The quinoline-based allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are promising candidates for clinically useful antiviral agents. Studies using these compounds have highlighted the role of IN in both early and late stages of virus replication. However, dissecting the exact mechanism of action of the quinoline-based ALLINIs has been complicated by the multifunctional nature of these inhibitors because they both inhibit IN binding with its cofactor LEDGF/p75 and promote aberrant IN multimerization with similar potencies in vitro. Here we report design of small molecules that allowed us to probe the role of HIV-1 IN multimerization independently from IN-LEDGF/p75 interactions in infected cells. We altered the rigid quinoline moiety in ALLINIs and designed pyridine-based molecules with a rotatable single bond to allow these compounds to bridge between interacting IN subunits optimally and promote oligomerization. The most potent pyridine-based inhibitor, KF116, potently (EC50 of 0.024 µM) blocked HIV-1 replication by inducing aberrant IN multimerization in virus particles, whereas it was not effective when added to target cells. Furthermore, KF116 inhibited the HIV-1 IN variant with the A128T substitution, which confers resistance to the majority of quinoline-based ALLINIs. A genome-wide HIV-1 integration site analysis demonstrated that addition of KF116 to target or producer cells did not affect LEDGF/p75-dependent HIV-1 integration in host chromosomes, indicating that this compound is not detectably inhibiting IN-LEDGF/p75 binding. These findings delineate the significance of correctly ordered IN structure for HIV-1 particle morphogenesis and demonstrate feasibility of exploiting IN multimerization as a therapeutic target. Furthermore, pyridine-based compounds present a novel class of multimerization selective IN inhibitors as investigational probes for HIV-1 molecular biology.
Background:The A128T substitution in HIV-1 integrase (IN) confers resistance to allosteric integrase inhibitors (ALLINIs). Results: The A128T substitution does not significantly alter ALLINI IC 50 values for IN-LEDGF/p75 binding but confers marked resistance to ALLINI-induced aberrant integrase multimerization. Conclusion: Allosteric perturbation of HIV-1 integrase multimerization underlies ALLINI antiviral activity. Significance: Our findings underscore the mechanism of ALLINI action and will facilitate development of second-generation compounds.
Eight new compounds, including two cyclopenta[b]benzopyran derivatives (1, 2), two cyclopenta[b]benzofuran derivatives (3, 4), three cycloartane triterpenoids (5–7), and an apocarotenoid (8), together with 16 known compounds, were isolated from the chloroform-soluble partitions of separate methanol extracts of a combination of the fruits leaves and twigs, and of the roots of Aglaia perviridis collected in Vietnam. Isolation work was monitored using human colon cancer cells (HT-29) and facilitated with an LC/MS dereplication procedure. The structures of the new compounds (1–8) were determined on the basis of spectroscopic data interpretation. The Mosher ester method was employed to determine the absolute configurations of 5–7, and the absolute configurations of the 9,10-diol unit of compound 8 was established by a dimolybdenum tetraacetate [Mo2(AcO)4] induced circular dichroism (ICD) procedure. Seven known rocaglate derivatives (9–15) exhibited significant cytotoxicity against the HT-29 cell line, with rocaglaol (9) being the most potent (ED50 0.0007 µM). The new compounds 2–4 were also active against this cell line, with ED50 values ranging from 0.46 to 4.7 µM. The cytotoxic compounds were evaluated against a normal colon cell line, CCD-112CoN. In addition, the new compound perviridicin B (2), three known rocaglate derivatives (9, 11, 12), as well as a known sesquiterpene, 2-oxaisodauc-5-en-12-al (17), showed significant NF-κB (p65) inhibitory activity in an ELISA assay.
BackgroundAllosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an important new class of anti-HIV-1 agents. ALLINIs bind at the IN catalytic core domain (CCD) dimer interface occupying the principal binding pocket of its cellular cofactor LEDGF/p75. Consequently, ALLINIs inhibit HIV-1 IN interaction with LEDGF/p75 as well as promote aberrant IN multimerization. Selection of viral strains emerging under the inhibitor pressure has revealed mutations at the IN dimer interface near the inhibitor binding site.ResultsWe have investigated the effects of one of the most prevalent substitutions, H171T IN, selected under increasing pressure of ALLINI BI-D. Virus containing the H171T IN substitution exhibited an ~68-fold resistance to BI-D treatment in infected cells. These results correlated with ~84-fold reduced affinity for BI-D binding to recombinant H171T IN CCD protein compared to its wild type (WT) counterpart. However, the H171T IN substitution only modestly affected IN-LEDGF/p75 binding and allowed HIV-1 containing this substitution to replicate at near WT levels. The x-ray crystal structures of BI-D binding to WT and H171T IN CCD dimers coupled with binding free energy calculations revealed the importance of the Nδ- protonated imidazole group of His171 for hydrogen bonding to the BI-D tert-butoxy ether oxygen and establishing electrostatic interactions with the inhibitor carboxylic acid, whereas these interactions were compromised upon substitution to Thr171.ConclusionsOur findings reveal a distinct mechanism of resistance for the H171T IN mutation to ALLINI BI-D and indicate a previously undescribed role of the His171 side chain for binding the inhibitor.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-014-0100-1) contains supplementary material, which is available to authorized users.
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