Document Detail


A high-throughput screening fluorescence polarization assay for fatty acid adenylating enzymes in Mycobacterium tuberculosis.
MedLine Citation:
PMID:  21771578     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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 K(D) 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.
Authors:
Kimberly D Grimes; Courtney C Aldrich
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2011-07-02
Journal Detail:
Title:  Analytical biochemistry     Volume:  417     ISSN:  1096-0309     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2011 Oct 
Date Detail:
Created Date:  2011-08-08     Completed Date:  2011-11-28     Revised Date:  2013-06-28    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  264-73     Citation Subset:  IM    
Copyright Information:
Copyright © 2011 Elsevier Inc. All rights reserved.
Affiliation:
Center for Drug Design, University of Minnesota, Minneapolis, MN, USA.
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MeSH Terms
Descriptor/Qualifier:
Carbon-Sulfur Ligases / antagonists & inhibitors,  chemistry*,  genetics
Catalytic Domain
Fatty Acids / chemistry,  metabolism
Fluorescence Polarization / methods*
Fluorescent Dyes / chemical synthesis
High-Throughput Screening Assays / methods*
Lipid Metabolism
Mycobacterium tuberculosis / enzymology*
Grant Support
ID/Acronym/Agency:
NS066415/NS/NINDS NIH HHS; R21 NS066415/NS/NINDS NIH HHS; R21 NS066415-01/NS/NINDS NIH HHS
Chemical
Reg. No./Substance:
0/Fatty Acids; 0/Fluorescent Dyes; EC 6.2.-/Carbon-Sulfur Ligases; EC 6.2.1.-/FadD28 protein, Mycobacterium tuberculosis
Comments/Corrections

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