Abstract
BackgroundTenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and efavirenz (EFV) are the three components of the once-daily, single tablet regimen (Atripla) for treatment of HIV-1 infection. Previous cell culture studies have demonstrated that the double combination of tenofovir (TFV), the parent drug of TDF, and FTC were additive to synergistic in their anti-HIV activity, which correlated with increased levels of intracellular phosphorylation of both compounds.ResultsIn this study, we demonstrated the combinations of TFV+FTC, TFV+EFV, FTC+EFV, and TFV+FTC+EFV synergistically inhibit HIV replication in cell culture and synergistically inhibit HIV-1 reverse transcriptase (RT) catalyzed DNA synthesis in biochemical assays. Several different methods were applied to define synergy including median-effect analysis, MacSynergy®II and quantitative isobologram analysis. We demonstrated that the enhanced formation of dead-end complexes (DEC) by HIV-1 RT and TFV-terminated DNA in the presence of FTC-triphosphate (TP) could contribute to the synergy observed for the combination of TFV+FTC, possibly through reduced terminal NRTI excision. Furthermore, we showed that EFV facilitated efficient formation of stable, DEC-like complexes by TFV- or FTC-monophosphate (MP)-terminated DNA and this can contribute to the synergistic inhibition of HIV-1 RT by TFV-diphosphate (DP)+EFV and FTC-TP+EFV combinations.ConclusionThis study demonstrated a clear correlation between the synergistic antiviral activities of TFV+FTC, TFV+EFV, FTC+EFV, and TFV+FTC+EFV combinations and synergistic HIV-1 RT inhibition at the enzymatic level. We propose the molecular mechanisms for the TFV+FTC+EFV synergy to be a combination of increased levels of the active metabolites TFV-DP and FTC-TP and enhanced DEC formation by a chain-terminated DNA and HIV-1 RT in the presence of the second and the third drug in the combination. This study furthers the understanding of the longstanding observations of synergistic anti-HIV-1 effects of many NRTI+NNRTI and certain NRTI+NRTI combinations in cell culture, and provides biochemical evidence that combinations of anti-HIV agents can increase the intracellular drug efficacy, without increasing the extracellular drug concentrations.
Highlights
Tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and efavirenz (EFV) are the three components of the once-daily, single tablet regimen (Atripla) for treatment of HIV-1 infection
We demonstrated that HIV-1 reverse transcriptase (RT) and TFV-terminated DNA form dead-end complex (DEC) in the presence of FTC-TP, which could contribute to the synergistic inhibition of HIV-1 RT by TFV-DP+FTC-TP at the enzymatic level
Based on our current study and the previously reported findings, we propose that there are at least three independent factors may contribute to synergistic effects of nucleotide reverse transcriptase (RT) inhibitor (NRTI) and non-nucleoside RT inhibitor (NNRTI): (1) diminished ATP binding in the presence of NNRTIs decreases efficiency of excision for NRTI terminator [18,20]; (2) increased RNase H activity in the presence of NNRTIs can diminish opportunities for NRTI excision [21]; and (3) as shown in our work, NNRTImediated stable complex formation prolongs and enhances the chain-termination effects of NRTIs
Summary
Tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and efavirenz (EFV) are the three components of the once-daily, single tablet regimen (Atripla) for treatment of HIV-1 infection. Previous cell culture studies have demonstrated that the double combination of tenofovir (TFV), the parent drug of TDF, and FTC were additive to synergistic in their anti-HIV activity, which correlated with increased levels of intracellular phosphorylation of both compounds. Resembling the natural dNTPs, the active metabolites of NRTIs serve as alternative substrates for HIV-1 RT during viral DNA synthesis, which results in chain-termination of DNA elongation due to the lack of the 3'-hydroxy moiety. NNRTI inhibit HIV-1 replication through multiple mechanisms [5], but mainly by inducing conformational changes within HIV-1 RT at the polymerase active site which significantly slow down viral DNA synthesis but have no effect on the binding affinity of natural dNTP and primer/template [6]
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