Effective therapeutics are urgently needed to counter infection and improve outcomes for patients suffering from COVID-19 and to combat this pandemic. Manipulation of epigenetic machinery to influence viral infectivity of host cells is a relatively unexplored area. The bromodomain and extraterminal (BET) family of epigenetic readers have been reported to modulate SARS-CoV-2 infection. Herein, we demonstrate apabetalone, the most clinical advanced BET inhibitor, downregulates expression of cell surface receptors involved in SARS-CoV-2 entry, including angiotensin-converting enzyme 2 (ACE2) and dipeptidyl-peptidase 4 (DPP4 or CD26) in SARS-CoV-2 permissive cells. Moreover, we show that apabetalone inhibits SARS-CoV-2 infection in vitro to levels comparable to those of antiviral agents. Taken together, our study supports further evaluation of apabetalone to treat COVID-19, either alone or in combination with emerging therapeutics.

Bromodomain and extraterminal proteins (BETs) are more than just epigenetic regulators of transcription. Here we highlight a new role for the BET protein BRD4 in the maintenance of higher order chromatin structure at Topologically Associating Domain Boundaries (TADBs). BD2-selective and pan (non-selective) BET inhibitors (BETi) differentially support chromatin structure, selectively affecting transcription and cell viability. Using RNA-seq and BRD4 ChIP-seq, the differential effect of BETi treatment on the transcriptome and BRD4 chromatin occupancy of human aortic endothelial cells from diabetic patients (dHAECs) stimulated with TNFα was evaluated. Chromatin decondensation and DNA fragmentation was assessed by immunofluorescence imaging and quantification. Key dHAEC findings were verified in proliferating monocyte-like THP-1 cells using real time-PCR, BRD4 co-immunoprecipitation studies, western blots, proliferation and apoptosis assays. We discovered that 1) BRD4 co-localizes with Ying-Yang 1 (YY1) at TADBs, critical chromatin structure complexes proximal to many DNA repair genes. 2) BD2-selective BETi enrich BRD4/YY1 associations, while pan-BETi do not. 3) Failure to support chromatin structures through BRD4/YY1 enrichment inhibits DNA repair gene transcription, which induces DNA damage responses, and causes widespread chromatin decondensation, DNA fragmentation, and apoptosis. 4) BD2-selective BETi maintain high order chromatin structure and cell viability, while reducing deleterious pro-inflammatory transcription. BRD4 plays a previously unrecognized role at TADBs. BETi differentially impact TADB stability. Our results provide translational insight for the development of BETi as therapeutics for a range of diseases including CVD, chronic kidney disease, cancer, and COVID-19.

Epigenetic mechanisms are implicated in transcriptional programs driving chronic kidney disease (CKD). Apabetalone is an orally available inhibitor of bromodomain and extraterminal (BET) proteins, which are epigenetic readers that modulate gene expression. In the phase 3 BETonMACE trial, apabetalone reduced risk of major adverse cardiac events (MACE) by 50% in the CKD subpopulation, indicating favorable effects along the kidney–heart axis. Activation of human renal mesangial cells (HRMCs) to a contractile phenotype that overproduces extracellular matrix (ECM) and inflammatory cytokines, and promotes calcification, frequently accompanies CKD to drive pathology. Here, we show apabetalone downregulated HRMC activation with TGF-β1 stimulation by suppressing TGF-β1-induced α-smooth muscle actin (α-SMA) expression, α-SMA assembly into stress fibers, enhanced contraction, collagen overproduction, and expression of key drivers of fibrosis, inflammation, or calcification including thrombospondin, fibronectin, periostin, SPARC, interleukin 6, and alkaline phosphatase. Lipopolysaccharide-stimulated expression of inflammatory genes IL6, IL1B, and PTGS2 was also suppressed. Transcriptomics confirmed apabetalone affected gene sets of ECM remodeling and integrins. Clinical translation of in vitro results was indicated in CKD patients where a single dose of apabetalone reduced plasma levels of key pro-fibrotic and inflammatory markers, and indicated inhibition of TGF-β1 signaling. While plasma proteins cannot be traced to the kidney alone, anti-fibrotic and anti-inflammatory effects of apabetalone identified in this study are consistent with the observed decrease in cardiovascular risk in CKD patients.

Facioscapulohumeral dystrophy (FSHD) is a muscle disease caused by inappropriate expression of the double homeobox 4 (DUX4) gene in skeletal muscle, and its downstream activation of pro-apoptotic transcriptional programs. Inhibitors of DUX4 expression have the potential to treat FSHD. Apabetalone is a clinical-stage bromodomain and extra-terminal (BET) inhibitor, selective for the second bromodomain on BET proteins. Using primary human skeletal muscle cells from FSHD type 1 patients, we evaluated apabetalone for its ability to counter DUX4′s deleterious effects and compared it with the pan-BET inhibitor JQ1, and the p38 MAPK inhibitor—and DUX4 transcriptional repressor—losmapimod. We applied RNA-sequencing and bioinformatic analysis to detect treatment-associated impacts on the transcriptome of these cells. Apabetalone inhibited the expression of DUX4 downstream markers, reversing hallmarks of FSHD gene expression in differentiated muscle cells. JQ1, but not apabetalone, was found to induce apoptosis. While both BET inhibitors modestly impacted differentiation marker expression, they did not affect myotube fusion. Losmapimod also reduced expression of DUX4 target genes but differed in its impact on FSHD-associated pathways. These findings demonstrate that apabetalone inhibits DUX4 target gene expression and reverses transcriptional programs that contribute to FSHD pathology, making this drug a promising candidate therapeutic for FSHD.

Background/Objectives: Obesity can cause metabolic disorders and hepatic steatosis. Continuous hepatic influx of dietary lipids leads to adaptive metabolic changes, including increased fatty acid oxidation (FAO) and ketogenesis. However, these adaptations are not enough to counter the detrimental accumulation of lipids, resulting in hepatic steatosis. Apabetalone is a clinical-stage Bromodomain and Extra-Terminal domain inhibitor (BETi) that attenuated the increase in hepatic fibrosis score (FS) and reduced the rate of ischemic major adverse cardiovascular events and hospitalizations for heart failure in a subgroup of patients having a high likelihood of Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) in the phase 3 clinical trial, BETonMACE. Methods: To analyze apabetalone’s effects on lipid and ketone metabolism, RNA seq, Oil Red staining and triglyceride quantification were performed in livers from a mouse model of diabetes-induced obesity and ketones were measured in plasma. Results: Mice fed a high-fat diet (HFD) were obese and demonstrated liver steatosis. Apabetalone treatment maintained the beneficial metabolic adaptation induced by HFD (increased FAO) and decreased hepatic triglycerides and lipid droplets. This inhibition of lipid anabolism redirected substrates to FAO and ketogenesis, resulting in increased plasma ketones, showing for the first time the role of BETi in ketogenesis. In the heart, apabetalone treatment reduced cardiac oxidative stress and plasma NT-proBNP levels. Conclusions: Apabetalone improves hepatic lipid handling, favoring ketogenesis. As ketones have demonstrated beneficial effects on cardiac function, increased ketones induced by apabetalone may not only contribute to the observed attenuation of FS in patients, but also a reduction in cardiac events among patients with high likelihood of MASLD, as well as in the overall trial population.