pHIV-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 RT9s ability to excise the chain-terminating AZT-MP (AZT-monophosphate) moiety from the chain-terminated DNA primer. Although AZT-MP excision occurs at the enzyme9s 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 nucleosideucleotide analogues, and may help to delay the onset of drug resistance. In the present study, we have developed a FRET (F?rster 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./p
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