Rational approaches for the design of effective human immunodeficiency virus type 1 nonnucleoside reverse transcriptase inhibitors.

The binding of several classes of nonnucleoside reverse transcriptase inhibitors (NNRTIs) to wild-type (wtRT) and K103N mutant (mRT) human immunodeficiency virus type 1 (HIV-1) reverse transcriptase is studied by molecular dynamics and energy decomposition techniques. The imidoylthiourea (ITU), diaryltriazine (DATA), and diarylpyrimidine (DAPY) NNRTIs studied maintain the hydrogen bond with Lys101 during the 3 ns molecular dynamics trajectories. When bound to mRT, all the DAPYs studied establish hydrogen bonds with Glu138; among these, those of the potent inhibitors TMC120 and TMC125 are water-mediated. The molecular interactions of the NNRTIs in the binding pocket are correlated to the drugs' potency. Quantitative free energy analyses show a linear relationship between the van der Waals energetic component and the potency against wtRT. The molecular basis of the interaction between NNRTIs and RT presented here provide quantitative approaches for the design of novel effective anti-HIV drugs.

Development of a new methodology for screening of human immunodeficiency virus type 1 microbicides based on real-time PCR quantification.

Potential topical retrovirucides or vaginal microbicides against human immunodeficiency virus type 1 (HIV-1) include nonnucleoside reverse transcriptase inhibitors (NNRTIs). To be successful, such agents have to be highly active against cell-free virions. In the present study, we developed a new real-time PCR-based assay to measure the natural endogenous reverse transcription (NERT) activity directly on intact HIV-1 particles in the presence of reverse transcriptase (RT) inhibitors. We further evaluated the permeability to nevirapine (NVP) and efavirenz (EFV) and their retention within nascent viral particles. We also demonstrated the NVP and EFV inhibitory effects on NERT activity and the impact of resistance mutations measured directly by this new strategy. Furthermore, the results showed a clear correlation between NERT activity and classical infectivity assays. The 50% inhibitory concentrations (IC50s) of NVP and EFV were demonstrated to be up to 100-fold higher for cell-free than for cell-associated virions, suggesting that cell-free virions are less permeable to these drugs. Our results suggest that NVP and EFV penetrate both the envelope and the capsid of HIV-1 particles and readily inactivate cell-free virions. However, the characteristics of these NNRTIs, such as lower permeability and lower retention during washing procedures, in cell-free virions reduce their efficacies as microbicides. Here, we demonstrate the usefulness of the NERT real-time PCR as an assay for screening novel antiretroviral compounds with unique mechanisms of action.