Development of a pterin-based fluorescent probe for screening dihydropteroate synthase.

Ying Zhao, Dalia Hammoudeh, Wenwei Lin, Sourav Das, Mi-Kyung Yun, Zhenmei Li, Elizabeth Griffith, Taosheng Chen, Stephen W White, Richard E Lee

Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, United States.

Bioconjugate chemistry 2011 Oct 19

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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.

Citation

Ying Zhao, Dalia Hammoudeh, Wenwei Lin, Sourav Das, Mi-Kyung Yun, Zhenmei Li, Elizabeth Griffith, Taosheng Chen, Stephen W White, Richard E Lee. Development of a pterin-based fluorescent probe for screening dihydropteroate synthase. Bioconjugate chemistry. 2011 Oct 19;22(10):2110-7