Selection of Chymotrypsin Inhibitors from a Conformationally-constrained Combinatorial Peptide Library

Abstract

A synthetic library of cyclic peptides was constructed utilizing the anti-tryptic loop region of the Bowman-Birk inhibitor, D4 from Macrotyloma axillare, as a template. The loop region of this proteinase inhibitor was reproduced by an 11 residue sequence, conformationally constrained by the presence of a disulfide bridge, to act as a mimetic of the functional reactive site region of this protein. This sequence, plus a pentaglycine spacer arm, was used to create a “one bead, one peptide” combinatorial library after on-resin deprotection and cyclization. Randomization at three positions considered to be important for proteinase specificity (P 2, P 1and P′ 2) with the genetically coded amino acids (minus cysteine) plus norleucine generated 8000 permutations. Screening this library with biotinylated α-chymotrypsin under appropriate con ditions revealed a small number (<0.05%) of beads that selectively bound the labeled proteinase. The sequences present on these active beads were determined, and found to have a well-defined consensus. Analysis of chymotrypsin inhibition in solution using re-synthesized peptides reveals that the sequences identified are potent inhibitors with K ivalues in the nanomolar range. These results show that directed randomization of the canonical loop is a powerful way of generating proteinase inhibitors with targeted specificities. Incorporation of selective random changes within a defined structural framework is found to be an effective means of generating variation in large synthetic systems. The functional basis for inhibition by the identified sequences is discussed.