Discovery of potent inhibitors of human β-tryptase from pre-equilibrated dynamic combinatorial libraries

Pre-equilibrated dynamic combinatorial libraries based on acyl hydrazone interchange of peptide-derived hydrazides and di- and tri-aldehydes have been used to discover potent inhibitors with nanomolar affinities for β-tryptase. To identify potent inhibitors the activity of the full library containing 95 members was compared with those of sub-libraries in which individual building blocks were missing. The most active library members contain a rigid central aromatic scaffold with three cationic peptide arms. The arms of the best inhibitors also contained a tailor-made GCP oxoanion binding motif attached to a lysine side chain. The most potent tri-armed hydrazones with peptide arms GKWR or GKWK(GCP) were shown to inhibit β-tryptase (K_ica. 10-20 nM) reversibly, non-competitively and selectively (compared to related serine proteases, e.g. trypsin and chymotrypsin), most likely by binding to the protein surface, also in agreement with molecular modelling calculations. These new inhibitors are one order of magnitude more efficient than related tetravalent inhibitors obtained from previous work on a split-mix-combinatorial library and were identified with significantly less effort, demonstrating the usefulness of this approach for the identification of enzyme inhibitors in general.