The SFTI-1 is a natural 14-residue cyclic peptide whose structure is further stabilized by a disulfide bridge and the reactive site loop resembles those of the Bowman-Birk protease inhibitors. Substitution of Lys-5 in SFTI-1 by some amino acids or N-substituted glycine residues has been described as an effective means to generate serine protease inhibitors of novel specificities. Thus, we undertook the design and synthesis of SFTI-1 analogues as inhibitors with improved specificity and selectivity for targeting two proteases: an Ang II-forming elastase-2 and a  C. durissus venom thrombin-like enzyme. The SFTI-1 analogues were synthesized by microwave-assisted solid phase peptide synthesis using standard Fmoc protocols, and the disulfide bridge formation was carried out in DMSO/acetic acid solution. The concentration and purity of each inhibitor isolated by reversed phase HPLC were assessed by amino acid analysis following acid hydrolysis. Trypsin, chymotrypsin, rat elastase-2, and thrombin-like enzyme were assayed with commercially available chromogenic substrates in the presence or absence of 0.1 然 and 1.0 然 of each inhibitor. Trypsin was fully inhibited by 0.1 然 of Arg-5 SFTI-1 and 88% by 0.1 然 of the SFTI-1 itself. Chymotrypsin was inhibited by 0.1 然 of Phe-5 SFTI-1 (100%) and Leu-5 SFTI-1 (65%). Purified thrombin-like enzyme, a protease with primary specificity directed to Arg residues, was refractory to the action of all SFTI-1 analogues tested, including the Arg-5 SFTI-1 and Lys-5 SFTI-1. These results corroborate previous findings describing the inefficacy of some plant trypsin inhibitors against snake venom coagulating enzymes. Rat elastase-2 activity was partially blocked upon incubation in the presence of  0.1 然 of three of the inhibitors tested: Phe-5 SFTI-1 (71%), Leu-5 SFTI-1 (40%) and Lys-5 SFTI-1 (20%). Although these results indicate that the SFTI-1 analogues are the best competitive  inhibitors for the elastase-2 described so far, the observed lack of selectivity may prevent their use as tools in pharmacological experiments aiming at unambiguous discrimination between elastase-2 and chymase, two of the Ang II-forming enzymes in the rat vasculature.

CAPES, CNPq, FAPESP