Recruiting Zn2+ to mediate potent, specific inhibition of serine proteases

As regulators of ubiquitous biological processes, serine proteases can caus e disease states when inappropriately expressed or regulated, and are thus rational targets for inhibition by drugs. Recently we described a new inhib ition mechanism applicable for the development of potent, selective small m olecule serine protease inhibitors that recruit physiological Zn2+ to media te high affinity (sub-nanomolar) binding. To demonstrate some of the struct ural principles by which the selectivity of Zn2+ mediated serine protease i nhibitors can be developed toward or against a particular target, here we d etermine and describe the structures of thrombin-BABIM-Zn2+, -keto-BABIM-Zn 2+, and -hemi-BABIM-Zn2+ (where BABIM is bis(5-amidino-2-benzimidazolyl)met hane, keto-BABIM is bis(5-amidino-2-benzimidazolyl)methane ketone, and hemi -BABIM is (5-amidino-2-benzimidazolyl)(2-benzimidazolyl) and compare them w ith the corresponding trypsin-inhibitor-Zn2+ complexes. Inhibitor binding i s mediated by a Zn ion tetrahedrally coordinated by two benzimidazole nitro gen atoms of the inhibitor, by N-His57(epsilon 2), and by O-Ser195(gamma). The structures of Zn2+-free trypsin-BABIM and -hemi-BABIM were also determi ned at selected pH values for comparison with the corresponding Zn2+-mediat ed complexes. To assess some of the physiological parameters important for harnessing Zn2+ as a co-inhibitor, crystal structures at multiple PH and [Z n2+] values were determined for trypsin-keto-BABIM. The K-d value of Zn2+ f or the binary trypsin-keto-BABIM complex was estimated to be <12 nM at pH 7 .06 by crystallographic determination of the occupancy of bound Zn2+ in hyp sin-keto-BABIM crystals soaked at this pH in synthetic mother liquor contai ning inhibitor and 100 nM Zn2+. In synthetic mother liquor saturated in Zn2 +, trypsin bound keto-BABIM is unhydrated at PH 9.00 and 9.93, and has an s p2 hybridized ketone carbon bridging the 5-amidinobenzimidazoles, whereas a t pH 7.00 and 8.00 it undergoes hydration and a change in geometry upon add ition of water to the bridging carbonyl group. To show how Zn2+ could be re cruited as a co-inhibitor of other enzymes, a method was developed for loca ting in protein crystals Zn2+ binding sites where design of Zn2+ mediated l igands can be attempted. Thus, by soaking trypsin crystals in high concentr ations of Zn2+ in the absence of a molecular inhibitor, the site where Zn2 mediates binding of BABIM and analogs was identified, as well as another Z n2+ binding site. (C) 1999 Academic Press.