Non-combinatorial library screening reveals subsite cooperativity and identifies new peptide substrates for human neutrophil elastase

D Leahy, X Chen, J Van Haeften and JM Harris

Queensland University of Technology, Institute of Health and Biomedical Innovation, Brisbane, QLD 4059, Australia

Human neutrophil elastase (HNE) has a well described bactericidal role and is also a key driver of the inflammation and parenchymal tissue damage that ultimately causes Chronic Obstructive Pulmonary Disease. To understand the enzyme's substrate selectivity and identify potential motifs for inhibitor design we synthesised a non-combinatorial (sparse matrix) library of tetra peptide substrates to screen against HNE. Sparse matrix libraries differ from the more common positional scanning approach in that they are able to discern positional enhancement or hindrance from the interaction of proximal amino acid sidechains in a given substrate. In particular we found a marked preference for Methionine sulfoxide as the second amino acid of the tetra peptide. This selectivity together with the profile's evident strong subsite cooperativity led us to carry out molecular dynamic simulation of preferred substrates in complex with HNE and identify the key elements of the HNE/substrate interface driving selectivity. Additionally, comparison of the most favoured substrates with known bacterial protein substrates of HNE revealed potential cleavage points and suggests a connection between the hypohalous acid and protease activities that macrophages use to destroy invading bacteria.