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

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 (IC50 = 0.27 nM), camostat (IC50 = 6.2 nM), FOY-251 (IC50 = 33.3 nM), and gabexate
(IC50 = 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.