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 pre-clinical 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. This fusion event is proceeded by release of viral RNA within the host cell. TMPRSS2 has an extracellular protease domain capable of cleaving the spike protein to initiate membrane fusion. Additionally, knock-out studies in mice have demonstrated reduced infection in the absence of TMPRSS2 with no detectable physiological impact; thus, TMPRSS2 is an attractive target for therapeutic development. 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 for pancreatitis due to their inhibition of trypsin-like proteases.Nafamostat and camostat are currently in clinical trials against COVID19. The rank order potency for the three inhibitors is: nafamostat (IC50 = 0.27 nM), camostat (IC50 = 6.2 nM) and gabexate (IC50 = 130 nM). Further profiling of these three inhibitors against a panel of proteases provides insight into selectivity and potency.

Molecular mechanism of SARS-CoV-2 cell entry inhibition via TMPRSS2 by Camostat and Nafamostat mesylate

“…1C). Our results are consistent with the finding that both drugs inhibit cell entry of SARS-CoV-2 and other coronaviruses, and that nafamostat is the more potent inhibitor [17,18,20].…”

“…Our binding assays provide evidence that both inhibitors directly act on TMPRSS2 and that nafamostat is more potent compared to camostat, and this qualitative difference is in agreement with complementary in vitro assays on purified protein [20] cellentry assays [17,18]. We note that the absolute IC50s values differ between these three assay types, reflecting differences in experimental conditions and which function is being inhibited and measured.…”

“…Both guanidinobenzoate-containing drugs are approved in Japan and have been demonstrated to inhibit SARS-CoV-2 cell-entry [10,17,18]. We report experimental measurements demonstrating that these drugs inhibit TMPRSS2 activity by using our recently established cell-based assays [19], consistent with in vitro enzymatic TMPRSS2 activity assays [20].…”

Functional and druggability analysis of the SARS-CoV-2 proteome

“…There are at the least three ACE2-inhibitors (Captopril, Lisinopril, Losartan), but this option is not very appealing due to blood pressure decreasing side-effects. The inhibition of TMPRSS2 is also being explored, by blocking the proteolytic priming of the S2 fragment, necessary to induce the membrane fusion; the inhibitors Nafamostat [176], Gabexate, and Calmostat [177], have been shown to inhibit viral production in cell cultures, and will be tested in humans; however, their low half-life opens the door to developing more efficient new drugs (cf. Ref.…”

“…Recently the SARS-CoV-2 orf3a structure was solved by cryo-EM (PDB 6XDC) [199]. The Nterminus (amino acids 1-39), the C-terminus (239-275) and a short loop (175)(176)(177)(178)(179)(180) were not observed, probably due to molecular disorder. Orf3a was solved as a homodimer, although the Authors were able to reconstruct the tetramer at a lower final resolution of ~6.5 Å, also showing a model of the neighboring dimers [199], and inferring from this model that residues W131, R134, K136, H150, T151, N152, C153, and D155 were involved in a network of interactions that would mediate tetramerization (Figure 11a).…”