title: Novel high-throughput screen identifies an HIV-1 reverse transcriptase inhibitor with a unique mechanism of action.
abstract: HIV-1 resistance to zidovudine [AZT (azidothymidine)] is associated with selection of the mutations M41L, D67N, K70R, L210W, T215F/Y and K219Q/E in RT (reverse transcriptase). These mutations decrease HIV-1 susceptibility to AZT by augmenting RT's ability to excise the chain-terminating AZT-MP (AZT-monophosphate) moiety from the chain-terminated DNA primer. Although AZT-MP excision occurs at the enzyme's polymerase active site, it is mechanistically distinct from the DNA polymerase reaction. Consequently, this activity represents a novel target for drug discovery, and inhibitors that target this activity may increase the efficacy of nucleoside/nucleotide analogues, and may help to delay the onset of drug resistance. In the present study, we have developed a FRET (Förster resonance energy transfer)-based high-throughput screening assay for the AZT-MP excision activity of RT. This assay is sensitive and robust, and demonstrates a signal-to-noise ratio of 3.3 and a Z' factor of 0.69. We screened three chemical libraries (7265 compounds) using this assay, and identified APEX57219 {3,3'-[(3-carboxy-4-oxo-2,5-cyclohexadien-1-ylidene)methylene]bis[6-hydroxybenzoic acid]} as the most promising hit. APEX57219 displays a unique activity profile against wild-type and drug-resistant HIV-1 RT, and was found to inhibit virus replication at the level of reverse transcription. Mechanistic analyses revealed that APEX57219 blocked the interaction between RT and the nucleic acid substrate.
authors: *Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.
authors: Chih-Wei
authors: CW
authors: Sheen
authors: *Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.
authors: Onur
authors: O
authors: Alptürk
authors: *Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.
authors: Nicolas
authors: N
authors: Sluis-Cremer
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D054303:HIV Reverse Transcriptase / Q000187:drug effects / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D007700:Kinetics; D018894:Reverse Transcriptase Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D012459:Salicylates / Q000302:isolation & purification* / Q000494:pharmacology; D014779:Virus Replication / Q000187:drug effects
chemical_list: C000592581:3,3'-((3-carboxy-4-oxo-2,5-cyclohexadien-1-ylidene)methylene)bis(6-hydroxybenzoic acid); D018894:Reverse Transcriptase Inhibitors; D012459:Salicylates; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase