Development of a mechanism-based assay for tissue transglutaminase—results of a high-throughput screen and discovery of inhibitors
In: Analytical Biochemistry, Jg. 338 (2005-03-15), Heft 2, S. 237-244
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Zugriff:
Abstract: Tissue transglutaminase (TGase) is a Ca2+-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-CH2-CH2-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 Ca2+ 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. [Copyright &y& Elsevier]
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Development of a mechanism-based assay for tissue transglutaminase—results of a high-throughput screen and discovery of inhibitors
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Autor/in / Beteiligte Person: | Case, April ; Ni, Jake ; Yeh, Li-An ; Stein, Ross L. |
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Zeitschrift: | Analytical Biochemistry, Jg. 338 (2005-03-15), Heft 2, S. 237-244 |
Veröffentlichung: | 2005 |
Medientyp: | academicJournal |
ISSN: | 0003-2697 (print) |
DOI: | 10.1016/j.ab.2004.09.047 |
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