Peptides isolated from the Cucurbitaceae Ecballium elaterium (squirting cucumber) were found to be very potent inhibitors of trypsin (Kd = 10-12 M). Among them, EETI II (Ecballium elaterium trypsin inhibitor II) is the most interesting one: it is a 28-residue peptide containing 6 cysteines engaged in 3 disulfide bridge. Its primary structure was determined and its total synthesis achieved, allowing a thorough study of this molecule which belongs to the newly discovered family of serine protease inhibitors isolated from Cucurbitaceae. The solid-phase synthesis of EETI II was achieved in very satisfactory yields, and modifications of its active site led to analogs capable of inhibiting α-chymotrypsin or elastase. It there- dimensional structure was determined by 2D NMR, distance geometry calculations and X-ray crystallography of its complex with porcine trypsin. From these studies, the disulfide bond connectivities were found to correspond to those suggested by amino acid sequencing. Molecular modeling of the peptide and comparison with other compounds with similar molecular topology, especially with CPI, a carboxypeptidase inhibitor found in potato leaves, led to the design and synthesis of a chimaeric molecule bearing inhibitory properties against trypsin, a serine protease, and carboxypeptidase A, a zinc-protease. Conformational studies of the major intermediate accumulating during the folding process showed that it contains 2 native disulfide bridges and leads directly to the naturel compound, unlike BPTI and CPI which fold through several non-native two-disulfide intermediates.
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