title: Mono-sulfonated tetrazolium salt based NAD(P)H detection reagents suitable for dehydrogenase and real-time cell viability assays.
abstract: Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and is important for several biological processes. For GDH inhibitor screening, we developed a novel mono-sulfonated tetrazolium salt (EZMTT), which can be synthesized using H2O2 oxidation and purified easily on silica gel in large quantities. The EZMTT detection method showed linear dose responses to NAD(P)H, dehydrogenase concentration and cell numbers. In E. coli GDH assay, the EZMTT method showed excellent assay reproducibility with a Z factor of 0.9 and caused no false positives in the presence of antioxidants (such as BME). Using the EZMTT-formazan-NAD(P)H system, we showed that EGCG is a potent E. coli GDH inhibitor (IC50 45 nM) and identified that Ebselen, a multifunctional thioredoxin reductase inhibitor, inactivated E. coli GDH (IC50 213 nM). In cell-based assays at 0.5 mM tetrazolium concentration, EZMTT showed essentially no toxicity after a 3-day incubation, whereas 40% of inhibition was observed for WST-8. In conclusion, EZMTT is a novel tetrazolium salt which provides improved features that are suitable for dehydrogenases and real-time cell-based high-throughput screening (HTS).
authors.affiliation: College of Pharmaceutical Science, Collaborative Innovation Center of Yangtza River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou, China; Department of Urology, Zhejiang Cancer Hospital, Hangzhou, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: College of Pharmaceutical Science, Collaborative Innovation Center of Yangtza River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou, China.
authors.forename: Min
authors.initials: M
authors.lastname: Zhu
authors.affiliation: College of Pharmaceutical Science, Collaborative Innovation Center of Yangtza River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.affiliation: College of Pharmaceutical Science, Collaborative Innovation Center of Yangtza River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou, China.
authors.forename: Danhui
authors.initials: D
authors.lastname: Cao
authors.affiliation: Hangzhou Jennifer Biotech. Inc., Hangzhou, China.
authors.forename: Jennifer Jin
authors.initials: JJ
authors.lastname: Ruan
authors.affiliation: College of Pharmaceutical Science, Collaborative Innovation Center of Yangtza River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou, China.
authors.forename: Weike
authors.initials: W
authors.lastname: Su
authors.affiliation: College of Pharmaceutical Science, Collaborative Innovation Center of Yangtza River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou, China. Electronic address: ruanbf@zjut.edu.cn.
authors.forename: Benfang Helen
authors.initials: BH
authors.lastname: Ruan
mesh_terms: D000072283:A549 Cells; D002470:Cell Survival; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D013778:Tetrazolium Salts / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology
chemical_list: D029968:Escherichia coli Proteins; D013778:Tetrazolium Salts; D009249:NADP; D005969:Glutamate Dehydrogenase
keywords: Cell proliferation; Cell toxicity; Dehydrogenase; High through-put screening; Mono-sulfonated tetrazolium salt; NAD(P)H assay