Novel high-throughput screen identifies an HIV-1 reverse transcriptase inhibitor with a unique mechanism of action

• Published in: Biochemical journal Vol. 462; no. 3; p. 425
• Main Authors: Sheen, Chih-Wei, Alptürk, Onur, Sluis-Cremer, Nicolas
• Format: Journal Article
• Language: English
• Published: England 15.09.2014
• Subjects: Fluorescence Resonance Energy Transfer; High-Throughput Screening Assays - methods; HIV Reverse Transcriptase - drug effects; HIV Reverse Transcriptase - metabolism; Kinetics; Reverse Transcriptase Inhibitors - isolation & purification; Reverse Transcriptase Inhibitors - pharmacology; Salicylates - isolation & purification; Salicylates - pharmacology; Virus Replication - drug effects
• ISSN: 1470-8728
• DOI: 10.1042/BJ20140365

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.