Expression and purification of human mPGES-1 in E. coli and identification of inhibitory compounds from a drug-library.
Human microsomal prostaglandin E synthase-1 (mPGES-1) is a membrane associated protein that catalyzes the conversion of prostaglandin H(2) (PGH(2)) into prostaglandin E(2) (PGE(2)). In this study, the expression of human mPGES-1 in E. coli was significantly enhanced by modifying the utility of specific codons and the recombinant mPGES-1 was efficiently purified to homogeneity. The K(m) and V(max) of the purified enzyme were determined and the trimeric state characterized by chemical cross-linking with glutaraldehyde. The purified mPGES-1 was used for the screening of a chemical library of bioactive or drug compounds to identify novel inhibitors, and oxacillin and dyphylline were identified as moderately inhibiting mPGES-1 with IC(50) values of 100 and 200 microM, respectively. As these compounds competitively inhibited the catalysis of PGH(2), their binding sites appeared to be located near the PGH2 binding pocket.
Kim
Woo-Il
WI
Department of Chemistry, Kookmin University, Seoul, Korea.
Choi
Kyung-A
KA
Do
Hyun-Soo
HS
Yu
Yeon Gyu
YG
D001667:Binding, Competitive; D004353:Drug Evaluation, Preclinical; D004400:Dyphylline / Q000493:pharmacokinetics; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D020128:Inhibitory Concentration 50; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010068:Oxacillin / Q000493:pharmacokinetics; D000071876:Prostaglandin-E Synthases; D054852:Small Molecule Libraries / Q000032:analysis*; D014169:Transformation, Bacterial
D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; D004400:Dyphylline; D019746:Intramolecular Oxidoreductases; C000605862:PTGES protein, human; D000071876:Prostaglandin-E Synthases; D010068:Oxacillin