Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation

Abstract

Integration is essential for HIV-1 replication, and the viral integrase (IN) protein is an important therapeutic target. Allosteric IN inhibitors (ALLINIs) that engage the IN dimer interface at the binding site for the host protein lens epithelium-derived growth factor (LEDGF)/ transcriptional coactivator p75 are an emerging class of small molecule antagonists. Consistent with the inhibition of a multivalent drug target, ALLINIs display steep antiviral dose-response curves ex vivo. ALLINIs multimerize IN protein and concordantly block its assembly with viral DNA in vitro, indicating that the disruption of two integration-associated functions, IN catalysis and the IN-LEDGF/p75 interaction, determines the multimode mechanism of ALLINI action. We now demonstrate that ALLINI potency is unexpectedly accounted for during the late phase of HIV-1 replication. The compounds promote virion IN multimerization and, reminiscent of class II IN mutations, block the formation of the electron-dense viral core and inhibit reverse transcription and integration in subsequently infected target cells. Mature virions are recalcitrant to ALLINI treatment, and compound potency during virus production is independent of the level of LEDGF/p75 expression. We conclude that cooperative multimerization of IN by ALLINIs together with the inability for LEDGF/p75 to effectively engage the virus during its egress from cells underscores the multimodal mechanism of ALLINI action. Our results highlight the versatile nature of allosteric inhibitors to primarily inhibit viral replication at a step that is distinct from the catalytic requirement for the target enzyme. The vulnerability of IN to small molecules during the late phase of HIV-1 replication unveils a pharmacological Achilles' heel for exploitation in clinical ALLINI development. AIDS | antiretroviral therapy

A Critical Role of the C-terminal Segment for Allosteric Inhibitor-induced Aberrant Multimerization of HIV-1 Integrase

“…[33][34][35][36]. These compounds bind at the IN CCD dimer interface occupying the principal LEDGF/p75 binding pocket (12,(37)(38)(39)(40)(41)(42). Similar to LEDGF/p75 Asp-366, the ALLINI carboxylic acid hydrogen bonds with the main chain nitrogens of residues Glu-170 and His-171 of one IN subunit.…”

“…In infected cells, ALLINIs impair both the early and late stage of HIV-1 replication underscoring the multifunctional nature of IN during HIV-1 replication (12,39,44,45). When added to target cells, ALLINIs block 3Ј-processing as well as reduce LEDGF/p75-mediated integration into active transcription units (39,46).…”

“…When added to target cells, ALLINIs block 3Ј-processing as well as reduce LEDGF/p75-mediated integration into active transcription units (39,46). Knockdown or knock-out of endogenous LEDGF/p75 significantly enhanced the potencies of these inhibitors in target cells, suggesting that there is competition between the cellular protein and ALLINIs for binding to HIV-1 IN during early steps of viral replication (12,47,48). However, the ALLINI antiviral potency is determined primarily through inhibiting the late stage of HIV-1 replication (12,41,42,44,45,48).…”

“…Knockdown or knock-out of endogenous LEDGF/p75 significantly enhanced the potencies of these inhibitors in target cells, suggesting that there is competition between the cellular protein and ALLINIs for binding to HIV-1 IN during early steps of viral replication (12,47,48). However, the ALLINI antiviral potency is determined primarily through inhibiting the late stage of HIV-1 replication (12,41,42,44,45,48). In producer cells, ALLINIs potently promote aberrant, higher-order multimerization of IN during virus maturation resulting in eccentric, non-infectious cores reminiscent to the phenotype seen with some class II IN mutants (8 -12, 44, 45).…”

“…In producer cells, ALLINIs potently promote aberrant, higher-order multimerization of IN during virus maturation resulting in eccentric, non-infectious cores reminiscent to the phenotype seen with some class II IN mutants (8 -12, 44, 45). In contrast with target cells, the absence of LEDGF/p75 had no effect on ALLINI potencies in the virus producer cells (12,44,47,48). The recent discovery of pyridine-based multimerization selective IN inhibitors, which potently promoted aberrant IN multimerization in vitro and in infected cells but were not effective inhibitors of IN-LEDGF/p75 interactions, have further highlighted IN multimerization as a plausible antiviral target (46).…”