Synthesis of prostaglandin F ethanolamide by prostaglandin F synthase and identification of Bimatoprost as a potent inhibitor of the enzyme: new enzyme assay method using LC/ESI/MS

Prostaglandin (PG) D2 ethanolamide (prostamide D2) was reduced to 9α,11β-PGF2 ethanolamide (9α,11β-prostamide F2) by PGF synthase, which also catalyzes the reduction of PGH2 and PGD2 to PGF2α and 9α,11β-PGF2, respectively. These enzyme activities were measured by a new method, the liquid chromatographic–electrospray ionization–mass spectrometry (LC/ESI/MS) technique, which could simultaneously detect the substrate and all products. PGF2α, 9α,11β-PGF2, PGD2, PGH2, 9α,11β-prostamide F2, and prostamide D2 were separated on a TSKgel ODS 80Ts column, ionized by electrospray, and detected in the negative mode. Selected ion monitoring (SIM) of m/z 353 ([M–H]−), 353 ([M–H]−), 351 ([M–H]−), 333 ([M–H–H2O]−), 456 ([M+59]−), and m/z 358 ([M−37]−) was used for quantifying PGF2α, 9α,11β-PGF2, PGD2, PGH2, 9α,11β-prostamide F2, and prostamide D2, respectively. The detection limit for PGF2α and 9α,11β-PGF2 was 0.01 pmol; that for PGH2 and PGD2, 0.1 pmol; and that for prostamide D2 and 9α,11β-prostamide F2, 0.5 and 0.03 pmol, respectively. The LC/ESI/MS technique for measuring PGF synthase activity showed higher sensitivity than other methods. Using this method, we found that Bimatoprost, the ethyl amide analog of 17-phenyl-trinor PGF2α and an anti-glaucoma agent, inhibited all three reductase activities of PGF synthase when used at a low concentration. These results suggest that Bimatoprost also behaves as a potent PGF synthase inhibitor in addition to having prostamide-like activity.