Optimization of Furin Inhibitors To Protect against the Activation of Influenza Hemagglutinin H5 and Shiga Toxin Hugo Gagnon, † Sophie Beauchemin, ‡ Anna Kwiatkowska, † Fre ́ de ́ ric Couture, † Franc ̧ ois D’Anjou, † Christine Levesque, † Fre ́ de ́ rik Dufour, † Adamy Roberge Desbiens, § Rolland Vaillancourt, § Sylvain Bernard, ‡ Roxane Desjardins, † Franc ̧ ois Malouin,* ,§ Yves L. Dory,* ,‡ and Robert Day* ,† † Institut de Pharmacologie de Sherbrooke (IPS) and De ́ partement de Chirurgie/Urologie, Faculte ́ de Me ́ decine et des Sciences de la Sante ́ (FMSS), Universite ́ de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Que ́ bec J1H 5N4, Canada ‡ De ́ partement de Chimie, Faculte ́ des Sciences, Institut de Pharmacologie de Sherbrooke (IPS), Universite ́ de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Que ́ bec J1H 5N4, Canada § De ́ partement de Biologie, Faculte ́ des Sciences, Centre d’E ́ tude et de Valorisation de la Diversite ́ Microbienne (CEVDM), Universite ́ de Sherbrooke, Sherbrooke, Quebec, J1K 2R1, Canada * S Supporting Information ABSTRACT: Proprotein convertases (PCs) are crucial in the processing and entry of viral or bacterial protein precursors and confer increased infectivity of pathogens bearing a PC activation site, which results in increased symptom severity and lethality. Previously, we developed a nanomolar peptide inhibitor of PCs to prevent PC activation of infectious agents. Herein, we describe a peptidomimetic approach that increases the stability of this inhibitor for use in vivo to prevent systemic infections and cellular damage, such as that caused by influenza H5N1 and Shiga toxin. The addition of azaβ 3 -amino acids to both termini of the peptide successfully prevented influenza hemagglutinin 5 fusogenicity and Shiga toxin Vero toxicity in cell-based assays. The results from a cell-based model using stable shRNA-induced proprotein convertase knockdown indicate that only furin is the major proprotein convertase required for HA5 cleavage. ■ INTRODUCTION The observed effectiveness of many anti-infective agents often rapidly decreases because of increased pathogen resistance. 1-3 Targeting host cell proteins has been proposed for various pathogens as an alternative approach to developing new anti- infective agents to prevent such occurrences. 4-6 The proprotein convertases (PCs), which belong to the family of serine proteases, have been suggested as potential therapeutic targets against various pathogens. 7,8 Seven of nine PCs (i.e., furin, PACE4, PC1/3, PC2, PC4, PC5/6, and PC7) recognize a consensus cleavage site, R-X-R/K-R↓, in precursor proteins. 9 These widely expressed proteases 10-13 primarily activate precursor proteins and produce smaller bioactive products. 14,15 Many reports have implicated PCs in the maturation of viral (e.g., highly pathogenic avian influenza (HPAI) hemagglutinin (HA) 16-18 ) or bacterial toxins (e.g., Shiga toxin, Stx 19 ). The processing of pathogen precursor proteins by PCs results in increased pathogen infectivity, symptom severity, and lethality. 17,19-21 Approximately 60% of human HPAI cases caused by H5N1 result in lethal infections, with spread of the infection to organs typically unreached by the virus itself. 22 These highly infectious characteristics are associated with the gain of a multibasic (RRRKKR) cleavage site in HA that is recognized by PCs (furin, PC5/6, and PC7). 7,16-18,20,22-27 Upon cleavage of an HA-containing multibasic cleavage site present in the TGN, HA allows viral membrane fusion with the host cell membrane, which results in the release of viral genetic material and viral replication. 5,21,28-30 Similarly, the Shiga toxins produced by Shigella dysenteriae and Shiga-like toxin- producing Escherichia coli (STEC) are examples of PC-activated bacterial toxins. 31,32 Shiga toxins contain an RXXR sequence in a loop stabilized by a disulfide bond that is very sensitive to cleavage by trypsin and PCs. 8,33,34 Furin was proposed as the PC responsible for this cleavage, which occurs at low pH in the trans-Golgi network or in the endosomes, resulting in a rapid intoxication of cells. 35 These viral and bacterial targets are particularly attractive for use in preventing future pandemics and the associated economic effects of these pathogens. The influenza virus rearrangements responsible for recent pandemic episodes were unpredictable, 1 and the acquisition of this multibasic cleavage site by human influenza could lead to virulent influenza pandemics. 36-38 Foodborne STEC, which is often associated with undercooked ground beef and contami- Received: April 29, 2013 Published: December 10, 2013 Article pubs.acs.org/jmc © 2013 American Chemical Society 29 dx.doi.org/10.1021/jm400633d | J. Med. Chem. 2014, 57, 29-41