In Silico and in Vitro Selection Method of Peptide Substrates for Protease Selectivity

Matrix metalloproteinases (MMPs) are central to extracellular matrix remodeling, and their upregulation is involved in numerous pathologies such as wound chronicity, carcinomas, or osteoarthritis. Sensing and monitoring MMP activity or exploiting their hydrolytic action as a trigger for targeted drug delivery represents promising avenues for innovative biomedical applications. Herein, we present a novel methodology to identify MMP-13-cleavable peptides that are optimized for both affinity and selectivity. To this aim, we developed a software named "CleavInsight" to generate a library of optimized peptide substrates considering complex biological environments alongside a "Competitive Substrate Fluorescence Assay" (CSFA). CSFA is a time-saving enzymatic assay that determines the affinity and selectivity of the synthesized peptides. We applied our methodology to osteoarthritic synovial fluid as a representative biological medium. The 24-candidate peptide sequences generated by the software were synthesized and tested in the CSFA with MMP-13. From this initial set, IC₅₀ values were calculated for the 8 peptides with the highest affinity, and the peptides were also screened for selectivity against MMP-9. The combination of CleavInsight software with the time-efficient CSFA constitutes a reliable toolkit for developing protease-responsive systems.