Structural analysis of an HIV-1 protease I47A mutant resistant to the protease inhibitor lopinavir

Abstract

We have identified a rare HIV-1 protease (PR) mutation, I47A, associated with a high level of resistance to the protease inhibitor lopinavir (LPV) and with hypersusceptibility to the protease inhibitor saquinavir (SQV). The I47A mutation was found in 99 of 112,198 clinical specimens genotyped after LPV became available in late 2000, but in none of 24,426 clinical samples genotyped from 1998 to October 2000. Phenotypic data obtained for five I47A mutants showed unexpected resistance to LPV (86- to >110-fold) and hypersusceptibility to SQV (0.1- to 0.7-fold). Molecular modeling and energy calculations for these mutants using our structural phenotyping methodology showed an increase in the binding energy of LPV by 1.9-3.1 kcal/mol with respect to the wild type complex, corresponding to a 20- to >100-fold decrease in binding affinity, consistent with the observed high levels of LPV resistance. In the WT PR-LPV complex, the Ile 47 side chain is positioned close to the phenoxyacetyl moiety of LPV and its van der Waals interactions contribute significantly to the ligand binding. These interactions are lost for the smaller Ala 47 residue. Calculated binding energy changes for SQV ranged from -0.4 to -1.2 kcal/mol. In the mutant I47A PR-SQV complexes, the PR flaps are packed more tightly around SQV than in the WT complex, resulting in the formation of additional hydrogen bonds that increase binding affinity of SQV consistent with phenotypic hypersusceptibility. The emergence of mutations at PR residue 47 strongly correlates with increasing prescriptions of LPV (Spearman correlation r(s) = 0.96, P < .0001).

Figure 1.

Structure of HIV-1 protease inhibitors saquinavir (SQV) and lopinavir (LPV).

Figure 2.

Comparison of the percentage of protease resistant viruses containing Val or Ala substitutions in position 47 among the clinical HIV-1 variants submitted for testing between 1998 and 2004 (♦) with the number of LPV/r prescriptions issued for the given period (▴). Only PR variants that were predicted to be resistant at least to one protease inhibitor were included in this analysis.

Figure 3.

Lopinavir complexes with the wild-type HIV-1 PR (a model derived from the crystal structure 1mui) (yellow) and with the 175236 I47A mutant PR variant (magenta). The ligand and the flap water molecule are shown as stick models colored by atom type. Mutated residues are labeled in blue; other important PR residues are labeled in red. Ligand–protein hydrogen bonds are shown as dashed lines, colored by donor–acceptor distance from blue for the shortest H-bonds to green to red for the longer ones.

Figure 4.

Interaction of the Lopinavir phenoxyacetyl moiety with the S2′ site of wild-type PR and of the I47A mutant PR variant 175236. The ligand and the flap water molecule are shown as stick models colored by atom type. Wild-type PR residues surrounding the phenoxyacetyl moiety are shown as yellow sticks and labeled in red. V32 and A47 residues of the mutant PR are shown as magenta sticks and labeled in blue.

Figure 5.

Saquinavir complexes with the wild-type HIV-1 PR (model based on the crystal structure 1hxb) (yellow) and with the mutant I47A PR variant 175236 (magenta). The ligands and flap water molecules are shown as stick models, colored by atom type for the mutant complex and in yellow for the WT complex. Mutated residues located close to the ligand are labeled in blue; other important PR residues are labeled in red. Ligand–protein hydrogen bonds are shown colored by donor–acceptor distance from blue for the shortest H-bonds to green to red for the longer ones.