TDK conference 2024

It has been well established that various corona- and influenza viruses undergo activation by type II transmembrane serine proteases for entry into host cells, making these host-cell proteases attractive pharmacological targets for the treatment of respiratory infections caused by these viruses. Additionally, due to the shortage of other effective pharmaceutical compounds, virus mutations, and the looming threat of future airborne pandemics, novel drugs for the treatment of viral infections are imperative for public health. In this study, seven different 3-amidinophenylalanine-based inhibitors (MI-21, MI-463, MI-472, MI-485, MI-1903, and MI-1904) of host-cell proteases matriptase and TMPRSS2 were investigated. The cytotoxic effects and inhibitory capacity of the protease inhibitors on cytochrome P450 (CYP) enzymes were evaluated with fluorometric assays. Metabolic depletion rates of each inhibitor were determined over a 120-min period by liquid chromatography-tandem mass spectrometry in primary human hepatocytes, and various pharmacokinetic (PK) parameters, including half-life, intrinsic clearance, hepatic extraction ratio, and bioavailability, were calculated. Furthermore, the antiviral efficacy of selected protease inhibitors against influenza A virus subtypes H1N1 and H9N2 was evaluated in vitro as well. Except for MI-21, all other inhibitors significantly inhibited CYP3A4, and none of the compounds showed inhibitory activity on enzymes CYP1A2, CYP2C9, CYP2C19, and CYP2D6. Analysis of calculated PK parameters revealed that inhibitors MI-463, MI-472, MI-485, MI-1900, and MI-1904 boasted better stability than MI-21 and MI-1903, and these results were complimented by the similar findings of the depletion study, in which MI-21 and MI-1903 retained the lowest % of their parent compounds after 120 min. After a 24-hour incubation period, inhibitors MI-463 and MI-1900 displayed significant anti-H1N1 properties and MI-463 also showed robust antiviral effects against H9N2 at 20 and 50 µM. Our findings suggest that MI-463 and MI-1900 can be used to prevent entry of these viruses into cells by suppression of host matriptase/TMPRSS2.