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

Virologic outcome at Week 48 and specific TDR at Baseline known to affect NRTI Backbone.

<p>Thymidine Analog Mutations (TAMs); Virologic Success at week 48 (VS), Virologic Failure at week 48 (VF). Transmitted Drug Resistance Mutations (TDRs); 9 subjects with M184V/I +/− TAMs and/or +/− K65R at baseline. 16 subjects with >1 TAMs at baseline. 2 subjects with K65R at baseline.</p

Prevalence of transmitted drug resistance (TDR) mutations by Ultra-deep Sequencing (UDS) and by standard genotyping (SG).

<p>N(<b>t</b>)RTI – nucleoside(tide) reverse transcriptase inhibitor; NNRTI – non-nucleoside reverse transcriptase inhibitor, PI – protease inhibitor. 141 subjects with UDS data; 147 subjects with standard genotypic data.</p

Specific TDR patterns and levels for subjects with a M184V/I and/or K65R +/− TAMs.

<p>VF: Virologic Failure VS: Virologic Success NRTI: Nucleoside Reverse Transcriptase Inhibitor NNRTI: Non-Nucleoside Reverse Transcriptase Inhibitor PI: Protease Inhibitor. TAMs: Thymidine Analogue Mutations. HIV VL: HIV Viral Load. TDRs: Transmitted Drug Resistance Mutations. 6-LPV^# has both a M184V and a K65R TDR. <b>Bold: M184V, TAMs, Major PI mutations according to HIV Stanford Database.</b></p

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

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

Transmitted Drug Resistance by ARV Class and Virologic Outcome.

<p>Transmitted Drug Resistance by ARV Class and Virologic Outcome: Note: TDRs are not mutually exclusive (i.e. a subject could have a TDR from more than one ARV class). VS: Virologic Success, VF: Virologic Failure N(t)RTI(s)- nucleoside(tide) reverse transcriptase inhibitor; NNRTI – non-nucleoside reverse transcriptase inhibitor, PI – protease inhibitor. Any: Any transmitted drug resistance mutations. Any TDR: p-value = 0.35, NRTI: p-value = 1, NNRTI: p-value = 1, PI: p-value = 0.02.</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

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