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Maturation Inhibitor Mechanistic Studies - Differential Inhibition of Gag Polymorphs
Zeyu Lin1, Joseph Cantone1, Tricia Protack1, Dieter Drexler1, Beata Nowicka-Sans1, Yuan Tian2, Zheng Liu1, Mark Krystal1, Alicia Regueiro-Ren1, Ira B. Dicker1
1 Virology, Bristol-Myers Squibb Co, Wallingford, CT, United States. 2 Department of Chemical Synthesis, Bristol-Myers Squibb Co, Princeton, NJ, United States.
Background: HIV-1 maturation inhibitors (MIs) are a class of inhibitors that may be effective in the treatment of HIV-1. MIs disrupt the final step in the HIV-1 protease-mediated cleavage of the HIV-1 Gag polyprotein, between capsid (CA) and spacer peptide 1 (SP1), which is responsible for a major conformational rearrangement of viral proteins within the virion, leading to the production of infectious virus. An MI, bevirimat, previously terminated from development as a result of inadequate coverage of polymorphic Gag variants present in the general HIV-1 population, was used as a model compound. Understanding the mechanism of action of MIs in greater detail may be of value to help guide the development of MIs with improved genotypic coverage.
Methods: In this study, we developed a novel LC/MS assay using assembled Gag virus-like particles (VLPs) to quantitatively characterize the kinetics of CA/SP1 cleavage of a family of Gag variant genotypes. These variant VLPs contained site-directed mutations that alter susceptibility of HIV-1 to bevirimat. This method was also used to study inhibition by bevirimat and a second MI in this system. Secondarily, we used a radioligand binding assay to measure the kinetics of dissociation of bevirimat and this second MI to the same Gag variant VLPs.
Results: The LC/MS cleavage assay allows for the simultaneous quantization of both cleavage steps leading to the production of free SP1 (SP1/nucleocapsid and CA/SP1) while monitoring the effects of MIs on these processes. The innate rates of cleavage at CA/SP1 roughly correlate inversely with the ability of bevirimat to inhibit these HIV-1 polymorphic viruses in antiviral assays; genotypes with more rapid CA/SP1 cleavage profiles are less sensitive to bevirimat. The MI kinetic dissociation data indicate that improvements to polymorphic antiviral activity arise from increases in MI dissociation half-lives toward those polymorphs.
Conclusions: Together, the innate polymorph cleavage rates at CA/SP1 and the MI-specific kinetic dissociation data suggest a model for MI inhibition of HIV-1 protease mediated CA/SP1 cleavage that can be used to quantify MI antiviral behavior as a function of both MI and Gag polymorphs.