title: Fluorescent techniques for discovery and characterization of phosphopantetheinyl transferase inhibitors.
abstract: Phosphopantetheinyl transferase (PPTase; E.C. 2.7.8.-) activates biosynthetic pathways that synthesize both primary and secondary metabolites in bacteria. Inhibitors of these enzymes have the potential to serve as antibiotic compounds that function through a unique mode of action and possess clinical utility. Here we report a direct and continuous assay for this enzyme class based upon monitoring polarization of a fluorescent phosphopantetheine analog as it is transferred from a low-molecular weight CoA substrate to higher-molecular weight protein acceptor. We demonstrate the utility of this method for the biochemical characterization of PPTase Sfp, a canonical representative from this class. We also establish the portability of this technique to other homologs by adapting the assay to function with the human PPTase, a target for which a microplate detection method does not currently exist. Comparison of these targets provides a basis to predict the therapeutic index of inhibitor candidates and offers a valuable characterization of enzyme activity.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego (UCSD), La Jolla, CA, USA.
authors.forename: Nicolas M
authors.initials: NM
authors.lastname: Kosa
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego (UCSD), La Jolla, CA, USA.
authors.forename: Timothy L
authors.initials: TL
authors.lastname: Foley
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego (UCSD), La Jolla, CA, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Burkart
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects* / Q000201:enzymology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors*; D055808:Drug Discovery / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005454:Fluorescence Polarization / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D008970:Molecular Weight; D017855:Transferases (Other Substituted Phosphate Groups) / Q000037:antagonists & inhibitors*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; C082662:phosphopantetheinyl transferase; D017855:Transferases (Other Substituted Phosphate Groups)