Remarkable selectivity enhancements of exceptionally small inhibitors are achieved toward the uPA target over the highly similar tPA anti-target through a single atom substitution on an otherwise relatively non-selective scaffold. Overall selectivities for uPA over tPA as high as 980-fold at physiological pH were realized. The increase in selectivity results from the displacement of a single bound water molecule common to the S1 site of both the uPA target and the tPA anti-target because of the ensuing deficit in hydrogen bonding of the arylamidine inhibitor when bound in the Ala190 protease anti-target.

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Tryptase, a serine protease released exclusively from activated mast cells, has been implicated as a potential causative agent in asthma. Enzymatically active tryptase is comprised of four subunits, and heparin stabilizes the associated tetramer. Lactoferrin, a cationic protein released from activated neutrophils, binds tightly to heparin, therefore we investigated lactoferrin as an inhibitor of tryptase and found that it is both a potent (Ki' is 24 nM) and selective inhibitor. Size exclusion chromatography studies revealed that lactoferrin disrupted the quaternary structure of active tryptase. Lactoferrin was tested in an allergic sheep model of asthma; aerosolized lactoferrin (10 mg in 3 ml phosphate-buffered saline, 0.5 h before as well as 4 and 24 h after inhalation challenge by Ascaris suum) abolished both late-phase bronchoconstriction (no significant increase in specific lung resistance 4 to 8 h following provocation, p < 0.05 versus vehicle treatment) and airway hyperresponsiveness (no detectable increase in airway sensitivity to carbachol challenge 24 h after antigen challenge, p < 0.05 versus vehicle). These data suggest tryptase involvement in both late-phase bronchoconstriction and airway hyperreactivity and furthermore suggest that a physiological function of neutrophil lactoferrin is the inhibition of tryptase released from mast cells.

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