Binding kinetics of highly mutated HIV-1 subtype C protease inhibition by Lopinavir and Darunavir in the face of altered conformational dynamics.

Highly mutated HIV-1 protease (PR) compromises the efficacy of lopinavir (LPV) and darunavir (DRV) used to formulate salvage regimens in HIV/AIDS management. Here, we report the kinetics of inhibition of lopinavir (LPV) and darunavir (DRV) on highly mutated South African HIV-1 subtype C PR obtained from clinical isolates. The wild-type and mutant South African HIV-1 subtype C PR were cloned and purified. Enzyme inhibition assays and fluorescence spectroscopy were utilized to determine the binding kinetics of LPV and DRV with the wild-type and mutant HIV-1 PR variants. Like DRV, the results of this study show that LPV has a mixed-type inhibition mechanism, which indicates the possibility of a second binding site on HIV-1 PR. Both LPV and DRV poorly inhibited the highly mutated HIV-1 PR variants and had a markedly increased dissociation rate cons bound to the mutant variants compared to the wild type. The fast dissociation of these inhibitors translated into a short residence time of the inhibitor bound to the mutant HIV-1 PR variants. Fluorescent spectroscopy showed that the changes in the tertiary structure of the mutant HIV-1 PR variants were associated with a more open conformation. This open conformation was associated with altered conformational dynamics, which may have resulted in the loss of tight binding of LPV and DRV. This study's findings provide insight into the mechanism of resistance to LPV and DRV by highly mutated HIV-1 PR and provide information supporting the use of binding kinetics measurement in understanding HIV-1 PR inhibitor drug resistance evolution.