Mechanistic Studies and Modeling Reveal the Origin of Differential Inhibition of Gag Polymorphic Viruses by HIV-1 Maturation Inhibitors

Abstract

HIV-1 maturation inhibitors (MIs) disrupt the final step in the HIV-1 protease-mediated cleavage of the Gag polyprotein between capsid p24 capsid (CA) and spacer peptide 1 (SP1), leading to the production of infectious virus. BMS-955176 is a second generation MI with improved antiviral activity toward polymorphic Gag variants compared to a first generation MI bevirimat (BVM). The underlying mechanistic reasons for the differences in polymorphic coverage were studied using antiviral assays, an LC/MS assay that quantitatively characterizes CA/SP1 cleavage kinetics of virus like particles (VLPs) and a radiolabel binding assay to determine VLP/MI affinities and dissociation kinetics. Antiviral assay data indicates that BVM does not achieve 100% inhibition of certain polymorphs, even at saturating concentrations. This results in the breakthrough of infectious virus (partial antagonism) regardless of BVM concentration. Reduced maximal percent inhibition (MPI) values for BVM correlated with elevated EC₅₀ values, while rates of HIV-1 protease cleavage at CA/SP1 correlated inversely with the ability of BVM to inhibit HIV-1 Gag polymorphic viruses: genotypes with more rapid CA/SP1 cleavage kinetics were less sensitive to BVM. In vitro inhibition of wild type VLP CA/SP1 cleavage by BVM was not maintained at longer cleavage times. BMS-955176 exhibited greatly improved MPI against polymorphic Gag viruses, binds to Gag polymorphs with higher affinity/longer dissociation half-lives and exhibits greater time-independent inhibition of CA/SP1 cleavage compared to BVM. Virological (MPI) and biochemical (CA/SP1 cleavage rates, MI-specific Gag affinities) data were used to create an integrated semi-quantitative model that quantifies CA/SP1 cleavage rates as a function of both MI and Gag polymorph. The model outputs are in accord with in vitro antiviral observations and correlate with observed in vivo MI efficacies. Overall, these findings may be useful to further understand antiviral profiles and clinical responses of MIs at a basic level, potentially facilitating further improvements to MI potency and coverage. HIV-1 continues to be a serious health threat, with nearly 40 million infected individuals worldwide. Despite effective treatment options, issues with resistance and drug toxicities illustrate the need for new drugs with novel mechanisms. Maturation inhibitors (MIs) block a key protease cleavage within its target, preventing formation of infectious HIV-1 virus. A first generation MI, (bevirimat), failed in clinical studies due to lack of broad spectrum activity, a result of amino acid polymorphisms around the site of action. BMS-955176 (GSK3532795) is a second generation MI active against these polymorphisms, and is currently in a Phase 2b study. We used a combination of antiviral and novel biochemical approaches to understand the mechanism for these spectrum differences. We find that while bevirimat exhibits incomplete antiviral activity, even at saturating drug concentrations, BMS-955176 exhibits greater ability to maximally inhibit these viruses, in part due to higher affinity for its target. These data were integrated into a semi-quantitative kinetic model whose outputs are in accord with in vitro antiviral observations and correlate with observed in vivo MI efficacies and the results of recent crystal and cryo-electron tomography structures. Our findings offer insights into MI activity and mechanism and may prove useful to help guide development of new MIs, with potential applicability to other virus systems and inhibitors.