Klonierung, Expression und enzymkinetische Charakterisierung der hämagglutininspaltenden Protease TMPRSS2
TMPRSS2 ist eine Typ II transmembrane Serinprotease mit einer multidomänen Struktur, die das Oberflächenglykoprotein Hämagglutinin (HA) der Influenzaviren mit einer monobasischen Schnittstelle spaltet. Dies ist eine Voraussetzung für die Virusfusion und Vermehrung. Weiterhin aktiviert sie das Fusion...
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Table of Contents: 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.