Optimization of Furin Inhibitors To Protect against the Activation of Influenza Hemagglutinin H5 and Shiga Toxin

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.

Identification of potent and compartment-selective small molecule furin inhibitors using cell-based assays

“…While the non-selective irreversible convertase inhibitor dec-RVKR-CMK decreases skin tumor progression [57], this highly reactive compound exhibits significant cell toxicity [58]. The most potent peptide-based convertase inhibitors are likely the multi-Leu peptides developed by the Day group, which block viral infection [59] as well as tumor growth [28, 29, 60]; and the peptide analogs recently developed by the Steinmetzer group [25, 61] which potently inhibit bacterial toxin activation. However, because peptide-based drugs suffer from potential stability problems, several groups have instead sought small molecule inhibitors.…”

“…In fact, various peptide-based furin inhibitors have been used to block and/or delay anthrax toxemia both in cells and in live animals by inhibiting furin-catalyzed processing of the anthrax PA protein [32, 62, 63]. Additionally, furin has also emerged as a target for the prevention of the cytotoxic activation of Pseudomonas aeruginosa exotoxin A and Shiga toxin [25, 40, 59, 64]. Thus, the general convertase inhibitor nona-D-arginine peptide [65, 66] provides efficient topical treatment of severe corneal keratitis caused by Pseudomonas aeruginosa , particularly when combined with the antibiotic ciprofloxacin [67].…”

Novel Furin Inhibitors with Potent Anti‐infectious Activity

“…[17] This approach was furthere laborated by other groups using different P1 residues and N-terminal elongations. [18,19] Further improved potencies in the low picomolar range have been achieved by combination with basic P5 residues. [20] Crystal structures of thesei nhibitors in complex with human furin have revealed numerousk ey interactions contributing to the strong in vitro potencyo ft hese analogues.…”

Structural Determinants of Furin Inhibitors Derived from Influenza Hemagglutinin

“…Studies have shown that the hemagglutinin of avian influenza virus A contains a TPRERRRKKRTGL sequence, which is a furin cleavage site. The optimization of TPRERRRKKRTGL peptide led to the discovery of a potent inhibitor with the following sequence: Ac-RARRRKKRT-NH2 [4,5]. In the present work, the structural determinants for furin inhibition were investigated using positional-scanning approach.…”