PMID- 20094656 OWN - NLM STAT- MEDLINE DCOM- 20100429 LR - 20211028 IS - 1742-2051 (Electronic) IS - 1742-206X (Print) IS - 1742-2051 (Linking) VI - 6 IP - 2 DP - 2010 Feb TI - A strategy to discover inhibitors of Bacillus subtilis surfactin-type phosphopantetheinyl transferase. PG - 365-75 LID - 10.1039/b913291k [doi] AB - Surfactin-type phosphopantetheinyl transferases (Sfp-PPTases) are responsible for modifying type I polyketide and non-ribosomal peptide synthases of prokaryotes and have been implicated in the activation of a variety of pathogen-associated virulence factors. As such, inhibitors of this enzyme class represent enticing leads for antibiotic development and can serve as tools in studies of bacterial metabolism. Currently, no small molecule inhibitors of Sfp-PPTase are known, highlighting the need for efficient methods for PPTase inhibitor identification and development. Herein, we present the design and implementation of a robust and miniaturized high-throughput kinetic assay for inhibitors of Sfp-PPTase using the substrate combination of rhodamine-labeled coenzyme A and Black Hole Quencher-2 labeled consensus acceptor peptide YbbR. Upon PPTase-catalyzed transfer of the rhodamine-labeled phosphopantetheinyl arm onto the acceptor peptide, the fluorescent donor and quencher are covalently joined and the fluorescence signal is reduced. This assay was miniaturized to a low 4 microL volume in 1536-well format and was used to screen the library of pharmacologically active compounds (LOPAC(1280)). Top inhibitors identified by the screen were further characterized in secondary assays, including protein phosphopantetheinylation detected by gel electrophoresis. The present assay enables the screening of large compound libraries against Sfp-PPTase in a robust and automated fashion and is applicable to designing assays for related transferase enzymes. FAU - Yasgar, Adam AU - Yasgar A AD - NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3370, USA. asimeono@mail.nih.gov FAU - Foley, Timothy L AU - Foley TL FAU - Jadhav, Ajit AU - Jadhav A FAU - Inglese, James AU - Inglese J FAU - Burkart, Michael D AU - Burkart MD FAU - Simeonov, Anton AU - Simeonov A LA - eng GR - R03 MH083266/MH/NIMH NIH HHS/United States GR - ZIA HG200319-06/ImNIH/Intramural NIH HHS/United States GR - 1R03MH083266/MH/NIMH NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, N.I.H., Intramural PT - Research Support, Non-U.S. Gov't DEP - 20091013 PL - England TA - Mol Biosyst JT - Molecular bioSystems JID - 101251620 RN - 0 (Bacterial Proteins) RN - 0 (Detergents) RN - 0 (Enzyme Inhibitors) RN - 0 (Rhodamines) RN - 0 (phosphopantetheinyl transferase) RN - EC 2.7.8.- (Transferases (Other Substituted Phosphate Groups)) RN - TPY09G7XIR (Fluorescein) SB - IM MH - Bacillus subtilis/*enzymology MH - Bacterial Proteins/*antagonists & inhibitors/metabolism MH - Detergents/chemistry MH - Drug Discovery/*methods MH - Drug Stability MH - Enzyme Inhibitors/chemistry/*pharmacology MH - Fluorescein/chemistry MH - Fluorescence Resonance Energy Transfer/methods MH - Models, Biological MH - Protein Binding MH - Rhodamines/chemistry MH - Substrate Specificity MH - Transferases (Other Substituted Phosphate Groups)/*antagonists & inhibitors/metabolism PMC - PMC2992954 MID - NIHMS247399 EDAT- 2010/01/23 06:00 MHDA- 2010/04/30 06:00 PMCR- 2010/11/29 CRDT- 2010/01/23 06:00 PHST- 2010/01/23 06:00 [entrez] PHST- 2010/01/23 06:00 [pubmed] PHST- 2010/04/30 06:00 [medline] PHST- 2010/11/29 00:00 [pmc-release] AID - 10.1039/b913291k [doi] PST - ppublish SO - Mol Biosyst. 2010 Feb;6(2):365-75. doi: 10.1039/b913291k. Epub 2009 Oct 13.