title: Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation.
abstract: Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation.
authors.affiliation: Drug Discovery Biology, Monash University, Parkville, VIC, Australia. Electronic address: laura.edgington-mitchell@monash.edu.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Edgington-Mitchell
authors.affiliation: Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Barlow
authors.affiliation: Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
authors.forename: Luigi
authors.initials: L
authors.lastname: Aurelio
authors.affiliation: Drug Discovery Biology, Monash University, Parkville, VIC, Australia.
authors.forename: Aminath
authors.initials: A
authors.lastname: Samha
authors.affiliation: Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.
authors.forename: Monika
authors.initials: M
authors.lastname: Szabo
authors.affiliation: Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia. Electronic address: bim.graham@monash.edu.
authors.forename: Bim
authors.initials: B
authors.lastname: Graham
authors.affiliation: Drug Discovery Biology, Monash University, Parkville, VIC, Australia; Department of Pharmacology and Therapeutics, University of Melbourne, Parkville, VIC, Australia. Electronic address: nigel.bunnett@monash.edu.
authors.forename: Nigel
authors.initials: N
authors.lastname: Bunnett
mesh_terms: D000818:Animals; D003092:Colitis / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D007249:Inflammation / Q000378:metabolism*; D051379:Mice; D015394:Molecular Structure; D063065:Organophosphonates / Q000138:chemical synthesis / Q000737:chemistry*; D010195:Pancreatitis / Q000378:metabolism; D057057:Serine Proteases / Q000032:analysis* / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D063065:Organophosphonates; D057057:Serine Proteases
keywords: Activity-based probes; Colitis; Diphenylphosphonate; Elastase; Fluorescent probes; Inflammation; Pancreatitis; Protease; Protease inhibitors; Trypsin