A Method for Identification of Anti-HIV Human miRNA Mimics and miRNA Inhibitors and Anti-HIV Pharmaceutical Compounds

Abstract

The present invention relates to methods for the identification of anti-HIV miRNAs and anti-HIV pharmaceutical compounds using high-throughput screening methods, comprising: transfecting reporter cells with a panel of miRNAs, infecting the reporter cells with HIV, screening the cells to identify miRNAs that modulate HIV infection and identifying the specific pathways, nucleic acids and/or polypeptides that are targeted by the miRNAs. The invention further provides for the identification and screening of anti-HIV pharmaceutical compounds having known activity against the specific pathways, nucleic acids and/or polypeptides that are targeted by the miRNAs for efficacy in the treatment of HIV. The invention also provides for the use of miRNA mimics, miRNA inhibitors and pharmaceutical compounds (including oncology drugs and kinase inhibitors) in the treatment and/or prevention of HIV infection.

What is claimed is: 
     
         1 . A method for the identification of pharmaceutical compounds targeting pathways associated with cancer, which are effective in the treatment of HIV infection, the method comprising the steps of:
 a) providing a first batch of reporter cells and providing a panel of miRNAs, wherein the reporter cells are divided into a plurality of samples and wherein each sample is transfected with an miRNA from the panel;   b) infecting the transfected sample with HIV;   c) screening the samples to identify one or more miRNA mimics or miRNA inhibitors which modulate HIV infection from the panel;   d) identifying a specific cellular pathway, a polynucleotide and/or a polypeptide which is targeted by the HIV modulating miRNA mimic or miRNA inhibitor from step c);   e) identifying one or more anti-cancer pharmaceutical compounds which have anti-cancer activity against the specific cellular pathway, polynucleotide and/or polypeptide of step d);   f) providing a second batch of reporter cells, wherein the second batch of reporter cells are divided into a plurality of samples and wherein each sample is treated with an anti-cancer pharmaceutical compound identified in step e);   g) infecting the second batch of samples with HIV, wherein the step of infecting the second batch of samples may occur before or after the treatment with the anti-cancer pharmaceutical compound; and   h) identifying an anti-cancer pharmaceutical compound which has pharmaceutical activity against HIV.   
     
     
         2 - 3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein the miRNAs are human miRNAs. 
     
     
         5 . The method of  claim 1 , wherein the miRNA which modulates HIV infection is selected from the group consisting of:
 i) HIV modulating miRNAs which are able to inhibit HIV entry into a cell;   ii) HIV modulating miRNA molecules which are able to suppress activation of the HIV Long Terminal Repeat (LTR) promoter;   iii) HIV modulating miRNA molecules which are able to sensitise uninfected cells to apoptosis in response to infection by HIV; and   iv) HIV modulating miRNA molecules which are able to promote activation of the HIV LTR promoter.   
     
     
         6 . The method of  claim 1 , wherein the HIV modulating miRNAs are selected from the group consisting of hsa-let-7a-5p, hsa-let-7d-5p, hsa-miR-23a, hsa-mir-29c*, hsa-miR-34c-3p, hsa-miR-92a-1-5p, hsa-mir-124a-3p, hsa-miR-124a, hsa-miR-125b-5p, hsa-miR-138, hsa-miR-146a, hsa-miR-149-3p, hsa-miR-150, hsa-miR-155, hsa-miR-193b-5p, hsa-miR-200c, hsa-miR-342-5p, hsa-miR-361-5p, hsa-miR-421, hsa-miR-423-3p, hsa-miR-504, hsa-miR-509-3p, hsa-miR-637, hsa-mir-650, hsa-miR-520d-5p, hsa-miR-1200, hsa-miR-1908, hsa-miR-1910, hsa-miR-2110, hsa-miR-3162, hsa-miR-3185, hsa-miR-3189, hsa-miR-3191, hsa-miR-3191-3p, hsa-miR-4259, and hsa-miR-4314. 
     
     
         7 . The method of  claim 1 , wherein the pharmaceutical compound of step g) is selected from the group consisting of:
 i) pharmaceutical compounds which are selectively toxic to cells infected with HIV;   ii) pharmaceutical compounds which are able to sensitize uninfected cells to apoptosis in response to infection by HIV;   iii) pharmaceutical compounds which are able to suppress activation of the HIV LTR promoter in cells infected with HIV;   iv) pharmaceutical compounds which are able to inhibit HIV entry into a cell;   v) pharmaceutical compounds which are able to prime uninfected cells to suppress HIV LTR promoter activity in response to infection by HIV.   
     
     
         8 . The method of  claim 1 , wherein the pharmaceutical compound of step g) is selected from an oncology drug or a kinase inhibitor. 
     
     
         9 . The method of  claim 8 , wherein the oncology drug is selected from the group consisting of abiraterone, aminolevulinic acid hydrochloride, cisplatin, dactinomycin, dexrazoxane, erlotinib hydrochloride, everolimus, floxuridine, gefitinib, ifosfamide, plicamycin, temozolomide, thalidomide, vemurafenib, vincristine sulfate, vorinostat, and pharmaceutically acceptable salts thereof. 
     
     
         10 . The method of  claim 8 , wherein the kinase inhibitor is selected from the group consisting of A-674563, AT7519, SNS-032, aurora A inhibitor I, crenolanib, foretinib, GSK2126458, NVP-BHG712, LDN193189, PIK-75, ponatinib, saracatinib, vargatef, and WZ4002. 
     
     
         11 . An miRNA mimic or an miRNA inhibitor selected from the group consisting of hsa-let-7a-5p, hsa-let-7d-5p, hsa-miR-23a, hsa-miR-92a-1-5p, hsa-mir-124a-3p, hsa-miR-124a, hsa-miR-138, hsa-miR-146a, hsa-miR-149-3p, hsa-miR-150, hsa-miR-193b-5p, hsa-miR-342-5p, hsa-miR-421, hsa-miR-423-3p, hsa-miR-509-3p, hsa-miR-637, hsa-mir-650, hsa-miR-520d-5p, hsa-miR-1908, hsa-miR-2110, hsa-miR-3162, hsa-miR-3185, hsa-miR-3189, hsa-miR-3191, hsa-miR-3191-3p, hsa-miR-4259, and hsa-miR-4314 for use in the suppression of HIV replication in a cell. 
     
     
         12 . An miRNA mimic or an miRNA inhibitor selected from the group consisting of hsa-mir-29c*, hsa-miR-193b-5p, hsa-miR-421, hsa-miR-1908, and hsa-miR-3189 for use in inducing apoptosis in a cell in response to HIV infection of the cell. 
     
     
         13 . An miRNA mimic or an miRNA inhibitor selected from the group consisting of hsa-miR-34c-3p, hsa-miR-125b-5p, hsa-miR-150, hsa-miR-155, hsa-miR-200c, hsa-miR-361-5p, hsa-miR-504, hsa-miR-1200 and hsa-miR-1910 for use in increasing the level of HIV replication in a cell. 
     
     
         14 . A compound or a pharmaceutically effective salt thereof selected from the group consisting of foretinib, GSK2126458, NVP-BHG712, ponatinib, saracatinib, temozolomide, thalidomide, vargatef, vincristine sulfate, and WZ 4002 for use in the prevention of HIV infection in a subject. 
     
     
         15 . A compound or a pharmaceutically acceptable salt thereof, selected from the group consisting of A-674563, dactinomycin, ifosfamide, cisplatin, dexrazoxane, everolimus and vemurafenib for use in the suppression of HIV infection in an HIV infected subject. 
     
     
         16 . A compound or a pharmaceutically acceptable salt thereof, selected from the group consisting of A-674563, abiraterone, aminolevulinic acid hydrochloride, AT7519, aurora A inhibitor I, crenolanib, erlotinib hydrochloride, floxuridine, gefitinib, LDN193189, PIK-75, plicamycin, SNS-032, and vorinostat for use in inducing apoptosis in a cell in response to HIV infection of the cell in a subject.