10.1021/bc200346e.s001 Ying Zhao Ying Zhao Dalia Hammoudeh Dalia Hammoudeh Wenwei Lin Wenwei Lin Sourav Das Sourav Das Mi-Kyung Yun Mi-Kyung Yun Zhenmei Li Zhenmei Li Elizabeth Griffith Elizabeth Griffith Taosheng Chen Taosheng Chen Stephen W. White Stephen W. White Richard E. Lee Richard E. Lee Development of a Pterin-Based Fluorescent Probe for Screening Dihydropteroate Synthase American Chemical Society 2011 sulfonamide class acid binding pocket pterin DHPS FP assay novel DHPS inhibitors Screening Dihydropteroate SynthaseDihydropteroate synthase phosphate release assays DMSO 2011-10-19 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Development_of_a_Pterin_Based_Fluorescent_Probe_for_Screening_Dihydropteroate_Synthase/2599159 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 <i>p</i>-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.