Abstract
Background/Objectives: Transmembrane serine proteases such as TMPRSS2 and matriptase have been identified as pivotal host factors in the influenza A infection due to their capacity to cleave the hemagglutinin and thereby facilitate viral activation. The inhibition of these enzymes has the potential to serve as an effective therapeutic strategy against numerous seasonal influenza strains. In our study, four 3-amidinophenylalanine-derived inhibitors were used to elucidate their antiviral efficacy, pharmacokinetic properties and affinities toward certain related trypsin-like serine proteases. Methods: K(i) values for TMPRSS2, matriptase, thrombin and factor Xa were determined using enzyme kinetic measurements. Cytochrome P450 3A (CYP3A) inhibitory activity was investigated using human liver microsomes, and protein binding was evaluated with human serum albumin and α(1)-acid glycoprotein. In vitro antiviral efficacy and cytotoxicity were determined in MDCK-II cells. Results: All compounds were non-cytotoxic and exhibited a relatively high affinity toward matriptase and bovine thrombin in the 10-30 nM concentration range. Among the inhibitors, MI-441 displayed the lowest K(i) value for TMPRSS2 (~60 nM). The weakest CYP3A inhibitory activity was observed for compounds MI-447 and MI-448. In addition, three of the four compounds (MI-441, MI-443 and MI-447) demonstrated significant antiviral activity. Conclusions: This study demonstrates that the investigated inhibitors exhibit a favorable safety profile, low binding to human serum albumin and pronounced antiviral activity against H1N1.