Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage

Abstract

BackgroundLEDGF/p75 (LEDGF) is the main cellular cofactor of HIV-1 integrase (IN). It acts as a tethering factor for IN, and targets the integration of HIV in actively transcribed gene regions of chromatin. A recently developed class of IN allosteric inhibitors can inhibit the LEDGF-IN interaction.ResultsWe describe a new series of IN-LEDGF allosteric inhibitors, the most active of which is Mut101. We determined the crystal structure of Mut101 in complex with IN and showed that the compound binds to the LEDGF-binding pocket, promoting conformational changes of IN which explain at the atomic level the allosteric effect of the IN/LEDGF interaction inhibitor on IN functions. In vitro, Mut101 inhibited both IN-LEDGF interaction and IN strand transfer activity while enhancing IN-IN interaction. Time of addition experiments indicated that Mut101 behaved as an integration inhibitor. Mut101 was fully active on HIV-1 mutants resistant to INSTIs and other classes of anti-HIV drugs, indicative that this compound has a new mode of action. However, we found that Mut101 also displayed a more potent antiretroviral activity at a post-integration step. Infectivity of viral particles produced in presence of Mut101 was severely decreased. This latter effect also required the binding of the compound to the LEDGF-binding pocket.ConclusionMut101 has dual anti-HIV-1 activity, at integration and post-integration steps of the viral replication cycle, by binding to a unique target on IN (the LEDGF-binding pocket). The post-integration block of HIV-1 replication in virus-producer cells is the mechanism by which Mut101 is most active as an antiretroviral. To explain this difference between Mut101 antiretroviral activity at integration and post-integration stages, we propose the following model: LEDGF is a nuclear, chromatin-bound protein that is absent in the cytoplasm. Therefore, LEDGF can outcompete compound binding to IN in the nucleus of target cells lowering its antiretroviral activity at integration, but not in the cytoplasm where post-integration production of infectious viral particles takes place.

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

“…At the same time, it should be noted that ALLINIs are more potent during virus particle maturation rather than during the early steps of HIV-1 replication (12,41,42,44,45). The competition between chromatin associated LEDGF/p75 and ALLINI binding to the HIV-1 IN dimer interface appears to be one significant factor reducing the inhibitor potency in target cells.…”

“…[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.…”

“…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).…”

Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif

“…This interaction targets viral integration into transcriptionally active regions of the host chromatin recognized by the LEDGF/p75 PWWP domain [17][18][19][20][21] . The important role of LEDGF/p75 in lentiviral replication triggered the development of small molecules that efficiently block HIV-1 replication, referred to as LEDGINs that target the LEDGF/p75-HIV IN interaction interface [22][23][24][25][26][27][28][29] . LEDGINs are currently under early clinical development.…”

Quantitative evaluation of the antiretroviral efficacy of dolutegravir

“…High slopes for HIV-1 protease inhibitors (12) and allosteric integrase inhibitors (ALLINIs) (13)(14)(15)(16)(17) reflect inhibition at multiple steps in the viral life cycle. The first-generation InSTI raltegravir has not shown activity at HIV-1 life cycle steps other than integration (13)(14)(15), but DTG has not been evaluated for such activity.…”