A Selective Irreversible Inhibitor of Furin Does Not Prevent <i>Pseudomonas Aeruginosa</i> Exotoxin A-Induced Airway Epithelial Cytotoxicity

<div><p>Many bacterial and viral pathogens (or their toxins), including <i>Pseudomonas aeruginosa</i> exotoxin A, require processing by host pro-protein convertases such as furin to cause disease. We report the development of a novel irreversible inhibitor of furin (QUB-F1) consisting of a diphenyl phosphonate electrophilic warhead coupled with a substrate-like peptide (RVKR), that also includes a biotin tag, to facilitate activity-based profiling/visualisation. QUB-F1 displays greater selectivity for furin, in comparison to a widely used exemplar compound (furin I) which has a chloromethylketone warhead coupled to RVKR, when tested against the serine trypsin-like proteases (trypsin, prostasin and matriptase), factor Xa and the cysteine protease cathepsin B. We demonstrate QUB-F1 does not prevent <i>P</i>. <i>aeruginosa</i> exotoxin A-induced airway epithelial cell toxicity; in contrast to furin I, despite inhibiting cell surface furin-like activity to a similar degree. This finding indicates additional proteases, which are sensitive to the more broad-spectrum furin I compound, may be involved in this process.</p></div

Irreversible inhibition and visualization of recombinant human furin by QUB-F1.

<p>Western blot analysis was performed under reducing and denaturing conditions demonstrating QUB-F1 (100 μM) is irreversibly bound to furin (0.4 μg). The inhibitor-proteases complex can be visualized using streptavidin-HRP, which detects the biotin reporter group on the compound.</p

Evaluation of the selectivity of action of QUB-F1 and furin I versus a selection of serine proteases.

<p>Trypsin (A), matriptase (B), prostasin (C), factor Xa (D), neutrophil elastase (E), proteinase 3 (F), cathepsin G (G) and chymotrypsin (H) activity was assessed using their cognate fluorogenic substrates in the presence or absence of 10 μM inhibitor (QUB-F1 or furin I) (N = 3). Data represent mean ± s.e.m.; ***P<0.001.</p