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.

Development of stavudine resistance was studied using human immunodeficiency virus type 1 isolates from 13 patients treated with stavudine for 18-22 months. Drug sensitivity testing on 11 of these pre- and posttherapy isolates identified only 2 posttreatment isolates with decreased stavudine sensitivity (ED50s < 4-fold higher than the average pretreatment ED50). Genotypic analysis of all 13 pairs of isolates identified multiple mutations in the reverse transcriptase (RT) gene. However, no genetic basis was identified to account for the observed changes in stavudine susceptibility. A recombinant virus containing the entire RT gene of the posttherapy isolate displaying the greatest resistance remained sensitive to stavudine. Five of the stavudine posttreatment isolates developed resistance (9- to 176-fold) to zidovudine, although the relationship between stavudine treatment and the appearance of zidovudine resistance remains unexplained. Analysis of 10 additional pairs of isolates did not confirm this relationship. The low frequency and modest degree of change in stavudine sensitivity following prolonged treatment is very encouraging.

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.

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 (G 2 T 1 C ؊1 A ؊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 G 2 :C 2 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) Gln 148 interacts with G 2 , T 1 , 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.Integration, the insertion of a double-stranded DNA copy of the viral RNA genome into the host genome, is absolutely required for HIV 2 replication (1). As such, integration presents an attractive target for the development of small molecule inhibitors that could be used to treat HIV. Several integrase (IN) inhibitors that are progressing through clinical development (2, 3) have been shown to lower viral load in infected patients.The virus-encoded IN protein catalyzes two essential activities in the viral life cycle, 3Ј-processing and strand transfer. The 3Ј-processing reaction cleaves off the final two bases (5Ј-GT dinucleotide) from the 3Ј ends of the viral LTR. The strand transfer activity catalyzes the concerted insertion of the two viral 3Ј ends into the host genome with a 5-bp separation. To accomplish this, IN simultaneously positions the two 3Ј-hydroxyls of the LTRs for nucleophilic attack onto the phosphodiester bonds of the genomic DNA (4). In vitro, both reactions are catalyzed by divalent magnesium or manganese, although magnesium is thought to be the actual metal cofactor in cells (5).Suitable in vitro systems for studying these processes have been described, in which the full-length protein is minimally required to direct both 3Ј-processing and strand transfer (6, 7). In these systems, IN is typically allowed to form an in situ complex with a model D...

Specific HIV integrase strand transfer inhibitors are thought to bind to the integrase active site, positioned to coordinate with two catalytic magnesium atoms in a pocket flanked by the end of the viral LTR. A structural role for the 3′ terminus of the viral LTR in the inhibitor-bound state has not previously been examined. This study describes the kinetics of binding of a specific strand transfer inhibitor to integrase variants assembled with systematic changes to the terminal 3′ adenosine. Kinetic experiments are consistent with a two-step binding model in which there are different functions for the terminal adenine base and the terminal deoxyribose sugar. Adenine seems to act as a "shield" which retards the rate of inhibitor association with the integrase active site, possibly by acting as an internal competitive inhibitor. The terminal deoxyribose is responsible for retarding the rate of inhibitor dissociation, either by sterically blocking inhibitor egress or by a direct interaction with the bound inhibitor. These findings further our understanding of the details of the inhibitor binding site of specific strand transfer inhibitors.