title: A Label-Free Continuous Fluorescence-Based Assay for Monitoring Ornithine Decarboxylase Activity with a Synthetic Putrescine Receptor.
abstract: Polyamines play an important role in cell growth, differentiation, and cancer development, and the biosynthetic pathway of polyamines is established as a drug target for the treatment of parasitic diseases, neoplasia, and cancer chemoprevention. The key enzyme in polyamine biosynthesis is ornithine decarboxylase (ODC). We report herein an analytical method for the continuous fluorescence monitoring of ODC activity based on the supramolecular receptor cucurbit[6]uril (CB6) and the fluorescent dye trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DSMI). CB6 has a significantly higher binding constant to the ODC product putrescine (>107M-1) than to the substrate L-ornithine (340 M-1). This enables real-time monitoring of the enzymatic reaction through a continuous fluorescence change caused by dye displacement from the macrocycle by the formed product, which allowed a straightforward determination of enzyme kinetic parameters ( kcat= 0.12 s-1and KM= 24 µM) and inhibition constants of the two ODC inhibitors α-difluoromethylornithine (DFMO) and epigallocatechin gallate (EGCG). The potential for high-throughput screening (HTS) was demonstrated by excellent Z' factors (>0.9) in a microplate reader format, and the sensitivity of the assay is comparable to or better than most established complementary methods, which invariably have the disadvantage of not being compatible with direct implementation and upscaling to HTS format in the drug discovery process.
authors.affiliation: 1 Department of Life Sciences and Chemistry, Jacobs University Bremen, Bremen, Germany.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Nilam
authors.affiliation: 2 Fraunhofer IME ScreeningPort, Hamburg, Germany.
authors.forename: Philip
authors.initials: P
authors.lastname: Gribbon
authors.affiliation: 2 Fraunhofer IME ScreeningPort, Hamburg, Germany.
authors.forename: Jeanette
authors.initials: J
authors.lastname: Reinshagen
authors.affiliation: 3 Institute of Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.|4 German Center for Cardiovascular Research (DZHK), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Cordts
authors.affiliation: 3 Institute of Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.|4 German Center for Cardiovascular Research (DZHK), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany.
authors.forename: Edzard
authors.initials: E
authors.lastname: Schwedhelm
authors.affiliation: 1 Department of Life Sciences and Chemistry, Jacobs University Bremen, Bremen, Germany.
authors.forename: Werner M
authors.initials: WM
authors.lastname: Nau
authors.affiliation: 1 Department of Life Sciences and Chemistry, Jacobs University Bremen, Bremen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Hennig
mesh_terms: D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D000518:Eflornithine / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D009952:Ornithine / Q000378:metabolism*; D009955:Ornithine Decarboxylase / Q000378:metabolism*; D065108:Ornithine Decarboxylase Inhibitors / Q000494:pharmacology*; D011073:Polyamines / Q000494:pharmacology; D011700:Putrescine / Q000378:metabolism*; D062165:Receptors, Artificial / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D065108:Ornithine Decarboxylase Inhibitors; D011073:Polyamines; D062165:Receptors, Artificial; D009952:Ornithine; D009955:Ornithine Decarboxylase; D011700:Putrescine; D000518:Eflornithine
keywords: decarboxylases; fluorescence methods; polyamines; supramolecular tandem assay