Transglutaminases (TGs) are enzymes that catalyze post-translational modifications of glutamine residues in proteins, via the formation of isopeptide bonds, esterification and deamination. The reaction produces insoluble macromolecular complexes and protein aggregates. Within the TG family of enzyme, tissue-specific Transglutaminase-2 (TG2) expression is proven to be a clinical target for multiple types of cancer and neurodegenerative diseases, including Alzheimer's and Huntington's diseases. Inhibition of TG2, with a Cystamine-derived drug candidate (currently in phase-II clinical trial for Huntington's disease), has shown to have neuroprotective effects in models of neurodegeneration. Further, TG2 interacts with retinoblastoma protein (Rb), GTPase, and protein kinases through serine/threonine phosphorylation in cancer. Therefore, TG2 is a clinical validated candidate in these pathological diseases, and is a highly relevant therapeutic biomarker. Assay Genie's Transglutaminase Inhibitor Screening Assay Kit utilizes the catalytic activity of TG2, in a deamidation reaction between donor and acceptor substrates, to generate a hydroxamate product. The stop solution reacts with the hydroxamate product, forming a purple complex that can be measured at 525 nm. In the presence of TG2 inhibitor, the enzyme loses its ability to catalyze the deamidation reaction, leading to a decrease in color development and a lower absorbance OD reading. This assay kit is simple and can be used to identify and characterize TG2 inhibitors in a high-throughput format.

Figure: Inhibition of TG2 Enzyme activity by Cystamine. Cystamine was pre-incubated with TG2 enzyme for 15 minutes at 37°C. Substrate was added and incubated for 2 hours at 37°C. Assays are performed following the kit protocol. IC 50 of Cystamine was determined to be 37.5 ± 2.6 µM. Assays are performed following the kit protocol.