The human immunodeficiency virus type 1 (HIV-1) protease (PR) is one of five viral targets of highly active anti-retroviral therapy (HAART) administered to the 34 million individuals living with HIV. The viral protease is a critical targetas it is responsible for virion maturation via processing of the Gag-Pol polypeptide. Patient adherence to HAART regimens is crucial, as non-adherence leads to continuous viral replication. Viral replication escape and subsequent rebound impedes HAART treatment by allowing for the growth of viral populations bearing various resistant mutations. The mutations within viral drug targets, including the viral protease, allow for inhibition evasion and continued biological function. Understanding the mechanisms underlying severe drug resistance is a major hindrance in inhibitor development. Increasing mutation number is not directly proportional to the severity of resistance, suggesting that resistance is not simply additive but that it is interdependent. I propose that the culmination of physical amino acid properties, locations, and combinations of resistant mutations underlie interdependent nature of multi-drug resistance. To probe potential patterns that underlie the interdependent mechanisms of resistance in the viral protease, I will use a panel of five multi-drug resistance (MDR) proteases derived from patients. The proteases in this panel bear between 19-26 mutations each and are resistant to even the most potent protease inhibitors (PIs). Using an array of biochemical and biophysical techniques, I will determine the in vitro inhibition and thermodynamic profiles for each of the proteases in th panel. I will use X-Ray crystallography and molecular dynamics simulations to structurally and dynamically characterize the physical aspects of interdependent resistance patterns. In addition to the protease variants, I have also obtained their cognate substrates from NC to p6 of Gag (residues 407-488). I will use the patient-derived proteases and their corresponding substrates to determine how substrate recognition and processing is allowed to continue in the presence of inhibitors using the techniques described above. Discerning and taking advantage of the mechanisms that underlie multi-drug resistance in viral targets could provide the tools necessary to proactively meliorate both current treatment and inhibitor design for HIV-1 targets.

Public Health Relevance

Although anti-retroviral therapy is effective for the majority of the 34 million individuals living with the human immunodeficiency virus worldwide, there is a small population for which the inhibitors administered are no longer effective. Understanding the underlying mutation patterns and essentially the algorithm used by the virus to confer cross-resistance to multiple protease inhibitors may provide a means to effectively improve current therapeutics such that they can withstand to the emergence of multiple mutations.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1)
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Gaillard, Shawn R
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University of Massachusetts Medical School Worcester
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United States
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Ragland, Debra A; Whitfield, Troy W; Lee, Sook-Kyung et al. (2017) Elucidating the Interdependence of Drug Resistance from Combinations of Mutations. J Chem Theory Comput 13:5671-5682
Potempa, Marc; Nalivaika, Ellen; Ragland, Debra et al. (2015) A Direct Interaction with RNA Dramatically Enhances the Catalytic Activity of the HIV-1 Protease In Vitro. J Mol Biol 427:2360-78
Ragland, Debra A; Nalivaika, Ellen A; Nalam, Madhavi N L et al. (2014) Drug resistance conferred by mutations outside the active site through alterations in the dynamic and structural ensemble of HIV-1 protease. J Am Chem Soc 136:11956-63