A fluorescent assay suitable for inhibitor screening and vanin tissue quantification.
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.
Ruan
Benfang H
BH
Inflammation and Immunology, Pfizer Biotherapeutics R&D, Cambridge, MA 02140, USA. ruanb@wyeth.com
Cole
Derek C
DC
Wu
Paul
P
Quazi
Amira
A
Page
Karen
K
Wright
Jill F
JF
Huang
Nelson
N
Stock
Joseph R
JR
Nocka
Karl
K
Aulabaugh
Ann
A
Krykbaev
Rustem
R
Fitz
Lori J
LJ
Wolfman
Neil M
NM
Fleming
Margaret L
ML
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
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D058851:GPI-Linked Proteins; D011994:Recombinant Proteins; D010205:Pantothenic Acid; D000581:Amidohydrolases; C022314:pantetheinase