Structural Adaptation of Darunavir Analogs Against Primary Resistance Mutations in HIV-1 Protease

HIV-1 protease is one of the prime targets of agents used in antiretroviral therapy against HIV. However, under selective pressure of protease inhibitors, primary mutations at the active site weaken inhibitor binding to confer resistance. Darunavir (DRV) is the most potent HIV-1 protease inhibitor in clinic; resistance is limited, as DRV fits well within the substrate envelope. Nevertheless, resistance is observed due to hydrophobic changes at residues including I50, V82 and I84 that line the S1/S1 pocket within the active site. Through enzyme inhibition assays and a series of 12 crystal structures, we interrogated susceptibility of DRV and two potent analogs to primary S1 mutations. The analogs had modifications at the hydrophobic P1 moiety to better occupy the unexploited space in the S1 pocket where the primary mutations were located. Considerable losses of potency were observed against protease variants with I84V and I50V mutations for all three inhibitors. The crystal structures revealed an unexpected conformational change in the flap region of I50V protease bound to the analog with the largest P1 moiety, indicating interdependency between the S1 subsite and the flap region. Collective analysis of protease-inhibitor van der Waals (vdW) interactions in the crystal structures using principle component analysis indicated I84V mutation underlying the largest variation in the vdW contacts. Interestingly, the principle components were able to distinguish inhibitor identity and relative potency solely based on vdW interactions of active site residues in the crystal structures. Our results reveal the interplay between inhibitor P1 moiety and primary S1 mutations, as well as suggesting a novel method for distinguishing the interdependence of resistance through principle component analyses.