Compositions and methods for the identification of inhibitors of retroviral infection

Abstract

Methods of identifying inhibitors of retroviral propagation, tRNA used in the methods, and kits, including the tRNA, which can be used in the methods, are disclosed. Methods of treating or preventing retroviral infections by administering an effective amount of the inhibitors, and pharmaceutical compositions including the inhibitors, are also disclosed. The methods involve forming a mixture comprising a linear sequence of a tRNA anticodon stem loop fragment that is not capable of forming a stem-loop, a target nucleic acid molecule capable of binding to the tRNA anticodon stem loop fragment, and a test compound. The mixture is incubated under conditions that allow binding of the tRNA anticodon stem loop fragment and the target nucleic acid molecule in the absence of the test compound. Assays can then be performed that detect whether or not the test compound inhibits the binding of the tRNA anticodon stem loop fragment and the target nucleic acid molecule.

1 . A method of identifying an inhibitor of retroviral reverse transcription, comprising: forming a mixture comprising a linear sequence of a tRNA anticodon stem loop fragment that is not capable of forming a stem-loop, a target nucleic acid molecule capable of binding to the tRNA anticodon stem loop fragment, and a test compound, wherein the target nucleic acid molecule corresponds to a portion of a retroviral genome involved in reverse transcription; incubating the mixture under conditions that allow binding of the tRNA anticodon stem loop fragment and the target nucleic acid molecule in the absence of the test compound; and detecting whether or not the test compound inhibits the binding of the tRNA anticodon stem loop fragment and the target nucleic acid molecule, wherein the absence of binding of the tRNA ASL fragment and the target nucleic acid molecule is indicative of the test compound being an inhibitor of retroviral reverse transcription. 
     
     
         2 . The method of  claim 1 , wherein the tRNA anticodon stem loop fragment comprises a modified nucleotide at a position corresponding to positions 34, 37, or 39 of the full tRNA. 
     
     
         3 . The method of  claim 1 , wherein the tRNA anticodon stem loop fragment is a linear fragment having from 10 to 14 nucleotides. 
     
     
         4 . The method of  claim 3 , wherein the tRNA anticodon stem loop fragment is a linear fragment having from 11 to 13 nucleotides. 
     
     
         5 . The method of  claim 4 , wherein the tRNA anticodon stem loop fragment is a linear fragment having 12 nucleotides. 
     
     
         6 . The method of  claim 1 , wherein the tRNA anticodon stem loop fragment corresponds to a fragment of tRNA Lys . 
     
     
         7 . The method of  claim 1 , wherein the tRNA anticodon stem loop fragment comprises at least one modified nucleic acid. 
     
     
         8 . The method of  claim 1 , wherein the tRNA anticodon stem loop fragment consists essentially of a nucleic acid sequence corresponding to a fragment of an anticodon stem loop. 
     
     
         9 . The method of  claim 1 , wherein the target nucleic acid molecule comprises a nucleic acid sequence corresponding to a nucleic acid from a retroviral genome. 
     
     
         10 . The method of  claim 1 , wherein the target nucleic acid molecule comprises a nucleic acid having the sequence 5′- GCGGUGUAAAAG. 
     
     
         11 . The method of  claim 1 , wherein the mixture forming step includes the addition of one or more test compounds to the mixture. 
     
     
         12 . The method of  claim 11 , wherein the test compound is selected from the group consisting of peptides, peptide analogs, nucleic acids, nucleic acid analogs, antibodies, hormones, natural or synthetic compounds, organic compounds, and combinations thereof. 
     
     
         13 . The method of  claim 12 , wherein the nucleic acids are selected from the group consisting of antisense oligomers, siRNA, RNAi, and dsRNA. 
     
     
         14 . The method of  claim 1 , wherein detecting inhibition of binding indicates that the test compound is effective for inhibiting reverse transcription. 
     
     
         15 . A method of screening for inhibitors of retroviral propagation, comprising: forming a mixture comprising a linear sequence of a tRNA anticodon stem loop fragment, a target nucleic acid molecule capable of binding to the tRNA anticodon stem loop fragment, and a test compound, wherein the target nucleic acid molecule corresponds to a portion of a retroviral genome involved in retroviral propagation; incubating the mixture under conditions that allow binding of the tRNA anticodon stem loop fragment and the target nucleic acid molecule in the absence of the test compound; and detecting whether or not the test compound binds to the tRNA ASL fragment or the target nucleic acid molecule, wherein the binding is indicative of the test compound being an inhibitor of the propagation of a retrovirus. 
     
     
         16 . The method of  claim 15 , wherein the method comprises screening for inhibitors of retroviral reverse transcription. 
     
     
         17 . The method of  claim 16 , wherein the method comprises detecting whether or not the test compound inhibits the binding of the tRNA anticodon stem loop fragment and the target nucleic acid molecule. 
     
     
         18 . The method of  claim 15 , wherein the linear sequence of a tRNA anticodon stem loop fragment is not capable of forming a stem-loop. 
     
     
         19 . A method of identifying an inhibitor of binding of a tRNA ASL and a target nucleic acid molecule, comprising: forming a mixture comprising a linear sequence of a tRNA anticodon stem loop fragment that is not capable of forming a stem-loop, a target nucleic acid molecule capable of binding to the tRNA anticodon stem loop fragment, and a test compound, wherein the target nucleic acid molecule corresponds to a portion of a retroviral genome involved in reverse transcription; incubating the mixture under conditions that allow binding of the tRNA anticodon stem loop fragment and the target nucleic acid molecule in the absence of the test compound; and detecting whether or not the test compound inhibits the binding of the tRNA anticodon stem loop fragment and the target nucleic acid molecule, wherein the absence of binding of the tRNA and the target nucleic acid molecule is indicative of the test compound being an inhibitor of reverse transcription. 
     
     
         20 . The method of  claim 19 , wherein the tRNA anticodon stem loop fragment comprises a modified nucleotide at a position selected corresponding to positions 34, 37, or 39 of the full tRNA. 
     
     
         21 . The method of  claim 19 , wherein the tRNA anticodon stem loop fragment is a linear fragment having from 10 to 14 nucleotides. 
     
     
         22 . The method of  claim 19 , wherein the tRNA anticodon stem loop fragment consists essentially of a nucleic acid sequence corresponding to a fragment of an anticodon stem loop. 
     
     
         23 . The method of  claim 19 , wherein the target nucleic acid molecule comprises a nucleic acid sequence corresponding to a fragment of a retroviral genome. 
     
     
         24 . The method of  claim 19 , wherein the target nucleic acid molecule comprises a nucleic acid having the sequence GCGGUGUAAAAG. 
     
     
         25 . A kit for screening inhibitors of retroviral reverse transcription comprising: a nucleic acid molecule consisting essentially of a linear sequence of a tRNA anticodon stem loop fragment; and a detectable label.