Identification of the first synthetic inhibitors of the type II transmembrane serine protease TMPRSS2 suitable for inhibition of influenza virus activation

Abstract

TMPRSS2 (transmembrane serine proteinase 2) is a multidomain type II transmembrane serine protease that cleaves the surface glycoprotein HA (haemagglutinin) of influenza viruses with a monobasic cleavage site, which is a prerequisite for virus fusion and propagation. Furthermore, it activates the fusion protein F of the human metapneumovirus and the spike protein S of the SARS-CoV (severe acute respiratory syndrome coronavirus). Increased TMPRSS2 expression was also described in several tumour entities. Therefore TMPRSS2 emerged as a potential target for drug design. The catalytic domain of TMPRSS2 was expressed in Escherichia coli and used for an inhibitor screen with previously synthesized inhibitors of various trypsin-like serine proteases. Two inhibitor types were identified which inhibit TMPRSS2 in the nanomolar range. The first series comprises substrate analogue inhibitors containing a 4-amidinobenzylamide moiety at the P1 position, whereby some of these analogues possess inhibition constants of approximately 20 nM. An improved potency was found for a second type derived from sulfonylated 3-amindinophenylalanylamide derivatives. The most potent derivative of this series inhibits TMPRSS2 with a K(i) value of 0.9 nM and showed an efficient blockage of influenza virus propagation in human airway epithelial cells. On the basis of the inhibitor studies, a series of new fluorogenic substrates containing a D-arginine residue at the P3 position was synthesized, some of them were efficiently cleaved by TMPRSS2.

In vitro characterization of TMPRSS2 inhibition in IPEC-J2 cells

“…Calu-3 cells infected with H1N1 or H3N2 influenza viruses were treated with I-432 and related TMPRSS2 inhibitors at 50 mM for 48 h incubation time and it was found that the viral replication was significantly reduced. So far, the most potent inhibitor for suppression of H1N1 and H3N2 influenza viral spread in Calu-3 cells is I-432, which was also applied in our present study 17 .…”

“…Suppression of HA cleavage and the inhibition of influenza virus spread in TMPRSS2-expressing MDCK cells by treatment with hydrophobic decanoylated peptide mimetic protease inhibitor, I-3 was observed after exposure of cells to A/Hamburg/09 (H1N1) 14,16 . I-432 was found to be one of the most potent 3-amidinophenylalanine-derived inhibitors of TMPRSS2 and matriptase 17 and it was also shown that I-432 at 50 mM did not affect cell viability in IPEC-J2 cells after 48 h treatment 18 .…”

“…Prior to the protease activity measurements, the cells were treated with I-432 at 10, 25 and 50 mM for 48 h. Cells were washed with phenol red-free DMEM and the protease activity was assessed by incubation of cells with the fluorogenic substrate Mes-D-Arg-Pro-Arg-AMC Â 2 TFA (I-507) 17 . I-507 was used at the final concentration of 200 mM in 50 mM PBS (pH ¼ 7.4) for 30 min at 37 C. Hydrolysis of the peptide was monitored by the measurements of fluorescence intensity using a Victor X2 2030 fluorescence spectrometer at ex ¼ 380 nm and em ¼ 460 nm.…”

“…Improved substrate properties were found for the fluorogenic AMC derivative I-507 (Mes-D-Arg-Pro-Arg-AMC K M ¼ 2.9 mM), which is more efficiently cleaved and was applied for a first inhibitor screening. In that work various substrateanalogue structures containing a 4-amidinobenzylamide as P1 residue and several arylsulfonylated amides of 3-amidinophenylalanine could be identified as potent TMPRSS2 inhibitors 17 .…”

Interaction exists between matriptase inhibitors and intestinal epithelial cells

“…Therefore, effective inhibitors of matriptase could be potential drugs for these diseases and pathogenic conditions. This led to the development of various types of matriptase inhibitors including the sulfonylated 3-amidinophenylalanine derivatives [17][18][19][20][21] and the substrate-analogue ketobenzothiazole derivatives derived from the known P4-P1 Arg-Gln-Ala-Arg substrate sequence at the autocatalytic activation site of matriptase 22,23 . Another selective small molecule matriptase inhibitor, the arginal derivative CVS-3983, could suppress the growth of androgen-independent prostate tumor xenografts in vivo 24 .…”