column (300 μm inner diameter x 15 cm).

Gradient separations consisted of a 2-minute isocratic step at 95% water and 5% acetonitrile (both phases contain 0.1 % formic acid). The organic phase was increased to 20% acetonitrile over 8 minutes and then increased to 90% acetonitrile over 25 minutes; held at 90% acetonitrile for 8 minutes and then increased to 95% in 2 minutes; finally they were returned to the initial conditions in 10 minutes (total acquisition time 45 minutes with a 10 minute recycle time). Tandem mass spectra were acquired in a data-dependent manner. Three microscans were averaged to generate the data-dependent full-scan spectrum. The most intense ion was subjected to tandem mass spectrometry, and three microscans were averaged to produce the MS/MS spectrum. Masses subjected to the MS/MS scan were placed on an exclusion list for 2 minutes. Resulting MS-MS spectra were searched against mammalian proteins in the Swiss-Prot database using the Mascot search engine (Matrix Science).

3D model of the human vimentin fragment.

[00118] The initial coordinates of 2B human vimentin fragment used in our computational studies came from the X-ray crystal structure (pdb code: 1 gk4.pdb) deposited in the Protein Data Bank. To encompass the structure of the protein environment surrounding the active residue Cys328, the missing residues of the

2A fragment (i.e. residues #313 to #327 in A and B helices) were built using the α-helical template structure and the automated homology modeling tool Modeler/lnsightll software

(Accelrys, Inc.). Then, the best 3D model was solvated in water and refined by performing a long-time molecular dynamics (MD) simulation in water.

Molecular docking.

[00119] To explore the possible vimentin-ligand binding mode, the first step was to dock the ligand, i.e. WFA or 12-D WS, to vimentin tetramer fragment by virtue of their geometric complementarity. The molecular docking for each vimentin-ligand binding was carried out in the same way as previously done when studying other protein-ligand binding systems, as disclosed in Hamza, A. and Zhan, C-G., "How Can (-)- Epigallocatechin Gallate from Green Tea Prevent HIV-1 Infection? Mechanistic Insights from Computational Modeling and the Implication for Rational Design of Anti-HIV-1 Entry Inhibitors," J. Phys. Chem. B 1 10, 2910-2917 (2006), herein incorporated by reference. [00120] A ligand-binding site was defined as that consisting of the residues within a sphere (with a radius of 20 A) centered at Cys328 residue. The ligand was initially positioned at ~10 A in front of Cys328 of the binding site. The initial docking calculations were performed on the ligand with the vimentin fragment binding site using the 'automatic docking' Affinity module of the lnsightll package (Accelrys, Inc.). The Affinity methodology uses a combination of Monte Carlo type and simulated Annealing procedures to dock the guest molecule (the ligand) to the host (the receptor). The vimentin-ligand binding structure obtained from the initial docking was further refined by performing an MD simulation in a water bath.

Western Blotting Experiments.

[00121] After treatments, cells were washed in PBS and extracts having equal amounts of proteins were subjected to SDS-PAGE on 4-20% Ths-glycine gels (BioEpress) and transferred to Immun-Blot PVDF membrane (Bio-Rad) using standard techniques. Primary antibodies were diluted in 5% non-fat dry milk Tris buffered saline, 0.02% Tween-20 (NFDM-TBST) at the concentration of 1 :500, and secondary antibodies were used at 1 :1000 dilution. Blots were extensively washed in TBST buffer and developed using enhanced chemiluminescence method (Amersham) and exposed to x-ray film.

Transfection Studies.

[00122] MCF-7 cells were transfected with a pCMV6-XL5 vector containing the human vimentin cDNA under CMV promoter according to vendor instructions (Origene). Control samples were transfected with an empty vector (PCMV6-XL4). Transfected cells were allowed to recover for 12 hours and subsequently treated with 2μM WFA for 18 hours. Equal amounts of protein lysates were then subjected to western blotting and probed with mouse monoclonal antibody against ubiquitin proteins.

Protein Transduction Studies.

[00123] Tetramehc vimentin (0.5 μg) (Cytoskeleton) was incubated with 10μM WFA or an equivalent amount of vehicle (DMSO) for 1 hour at 37°C to form protein-WFA adducts. Vimentin-WFA or vimentin alone (0.5 μg) were mixed with the CHARIOT™ protein transduction reagent (Active Motif) and incubated for 30 minutes at 24°C to form complex according to instructions of manufacturer. The protein-CHARIOT™ complex was subsequently added to BAECs in serum-free medium and incubated for 1 hour at 37°C-5% CO2 conditions. Fresh complete medium was then added and cells were incubated for an additional 18 hours under normal culture conditions. Cells were processed for immunohistochemistry analysis, as described.

Apoptosis by Flow Cytometry.

[00124] To assess the apoptosis activity of WFA, embryonic fibroblast vimentin- deficient cell lines (Vim^"'^"; MFT-16) and wild-type (Vim^+/+; MFT-6) cells were treated with 5 μM WFA or an equivalent amount of vehicle (DMSO) for 24 hours at 37°C-5% CO₂ conditions in complete medium. Apoptotic cells were measured by using the VYBRANT™ Apoptosis Assay Kit (Molecular Probes) according to the manufacturer's instructions. Flow cytometric analysis was conducted at the University of Kentucky Core Flow Cytometry Center. Cell Staining Procedures and Fluorescence Imaging. [00125] After treatments, BAECs were washed with PBS and fixed with 4% paraformaldehyde for 5 minutes. Cells were then permeabilized with 0.1 % Thton-X in PBS for 20 minutes at 4°C and blocked for 30 minutes in 3% BSA to prevent nonspecific binding. Rabbit polyclonal vimentin antibody (Vim) or mouse monoclonal vimentin antibody (V9) was applied to cells for 1 hour at 24°C at 1 :400 dilution in PBS. After extensive washes with PBS, cells were incubated with anti-rabbit FITC-conjugated secondary antibody (1 :500) or anti-mouse Texas Red-conjugated secondary antibody (1 :500) for 30 minutes. After washing three times with PBS, cells labeled with Vim-antibody were incubated with phalloidin conjugated to Rhodamine (1 :200) for 20 minutes. After extensive washes (1-2 hours), cells were visualized using a Nikon TE2000 microscope.

Transmission Electron Microscopy of Vimentin Filaments.

[00126] Tetramehc hamster vimentin was subjected to in vitro filament formation assays using vendor-supplied reagents and instructions (Vimentin Filament Biochemistry Kit, Denver, CO). Vimentin (0.5 mg/ml) was mixed with WFA (5 μM or 25 μM), DMSO or 12D-WS (25 μM) in filament polymerization buffer (170 mM NaCI final concentration) and incubated for 1 hour at 37°C. Protein was immediately fixed in 0.5% glutarldehyde and stained with uranyl acetate and applied to copper grids for EM staining (University of Kentucky Core Microscopy and Imaging Facilities). Over 100 grids for each treatment were viewed at 80 kV on a FEI Biotwin 12 transmission electron microscope and 25 representative images were collected. The experiment was repeated in entirety.

Enzyme Kinetic Studies.

[00127] /(association values were determined as follows. Inhibitors were mixed with a fluorogenic peptide substrate and assay buffer (20 mM Tris (pH 8.0), 0.5 mM EDTA, and 0.035% SDS) in a 96-well plate. The chymotrypsin-like activity was assayed using the fluorogenic peptide substrates Suc-Leu-Leu-Val-Tyr-AMC (Sigma-Aldhch). Hydrolysis was initiated by the addition of bovine 2OS proteasome, and the reaction was followed by fluorescence (360-nm excitation/460-nm detection) using a Microplate Fluorescence Reader (FL600; Bio-Tek Instruments, Inc., Winnoski, VT) employing the software KC4 v.2.5 (Bio-Tek Instruments, Inc., Winooski, VT). Reactions were allowed to proceed for 60-90 minutes, and fluorescence data were collected every 1 minute. Fluorescence was quantified as arbitrary units and progression curves were plotted for each reaction as a function of time. k_Obse_rved/[l] values were obtained using PRISM program by nonlinear least squares fit of the data to the following equation: fluorescence = v_st + [{v₀ - Vs)/k_Observed][1 - exp(-k_observed t)], where Vo and v_s are the initial and final velocities, respectively, and /(observed is the reaction rate constant. The range of inhibitor concentrations tested was chosen so that several half-lives could be observed during the course of the measurement. Reactions were performed using inhibitor concentrations that were <100-fold of those of the proteasome assayed.

Corneal Neovascularization Assays in Mice.

[00128] Vimentin homozygous-deficient mice (vim^"'^") and vimentin-heterozygous- deficient mice (vim^+/") in the 129/Svev background were obtained from David Markovitz (University of Michigan Medical Center) and breeding colonies established at the University of Kentucky. All mice were housed in specific pathogen-free cages in designated lab animal housing facilities. Age-matched littermates were genotyped by polymerase chain reaction, as described, and vim^"'^" and vim^+/+ mice were employed for corneal vascularization experiments. In brief, mice between 4-6 weeks of age were anesthetized by intraperiteoneal injection (i.p.) of ketamine and xylazine. Corneas were topically anesthetized by application of proparacain drop and 1 microliter drop of dilute

0.15 M sodium hydroxide was applied for 1 minute. The cornea was immediately washed extensively in saline solution and corneal and limbal epithelium gently removed by scraping with a blunt Tooke corneal knife. The cornea was topically treated with Atropine eye drop and covered with tobramycin and erythromycin antibiotic eye ointment. Upon recovery from anesthesia, mice were replaced in cages and monitored by trained personnel for resumption of normal activity. WFA (2 mg/kg solubilized in DMSO) or vehicle (DMSO) was injected intraperiteoneally in some groups of mice after their recovery from corneal injury, and subsequently every day after for a period of 10 days. Mice were humanely sacrificed and eyes enucleated. Mouse eyes were washed in PBS and dissected in half to obtain anterior segment half. The scleral tissue was carefully removed and corneal buttons were prepared. Corneal tissues were fixed in 100% acetone for 20 minutes, washed in PBS for 1 hour and blocked for 18 hours in 1 % BSA- PBS at 4°C. Cornea whole mount staining was performed by incubating tissues in FITC- conjugated rat anti-mouse CD31 antibody (BD Pharmingen; 1 :333 dilution in 1 % BSA- PBS) for 12 hours. Excess stain was washed away by incubation for 24 hours at 4°C in 1 % BSA-PBS. Corneal whole mounts were affixed to glass slides, cover-slipped and photographed on a Nikon TE2000 microscope. Fluorescence was quantified by importing images in NIH ImageJ, as taught by Ambati, B. K. et al., Sustained inhibition of corneal neovascularization by genetic ablation of CCR5, Invest. Ophthalmol. Vis. Sci. 44, 590-593 (2003), hereinafter incorporated by reference.

[00129] Type-Ill intermediate filament proteins vimentin, desmin and GFAP include conserved WFA-binding cysteine amino acid sites. Figure 6a depicts a comparison of human type-Ill intermediate filament protein sequences for human vimentin, desmin and GFAP, SEQ ID NOs:15-17, respectively, where the boldfaced letters contain the WFA-binding unique cysteine amino acid site present in the 2B rod domain conserved motif of these proteins. Figure 6b shows the WFA binding sequence for mouse vimentin, desmin and GFAP, showing that this motif is conserved among the aforementioned three type-Ill intermediate filament proteins. Accordingly, WFA and its analogs, which target one of these type-Ill intermediate filament proteins, will target other type-Ill intermediate filament proteins having the conserved binding region. Therefore, WFA and its analogs can be used to target type-Ill intermediate filament proteins and thereby treat disorders associated with aberrant levels or altered forms of the type-Ill intermediate filament proteins.

[00130] Referring now to Figures 7a-7f, the stained slides shown in Figures 7a-7f demonstrate that WFA binds to mouse cardiac cells expressing desmin and thus provides evidence which supports the use of WFA and its analogs to bind type-Ill intermediate filament proteins, and thereby treat disorders associated with desmin production.

[00131] The experiments which produced the results shown in Figures 7a-7f were taken from mouse hearts freshly collected and frozen cryosections 5 μm thick placed on glass slides. Slides were wetted in phosphate buffered saline (PBS) for 1 minute and incubated with streptavidin (1 :100 diluted in PBS) to block endogenous biotinylated proteins. Slides were washed in PBS and then incubated with 500 nM WFA-biotin for 30 min at room temperature, followed by washes in PBS. The slides were fixed in 100% acetone for 5 min on ice, washed in PBS and blocked with 2% goat serum/1 % bovine serum albumin/0.5% Triton X-100/0.01 % Tween 20 in PBS for 30 min. The slides were incubated with primary antibody against desmin (1 :50 dilution) for 1 h. The slides were washed for 30 min, incubated with streptavidin-FITC conjugate (1 :5000 dilution) and anti- rabbit secondary antibody-Texas red conjugate (1 :1000 dilution) for 30 min. The slides were washed exhaustively with PBS for 3 h, and cover slipped with mounting medium containing DAPI and viewed on a Nikon TE2000 fluorescent microscope. Digital photographs using a 4OX objective were taken and overlay of WFA binding pattern signals (Figure 7a) with desmin expression (Figure 7b) show extensive overlap (Figure 7c) in the cardiac muscles of heart tissue. Enlarged images of WFA binding pattern signals (Figure 7d) with corresponding desmin expression (Figure 7e) and color overlap (Figure 7f) reveal that cardiac muscle striations of heart tissues are labeled, confirming WFA binding to desmin. Nuclei stained with DAPI are in blue (Figures 7c and 7f). [00132] Referring now to Figure 8, the western blot shown demonstrates that treatment with WFA strongly reduces protein ubiquitination, thus demonstrating that dual targeting of injury-induced expression of vimentin and desmin provide a potent drug effect of WFA on the ubiquitin proteasome pathway. Wild type (Vim+/+) and vimentin- deficient (Vim-/-) SVeV mice were subjected to mild alkali burn (0.15 N NaOH) with epithelial and limbal scrape injury to the cornea. Mice were either treated with vehicle or with WFA (2.5 mg/kg/d) each day for 7 days. Corneal tissue from each group (n= 4) was collected and total protein extracted. Equal amount of protein was subjected to SDS- polyacrylamide gel electrophoresis and protein was blotted on nylon membrane. The blots were probed with monoclonal antibody against ubiquitin. WFA treatment strongly reduced protein ubiquitination in wild-type Vim+/+ mice and to a lesser extent in Vim-/- mice compared to respective vehicle treated control groups, revealing that in the cornea the dual targeting of injury-induced expression of vimentin and desmin may be responsible for the potent drug effect of WFA on the ubiquitin proteosome pathway. [00133] Referring now to the experiments with data shown in Figures 9-1 1 , astrocytes in culture can express vimentin in addition to GFAP. This unique feature allows for assessing the expression of both these IF proteins in this model in response to drug treatment. To determine whether WFA targets the expression of GFAP in astrocytes, a rat astrocyte brain-derived cell culture model (ScienCell Research Laboratories, Carlsbad, CA) was used. WFA activity was investigated in the presence and absence of inflammatory stimulus from the cytokine TNF-α, which is known to induce stress response in astrocytes.

[00134] Referring now specifically to Figures 9a and 9b, western blot analysis demonstrated that WFA dose-dependently caused downregulation of soluble GFAP expression in astrocytes, with 2 μM potently causing its expression to be abrogated (Figure 9a). The blots were produced from astrocytes plated in medium containing 10% fetal bovine serum. Others were additionally incubated with 5 ng/ml TNF-α for a period of 18 h. Both untreated and TNF-treated cell cultures were subsequently treated for 2 h with different doses of WFA. Cell extracts were prepared in Tris buffer containing 1 % NP-40 and 200 mM NaCI to isolate soluble GFAP pools. Equal amounts of protein were subjected to 4-20% PAGE and blotted onto nylon membrane. The blot was probed with antibody against GFAP (1 :20,000 dilution; Abeam, Cambridge, MA) and developed using ECL with exposure to x-ray film.

[00135] In Figure 9a, the GFAP band is indicated by the arrowhead, while a nonspecific (ns) band that reveals equal protein loading is indicated by the small arrow. Blots were re-probed with anti-vimentin antibody (1 :1000 dilution; Abeam, Cambridge, MA) to identify WFA activity on vimentin expression in astrocytes, as shown in Figure 9b. [00136] Interestingly, in the presence of TNF-α, WFA activity was more pronounced compared to that in unstimulated cells, as revealed by the 500 nM WFA dose potently downregulating GFAP expression in TNF-treated cells compared to similar dose in non- stimulated cells. These results provide evidence that WFA is more effective in targeting expression of GFAP under stress conditions.

[00137] As shown in Figure 9b, when these blots were reprobed for expression of vimentin, WFA similarly downregulated vimentin expression in a dose-related manner, with TNF-stimulated cells showing a greater response to WFA. [00138] The dose-response activity of WFA in astrocytes was further investigated by immunofluorescence staining for GFAP and vimentin expression. Astrocytes were plated on glass cover slips and treated with different doses of WFA for 18 h. WFA treatment caused GFAP to be localized to the perinuclear region, with cells starting to show this phenotype at 200 nM and this pattern becoming more prominent in the majority of cells at 1 μM, which was different from the elaborate cytoskeletal decoration by GFAP filaments in untreated astrocytes (as shown in Figures 10a-10d). Specifically, astrocytes were plated on glass slides in medium containing 10% fetal bovine serum in the presence and absence of WFA for 18 h. Cells were fixed, permeabilized and stained with anti-GFAP antibody (1 :1000 dilution; Abeam, Cambridge, MA). Figure 10a shows vehicle treated; Figure 10b shows 200 nM WFA; Figure 10c shows 1 μM WFA and Figure 10d shows 5 μM WFA. Nuclei were stained with DAPI and cells were photographed on a Nikon TE2000 fluorescent microscope using 3OX objective. At the higher dose of 5 μM, cell shape was dramatically altered with reduction in lengths of cell processes and their numbers. GFAP expression was found to be highly condensed and the staining taking on a diffuse punctate pattern.

[00139] When astrocytes were stained for vimentin, a similar dose-related pattern of WFA activity on vimentin distribution in cells, with drug activity becoming pronounced only over 1 μM. Specifically, astrocytes were plated on glass slides in medium containing 10% fetal bovine serum in the presence and absence of WFA for 18 h. Cells were fixed, permeabilized and stained with anti-vimentin antibody (1 :50 dilution; SantaCruz Biotechnology, CA). Figure 1 1 a shows vehicle treated; Figure 1 1 b shows 200 nM WFA; Figure 1 1 c shows 1 μM WFA and Figure 1 1 d shows 5 μM WFA. Cells were photographed on a Nikon TE2000 fluorescent microscope using 3OX objective. Thus, the dose-related activity of WFA on astrocyte morphology and GFAP and vimentin expression provide evidence that withanolides such as WFA are a new class of inhibitors of astrocytes, which can be employed to alter IF protein overexpression that is linked to fibrosis, gliosis and scar formation. Collectively, this data provides evidence that WFA can target type-Ill IF proteins, which underscore the broader implications of the pharmacological activity of this inhibitor.

[00140] Referring now to Figures 12a-12d and Figures 13a-13b, the angiofibrotic expression of heme oxygenase-1 (HO-1 ) is abrogated by WFA treatment. Evidence is provided by experiments in which C57BL/6 mice were subjected to inflammatory corneal angiogenesis induction by alkali treatment and epithelial debridement and treated in groups of four by i.p. injection with DMSO vehicle (Veh) or WFA (2.5 mg/kg/d) for 8 days after injury. Frozen sections from whole eyes were obtained and stained with antibody to HO-1 (1 :400 dilution; Santa Cruz Biotechnology, CA) with secondary FITC-conjugated antibody and imaged on a Leica confocal microscope using a 4OX objective. Figures 12a-12d are representative images of FITC staining overlayed on Nomarski images of respective corneal sections which show no expression of HO-1 in uninjured cornea (UnC, Figure 12a), no staining in injured corneas of vehicle-treated mice in the absence of primary antibody (Veh-Ab, Figure 12b), strong staining in corneal stroma in vehicle- treated mice in the presence of primary antibody (Veh; arrows, Figure 12c) and absence of staining in WFA-treated mice in the presence of HO-1 primary antibody (WA, Figure 12d).

[00141] Figures 13a and 13b are micrographs of frozen sections stained with CD31-FITC conjugated antibody (1 :1000 dilution) and imaged with 4OX objective. Representative FITC images were overlayed on Nomarski images of their respective corneal sections. The presence of corneal vessels in the stroma of vehicle-treated sample (Veh, Figure 13b) is abundant compared to that in WFA-treated sample (WA, Figure 13a).

[00142] Figures 14a-14c are micrographs demonstrating that WFA promotes recovery of TKT antigenicity, where corneal transketolase (TKT) expression is retained in injured corneas by WFA treatment. The micrographs were generated using C57BL/6 mice subjected to inflammatory corneal angiogenesis induction by alkali treatment and epithelial debridement. They were treated in groups of four by i.p. injection with DMSO vehicle (Veh) or 2.5 mg/kg/d WFA (WA) and whole eyes were isolated on 4 d post-injury. Frozen sections were stained with rabbit anti-mouse TKT antibody and detected with a FITC-conjugated secondary antibody. The fluorescence stained corneal sections were imaged on a Leica confocal microscope. The FITC images overlayed on Nomarski images of their respective corneal sections show abundant expression of TKT in uninjured corneas (Figure 14a) with retention of TKT antigenicity in WFA-treated injury healing corneas (Figure 14b), and TKT antigen loss in corneas of vehicle-treated mice (Figure 14c).

[00143] Referring now to Figures 15a-15c, western blots were used to show that WFA targets the intermediate filaments, vimentin and GFAP, in astrocytes. The blots of Figures 15a-15c were generated by cultuhng primary astrocytes derived from rat brain (ScienCell Research Laboratories) between passage 3 to 6 which were preincubated with vehicle (-) or with 10 μM WFA for 30 minutes and subsequently with 5 μM biotinylated withafehn A (WFA-B) for 2 hours. Cell lysates were prepared in 1 % Triton X-100 buffer and purified over NEUTRAVIDIN^® affinity columns and subjected to SDS-PAGE. The protein blots were developed with Streptavidin-HRP. The biotin label is incorporated in the 56 kDa and 50 kDa protein in a WFA-competitive manner (Figure 15a), where the presence of an endogenous 70 kDa biotinylated band is unaffected by WFA (arrowhead). In such blots probed with antibody to vimentin (V9 mouse monoclonal; Santa Cruz Biotechnology; Figure 15b), and subsequently to GFAP (rabbit polyclonal; Abeam; Figure 15c), it is demonstrated that WFA-B binding identifies both vimentin and GFAP targets in a WFA-competitive manner.

[00144] Referring now to Figure 16, the western blot shows that WFA modulates TNF-α-induced ubiquitination in astrocytes. Cultured rat brain astrocytes were incubated in the absence and presence of inflammatory cytokine tumor necrosis factor (TNF)-α (5 ng/ml) and in the absence and presence of different doses of WFA for 18 h. Cell extracts were isolated and subjected to SDS-PAGE and proteins were blotted. The blots were probed with a monoclonal antibody to ubiquitin (Santa Cruz Biotechnology) and exposed to x-ray film. The results of experimentation identify the dose-related effect of WFA to either up or downregulate ubiquitination levels in activated astrocytes. [00145] Referring now to Figures 17a-17d, the series of stains provides evidence that WFA induces GFAP fragmentation in glial cells of alkali injured mice eyes during retinal gliosis. Mice (wild-type 129 Svev and vimentin KO in 129 Svev background) were subjected to alkali treatment (0.15 M sodium hydroxide for 1 min) followed by corneal epithelial debridement. They were treated in groups of four by i.p injection with DMSO vehicle (Veh) or 2 mg/kg/d withaferin A (WFA) and whole eyes were isolated on 8 d post- injury. Eyes were subjected to cryosectioning and sections were fixed. Sections were stained with rabbit polyclonal antibody against GFAP (Abeam) and vimentin (3B4 mouse monoclonal, Abeam). Secondary-FITC conjugated antibody against rabbit IgG and Cy3- conjugated antibody against mouse IgG were employed to identify GFAP and vimentin, respectively. Epifluorescence was detected on a Nikon TE2000 microscope and digital images from representative areas of the retina were obtained. Uninjured eyes of wild- type mice (non-injured WT, Figure 17a) showed basal GFAP expression localized to ganglion cell layer, whereas in injured wild-type mice treated with vehicle (injured WT- Veh, Figure 17b) there is tremendous upregulation of GFAP expression in glial cells (Muller and astrocytes) that transverse the photoreceptor cell layers to the outer nuclear layer. In injured wild-type mice treated with WFA (injured WT-WFA, Figure 17c), GFAP expression in glial cells appears to be both downregulated and highly fragmented (GCL: ganglion cell layer, IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer). Remarkably, this fragmented staining pattern for GFAP appears similar to that of injured vimentin-deficient mice treated with vehicle (injured Vim KO-Veh, Figure 17d) and that of injured vimentin-deficient mice treated with WFA (injured Vim KO-WFA), as shown in Figure 18b, as compared with injured WT-WFA of Figure 18a.

[00146] Referring now to Figure 19, the stains provide evidence that WFA downregulates vimentin expression in glial cells of akali injured mouse eyes during retinal gliosis. The stains of Figure 19 were produced from tissue sections from wild-type mice treated with vehicle (panels A and C) or WFA. Panels B and D were also co- stained for vimentin. Vimentin expression in injured wild-type vehicle-treated mice (injured WT-Veh, panel C) is observed in GCL and to the outer nuclear layer (ONL), but in injured wild-type WFA-treated mice (injured WT-WFA, panel D), there is a striking abrogation of vimentin expression in neural retina. Staining, however, for vimentin is restricted to preexisting retinal blood vessels (arrows, panel D). Referring now to Figure 20, this feature is corroborated by staining sections with antibody to CD31 , which shows similar staining in resident capillaries of retinas of WFA-treated injured wild-type mice (injured WT-WFA; arrows, Figure 20, panel B) and retinas of non-injured wild-type mice (non-injured WT; arrows, Figure 20, panel A).

[00147] Referring now to Figure 21 , the depicted stains show differential targeting of GFAP and vimentin by WFA in glial cells during retinal gliosis. Tissue sections from wild-type and vimentin KO mice from uninjured and alkali injured treatment groups were doubly stained for vimentin and GFAP. Sections were counterstained with DAPI to show the photoreceptors cell nuclei in the inner nuclear layer (INL) and outer nuclear layer (ONL). Wild-type non-injured (non-injured WT, panel A) mice show little overlap of GFAP (green) and vimentin (red) in GCL and IPL, whereas there is significant overlap of GFAP and vimentin staining that is highly accentuated in GCL and IPL in vehicle-treated injured wild-type (injured WT-Veh, panel B) mice. As noted previously, WFA potently abrogates gliosis-induced vimentin expression and causes GFAP expression to be reduced and fragmented (injured WT-WFA, panel C), which appears phenotypically similar to that observed in injured vimentin KO mice treated with vehicle (Injured Vim KO- Veh, panel D).

[00148] It will now be clear to one skilled in the art that the data collected and described herein provides evidence that during astrocyte activation and retinal gliosis WFA drug activity causes downregulation of vimentin expression and GFAP fragmentation, which is a phenotype that mirrors closely what is found in genetically- ablated vimentin deficient mice. [00149] It will now be clear to one of ordinary skill in the art that the use of small molecules which bind to type-Ill intermediate filament proteins associated with various disorders and diseases will be an effective treatment of those disorders and diseases. From Schroder, Bar, Banwell, Liu April 2006, Liu Feb. 2006, Pekny, Faijerson, Nakazawa, Hartmann, Eng, Brenner, Ermakova, Stamatakis, Gharbi, Van Beijnum and the experimental data disclosed herein, one skilled in the art will know that the binding of WFA or one of its analogs will affect the metabolic pathway of the type-Ill filament associated with the respective disease. Thus, binding WFA or its analogs to the type-Ill filament protein is an effective treatment of those diseases associated with the type-Ill filament protein.

[00150] Although the invention has been described in considerable detail with respect to preferred embodiments, it will be apparent that the invention is capable of numerous modifications and variations, apparent to those skilled in the art, without departing from the spirit and scope of the claims.

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Claims

1. A small molecule screening method comprising: generating an affinity labeled withanolide analog by binding withanolide compound to an affinity tag via a linker group; introducing the affinity labeled withanolide analog to a cell culture that has been exposed to small molecule drug candidates; and contacting the affinity labeled withanolide analog with one of:

(i) a purified protein that has been exposed to one or more small molecule drug candidates;

(ii) a cell extract that has been exposed to one or more small molecule drug candidates; and

(iii) a protein mixture that has been exposed to one or more small molecule drug candidates.

2. The method of claim 1 , wherein the affinity tag is a biotin moiety.

3. The method of claim 1 , wherein the withanolide with linker group is selected from the group of structures consisting of:

where:

R' is a methyl group of a phenyl group;

R" is a methyl group, ethyl group or a propyl group;

R'" is an amino acid; and

(a) Ra, Rb & Rc are -OH; or

(b) Ra, Rb and Rc are independently -O-Rd-Re,

Rd is a straight or branched alkyl with up to 12 carbons or aralkyl, Re is -OH₁-NH₂, -Cl, Br, -I, -F, CF₃, or biotin, digoxigenin, BODIFY (δ-chloromethyW^-difluoro-i ^δJ-tetramethyW-bora-Sa^a-diaza-s-indacene) succinate, or radioactive ligand; or

(c) Ra, Rb & Rc are independently -O-C(=O)-Rd-Re,

Rd is a straight or branched alkyl with up to 12 carbons or aralkyl, (i) Re is -OH₁-NH₂, -Cl, Br, -I, -F, CF₃ or biotin, digoxigenin, BODIFY (8- chloromethyl-4, 4-difluoro-1 , 3, 5, 7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) succinate, or radioactive ligand -O-C(=O)-R-Rd, where R is mono- di- tri- ehyleneglycol; or

(ii) Re is OH, -NH₂, -Cl, Br, -I, -F, CF₃ or biotin, digoxigenin, BODIFY (8- chloromethyl-4, 4-difluoro-1 , 3, 5, 7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) succinate, or radioactive ligand; or

(d) Ra, Rb & Rc are independently -O-C(=O)-X-NH-Re where X is a straight or branched alkyl with up to 12 carbons or mono, di- tri- ehyleneglycol,

Re is -OH₁-NH₂, -Cl₁ Br₁ -I₁ -F₁ CF₃ or biotin, digoxigenin, BODIFY (8-chloromethyl- 4,4-difluoro-1 ,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) succinate, or radioactive ligand or Rd is -(C=O)-Re,

Re is Cy5.5 acetate, Fluorescein acetate, 2-Naphthoxy acetate, Benzoyl, Benzoyl benzyl acetate, phloro-acetophenone acetate, 4-methoxy-2-hydroxy-benzoate, Alexa succinate, Coumahn acetate, 1-naphthyl, 1 -, or 1 ,3- or, 1 ,3,5-methoxy-benzyl, 1 to 5 fluoro-benzyl or piperazynyl; or (e) Ra, Rb & Rc are independently -O-C(=O)-X-Y-Z, where X is a straight or branched alkyl with up to 12 carbons or mono- di- tri- ehyleneglycol

(i) Y is penta, hexa, hepta and octapeptides comprising any combination of amino acids selected from the group consisting of Leu, Ala, Pro Tyr, lie, hydroxy proline, and Cys; and Z is (argine)8, Z = -OH, -O-benzyl, -NH₂; or

(ii) Y is (argine)8, and Z = penta, hexa, hepta and octapeptides comprising any combination of amino acids selected from the group consisting of Leu, Ala, Pro Tyr, He, hydroxy proline, and Cys.

4. The method of claim 1 , further comprising identifying the small molecule as a potential drug based on its binding to the target and preventing the withanolide analog from binding.

5. The method of claim 1 , further comprising identifying a target protein of the withanolide analog based on the binding of the target protein to the withanolide analog.

6. The method of claim 5, further comprising determining a target binding site of one or more of the small molecule candidates with the target protein.

7. The method of claim 1 , wherein at least one of the small molecule candidates comprises an analog of withafehn A.

8. The method of claim 1 , wherein the withanolide analog comprises an analog of withafehn A.

9. The method of claim 1 , wherein at least one small molecule candidate comprises a withanolide analog.

10. The method of claim 1 , wherein the affinity tag is a digoxigenin moiety.

1 1. The method of claim 1 , wherein the tag is a fluorescent moiety.

12. The method of claim 1 , wherein the tag is a BODIFY moiety.

13. The method of claim 1 , wherein the tag is a radioactive compound.

14. The method of claim 1 , wherein the tag is a luminescent reagent.

15. The method of claim 1 , wherein the tag is a quantum dot.

16. The method of claim 1 , further comprising generating a second small molecule, which binds to the target binding site based on the small molecule binding to a target protein.

17. A method for generating a small molecule probe comprising: generating an affinity labeled small molecule probe by binding a small molecule compound to an affinity tag via a linker group.

18. The method of claim 17, wherein the affinity tag is a biotin moiety.

19. The method of claim 17, wherein the linker group is a C-ι-C₂o long hydrocarbon chain linker.

20. The method of claim 17, wherein the small molecule probe is a WFA analog.

21. The method of claim 17, wherein the small molecule probe is Withafehn A.

22. The method of claim 17, wherein a target of the small molecule probe is vimentin.

23. The method of claim 17, wherein a target of the small molecule probe is an intermediate filament protein.

24. The method of claim 17, wherein the target of the small molecule probe is a type-Ill fragment of a type-Ill intermediate filament protein.

25. An affinity labeled screening withanolide analog comprising a withanolide compound covalently bonded to a linker molecule which is covalently bonded to an affinity moiety tag.

26. The affinity labeled screening compound of claim 25, wherein the affinity tag is a biotin moiety.

27. The affinity labeled screening withanolide of claim 25, wherein the linker group is a Ci-C₂o long hydrocarbon chain linker.

28. The affinity labeled screening withanolide of claim 25, wherein the withanolide analog is a WFA analog.

29. The affinity labeled screening withanolide of claim 26, wherein a target of the withanolide analog is vimentin.

30. The affinity labeled screening withanolide of claim 26, wherein a target of the withanolide analog is a type-Ill intermediate filament protein.

31. The affinity labeled screening withanolide of claim 26, wherein a target of the withanolide analog is a fragment of an intermediate filament.

32. A method for treating human and animal disorders characterized by aberrant or altered levels of vimentin comprising administering an effective amount of withafehn A or an analog thereof to an individual in need of treatment therefrom to bind to vimentin, thereby treating the disease associated with aberrant or altered levels of vimentin.

33. The method of claim 32, wherein the binding of vimentin with withafehn A or an analog thereof limits or inhibits angiogenesis associated with vimentin.

34. The method of claim 32, wherein the human or animal disorders associated with aberrant or altered levels of vimentin are selected from the group consisting of epithelial and breast cancer.

35. The method of claim 32, wherein the binding of vimentin with the withafehn A or an analog thereof limits or inhibits fibrosis associated with aberrant vimentin expression.

36. The method of claim 32, wherein the binding of vimentin with the withafehn A or an analog thereof limits or inhibits inflammation associated with aberrant vimentin expression.

37. The method of claim 32, wherein the binding of vimentin with the withafehn A or an analog thereof limits or inhibits organ transplant failure associated with aberrant vimentin expression.

38. The method of claim 32, wherein the binding of vimentin with the withafehn A or an analog thereof limits or inhibits scar tissue associated with aberrant vimentin expression.

39. The method of claim 32, wherein the withafehn A or an analog thereof is WFA.

40. A method for treating human and animal disorders characterized by aberrant or altered levels of type-Ill intermediate filament protein comprising administering an effective amount of withafehn A or an analog thereof to an individual in need of treatment therefrom to bind to the type-Ill intermediate filament protein, thereby treating the disease associated with aberrant or altered levels of the type-Ill intermediate filament protein.

41. The method of claim 40, wherein the binding of type-Ill intermediate filament protein with the withafehn A or an analog thereof limits or inhibits angiogenesis associated with the type-Ill intermediate filament protein.

42. The method of claim 40, wherein the human or animal disorders associated with aberrant or altered levels of type-Ill intermediate filament protein are selected from the group consisting of epithelial and breast cancer.

43. The method of claim 40, wherein the binding of type-Ill intermediate filament protein with the withafehn A or an analog thereof limits or inhibits fibrosis associated with aberrant the type-Ill intermediate filament protein expression.

44. The method of claim 40, wherein the binding of type-Ill intermediate filament protein with the withafehn A or an analog thereof limits or inhibits inflammation associated with aberrant type-Ill intermediate filament protein expression.

45. The method of claim 40, wherein the binding of a type-Ill intermediate filament protein with the withafehn A or an analog thereof limits or inhibits organ transplant failure associated with aberrant the type-Ill intermediate filament protein expression.

46. The method of claim 40, wherein the binding of a type-Ill intermediate filament protein with the withafehn A or an analog thereof limits or inhibits scar tissue associated with aberrant type-Ill intermediate filament protein expression.

47. The method of claim 40, wherein the type-Ill intermediate filament protein is selected from the group consisting of vimentin, desmin, GFAP, and periphehn.

48. The method of claim 40, wherein the type-Ill intermediate filament protein is desmin and the disorder characterized by aberrant levels of desmin is selected from the group consisting of cardiac disease, cardiomyopathies, desmin-related myopathies and musculoskeletal disorders, wherein binding of the withafehn A to desmin treats the selected disorder.

49. The method of claim 40, wherein the type-Ill intermediate filament protein is GFAP and the disorder characterized by aberrant levels of GFAP is selected from disorders associated with:

(i) inflammatory reactive astrocytes comprising of neurological diseases, enteric neurogliopathies, spinal and neuronmuscular injuries; and

(ii) disease associated with glial cells expressing aberrant levels or altered forms GFAP consisting of gliosis, motor neuron degeneration and scar formation, neural cell and stem cell transplant failure, early and late forms of age-related macular degeneration, macular edema, retinal detachment, proliferative diabetic retinopathy, retinopathy of prematurity, glaucoma, proliferative vitreoretinopathy, retinitis pigmentosa, uveitis, retinoblastoma, gliomas, glioblastoma, glial tumors, optic nerve damage, retinal ischemia, bomb blast injury, chemical injury, thermal burns, viral infections, Alexander disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, traumatic brain injury, developmental disorders, anxiety, migraine, schizophrenia, bipolar disorders, addiction, depression and mood disorders, stroke, and epilepsy.

50. The method of claim 40, wherein the disorders treated are selected from diseases associated with altered levels of the type III intermediate filament proteins consisting of angiofibroic diseases, macular edema, proliferative diabetic retinopathy, macular degeneration, neovascular glaucoma, corneal neovascularization, and endometriosis, diseases with scar tissue formation, keloids, kidney fibrosis, pulmonary fibrosis, cardiac fibrosis, chemotherapy/radiation induced lung fibrosis, pancreatitis, inflammatory bowel disease, Crohn's disease, necrotizing enterocolitis, hypertrophic scar, nodular fasciitis, eosinophilic fasciitis, Dupuytren's contracture, general fibrosis syndrome, characterized by replacement of normal muscle tissue by fibrous tissue in varying degrees, retroperitoneal fibrosis, liver fibrosis, and acute fibrosis, chronic inflammation such as Crohn's disease, ulcerative colitis, psoriasis, sarcoidosis, and rheumatoid arthritis, and organ transplant failure.

51. A method to target type III intermediate filaments, said method comprising contacting type III intermediate filaments with a chemical toxin or radioactive agent linked to withafehn A, withanolide or analog to thereby deactivate the type III intermediate filament.