Background and Purpose Proteolytic balance is dysregulated in many diseases, with proteases playing critical roles in pathological pathways. A high level of Trypsin-3 expression has been implicated as a significant mediator of tumour progression and metastasis and this protease is associated with poor prognosis for patient in various cancers. Therefore, Trypsin-3 inhibition has emerged as a promising therapeutic target. However, no physiological or pharmacological inhibitor has yet been described that specifically target Trypsin-3. A major challenge in developing druggable inhibitor for this protease lies in achieving enough selectivity, as proteases belong to a large enzymatic family with close homologues that share similarities in their three-dimensional folding of their active conformation. Experimental Approach An advanced screening strategy of a large library of synthetic humanized nanobodies was employed to isolate highly selective recombinant antibodies targeting the active conformation of Trypsin-3. Among five hits, we combined two domains with distinct paratopes and inhibitory mechanisms to generate a macrodrug candidate capable to efficiently block the Trypsin-3 activity. Key Results This bispecific nanobody demonstrated exceptionally high selectivity and affinity towards Trypsin-3 in vitro, as well as a strong ability to inhibit cancer cell migration ex vivo on PC-3 cancer cell line. Conclusion and Implications. This study underscores the versatility and potential of synthetic nanobody engineering in the development of very selective protease inhibitors, paving the way for their consideration as drug candidates for clinical development.

Background and purpose Chymotrypsin is a serine protease produced by the pancreas and secreted into the lumen of the small intestine, where it digests food proteins. Due to its presence in the gut lumen, we hypothesized that chymotrypsin activity may be found close to epithelial cells and signals to them via Protease-activated receptors (PARs). We deciphered molecular pharmacology mechanisms for chymotrypsin signaling in intestinal epithelial cells. Experimental approaches The presence and activity of chymotrypsin were evaluated by western blot (WB) and enzymatic activity tests in luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally-tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. To study the pharmacology of chymotrypsin signals, we investigated calcium signaling and ERK1/2 activation using calcium mobilization assays and WB in CMT93 intestinal epithelial cells. Key results We found that chymotrypsin was present and active in the vicinity of the murine and human colonic epithelium. Molecular pharmacology studies evidenced that chymotrypsin cleaved both PARs receptors. While chymotrypsin activated calcium and ERK1/2 signaling pathways through PAR2, it disarmed PAR1, preventing further activation by its canonical agonist thrombin. CONCLUSION Our work suggests that the function of chymotrypsin in the gut lumen goes well beyond a simple digestive role. Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.