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. 2016 Nov 4;291(45):23569-23577.
doi: 10.1074/jbc.M116.753384. Epub 2016 Sep 19.

A New Class of Allosteric HIV-1 Integrase Inhibitors Identified by Crystallographic Fragment Screening of the Catalytic Core Domain

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Free PMC article

A New Class of Allosteric HIV-1 Integrase Inhibitors Identified by Crystallographic Fragment Screening of the Catalytic Core Domain

Disha Patel et al. J Biol Chem. .
Free PMC article

Abstract

HIV-1 integrase (IN) is essential for virus replication and represents an important multifunctional therapeutic target. Recently discovered quinoline-based allosteric IN inhibitors (ALLINIs) potently impair HIV-1 replication and are currently in clinical trials. ALLINIs exhibit a multimodal mechanism of action by inducing aberrant IN multimerization during virion morphogenesis and by competing with IN for binding to its cognate cellular cofactor LEDGF/p75 during early steps of HIV-1 infection. However, quinoline-based ALLINIs impose a low genetic barrier for the evolution of resistant phenotypes, which highlights a need for discovery of second-generation inhibitors. Using crystallographic screening of a library of 971 fragments against the HIV-1 IN catalytic core domain (CCD) followed by a fragment expansion approach, we have identified thiophenecarboxylic acid derivatives that bind at the CCD-CCD dimer interface at the principal lens epithelium-derived growth factor (LEDGF)/p75 binding pocket. The most active derivative (5) inhibited LEDGF/p75-dependent HIV-1 IN activity in vitro with an IC50 of 72 μm and impaired HIV-1 infection of T cells at an EC50 of 36 μm The identified lead compound, with a relatively small molecular weight (221 Da), provides an optimal building block for developing a new class of inhibitors. Furthermore, although structurally distinct thiophenecarboxylic acid derivatives target a similar pocket at the IN dimer interface as the quinoline-based ALLINIs, the lead compound, 5, inhibited IN mutants that confer resistance to quinoline-based compounds. Collectively, our findings provide a plausible path for structure-based development of second-generation ALLINIs.

Keywords: HIV-1; HIV-1 integrase; X-ray crystallography; allosteric inhibitor; drug discovery; drug screening; fragment screening; human immunodeficiency virus (HIV); integrase; medicinal chemistry.

Figures

FIGURE 1.
FIGURE 1.
Higher concentration soaks reveal binding of 1 to the LEDGF/p75 binding site at the CCD dimer interface. Stronger electron density for 1 bound at the LEDGF/p75 site (2FoFc map, contoured at 1.0σ) was observed with increasing concentration.
FIGURE 2.
FIGURE 2.
Crystal structure of 1 bound to the IN CCD dimer. Left, crystal structure of 1 bound to a crystal contact site (red spheres) and the LEDGF/p75 binding site (green spheres) within the IN CCD dimer (yellow and cyan ribbons, PDB code 5KRS). Right, detailed view of 1 (green) bound to the LEDGF/p75 binding site. Monomers are shown as yellow and cyan ribbons; residues defining the binding site are shown as sticks (thin sticks, apo residue conformations); the bridging water molecules are shown as red spheres. Dashed lines represent hydrogen bonds (black).
FIGURE 3.
FIGURE 3.
In vitro inhibitory activities of compounds 1, 5, and 8. A, dose-dependent effects of the indicated compounds on LEDGF/p75-dependent integration assay. B, comparative effects of the indicated fragments and ALLINI BI-D on inducing aberrant IN multimerization. C, zoomed-in view of the results in B showing the dose-dependent effects of the indicated fragments on promoting higher-order IN multimerization. D, summary of inhibitory potencies of the fragments in indicated HTRF-based assays. E, inhibitory activities of 5 in LEDGF/p75-dependent integration assays catalyzed by wild-type, A128T, and H171T INs.
FIGURE 4.
FIGURE 4.
A, BI-D and compound 5 dose-response curves in treated SupT1 cells (acute or early phase of HIV-1 replication). B, HEK293T cells producing HIV-Luc were treated with the indicated concentrations of compounds. Luciferase assays were conducted using SupT1 cell extracts 2 days after HIV-Luc infection. C, tabulation of EC50 values under the different conditions of HIV-1 infection and CC50 values for compound cytotoxicity, as determined by WST-1 cellular proliferation assay.
FIGURE 5.
FIGURE 5.
A, crystallographic structure of 8 bound to IN CCD dimer (PDB code 5KRT). Dashed lines represent hydrogen (black) and halogen (green) bonding interactions. B, hydrated docking result of 5 bound to PDB: 5KRS; dashed lines represent hydrogen (black) bond interactions. C, overlay of the crystal structure of BI-D (PDB code 4IDL, green) and docked pose of 5 (gray) bound to the IN CCD dimer (yellow, monomer A; cyan, monomer B).

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