Roberts G, Porter D, Boone L, Chan J, Martin-Carpenter L, Gerondelis P, Short S, Weaver K; Conference on Retroviruses and Opportunistic Infections (11th : 2004 : San Francisco, Calif.).
Program Abstr Conf Retrovir Oppor Infect 11th 2004 San Franc Calif. 2004 Feb 8-11; 11: abstract no. 529.
GlaxoSmithKline, Research Triangle Park, NC, USA
BACKGROUND: The NNRTI candidate GW695634, in phase I clinical development, is the prodrug of GW678248. Antiviral activity of the prodrug was examined. The interactions of GW695634 and GW678248 with HIV-1 reverse transcriptase (RT) were investigated by spectroscopic techniques. Inhibition of primer unblocking was also investigated.METHODS: Virus assays were performed in HeLa CD4, MT4, and PBMC. The kinetics of inhibition of RT activity were determined with continuous time-resolved fluorescence energy transfer assay in which Cy5-dUMP was incorporated into 5' europium-labelled primer template. The values of the dissociation constants in the absence of catalysis were determined by monitoring fluorescence of the hexachlorofluorescene moiety of the primer:template (PrTp), HF15X:20. Wild-type (wt) and mutant enzymes examined were RT(wt), RT(D67N/K70R/T215Y/K219Q), RT(L100I), RT(K103N), RT(V106A), RT(V106I), RT(V106A/Y181C), RT(V108I/Y181C), RT(V108I), RT(E138K), RT(Y181C), RT(Y188C), and RT(P236L). Primer unblocking activity was measured by following the loss of 3H-AZT-MP from chain-terminated primer template in a filter paper binding assay.RESULTS: GW678248, the active parent, exhibited greater overall potency than did GW695634 for wild type virus (0.4 to 1.0 nM vs 18 to 240 nM, respectively) and enzyme (IC50 of 1.8 nM vs 5.7 nM, respectively). GW678248 bound tightly to free RT and RT/PrTp. Representative time-courses for the binding reaction with RT(wt)/PrTp and selected concentrations of GW678248 and GW695634 were determined. The rate of dissociation of GW678248 from RT/PrTp/GW678248 was very slow, with values ranging between <0.00002 s-1 and 0.005 s-1. Primer extension and unblocking activities were inhibited by GW678248.CONCLUSIONS: Comparison of antiviral activity indicates that GW678248 is 25- to 400-fold more potent than GW695634 depending on the cell and assay system. GW678248 bound tightly to mutant RT that are resistant to other NNRTI. The rank order of the sensitivity of purified mutant RT mirrored cell culture results. GW678248 inhibited the primer unblocking reaction that has been proposed to contribute to AZT resistance associated with the quad mutant, D67N/K70R/T215Y/K219Q.
Publication Types:
Keywords:
- Antiviral Agents
- DNA Primers
- Kinetics
- Thermodynamics
- Zidovudine
- antagonists & inhibitors
- metabolism
- pharmacokinetics
- reverse transcriptase, Human immunodeficiency virus 1
Other ID:
UI: 102271491
From Meeting Abstracts