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Divergent Inhibitor Susceptibility among Airway Lumen-Accessible Tryptic Proteases.

Nimishakavi S, Raymond WW, Gruenert DC, Caughey GH - PLoS ONE (2015)

Bottom Line: We hypothesized that these inhibitors inactivate a variety of airway protease targets, potentially with bystander effects.Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Product inhibition by 6-amidino-2-naphthol.These graphs compare inhibitory potency of nafamostat with that of its liberated cleavage product, 6-amidino-2-naphthol, versus matriptase and β-tryptase.
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pone.0141169.g004: Product inhibition by 6-amidino-2-naphthol.These graphs compare inhibitory potency of nafamostat with that of its liberated cleavage product, 6-amidino-2-naphthol, versus matriptase and β-tryptase.

Mentions: As shown in Fig 4, the IC50 of nafamostat’s leaving group, 6-amidino-2-naphthol, was >104-fold higher for matriptase and β-tryptase than the IC50 of nafamostat itself. Although one potential mechanism of high potency of a cleavable inhibitor is generation of a leaving group that is a more potent competitive inhibitor than is the uncleaved parent compound, the very high IC50 of 6-amidino-2-naphthol relative to that of nafamostat itself suggests that this is not the case. Therefore, inhibition by the non-acyl component of hydrolyzed nafamostat does not explain nafamostat’s high potency upon cleavage by these enzymes and, given the very low rates of nafamostat turnover, 6-amidino-2-naphthol would be unlikely to achieve the concentrations needed to reduce matriptase and β-tryptase activity.


Divergent Inhibitor Susceptibility among Airway Lumen-Accessible Tryptic Proteases.

Nimishakavi S, Raymond WW, Gruenert DC, Caughey GH - PLoS ONE (2015)

Product inhibition by 6-amidino-2-naphthol.These graphs compare inhibitory potency of nafamostat with that of its liberated cleavage product, 6-amidino-2-naphthol, versus matriptase and β-tryptase.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4612780&req=5

pone.0141169.g004: Product inhibition by 6-amidino-2-naphthol.These graphs compare inhibitory potency of nafamostat with that of its liberated cleavage product, 6-amidino-2-naphthol, versus matriptase and β-tryptase.
Mentions: As shown in Fig 4, the IC50 of nafamostat’s leaving group, 6-amidino-2-naphthol, was >104-fold higher for matriptase and β-tryptase than the IC50 of nafamostat itself. Although one potential mechanism of high potency of a cleavable inhibitor is generation of a leaving group that is a more potent competitive inhibitor than is the uncleaved parent compound, the very high IC50 of 6-amidino-2-naphthol relative to that of nafamostat itself suggests that this is not the case. Therefore, inhibition by the non-acyl component of hydrolyzed nafamostat does not explain nafamostat’s high potency upon cleavage by these enzymes and, given the very low rates of nafamostat turnover, 6-amidino-2-naphthol would be unlikely to achieve the concentrations needed to reduce matriptase and β-tryptase activity.

Bottom Line: We hypothesized that these inhibitors inactivate a variety of airway protease targets, potentially with bystander effects.Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus