Synthesis of a mixture of cyclic peptides based on the Bowman‐Birk reactive site loop to screen for serine protease inhibitors

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Abstract
A peptide mixture containing 21 peptide sequences has been constructed to test the Bowman-Birk inhibitor reactive-site loop motif as the basis of inhibition for a range of serine proteases. The 21 peptides are all based on an 11 amino acid sequence designed from a Bowman-Birk like inhibitor reactive-site loop. Variation has been introduced at the P1 site of the loop, which has been randomised to include all the natural l-amino acids (except for cysteine), plus the non-natural l-amino acids ornithine and norleucine. The mixture of peptides was screened for specific binding to immobilised porcine pancreatic elastase, subtilisin BPN′, α-chymotrypsin, trypsin, anhydro-α-chymotrypsin and anhydrotrypsin. Five peptides from the mixture bind to α-chymotrypsin, two of which also bind to anhydro-α-chymotrypsin, and two peptides bind trypsin, neither of which binds to anhydro-trypsin. The competitive inhibition constants (K1) and the rates of proteolytic hydrolysis of the individual peptides with their respective enzymes were determined. The rates of hydrolysis were found to vary widely and show little correlation with the K1 values. In the case of the α-chymotrypsin inhibitors, the peptides with the lowest Ki (0.1–0.05 mM) were the only peptides that bound to anhydro-α-chymotrypsin. However, no peptides bound to anhydrotrypsin, suggesting a fundamental difference in the way that α-chymotrypsin and trypsin are inhibited by these cyclic peptides.