title: A fluorescent assay suitable for inhibitor screening and vanin tissue quantification.
abstract: Vanin-1 is a pantetheinase that catalyzes the hydrolysis of pantetheine to produce pantothenic acid (vitamin B5) and cysteamine. Reported here is a highly sensitive fluorescent assay using a novel fluorescently labeled pantothenate derivative. The assay has been used for characterization of a soluble version of human vanin-1 recombinant protein, identification and characterization of hits from high-throughput screening (HTS), and quantification of vanin pantothenase activity in cell lines and tissues. Under optimized assay conditions, we quantified vanin pantothenase activity in tissue lysate and found low activity in lung and liver but high activity in kidney. We demonstrated that the purified recombinant vanin-1 consisting of the extracellular portion without the glycosylphosphatidylinositol (GPI) linker was highly active with an apparent K(m) of 28 microM for pantothenate-7-amino-4-methylcoumarin (pantothenate-AMC), which was converted to pantothenic acid and AMC based on liquid chromatography-mass spectrometry (LC-MS) analysis. The assay also performed well in a 384-well microplate format under initial rate conditions (10% conversion) with a signal-to-background ratio (S/B) of 7 and a Z factor of 0.75. Preliminary screening of a library of 1280 pharmaceutically active compounds identified inhibitors with novel chemical scaffolds. This assay will be a powerful tool for target validation and drug lead identification and characterization.
authors.affiliation: Inflammation and Immunology, Pfizer Biotherapeutics R&D, Cambridge, MA 02140, USA. ruanb@wyeth.com
authors.forename: Benfang H
authors.initials: BH
authors.lastname: Ruan
authors.forename: Derek C
authors.initials: DC
authors.lastname: Cole
authors.forename: Paul
authors.initials: P
authors.lastname: Wu
authors.forename: Amira
authors.initials: A
authors.lastname: Quazi
authors.forename: Karen
authors.initials: K
authors.lastname: Page
authors.forename: Jill F
authors.initials: JF
authors.lastname: Wright
authors.forename: Nelson
authors.initials: N
authors.lastname: Huang
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Stock
authors.forename: Karl
authors.initials: K
authors.lastname: Nocka
authors.forename: Ann
authors.initials: A
authors.lastname: Aulabaugh
authors.forename: Rustem
authors.initials: R
authors.lastname: Krykbaev
authors.forename: Lori J
authors.initials: LJ
authors.lastname: Fitz
authors.forename: Neil M
authors.initials: NM
authors.lastname: Wolfman
authors.forename: Margaret L
authors.initials: ML
authors.lastname: Fleming
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry; D058851:GPI-Linked Proteins; D057166:High-Throughput Screening Assays; D006801:Humans; D007668:Kidney / Q000201:enzymology; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D010205:Pantothenic Acid / Q000737:chemistry; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D058851:GPI-Linked Proteins; D011994:Recombinant Proteins; D010205:Pantothenic Acid; D000581:Amidohydrolases; C022314:pantetheinase