Author ORCID Identifier

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Irene Weber

Second Advisor

Robert Harrison

Third Advisor

William Walthall


HIV/AIDS continues to be a public health threat, with about 1 million people newly infected each year and nearly 40 million deaths from HIV/AIDS since 1980. HIV-1 protease (PR) is an important drug target for HIV antiretroviral therapy (ART). PR catalyzes the final step in the HIV-1 life cycle and is necessary for HIV to become infectious. Nine inhibitors of PR have been FDA approved for use in ART since 1995, but resistance to PR inhibitors is a growing problem. Several drug resistant mutants of PR have previously been studied to determine mechanisms of resistance and inform design of novel PR inhibitors. This research investigates two highly drug resistant mutants of PR, PR20 and PRS17, that are resistant to all clinical PR inhibitors but have different drug resistance mechanisms. PR20 evades PR inhibitors via a cluster of mutations in the active site that alter inhibitor binding. PRS17 has two mutations in the active site (V82S and G48V) and a unique curled flap conformation that starts at V48 and continues through G52. A revertant mutant of PRS17 (V48G) was characterized to examine the contribution of the G48V mutation to drug resistance.

Novel antiviral inhibitors GRL-0489A and GRL-0739A show similar inhibition to darunavir. Analysis of active site hydrogen bonding in PR20 structures showed no major differences versus PR20/DRV.

Enzymes kinetic assays showed PR inhibitors were more effective for revertant mutant PRS17V48G than for PRS17. Crystal structures were solved for the revertant with and without inhibitor. The curled flap conformation seen in PRS17 was reversed in PRS17V48G. Molecular dynamics (MD) simulations of open flap inhibitor-less PRS17V48G revealed the flaps were less dynamic than in PRS17. Structural analysis, MD simulations and enzyme kinetics assays show that PRS17V48G is more stable and more susceptible to inhibitors than PRS17. These data can be used to inform design of novel protease inhibitors to target PR mutants that are not resistant due to active site mutations.


File Upload Confirmation