The mechanism of H171T resistance reveals the importance of Nδ-protonated His171 for the binding of allosteric inhibitor BI-D to HIV-1 integrase

Background: Allosteric 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. Results: We 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. Conclusions: Our 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 material The online version of this article (doi:10.1186/s12977-014-0100-1) contains supplementary material, which is available to authorized users

Indole-Based Allosteric Inhibitors of HIV-1 Integrase

Employing a scaffold hopping approach, a series of allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) have been synthesized based on an indole scaffold. These compounds incorporate the key elements utilized in quinoline-based ALLINIs for binding to the IN dimer interface at the principal LEDGF/p75 binding pocket. The most potent of these compounds displayed good activity in the LEDGF/p75 dependent integration assay (IC50 = 4.5 mu M) and, as predicted based on the geometry of the five- versus six-membered ring, retained activity against the A128T IN mutant that confers resistance to many quinoline-based ALLINIs. (C) 2016 Elsevier Ltd. All rights reserved

A New Class of Multimerization Selective Inhibitors of HIV-1 Integrase

<div><p>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 <i>in vitro</i>. 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.</p></div

Genotype of HIV-1 variants selected in cell culture in the presence of KF116.

<p>(<b>A</b>) Mutations in the HIV-1_NL4-3 IN gene of resistant viruses selected with KF116. Clonal sequencing of viral passage was carried out at passages 5 and 10, respectively. Eighty-two clones from each viral passage were sequenced using three sequencing primers covering the entire IN gene. Percentage of IN mutations for a given passage are indicated. Passage 5 corresponds to 50 days of selection with the KF116 concentration reaching 0.8 µM. Passage 10 corresponds to 100 days of selection with the KF116 concentration reaching 25.6 µM. (<b>B</b>) Crystal structure of KF116 bound to HIV-1 IN CCD dimer indicating the Thr-124, Val-165 and Thr-174 residues. The IN subunit 1 and 2 are colored in cyan and green, respectively. KF116 is shown in magenta.</p

Activities of HIV-1 IN inhibitors.

<p>Data for IC₅₀ and EC₅₀ are given as the mean ± SD from at least three independent experiments.</p><p>CC₅₀ values of >100 µM indicates that the respective inhibitors were not cytotoxic at the tested concentrations upto 100 µM.</p>a<p>BI-1001 EC₅₀ value and the assay method have been described elsewhere <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004171#ppat.1004171-Kessl3" target="_blank">[33]</a>.</p

Crystal structures of LEDGF/IBD (A), BI-1001 (B), KF115 (C), and KF116 (D) bound to HIV-1 IN CCD.

<p>The IN subunit 1 and 2 are colored in cyan and green, respectively. LEDGF/IBD loop (amino acids 365–368) is shown in dark blue. BI-1001 is shown in orange. KF115 is shown in red. KF116 is shown in magenta. The hydrogen bonds between the IN subunit and the LEDGF/IBD or the indicated inhibitors are shown by black dashed lines. Side chains of HIV-1 IN residues A128T and T125 in subunit 1, and E170, H171 and T174 in subunit 2 are shown.</p

Chemical structures of ALLINIs and MINIs.

<p>Chemical structures of ALLINIs and MINIs.</p

Virions produced in the presence of KF116 are defective in reverse transcription.

<p>(<b>A–E</b>) VSV-G pseudotyped HIV-1-Luc produced in the presence of DMSO, 1 µM KF116, or 1 µM RAL were used to infect HEK293T cells. Infected cells were harvested at the indicated times and subjected to quantitative PCR (qPCR) or luciferase assay. Graphs indicate the amount of PCR products relative to non-treated (DMSO) sample at 6 h post-infection for (<b>A</b>) early reverse transcription (Early RT), (<b>B</b>) late reverse transcription (Late RT) and (<b>C</b>) 2-LTR circles (2-LTRs) products. (<b>D</b>) Bar graphs indicate the integrated provirus relative to non-treated (DMSO) control at 7 days post-infection. (<b>E</b>) Aliquots of infected cells were harvested and luciferase assay was performed at 48 h post-infection. The luciferase signal obtained for the non-treated (DMSO) sample was set to 100%. All graphs represent mean ± SD (<i>n</i> = 3). (<b>F–J</b>) HEK293T cells were treated with DMSO, 1 µM KF116, or 1 µM RAL and then infected with VSV-G pseudotyped HIV-1-Luc. Infected cells were harvested at the indicated times and subjected to qPCR or luciferase assay. Graphs indicate the amount of PCR products relative to non-treated (DMSO) sample at 6 h post-infection for (<b>F</b>) early reverse transcription (Early RT), (<b>G</b>) late reverse transcription (Late RT) and (<b>H</b>) 2-LTR circles (2-LTRs) products. (<b>I</b>) Bar graphs indicate the integrated provirus relative to non-treated (DMSO) sample at 7 days post-infection. (<b>J</b>) Aliquots of infected cells were harvested and luciferase assay was performed at 48 h post-infection. The luciferase signal obtained for the non-treated (DMSO) sample was set to 100%. All graphs represent mean ± SD (<i>n</i> = 3).</p

LEDGF/p75 expression does not affect KF116 potency during late stage of HIV-1 replication.

<p>(<b>A</b>) Equivalent whole cell lysates from the clonal TALEN-derived <i>PSIP</i>1 KO cell line (indicated as “KO”) and parental wild type HEK293T cell line (indicated as “WT”) were subjected to SDS-PAGE and immunoblotted for LEDGF/p75 and a GAPDH control to verify knockdown of LEDGF/p75 protein. (<b>B</b>) Dose-response curves representing the antiviral assays performed in WT or KO cell lines under the indicated conditions of drug treatment. For producer cell treatment, the VSV-G pseudotyped HIV-1-Luc progeny virions were prepared in the indicated cell line in the presence of KF116 and were then used to infect untreated HEK293T cells. For target cell treatment, KF116 was added directly to the indicated cell line and the cells were infected with untreated VSV-G pseudotyped HIV-1-Luc virions. (<b>C</b>) EC₅₀ values for the indicated antiviral assays. Results represent mean ± SD from three independent experiments.</p