Biochemical Mechanism of HIV-1 Resistance to Rilpivirine.

Rilpivirine (RPV) is a second generation nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) that efficiently inhibits HIV-1 resistant to first generation NNRTIs. Virological failure during therapy with RPV and emtricitabine (FTC) is associated with the appearance of E138K and M184I mutations in RT. Here we investigate the biochemical mechanism of RT inhibition and resistance to RPV. We used two transient kinetics approaches (quench-flow and stopped-flow) to determine how subunit-specific mutations in RT p66 or p51 affect association and dissociation of RPV to RT, as well as their impact on binding of dNTP and DNA and the catalytic incorporation of nucleotide. We compared wild-type (WT) to four subunit-specific RT mutants, p66(M184I)/p51(WT), p66(E138K)/p51(E138K), p66(E138K-M184I)/p51(E138K), and p66(M184I)/p51(E138K). 184I in p66 (p66(184I)) decreased the catalytic efficiency of RT (k(pol)/K(d.dNTP)), primarily through a decrease in dNTP binding (K(d.dNTP)). 138K either in both subunits or in p51 alone abrogated the negative effect of p66(184I) by restoring dNTP binding. Furthermore, p51(138K) reduced RPV susceptibility by altering the ratio of RPV dissociation to RPV association, resulting in a net reduction in RPV equilibrium binding affinity (K(d.RPV) = k(off.RPV)/k(on.RPV)). Quantum mechanics / molecular mechanics hybrid molecular modeling revealed that p51E138K affects access to the RPV binding site by disrupting the salt bridge between p51(E138) and p66(K101). p66(184I) caused repositioning of the Y183 active site residue and decreased the efficiency of RT, whereas addition of p51(138K) restored Y183 to a WT-like conformation, thus abrogating the 184I-induced functional defects.