Discovery of Bacterial Fatty Acid Synthase Type II Inhibitors Using a Novel Cellular Bioluminescent Reporter Assay

Abstract

bNovel, 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 [CC 50 ] > 80 M). The most potent SABA analogs had MICs of 0.5 to 7.0 M (0.2 to 3.0 g/ml) against an efflux-deficient Escherichia coli (⌬tolC) 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. Pseudomonas aeruginosa is a highly virulent, persistent human pathogen with both acquired and intrinsic drug resistances. It is the most common cause of nosocomial pneumonia, causing 15% to 20% of hospital-acquired pneumonias (1), and up to 75% of patients in intensive care units are colonized with this pathogen (2). P. aeruginosa is also becoming a major cause of communityacquired pneumonia in severely ill patients (3). An astounding 30% of clinical isolates from critically ill patients are resistant to three or more drugs, which leads to treatment failure (4). The discovery and development of new classes of antibiotics, which are not subject to existing target-based resistance mechanisms, is an important strategy in combating drug resistance, and targeting unexploited or underexploited essential bacterial pathways has been a successful strategy for discovering new compound classes (5, 6).This study focused on the fatty acid synthesis type II (FASII) pathway in P. aeruginosa. While most bacteria utilize exogenous fatty acids for phospholipid synthesis (7), the FASII pathway is absolutely essential for bacterial membrane biogenesis in Gramnegative bacteria because the hydroxyl-fatty acid constituents of LPS cannot be obtained from an extracellular source (8). FASII inhibitors also block the production of Gram-negative signaling molecules, including homoserine lactones and hydroxyquinolines, which are important to establish and maintain P. aeruginosa virulence (9). This FASII macromolecular synthesis pathway is conserved and essential in Gram-negative bacteria but is absent from the mammalian cytopl...

Optimization and Mechanistic Characterization of Pyridopyrimidine Inhibitors of Bacterial Biotin Carboxylase

“…[6][7][8][9][10][11] The first committed step in this pathway is catalyzed by an essential cytoplasmic acetyl-CoA carboxylase multienzyme complex 12 (see also Supporting Information Text S1), and inhibitors of the BC active site of this complex have shown antibacterial activity against several Gram-negative species. 5,[13][14][15][16][17] The BC active site is highly conserved across a number of bacterial pathogens, including P. aeruginosa, E. coli, and H. influenzae. 18 While 1 lacks the potency to become a therapeutic agent, it is a promising starting point for medicinal chemistry efforts because of its selective on-target activity, low molecular weight, and available co-crystal structures with BC that enable structure-based molecular design.…”

Mechanism of biotin carboxylase inhibition by ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulphonylamino]benzoate

“…In 2015 a group from Microbiotix, Inc. developed an in vivo high-throughput screen capable of identifying FASII inhibitors in Pseudomonas aeruginosa and Escherichia coli 28 . That screen identified five compounds that selectively inhibit FASII 28 . Two of those compounds share a sulfonamidobenzamide (SABA) core structure 28 .…”

“…That screen identified five compounds that selectively inhibit FASII 28 . Two of those compounds share a sulfonamidobenzamide (SABA) core structure 28 . The more potent of those two compounds is ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulfonylamino]benzoate (SABA1) ( Figure 2 ).…”

“…The more potent of those two compounds is ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulfonylamino]benzoate (SABA1) ( Figure 2 ). SABA1 has a MIC of 0.45–0.9 µM against efflux compromised E. coli ( ΔtolC::tet ) and prolongs the doubling time of the efflux compromised P. aeruginosa strain PAO397 by 30% 28 . They determined that the antibacterial action of SABA1 is due to inhibition of ACC.…”

“…They determined that the antibacterial action of SABA1 is due to inhibition of ACC. It has an IC 50 of 4.0 µM against E. coli ACC 28 and mapping of resistant mutations suggests that SABA1 targets BC 28 .…”