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Improved peptide-based inhibitors of human beta tryptase were discovered using information gleaned from tripeptide library screening and structure-guided design methods, including fragment screening. Our efforts sought to improve this class of inhibitors by replacing the traditional Lys or Arg P1 element. The optimized compounds display low nanomolar potency against the mast cell target and several hundred-fold selectivity with respect to serine protease off targets. Thus, replacement of Lys/Arg at P1 in a peptide-like scaffold does not need to be accompanied by a loss in target affinity.

Serine proteases play a role in a variety of disease states and thus are attractive targets for therapeutic intervention. We report the kinetic characterization of a class of serine protease inhibitors whose potencies and selectivities are dramatically enhanced in the presence of Zn(II). The structural basis for Zn(II)-mediated inhibition of trypsin-like proteases has recently been reported [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612]. A case study of the kinetic behavior of human tryptase inhibitors is provided to illustrate the general phenomenon of Zn(II)-mediated inhibition. Tryptase, Zn(II), and the inhibitor form a ternary complex which exhibits classic tight-binding inhibition. The half-life for release of inhibitor from the tryptase-Zn(II)-inhibitor complex has been measured for a number of inhibitors. Consistent with tight-binding behavior, potent tryptase inhibitors are characterized by extremely slow rates of dissociation from the ternary complex with half-lives on the order of hours. A model of human serum, designed to reproduce physiological levels of Zn(II), has been employed to evaluate the performance of Zn(II)-potentiated tryptase inhibitors under physiological conditions. We demonstrate that Zn(II)-mediated inhibition can be achieved at physiological Zn(II) levels.

The streptavidin-bound crystal structures of two disulfide-bridge cyclic peptides (cyclo-Ac-[CHPQGPPC]-NH2 and cyclo-Ac-[CHPQFC]-NH2) and of a linear peptide (FSHPQNT) were determined, as well as the structure of apostreptavidin (streptavidin-sulfate). Both the linear and disulfide-bridged cyclic peptides studied share a common HPQ conformation and make common interactions with streptavidin, although significant differences in structures and interactions occur for flanking residues among the complexes. The conformation of the linear peptide in the crystal structure of streptavidin-FSHPQNT was found to differ from that in the same complex published [Weber, P. C., Pantoliano, M. W., & Thompson, L. D. (1992) Biochemistry 31, 9350-9354]. In the present investigation, the HPQNT portion of the ligand is well-defined with some density defining the Phe, whereas in the investigation of Weber et al. only the HPQ segment of the bound peptide could be interpreted. Both bound cyclic peptides adopt a beta-turn involving an H-bond between the His main chain carbonyl and the main chain amide NH of the i+3 residue. In the streptavidin-bound cyclo-Ac-[CHPQFC]-NH2 structure, there is an additional H-bond, indicative of alpha-helix, between the main chain His carbonyl and the main chain C-terminal Cys amide NH group. Binding interactions for both cyclic and linear peptides include direct H-bonds, H-bonds mediated by tightly bound water molecules, and hydrophobic interactions. The above structures and that of streptavidin-biotin in the literature are compared and discussed in the context of structure-based ligand design.

A novel class of mechanism-based inhibitors of the serine proteases is developed using epitaxial selection. Tripeptide boronates esterified by an alcohol or alcohols at the boron retain the tight binding to trypsin-like enzymes associated with transition-state analogs and incorporate additional groups that can be utilized for selectivity between proteases. Formed by reaction of a series of alcohols with the inhibitor boronate oxygen(s), the most structurally compatible alcohol-derivatized inhibitors are either selected by binding to the enzyme (epitaxial selection) or assembled by epitaxial reaction on the enzyme surface. Mass spectrometry of the derivatized boronates and X-ray crystallography of the complexes identify the chemical structures and the three-dimensional interactions of inhibitors generated. This scheme also engineers novel, potent (Ki approximately 7 nM), and more specific inhibitors of individual serine proteases, by derivitizations of compounds obtained by epitaxial selection.