Entecavir Exhibits Inhibitory Activity against Human Immunodeficiency Virus under Conditions of Reduced Viral Challenge▿

Entecavir (ETV) was developed for the treatment of chronic hepatitis B virus (HBV) infection and is globally approved for that indication. Initial preclinical studies indicated that ETV had no significant activity against human immunodeficiency virus type 1 (HIV-1) in cultured cell lines at physiologically relevant ETV concentrations, using traditional anti-HIV assays. In response to recent clinical observations of anti-HIV activity of ETV in HIV/HBV-coinfected patients not receiving highly active antiretroviral therapy (HAART), additional investigative studies were conducted to expand upon earlier results. An extended panel of HIV-1 laboratory and clinical strains and cell types was tested against ETV, along with a comparison of assay methodologies and resistance profiling. These latest studies confirmed that ETV has only weak activity against HIV, using established assay systems. However, a >100-fold enhancement of antiviral activity (equivalent to the antiviral activity of lamivudine) could be obtained when assay conditions were modified to reduce the initial viral challenge. Also, the selection of a M184I virus variant during the passage of HIV-1 at high concentrations of ETV confirmed that ETV can exert inhibitory pressure on the virus. These findings may have a significant impact on how future assays are performed with compounds to be used in patients infected with HIV. These results support the recommendation that ETV therapy should be administered in concert with HAART for HIV/HBV-coinfected patients

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

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

Antiviral Activity of MIs Toward Gag Polymorphic Viruses

<p>Antiviral Activity of MIs Toward Gag Polymorphic Viruses</p

Representative antiviral dose-response plots for selected MIs in a multiple cycle antiviral assay.

<p>Representative examples of antiviral inhibition curves in a multiple cycle replication assay for A) BVM, B) BMS-1 and C) BMS-955176 toward WT, V362I, V370A and ΔV370 viruses. Maximal percent inhibition (MPI) values were calculated from the plateaus in inhibition at the highest inhibitor concentration, and tabulated and normalized for multiple experiments in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005990#ppat.1005990.t002" target="_blank">Table 2</a> using the equation MPI = (1 - (signal from the average at the two highest drug concentrations/signal from no drug control) * 100). Units are in <i>Renilla</i> luciferase relative light units (RLU). In cases where there is no curve, and thus no plateau <i>per se</i>, the same calculation method was employed using the two values at the highest compound concentration (6 μM).</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