Discovery of ligands binding to HIV-1 reverse transcriptase and capsid protein using X-ray crystallography, fragment screening, and covalent small molecule screening

Human immunodeficiency virus (HIV) infection is a global epidemic, with 37.7 million infected patients as of 2021. Over the past few decades, antiretroviral therapy (ART) for treatment of HIV-1 has been revolutionary in reducing HIV-related morbidity and mortality. However, it suffers from issues such as lack of drug adherence and accumulation of drug resistance mutations. At the same time, while there have been vast strides in our understanding of viral infection, some aspects of viral replication like uncoating of the viral capsid core inside host cells, interaction with host factors, immune response, and HIV latency are not well-understood. Alternative strategies are needed that can target the HIV in novel ways, to help understand its mechanism of action and develop better inhibitors of viral replication.Fragment screening and covalent small-molecule screening have emerged as useful tools for finding binders of drug targets, that can be developed into probes, inhibitors and even drugs. Fragment screening employs libraries of molecules that are smaller than drugs (“fragments”, molecular weight ~ 100 - 300 Da), that can be used to experimentally identify ligand-binding sites on drug targets. This can be done by a variety of biophysical, biochemical, and structural methods. Due to their small size and low molecular complexity, fragments can access sites on proteins that may not be accessible to larger drug-like binders. Fragment binders can then be grown into larger inhibitors or probes. This strategy has yielded six FDA-approved drugs over the past decade, and has been being increasingly employed by pharmaceutical companies to develop drugs against novel targets. Covalent small-molecule screening uses libraries of mildly electrophilic compounds that may bind covalently to reactive groups on proteins. Targeting specific residues on proteins that act as nucleophilic centers for these electrophilic binders can lead to selective binders. This technique has the advantage of yielding high potency binders, with prolonged drug action. This thesis discusses the use of fragment screening and covalent small-molecule screening to discover binders of HIV-related proteins. The design of a specialized fragment library for X-ray crystallographic fragment screening (the “Halo Library”) is described. Proof-of-concept testing has been carried out with crystals of HIV-1 reverse transcriptase (RT), with promising results. Fragment screening was carried out with HIV-1 capsid protein, which plays an important structural role in viral replication. Novel binders have been discovered, paving the way for development of useful inhibitors of viral replication. HIV-1 is subdivided into clades A-K, and multiple circulating recombinant factors (CRFs) like clade AG. The use of covalent small molecule screening against HIV-1 RT belonging to clade AG, which shares a low sequence homology (90%) with the well-researched clade B, is employed to discover covalent binders. The various clades of RT are poorly understood, and the use of covalent chemistry to find binders of HIV-1 RT Clade AG may assist with understanding inter-clade variation in viral action, and develop tailored probes and inhibitors. A few such covalent binders were discovered in this study.Ph.D.Includes bibliographical reference