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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: Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase.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.

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

Comparison of inhibitor potencies.Data are expressed as percentage of activity relative to no-inhibitor control activity versus log10 ratios of inhibitor to protease concentration. The proteases tested are human airway trypsin-like protease (HAT), matriptase, prostasin and β-tryptase. The dashed vertical line marks the inhibitor/enzyme ratio at which 50% inhibition is predicted for inactivation with 1:1 stoichiometry. Aprotinin achieves near-stoichiometric inhibition of prostasin and matriptase, whereas nafamostat achieves near-stoichiometric inhibition of tryptase and matriptase. N = 3–4; error bars show ± S.D.
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pone.0141169.g002: Comparison of inhibitor potencies.Data are expressed as percentage of activity relative to no-inhibitor control activity versus log10 ratios of inhibitor to protease concentration. The proteases tested are human airway trypsin-like protease (HAT), matriptase, prostasin and β-tryptase. The dashed vertical line marks the inhibitor/enzyme ratio at which 50% inhibition is predicted for inactivation with 1:1 stoichiometry. Aprotinin achieves near-stoichiometric inhibition of prostasin and matriptase, whereas nafamostat achieves near-stoichiometric inhibition of tryptase and matriptase. N = 3–4; error bars show ± S.D.

Mentions: The in vitro, steady state susceptibility of four human airway proteases (HAT, matriptase, prostasin and β-tryptase) to inactivation varied depending on the inhibitor studied. Fig 2 compares the potency of individual inhibitors versus the four proteases. Fig 3 compares the inhibitor susceptibility of individual proteases versus the five inhibitors. To facilitate comparison of potencies over a range of inhibitor and protease concentrations, the data are graphed as percentage of uninhibited enzyme activity versus log of the ratio of inhibitor concentration to starting concentration of active enzyme (log10 ([inhibitor]/[enzyme])). In such plots, activity is 0% when log10 ([inhibitor]/[enzyme]) = 0 for an inhibitor that inactivates a protease with full potency and 1:1 stoichiometry. The normalized IC50 for such an inhibitor would be log10 (0.5) = -0.3. The observed IC50 data are shown in Table 2.


Divergent Inhibitor Susceptibility among Airway Lumen-Accessible Tryptic Proteases.

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

Comparison of inhibitor potencies.Data are expressed as percentage of activity relative to no-inhibitor control activity versus log10 ratios of inhibitor to protease concentration. The proteases tested are human airway trypsin-like protease (HAT), matriptase, prostasin and β-tryptase. The dashed vertical line marks the inhibitor/enzyme ratio at which 50% inhibition is predicted for inactivation with 1:1 stoichiometry. Aprotinin achieves near-stoichiometric inhibition of prostasin and matriptase, whereas nafamostat achieves near-stoichiometric inhibition of tryptase and matriptase. N = 3–4; error bars show ± S.D.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141169.g002: Comparison of inhibitor potencies.Data are expressed as percentage of activity relative to no-inhibitor control activity versus log10 ratios of inhibitor to protease concentration. The proteases tested are human airway trypsin-like protease (HAT), matriptase, prostasin and β-tryptase. The dashed vertical line marks the inhibitor/enzyme ratio at which 50% inhibition is predicted for inactivation with 1:1 stoichiometry. Aprotinin achieves near-stoichiometric inhibition of prostasin and matriptase, whereas nafamostat achieves near-stoichiometric inhibition of tryptase and matriptase. N = 3–4; error bars show ± S.D.
Mentions: The in vitro, steady state susceptibility of four human airway proteases (HAT, matriptase, prostasin and β-tryptase) to inactivation varied depending on the inhibitor studied. Fig 2 compares the potency of individual inhibitors versus the four proteases. Fig 3 compares the inhibitor susceptibility of individual proteases versus the five inhibitors. To facilitate comparison of potencies over a range of inhibitor and protease concentrations, the data are graphed as percentage of uninhibited enzyme activity versus log of the ratio of inhibitor concentration to starting concentration of active enzyme (log10 ([inhibitor]/[enzyme])). In such plots, activity is 0% when log10 ([inhibitor]/[enzyme]) = 0 for an inhibitor that inactivates a protease with full potency and 1:1 stoichiometry. The normalized IC50 for such an inhibitor would be log10 (0.5) = -0.3. The observed IC50 data are shown in Table 2.

Bottom Line: Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase.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.

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