Microrna modulators and method for identifying and using the same

Abstract

The present invention is a method for identifying agents which modulate microRNA activity. The invention involves contacting a cell harboring a microRNA and a microRNA binding sequence, which is operably linked to a nucleic acid molecule encoding a reporter protein, with a test agent and determining whether the test agent increases or decreases the expression of the reporter protein thereby identifying a microRNA modulator. Antagonists identified by this screening assay are provided, as are methods for using the same to inhibit microRNA activity and prevent or treat disease.

1 . A method for identifying a microRNA modulator comprising contacting a cell harboring:
 (a) a microRNA, and   (b) a microRNA binding sequence, which is operably linked to a nucleic acid molecule encoding a reporter protein,   with a test agent and determining whether the test agent increases or decreases the expression of the reporter protein thereby identifying a microRNA modulator.   
     
     
         2 . The method of  claim 1 , wherein the microRNA is from a mammalian cell. 
     
     
         3 . The method of  claim 2 , wherein the microRNA is from a human cell. 
     
     
         4 . The method of  claim 3 , wherein the microRNA is selected from the group of miR-17, miR-19a, miR-21, miR-30C, miR-31, miR-34b, miR-34c, miR-127, miR-136, miR-141, miR-142-3p, miR-142-5p, miR-143, miR-144, miR-145, miR-150, miR-200b, miR-200c, miR-221, miR-222, miR-373, miR-376a, miR-451, miR-486 and miR-520C. 
     
     
         5 . The method of  claim 1 , wherein the microRNA binding sequence is selected from the group of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12. 
     
     
         6 . The method of  claim 1 , wherein the microRNA is endogenous to the cell. 
     
     
         7 . The method of  claim 1 , wherein the cell is genetically-engineered to express the microRNA. 
     
     
         8 . The method of  claim 1 , wherein the reporter protein is selected from the group of green fluorescent protein, red fluorescent protein, yellow fluorescent protein, luciferase, beta-galactosidase, and beta-glucuronidase. 
     
     
         9 . The method of  claim 1 , wherein the microRNA binding sequence is downstream of the reporter protein coding sequence. 
     
     
         10 . The method of  claim 1 , wherein the microRNA binding sequence is upstream of the reporter protein coding sequence. 
     
     
         11 . The method of  claim 1 , wherein the test agent is selected from a protein, polypeptide, peptide, nucleic acid, oligonucleotide, carbohydrate, lipid, synthetic molecule, semi-synthetic molecule, and purified natural product. 
     
     
         12 . The method of  claim 1 , wherein the test agent is not a nucleic acid or nucleic acid molecule. 
     
     
         13 . A modulator identified by the method of  claim 1 . 
     
     
         14 . The modulator of  claim 13 , wherein said modulator is an antagonist. 
     
     
         15 . The modulator of  claim 14 , wherein the antagonist comprises a compound of Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11 or Table 12. 
     
     
         16 . The modulator of  claim 13 , wherein said modulator is an agonist. 
     
     
         17 . An miR-21 antagonist comprising 
       
         
           
           
               
               
           
         
       
       wherein each R is independently H, amino, hydroxyl, thiol, amide, aryl, heteroaryl, allyl, propargyl, alkyl, silyl, halogen, or nitro, with the proviso that the antagonist is not Compound 3. 
     
     
         18 . The miR-21 antagonist of  claim 17 , wherein said compound is selected from the group of Compound 1 and Compound 2. 
     
     
         19 . A pharmaceutical composition comprising a miR-21 antagonist of  claim 17  in admixture with a pharmaceutically acceptable carrier. 
     
     
         20 . A method for inhibiting the activity of miR-21 microRNA comprising contacting a cell which expresses miR-21 with a diazobenzene miR-21 antagonist thereby inhibiting the activity of the miR-21 microRNA. 
     
     
         21 . A method for treating a disease or condition associated with miR-21 comprising administering to a subject in need of treatment of a disease or condition associated with miR-21 with a diazobenzene miR-21 antagonist thereby treating the disease or condition. 
     
     
         22 . The method of  claim 21 , wherein said disease or condition is cancer or myocardial disease. 
     
     
         23 . The method of  claim 21 , wherein the diazobenzene miR-21 antagonist is administered in combination with one or more antiproliferative agents. 
     
     
         24 . The method of  claim 23 , wherein the antiproliferative agents are selected from the group of antimetabolites, vinca alkaloids, agents that inhibit NF-kappaB, agents that affect protein synthesis, antibiotics, hormone antagonists, nucleic acid damaging agents, intercalating agents, topoisomerase inhibitors, antibodies, and metal coordination complexes. 
     
     
         25 . An miR-21 antagonist comprising 
       
         
           
           
               
               
           
         
       
       wherein each R is independently H, amino, hydroxyl, thiol, amide, aryl, heteroaryl, allyl, propargyl, alkyl, silyl, halogen, or nitro, with the proviso that the antagonist is not Compound 4. 
     
     
         26 . The miR-21 antagonist of  claim 25 , wherein said compound is Compound 5. 
     
     
         27 . A pharmaceutical composition comprising a miR-21 antagonist of  claim 25  in admixture with a pharmaceutically acceptable carrier. 
     
     
         28 . A method for inhibiting the activity of miR-21 microRNA comprising contacting a cell which expresses miR-21 with an indenoisoquinoline miR-21 antagonist thereby inhibiting the activity of the miR-21 microRNA. 
     
     
         29 . The method of  claim 28 , wherein the indenoisoquinoline miR-21 antagonist is selected from the group of Compound 4 or Compound 5. 
     
     
         30 . A method for treating a disease or condition associated with miR-21 comprising administering to a subject in need of treatment of a disease or condition associated with miR-21 with an indenoisoquinoline miR-21 antagonist thereby treating the disease or condition. 
     
     
         31 . The method of  claim 30 , wherein the indenoisoquinoline miR-21 antagonist is selected from the group of Compound 4 or Compound 5. 
     
     
         32 . The method of  claim 30 , wherein said disease or condition is cancer or myocardial disease. 
     
     
         33 . The method of  claim 30 , wherein the indenoisoquinoline miR-21 antagonist is administered in combination with one or more antiproliferative agents. 
     
     
         34 . The method of  claim 33 , wherein the antiproliferative agents are selected from the group of antimetabolites, vinca alkaloids, agents that inhibit NF-kappaB, agents that affect protein synthesis, antibiotics, hormone antagonists, nucleic acid damaging agents, intercalating agents, topoisomerase inhibitors, antibodies, and metal coordination complexes. 
     
     
         35 . An miR-21 antagonist comprising 
       
         
           
           
               
               
           
         
       
       wherein each R is independently H, amino, hydroxyl, thiol, amide, aryl, heteroaryl, allyl, propargyl, alkyl, silyl, halogen, or nitro, with the proviso that the antagonist is not Compound 6, 7, 8 or estrone. 
     
     
         36 . A pharmaceutical composition comprising a miR-21 antagonist of  claim 35  in admixture with a pharmaceutically acceptable carrier. 
     
     
         37 . A method for inhibiting the activity of miR-21 microRNA comprising contacting a cell which expresses miR-21 with a cyclopentaphenanthrene miR-21 antagonist thereby inhibiting the activity of the miR-21 microRNA. 
     
     
         38 . The method of  claim 37 , wherein the cyclopentaphenanthrene miR-21 antagonist is selected from the group of Compound 6, Compound 7, Compound 8 or estrone. 
     
     
         39 . A method for treating a disease or condition associated with miR-21 comprising administering to a subject in need of treatment of a disease or condition associated with miR-21 with a cyclopentaphenanthrene miR-21 antagonist thereby treating the disease or condition. 
     
     
         40 . The method of  claim 39 , wherein the cyclopentaphenanthrene miR-21 antagonist is selected from the group of Compound 6, Compound 7, Compound 8 or estrone. 
     
     
         41 . The method of  claim 39 , wherein said disease or condition is cancer or myocardial disease. 
     
     
         42 . The method of  claim 39 , wherein the cyclopentaphenanthrene miR-21 antagonist is administered in combination with one or more antiproliferative agents. 
     
     
         43 . The method of  claim 42 , wherein the antiproliferative agents are selected from the group of antimetabolites, vinca alkaloids, agents that inhibit NF-kappaB, agents that affect protein synthesis, antibiotics, hormone antagonists, nucleic acid damaging agents, intercalating agents, topoisomerase inhibitors, antibodies, and metal coordination complexes.