Bromodomain and Extraterminal Protein Inhibitor, Apabetalone (RVX-208), Reduces ACE2 Expression and Attenuates SARS-Cov-2 Infection In Vitro

Abstract

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.

Keywords: BET proteins; COVID-19; SARS-CoV-2; angiotensin-converting enzyme 2 (ACE2); apabetalone.

Figure 1

BETi treatment downregulated ACE2 transcript levels in various cell types. Gene expression of hACE2 (A,C–E) or RhACE2 (B) was measured by TaqMan real-time PCR. Calu-3 (A) were treated for 48 h, Vero E6 (B) were treated for 24 h, HepG2 (C), and Huh-7 (D) cells were treated for 96 h. Primary human hepatocytes (n = 3 donors) (E) were treated for 48 h. Error bars represent SD. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA followed by Dunnett’s multiple comparison test.

Figure 2

BETi treatment reduced both surface and total ACE2 protein levels. (A) Immunoblot of total ACE2 protein in Calu-3 cells following BETi treatment for 48 h. Values below the blots indicate band quantification via densitometric analysis (ACE2/β-ACTIN) and are represented as fold change to vehicle-treated cells (0.05% v/v DMSO). (B) Immunoblot of total ACE2 protein in Vero E6 cells following BETi treatment for 48 h. Values below the blot indicate band quantification via densitometric analysis (ACE2/GAPDH) and are represented as fold change to vehicle-treated cells (0.1% v/v DMSO). (C) Representative histogram from flow cytometry showing overlay of cell surface ACE2 on Calu-3 cells following 48 h of the indicated treatments. (D–E) Quantification of ACE2 protein levels on Calu-3 (D; n = 3) or Vero E6 (E, n = 5) following BETi treatment for 48 h. ACE2 surface expression is shown as mean fluorescent intensity (MFI) ratio to isotype control (D–E). Error bars represent SD. * p < 0.05, *** p < 0.001, one-way ANOVA followed by Dunnett’s multiple comparison test.

Figure 3

BET inhibition decreased SARS-CoV-2 spike RBD binding to Calu-3 and Vero E6. (A) A histogram overlay representing the binding of SARS-CoV-2 spike RBD Fc fusion protein to Calu-3 following 48 h of apabetalone or JQ1 treatment at the indicated concentrations. (B,C) Quantification of BETi-driven changes to SARS-CoV-2 spike RBD binding to Calu-3 (B; n = 3) or Vero E6 (C; n = 3) cells following 48 h of treatment. Binding results are shown as mean fluorescent intensity (MFI) ratio to Fc control. Error bars represent SD. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA followed by Dunnett’s multiple comparison test.

Figure 4

BETi compounds diminished SARS-CoV-2 infection in Calu-3. (A) Schematic illustration of SARS-CoV-2 infection assay design. Briefly, cells were pre-treated with BETi (apabetalone or JQ1), comparator/control compounds (camostat mesylate (camostat or remdesivir) or vehicle control (DMSO 0.1% v/v) for 48 h. Treatments were washed off and cells were then infected with SARS-CoV-2 at an MOI of 0.1. 48 h post-infection, Calu-3 cells were fixed and stained for spike protein. (B) Percent inhibition of viral infectivity by BETi pre-treatment in Calu-3 cells (blue bars). (C) Compound-induced cytotoxicity in viral infected Calu-3 cells is shown in grey bars (n = 3 independent experiments). (D) Representative immunofluorescence images of virus infection in Calu-3 cells for the treatments and indicated concentrations. Scale bar = 100 μm. Error bars represent SEM. ** p < 0.01, *** p < 0.001, one-way ANOVA followed by Dunnett’s multiple comparison test.