Himadri Samanta

Changes to the HIV Long Terminal Repeat and to HIV Integrase Differentially Impact HIV Integrase Assembly, Activity, and the Binding of Strand Transfer Inhibitors

Human immunodeficiency virus (HIV) integrase enzyme is required for the integration of viral DNA into the host cell chromosome. Integrase complex assembly and subsequent strand transfer catalysis are mediated by specific interactions between integrase and bases at the end of the viral long terminal repeat (LTR). The strand transfer reaction can be blocked by the action of small molecule inhibitors, thought to bind in the vicinity of the viral LTR termini. This study examines the contributions of the terminal four bases of the nonprocessed strand (G2T1C–1A–2) of the HIV LTR on complex assembly, specific strand transfer activity, and inhibitor binding. Base substitutions and abasic replacements at the LTR terminus provided a means to probe the importance of each nucleotide on the different functions. An approach is described wherein the specific strand transfer activity for each integrase/LTR variant is derived by normalizing strand transfer activity to the concentration of active sites. The key findings of this study are as follows. 1) The G2:C2 base pair is necessary for efficient assembly of the complex and for maintenance of an active site architecture, which has high affinity for strand transfer inhibitors. 2) Inhibitor-resistant enzymes exhibit greatly increased sensitivity to LTR changes. 3) The strand transfer and inhibitor binding defects of a Q148R mutant are due to a decreased affinity of the complex for magnesium. 4) Gln148 interacts with G2, T1, and C–1 at the 5′ end of the viral LTR, with these four determinants playing important and overlapping roles in assembly, strand transfer catalysis and high affinity inhibitor binding.

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors *

In this study, eight different HIV-1 integrase proteins containing mutations observed in strand transfer inhibitor-resistant viruses were expressed, purified, and used for detailed enzymatic analyses. All the variants examined were impaired for strand transfer activity compared with the wild type enzyme, with relative catalytic efficiencies (kp/Km) ranging from 0.6 to 50% of wild type. The origin of the reduced strand transfer efficiencies of the variant enzymes was predominantly because of poorer catalytic turnover (kp) values. However, smaller second-order effects were caused by up to 4-fold increases in Km values for target DNA utilization in some of the variants. All the variants were less efficient than the wild type enzyme in assembling on the viral long terminal repeat, as each variant required more protein than wild type to attain maximal activity. In addition, the variant integrases displayed up to 8-fold reductions in their catalytic efficiencies for 3′-processing. The Q148R variant was the most defective enzyme. The molecular basis for resistance of these enzymes was shown to be due to lower affinity binding of the strand transfer inhibitor to the integrase complex, a consequence of faster dissociation rates. In the case of the Q148R variant, the origin of reduced compound affinity lies in alterations to the active site that reduce the binding of a catalytically essential magnesium ion. Finally, except for T66I, variant viruses harboring the resistance-inducing substitutions were defective for viral integration.

Identification and Characterization of BMS-955176, a Second-Generation HIV-1 Maturation Inhibitor with Improved Potency, Antiviral Spectrum, and Gag Polymorphic Coverage

ABSTRACT BMS-955176 is a second-generation human immunodeficiency virus type 1 (HIV-1) maturation inhibitor (MI). A first-generation MI, bevirimat, showed clinical efficacy in early-phase studies, but ∼50% of subjects had viruses with reduced susceptibility associated with naturally occurring polymorphisms in Gag near the site of MI action. MI potency was optimized using a panel of engineered reporter viruses containing site-directed polymorphic changes in Gag that reduce susceptibility to bevirimat (including V362I, V370A/M/Δ, and T371A/Δ), leading incrementally to the identification of BMS-955176. BMS-955176 exhibits potent activity (50% effective concentration [EC50], 3.9 ± 3.4 nM [mean ± standard deviation]) toward a library (n = 87) of gag/pr recombinant viruses representing 96.5% of subtype B polymorphic Gag diversity near the CA/SP1 cleavage site. BMS-955176 exhibited a median EC50 of 21 nM toward a library of subtype B clinical isolates assayed in peripheral blood mononuclear cells (PBMCs). Potent activity was maintained against a panel of reverse transcriptase, protease, and integrase inhibitor-resistant viruses, with EC50s similar to those for the wild-type virus. A 5.4-fold reduction in EC50 occurred in the presence of 40% human serum plus 27 mg/ml of human serum albumin (HSA), which corresponded well to an in vitro measurement of 86% human serum binding. Time-of-addition and pseudotype reporter virus studies confirm a mechanism of action for the compound that occurs late in the virus replication cycle. BMS-955176 inhibits HIV-1 protease cleavage at the CA/SP1 junction within Gag in virus-like particles (VLPs) and in HIV-1-infected cells, and it binds reversibly and with high affinity to assembled Gag in purified HIV-1 VLPs. Finally, in vitro combination studies showed no antagonistic interactions with representative antiretrovirals (ARVs) of other mechanistic classes. In conclusion, BMS-955176 is a second-generation MI with potent in vitro anti-HIV-1 activity and a greatly improved preclinical profile compared to that of bevirimat.

Discovery of BMS-955176, a Second Generation HIV-1 Maturation Inhibitor with Broad Spectrum Antiviral Activity

HIV-1 maturation inhibition (MI) has been clinically validated as an approach to the control of HIV-1 infection. However, identifying an MI with both broad polymorphic spectrum coverage and good oral exposure has been challenging. Herein, we describe the design, synthesis, and preclinical characterization of a potent, orally active, second generation HIV-1 MI, BMS-955176 (2), which is currently in Phase IIb clinical trials as part of a combination antiretroviral regimen.