Intro: The emergence of a highly infectious SARS-CoV-2 has resulted in a global pandemic. Moreover, the emergence of SARS-CoV-2 variants possessing spike protein mutations raise concerns of vaccine resistance. TMPRSS2, a serine protease, processes the spike protein for SARS-CoV-2 entry. We hypothesized that exploiting the substrate specificity of TMPRSS2 could lead to specific and potent inhibitors of TMPRSS2 with efficacy against newly emergent strains. Methods: 1200 tetrapeptides possessing a ketobenzothiazole or ketothiazole were docked to a homology model of TMPRSS2 using the Molecular Operating Environment software. The top hit, NH2-Nle-Gln-2Nal-Arg-Ketobenzothiazole (1) was synthesized via coupling of the Arg(Mtr)-Ketobenzothiazole and Boc-Nle-Gln(Trt)-2Nal with subsequent global deprotection. In vitro competitive assays were performed using TMPRSS2, expressed in Sf9 insect cells, and various plasma serine proteases, harvested from plasma. Cell viability assays were performed on HPL1D cells. Wild-type and N501Y HexaPro spike proteins were expressed in Expi293 cells. Pseudoviral entry studies were performed using Calu3 cells. Results: The highest-ranking inhibitor (1) exhibited a tight-binding inhibitor profile with a Ki of 6±2 nM. It showed no inhibitory activity towards Factor IIa, Factor VIIa, APC, or tPA, whereas Nafamostat, a benchmark TMPRSS2 inhibitor, showed varying degrees of inhibition of these enzymes. While 1 inhibited plasmin, Nafamostat had an approximate 100-fold lower IC50 value. Coagulation testing showed, at 1 μM, 1 had showed no effect on clinical coagulation parameters, whereas Nafamostat had a significant increase in partial thromboplastin time. 1 showed no cytotoxicity up to 20 μM in HPL1D cells over 96 hours. 1 and Nafamostat inhibited TMPRSS2-mediated cleavage of the HexaPro spike protein at IC50 values of 0.27 and 0.39 μM. Inhibition of the TMPRSS2-mediated cleavage of N501Y HexaPro by 1 showed no significant difference with an IC50 value of 0.23 μM. Pseudoviral entry assays showed an inhibition by 1 and Nafamostat with an IC50 of 0.59 and 0.30 nM. Conclusion: NH2-Nle-Gln-2Nal-Arg-Ketobenzothiazole represents a starting point in the development of potent inhibitors of TMPRSS2 with a lower risk of antiviral toxicity to target potential variants of SARS-CoV-2.

Development and characterization of a novel and selective TMPRSS2 inhibitor as a potential COVID19 treatment strategy

ACS Spring Meeting 2021

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