title: An Efficient and Economical Assay to Screen for Triclosan Binding to FabI.
abstract: Triclosan is an effective inhibitor for enoyl acyl carrier protein reductase (ENR) in fatty acid biosynthesis. Triclosan-resistant mutants of ENR have emerged. Thus, it is important to detect these triclosan-resistant mutations in ENR. Generally, enzyme activity assays on the mutants are used to determine the effect of triclosan on ENR activity. Since the substrates are linked to acyl carrier protein (ACP), the assays are challenging due to the need to prepare the ACP and link it to the substrates. Non-ACP-linked (coenzyme A [CoA]-linked) substrates can be used in some ENR, but not in all. Consequently, screening for triclosan-resistant mutants is also challenging. We have developed a simple thermal shift assay, which does not use ACP-linked substrates, to determine the binding ability of triclosan to the ENR active site, and thus it can be used for screening for triclosan-resistant mutants. Staphylococcus aureus FabI enzyme and its mutants were used to demonstrate the binding ability of triclosan with NADP(+) to FabI. The direct correlation between the binding ability and enzyme activity was demonstrated with Francisella tularensis FabI. This method may also be applied to select effective triclosan analogues that inhibit ENR activity.
authors.affiliation: University of Illinois at Chicago, Chicago, IL, USA.
authors.forename: Robel D
authors.initials: RD
authors.lastname: Demissie
authors.affiliation: University of Illinois at Chicago, Chicago, IL, USA.
authors.forename: Pauline
authors.initials: P
authors.lastname: Kabre
authors.affiliation: University of Illinois at Chicago, Chicago, IL, USA.
authors.forename: Micheal L
authors.initials: ML
authors.lastname: Tuntland
authors.affiliation: University of Illinois at Chicago, Chicago, IL, USA lfung@uic.edu.
authors.forename: Leslie W-M
authors.initials: LW
authors.lastname: Fung
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D024881:Drug Resistance, Bacterial / Q000235:genetics; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005604:Francisella tularensis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D057166:High-Throughput Screening Assays; D009154:Mutation; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013211:Staphylococcus aureus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D013696:Temperature; D014260:Triclosan / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D014260:Triclosan; D009249:NADP; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)
keywords: antibacterial drugs; fluorescence methods; high-content screening; ligand binding; pharmacology; protein chemistry; protein labeling; proteomics; receptor binding