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.