Methods for identifying small molecules that modulate premature translation termination and nonsense mediated mrna decay

• US 20060257866A1
• Filed: 07/24/2003
• Published: 11/16/2006
• Est. Priority Date: 07/24/2002
• Status: Active Grant

• Current Assignee: PTC Therapeutics, Inc.
• Original Assignee: PTC Therapeutics, Inc.
• Inventors: Welch, Ellen M., Almstead, Neil Gregory, Rando, Robert F., Pellegrini, Mathew C.
• Granted Patent: US 7,927,791 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

The present invention relates to a method for screening and identifying compounds that modulate premature translation termination and/or nonsense-mediated messenger ribonucleic acid (“mRNA”) by interacting with a preselected target ribonucleic acid (“RNA”). In particular, the present invention relates to identifying compounds that bind to regions of the 28S ribosomal RNA (“rRNA”) and analogs thereof. Direct, noncompetitive binding assays are advantageously used to screen libraries of compounds for those that selectively bind to a preselected target RNA. Binding of target RNA molecules to a particular compound is detected using any physical method that measures the altered physical property of the target RNA bound to a compound. The structure of the compound attached to the labeled RNA is also determined. The methods used will depend, in part, on the nature of the library screened. The methods of the present invention provide a simple, sensitive assay for high-throughput screening of libraries of compounds to identify pharmaceutical leads.

Claims

1. A method for identifying a compound that binds to a target RNA, said method comprising

(a) contacting a detectably labeled target RNA molecule with a library of compounds under conditions that permit direct binding of the labeled target RNA to a member of the library of compounds and the formation of a detectably labeled target RNA:

compound complex, wherein the target RNA is a region of 28S rRNA or contains a premature stop codon; and

(b) detecting the formation of a detectably labeled target RNA;

compound complex.

6. The method of claim 1, 2, 3, 4 or 5, wherein each compound in the library is attached to a solid support.

7. The method of claim 6, wherein the solid support is a silica gel, a resin, a derivatived plastic film, a glass bead, cotton, a plastic bead, a polystyrene bead, an aluminum gel, a glass slide or a polysaccharide.

8. The method of claim 1, 2, 3, 4 or 5, wherein the library of compounds is attached to a chip.

9. The method of claim 1, 2, 3, 4 or 5, wherein the detectably labeled RNA is labeled with a fluorescent dye, phosphorescent dye, ultraviolet dye, infrared dye, visible dye, radiolabel, enzyme, spectroscopic colorimetric label, affinity tag, or nanoparticle.

10. The method of claim 1, 2, 3, 4 or 5, wherein the compound is a combinatorial library of compounds comprising peptoids;

random biooligomers;

diversomers such as hydantoins, benzodiazepines and dipeptides;

vinylogous polypeptides;

nonpeptidal peptidomimetics;

oligocarbamates;

peptidyl phosphonates;

peptide nucleic acid libraries;

antibody libraries;

carbohydrate libraries;

or small organic molecule libraries.

11. The method of claim 10, wherein the small organic molecule libraries are libraries of benzodiazepines, isoprenoids, thiazolidinones, metathiazanones, pyrrolidines, morpholino compounds, or diazepindiones.

12. The method of claim 1, 2, 3, 4 or 5, wherein the detectably labeled target RNA:

compound complex is detected by electrophoresis, fluorescence spectroscopy, surface plasmon resonance, mass spectrometry, scintillation, proximity assay, structure-activity relationships (“

SAR”

) by NMR spectroscopy, size exclusion chromatography, affinity chromatography, or nanoparticle aggregation.

13. The method of claim 1, 2, 3, 4 or 5, wherein the method further comprises determining the structure of the compound.

14. The method of claim 13, wherein the structure of the compound is determined by mass spectroscopy, NMR, X-ray crystallography, Edman degradation or vibration spectroscopy.

15. The method of claim 1, 2, 3, 4 or 5, wherein the premature stop codon is UAG, UGA or UAA.

2. A method for identifying a compound to test for its ability to modulate premature translation termination or nonsense-mediated mRNA decay, said method comprising:

(a) contacting a detectably labeled target RNA molecule with a library of compounds under conditions that permit direct binding of the labeled target RNA to a member of the library of compounds and the formation of a detectably labeled target RNA;

compound complex, wherein the target RNA is a region of 28S rRNA or contains a premature stop codon; and

(b) detecting a detectably labeled target RNA;

compound complex formed in step(a), so that if a target RNA;

compound complex is detected then the compound identified is tested for its ability to modulate premature translation or nonsense-mediated mRNA delay.

3. A method for identifying a compound that binds to a target RNA, said method comprising detecting the formation of a detectably labeled target RNA:

compound complex formed from contacting a detectably labeled RNA with a member of a library of compounds under conditions that permit direct binding of the labeled target RNA to a member of the library of compounds and the formation of a detectably labeled target RNA;

compound complex, wherein the target RNA is a region of 28S rRNA or contains a premature stop codon.

4. A method of identifying a compound that modulates premature translation termination or nonsense-mediated mRNA decay, said method comprising:

(a) contacting a detectably labeled target RNA molecule with a library of compounds under conditions that permit direct binding of the labeled target RNA to a member of the library of compounds and the formation of a detectably labeled target RNA;

compound complex, wherein the target RNA is a region of 28S rRNA or contains a premature stop codon; and

(b) detecting a detectably labeled target RNA;

compound complex formed in step(a), so that if a target RNA;

compound complex is detected, then

(c) contacting the compound with a cell-free translation mixture and a nucleic acid sequence comprising a regulatory element operably linked to a reporter gene, wherein the reporter gene contains a premature stop codon; and

(d) detecting the expression of the reporter gene, wherein a compound that modulates premature translation termination or nonsense-mediated mRNA decay is identified if the expression of the reporter gene in the presence of the compound is altered relative to the expression of the reporter gene in the absence of the compound or the presence of a negative control.

5. A method of identifying a compound that modulates premature translation termination or nonsense-mediated mRNA decay, said method comprising:

(a) contacting a detectably labeled target RNA molecule with a library of compounds under conditions that permit direct binding of the labeled target RNA to a member of the library of compounds and the formation of a detectably labeled target RNA;

compound complex, wherein the target RNA is a region of 28S rRNA or contains a premature stop codon; and

(b) detecting a detectably labeled target RNA;

compound complex formed in step(a), so that if a target RNA;

compound complex is detected, then

(c) contacting the compound with a cell containing a nucleic acid sequence comprising a regulatory element operably linked to a reporter gene, wherein the reporter gene contains a premature stop codon; and

(d) detecting the expression of the reporter gene, wherein a compound that modulates premature translation termination or nonsense-mediated mRNA decay is identified if the expression of the reporter gene in the presence of the compound is altered relative to the expression of the reporter gene in the absence of the compound or the presence of a negative control.