title: Development of a mechanism-based assay for tissue transglutaminase--results of a high-throughput screen and discovery of inhibitors.
abstract: Tissue transglutaminase (TGase) is a Ca(2+)-dependent enzyme that catalyzes cross-linking of intracellular proteins through a mechanism that involves isopeptide bond formation between Gln and Lys residues. In addition to its transamidation activity, TGase can bind guanosine 5'-triphosphate (GTP) and does so in a manner that is antagonized by calcium. Once bound, GTP undergoes hydrolysis to form guanosine 5'-diphosphate and inorganic phosphate. TGase is thought to play a pathogenic role in neurodegenerative diseases by promoting aggregation of disease-specific proteins that accumulate in these disorders. Thus, this enzyme represents a viable target for drug discovery. We now report the development of a mechanism-based assay for TGase and the results of a screen using this assay in which we tested 56,500 drug-like molecules for their ability to inhibit TGase. In this assay, the Gln- and Lys-donating substrates are N,N-dimethylated casein (NMC) and N-Boc-Lys-NH-CH(2)-CH(2)-NH-dansyl (KXD), respectively. Through a combination of steady state kinetic experiments and reaction progress curve simulations, we were able to calculate values for the initial concentrations of NMC, KXD, and Ca(2+) that would produce a steady state situation in which all thermodynamically significant forms of substrate-bound TGase exist in equal concentration. Under these conditions, the assay is sensitive to both competitive and mixed active-site inhibitors and to inhibitors that bind to the GTP site. The assay was optimized for automated screening in 384-well format and was then used to test our compound library. From among these compounds, 104 authentic hits that represent several mechanistic classes were identified.
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard Center for Neurodegeneration and Repair, Cambridge, MA 02139, USA.
authors.forename: April
authors.initials: A
authors.lastname: Case
authors.forename: Jake
authors.initials: J
authors.lastname: Ni
authors.forename: Li-An
authors.initials: LA
authors.lastname: Yeh
authors.forename: Ross L
authors.initials: RL
authors.lastname: Stein
mesh_terms: D002364:Caseins / Q000737:chemistry; D003619:Dansyl Compounds / Q000737:chemistry; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors*; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases / Q000037:antagonists & inhibitors*
chemical_list: D002364:Caseins; D003619:Dansyl Compounds; D004791:Enzyme Inhibitors; C002087:N,N-dimethylcasein; C040741:epsilon-tert-butyloxycarbonyl-lysine; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins; D008239:Lysine