Proteolytic cleavage of the influenza virus surface glycoprotein hemagglutinin (HA) by host cell proteases is crucial for infectivity and virus spread. The proteases HAT (human airway trypsin-like protease) and TMPRSS2 (transmembrane protease serine S1 member 2) known to be present in the human airways were previously identified as proteases that cleave HA. We studied subcellular localization of HA cleavage and cleavage inhibition of seasonal influenza virus A/Memphis/14/96 (H1N1) and pandemic virus A/Hamburg/5/ 2009 (H1N1) in MDCK cells that express HAT and TMPRSS2 under doxycycline-induced transcriptional activation. We made the following observations: (i) HA is cleaved by membrane-bound TMPRSS2 and HAT and not by soluble forms released into the supernatant; (ii) HAT cleaves newly synthesized HA before or during the release of progeny virions and HA of incoming viruses prior to endocytosis at the cell surface, whereas TMPRSS2 cleaves newly synthesized HA within the cell and is not able to support the proteolytic activation of HA of incoming virions; and (iii) cleavage activation of HA and virus spread in TMPRSS2-and HATexpressing cells can be suppressed by peptide mimetic protease inhibitors. The further development of these inhibitors could lead to new drugs for influenza treatment.Human influenza viruses cause acute infection of the respiratory tract that affects millions of people during seasonal outbreaks every year. Furthermore, the emergence of a new influenza virus for which there is little or no immunity in the human population may provoke an influenza pandemic, as is the case with the currently circulating swine origin H1N1 influenza A virus.Influenza virus replication is initiated by the surface glycoprotein hemagglutinin (HA) that mediates binding to sialic acid-containing cell surface receptors and fusion of the viral envelope with the endosomal membrane. HA is synthesized as a precursor protein HA0 and needs to be cleaved by a host cell protease into the subunits HA1 and HA2 to gain its fusion capacity (10,20,37). Proteolytic cleavage of HA0 enables HA to undergo conformational changes at low pH that expose the N-terminal hydrophobic fusion peptide of HA2 and trigger membrane fusion (36). HA0 of most avian and mammalian influenza viruses contains a single arginine, rarely a single lysine, at the cleavage site. In general, activation of HA0 with a monobasic cleavage site was assumed to occur extracellularly when virions are already released from the cells, and trypsin (21, 22), as well as several trypsin-like proteases such as plasmin (12, 23, 24), tryptase Clara from rat bronchiolar epithelial Clara cells, mast cell tryptase from porcine lung (19), and a protease similar to blood clotting factor Xa from chicken allantoic fluid (13), have been identified as HA-activating enzymes in vitro. Furthermore, some bacterial proteases were shown to support proteolytic activation of HA, too (32, 41). However, the proteases responsible for HA cleavage in the human airways were only poorly defined until recent...

Furin belongs to the family of proprotein convertases (PCs) and is involved in numerous normal physiological and pathogenic processes, such as viral propagation, bacterial toxin activation, cancer and metastasis. Furin and related furin-like PCs cleave their substrates at characteristic multibasic consensus sequences, preferentially after an arginine residue. By incorporation of decarboxylated arginine mimetics in P1 position of substrate analogue peptidic inhibitors we could identify highly potent furin inhibitors. The most potent compound, phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide (15), inhibits furin with a K i -value of 0.81 nM and has also comparable affinity to other PCs like PC1/3, PACE4, and PC5/6, whereas PC2 and PC7 or trypsin-like serine proteases were poorly affected. In fowl plague virus (influenza A, H7N1)-infected MDCK cells inhibitor 15 reduced proteolytic hemagglutinin cleavage and was able to reduce virus propagation in a long term infection test. Molecular modelling revealed several key interactions of the 4-amidinobenzylamide residue in the S1 pocket of furin contributing to the excellent affinity of these inhibitors.

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.

Well-ordered water molecules are displaced from thrombin's hydrophobic S3/4-pocket by P3-varied ligands (Gly, d-Ala, d-Val, d-Leu to d-Cha with increased hydrophobicity and steric requirement). Two series with 2-(aminomethyl)-5-chlorobenzylamide and 4-amidinobenzylamide at P1 were examined by ITC and crystallography. Although experiencing different interactions in S1, they display almost equal potency. For both scaffolds the terminal benzylsulfonyl substituent differs in binding, whereas the increasingly bulky P3-groups address S3/4 pocket similarly. Small substituents leave the solvation pattern unperturbed as found in the uncomplexed enzyme while increasingly larger ones stepwise displace the waters. Medium-sized groups show patterns with partially occupied waters. The overall 40-fold affinity enhancement correlates with water displacement and growing number of van der Waals contacts and is mainly attributed to favorable entropy. Both Gly derivatives deviate from the series and adopt different binding modes. Nonetheless, their thermodynamic signatures are virtually identical with the homologous d-Ala derivatives. Accordingly, unchanged thermodynamic profiles are no reliable indicator for conserved binding modes.

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