Source: Dissertation Abstracts International, Volume: 78-07(E), Section: B.

Adviser: Karen S. Anderson.

Human immunodeficiency virus (HIV) is the etiological agent of acquired immunodeficiency syndrome (AIDS). Comprising half of the FDA-approved drugs used to treat HIV, reverse transcriptase (RT) inhibitors are a cornerstone of antiretroviral therapy (ART). There are two classes of reverse transcriptase inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs), which terminate viral DNA synthesis, and non-nucleoside reverse transcriptase inhibitors (NNRTIs), which are allosteric noncompetitive inhibitors that disrupt catalysis.

The effective treatment of HIV is plagued by high rates of viral infection, the error prone nature of RT, and the rapid selection of resistant viral variants. Further complicating treatment is the need for patient compliance to a lifelong ART drug regimen to maintain low viral loads. Collectively, this necessitates the continuous development of novel, chemically diverse antiretroviral inhibitors with favorable physiochemical properties and improved resistance profiles as well as a greater understanding of the molecular mechanisms of host toxicity the many patients requiring decades of treatment suffer from.

The overarching goal of this dissertation is to further our understanding of the molecular mechanisms of inhibition, resistance, and host toxicity of nucleoside and non-nucleoside reverse transcriptase inhibitors used in treating HIV infected patients. Towards this end, structural and kinetic mechanistic analyses are used to understand the molecular basis of inhibition for two diaryltriazine NNRTIs that were rationally designed to maintain potency, have high barriers to drug resistance, and possess desirable physiochemical properties. This analysis will facilitate future development of NNRTIs with superior pharmacological profiles that yield greater bioavailability and ease of formulation to improve patient adherence to dosing regimens. In addition, the identification and characterization of immutable residues within the non-nucleoside inhibitor binding pocket (NNIBP) presents a formidable strategy for developing new inhibitors that combat the development of drug resistance. Pre-steady-state kinetic analyses of RT are conducted to elucidate the catalytic role of proline95 as a conserved, immutable residue of the NNIBP and provide a molecular basis for the extreme potency of a novel catechol diether class of NNRTIs.

Alternative mechanisms of host toxicity that stem from the requirement of lifelong administration of ART are pursued. Pre-steady-state kinetics is used to examine the link between mutations in human mitochondrial polymerase y (pol y) and the development of mitochondrial toxicity to understand whether certain mutations may predispose patients to the development of host toxicity. This investigation will guide the practice of personalized medicine to circumvent the development of NRTI-associated toxicities. As rational drug design and the iterative development of ART have improved the side-effect profiles of the most recent nucleotide analogs, continued reports of patients experiencing side effects and toxicity suggest alternative mechanisms of toxicity. To address this possibility and in an effort to identify such alternative mechanisms of host toxicity, the first pre-steady-state kinetic analysis of human PrimPol, a recently discovered primase-polymerase active in the nucleus and mitochondria, is performed. Its mechanism of polymerization and incorporation of natural dNTP and NRTI substrates to inform its potential as a perpetrator of NRTI-associated toxicity are described. These studies reveal that nucleotide selectivity limits chemical catalysis while release of the elongated DNA product is the overall rate-limiting step. PrimPol incorporates 4 of the 8 FDA-approved antiviral NRTIs with a kinetic profile distinct from pol y that may manifest in toxicity.

Collectively the findings presented herein provide insight into the development of novel ART with improved physiochemical properties, higher barriers to the development of drug resistance, and reduced side-effect and host toxicity profiles.