title: High-throughput screen for inhibitors of 1-deoxy-d-xylulose 5-phosphate reductoisomerase by surrogate ligand competition.
abstract: 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is a key enzyme in a biosynthetic pathway for isoprenoids that is unique to eubacteria and plants. Dxr catalyzes the rearrangement and NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate. The authors have purified Escherichia coli Dxr and devised a high-throughput screen (HTS) for compounds that bind to this enzyme at a functional site. Evidence is presented that the surrogate ligand directly binds or allosterically affects both the D-1-deoxyxylulose 5-phosphate (DXP) and NADPH binding sites. Compounds that bind at either or both sites that compete for binding with the surrogate ligand register as hits. The time-resolved fluorescence-based assay represents an improvement over the Dxr enzyme assay that relies on relatively insensitive measurements of NADPH oxidation. Screening 32,000 compounds from a diverse historical library, the authors obtained 89 potent inhibitors in the surrogate ligand competition assay. The results presented here suggest that peptide surrogate ligands may be useful in formatting HTS for proteins with difficult biochemical assays or targets of unknown function.
authors.affiliation: Karo Bio USA, Inc., Durham, NC, USA.
authors.forename: Elizabeth B
authors.initials: EB
authors.lastname: Gottlin
authors.forename: R Edward
authors.initials: RE
authors.lastname: Benson
authors.forename: Scott
authors.initials: S
authors.lastname: Conary
authors.forename: Brett
authors.initials: B
authors.lastname: Antonio
authors.forename: Kellie
authors.initials: K
authors.lastname: Duke
authors.forename: E Sturgis
authors.initials: ES
authors.lastname: Payne
authors.forename: S Salman
authors.initials: SS
authors.lastname: Ashraf
authors.forename: Dale J
authors.initials: DJ
authors.lastname: Christensen
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000037:antagonists & inhibitors*; D001665:Binding Sites; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004345:Drug Industry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005063:Europium / Q000737:chemistry; D005578:Fosfomycin / Q000031:analogs & derivatives* / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008024:Ligands; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D009249:NADP / Q000737:chemistry; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D019809:Streptavidin / Q000737:chemistry; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D008024:Ligands; D009097:Multienzyme Complexes; D019151:Peptide Library; D010455:Peptides; D005578:Fosfomycin; D005063:Europium; D009249:NADP; C024640:fosmidomycin; D019809:Streptavidin; D010088:Oxidoreductases; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D019747:Aldose-Ketose Isomerases