Development of fluorogenic substrates for colorectal tumor-related neuropeptidases for activity-based diagnosis.

The M3 metalloproteases, neurolysin and THOP1, are neuropeptidases that are expressed in various tissues and metabolize neuropeptides, such as neurotensin. The biological roles of these enzymes are not well characterized, partially because the chemical tools to analyse their activities are not well developed. Here, we developed a fluorogenic substrate probe for neurolysin and thimet oligopeptidase 1 (THOP1), which enabled the analysis of enzymatic activity changes in tissue and plasma samples. In particular, the probe was useful for studying enzyme activities in a single-molecule enzyme assay platform, which can detect enzyme activity with high sensitivity. We detected the activity of neurolysin in plasma samples and revealed higher enzyme activity in the blood samples of patients with colorectal tumor. The result indicated that single-molecule neurolysin activity is a promising candidate for a blood biomarker for colorectal cancer diagnosis.

Development of a Novel Intraocular-Pressure-Lowering Therapy Targeting ATX.

Elevated intraocular pressure (IOP) is the major cause of glaucoma, which is the second leading cause of blindness. However, current glaucoma treatments cannot completely regulate IOP and progression of glaucoma. Our group recently found that autotaxin (ATX) activity in human aqueous humor (AH) was positively correlated with increased IOP in various subtypes of glaucoma. To develop new IOP-lowering treatments, we generated a novel ATX inhibitor as an ophthalmic drug by high-throughput screening, followed by inhibitor optimization. Administration of the optimized ATX inhibitor (Aiprenon) reduced IOP in laser-treated mice exhibiting elevated IOP and higher level of ATX activity in AH and normal mice in vivo. The stimulation of ATX induced outflow resistance in the trabecular pathway; however, administration of Aiprenon recovered the outflow resistance in vitro. The in vitro experiments implied that the IOP-lowering effect of Aiprenon could be correlated with the altered cellular behavior of trabecular meshwork (TM) and Schlemm's canal endothelial (SC) cells. Overall, our findings showed that ATX had major impact in regulating IOP as a target molecule, and potent ATX inhibitors such as Aiprenon could be a promising therapeutic approach for lowering IOP.