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

<div><p>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. <i>In vitro</i> 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 <i>in vitro</i> antiviral observations and correlate with observed <i>in vivo</i> 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.</p></div

Schematic for Cleavage of CA/SP1.

<p>A) Schematic for the processing HIV Gag at CA/SP1 and SP1/NC sites by HIV-protease. B) Detail of the cleavage region around CA/SP1 showing sites for HIV-1 protease cleavage (H1 and H2) and sites for subsequent cleavage by trypsin (T).</p

Inhibition of HIV-1 protease mediated CA/SP1 cleavage by MIs.

<p>A) BVM, B) BMS-955176: Inhibition of CA/SP1 cleavage of WT, ΔV370 and A364V VLPs <i>in vitro</i> as monitored by LC/MS analysis (Materials and Methods); values are an average of 9 replicates; bars are SEM; MI concentrations: 3 μM.</p

Modeling of the rate of CA/SP1 cleavage of HIV-1 WT, V370A, V362I and ΔV370 VLP in the presence of 300 nM MI.

<p>Modeled fractional rate of production of SP1 peptide from Gag VLP cleavage using model 2a at 300 nM MI, as noted in text; no MI: diamonds; BVM: squares; BMS-955176: triangles; y-axis: fraction of CA/SP1 cleavage is a surrogate for production of mature virus, as indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005990#ppat.1005990.g005" target="_blank">Fig 5</a>; A) WT; B) V370A; C)V362I; D) ΔV370</p

Rates of cleavage of WT and Gag polymorphic VLPs at CA/SP1 by HIV-1 protease <i>in vitro</i>

<p>Rates of cleavage of WT and Gag polymorphic VLPs at CA/SP1 by HIV-1 protease <i>in vitro</i></p

Scheme for inhibition of HIV-1 infectivity by MIs.

<p>Models that depict the inhibition of mature virus formation by HIV-1 maturation inhibitors. Immature virus B is in equilibrium for binding to MI to produce MI-bound immature form A. In model 1, HIV protease can only cleave immature virus (form B) in the absence of MIs. The innate cleavage rate constant (<i>k</i>₁) is a function of Gag polymorphs. In model 2, HIV-1 protease may cleave CA/SP1 in either form A (bound) or form B (unbound) to produce mature virus C, thus the former pathway provides an escape mechanism for the formation of mature virus in the presence of MIs with rate constant <i>k</i>₂. This second cleavage rate constant (<i>k</i>₂) is a function of both MIs and Gag polymorph, and is derived from inclusion of MPI values from multiple (model 2a) or single (model 2b) cycle antiviral assays (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005990#ppat.1005990.t002" target="_blank">Table 2</a>).</p

Calculated (Model 2a) <i>vs</i>. observed responses for BMS-955176 and BVM vs. clinical results.

<p>Model 2a reduction in rate of CA/SP1 cleavage of WT, V362I and V370A (surrogates for polymorphic viruses, as described in the text) at 300 nM MI, <i>vs</i>. observed reduction in HIV-1 log₁₀ c/mL RNA in proof of confidence (POC) studies; WT = wild type; BMS-955176 polymorphic clinical subtype B = changes at positions Gag 362, 369, 370 or 371; BVM polymorphic clinical = changes at positions Gag 369, 370 or 371; BMS-955176 clinical POC study, 40 mg maximal median HIV-1 log₁₀ RNA reduction; BVM clinical POC study, 250–400 mg.</p

Modeling effects of 300 nM MI on log₁₀ reduction in rate of CA/SP1 cleavage/predicted reduction in viral RNA.

<p>Modeling effects of 300 nM MI on log₁₀ reduction in rate of CA/SP1 cleavage/predicted reduction in viral RNA.</p

Antiviral profiles of selected ARVs of differing mechanism of action in the SC Assay.

<p>A) Test compound added during transfection to produce LAI pseudotyped NLRepRlucP373 Δ<i>env</i> and evaluated for activity by infections in stage 2, as described in text; B) Virus produced by transfection, test compound added at the second stage of virus infection, as described in text; BMS-955176 (MI, filled circles); BVM (MI, open circles); nelfinavir (protease inhibitor, triangles), BMS-378806 (HIV attachment inhibitor, squares).</p

Affinities of BMS-955176 and BVM toward HIV-1 VLPs.

<p>Affinities of BMS-955176 and BVM toward HIV-1 VLPs.</p