PK/PD investigation of antiviral host matriptase/TMPRSS2 inhibitors in cell models.

Certain corona- and influenza viruses utilize type II transmembrane serine proteases for cell entry, making these enzymes potential drug targets for the treatment of viral respiratory infections. In this study, the cytotoxicity and inhibitory effects of seven matriptase/TMPRSS2 inhibitors (MI-21, MI-463, MI-472, MI-485, MI-1900, MI-1903, and MI-1904) on cytochrome P450 enzymes were evaluated using fluorometric assays. Additionally, their antiviral activity against influenza A virus subtypes H1N1 and H9N2 was assessed. The metabolic depletion rates of these inhibitors in human primary hepatocytes were determined over a 120-min period by LC-MS/MS, and PK parameters were calculated. The tested compounds, with the exception of MI-21, displayed potent inhibition of CYP3A4, while all compounds lacked inhibitory effects on CYP1A2, CYP2C9, CYP2C19, and CYP2D6. The differences between the CYP3A4 activity within the series were rationalized by ligand docking. Elucidation of PK parameters showed that inhibitors MI-463, MI-472, MI-485, MI-1900 and MI-1904 were more stable compounds than MI-21 and MI-1903. Anti-H1N1 properties of inhibitors MI-463 and MI-1900 and anti-H9N2 effects of MI-463 were shown at 20 and 50 µM after 24 h incubation with the inhibitors, suggesting that these inhibitors can be applied to block entry of these viruses by suppressing host matriptase/TMPRSS2-mediated cleavage.

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

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.