Vaccine 30 (2012) 7374–7380 Contents lists available at SciVerse ScienceDirect Vaccine j ourna l ho me pag e: www.elsevier.com/locate/vaccine Hemagglutinin activating host cell proteases provide promising drug targets for the treatment of influenza A and B virus infections Eva Böttcher-Friebertshäuser a,∗ , Yinghui Lu a , Daniela Meyer b , Frank Sielaff b , Torsten Steinmetzer b , Hans-Dieter Klenk a , Wolfgang Garten a a Institute of Virology, Philipps-University Marburg, Marburg, Germany b Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marburg, Germany a r t i c l e i n f o Article history: Received 29 December 2011 Received in revised form 30 April 2012 Accepted 1 October 2012 Available online 13 October 2012 Keywords: Influenza Hemagglutinin Proteolytic activation TMPRSS2 Peptide mimetic protease inhibitors a b s t r a c t Cleavage of the influenza virus hemagglutinin (HA) by host cell proteases is crucial for infectivity and spread of the virus. Some years ago, we identified TMPRSS2 and HAT from human airways as activating proteases of influenza A viruses containing a monobasic HA cleavage site. Therefore, these proteases are considered as potential drug targets. In this report, first we show that HA of influenza B virus is activated by TMPRSS2 and HAT, too. We further demonstrate that benzylsulfonyl-d-arginine-proline- 4-amidinobenzylamide (BAPA), which is a potent inhibitor of HAT and TMPRSS2, efficiently suppresses virus propagation in TMPRSS2-expressing human airway epithelial cells by inhibition of HA cleavage. BAPA treatment reduced virus titers of different influenza A and B viruses more than 1000-fold and delayed virus propagation by 24–48 h at non-cytotoxic concentrations. A combination of BAPA with the neuraminidase (NA) inhibitor oseltamivir carboxylate efficiently blocked influenza virus replication in airway epithelial cells at remarkable lower concentrations for each compound than treatment with either inhibitor alone. Our studies provide a novel and potent approach for influenza chemotherapy that should be considered for influenza treatment. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction Influenza (flu) is a highly contagious acute infection of the respi- ratory tract that affects millions of people during annual epidemics and occasional occurring pandemics. Seasonal outbreaks are caused by both influenza A and B virus strains that co-circulate with varying predominance and may give rise to severe morbidity and mortality equally. Furthermore, the emergence of a new influenza A virus for which there is little or no immunity in the human popu- lation may provoke an influenza pandemic as has been the case in 1918, 1957, 1968 and recently in 2009. Influenza viruses are enveloped viruses and contain two spike glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA). The HA mediates binding to N-acetyl-neuraminic acid con- taining cell surface receptors and fusion of viral and endosomal membranes, in order to release the viral genome into the host cell [8]. The NA cleaves N-acetyl-neuraminic acid from carbohydrate moieties facilitating entrance and egress of the virus [8,20]. The HA is synthesised as a fusion incompetent precursor pro- tein HA0 that is post-translationally cleaved by a host cell protease ∗ Corresponding author. Tel.: +49 64212866019. E-mail address: boettch6@staff.uni-marburg.de (E. Böttcher-Friebertshäuser). into the disulfide-linked subunits HA1 and HA2 [9,27]. Cleavage of HA0 is a prerequisite to undergo conformational changes at low pH in the endosome that trigger membrane fusion and, there- fore, essential for viral infectivity. The amino acid sequence at the HA cleavage site varies between viral strains, and can affect tis- sue tropism, spread and pathogenicity of the virus [9,27]. Most avian and mammalian influenza A viruses, including the sub- types H1 and H3 that currently infect humans, and influenza B viruses, contain a single arginine (R) at the HA cleavage site and are activated by trypsin in vitro [17,18]. Relevant trypsin- like proteases are present in a limited number of tissues such as the respiratory- and the intestinal tract, thereby restricting virus infection to these tissues. We demonstrated that the type II trans- membrane proteases (TTSP) TMPRSS2 (transmembrane protease, serine S1 member 2) and HAT (human airway trypsin-like protease) present in the human respiratory epithelium cleave influenza A virus HA at a monobasic cleavage site [2]. Recently, the TMPRSS2- related protease TMPRSS4 was shown to cleave HA with monobasic cleavage site, too [6]. In contrast, the HA of highly pathogenic avian influenza viruses of subtypes H5 and H7 is cleaved after the multibasic motif R-X-R/K-R by the subtilisin-like proteases furin and proprotein convertase 5/6 (PC5/6) [9,14,28]. The ubiquity of these enzymes mediates systemic infection with an often fatal outcome. 0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2012.10.001