title: Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase.
abstract: Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS.
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD15EH, UK.
authors.forename: Emma J
authors.initials: EJ
authors.lastname: Shanks
authors.forename: Han B
authors.initials: HB
authors.lastname: Ong
authors.forename: David A
authors.initials: DA
authors.lastname: Robinson
authors.forename: Stephen
authors.initials: S
authors.lastname: Thompson
authors.forename: Natasha
authors.initials: N
authors.lastname: Sienkiewicz
authors.forename: Alan H
authors.initials: AH
authors.lastname: Fairlamb
authors.forename: Julie A
authors.initials: JA
authors.lastname: Frearson
mesh_terms: D020134:Catalytic Domain; D003124:Colorimetry / Q000379:methods*; D018360:Crystallography, X-Ray; D045304:Cytochromes c / Q000378:metabolism*; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology
chemical_list: D009249:NADP; D045304:Cytochromes c; D010088:Oxidoreductases; C085592:pteridine reductase; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate