In vitro interaction of potential antiviral TMPRSS2 inhibitors with human serum albumin and cytochrome P 450 isoenzymes

Abstract

The interactions of four sulfonylated Phe(3-Am)-derived inhibitors (MI-432, MI-463, MI-482 and MI-1900) of type II transmembrane serine proteases (TTSP) such as transmembrane protease serine 2 (TMPRSS2) were examined with serum albumin and cytochrome P450 (CYP) isoenzymes. Complex formation with albumin was investigated using fluorescence spectroscopy. Furthermore, microsomal hepatic CYP1A2, 2C9, 2C19 and 3A4 activities in presence of these inhibitors were determined using fluorometric assays. The inhibitory effects of these compounds on human recombinant CYP3A4 enzyme were also examined. In addition, microsomal stability assays (60-min long) were performed using an UPLC-MS/MS method to determine depletion percentage values of each compound. The inhibitors showed no or only weak interactions with albumin, and did not inhibit CYP1A2, 2C9 and 2C19. However, the compounds tested proved to be potent inhibitors of CYP3A4 in both assays performed. Within one hour, 20%, 12%, 14% and 25% of inhibitors MI-432, MI-463, MI-482 and MI-1900, respectively, were degraded. As essential host cell factor for the replication of the pandemic SARS-CoV-2, the TTSP TMPRSS2 emerged as an important target in drug design. Our study provides further preclinical data on the characterization of this type of inhibitors for numerous trypsin-like serine proteases.

In vitro characterization of the furin inhibitor MI-1851: Albumin binding, interaction with cytochrome P450 enzymes and cytotoxicity

“…These observations are also in agreement with the current experimental results ( Fig. 4 B) and the recently reported data [21] in regard to the microsomal CYP3A4 inhibition by MI-1851 and the other MI compounds listed. These findings suggest that MI-1851 is only a weak inhibitor of CYP3A4 and presumably it does not modify significantly the CYP3A4-mediated biotransformation of other drugs.…”

“…Nevertheless, in the in vitro assay with human recombinant CYP3A4, the furin inhibitor caused only slight inhibition of testosterone hydroxylation even at 20 μM concentration ( Fig. 4 A), while other benzamidine derivatives tested previously (MI-432, MI-463, MI-482 and MI-1900) showed much stronger inhibitory effects (IC 50 = 2–12 μM) [21] . These observations are also in agreement with the current experimental results ( Fig.…”

“…Based on spectroscopic and ultrafiltration studies, inhibitor MI-1851 does not form stable complexes with HSA and it does not affect significantly the albumin-binding of site I and site II ligands. Our previous investigation with benzamidine-derived inhibitors of trypsin-like serine proteases like compounds MI-432, MI-463, MI-482 and MI-1900 showed also no or only weak interactions with HSA [21] . Thus, it is reasonable to hypothesize that MI-1851 does not interfere with the albumin-binding of other medications.…”

“…In the frame of in vitro studies of 3-amidinophenylalanine (Phe(3-Am))-derived TMPRSS2 inhibitors, it was demonstrated that MI-1900 and its structural analog MI-1907 (up to 50 μM) did not cause damage to human intestinal epithelial cells and PHHs (based on 24 h cytotoxicity assays). Furthermore, four Phe(3-Am) derivatives (MI-432, MI-463, MI-482 and MI-1900) appeared to be potent inhibitors of CYP3A4 at the nanomolar or low micromolar range in microsomal and recombinant enzyme experiments [21] , [22] .…”

“…CYP3A4 assay with the human recombinant enzyme (CypExpress™ 3A4 Cytochrome P450 human kit) and with the FDA-recommended substrate testosterone as well as the quantification of the substrate and the product (6β-hydroxytestosterone) were performed as described in our recent study [21] .…”