Application of Intramolecular O-to-N Phosphoryl Transfer Reaction to Design Fluorogenic Probes to Detect Activities of Enzymes That Metabolize Short Peptides and Acylamino Acids.

We propose the design strategy of fluorogenic probes of proteases/peptidases and acylamino acid hydrolases utilizing an intramolecular O-to-N phosphoryl transfer reaction, in which the main chain of peptides or amino acids is retained from the natural substrate but the side chain was designed to attach the fluorophore. The strategy is useful to design fluorogenic probes for peptidases/proteases that do not prefer the main chain modification and acylamino acid hydrolases. We have developed the fluorogenic substrates for GGT5, GGCT, and PM20D1 and have performed the screening of PM20D1 inhibitors/activators to characterize the compounds that modify the activity of PM20D1.

Single-Molecule Oxidoreductase Activity Analysis for Activity-Based Diagnosis Based on Proteoform Alterations.

We developed a single-molecule enzyme activity assay platform for NAD(P)-dependent oxidoreductases, leveraging a new NAD(P)H-responsive fluorogenic probe optimized for microdevice-based fluorometric detection. This platform enabled the detection of enzyme activities in blood and cerebrospinal fluid (CSF), including lactate dehydrogenase, glucose-6-phosphate dehydrogenase, and hexokinases. We demonstrate its potential for activity-based diagnosis by detecting altered populations of enzyme activity species in blood and CSF from liver damage in brain tumor patients.

Identification of activity-based biomarkers for early-stage pancreatic tumors in blood using single-molecule enzyme activity screening.

Single-molecule enzyme activity-based enzyme profiling (SEAP) is a methodology to globally analyze protein functions in living samples at the single-molecule level. It has been previously applied to detect functional alterations in phosphatases and glycosidases. Here, we expand the potential for activity-based biomarker discovery by developing a semi-automated synthesis platform for fluorogenic probes that can detect various peptidases and protease activities at the single-molecule level.

Thioester-Based Coupled Fluorogenic Assays in Microdevice for the Detection of Single-Molecule Enzyme Activities of Esterases with Specified Substrate Recognition.

Single-molecule enzyme activity assay is a platform that enables the analysis of enzyme activities at single proteoform level. The limitation of the targetable enzymes is the major drawback of the assay, but the general assay platform is reported to study single-molecule enzyme activities of esterases based on the coupled assay using thioesters as substrate analogues. The coupled assay is realized by developing highly water-soluble thiol-reacting probes based on phosphonate-substituted boron dipyrromethene (BODIPY).