Development of pathway-oriented screening to identify compounds to control 2-methylglyoxal metabolism in tumor cells.

Controlling tumor-specific alterations in metabolic pathways is a useful strategy for treating tumors. The glyoxalase pathway, which metabolizes the toxic electrophile 2-methylglyoxal (MG), is thought to contribute to tumor pathology. We developed a live cell-based high-throughput screening system that monitors the metabolism of MG to generate D-lactate by glyoxalase I and II (GLO1 and GLO2). It utilizes an extracellular coupled assay that uses D-lactate to generate NAD(P)H, which is detected by a selective fluorogenic probe designed to respond exclusively to extracellular NAD(P)H. This metabolic pathway-oriented screening is able to identify compounds that control MG metabolism in live cells, and we have discovered compounds that can directly or indirectly inhibit glyoxalase activities in small cell lung carcinoma cells.

Establishment of live-cell-based coupled assay system for identification of compounds to modulate metabolic activities of cells.

In this study, we present a live-cell-based fluorometric coupled assay system to identify the compounds that can regulate the targeted metabolic pathways in live cells. The assay is established through targeting specific metabolic pathways and using "input" and "output" metabolite pairs. The changes in the extracellular output that are generated and released into the extracellular media from the input are assessed as the activity of the pathway. The screening for the glycolytic pathway and amino acid metabolism reveals the activities of the present drugs, 6-BIO and regorafenib, that regulate the metabolic fate of tumor cells.

Metabolic-Pathway-Oriented Screening Targeting S-Adenosyl-l-methionine Reveals the Epigenetic Remodeling Activities of Naturally Occurring Catechols.

Methyl transfer reactions play important roles in many biological phenomena, wherein the methylation cofactor S-adenosyl-l-methionine (SAM) serves as the important currency to orchestrate those reactions. We have developed a fluorescent-probe-based high-throughput screening (HTS) system to search for the compounds that control cellular SAM levels. HTS with a drug repositioning library revealed the importance of catechol-O-methyltransferase (COMT) and its substrates in controlling the SAM concentrations and histone methylation levels in colorectal tumor cells.

Development of Chemical Tools to Monitor and Control Isoaspartyl Peptide Methyltransferase Activity.

We have established a coupled assay system targeting protein l-isoaspartyl methyltransferase (PIMT), a key enzyme in the metabolism of isoaspartyl peptides and proteins. The system utilizes a fluorogenic peptide probe containing an isoaspartyl residue at the P1' position of the caspase-3 recognition sequence. Following PIMT-catalyzed methyl transfer reaction, the methylated probe is specifically cleaved by caspase-3 to give fluorescence activation. High-throughput screening of our chemical library with this assay system identified PIMT inhibitors that may be useful as leads in the design of chemical probes for controlling PIMT activity.