title: A high-throughput fluorescence-based assay for Plasmodium dihydroorotate dehydrogenase inhibitor screening.
abstract: Plasmodium dihydroorotate dehydrogenase (DHODH) is a mitochondrial membrane-associated flavoenzyme that catalyzes the rate-limiting step of de novo pyrimidine biosynthesis. DHODH is a validated target for malaria, and DSM265, a potent inhibitor, is currently in clinical trials. The enzyme catalyzes the oxidation of dihydroorotate to orotate using flavin mononucleotide (FMN) as cofactor in the first half of the reaction. Reoxidation of FMN to regenerate the active enzyme is mediated by ubiquinone (CoQD), which is the physiological final electron acceptor and second substrate of the reaction. We have developed a fluorescence-based high-throughput enzymatic assay to find DHODH inhibitors. In this assay, the CoQD has been replaced by a redox-sensitive fluorogenic dye, resazurin, which changes to a fluorescent state on reduction to resorufin. Remarkably, the assay sensitivity to find competitive inhibitors of the second substrate is higher than that reported for the standard colorimetric assay. It is amenable to 1536-well plates with Z' values close to 0.8. The fact that the human enzyme can also be assayed in the same format opens additional applications of this assay to the discovery of inhibitors to treat cancer, transplant rejection, autoimmune diseases, and other diseases mediated by rapid cellular growth.
authors.affiliation: Platform Technology and Science Tres Cantos, GlaxoSmithKline, Centro de Investigación Básica, 28760, Tres Cantos, Spain.
authors.forename: Iván
authors.initials: I
authors.lastname: Caballero
authors.affiliation: Tres Cantos Medicines Development Campus, Diseases of the Developing World, GlaxoSmithKline, 28760, Tres Cantos, Spain.
authors.forename: María José
authors.initials: MJ
authors.lastname: Lafuente
authors.affiliation: Tres Cantos Medicines Development Campus, Diseases of the Developing World, GlaxoSmithKline, 28760, Tres Cantos, Spain.
authors.forename: Francisco-Javier
authors.initials: FJ
authors.lastname: Gamo
authors.affiliation: Platform Technology and Science Tres Cantos, GlaxoSmithKline, Centro de Investigación Básica, 28760, Tres Cantos, Spain. Electronic address: concha.cid@gsk.com.
authors.forename: Concepción
authors.initials: C
authors.lastname: Cid
mesh_terms: D000090722:Dihydroorotate Dehydrogenase; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000737:chemistry* / Q000494:pharmacology; D005453:Fluorescence; D057166:High-Throughput Screening Assays; D006801:Humans; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010961:Plasmodium / Q000201:enzymology*; D013329:Structure-Activity Relationship
chemical_list: D000090722:Dihydroorotate Dehydrogenase; D004791:Enzyme Inhibitors; D044925:Oxidoreductases Acting on CH-CH Group Donors
keywords: Assay development; DHODH; Fluorescence; HTS; Malaria; Resazurin