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


Sheen C., ALPTUERK O., SLUIS-CREMER N.

BIOCHEMICAL JOURNAL, cilt.462, ss.425-432, 2014 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 462
  • Basım Tarihi: 2014
  • Doi Numarası: 10.1042/bj20140365
  • Dergi Adı: BIOCHEMICAL JOURNAL
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Sayfa Sayıları: ss.425-432
  • İstanbul Teknik Üniversitesi Adresli: Evet

Özet

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 AZTMP (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 (Forster 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.