A high-throughput screen for TMPRSS2 expression identifies FDA-approved compounds that can limit SARS-CoV-2 entry

Abstract

SARS-CoV-2 (2019-nCoV) is the pathogenic coronavirus responsible for the global pandemic of COVID-19 disease. The Spike (S) protein of SARS-CoV-2 attaches to host lung epithelial cells through the cell surface receptor ACE2, a process dependent on host proteases including TMPRSS2. Here, we identify small molecules that reduce surface expression of TMPRSS2 using a library of 2,560 FDA-approved or current clinical trial compounds. We identify homoharringtonine and halofuginone as the most attractive agents, reducing endogenous TMPRSS2 expression at sub-micromolar concentrations. These effects appear to be mediated by a drug-induced alteration in TMPRSS2 protein stability. We further demonstrate that halofuginone modulates TMPRSS2 levels through proteasomal-mediated degradation that involves the E3 ubiquitin ligase component DDB1- and CUL4-associated factor 1 (DCAF1). Finally, cells exposed to homoharringtonine and halofuginone, at concentrations of drug known to be achievable in human plasma, demonstrate marked resistance to SARS-CoV-2 infection in both live and pseudoviral in vitro models. Given the safety and pharmacokinetic data already available for the compounds identified in our screen, these results should help expedite the rational design of human clinical trials designed to combat active COVID-19 infection.

ACE2, the Counter-Regulatory Renin–Angiotensin System Axis and COVID-19 Severity

“…The level of viral RNA in HAI-2 KD cells was approximately 40 times greater than that in control cells, indicating that the endogenous level of HAI-2 in Calu-3 cells alleviated SARS-CoV-2 infection [97]. In accordance with the previous findings, some small molecules (e.g., homoharringtonine and halofuginone) that reduce surface expression of TMPRSS2 render cells exposed to them at drug concentrations known to be achievable in human plasma markedly resistant to SARS-CoV-2 infection in both live and pseudoviral in vitro models [98].…”

Discovery of potential anti-SARS-CoV-2 drugs based on large-scale screening in vitro and effect evaluation in vivo

“…Masitinib, an inhibitor of Kit (c-Kit) and PDGFRα/β, inhibited the 3CL pro main proteases of coronaviruses and picornaviruses and was effective in reducing SARS-CoV-2 replication in mice (Drayman et al, 2021). Similar to reports that drug screening is highly dependent on the chosen cell lines and infection conditions (Mirabelli et al, 2021), 17 of the 24 compounds were shown to inhibit SARS-CoV-2 infection in HeLa-ACE2 cells, including reported compounds with broad-spectrum antiviral activity, such as halofuginone, homoharringtonine, salinomycin sodium salt, ouabain, quinacrine 2HCl, and three kinds of dopamine receptor inhibitors (including trifluoperazine 2HCl, pimozide, and prochlorperazine dimaleate salt) (Chen et al, 2021;Ju et al, 2021;Nemerow and Cooper, 1984;Ochiai et al, 1991;Otręba et al, 2020). Dopamine receptor inhibitors have demonstrated broad-spectrum antiviral activity, including against influenza virus, herpes virus, hepatitis C virus, and SARS-CoV-2 (Berber and Doluca, 2021;Nemerow and Cooper, 1984;Ochiai et al, 1991;Otręba et al, 2020).…”

The Transmembrane Protease TMPRSS2 as a Therapeutic Target for COVID-19 Treatment

“…The two most active compounds, halofuginone and homoharringtonine, significantly reduced SARS-CoV-2 replication in immortalized Calu-3 lung cells [108]. Mechanistically, halofuginone impairs TMPRSS2 protein stability by increasing proteasomal degradation and affects translation due to its function as glutamyl-prolyl-tRNA inhibitor [108,109]. Homoharringtonine interferes with protein biosynthesis through blockade of the large ribosomal subunit and has been shown to inhibit a variety of TMPRSS2-dependent (PEDV, Dong 2018) and -independent (VSV, HSV-1, RV; VZV, MHV) viruses [110][111][112].…”

“…In an in vitro screening approach, Chen and colleagues identified therapeutic compounds that reduce TMPRSS2 protein expression in the low-to sub-micromolar range. The two most active compounds, halofuginone and homoharringtonine, significantly reduced SARS-CoV-2 replication in immortalized Calu-3 lung cells [108]. Mechanistically, halofuginone impairs TMPRSS2 protein stability by increasing proteasomal degradation and affects translation due to its function as glutamyl-prolyl-tRNA inhibitor [108,109].…”