title: Development of a high-throughput assay to measure histidine decarboxylase activity.
abstract: Histamine is a well-known mediator of allergic, inflammatory, and neurological responses. More recent studies suggest a role for histamine and its receptors in a wide range of biological processes, including T-cell maturation and bone remodeling. Histamine serum levels are regulated mainly by the activity of the histamine-synthesizing enzyme histidine decarboxylase (HDC). Despite the importance of this enzyme in many physiological processes, very few potent HDC inhibitors have been identified. HDC assays suitable for high-throughput screening have not been reported. The authors describe the development of a fluorescence polarization assay to measure HDC enzymatic activity. They used a fluorescein-histamine probe that binds with high affinity to an antihistamine antibody for detection. Importantly, they show that probe binding is fully competed by histamine, but no competition by the HDC substrate histidine was observed. The automated assay was performed in a total volume of 60 muL, had an assay window of 80 to 100 mP, and had a Z' factor of 0.6 to 0.7. This assay provides new tools to study HDC activity and pharmacological modulation of histamine levels.
authors.affiliation: Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, USA.
authors.forename: E Michael
authors.initials: EM
authors.lastname: August
authors.forename: Lori
authors.initials: L
authors.lastname: Patnaude
authors.forename: Jerry
authors.initials: J
authors.lastname: Hopkins
authors.forename: Joey
authors.initials: J
authors.lastname: Studts
authors.forename: Elda
authors.initials: E
authors.lastname: Gautschi
authors.forename: Anthony
authors.initials: A
authors.lastname: Shrutkowski
authors.forename: Anthony
authors.initials: A
authors.lastname: Kronkaitis
authors.forename: Martha
authors.initials: M
authors.lastname: Brown
authors.forename: Alisa
authors.initials: A
authors.lastname: Kabcenell
authors.forename: Daniel
authors.initials: D
authors.lastname: Rajotte
mesh_terms: D004791:Enzyme Inhibitors / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D006632:Histamine / Q000378:metabolism; D006633:Histamine Antagonists / Q000276:immunology; D006639:Histidine / Q000378:metabolism; D006640:Histidine Decarboxylase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D020128:Inhibitory Concentration 50; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D006633:Histamine Antagonists; D006639:Histidine; D006632:Histamine; D006640:Histidine Decarboxylase; D016650:Fluorescein-5-isothiocyanate