Claims for Patent: 6,503,713

Summary for Patent: 6,503,713

Title:	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.
Inventor(s):	Rana; Tariq M (Piscataway, NJ)
Assignee:	University of Medicine and Dentistry of New Jersey (New Brunswick, NJ)
Application Number:	09/679,451
Patent Claims:	1. A method for identifying a test compound that binds to a target RNA molecule, 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, wherein each solid support has substantially one type of test compound attached to its surface under conditions that permit direct binding of the dye-labeled RNA to a solid-support attached test compound so that a selected a dye-labeled target RNA:support-attached test compound complex is formed; 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. 2. A method for identifying a test compound that binds to a target RNA molecule, comprising the step of determining the structure of a test compound of an RNA:support-attached test compound complex formed from contacting an aqueous solution of a dye-labeled target RNA molecule with a plurality of test compounds attached to a plurality of solid supports, wherein each solid support has substantially one type of test compound attached to its surface under conditions that permit direct binding of the dye-labeled RNA to a solid-support attached test compound so that a selected a dye-labeled target RNA:support-attached test compound complex is formed and wherein said dye-labeled target RNA:support-attached test compound is separated from uncomplexed target RNA molecules and test compounds, wherein said structure is determined by decoding said solid support. 3. The method of claim 1, 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.sub.2. 4. The method of claim 2, 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.sub.2. 5. The method of claim 1, wherein the aqueous solution comprises a buffer. 6. The method of claim 2, wherein the aqueous solution comprises a buffer. 7. The method of claim 1, wherein the aqueous solution comprises a detergent or surfactant. 8. The method of claim 2, wherein the aqueous solution comprises a detergent or surfactant. 9. The method of claim 1, wherein the aqueous solution comprises an unlabeled target RNA molecule having at least one mutation at the binding site of said test compound. 10. The method of claim 2, wherein the aqueous solution comprises an unlabeled target RNA molecule having at least one mutation at the binding site of said test compound. 11. The method of claim 1, wherein the dye of the dye-labeled target RNA molecule is selected from the group consisting of a fluorescent dye, a phosphorescent dye, an ultraviolet dye, an infrared dye, and a visible dye. 12. The method of claim 2, wherein the dye of the dye-labeled target RNA molecule is selected from the group consisting of a fluorescent dye, a phosphorescent dye, an ultraviolet dye, an infrared dye and a visible dye. 13. The method of claim 11, wherein said target RNA is labeled with a visible dye. 14. The method of claim 12, wherein said target RNA is labeled with a visible dye. 15. The method of claim 13, wherein the visible dye is disperse red. 16. The method of claim 14, wherein the visible dye is disperse red. 17. The method of claim 1, wherein said solid support is selected from the group consisting of a silica gel, a resin, a derivatized plastic film, a glass bead, cotton, a plastic bead, a polystyrene bead, an alumina gel, and a polysaccharide. 18. The method of claim 2, wherein said solid support is selected from the group consisting of a silica gel, a resin, a derivatized plastic film, a glass bead, cotton, a plastic bead, a polystyrene bead, an alumina gel, and a polysaccharide. 19. The method of claim 1, wherein determining the structure of substantially one type of test compound comprises using a direct technique selected from the group consisting of nucleic acid sequencing of the test compound, peptide sequencing of the test compound, NMR, Fourier transform infra-red spectroscopy, Fourier transform Raman spectroscopy, electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry, HPLC, and X-ray photoelectron spectroscopy. 20. The method of claim 2, wherein determining the structure of substantially one type of test compound comprises using a direct technique selected from the group consisting of nucleic acid sequencing of the test compound, peptide sequencing of the test compound, NMR, Fourier transform infra-red spectroscopy, Fourier transform Raman spectroscopy, electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry, HPLC, and X-ray photoelectron spectroscopy. 21. The method of claim 1, wherein determining the structure of substantially one type of test compound comprises using an indirect technique selected from the group consisting of nucleic acid sequencing of a molecular tag, peptide sequencing of a molecular tag, indexing using readable molecular tags, use of laser optical synthesis chips, and use of fluorescently labeled solid supports. 22. The method of claim 2, wherein determining the structure of substantially one type of test compound comprises using an indirect technique selected from the group consisting of nucleic acid sequencing of a molecular tag, peptide sequencing of a molecular tag, indexing using readable molecular tags, use of laser optical synthesis chips, and use of fluorescently labeled solid supports. 23. The method of claim 1, wherein the test compound is selected from the group consisting of peptides, peptide analogs, nucleic acids, nucleic acid analogs, PNAs, hormones, antigens, synthetic or naturally occurring drugs, opiates, dopamine, serotonin, catecholamines, thrombin, acetylcholine, prostaglandins, organic molecules, pheromones, adenosine, sucrose, glucose, lactose and galactose. 24. The method of claim 2, wherein the test compound is selected from the group consisting of peptides, peptide analogs, nucleic acids, nucleic acid analogs, hormones, antigens, synthetic or naturally occurring drugs, opiates, dopamine, PNAs, serotonin, catecholamines, thrombin, acetylcholine, prostaglandins, organic molecules, pheromones, adenosine, sucrose, glucose, lactose and galactose. 25. The method of claim 1, wherein the target RNA is selected from the group consisting of a mammalian RNA, a viral RNA, a bacterial RNA, a protozoal RNA, and a fungal RNA. 26. The method of claim 2, wherein the target RNA is selected from the group consisting of a mammalian RNA, a viral RNA, a bacterial RNA, a protozoal RNA, and a fungal RNA. 27. The method of claim 1, wherein the target RNA is selected from the group consisting of a messenger RNA, a transfer RNA, a ribosomal RNA and an enzymatic RNA. 28. The method of claim 2, wherein the target RNA is selected from the group consisting of a messenger RNA, a transfer RNA, a ribosomal RNA and an enzymatic RNA. 29. The method of claim 27, wherein said messenger RNA encodes a protein selected from the group consisting of 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-.beta., TGF-.alpha., VEGF, interferon, TNF-.alpha., TNF-.beta., cathepsin K, cytochrome p-450, farnesyl 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-.alpha. reductase, 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, antibodies, 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, GTP-binding regulatory proteins, interleukin receptors, ion channel receptors, leukotriene receptor antagonists, lipoprotein receptors, opioid pain receptors, substance P receptors, retinoic acid and retinoid receptors, steroid receptors, T-cell receptors, thyroid hormone receptors, TNF receptors, tissue plasminogen activator; transmembrane receptors, calcium pump, proton pump, Na/Ca exchanger, MRP 1, MRP2, P170, LRP, cMOAT, transferrin, APC, brcal, brca2, DCC, MCC, MTS1, NF1, NF2, nm23, p53 and Rb. 30. The method of claim 28, wherein said target RNA is a messenger RNA that encodes a protein selected from the group consisting of 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, MTS 1, 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-.beta., TGF-.alpha., VEGF, interferon, TNF-.alpha., TNF-.beta., cathepsin K, cytochrome p-.sup.4 50, farnesyl 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-.alpha. reductase, 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, antibodies, 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, GTP-binding regulatory proteins, interleukin receptors, ion channel receptors, leukotriene receptor antagonists, lipoprotein receptors, opioid pain receptors, substance P receptors, retinoic acid and retinoid receptors, steroid receptors, T-cell receptors, thyroid hormone receptors, TNF receptors, tissue plasminogen activator; transmembrane receptors, calcium pump, proton pump, Na/Ca exchanger, MRP 1, MRP2, P170, LRP, cMOAT, transferrin, APC, brcal, brca2, DCC, MCC, MTSI, NF1, NF2, nm23, p53 and Rb. 31. A method for forming a target RNA:test compound complex, comprising the step of contacting a target RNA molecule with the test compound identified from the method of claim 1. 32. The method of claim 31, wherein the contacting occurs in the presence of an aqueous solution. 33. The method of claim 32, 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.sub.2. 34. The method of claim 32, wherein the aqueous solution comprises a buffer. 35. The method of claim 32, wherein the aqueous solution comprises a detergent or surfactant. 36. The method of claim 31, wherein the step of contacting a target RNA with the test compound displaces a ligand of said target RNA. 37. The method of claim 31, wherein the step of contacting a target RNA with the test compound occurs in vivo. 38. A method for increasing or decreasing the production of a protein comprising the step of contacting a target messenger RNA molecule that encodes said protein with the test compound identified from the method of claim 1. 39. The method of claim 38, wherein the test compound is selected from the group consisting of peptides, peptide analogs, nucleic acids, nucleic acid analogs, hormones, antigens, synthetic or naturally occurring drugs, opiates, dopamine, PNAs, serotonin, catecholamines, thrombin, acetylcholine, prostaglandins, organic molecules, pheromones, adenosine, sucrose, glucose, lactose and galactose. 40. The method of claim 38, wherein the target RNA is selected from the group consisting of a mammalian RNA, a viral RNA, a bacterial RNA, a protozoal RNA, and a fungal RNA. 41. The method of claim 40, wherein the target RNA is selected from the group consisting of a messenger RNA, a transfer RNA, a ribosomal RNA and an enzymatic RNA. 42. The method of claim 41, wherein the messenger RNA encodes a protein selected from the group consisting of amyloid protein, amyloid precursor protein, angiostatin, endostatin, METH-1, METH-2, Factor IX, Factor VIII, collagen, cyclin dependent kinase, cyclin D1, cyclin E, WAF1, 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-.beta., TGF-.alpha., VEGF, interferon, TNF-.alpha., TNF-.beta., 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-.alpha. reductase, 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, antibodies, 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, GTP-binding regulatory proteins, interleukin receptors, ion channel receptors, leukotriene receptor antagonists, lipoprotein receptors, opioid pain receptors, substance P receptors, retinoic acid and retinoid receptors, steroid receptors, T-cell receptors, thyroid hormone receptors, TNF receptors, tissue plasminogen activator; transmembrane receptors, calcium pump, proton pump, Na/Ca exchanger, MRP 1, MRP2, P170, LRP, cMOAT, transferrin, APC, brcal, brca2, DCC, MCC, MTS1, NF1, NF2, nm23, p53 and Rb. 43. The method of claim 38, wherein increasing or decreasing the production of a protein interferes with the progression of a disease associated with decreasing or increasing the production of said protein, respectively. 44. The method of claim 43, wherein the disease is selected from the group consisting of amyloidosis, hemophilia, Alzheimer's disease, atherosclerosis, cancer, giantism, dwarfism, hypothyroidism, hyperthyroidism, inflammation, cystic fibrosis, autoimmune disorders, diabetes, aging, obesity, neurodegenerative disorders, and Parkinson's disease. 45. The method of claim 43, wherein said disease is caused by a bacteria, a fungus, a protozoa, or a virus. 46. The method of claim 45, wherein the disease is selected from the group consisting of HIV infection, AIDS, human T-cell leukemia, SIV infection, FIV infection, feline leukemia, hepatitis A, hepatitis B, hepatitis C, Dengue fever, malaria, rotavirus infection, severe acute gastroenteritis, diarrhea, encephalitis, hemorrhagic fever, syphilis, legionella, whooping cough, gonorrhea, sepsis, influenza, pneumonia, tinea infection, candida infection, and meningitis. 47. The method of claim 23, wherein the test compound is selected from the group consisting of: H.sub.2 N-(L)Lys-(D)Lys-(L)Asn-OH, H.sub.2 N-(L)Lys-(D)Lys-(D)Asn-OH, H.sub.2 N-(L)Lys-(L)Lys-(L)Asn-OH, H.sub.2 N-(L)Arg-(D)Lys-(L)Asn-OH, H.sub.2 N-(L)Arg-(D)Lys-(L)Val-OH, H.sub.2 N-(L)Arg-(D)Lys-(L)Arg-OH, H.sub.2 N-(D)Thr-(D)Lys-(L)Asn-OH, and H.sub.2 N-(D)Thr-(D)Lys-(L)Phe-OH. 48. The method of claim 24, wherein the test compound is selected from the group consisting of: H.sub.2 N-(L)Lys-(D)Lys-(L)Asn-OH, H.sub.2 N-(L)Lys-(D)Lys-(D)Asn-OH, H.sub.2 N-(L)Lys-(L)Lys-(L)Asn-OH, H.sub.2 N-(L)Arg-(D)Lys-(L)Asn-OH, H.sub.2 N-(L)Arg-(D)Lys-(L)Val-OH, H.sub.2 N-(L)Arg-(D)Lys-(L)Arg-OH, H.sub.2 N-(D)Thr-(D)Lys-(L)Asn-OH, and H.sub.2 N-(D)Thr-(D)Lys-(L)Phe-OH. 49. The method of claim 27, wherein the target RNA is HIV TAR RNA. 50. The method of claim 28, wherein the target RNA is HIV TAR RNA.

Details for Patent 6,503,713

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Nps Pharmaceuticals, Inc.	NATPARA	parathyroid hormone	For Injection	125511	01/23/2015		2019-10-04