Resistance of HIV Type 1 to Proteinase Inhibitor Ro 31-8959

Abstract

During replication of human immunodeficiency virus type 1 (HIV-1), proteolytic cleavage of Gag and Gag-Pol precursor proteins into different functional protein subunits is catalyzed by the viral proteinase, and this enzyme is the target of the antiviral proteinase inhibitor, Ro 31-8959. We investigated in vitro which HIV mutants with reduced sensitivity to Ro 31-8959 emerged during proteinase inhibition treatment; from three different HIV-1 strains, comparable progeny virus resistant to proteinase inhibitor were found, whereas the same experimental protocol detected no resistant HIV-2 mutants. Molecular analysis of the mutations underlying resistance revealed a multistep mechanism in which an amino acid exchange was common to all resistant isolates, and in all experiments preceded further exchanges at position 90 (leucine to methionine) and/or at position 54 (isoleucine to valine). For wild-type strains the 90% inhibitory concentrations of Ro 31-8959 were close to 20 nM, whereas HIV-1 mutants with all 3 amino acid exchanges had more than 50-fold increased 90% inhibitory concentrations (above 1000 nM). The primary event (Gly-48 to valine) occurs at the hinge of the flaps of the proteinase, thus hampering entry of the inhibitor to the active center and suggesting steric hindrance. Detailed knowledge of this stereotypic process could open inhibitor design, thus preventing conceivable escape of resistant virus on proteinase inhibitor action.

Anti-Human Immunodeficiency Virus Activity, Bioavailability and Drug Resistance Profile of the Novel Proteinase Inhibitor MDL 74,695

“…The cross-resistance experiments conducted with the viruses selected in this study and the three proteinase inhibitors MDL 74,695, saquinavir and indinavir revealed which amino acid exchanges were most important for conferring resistance to each of the compounds. It was apparent that a single glycine to valine amino acid substitution at position 48 was important for conferring resistance to saquinavir, whilst an additional leucine to methionine amino acid exchange at position 90 increased the level of resistance, as reported previously (Jacobsen et al, 1995;Eberle et al, 1995;Maschera et al, 1995). However, virus with these two mutations remained sensitive to both MDL 74,695 and indinavir, despite the fact that virus with a single G48V exchange was selected for after growth of HIV in the presence ofMDL 74,695.…”

“…Structural modelling with MDL 74,695 is desirable to confirm this speculation but has not been carried out as yet. The mutations identified for saquinavir in the current study were the same as those previously reported for this drug (Jacobsen et al, 1995;Eberle et al, 1995). We do not consider the more rapid development of resistance to saquinavir (passage 17) compared with MDL 74,695 (passage 20) in this study to be signifIcant.…”

“…These exchanges were identical in the four different viruses that were sequenced and were the same as previously reported for saquinavir (Eberle et al, 1995), Of four viruses with reduced sensitivity to MDL 74,695 that were sequenced, two had three identical nucleotide exchanges. These were an ATG to ATA exchange at position 46 (conservative), an ATT to GTT exchange at position 50 (isoleucine to valine) and a GTC to ATC exchange at position 82 (valine to isoleucine), One virus had a single GGG to GTG exchange at position 48 (glycine to valine) and the fourth virus had the …”

“…The amino acid exchanges we identified in the viruses with reduced sensitivity to MDL 74,695 had all previously been seen with Anti-HIV activity, bioavailability and drug resistance effects of MOL74,695 other proteinase inhibitors (Mellors et al, 1995). The glycine to valine exchange at position 48 was reported for saquinavir (Eberle et al, 1995) and XlVI323 (King et al, 1995) and the isoleucine to valine exchange at position 50 has been seen with VB 11,328 and VX-478 (Tisdale et al, 1995;Partaledis et al, 1995). These residues are in the highly flexible and exposed surface loops which cover the substrate binding cleft (Wlodawer & Erickson, 1993).…”

“…As expected MDL 74,695 showed equivalent activity against virus isolates resistant to RT inhibitors, but as with all other classes of proteinase inhibitor that have been documented (Otto et al, 1993b;Craig et al, 1993;Dianzani etal., 1993;El-Farrash etal., 1994;Ho etal., 1994;Kaplan et al, 1994;Tisdale et al, 1995;King et al, 1995;Eberle et al, 1995;Markowitz et al, 1995b), drug resistant mutants were rapidly selected for in cell culture. The amino acid exchanges we identified in the viruses with reduced sensitivity to MDL 74,695 had all previously been seen with Anti-HIV activity, bioavailability and drug resistance effects of MOL74,695 other proteinase inhibitors (Mellors et al, 1995).…”