Oxyanion-mediated inhibition of serine proteases

Novel aryl derivatives of benzamidine were synthesized and tested for their inhibitory potency against bovine trypsin, rat skin tryptase, human recomb inant granzyme A, human thrombin, and human plasma kallikrein. All compound s show competitive inhibition against these proteases with K-i values in th e micromolar range. X-ray structures were determined to 1.8 Angstrom resolu tion for trypsin complexed with two of the para-substituted benzamidine der ivatives, 1-(4-amidinophenyl)-3-(4-chlorophenyl)urea (ACPU) and 1-(4-amidin ophenyl)-3-(4-phenoxyphenyl)urea (APPU). Although the inhibitors do not eng age in direct and specific interactions outside the S1 pocket, they do form intimate indirect contacts with the active site of trypsin. The inhibitors are linked to the enzyme by a sulfate ion that forms an intricate network of three-centered hydrogen bonds. Comparison of these structures with other serine protease structures with noncovalently bound oxyanions reveals a pa ir of highly conserved oxyanion-binding sites in the active site. The posit ions of noncovalently bound oxyanions, such as the oxygen atoms of sulfate, are distinct from the positions of covalent oxyanions of tetrahedral inter mediates. Noncovalent oxyanion positions are outside the "oxyanion hole." K inetics data suggest that protonation stabilizes the ternary inhibitor/oxya nion/protease complex. In sum, both cations and anions can mediate Ki. Cati on mediation of potency of competitive inhibitors of serine proteases was p reviously reported by Stroud and co-workers [Katz, B. A., Clark, J. M., Fin er-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Mo ore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612].