Search

PBO (pyrrolobenzoxazepinone) derivatives are non-nucleoside reverse transcriptase inhibitors (NNRTIs), which display a selective interaction with the catalytic ternary complex of HIV-1 reverse transcriptase (RT) and its substrates. In order to develop novel PBOs with improved resistance profiles, we synthesised additional PBO derivatives, specifically designed to target highly conserved residues in the beta12-beta13 hairpin, the so-called "primer grip" region of HIV-1 RT. Here, we investigated the biochemical and enzymological mechanism of inhibition of HIV-1 RT wild type and carrying NNRTIs-resistance mutations, by these derivatives. Our kinetic analysis indicates that the ability of PBOs to selectively target the catalytic ternary complex of RT with its substrates directly correlates with greatly reduced sensitivity to NNRTIs-resistance mutations, particularly the K103N substitution. Molecular modeling and docking studies provided an explanation for this correlation at the structural level.

The non-nucleoside reverse transcriptase (RT) inhibitor (NNRTI)-binding pocket of HIV-1 RT spans codons 100-110, 180-190 and 220-240 and mutations in these domains are responsible for HIV-1 NNRTI resistance. Recombinant HIV-1 strains carrying G190S/A/E, G190S+T215Y, T215Y and K103N mutations were constructed to evaluate susceptibility to both NNRTIs and nucleoside RT inhibitors (NRTIs). In addition, purified recombinant RT enzymes were obtained to determine the degree of in vitro inhibition by drugs of both classes. High-level resistance to nevirapine and moderate level resistance to both stavudine and zidovudine were associated with G190S/A/E substitutions. The simultaneous presence of G190S and T215Y decreased stavudine and zidovudine susceptibility more than T215Y alone. On the other hand, G190S was associated with a marked decrease in RT catalytic efficiency, while T215Y showed a more limited effect. Interestingly, the simultaneous presence of G190S and T215Y was associated with a reduction in the impairment of the G190S-mutated enzyme. Mutations in the HIV-1 RT NNRTI binding pocket may be associated with cross-resistance to NRTI. Selection of double mutants, with further decrease in NRTI susceptibility, might be favoured by the compensatory effect of T215Y on the reduction of RT catalytic efficiency associated with G190S.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) derivatives with D113E, Y115F, F116Y, Q151E/N, and M184V mutations were studied for their phosphorolysis-mediated resistance to the nucleoside RT inhibitors (NRTIs) zidovudine and stavudine and for their inhibition by the nonnucleoside analogs (NNRTIs) efavirenz and nevirapine. The results presented here indicate that these single amino acid substitutions within the nucleotide binding pocket of the viral RT can independently affect different enzymatic properties, such as catalytic efficiency, drug binding, and phosphorolytic activity. Moreover, small local alterations of the physicochemical properties of the microenvironment around the active site can have profound effects on some NRTIs while hardly affecting other ones. In conclusion, even though different mutations within the nucleotide binding pocket of HIV-1 RT can result in a common phenotype (i.e., drug resistance), the molecular mechanisms underlying this phenotype can be very different. Moreover, the same mutation can give rise to different phenotypes depending on the nature of the substrates and/or inhibitors.