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10 Nov 2015

Lucile Moynié1, Anthony G. Hope2, Kara Finzel3, Jason Schmidberger4, Stuart M. Leckie1, Gunter Schneider4, Michael D. Burkart3, Andrew D. Smith1, David W. Gray2, James H. Naismith1, 5,

1 BSRC & EaStChem, North Haugh, The University, St Andrews KY16 9ST, UK
2 The Drug Discovery Unit, James Black Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK
3 Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA
4 Department of Medical Biochemistry & Biophysics, Karolinska Institutet, S-171 77, Stockholm, Sweden
5 State Key Laboratory of Biotherapy, Sichuan University, China


•FabA is a promising drug target in Gram negative bacteria
•high throughput coupled assay for FabA using a substrate analogue has been developed
•new class of non-covalent inhibitor has been identified using the assay
•the compound inhibits the biological activity of FabA
•crystal structure of the compound bound to FabA has been determined


Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathway(s) 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 (ACP) 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 FAS II 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 E. coli ACP and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalizes affinity and suggests future design opportunities. Employing NAC fatty acid mimics to developing further high throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials.