High-Precision, Room Temperature Screening Assay for Inhibitors of Microsomal Prostaglandin E Synthase-1

被引：2

作者：

Andersson, Susanne ^{[1
]}

Norman, Mattias ^{[1
]}

Olsson, Rolf ^{[1
]}

Smith, Robin ^{[1
]}

Liu, Gang ^{[1
]}

Nord, Johan ^{[1
]}

机构：

[1] AstraZeneca, CNS Pain iMED, Dept Neurosci, S-15185 Sodertalje, Sweden

来源：

JOURNAL OF BIOMOLECULAR SCREENING | 2012年 / 17卷 / 10期

关键词：

automation or robotics; liquid handling; enzyme assays or enzyme kinetics; fluorescence methods; inflammation; autoimmune diseases; INDUCIBLE ENZYME; IDENTIFICATION;

D O I：

10.1177/1087057112456424

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Microsomal prostaglandin E synthase-1 (mPGES-1) is the major enzyme catalyzing the isomerization of prostaglandin (PG) H 2 to PGE(2). Here we report the development of a robust and practical automated assay in a 384-well format for room temperature screening of mPGES-1 inhibitors with high precision and low reagent consumption. The assay should enable precise structure-activity relationship development. It uses acetonitrile as solvent for PGH(2), FeCl2/citrate as stop reagent, and a short reaction time. Combined with high-precision liquid transfer and extensive mixing after addition of reactants, these properties let the assay reach Z' > 0.7 and high reproducibility of inhibitor IC50 values. Thorough investigation of the quality of mixing in all liquid transfer steps proved crucial for reaching high-precision performance.

引用

页码：1372 / 1378

页数：7

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