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Browsing Virology by Author "Eche, Simeon."

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    Biochemical characterization of highly mutated South African HIV-1 subtype C protease.
    (2021) Eche, Simeon.; Gordon, Michelle Lucille.
    Understanding the underlying molecular mechanism of HIV-1 protease (PR) inhibition by HIV-1 protease inhibitors (PIs) is essential to gain mechanistic insight into the evolution of resistance to HIV-1 PIs. HIV-1 PIs have improved patient care management, but the accumulation of drug resistance mutations in the HIV-1 PR gene diminishes their inhibitory capacity. The current study investigated the kinetic and structural characteristics of highly mutated South African HIV-1 subtype C PR from clinical isolates obtained from individuals failing a lopinavir (LPV) inclusive regimen at the point of switch to darunavir (DRV) based therapy. In this study, enzyme activity and inhibition assays were used to determine the biochemical fitness of HIV-1 PR variants and the inhibitory constants of HIV-1 PIs for drug-resistant HIV-1 subtype C proteases. The mechanistic insight into the impact of the accumulated drug resistance mutations on the HIV-1 PR structure and its interaction with LPV and DRV was obtained using fluorescence spectroscopy and molecular dynamic simulation. The study showed that the unfavorable binding landscape caused by the accumulation of drug-resistance mutations resulting from LPV associated drug pressure would shape the outcome of DRV-based therapy after a switch in the treatment regimen. This is related to the distortion of the HIV-1 PR structure associated with increased solvent exposure and instability of the HIV-1 PR dimer caused by these mutations leading to a shorter lifetime of the enzyme-inhibitor complex. Analysis of the binding kinetics of LPV and DRV with the HIV-1 PR variants showed that the drug resistance mutations caused an imbalance between the association and dissociation rate constants favoring a fast dissociation rate. The latter resulted in a reduced inhibitor residence time. Our findings showed that LPV had a longer residence time than DRV when bound to the HIV-1 PR variants; this shows LPV can be a suitable platform for developing newer HIV-1 PIs with a longer residence time. However, the enzyme inhibition mechanism shows both LPV and DRV act via a two-step tight-binding mixed inhibition mechanism, suggesting the existence of a second binding site on HIV-1 PR for these inhibitors. The information provided in this thesis adds to existing knowledge about HIV-1 PI drug resistance and for the design of novel HIV-1 PIs with the potential to evade drug resistance mutations.