Discovery of new neutrophil elastase inhibitors through Meloidae genome and transcriptome analyses

Neutrophil elastase (NE), a serine protease secreted by activated neutrophils, is a key regulator of inflammation and tissue damage. Among natural NE regulators, Kunitz-type serine protease inhibitors have attracted considerable attention for their therapeutic potential. In this study, integrated transcriptomic and genomic analyses of the blister beetles Lydus trimaculatus and Mylabris variabilis, led to the identification of four novel Kunitz-type inhibitors, Lyd_37798, Myl_35212i1, Myl_17096, and Myl_35212i2 with Ki values against NE of 32.36 nM, 76.45 nM, 154.5 nM, and 754.3 nM, respectively. While all peptides show a conserved Kunitz scaffold, they differ in their P1 residues, suggesting functional diversity. Notably, the most potent inhibitor, Lyd_37798, displays an uncommon aspartic acid at the P1 position, proposing new insights for the design of elastase-target drugs. Structural modelling demonstrated that these peptides bind NE with a reactive loop closely resembling that of the known elastase-specific inhibitor Elafin. Functionally, Lyd_37798 (P1 Asp) and Myl_17096 (P1 Lys) significantly suppressed the formation of neutrophil extracellular traps (NETs), reducing NET release by 89.2 % and 86.9 % respectively. Both peptides also revealed high specificity for NE, without affecting a broad spectrum of cellular ion channels, suggesting a favourable safety profile. Overall, this study highlights the unexploited potential of insect-derived Kunitz peptides as a valuable source of selective NE inhibitors and lays the foundation for their development as therapeutic drugs against NE-mediated inflammatory and degenerative diseases.