Divergent Inhibitor Susceptibility among Airway Lumen-Accessible Tryptic Proteases.
Tryptic serine proteases of bronchial epithelium regulate ion flux, barrier integrity, and allergic inflammation. Inhibition of some of these proteases is a strategy to improve mucociliary function in cystic fibrosis and asthmatic inflammation. Several inhibitors have been tested in pre-clinical animal models and humans. We hypothesized that these inhibitors inactivate a variety of airway protease targets, potentially with bystander effects. To establish relative potencies and modes of action, we compared inactivation of human prostasin, matriptase, airway trypsin-like protease (HAT), and β-tryptase by nafamostat, camostat, bis(5-amidino-2-benzimidazolyl)methane (BABIM), aprotinin, and benzamidine. Nafamostat achieved complete, nearly stoichiometric and very slowly reversible inhibition of matriptase and tryptase, but inhibited prostasin less potently and was weakest versus HAT. The IC50 of nafamostat's leaving group, 6-amidino-2-naphthol, was >104-fold higher than that of nafamostat itself, consistent with suicide rather than product inhibition as mechanisms of prolonged inactivation. Stoichiometric release of 6-amidino-2-naphthol allowed highly sensitive fluorometric estimation of active-site concentration in preparations of matriptase and tryptase. Camostat inactivated all enzymes but was less potent overall and weakest towards matriptase, which, however was strongly inhibited by BABIM. Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase. Benzamidine was universally weak. Thus, each inhibitor profile was distinct. Nafamostat, camostat and aprotinin markedly reduced tryptic activity on the apical surface of cystic fibrosis airway epithelial monolayers, suggesting prostasin as the major source of such activity and supporting strategies targeting prostasin for inactivation.
Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America; Veterans Affairs Medical Center, San Francisco, California, United States of America.
Shilpa
S
Nimishakavi
Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America; Veterans Affairs Medical Center, San Francisco, California, United States of America.
Wilfred W
WW
Raymond
Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America; Department of Otolaryngology-Head and Neck Surgery, University of California San Francisco, San Francisco, California, United States of America; Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California San Francisco, San Francisco, California, United States of America; Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California, United States of America; Institute for Human Genetics, University of California San Francisco, San Francisco, California, United States of America; Department of Pediatrics, University of Vermont College of Medicine, Burlington, Vermont, United States of America.
Dieter C
DC
Gruenert
Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America; Veterans Affairs Medical Center, San Francisco, California, United States of America; Department of Medicine, University of California San Francisco, San Francisco, California, United States of America.
George H
GH
Caughey
D007611:Aprotinin / Q000494:pharmacology; D001980:Bronchi / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D020134:Catalytic Domain; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D004952:Esters; D016670:Gabexate / Q000031:analogs & derivatives / Q000494:pharmacology; D006146:Guanidines; D006801:Humans; D012697:Serine Endopeptidases / Q000737:chemistry*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D053802:Tryptases / Q000037:antagonists & inhibitors*; D000099271:Prostasin
D007611:Aprotinin; D004952:Esters; D016670:Gabexate; D006146:Guanidines; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D053802:Tryptases; D000099271:Prostasin; C034532:camostat; C485595:matriptase