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 beta-hairpin conformation when bound at the active site of bovine beta-trypsin. We illustrate here an approach to inhibitor design in which the beta 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 beta-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.

Sunflower Trypsin Inhibitor 1 Derivatives as Molecular Scaffolds for the Development of Novel Peptidic Radiopharmaceuticals

“…The small size and the exceptional rigidity and stability allow the use of SFTI as scaffold for the introduction of other peptide motifs. As demonstrated by Descours et al it is possible to graft smaller fragments of SFTI-1 onto a platform of D-Pro-L-Pro and to retain significant inhibitory activity [19]. Furthermore, an 11-residue peptide corresponding to the reactive-site loop of soybean Bowman-Birk inhibitor (BBI) has been optimized for inhibition of another proteinase via screening of a combinatorial library [20].…”

The Absolute Structural Requirement for a Proline in the P3′-position of Bowman-Birk Protease Inhibitors Is Surmounted in the Minimized SFTI-1 Scaffold

“…However, the lack of importance of other residues is surprising given that the conserved cis-proline at P3Ј has been found to be also critical for function in studies of related peptides. For instance, when the active sequence of SFTI-1 was grafted onto a D-Pro-L-Pro template and each residue in the binding loop was subsequently replaced with alanine, both Lys 5 and Pro 8 were found to be critical for activity, with substitution of Pro 8 having a greater effect on activity than substitution of Lys 5 (31). The binding loop template used for those studies was essentially a truncated SFTI-1 molecule in which the secondary loop is omitted, as illustrated in Fig.…”

“…This result is in contrast to previous studies on truncated derivatives that only contain the binding loop of SFTI-1 (27,31) and demonstrates that the secondary loop is a crucial stabilizing factor in SFTI-1. In particular, Asp 14 from the secondary loop is a key residue in maintaining the well defined native structure.…”

Interactions between trypsin and its peptidic inhibitors studied by isothermal titration calorimetry (ITC)

“…SFTI-1 is a backbone-cyclized peptide composed of 14 amino acids and bisected by one disulphide bridge into binding and secondary loops. SFTI-1 belongs to the well-characterized Bowman-Birk family of natural inhibitors and displays high affinity towards trypsin (based on the independent colorimetric analyses, the association constant K a = 1.1 9 10 10 M -1 [6] and inhibition constant K i = 0.1 nM [5], 1 nM [7] and 13 nM [8]). Moreover, we showed that both native bicyclic SFTI-1 and its analogue deprived of the cyclic backbone, but having the disulphide bond, have comparable inhibitory activity [6].…”