Compositions and methods of screening for compounds that modulate activity at a tweak binding site on a crd of fn14

Patent metadata

Field	Value
Publication number	US-2016146784-A1
Application number	US-201414904363-A
Country	US
Kind code	A1
Filing date	Jul 9, 2014
Priority date	Jul 9, 2013
Publication date	May 26, 2016
Grant date	—

Abstract

The present disclosure is directed to methods of screening a compound for modulating activity at a TNF-like weak inducer of apoptosis (TWEAK) binding site on a cysteine-rich domain (CRD) of fibroblast growth factor-inducible 14 (Fn14). The present disclosure also provides heterocyclic compounds and pharmaceutically acceptable salts thereof and methods for the prevention, treatment, and amelioration of cell proliferative disorders with these compounds.

First claim

1 - 47 . (canceled)

48 . A method of screening a compound for modulating activity at a TWEAK binding site on a CRD of Fn14, the method comprising:
a) screening a plurality of compounds using a cell-based luciferase reporter assay, wherein inhibition of luciferase signal following addition of each of the respective plurality of compounds and TWEAK validates the modulating activity of the respective compound at the TWEAK binding site on the CRD of Fn14; b) screening the compounds validated in step a) using a cell-based luciferase reporter assay, wherein a lack of inhibition of luciferase signal following addition of the compounds validated in step a) and purified tumor necrosis factor alpha (TNFα) confirms specificity of the modulating activity of the compounds validated in step a); and c) screening compounds validated and confirmed in steps a) and b) by measuring inhibition of migration of cells resulting from addition of the compounds validated and confirmed in steps a) and b) and purified TWEAK, wherein inhibition of migration of the cells validates the modulating activity of the compound at the TWEAK binding site on the CRD of Fn14.

49 . The method of claim 48 further comprising screening the compounds validated in step a) to assess non-specific cytotoxicity using a cell viability assay.

50 . The method of claim 48 , wherein compounds that are validated in step a) have an IC 50 of at least 10 μM.

51 . The method of claim 48 further comprising performing an ELISA assay with Fn14, a soluble form of TWEAK (sTWEAK), and the compounds validated in step c) to calculate the reduction in sTWEAK binding due to the addition of the compounds validated in step c) with a standard curve.

52 . The method according to claim 48 , wherein the cell-based luciferase reporter assay comprises human embryonic kidney cells.

53 . The method according to claim 48 , wherein the cell-based luciferase reporter assay comprises cells that stably co-express full length Fn14 and a firefly luciferase reporter driven by NF-κB response elements.

54 . The method according to claim 48 , wherein measuring inhibition of migration of cells comprises culturing cells in medium containing the compounds validated and confirmed in steps a) and b) and monitoring radial migration of the cells.

55 . The method of claim 54 , wherein the cells are glioblastoma or breast cancer cells.

56 . The method of claim 48 further comprising administering a compound identified in the screening method to a subject to modify the TNF-like weak inducer of apoptosis (TWEAK) fibroblast growth factor-inducible 14 (Fn14) pathway in the subject.

57 . (canceled)

58 . The method of claim 56 , wherein the compound or composition is in an amount sufficient to ameliorate or treat an autoimmune disease, a stroke, or a cardiovascular disease.

59 . The method of claim 58 , wherein the autoimmune disease is hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmune neutropenia, autoimmunocytopenia, antiphospholipid syndrome, dermatitis, gluten-sensitive enteropathy, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis, Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendo-crinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, myocarditis, IgA glomerulonephritis, dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism, systemic lupus erythematosus, discoid lupus, Goodpasture's syndrome, Pemphigus, Graves' Disease, Myasthenia Gravis, and insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, schleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, adrenergic drug resistance with asthma or cystic fibrosis, chronic active hepatitis, primary biliary cirrhosis, endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, an inflammatory disorder, a granulomatous disorder, an atrophic disorder, or an alloimmune disease.

60 . The method of claim 56 , wherein the compound or composition is in an amount sufficient to ameliorate or treat a cancer selected from the group consisting of a brain tumor, glioblastoma, breast cancer, prostate cancer, esophageal cancer, ovarian cancer, colon cancer, lung cancer, melanoma, pancreatic cancer, liver cancer, and renal cancer.

Assignees

• Translational Genomics Res Inst

Inventors

• Tran Nhan
• Meunice Nathalie
• Dhruv Harshil
• Yin Hongwei

Classifications

• A61K31/541

  Non-condensed thiazines containing further heterocyclic rings · CPC title

• A61K31/635

  having a heterocyclic ring, e.g. sulfadiazine · CPC title

• A61K31/433

  Thidiazoles · CPC title

• A61K31/542

  ortho- or peri-condensed with heterocyclic ring systems · CPC title

• G01N2500/10

  involving cells · CPC title