An Enzymatic TMPRSS2 Assay for Assessment of Clinical Candidates and Discovery of Inhibitors as Potential Treatment of COVID-19

Abstract

SARS-CoV-2 is the viral pathogen causing the COVID19 global pandemic. Consequently, much research has gone into the development of preclinical assays for the discovery of new or repurposing of FDA-approved therapies. Preventing viral entry into a host cell would be an effective antiviral strategy. One mechanism for SARS-CoV-2 entry occurs when the spike protein on the surface of SARS-CoV-2 binds to an ACE2 receptor followed by cleavage at two cut sites (“priming”) that causes a conformational change allowing for viral and host membrane fusion. TMPRSS2 has an extracellular protease domain capable of cleaving the spike protein to initiate membrane fusion. A validated inhibitor of TMPRSS2 protease activity would be a valuable tool for studying the impact TMPRSS2 has in viral entry and potentially be an effective antiviral therapeutic. To enable inhibitor discovery and profiling of FDA-approved therapeutics, we describe an assay for the biochemical screening of recombinant TMPRSS2 suitable for high throughput application. We demonstrate effectiveness to quantify inhibition down to subnanomolar concentrations by assessing the inhibition of camostat, nafamostat, and gabexate, clinically approved agents in Japan. Also, we profiled a camostat metabolite, FOY-251, and bromhexine hydrochloride, an FDA-approved mucolytic cough suppressant. The rank order potency for the compounds tested are nafamostat (IC 50 = 0.27 nM), camostat (IC 50 = 6.2 nM), FOY-251 (IC 50 = 33.3 nM), and gabexate (IC 50 = 130 nM). Bromhexine hydrochloride showed no inhibition of TMPRSS2. Further profiling of camostat, nafamostat, and gabexate against a panel of recombinant proteases provides insight into selectivity and potency.

SARS-CoV-2 and SARS-CoV spike-mediated cell-cell fusion differ in the requirements for receptor expression and proteolytic activation

“…We are fairly confident to postulate that these favorable results are unlikely due to inhibition of TMPRSS2, although we can’t fully exclude extremely weak activity. This view is supported by a recent study that found no effect of Bromhexine on TMPRSS2 activity (30). More likely, favorable patient outcomes are attributable to the beneficial effects of Bromhexine or its main metabolite Ambroxol on lung function, general defense mechanisms against airway infections, and inflammatory response (16–19, 50).…”

“…As fusion effectors, SARS1-S, SARS2-S as well as SARS2-S1/S2-mut and SARS2-S2′-AA were included. To test the effects of TMPRSS2 inhibition by small molecules on the activation of wt SARS2-S and the two mutants as well as SARS1-S, we incubated the different target cells with Bromhexine, reportedly a specific inhibitor of TMPRSS2 (15), the chemically related compound Ambroxol, or Camostat, an irreversible inhibitor of TMPRSS2 and many serine proteases in general (29, 30), at 50 µM (Fig. 3 A).…”

“…It is at the moment not clear by what mechanism of action Bromhexine modulates TMPRSS2 activity, if it does so, and we therefore cannot exclude that processing of some substrates is actually enhanced or altered instead of inhibited, as reported for several substrates (15, 44). Recently, another study also reported lack of inhibitory activity of Bromhexine against TMPRSS2 (30). Activity of Bromhexine against TMPRSS2-mediated receptor shedding, which could also explain our observations, was not observed, in contrast to Camostat that increased ACE2 expression levels in the presence of TMPRSS2 (Figs.…”

Drug Repurposing Screen for Compounds Inhibiting the Cytopathic Effect of SARS-CoV-2

“…A number of small molecules of interest for treating COVID-19 that are currently in clinical trials were not hits in our assay. For example, the TMPRSS2 inhibitors camostat and nafamstat are protease inhibitors approved in Japan for treating pancreatitis, and known to inhibit TMPRSS2 ( Shrimp et al, 2020 ). While TMPRSS2 is reported to be a mediator of SARS-CoV-2 cell entry, Vero E6 cells do not express TMPRSS2, so this class of compound are not active in the Vero E6 assay.…”

Effective drugs used to combat SARS-CoV-2 infection and the current status of vaccines

“…In order to uncover the molecular activity hypothesis of these medicines and provide evidence that can help advance them further, Tim Hempel et al have combined cell-based assays, detailed molecular experiments, and Markov modeling [ 95 ]. Prior studies provided evidence that both blockers work directly on Transmembrane serine protease 2 (TMPRSS2) and that Nafamostat is more active than Camostat, and this qualitative discrepancy is consistent with additional purified protein and cell-entry assays in addition to in vitro assays [ 28 , 96 , 97 ]. Tim Hempel et al observe that between such three assay forms, the absolute IC50 s amounts differ, indicating variations in experimental parameters and which feature has been inhibited and calculated.…”