Abstract
The multifunctional HIV-1 enzyme integrase interacts with viral DNA and its key cellular cofactor LEDGF to effectively integrate the reverse transcript into a host cell chromosome. These interactions are crucial for HIV-1 replication and present attractive targets for antiviral therapy. Recently, 2-(quinolin-3-yl) acetic acid derivatives were reported to selectively inhibit the integrase-LEDGF interaction in vitro and impair HIV-1 replication in infected cells. Here, we show that this class of compounds impairs both integrase-LEDGF binding and LEDGF-independent integrase catalytic activities with similar IC(50) values, defining them as bona fide allosteric inhibitors of integrase function. Furthermore, we show that 2-(quinolin-3-yl) acetic acid derivatives block the formation of the stable synaptic complex between integrase and viral DNA by allosterically stabilizing an inactive multimeric form of integrase. In addition, these compounds inhibit LEDGF binding to the stable synaptic complex. This multimode mechanism of action concordantly results in cooperative inhibition of the concerted integration of viral DNA ends in vitro and HIV-1 replication in cell culture. Our findings, coupled with the fact that high cooperativity of antiviral inhibitors correlates with their increased instantaneous inhibitory potential, an important clinical parameter, argue strongly that improved 2-(quinolin-3-yl) acetic acid derivatives could exhibit desirable clinical properties.
Highlights
HIV-1 integrase is an important antiretroviral target due to its essential role in virus replication [1]
LEDGIN-6 emerged through rational structure-based design to spatially mimic the interactions of LEDGF integrase binding domain (IBD) hot spot residues Ile-365 and Asp366 in their contacts with the integrase-catalytic core domain (CCD) dimer interface and has been reported to selectively inhibit the integraseLEDGF interaction (IC50 ϭ 1.37 M) but not integrase 3Ј-processing activity (IC50 Ͼ 250 M) [10]
We investigated the mechanism of action of two prototypes of a growing number of small molecule compounds that bind HIV-1 integrase distal from the enzyme active site
Summary
Chemical Synthesis of Integrase Inhibitors—2-(6-Chloro-2methyl-4-phenylquinolin-3-yl)pentanoic acid (LEDGIN-6) was prepared in six steps from commercially available 2-amino-5chloro-benzonitrile (Sigma-Aldrich) according to the scheme provided by Debyser and co-workers [10]. 2-(6-Bromo-4-(4chlorophenyl)-2-methylquinolin-3-yl)-2-methoxyacetic acid (BI-1001) was synthesized in five steps from commercially available 2-amino-4Ј-chlorobenzophenone (TCI America) through slight modification of the procedures reported in the patent [15]. 16802 JOURNAL OF BIOLOGICAL CHEMISTRY was preincubated with increasing concentrations of LEDGIN-6 or BI-1001 at room temperature for 30 min in 22 mM HEPES (pH 7.4) buffer containing 25.3 mM NaCl, 5.5 mM MgSO4, 11 mM DTT, 4.4 M ZnCl2 To this mixture, 1 M viral donor DNA (32-mer blunt-ended U5) and 600 ng of target (pBR322) DNAs were added. 10 nM N-terminally His-tagged integrase was pre-incubated in a binding buffer (150 mM NaCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mg/ml BSA, 25 mM Tris (pH 7.4)) with the tested compound for 30 min at room temperature, and 10 nM C-terminally FLAG-tagged LEDGF was added to the reaction. After incubation of HIV-1 integrase and inhibitor at room temperature for 1 h in 50 mM MOPS (pH 7.2) 50 mM NaCl, 10 mM MgCl2, 2 mM -mercaptoethanol, and 1% dimethyl sulfoxide, sypro orange was added to the final concentration of 0.1% (v/v). Where x is the inhibitor concentration, y is the percentage of inhibition, k is IC50 (or EC50 for antiviral measures), and n (or m for the virus data) is the Hill slope [28]
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