Methods for identifying RNA binding compounds

Abstract

The present invention relates to methods of screening for compounds that bind RNA molecules. In particular, the methods of the invention comprise screening a library of test compounds, each of which is attached to a solid support, with a dye-labeled RNA molecule to form a dye-labeled target RNA:support-attached test compound complex. By virtue of the dye label on the target RNA, the support becomes labeled and can be separated from unlabeled solid supports. The present invention further relates to methods of inhibiting an RNA-protein interaction, to methods of screening for compounds that increase or decrease the production of a protein, and to methods of screening for a compound that is capable of treating or preventing a disease whose progression is associated with an in vivo binding of a test compound to a target RNA.

1 - 62 . (canceled)  
     
     
         63 . A method for forming a dye-labeled target RNA:support-attached test compound complex, comprising contacting an aqueous solution of a dye-labeled target RNA molecule with a plurality of test compounds attached to a plurality of solid supports under conditions that permit direct binding of the dye-labeled RNA to a solid-support attached test compound so that a dye-labeled target RNA:support-attached test compound complex is formed, wherein each solid support has substantially one type of test compound attached to its surface.  
     
     
         64 . The method of  claim 63  further comprising selecting the dye-labeled target RNA:support-attached test compound formed from uncomplexed target RNA molecules and test compounds.  
     
     
         65 . The method of  claim 64  further comprising determining the structure of the test compound of the RNA:support-attached test compound complex by decoding said solid support.  
     
     
         66 . The method of  claim 63 , wherein the aqueous solution comprises from about 2 mM to about 20 mM KCl, from about 1 mM to about 100 mM NaCl, and from about 0 mM to about 20 mM MgCl 2 .  
     
     
         67 . The method of  claim 63 , wherein the aqueous solution comprises a buffer.  
     
     
         68 . The method of  claim 63 , wherein the aqueous solution comprises a detergent or surfactant.  
     
     
         69 . The method of  claim 63 , wherein the aqueous solution comprises an unlabeled target RNA molecule having at least one mutation at the binding site of said test compound.  
     
     
         70 . The method of  claim 63 , wherein the dye of the dye-labeled target RNA molecule is a fluorescent dye, a phosphorescent dye, an ultraviolet dye, an infrared dye, or a visible dye.  
     
     
         71 . The method of  claim 63 , wherein said solid support is a silica gel, a resin, a derivatized plastic film, a glass bead, cotton, a plastic bead, a polystyrene bead, an alumina gel, or a polysaccharide.  
     
     
         72 . The method of  claim 63 , wherein the target RNA is a mammalian RNA, a viral RNA, a bacterial RNA, a protozoal RNA, or a fungal RNA.  
     
     
         73 . The method of  claim 63 , wherein the target RNA is a messenger RNA, a transfer RNA, a ribosomal RNA or an enzymatic RNA.  
     
     
         74 . The method of  claim 73 , wherein said messenger RNA encodes amyloid protein, amyloid precursor protein, angiostatin, endostatin, METH-1, METH-2, Factor IX, Factor VIII, collagen, cyclin dependent kinase, cyclin D1, cyclin E, WAF 1, cdk4 inhibitor, MTS1, cystic fibrosis transmembrane conductance regulator gene, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, erythropoietin, G-CSF, GM-CSF, M-CSF, SCF, thrombopoietin, BNDF, BMP, GGRP, EGF, FGF, GDNF, GGF, HGF, IGF-1, IGF-2, KGF, myotrophin, NGF, OSM, PDGF, somatotrophin, TGF-β, TGF-α, VEGF, interferon, TNF-α, TNF-β, cathepsin K, cytochrome p-450, famesyl transferase, glutathione-s transferase, heparanase, HMG CoA synthetase, n-acetyltransferase, phenylalanine hydroxylase, phosphodiesterase, ras carboxyl-terminal protease, telomerase, TNF converting enzyme, E-cadherin, N-cadherin, selectin, CD40, 5-αreductase, atrial natriuretic factor, calcitonin, corticotrophin releasing factor, glucagon, gonadotropin, gonadotropin releasing hormone, growth hormone, growth hormone releasing factor, somatotropin, insulin, leptin, luteinizing hormone, luteinizing hormone releasing hormone, parathyroid hormone, thyroid hormone, thyroid stimulating hormone, an antibody, CTLA4, hemagglutinin, MHC proteins, VLA-4, kallikrein-kininogen-kinin system, CD4, sis, hst, ras, abl, mos, myc, fos, jun, H-ras, ki-ras, c-fms, bcl-2, L-myc, c-myc, gip, gsp, HER-2, bombesin receptor, estrogen receptor, GABA receptor, EGFR, PDGFR, FGFR, NGFR, a GTP-binding regulatory protein, an interleukin receptor, an ion channel receptor, a leukotriene receptor antagonist, a lipoprotein receptor, an opioid pain receptor, a substance P receptor, a retinoic acid or retinoid receptor, a steroid receptor, a T-cell receptor, a thyroid hormone receptor, a TNF receptor, tissue plasminogen activator; a transmembrane receptor, calcium pump, proton pump, Na/Ca exchanger, MRP  1 , MRP 2 , P170, LRP, cMOAT, transferrin, APC, brcal, brca2, DCC, MCC, MTS1, NF1, NF2, nm23, p53 or Rb.  
     
     
         75 . The method of  claim 63 , wherein the target RNA is HIV TAR RNA.  
     
     
         76 . The method of  claim 63 , wherein the test compound is: 
         H 2 N-(L)Lys-(D)Lys-(L)Asn-OH, H 2 N-(L)Lys-(D)Lys-(D)Asn-OH, H 2 N-(L)Lys-(L)Lys-(L)Asn-OH, H 2 N-(L)Arg-(D)Lys-(L)Asn-OH, H 2 N-(L)Arg-(D))Lys-(L)Val-OH, H 2 N-(L)Arg-(D)Lys-(L)Arg-OH, H 2 N-(D)Thr-(D)Lys-(L)Asn-OH, or H 2 N-(D)Thr-(D)Lys-(L)Phe-OH. 
     
     
         77 . A method for treating or preventing a disease whose progression is associated with the binding of a target RNA with one or more in vivo ligands, said method comprising administering to a patient in need of such treatment or prevention an effective amount of a test compound, or a pharmaceutically acceptable salt thereof, identified according to a method for identifying a test compound, comprising the steps of: 
 (a) contacting an aqueous solution of a dye-labeled target RNA molecule with a plurality of test compounds attached to a plurality of solid supports under conditions that permit direct binding of the dye-labeled RNA to a solid-support attached test compound so that a dye-labeled target RNA:support-attached test compound complex is formed, wherein each solid support has substantially one type of test compound attached to its surface;    (b) selecting the dye-labeled target RNA:support-attached test compound formed in step (a) from uncomplexed target RNA molecules and test compounds; and    (c) determining the structure of the test compound of the RNA:support-attached test compound complex selected in step (b) by decoding said solid support.    
     
     
         78 . A method for treating or preventing a disease whose progression is associated with the binding of a target RNA with one or more in vivo ligands, said method comprising administering to a patient in need of such treatment or prevention an effective amount of a test compound, or a pharmaceutically acceptable salt thereof, identified according to a method for identifying a test compound, comprising determining the structure of a test compound of an RNA:support-attached test compound complex formed by contacting an aqueous solution of a dye-labeled target RNA molecule with a plurality of test compounds attached to a plurality of solid supports under conditions that permit direct binding of the dye-labeled RNA to a solid-support attached test compound so that a selected dye-labeled target RNA:support-attached test compound complex is formed, wherein each solid support has substantially one type of test compound attached to its surface.  
     
     
         79 . The method of  claim 77  or  78 , wherein the test compound is administered intradermally, intramusclarly, intraperitoneally, intravenously, subcutaneously, intranasally, epidurally, orally, sublingually, intracerebrally, intravaginally, transdermally, rectally, by inhalation or topically.  
     
     
         80 . The method of  claim 77  or  78 , wherein the patient is a mammal.  
     
     
         81 . The method of  claim 77  or  78 , wherein the patient is a human.  
     
     
         82 . The method of  claim 77  or  78 , wherein the disease is amyloidosis, hemophilia, Alzheimer's disease, atherosclerosis, cancer, giantism, dwarfism, hypothyroidism, hyperthyroidism, inflammation, cystic fibrosis, an autoimmune disorder, diabetes, aging, obesity, a neurodegenerative disorder, or Parkinson's disease.  
     
     
         83 . The method of  claim 77  or  78 , wherein the disease is caused by a bacterial, a fungal, a protozoan, or a viral infection.  
     
     
         84 . The method of  claim 77  or  78 , wherein the disease is AIDS, human T-cell leukemia, feline leukemia, hepatitis A, hepatitis B, hepatitis C, Dengue fever, malaria, severe acute gastroenteritis, diarrhea, encephalitis, hemorrhagic fever, syphilis, legionella, whooping cough, gonorrhea, sepsis, influenza, pneumonia, or meningitis.  
     
     
         85 . The method of  claim 77  or  78 , wherein the test compound is: 
         H 2 N-(L)Lys-(D)Lys-(L)Asn-OH, H 2 N-(L)Lys-(D)Lys-(D)Asn-OH, H 2 N-(L)Lys-(L)Lys-(L)Asn-OH, H 2 N-(L)Arg-(D)Lys-(L)Asn-OH, H 2 N-(L)Arg-(D)Lys-(L)Val-OH, H 2 N-(L)Arg-(D)Lys-(L)Arg-OH, H 2 N-(D)Thr-(D)Lys-(L)Asn-OH, or H 2 N-(D)Thr-(D)Lys-(L)Phe-OH.