Establishment of a cell-based drug screening system for identifying selective down-regulators of mPGES-1

Abstract.

Objective: To establish a drug screening system based on transcriptional regulation of microsomal PGE₂ synthase 1 (mPGES-1), cyclooxygenase 1 (COX-1) and 2 (COX-2) for discovering selective down-regulators of mPGES-1.

Methods: The upstream regulatory sequences of mPGES-1, COX-1, COX-2 were respectively cloned into pGL3B-neo vector containing luciferase gene and neomycin resistance gene (the pGL3B-neo vector had been previously constructed by cloning the neomycin resistance gene into the Sal I site of the pGL3-Basic vector). After that, the recombinant reporter gene vectors pGL3B-neo/mPGES-1, pGL3B-neo/COX-1, pGL3B-neo/COX-2 were respectively transfected into A549 cells and therefore stable cell lines, namely M 1, M 2 and M 3, were obtained. Samples were detected then by testing luciferase activity of M 1, M 2 and M 3 cells in microtiter wells to identify compounds that can selectively down-regulate mPGES-1 expression. Through luciferase activity testing, the compounds which had more than 40 % inhibition ratio on M 1 and less than 20 % inhibition ratio on M 2 and M 3 cells could be regarded as hits.

Results: Using the cell-based reporter gene assay, we screened compounds for selectively down-regulation of mPGES-1 expression and several compounds were discovered.

Conclusion: A cell-based drug screening system was established to screen selective down-regulators of mPGES-1 expression, and compound CM188 was identified, which might become a lead compound for novel anti-arthritic drugs.