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BET inhibitors RVX-208 and PFI-1 reactivate HIV-1 from latency.

ABSTRACT: Persistent latent reservoir in resting CD4+ T cells is a major obstacle in curing HIV-1 infection. Effective strategies for eradication of the HIV-1 reservoir are urgently needed. We report here for the first time that two BET inhibitors, RVX-208, which has entered phase II clinical trials for diverse cardiovascular disorders, and PFI-1, which has been widely studied in oncology, can reactivate HIV-1 from latency. RVX-208 and PFI-1 treatment alone or in combination with other latency reversing agents efficiently reactivated HIV-1 transcription through an up-regulation of P-TEFb by increasing CDK9 Thr-186 phosphorylation in latently infected Jurkat T cells in vitro. The two BET inhibitors also reactivated HIV-1 transcription in cART treated patient-derived resting CD4+ T cells ex vivo, without influence on global immune cell activation. Our findings, in combination with previous reports, further confirm that BET inhibitors are a group of leading compounds for combating HIV-1 latency for viral eradication.

SUBMITTER: Lu P

PROVIDER: S-EPMC5709369 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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BET inhibitors RVX-208 and PFI-1 reactivate HIV-1 from latency.

Lu Panpan P Shen Yinzhong Y Yang He H Wang Yanan Y Jiang Zhengtao Z Yang Xinyi X Zhong Yangcheng Y Pan Hanyu H Xu Jianqing J Lu Hongzhou H Zhu Huanzhang H

Scientific reports 20171130 1

Persistent latent reservoir in resting CD4+ T cells is a major obstacle in curing HIV-1 infection. Effective strategies for eradication of the HIV-1 reservoir are urgently needed. We report here for the first time that two BET inhibitors, RVX-208, which has entered phase II clinical trials for diverse cardiovascular disorders, and PFI-1, which has been widely studied in oncology, can reactivate HIV-1 from latency. RVX-208 and PFI-1 treatment alone or in combination with other latency reversing a ...[more]

PMID: 29192216

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Project description:Bromodomains have emerged as attractive candidates for the development of inhibitors targeting gene transcription. Inhibitors of the bromo-and-extra-terminal (BET) family recently showed promising activity in diverse disease models. However, the pleiotropic nature of BET proteins regulating tissue specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. Here we report that RVX-208, a compound currently in phase II clinical trials, is a BET bromodomain inhibitor specific for second bromodomains (BD2). Co-crystal structures revealed binding modes of RVX-208 and its synthetic precursor and fluorescent recovery after photobleaching demonstrated that RVX-208 displaces BET proteins from chromatin. However, gene expression data showed that BD2 inhibition only modestly affects BET-dependent gene transcription. Our data demonstrate the feasibility of specific targeting within the BET family resulting in different transcriptional outcomes and highlight the importance of BD1 in transcriptional regulation HepG2 Cells were treated with eitther DMSO or 0.5uM JQ1 or 5uM RVX-208. Three samples per condition, total of nine samples. Inhbitor treatment was carried out for 4h before RNA was extracted. HepG2 cells (ATCC: HB-8065) were maintained in M-NM-1-MEM (Cat.#BE12-169F; BioWhittaker) supplemented with 10 % heat-inactivated foetal calf serum (PAA #A15-152), non-essential amino acids (Cat. #M7145; Sigma), glutamine (Cat.#M11-004; PAA), and vitamins (Cat.#M6895; Sigma). Cells were grown at 37 M-BM-0C in a humidified cabinet at 5 % CO2 (Heraeus Function Line). For experiments, cells were seeded the day prior to treatment at 2x105/ml. Treatments were performed for 4 h so that a final concentration of 0.1 % DMSO (Cat.#D1435; Sigma) was achieved. At harvest, cells were washed once with PBS (Cat.#H15-002; PAA), and lysed in situ using RLT buffer supplemented with 10 M-NM-<l/ml M-NM-2-mercaptoethanol (Cat.#M7522; Sigma). Total RNA was extracted and prepared using RNeasy columns (Cat.#74106 plus; Qiagen) including a Qia shredding step (Cat.#79656; Qiagen) and an on-column DNAse digestion (Cat.#EN0521; Fermentas), according to the manufacturerM-bM-^@M-^Ys instructions. The resulting RNA was quantified and quality controlled using a Nanodrop spectrophotometer (model ND1000; Thermo Fisher). RNA integrity was assessed on a BioAnalyzer (model G2938C; Agilent Laboratories, USA) and all samples had a RNA Integrity Number (RIN) M-bM-^IM-% 9. Labelled sense ssDNA for hybridization was generated from 200 ng starting RNA with the Ambion WT expression kit (Cat.#4411973; Ambion) and the Affymetrix GeneChip WT Terminal Labelling and Controls Kit (Cat.#901525; Affymetrix) according to the manufacturerM-bM-^@M-^Ys instructions. The distribution of fragmented sense ssDNA lengths was measured on the BioAnalyser. The fragmented ssDNA was labelled and hybridized for 17 hours at 45 M-BM-0C on the Affymetrix GeneChip Human Gene 1.0 ST Array (Affymetrix). Chips were processed on an Affymetrix GeneChip Fluidics Station 450 and Scanner 3000 and the affymetrix Command Console (v.3.2.4; Affymetrix) was used to generate CEL files.

Project description:BackgroundApabetalone (RVX-208) is a bromodomain and extraterminal protein inhibitor (BETi) that in phase II trials reduced the relative risk (RR) of major adverse cardiac events (MACE) in patients with cardiovascular disease (CVD) by 44% and in diabetic CVD patients by 57% on top of statins. A phase III trial, BETonMACE, is currently assessing apabetalone's ability to reduce MACE in statin-treated post-acute coronary syndrome type 2 diabetic CVD patients with low high-density lipoprotein C. The leading cause of MACE is atherosclerosis, driven by dysfunctional lipid metabolism and chronic vascular inflammation (VI). In vitro studies have implicated the BET protein BRD4 as an epigenetic driver of inflammation and atherogenesis, suggesting that BETi may be clinically effective in combating VI. Here, we assessed apabetalone's ability to regulate inflammation-driven gene expression and cell adhesion in vitro and investigated the mechanism by which apabetalone suppresses expression. The clinical impact of apabetalone on mediators of VI was assessed with proteomic analysis of phase II CVD patient plasma.ResultsIn vitro, apabetalone prevented inflammatory (TNFα, LPS, or IL-1β) induction of key factors that drive endothelial activation, monocyte recruitment, adhesion, and plaque destabilization. BRD4 abundance on inflammatory and adhesion gene promoters and enhancers was reduced by apabetalone. BRD2-4 degradation by MZ-1 also prevented TNFα-induced transcription of monocyte and endothelial cell adhesion molecules and inflammatory mediators, confirming BET-dependent regulation. Transcriptional regulation by apabetalone translated into a reduction in monocyte adhesion to an endothelial monolayer. In a phase II trial, apabetalone treatment reduced the abundance of multiple VI mediators in the plasma of CVD patients (SOMAscan® 1.3 k). These proteins correlate with CVD risk and include adhesion molecules, cytokines, and metalloproteinases. Ingenuity® Pathway Analysis (IPA®) predicted that apabetalone inhibits pro-atherogenic regulators and pathways and prevents disease states arising from leukocyte recruitment.ConclusionsApabetalone suppressed gene expression of VI mediators in monocytes and endothelial cells by inhibiting BET-dependent transcription induced by multiple inflammatory stimuli. In CVD patients, apabetalone treatment reduced circulating levels of VI mediators, an outcome conducive with atherosclerotic plaque stabilization and MACE reduction. Inhibition of inflammatory and adhesion molecule gene expression by apabetalone is predicted to contribute to MACE reduction in the phase III BETonMACE trial.

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BET inhibitors RVX-208 and PFI-1 reactivate HIV-1 from latency.

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BET inhibitors RVX-208 and PFI-1 reactivate HIV-1 from latency.

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