Optimization of fluorogenic CatS substrates for monitoring cancer-associated proteolytic activity

Cancer represents a major global health, social, and economic burden. In many regions of the world, cancer is the second leading cause of death after cardiovascular diseases. Using the "mix and split" method of combinatorial chemistry, we performed cathepsin S (CatS) substrate selectivity profiling at non-primed and primed positions. We performed the deconvolution of peptide libraries by iterative method. Enzymatic activity was determined by measuring absorbance or fluorescence. A fluorogenic substrate for monitoring CatS-related proteolytic activity ABZ-Lys-Phe-Met-Arg-Leu-Leu-Phe-Tyr (3-NO₂)-NH₂ was optimized. It was used to determine enzyme activity in urine samples from patients with confirmed different types of cancers (n = 19). No detectable proteolytic activity above background levels was observed in urine samples collected from healthy volunteers. The obtained results suggest that elevated CatS-related proteolytic activity may be detected in urine samples collected from patients with selected cancers. However, the present study was not designed to investigate the mechanistic role of CatS in carcinogenesis, but rather to demonstrate that such activity can be measured in biological samples using the optimized substrate. The observed proteolytic activity in urine samples may result from the presence of CatS, among other enzymes, as well as other enzymes that are directly related to the type of disease. We obtained a selective substrate for monitoring CatS-related proteolytic activity that can be applied in studies using biological samples. The presented results constitute a preliminary proof-of-concept and indicate the potential usefulness of urine-based enzymatic assays in future cancer biomarker studies. Further validation on larger and clinically characterized cohorts is required.