Abstract

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a very promising new class of anti-HIV-1 agents that exhibit a multimodal mechanism of action by allosterically modulating IN multimerization and interfering with IN-lens epithelium-derived growth factor (LEDGF)/p75 binding. Selection of viral strains under ALLINI pressure has revealed an A128T substitution in HIV-1 IN as a primary mechanism of resistance. Here, we elucidated the structural and mechanistic basis for this resistance. The A128T substitution did not affect the hydrogen bonding between ALLINI and IN that mimics the IN-LEDGF/p75 interaction but instead altered the positioning of the inhibitor at the IN dimer interface. Consequently, the A128T substitution had only a minor effect on the ALLINI IC50 values for IN-LEDGF/p75 binding. Instead, ALLINIs markedly altered the multimerization of IN by promoting aberrant higher order WT (but not A128T) IN oligomers. Accordingly, WT IN catalytic activities and HIV-1 replication were potently inhibited by ALLINIs, whereas the A128T substitution in IN resulted in significant resistance to the inhibitors both in vitro and in cell culture assays. The differential multimerization of WT and A128T INs induced by ALLINIs correlated with the differences in infectivity of HIV-1 progeny virions. We conclude that ALLINIs primarily target IN multimerization rather than IN-LEDGF/p75 binding. Our findings provide the structural foundations for developing improved ALLINIs with increased potency and decreased potential to select for drug resistance.

Highlights

  • The A128T substitution in HIV-1 integrase (IN) confers resistance to allosteric integrase inhibitors (ALLINIs)

  • To examine the effects of the A128T substitution on HIV-1 IN function, we compared the catalytic activities of purified recombinant WT and mutant proteins

  • ALLINI-1 was identified by Boehringer Ingelheim through a high-throughput screen for IN 3Ј-processing activity [13], and its multimodal mechanism of action has been elucidated by our group [14]

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Summary

Background

The A128T substitution in HIV-1 integrase (IN) confers resistance to allosteric integrase inhibitors (ALLINIs). WT IN catalytic activities and HIV-1 replication were potently inhibited by ALLINIs, whereas the A128T substitution in IN resulted in significant resistance to the inhibitors both in vitro and in cell culture assays. Subsequent studies from our group and others demonstrated that 2-(quinolin-3yl)acetic acid derivatives exhibit a multimodal mechanism of action by allosterically modulating the IN structure, which affects both IN-LEDGF/p75 binding and catalytic activity (14 – 16). We have investigated the structural and mechanistic properties for the resistance of A128T IN to ALLINIs. Strikingly, the A128T substitution only modestly affected ALLINI IC50 values for IN-LEDGF/p75 binding but markedly altered the multimerization of IN in the presence of the inhibitors. Our studies highlight that aberrant IN multimerization is the primary target of this class of inhibitors and provide the structural foundations for the development of second-generation ALLINIs with increased potency and decreased potential to select for drug resistance

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