Structure-activity relationships of HIV-1 reverse transcriptase with small molecules: searching for inhibitors with new mechanisms of action
Creators: name: Losada, Natalie M.
Publisher: name: No Publisher Supplied
Year: 2024

description: Four decades after the start of the epidemic, human immunodeficiency virus (HIV) and its resulting disease, acquired immunodeficiency syndrome (AIDS), remain a global issue. Since the discovery of HIV, more than 20 HIV drugs have been FDA approved and target almost every part of the viral replication cycle from entry to maturation. However, due to many factors including HIV’s high mutation rate, unequal access to therapies, and sometimes low adherence to medication, many drug-resistant mutants of HIV exist that are difficult to treat. There is an ongoing need for new drugs, ideally with new mechanisms of action, that can evade current and future drug resistance. HIV reverse transcriptase (RT) has proven to be a valuable target for HIV therapies because: i) it is a non-human protein, ii) it stops the replication cycle before the viral genome is permanently integrated into the host, and iii) as an enzyme it is relatively more conserved than structural proteins. Current FDA-approved RT drugs bind at only two locations: the polymerase active site (nucleos(t)ide RT inhibitors, NRTIs) and an allosteric site (non-nucleoside RT inhibitors, NNRTIs). This thesis represents a tour of RT inhibition mechanisms, accomplished by evaluating inhibitors binding at those and other binding pockets of RT, as well as initiating investigation of RT from another significant HIV subtype. In this work, structure-activity relationships between RT and multiple series of inhibitors are investigated using X-ray crystallography, biophysical assays, and in vitro biochemical assays. The conclusions of this work are enriched and supported by additional information from medicinal chemistry studies and cell-based assays performed by collaborators. In Chapter 2, collaborators from Shandong University use in vitro and cell based studies to develop a series of NNRTIs, based on the etravirine scaffold, that maintain potency against the NNRTI-resistant mutant, E138K. Crystal structures of wild-type and mutant RT with the lead compounds (IDs 6D, 7D, and 8V) provide a clear explanation of their potency against E138K RT. The salt bridge between E138 and K101 is disrupted with the E138K mutation and the mutant is less susceptible to the second-generation NNRTI, rilpivirine, but the compounds in this study salvage their interactions with RT to maintain potency. In Chapters 3 and 4, collaborators from the New York Blood Center developed HIV entry inhibitors, utilizing in vitro and cell-based studies, which led to the serendipitous discovery of inhibitors that also target HIV RT. Crystal structures of RT in complex with these compounds (lead compounds NBD-14189 and NBD-14270) reveal their unique binding site that bridges the two known drug binding sites on RT (Chapter 3). Additionally, biophysical assays (differential scanning fluorimetry), biochemical assays (polymerase and RNase H activity assays), and X-ray crystallographic studies of RT/DNA complexes with the lead compounds further the understanding of their mixed inhibition mechanism against RT (Chapter 4). In Chapter 5, X-ray crystallographic studies and biophysical studies were performed with HIV RT in complex with DNA and Compound A, a compound from a distinct class of RT inhibitors known as nucleotide-competing RT inhibitors (NcRTIs). The crystal structures reveal protein-DNA-inhibitor interactions that could potentially lead to a new class of HIV RT drugs. Additionally, biophysical experiments with compounds from Chapters 3 and 4 reveal that each compounds’ binding is mutually exclusive. In Chapter 6, a very prevalent and uncommonly studied subtype of HIV, subtype C, is investigated. Comparison of the crystal structure of subtype C RT in complex with DNA to its equivalent with subtype B RT reveals subtle differences in the RT enzyme. This crystal structure sets the groundwork for further development of more effective, subtype-specific treatments.