Klonierung, Expression und enzymkinetische Charakterisierung der hämagglutininspaltenden Protease TMPRSS2

TMPRSS2 is a multidomain type II transmembrane serine protease that cleaves the surface glycoprotein hemagglutinin (HA) of influenza viruses with 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 coronavirus. Increased TMPRSS2 expression was also described in several tumor entities. Therefore, TMPRSS2 emerged as a potential target for drug design. The catalytic domain of TMPRSS2 was expressed in E. 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 in P1 position, whereby some of these analogues possess inhibition constants around 20 nM. An improved potency was found for second type derived from sulfonylated 3-amindinophenylalanylamide derivates. The most potent derivative of this series inhibits TMPRSS2 with a Ki value of 0.9 nM and showed an efficient blockage of influenza virus propagation in human airway epithelial cells. Based on the inhibitor studies a series of new fluorogenic substrates containing a D-arginine residue in P3 position was synthesized, some of them were efficiently cleaved by TMPRSS2.