Nonnucleoside Reverse Transcriptase Inhibitor Hypersusceptibility and Resistance by Mutation of Residue 181 in HIV-1 Reverse Transcriptase.

Substitutions at residue Y181 in HIV-1 reverse transcriptase (RT), in particular, Y181C, Y181I, and Y181V, are associated with nonnucleoside RT inhibitor (NNRTI) cross-resistance. In this study, we used kinetic and thermodynamic approaches, in addition to molecular modeling, to gain insight into the mechanisms by which these substitutions confer resistance to nevirapine (NVP), efavirenz (EFV), and rilpivirine (RPV). Using pre-steady-state kinetics, we found that the dissociation constant ( ) values for inhibitor binding to the Y181C and Y181I RT-template/primer (T/P) complexes were significantly reduced.

Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening.

Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A.

Mechanism of allosteric inhibition of HIV-1 reverse transcriptase revealed by single-molecule and ensemble fluorescence.

Non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are routinely used to treat HIV-1 infection, yet their mechanism of action remains unclear despite intensive investigation. In this study, we developed complementary single-molecule fluorescence and ensemble fluorescence anisotropy approaches to discover how NNRTIs modulate the intra-molecular conformational changes and inter-molecular dynamics of RT-template/primer (T/P) and RT-T/P-dNTP complexes. We found that NNRTI binding to RT induces opening of the fingers and thumb subdomains, which increases the dynamic sliding motion of the enzyme on the T/P and reduces dNTP binding affinity.

Competitive fitness assays indicate that the E138A substitution in HIV-1 reverse transcriptase decreases in vitro susceptibility to emtricitabine.

We characterized the relative fitness of multiple nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-resistant HIV-1 variants in the presence of etravirine (ETV), rilpivirine (RPV), and/or the nucleoside RT inhibitor emtricitabine (FTC) by simultaneous competitive culture and 454 deep sequencing. The E138A substitution, typically associated with decreased virologic responses to ETV- and RPV-containing regimens, confers a clear fitness advantage to the virus in the presence of FTC and decreases FTC susceptibility 4.7-fold.

In vitro characterization of a sustained-release formulation for enfuvirtide.

Although approved by the U.S. Food and Drug Administration, enfuvirtide is rarely used in combination antiretroviral therapies (cART) to treat HIV-1 infection, primarily because of its intense dosing schedule that requires twice-daily subcutaneous injection.

Replication fitness of multiple nonnucleoside reverse transcriptase-resistant HIV-1 variants in the presence of etravirine measured by 454 deep sequencing.

We applied an efficient method to characterize the relative fitness levels of multiple nonnucleoside reverse transcriptase (NNRTI)-resistant HIV-1 variants by simultaneous competitive culture and 454 deep sequencing. Using this method, we show that the Y181V mutation in the HIV-1 reverse transcriptase in particular confers a clear selective advantage to the virus over 14 other NNRTI resistance mutations in the presence of etravirine in vitro.