A New Family of β-Hairpin Mimetics Based on a Trypsin Inhibitor from Sunflower Seeds

Abstract

The ability of proteases to regulate many aspects of cell function and defense accounts for the considerable interest in the design of novel protease inhibitors. There are many naturally occurring proteinaceous serine protease inhibitors, one of which is a 14 amino acid cyclic peptide from sunflower seeds that shows both sequence and conformational similarity with the trypsin-reactive loop of the Bowman–Birk family of serine protease inhibitors. This inhibitor adopts a β-hairpin conformation when bound at the active site of bovine β-trypsin. We illustrate here an approach to inhibitor design in which the β hairpin from the naturally occurring peptide is transplanted onto a hairpin-inducing template. Two mimetics with the sequences RC*TKSIPPIC*F (where C*C* is a disulfide) and TKSIPPI are studied, each mounted onto a D-Pro–L-Pro template. NMR studies revealed a well-defined β-hairpin conformation for each mimetic in aqueous solution; this conformation is closely related to the trypsin-bound conformation of the natural inhibitor and includes a cis-Ile–Pro peptide bond. Both mimetics inhibit trypsin in the mid nanomolar range. An alanine scan revealed the importance for inhibitory activity of the specificity-determining Lys residue and of the first but not the second Pro residue in the IPPI motif. Since these hairpin mimetics can be prepared by parallel combinatorial synthesis, this family of molecules may be a useful starting point for the discovery of other biologically or medicinally useful serine protease inhibitors.