A high-throughput screening fluorescence polarization assay for fatty acid adenylating enzymes in Mycobacterium tuberculosis

Kimberly D. Grimes, Courtney C. Aldrich

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), encodes for an astonishing 34 fatty acid adenylating enzymes (FadDs), which play key roles in lipid metabolism. FadDs involved in lipid biosynthesis are functionally nonredundant and serve to link fatty acid and polyketide synthesis to produce some of the most architecturally complex natural lipids including the essential mycolic acids as well as the virulence-conferring phthiocerol dimycocerosates, phenolic glycolipids, and mycobactins. Here we describe the systematic development and optimization of a fluorescence polarization assay to identify small molecule inhibitors as potential antitubercular agents. We fluorescently labeled a bisubstrate inhibitor to generate a fluorescent probe/tracer, which bound with a KD of 245 nM to FadD28. Next, we evaluated assay performance by competitive binding experiments with a series of known ligands and assessed the impact of control parameters including incubation time, stability of the signal, temperature, and DMSO concentration. As a final level of validation the LOPAC1280 library was screened in a 384-well plate format and the assay performed with a Z-factor of 0.75, demonstrating its readiness for high-throughput screening.

Original languageEnglish (US)
Pages (from-to)264-273
Number of pages10
JournalAnalytical Biochemistry
Volume417
Issue number2
DOIs
StatePublished - Oct 15 2011

Keywords

  • Adenylate-forming enzymes
  • Adenylation
  • Fluorescence polarization
  • High throughput screening
  • Mycobacterium tuberculosis

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