Identification of a small-molecule inhibitor of nardilysin and its pharmacological effects on transcriptional regulation and inflammatory arthritis.

Nardilysin (NRDC) is a zinc metallopeptidase of the M16 family that participates in diverse biological processes, including ectodomain shedding and antigen processing. NRDC also functions as a transcriptional coregulator, and this coregulatory activity depends on its metallopeptidase catalytic function. However, small-molecule inhibitors targeting NRDC have not been well characterized, limiting pharmacological investigation of its enzymatic function. Here we established a fluorescence-quenched peptide substrate assay suitable for high-throughput screening of NRDC protease activity. Using this assay, we screened 33,305 compounds and identified 140 candidate NRDC inhibitors. Through secondary screening, orthogonal validation and structure-activity relationship analysis, three inhibitor scaffolds were identified, from which compound R5-1 was selected for further characterization. Enzyme kinetic analysis demonstrated that R5-1 inhibits NRDC activity in a non-competitive manner, indicating reduced catalytic turnover rather than directly competing with substrate binding. Structural modeling and docking analysis further suggested that R5-1 binds to a distal allosteric site in NRDC. Pharmacological inhibition of NRDC with R5-1 attenuated NRDC-dependent transcriptional repression of PGC-1α, phenocopying the effect of catalytic inactivation. Furthermore, administration of R5-1 significantly ameliorated arthritis severity in a mouse model of autoimmune arthritis. Together, these findings identify R5-1 as a small-molecule inhibitor of NRDC and provide mechanistic insights linking NRDC protease activity to transcriptional regulation and inflammatory disease.