Inhibition of airway epithelial sodium channel (ENaC) function enhances mucociliary clearance (MCC). ENaC is positively regulated by channel-activating proteases (CAPs), and CAP inhibitors are therefore predicted to be beneficial in diseases associated with impaired MCC. The aims of the present study were to 1) identify low-molecular-weight inhibitors of airway CAPs and 2) to establish whether such CAP inhibitors would translate into a negative regulation of ENaC function in vivo, with a consequent enhancement of MCC. To this end, camostat, a trypsin-like protease inhibitor, provided a potent (IC 50 ϳ50 nM) and prolonged attenuation of ENaC function in human airway epithelial cell models that was reversible upon the addition of excess trypsin. In primary human bronchial epithelial cells, a potency order of placental bikunin Ͼ camostat Ͼ 4-guanidinobenzoic acid 4-carboxymethyl-phenyl ester Ͼ aprotinin Ͼ Ͼ soybean trypsin inhibitor ϭ ␣1-antitrypsin, was largely consistent with that observed for inhibition of prostasin, a molecular candidate for the airway CAP. In vivo, topical airway administration of camostat induced a potent and prolonged attenuation of ENaC activity in the guinea pig trachea (ED 50 ϭ 3 g/kg). When administered by aerosol inhalation in conscious sheep, camostat enhanced MCC out to at least 5 h after inhaled dosing. In summary, camostat attenuates ENaC function and enhances MCC, providing an opportunity for this approach toward the negative regulation of ENaC function to be tested therapeutically.

Structure-activity relationships in analogues of the irritant natural product capsaicin have previously been rationalized by subdivision of the molecule into three structural regions (A,B, and C). The hypothesis that resiniferatoxin (RTX), which is a high-potency ligand for the same receptor and which has superficial structural similarities with capsaicin, could be analogously subdivided has been investigated. The effects of making parallel changes in the two structural series have been studied in a cellular functional assay which is predictive of analgesic activity. Parallel structural changes in the two series lead to markedly different consequences on biological activity; the 3- and 4-position aryl substituents (corresponding to the capsaicin 'A-region') which are strictly required for activity in capsaicin analogues are not important in RTX analogues. The homovanillyl C-20 ester group in RTX (corresponding to the capsaicin 'B-region') is more potent than the corresponding amide, in contrast to the capsaicin analogues. Structural variations to the diterpene moiety suggest that the functionalized 5-membered diterpene ring of RTX is an important structural determinant for high potency. Modeling studies indicate that the 3D position of the alpha-hydroxy ketone moiety in the 5-membered ring is markedly different in the phorbol (inactive) analogues and RTX (active) series. This difference appears to be due to the influence of the strained ortho ester group in RTX, which acts as a local conformational constraint. The reduced activity of an analogue substituted in this region and the inactivity of a simplified analogue in which this unit is entirely removed support this conclusion.

1 Bradyzide is from a novel class of rodent-selective non-peptide B 2 bradykinin antagonists (1-(2-Nitrophenyl)thiosemicarbazides). 7 Bradyzide is orally available and blocks bradykinin-induced hypotension and plasma extravasation. 8 Bradyzide shows long-lasting oral activity in rodent models of in¯ammatory hyperalgesia, reversing Freund's complete adjuvant (FCA)-induced mechanical hyperalgesia in the rat knee joint (ED 50 , 0.84 mmol kg 71 ; duration of action 44 h). It is equipotent with morphine and diclofenac, and 1000 times more potent than paracetamol, its maximal eect exceeding that of the non-steroidal anti-in¯ammatory drugs (NSAIDs). Bradyzide does not exhibit tolerance when administered over 6 days. 9 In summary, bradyzide is a potent, orally active, antagonist of the B 2 bradykinin receptor, with selectivity for the rodent over the human receptor.

Supporting InformationThe supporting information is available free of charge on the ACS Publications website at DOI: Synthetic methods, characterization, crystallographic and computational modeling data.

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