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Inhibitor screening assay of microsomal prostaglandin E2 synthase-1 (mPGES-1) using coupled enzyme assay system and mammalian cell assay

2009년 8월 14일 14시 37분 05초
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목 <발표Ⅱ>
저자 및
최경아, 박성준, 유연규
국민대학교 생명나노화학과, Korea
mPGES-1 catalyzes the conversion of PGH2 into PGE2, which induced inflammatory response, fever, and pain. To establish an assay method of searching inhibitor of mPGES-1, a coupled enzyme assay system using mPGES-1 and 15-PGDH has been developed. In this assay, PGH2 was converted to PGE2 by mPGES-1 and then PGE2 was transformed to the 15-keto-PGE2 by 15-PGDH accompanying the production of NADH. During the reaction, spontaneous decomposition of PGH2 was prevented by PMA. To test this assay, the Km value of substrate and the IC50 value of MK-886 measured (150 µM and 3.4 µM, respectively) and then the result is close to both Km value and IC50 value as previously reported. To test the utility of this assay, the 160 compounds were assayed and Z´ factor that reflects both the assay signal dynamic range and data variation was determined. We demonstrated that the coupled enzyme assay system has a high Z’ value (0.86 ± 0.03), which is appropriate for a HTS assays. Using the assay system we have screened a fragment chemical library from Korea Chemical Bank and identified a few compounds that inhibit mPGES-1 activity. The inhibitory activity of these compounds was further confirmed by a cell-based assay method using A549 cells.