Hepatitis B virus (HBV) is a global epidemic leading to an estimated 820,000 deaths in 2019. Despite the existence of a vaccine, 296 million individuals worldwide are chronically infected. Nucleoside drugs are being used to treat HBV infections, but chronically infected individuals may still develop liver disease, cirrhosis, or cancer under existing therapy. The HBV capsid protein (Cp) has emerged as a next-generation antiviral target because it is involved in almost all stages of viral infection, and existing modulators of Cp assembly reduce or eliminate the viral minichromosome (covalently-closed circular DNA, cccDNA) in cellular assays. Two classes of capsid assembly effector agents (CAE) are under development represented in this study by GLS4 (heteroaryl-pyrimidine, HAP) and GLP-26 (glyoxamidopyrrolo derivative). Due to challenges in viral selection, it remains to be seen what mutant will be selected in culture and in humans that confers resistance to these drugs. It is the central hypothesis of this study that resistant mutations will decrease CAE binding while retaining native capsid assembly energetics. To test this hypothesis, we developed a computational approach to generate all possible mutations in the CAE binding pocket and predicted the change in binding affinities in bound form and unbound form. This method identified several resistant mutations, including most of those previously reported, and predicted antiviral activity against existing HBV strains. We used a 3-steps approach with residue scanning and MM/GBSA of Schrödinger suite. The best-ranked resistance mutations retained favorable assembly energetics with reduced affinity for the drug. Out of predicted mutations, the mutations accessible by single nucleotide polymorphism might emerge as resistant mutations against drug due to low genetic barrier. The mutations associated with cross-resistance to both GLS4 and GLP-26 were also identified. Most importantly, the computational approach indicated specific naturally occurring mutations in the HBV Cp protein resist GLS4, but these species remain susceptible to GLP-26 inhibition suggesting possible synergy. Several key mutations were identified that may emerge in vitro and in a clinical setting. The results also suggest that GLP-26 will possess broad anti-HBV activity, more so than GLS4. This study demonstrates the utility of computational resistance prediction in the context of drug discovery, which can be applied to many viral protiens.

Computational prediction of susceptibility and resistance for HBV capsid assembly effectors

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ACS Fall 2022

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