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The employment of HIV-1 protease (PR) inhibitors (PIs) in antiviral therapy has been successful in reducing mortality of HIV/AIDS patients. However, the long-term efficacy of PIs is challenged by the rapid emergence of drug-resistant mutants of PR. To understand the underlying mechanism of drug resistance, structures and activities of HIV-1 PR and its drug resistant mutants have been extensively studied. Here, PR mutants PRR8Q, PRD30N, PRI47V, PRI50V, PRI54M, PRV82A, and PRN88D/S bearing single substitutions have been investigated by crystallography and kinetics. GRL-0519 is a potent new antiviral inhibitor of HIV-1 PR that possesses tris-tetrahydrofuran (tris-THF) as the P2 ligand. The crystal structures of GRL-0519 were determined at resolutions of 1.06-1.49 Å in complex with the mutants PRR8Q, PRD30N, PRI50V, PRI54M, and PRV82A. I50V lost its interaction with inhibitor while V82Aand I54M compensated for the mutation through the main chain shift and flexibility of 80’s loop (residues 78-82), respectively. The structural changes may account for the worst inhibition of GRL-0519 for PRI50V (60-fold decrease relative to wild-type enzyme)and moderate inhibition for PRI54M and PRV82A (6-7-fold decrease). The large tris-THF group at P2 provides a good fit in the S2 subsite and may be effective against resistant virus with mutations of residues in this subsite. SQV and DRV are two clinical inhibitors that were designed to target the wild type PR and its drug resistant mutants, respectively. The crystal structures of PR mutants PRI47V, PRN88D/s in complex with DRV and mutants PRI47V and PRN88D in complex with SQV with resolutions of 1.13-1.72 Å were also analyzed. Mutation I47V gained more hydrophobic interactions with DRV and SQV. Interestingly, the structural changes did not affect the inhibition of both inhibitors for PRI47V (relative Ki is 0.7 and 1 for DRV and SQV, respectively). DRV and SQV showed 8-fold increase in Ki for PRN88D and only very subtle local changes have been observed on the structures. DRV induced 0.3 fold reduction in Ki for PRN88S and the distal structural changes have been transferred to the active site. This study provided fundamental information for understanding drug resistance and future design of potential antiviral drugs.

HIV-1 protease (PR) inhibitors (PIs) are important anti-HIV drugs for the treatment of AIDS and have shown great success in reducing mortality and prolonging the life of HIV-infected individuals. However, the rapid development of drug resistance is one of the major factors causing the reduced effectiveness of PIs. Consequently, various drug resistant mutants of HIV-1 PR have been extensively studied to gain insight into the mechanisms of drug resistance. In this study, the crystal structures, dimer stabilities, and kinetics data have been analyzed for wild type PR and over 10 resistant mutants including PRL24I, PRI32V, PRM46L, PRG48V, PRI50V, PRF53L, PRI54V, PRI54M, PRG73S and PRL90M. These mutations lie in varied structural regions of PR: adjacent to the active site, in the inhibitor binding site, the flap or at protein surface. The enzymatic activity and inhibition were altered in mutant PR to various degrees. Crystal structures of the mutants complexed with a substrate analog inhibitor or drugs indinavir, saquinavir and darunavir were determined at resolutions of 0.84 – 1.50 Å. Each mutant revealed distinct structural changes, which are usually located at the mutated residue, the flap and inhibitor binding sites. Moreover, darunavir was shown to bind to PR at a new site on the flap surface in PRI32V and PRM46L. The existence of this additional inhibitor binding site may explain the high effectiveness of darunavir on drug resistant mutants. Moreover, the unliganded structure PRF53L had a wider separation at the tips of the flaps than in unliganded wild type PR. The absence of flap interactions in PRF53L suggests a novel mechanism for drug resistance. Therefore, this study enhanced our understanding of the role of individual residues in the development of drug resistance and the structural basis of drug resistance mechanisms. Atomic resolution crystal structures are valuable for the design of more potent protease inhibitors to overcome the drug resistance problem.