Discovery of a small molecule agonist of phosphatidylinositol 3-kinase p110α that reactivates latent HIV-1

Combination antiretroviral therapy (cART) can effectively suppress HIV-1 replication, but the latent viral reservoir in resting memory CD4+ T cells is impervious to cART and represents a major barrier to curing HIV-1 infection. Reactivation of latent HIV-1 represents a possible strategy for elimination of this reservoir. In this study we describe the discovery of 1,2,9,10-tetramethoxy-7H-dibenzo[de,g]quinolin-7-one (57704) which reactivates latent HIV-1 in several cell-line models of latency (J89GFP, U1 and ACH-2). 57704 also increased HIV-1 expression in 3 of 4 CD8+-depleted blood mononuclear cell preparations isolated from HIV-1-infected individuals on suppressive cART. In contrast, vorinostat increased HIV-1 expression in only 1 of the 4 donors tested. Importantly, 57704 does not induce global T cell activation. Mechanistic studies revealed that 57704 reactivates latent HIV-1 via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. 57704 was found to be an agonist of PI3K with specificity to the p110a isoform, but not the p110β, δ or γ isoforms. Taken together, our work suggests that 57704 could serve as a scaffold for the development of more potent activators of latent HIV-1. Furthermore, it highlights the involvement of the PI3K/Akt pathway in the maintenance of HIV-1 latency. © 2014 Doyon et al

Latent HIV-1 reactivation activity of 57704, alone and in combination with other inducing agents, in the U1 and ACH2 cell line models of virus latency.

<p>HIV-1 expression was assessed by quantitative PCR analysis of early and late viral RNA transcripts. Where appropriate, TNFα was used as an activation control. (A) Latent HIV-1 reactivation of activity 57704 in the J89GFP cell line. An EC₅₀ of 10.3±1.2 µM was determined for 57704 in this cell line. (B) Latent HIV-1 reactivation activity of 57704 in the ACH2 cell line. (C) Latent HIV-1 reactivation of activity 57704 in the U1 cell line. (D) Graph of activation of latent HIV-1 early and late gene transcript expression in the ACH2 and U1 cell lines. EC₅₀ values were determined in Sigma Plot by nonlinear regression, as described previously. (E) Latent HIV-1 reactivation of 5 µM 57704 in combination with 1 µM prostratin in the U1 cell line. (F) Latent HIV-1 reactivation of 5 µM 57704 in combination with 5 µM SAHA in the U1 cell line. (G) Latent HIV-1 reactivation of 5 µM 57704 in combination with 2.5 µM DSF in the U1 cell line. (H) Latent HIV-1 reactivation of 5 µM 57704 in combination with 20 µM BIX-01294 in the U1 cell line. Data represent the mean ± standard deviation from 3 replicate experiments.</p

In vitro activity of 57704 and SAHA against class I HDAC isoforms.

<p>Data represent the mean ± standard deviation from 3 replicate experiments.</p

57704 reactivates latent HIV-1 via the PI3K/Akt signaling pathway.

<p>(A) Latent HIV-1 reactivation activity of prostratin in the absence (control) or presence of inhibitors of calcineurin (1 µM cyclosporin A), JNK (1 µM SP00125), PI3K (20 nM wortmannin), Akt (500 nM Akt Inhibitor IV) and PKC (500 nM Go6983). (B) Latent HIV-1 reactivation activity of 57704 in the absence (control) or presence of inhibitors of p38 MAPK (50 nM SB203580), calcineurin, JNK, PI3K, Akt, PKC and NF-κB (20 µM NF- κB activation inhibitor). (C) Western blot analysis of phosphorylated Akt in J89GFP treated with 5 µM 57704. (D) Latent HIV-1 reactivation activity of 57704 in the absence (control) or presence of a pan-inhibitor of PI3K (15 µM LY294002) or inhibitors targeting the p110α (2 µM PI-103), p110δ (5 µM IC87114), p110γ (2 µM AS-605240) or p110β (2 µM TGX-221) isoforms. (E) PI3K isoform transcript levels in cells treated with 5 µM 57704 or 5 µM 57704 SAHA.(F) Inhibition of the PI3K p110α, β, δ and γ isoforms by 100 nM wortmannin alone or in combination with 5 µM 57704 or 5 µM 57704 SAHA.</p

Latent HIV-1 reactivation activity of 57704 and T cell activation in CD8+-depleted MNC.

<p>(A) Quantitation of cell-free HIV-1 RNA in the culture supernatants of CD8+-depleted blood MNC incubated with 57704 or SAHA. (B) Surface expression of CD69, CD38 and HLA-DR after 24 h in purified T cells following exposure to 10 µg/mL PHA, 57704 or SAHA. (C) Surface expression of CD69, CD38 and HLA-DR after 5 days in purified T cells following exposure to 10 µg/mL PHA, 57704 or SAHA.</p

Comparison of methods to quantify inducible HIV-1 outgrowth

The quantitative viral outgrowth assay (qVOA) is the gold standard for measuring inducible, replication-competent HIV-1. Using MOLT4-R5 and SupT1-R5 cell lines instead of allogeneic blasts and HIV-1 RNA detection rather than p24 enzyme-immunoassay (EIA) has been proposed to improve the sensitivity of the qVOA. It is unclear, however, how these alternative approaches affect qVOA performance. We compared three qVOAs methods across 15 persons with HIV-1 on suppressive antiretroviral therapy and found that the MOLT4-R5 method yielded a significantly higher proportion of p24-positive wells (42%) than both the allogeneic blast (29%) and SupT1-R5 (32%) assays. Additionally, 5 of 7 qVOAs that were negative by p24 EIA showed viral outgrowth by HIV-1 RNA quantification (>10-fold increase within 7 days). These findings reveal the potential for underestimation of the latent, inducible reservoir by qVOA depending on the target cells used and the measure of viral outgrowth. Use of MOLT4-R5 cells with both p24 EIA and HIV-1 RNA to detect viral outgrowth was the most sensitive method

Integration in oncogenes plays only a minor role in determining the in vivo distribution of HIV integration sites before or during suppressive antiretroviral therapy.

HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 15,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 395,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, but modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 7 specific genes, and by clonal expansion. Clones in which a provirus integrated in an oncogene contributed to cell survival comprised only a small fraction of the clones persisting in on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist

<i>Ex vivo</i> activation of CD4⁺ T-cells from donors on suppressive ART can lead to sustained production of infectious HIV-1 from a subset of infected cells

<div><p>The fate of HIV-infected cells after reversal of proviral latency is not well characterized. Simonetti, <i>et al</i>. recently showed that CD4⁺ T-cells containing intact proviruses can clonally expand <i>in vivo</i> and produce low-level infectious viremia. We hypothesized that reversal of HIV latency by activation of CD4⁺ T-cells can lead to the expansion of a subset of virus-producing cells rather than their elimination. We established an <i>ex vivo</i> cell culture system involving stimulation of CD4⁺ T-cells from donors on suppressive antiretroviral therapy (ART) with PMA/ionomycin (day 1–7), followed by rest (day 7–21), and then repeat stimulation (day 21–28), always in the presence of high concentrations of raltegravir and efavirenz to effectively block new cycles of viral replication. HIV DNA and virion RNA in the supernatant were quantified by qPCR. Single genome sequencing (SGS) of p6-PR-RT was performed to genetically characterize proviruses and virion-associated genomic RNA. The replication-competence of the virions produced was determined by the viral outgrowth assay (VOA) and SGS of co-culture supernatants from multiple time points. Experiments were performed with purified CD4⁺ T-cells from five consecutively recruited donors who had been on suppressive ART for > 2 years. In all experiments, HIV RNA levels in supernatant increased following initial stimulation, decreased or remained stable during the rest period, and increased again with repeat stimulation. HIV DNA levels did not show a consistent pattern of change. SGS of proviruses revealed diverse outcomes of infected cell populations, ranging from their apparent elimination to persistence and expansion. Importantly, a subset of infected cells expanded and produced infectious virus continuously after stimulation. These findings underscore the complexity of eliminating reservoirs of HIV-infected cells and highlight the need for new strategies to kill HIV-infected cells before they can proliferate.</p></div