title: Discovery of bacterial fatty acid synthase type II inhibitors using a novel cellular bioluminescent reporter assay.
abstract: Novel, cellular, gain-of-signal, bioluminescent reporter assays for fatty acid synthesis type II (FASII) inhibitors were constructed in an efflux-deficient strain of Pseudomonas aeruginosa and based on the discovery that FASII genes in P. aeruginosa are coordinately upregulated in response to pathway disruption. A screen of 115,000 compounds identified a series of sulfonamidobenzamide (SABA) analogs, which generated strong luminescent signals in two FASII reporter strains but not in four control reporter strains designed to respond to inhibitors of pathways other than FASII. The SABA analogs selectively inhibited lipid biosynthesis in P. aeruginosa and exhibited minimal cytotoxicity to mammalian cells (50% cytotoxic concentration [CC50] ≥ 80 μM). The most potent SABA analogs had MICs of 0.5 to 7.0 μM (0.2 to 3.0 μg/ml) against an efflux-deficient Escherichia coli (ΔtolC) strain but had no detectable MIC against efflux-proficient E. coli or against P. aeruginosa (efflux deficient or proficient). Genetic, molecular genetic, and biochemical studies revealed that SABA analogs target the enzyme (AccC) catalyzing the biotin carboxylase half-reaction of the acetyl coenzyme A (acetyl-CoA) carboxylase step in the initiation phase of FASII in E. coli and P. aeruginosa. These results validate the capability and the sensitivity of this novel bioluminescent reporter screen to identify inhibitors of E. coli and P. aeruginosa FASII.
authors.affiliation: Microbiotix, Inc., Worcester, Massachusetts, USA.
authors.forename: Joselynn
authors.initials: J
authors.lastname: Wallace
authors.affiliation: Microbiotix, Inc., Worcester, Massachusetts, USA.
authors.forename: Nicholas O
authors.initials: NO
authors.lastname: Bowlin
authors.affiliation: Microbiotix, Inc., Worcester, Massachusetts, USA.
authors.forename: Debra M
authors.initials: DM
authors.lastname: Mills
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Panatda
authors.initials: P
authors.lastname: Saenkham
authors.affiliation: Microbiotix, Inc., Worcester, Massachusetts, USA.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Kwasny
authors.affiliation: Microbiotix, Inc., Worcester, Massachusetts, USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Opperman
authors.affiliation: Microbiotix, Inc., Worcester, Massachusetts, USA.
authors.forename: John D
authors.initials: JD
authors.lastname: Williams
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Charles O
authors.initials: CO
authors.lastname: Rock
authors.affiliation: Microbiotix, Inc., Worcester, Massachusetts, USA.
authors.forename: Terry L
authors.initials: TL
authors.lastname: Bowlin
authors.affiliation: Microbiotix, Inc., Worcester, Massachusetts, USA dmoir@microbiotix.com.
authors.forename: Donald T
authors.initials: DT
authors.lastname: Moir
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D054889:Fatty Acid Synthase, Type II / Q000037:antagonists & inhibitors*; D008826:Microbial Sensitivity Tests; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology
chemical_list: D000900:Anti-Bacterial Agents; D000105:Acetyl Coenzyme A; D054889:Fatty Acid Synthase, Type II