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 anti-HIV agent, 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:   (a) reverse transcription;   b) HIV translation of viral RNA to precursor proteins;   c) final packaging and assembly; or   d) retroviral primer recruitment;   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:   i) an inhibitor of retroviral reverse transcription,   ii) an inhibitor of HIV translation of viral RNA to precursor proteins,   iii) an inhibitor of HIV final packaging and assembly of new virions by the inhibition of secondary virus spread through the decreasing of viral budding, or   iv) an inhibitor of HIV's recruitment of the retroviral primer, human tRNA Lys3 , respectively,   wherein the tRNA anticodon stem loop fragment comprises the sequence 5′-GCUXUUAYZCUG, in which the X, Y, and Z refer to modified nucleosides, and optionally comprises a label.   
     
     
         2 - 4 . (canceled) 
     
     
         5 . A method of screening for inhibitors of retroviral propagation, comprising:
 a) obtaining
 i) a mixture containing: 
 a linear sequence of a tRNA anticodon stem loop fragment 
 a target nucleic acid molecule which corresponds to a portion of a retroviral genome involved in retroviral propagation, and capable of binding to the tRNA anticodon stem loop fragment, 
 an effector molecule that binds to the anticodon stem loop fragment capable of altering the molecular dynamics of the complex while not affecting the fragments ability to bind to a target nucleic acid molecule which corresponds to a portion of a retroviral genome involved in retroviral propagation; 
 an effector molecule capable of binding to the target RNA containing a detection signal enhancer to amplify the signal induced by binding and thus reducing the amount of material necessary for detection and 
 ii) a test compound, 
   b) incubating the target nucleic acid molecule in 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 compounds; and   c) 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,   wherein a positive determination is made by the detection of change in a spectral signal that is the result of disruption of the complex formation, and   wherein the tRNA anticodon stem loop fragment comprises the sequence 5′-GCUXUUAYZCUG, in which the X, Y, and Z refer to modified nucleosides, and optionally comprises a label.   
     
     
         6 . The method of any of  claim 1  or  5 , wherein the modified nucleosides are selected from the group consisting of unknown modified adenosine (?A), 1-methyladenosine (m1A), 2-methyladenosine (m2A), N6-isopentenyladenosine (i6A), 2-methylthio-N6-isopentenyladenosine (ms2i6A), N6-methyladenosine (m6A), N6-threonylcarbamoyladenosine (t6A), N6-methyl-N6 threonylcarbomoyladenosine (m6t6A), 2-methylthio-N-6-threonylcarbamoyladenosine (ms2t6A), 2′-O-methyladenosine I Inosine (Am), 1-methylinosine Ar(p) 2′-O-(5-phospho)ribosyladenosine (m1I), N 6 -(cis-hydroxyisopentenyl)adenosine (i06A), Unknown modified cytidine (?C), 2-thiocytidine (s2C), 2′-O-methylcytidine (Cm), N 4 -acetyl cytidine (ac4C), 5-methylcytidine (m5C), 3-methyl cytidine (m3C), lysidine (k2C), 5-formllcytidin (f5C), 2′-O-methyl-5-formylcytidin (f5 Cm), unknown modified guanosine (?G), 2′-O-(5phospho) ribosylguanosine (Gr(p)), I-methylguanosine (mIG), N2-methylguanosine (m2G), 2′-O-methylguanosine (Gm), N2 N2-dimethylguanosine (m22G), N2,N2,2′-O-trimethylguanosine (m22Gm), 7-methylguanosine (m7G), archaeosine (fa7d7G), queuosine (Q), mannosyl-queuosine (manQ), galactosyl-queuosine (galQ), wybutosine (yW), peroxywybutosine (O 2 yW), unknown modified uridine (?U), 5-methylaminomethyluridine (mnm5U), 2-thiouridine (s2U), 2′-O-methyluridine (Urn), 4-thiouridine (s4U), 5-carbamoylmethyluridine (ncm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), uridine 5-oxyacetic acid (cmO5U), 5-methoxyuridine (mo5U), 5-carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 3-(3-amino-3-carboxypropyl)uridine (acp3U), 5(carboxyhydroxymethyl)uridinemethyl ester (mchm5U), 5-carboxymethylaminomethyl-2′-β-methyluridine (cmnm5Um), 5-carbamoylmethyl-2′-O-methyluridine (ncm5Um), Dihydrouridine (D), pseudouridine (ψ), 1-methylpseudouridine (m1ψ), 2′-O-methylpseudouridine (ψm), ribosylthymine (m5U), 5-methyl-2-thiouridine (m5s2U), and 5,2′-O-dimethyluridine (m5Um) 
     
     
         7 . The method of any of  claim 1  or  5 , wherein the modified nucleosides are selected from the group consisting of mnm5s2U, mcm5s2U, ms2t6A, s2U, ψ, and t6A. 
     
     
         8 . The method of any of  claim 1  or  5 , wherein the tRNA anticodon stem loop fragment comprises the nucleic acid sequence 5′-CU(mnm5s2U)UU(ms2t6A)A(‘pseudo U)CUGC, GCU(mnm5s2U)UU(ms2t6A)A(pseudo U)CUG or CU(mnm5s2U)UU(ms2t6A)A(pseudoU)CUGC(fluorescein. 
     
     
         9 - 10 . (canceled) 
     
     
         11 . The method of any of  claim 1  or  5 , wherein the tRNA anticodon stem loop fragment consists essentially of the nucleic acid sequence 5′-CU(mnm5s2U)UU(ms2t6A) A(pseudoU)CUGC(fluorescein). 
     
     
         12 . The method of any of  claim 1  or  5 , wherein the tRNA anticodon stem loop fragment is a linear fragment having from 10 to 14 nucleotides. 
     
     
         13 - 14 . (canceled) 
     
     
         15 . The method of any of  claim 1  or  5 , wherein the mixture forming step includes the addition of one or more test compounds to the mixture. 
     
     
         16 . (canceled) 
     
     
         17 . The method of any of  claim 1  or  5 , wherein the nucleic acids are selected from the group consisting of antisense oligomers, siRNA, RNAi, and dsRNA. 
     
     
         18 . The method of any of  claim 1  or  5 , wherein the tRNA fragment further comprises a label. 
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 18 , wherein the label is selected from the group consisting of radioactive isotopes, dyes, fluorescent dyes, fluorophores, electron-dense reagents, enzymes and their substrates, biotin-streptavidin, digoxigenin, or hapten; and proteins for which antisera or monoclonal antibodies are available. 
     
     
         21 . The method of  claim 18 , wherein the label is an affinity tag. 
     
     
         22 . A method of capturing the isolated tRNA fragment of  claim 21 , comprising contacting the isolated tRNA fragment of claim  13 , wherein the label is an affinity tag, with a complimentary ligand coupled to a solid support that allows for the capture of the affinity tag-labeled tRNA fragment. 
     
     
         23 . The method of  claim 22 , wherein the affinity tags and complimentary partners are selected from the group consisting of biotin-streptavidin, complimentary nucleic acid fragments, aptamers, or haptens and proteins for which antisera or monoclonal antibodies are available. 
     
     
         24 . An isolated tRNA fragment comprising the sequence 5′-GCUXUUAYZCUG, in which the X, Y, and Z refer to modified nucleosides, optionally including a label. 
     
     
         25 . The tRNA fragment of  claim 24 , wherein the modified nucleosides are selected from the group consisting of unknown modified adenosine (?A), I-methyladenosine (m1A), 2-methyladenosine (m2A), N6-isopentenyladenosine (i6A), 2-methylthio-N-6-isopentenyladenosine (ms2i6A), N6-methyladenosine (m6A), N6-threonylcarbamoyladenosine (t6A), N6-methyl-N6 threonylcarbomoyladenosine (m6t6A), 2-methylthio-N-6-threonylcarbamoyladenosine (ms2t6A), 2′-O-methyladenosine I Inosine (Am), I-methylinosine Ar(p) 2′-O-(5-phospho)ribosyladenosine (m1I), N6-(cis-hydroxyisopentenyl)adenosine (i06A), Unknown modified cytidine (?C), 2-thiocytidine (s2C), 2′-O-methylcytidine (Cm), N4-acetylcytidine (ac4C), 5-methylcytidine (m5C), 3-methylcytidine (m3C), lysidine (k2C), 5-forml1cytidin (f5C), 2′-O-methyl-5-formylcytidin (f5Cm), unknown modified guanosine (?G), 2′-O-(5phospho) ribosylguanosine (Gr(p)), I-methylguanosine (mIG), N2-methylguanosine (m2G), 2′-O-methylguanosine (Gm), N2 N2-dimethylguanosine (m22G), N2,N2,2′-O-trimethylguanosine (m22Gm), 7-methylguanosine (m7G), archaeosine (fa7d7G), queuosine (Q), mannosyl-queuosine (manQ), galactosyl-queuosine (galQ), wybutosine (yW), peroxywybutosine (O 2 yW), unknown modified uridine (?U), 5-methylaminomethyluridine (mnm5U), 2-thiouridine (s2U), 2′-O-methyluridine (Urn), 4-thiouridine (s4U), 5-carbamoylmethyluridine (ncm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), uridine 5-oxyacetic acid (cmO5U), 5-methoxyuridine (mo5U), 5-carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 3-(3-amino-3-carboxypropyl)uridine (acp3U), 5 (carboxyhydroxymethyl)uridinemethyl ester (mchm5U), 5-carboxymethylaminomethyl-2′-O-methyluridine (cmnm5Um), 5-carbamoylmethyl-2′-β-methyluridine (ncm5Um), Dihydrouridine (D), pseudouridine (ψ), 1-methylpseudouridine (m1ψ), 2′-O-methylpseudouridine (ψm), ribosylthymine (m5U), 5-methyl-2-thiouridine (m5s2U), and 5,2′-O-dimethyluridine (m5Um) 
     
     
         26 . The tRNA fragment of  claim 24 , wherein the modified nucleosides are selected from the group consisting of mnm5s2U, mcm5s2U, ms2t6A, s2U, ψ, and t6A. 
     
     
         27 . An isolated tRNA fragment comprising the nucleic acid sequence 5′-CU(mnm5s2U)UU(ms2t6A)A(T)CUGC or 5′-GCU(mnm5s2U)UU(ms2t6A)A(T)CUG. 
     
     
         28 . (canceled) 
     
     
         29 . The isolated tRNA fragment of any of  claims 24 - 27 , further comprising a label. 
     
     
         30 - 31 . (canceled) 
     
     
         32 . The isolated tRNA fragment of  claim 29 , wherein the label is an affinity tag. 
     
     
         33 . A method of capturing the isolated tRNA fragment of  claim 32 , comprising contacting the isolated tRNA fragment of  claim 32 , wherein the label is an affinity tag, with a complimentary ligand coupled to a solid support that allows for the capture of the affinity tag-labeled tRNA fragment. 
     
     
         34 . (canceled) 
     
     
         35 . A kit comprising an isolated tRNA fragment of any of  claims 24 - 27 . 
     
     
         36 - 45 . (canceled) 
     
     
         46 . A method of treating or preventing a retroviral infection in a patient, comprising administering to the patient an effective treatment or preventive amount of a compound that inhibits the ability of any portion of the HIV genome involved in reverse transcription to bind to or associate with a host cell tRNA in the patient. 
     
     
         47 . The method of  claim 46 , wherein the portion of the HIV genome involved in reverse transcription is the 5′ un-translated region of the HIV genome, or wherein the portion of the host cell tRNA comprises human tRNA .Lys3 . 
     
     
         48 . The method of  claim 47 , wherein the 5′ un-translated region of the HIV genome comprises residues 157 to 169 of the 5′ un-translated region of HIV-1 
     
     
         49 . The method of  claim 47 , wherein the portion of the host cell tRNA comprises nucleotides 32-43 of human tRNA .Lys3 . 
     
     
         50 . The method of  claim 46 , wherein the binding or association of the host cell tRNA to a retroviral genome initiates, primes, or facilitates reverse transcription of the retroviral genome in the absence of the administered compound. 
     
     
         51 . A method of treating or preventing a retroviral infection in a patient, comprising:
 a) administering to the patient an effective treatment or preventative amount of a compound that directly interacts with nucleotides 32-43 of the patient's tRNA Lys3  such that binding of retroviral RNA to those nucleotides is inhibited, or   b) administering to the patient an effective treatment or preventative amount of a compound that directly interacts with nucleotides 157-169 of the retroviral RNA, such that binding of the patient's tRNA Lys3  to those nucleotides is inhibited, or   c) administering to the patient an effective amount of a compound the disrupts the RNA/RNA complex formed between the retroviral RNA and the patient's tRNA Lys3 .   
     
     
         52 . A method of treating or preventing a retroviral infection in a patient, comprising administering to a patient an effective treatment or preventative amount of a compound that binds to nucleotides 32-43 of the HIV primer human tRNA Lys3 , or to nucleotides 157-169 of the retroviral RNA, and competitively inhibits their binding, wherein in the absence of the compound, a primer binding complex of HIV would form, and in the presence of the compound, formation of the primer binding complex of HIV is inhibited. 
     
     
         53 . The method of  claim 46 , wherein the compound is a small molecule. 
     
     
         54 . The method of  claim 46 , wherein the compound inhibits retroviral reverse transcription, viral recruitment of the retroviral primer used in translation, human tRNA Lys3 , the final packaging and assembly of new virions, or the binding of a host cell tRNA to a predetermined target nucleic acid molecule in the retrovirus. 
     
     
         55 . The method of  claim 46 , further comprising the co-administration of a second antiretroviral agent. 
     
     
         56 . The method of  claim 55 , wherein the second antiretroviral agent is selected from the group consisting of NRTIs, NNRTIs, VAP anti-idiotypic antibodies, CD4 and CCR5 receptor inhibitors, entry inhibitors, antisense oligonucleotides, ribozymes, protease inhibitors, neuraminidase inhibitors, tyrosine kinase inhibitors, PI-3 kinase inhibitors, and Interferons. 
     
     
         57 . The method of  claim 46 , wherein the retrovirus is selected from the group consisting of Feline Immunodeficiency Virus (FIV), Simian Immunodeficiency Virus (SIV), Avian Leucosis Virus, Feline Leukemia Virus, Walleye Dermal Sarcoma Virus, Human T-Lymphotropic Virus, and Human Immunodeficiency Viruses (HIV). 
     
     
         58 . The method of  claim 57 , wherein the retrovirus is HIV, and the HIV is selected from the group consisting of HIV-I, HIV-II, HIV-III, and mutated versions thereof.