title: Development of a pterin-based fluorescent probe for screening dihydropteroate synthase.
abstract: Dihydropteroate synthase (DHPS) is the classical target of the sulfonamide class of antimicrobial agents, whose use has been limited by widespread resistance and pharmacological side effects. We have initiated a structure-based drug design approach for the development of novel DHPS inhibitors that bind to the highly conserved and structured pterin subsite rather than to the adjacent p-aminobenzoic acid binding pocket that is targeted by the sulfonamide class of antibiotics. To facilitate these studies, a robust pterin site-specific fluorescence polarization (FP) assay has been developed and is discussed herein. These studies include the design, synthesis, and characterization of two fluorescent probes, and the development and validation of a rapid DHPS FP assay. This assay has excellent DMSO tolerance and is highly reproducible as evidenced by a high Z' factor. This assay offers significant advantages over traditional radiometric or phosphate release assays against this target, and is suitable for site-specific high-throughput and fragment-based screening studies.
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, United States.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhao
authors.forename: Dalia
authors.initials: D
authors.lastname: Hammoudeh
authors.forename: Wenwei
authors.initials: W
authors.lastname: Lin
authors.forename: Sourav
authors.initials: S
authors.lastname: Das
authors.forename: Mi-Kyung
authors.initials: MK
authors.lastname: Yun
authors.forename: Zhenmei
authors.initials: Z
authors.lastname: Li
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Griffith
authors.forename: Taosheng
authors.initials: T
authors.lastname: Chen
authors.forename: Stephen W
authors.initials: SW
authors.lastname: White
authors.forename: Richard E
authors.initials: RE
authors.lastname: Lee
mesh_terms: D001408:Bacillus anthracis / Q000201:enzymology*; D001665:Binding Sites; D001667:Binding, Competitive; D004094:Dihydropteroate Synthase / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011622:Pterins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D005456:Fluorescent Dyes; D011622:Pterins; D004094:Dihydropteroate Synthase