In vitro testing of host-targeting small molecule antiviral matriptase/TMPRSS2 inhibitors in 2D and 3D cell-based assays

Biomed Pharmacother. 2023 Dec:168:115761. doi: 10.1016/j.biopha.2023.115761. Epub 2023 Oct 20.


The outbreak of coronavirus disease 2019 (COVID-19) pandemic strongly stimulated the development of small molecule antivirals selectively targeting type II transmembrane serine proteases (TTSP), required for the host-cell entry of numerous viruses. A set of 3-amidinophenylalanine derivatives (MI-21, MI-472, MI-477, MI-485, MI-1903 and MI-1904), which inhibit the cleavage of certain viral glycoproteins was characterized in 2D and 3D primary human hepatocyte models on collagen- and Matrigel-coating using a CCK-8 assay to evaluate their cytotoxicity, a resorufin-based method to detect redox imbalances, fluorescence and ultrafiltration experiments to evaluate their interactions with human serum albumin (HSA) and α-acidic glycoprotein (AGP), and luminescence measurement to assess CYP3A4 modulation. For elucidation of selectivity of the applied compounds towards matriptase, transmembrane serine protease 2 (TMPRRS2), thrombin and factor Xa (FXa) Ki values were determined. It was proven that cell viability was only deteriorated by inhibitor MI-1903, and redox status was not influenced by administration of the selected inhibitors at 50 µM for 24 h. MI-472 and MI-477 formed relatively stable complexes with AGP. CYP3A4 inhibition was found to be strong in PHHs exposed to all inhibitors with the exception of MI-21, which seems to be a promising drug candidate also due to its better selectivity towards matriptase and TMPRSS2 over the blood clotting proteases thrombin and FXa. Our in vitro pharmacokinetic screening with these inhibitors helps to select the compounds with the best selectivity and safety profile suitable for a further preclinical characterization without animal sacrifice.

Keywords: 3D Matrigel; Cytochrome P450 3A4; Primary human hepatocytes; Protein binding; SARS-CoV-2; TMPRSS2.

MeSH terms

  • Animals
  • Antiviral Agents* / pharmacology
  • Cytochrome P-450 CYP3A*
  • Humans
  • Serine Endopeptidases* / metabolism
  • Serine Proteinase Inhibitors* / pharmacology
  • Thrombin*


  • Antiviral Agents
  • Cytochrome P-450 CYP3A
  • matriptase
  • Serine Endopeptidases
  • Thrombin
  • TMPRSS2 protein, human
  • Serine Proteinase Inhibitors