title: A Substrate Mimic Allows High-Throughput Assay of the FabA Protein and Consequently the Identification of a Novel Inhibitor of Pseudomonas aeruginosa FabA.
abstract: Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathways that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-Acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FASII pathway, into a high-throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50=5.7±0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of Escherichia coli acyl carrier protein and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalises affinity and suggests future design opportunities. Employing NAC fatty acid mimics to develop further high-throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials.
authors.affiliation: Biomedical Sciences Research Complex and EaStCHEM, School of Chemistry, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, United Kingdom. Electronic address: lm108@st-andrews.ac.uk.
authors.forename: Lucile
authors.initials: L
authors.lastname: Moynié
authors.affiliation: The Drug Discovery Unit, James Black Complex, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom. Electronic address: a.g.hope@dundee.ac.uk.
authors.forename: Anthony G
authors.initials: AG
authors.lastname: Hope
authors.affiliation: Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA. Electronic address: kfinzel@ucsd.edu.
authors.forename: Kara
authors.initials: K
authors.lastname: Finzel
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden. Electronic address: jason.schmidberger@gmail.com.
authors.forename: Jason
authors.initials: J
authors.lastname: Schmidberger
authors.affiliation: Biomedical Sciences Research Complex and EaStCHEM, School of Chemistry, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, United Kingdom. Electronic address: leckiestm@gmail.com.
authors.forename: Stuart M
authors.initials: SM
authors.lastname: Leckie
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden. Electronic address: Gunter.Schneider@ki.se.
authors.forename: Gunter
authors.initials: G
authors.lastname: Schneider
authors.affiliation: Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA. Electronic address: mburkart@ucsd.edu.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Burkart
authors.affiliation: Biomedical Sciences Research Complex and EaStCHEM, School of Chemistry, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, United Kingdom. Electronic address: ads10@st-andrews.ac.uk.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Smith
authors.affiliation: The Drug Discovery Unit, James Black Complex, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom. Electronic address: d.w.gray@dundee.ac.uk.
authors.forename: David W
authors.initials: DW
authors.lastname: Gray
authors.affiliation: Biomedical Sciences Research Complex and EaStCHEM, School of Chemistry, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, United Kingdom; State Key Laboratory of Biotherapy, Sichuan University, Chengdu, Sichuan, China 610065. Electronic address: naismith@st-andrew.ac.uk.
authors.forename: James H
authors.initials: JH
authors.lastname: Naismith
mesh_terms: D018360:Crystallography, X-Ray; D003543:Cysteamine / Q000031:analogs & derivatives* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology; D054889:Fatty Acid Synthase, Type II / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D008958:Models, Molecular; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
chemical_list: D004791:Enzyme Inhibitors; C047122:3-hydroxydecanoic acid N-acetylcysteamine thioester; D003543:Cysteamine; D054889:Fatty Acid Synthase, Type II
keywords: HTS; co-complex; crystal structure; drug discovery; pathogen