A high throughput screen for TMPRSS2 expression identifies FDA-approved and clinically advanced 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 identified small molecules that can reduce surface expression of TMPRSS2 using a 2,700 FDA-approved or current clinical trial compounds. Among these, homoharringtonine and halofuginone appear the most potent 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, demonstrated marked resistance to SARS-CoV-2 pseudoviral infection. 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 COVID-19 infection.

SARS-CoV-2 spike protein 13-mer subdomain corresponds to the drug-binding domain of 3 glutamyl-propyl-tRNA synthetase 1 and is targetable by halofuginone

“…Our data support the ndings of Chen et al and Sandoval et al, showing that halofuginone blocks infection of ACE2-expressing cells in vitro (19,20); however, it extends their ndings by providing a clear mechanism. The concept of using a small molecule inhibitor to bind to the spike protein such that it cannot effectively engage the host cell presents a novel mechanism by which to minimize infection.…”

“…In particular, halofuginone has been shown to exert pleotropic effects on the immune system (18) and has received orphan drug designation by the FDA in March 2020 for the treatment of scleroderma. More recently, halofuginone has been shown to be a potent inhibitor of SARS-CoV-2 infection in vitro, through two distinct putative mechanisms: down regulation of TMPRSS2 or through the effects of amino acid starvation on genomic biosynthesis and heparin sulfate decoration of proteoglycans on the cell surface (19,20).…”

“…We subsequently undertook in vitro enzymatic experiments to see if the drug could inhibit binding of the spike protein to the ACE2 receptor, thereby restoring the enzymatic activity of ACE2. In this analysis, the receptor binding domain (RBD) of spike protein (consisting of amino acids 330-530) was used rather than complete spike protein so that we could restrict our ndings as tightly as possible to a minimal structure containing the 13-mer region (amino acids 482-494) and therefore de nitively conclude the mechanism by which the drug might be inhibiting infection as reported by Chen et al, 2020, andSandoval et al, 2021. Indeed, at the 0.01-0.1 nM range, halofuginone blocked the ability of the RBD to bind to ACE2, as evidenced by restoration of ACE2 activity in the context of spike protein RBD.…”

SARS-CoV-2 spike protein 13-mer subdomain corresponds to the drug-binding domain of glutamyl-propyl-tRNA synthetase 1 and is targetable by halofuginone

“…In particular, halofuginone has been shown to exert pleotropic effects on the immune system (18) and has received orphan drug designation by the FDA in March 2020 for the treatment of scleroderma. More recently, halofuginone has been shown to be a potent inhibitor of SARS-CoV-2 infection in vitro, through two distinct putative mechanisms: down regulation of TMPRSS2 or through the effects of amino acid starvation on genomic biosynthesis and heparin sulfate decoration of proteoglycans on the cell surface (19,20).…”

Scutellaria barbata D. Don Inhibits the Main Proteases (Mpro and TMPRSS2) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection

“…Of those three compounds, 10–hydroxyusambarensine was identified as the most effective, by docking into the S1-specificity pocket of TMPRSS2 [ 31 ]. Other natural compounds, including geranium, lemon essential oils, homoharringtonine, and halofuginone have also demonstrated TMPRSS2 inhibitory effects [ 30 , 53 ]. Of note, several derivatives of SB, including scutellarin, baicalein, wogonin, and luteolin have also been reported to inhibit M pro and TMPRSS2, further supporting the promising results reported herein.…”