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

Arch Biochem Biophys. 2004 Apr 15;424(2):128-36. doi: 10.1016/j.abb.2004.02.009.

Abstract

Prostaglandin (PG) D(2) ethanolamide (prostamide D(2)) was reduced to 9alpha,11beta-PGF(2) ethanolamide (9alpha,11beta-prostamide F(2)) by PGF synthase, which also catalyzes the reduction of PGH(2) and PGD(2) to PGF(2alpha) and 9alpha,11beta-PGF(2), 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. PGF(2alpha), 9alpha,11beta-PGF(2), PGD(2), PGH(2), 9alpha,11beta-prostamide F(2), and prostamide D(2) 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-H(2)O](-)), 456 ([M+59](-)), and m/z 358 ([M-37](-)) was used for quantifying PGF(2alpha), 9alpha,11beta-PGF(2), PGD(2), PGH(2), 9alpha,11beta-prostamide F(2), and prostamide D(2), respectively. The detection limit for PGF(2alpha) and 9alpha,11beta-PGF(2) was 0.01pmol; that for PGH(2) and PGD(2), 0.1pmol; and that for prostamide D(2) and 9alpha,11beta-prostamide F(2), 0.5 and 0.03pmol, 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 PGF(2alpha) 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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amides
  • Bimatoprost
  • Chromatography, Liquid / methods*
  • Cloprostenol / analogs & derivatives
  • Enzyme Inhibitors / chemistry
  • Enzyme Stability
  • Humans
  • Hydroxyprostaglandin Dehydrogenases / antagonists & inhibitors
  • Hydroxyprostaglandin Dehydrogenases / chemistry*
  • Lipids / chemistry*
  • Prostaglandin D2 / chemistry*
  • Prostaglandins F, Synthetic / chemical synthesis*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Restriction Mapping
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Substrate Specificity

Substances

  • Amides
  • Enzyme Inhibitors
  • Lipids
  • Prostaglandins F, Synthetic
  • Recombinant Proteins
  • Cloprostenol
  • Hydroxyprostaglandin Dehydrogenases
  • prostaglandin-F synthase
  • Bimatoprost
  • Prostaglandin D2