Macrocyclic peptides MCP-3 and MCP-4 potently and selectively inhibit human neutrophil serine protease 4

Neutrophil serine protease 4 (NSP4) is a unique and ancient member of the neutrophil serine protease family, distinguished by its strong preference for arginine cleavage. While highly expressed in myeloid precursor cells and involved in regulating histamine levels during mast cell maturation, its specific function within neutrophil biology remains largely unknown. Current understanding of NSP4 is limited by the lack of potent and selective tools, hindering investigations into its biological roles in various inflammatory and immune processes. Developing such inhibitors is crucial for elucidating NSP4's precise contributions to health and disease.

Researchers employed large mRNA-displayed libraries, encoding up to 10^13 unique macrocyclic peptides (MCPs) containing both natural and non-natural amino acids, to screen for inhibitors of human NSP4. The screening process aimed to identify compounds that could strongly inhibit recombinant and endogenous cell-derived NSP4 activity while maintaining high selectivity. Identified MCPs were then subjected to biochemical studies using activity-based probes and structural analysis. Crystal structures of the most potent inhibitors, MCP-3 and MCP-4, were determined to elucidate their binding mechanisms within the NSP4 active site.

Library screening successfully yielded six macrocyclic peptides that potently inhibited both recombinant and endogenous cell-derived NSP4 activity. Crucially, these MCPs demonstrated exceptional selectivity, showing no inhibitory effect against 13 structurally related proteases. > The two most potent inhibitors, MCP-3 and MCP-4, exhibited impressive binding affinities with KD values of 6.5 nM and 1.0 nM, respectively. Crystal structures confirmed that MCP-3 and MCP-4 bind directly to the NSP4 active site region, consistent with biochemical findings. Remarkably, these de novo generated MCPs mirrored the ring conformation, substrate-like interaction, and resistance to hydrolysis observed in natural serine protease inhibitors like the sunflower trypsin inhibitor-1, indicating a conserved inhibitory mechanism.