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.

In silico identification and in vitro evaluation of a protein‐protein interaction inhibitor of Escherichia coli fatty acid biosynthesis

“…Secondary assay confirmation. For secondary validation, we next turned to our previous work using a crosslinking based assay to orthogonally confirm the PPI inhibiting activity of suramin (Figure 3b) (Moynié et al., 2016). Here, EcACP is loaded with a phosphopantetheine analogue which bears a mechanism‐based reactive warhead, referred to as sulfonyl‐alkyne‐EcACP, that forms a covalent bond with the active site H70 on FabA resulting in a cross‐linked complex, EcACP=FabA (Figure S6).…”

“…Here, EcACP is loaded with a phosphopantetheine analogue which bears a mechanism‐based reactive warhead, referred to as sulfonyl‐alkyne‐EcACP, that forms a covalent bond with the active site H70 on FabA resulting in a cross‐linked complex, EcACP=FabA (Figure S6). We have previously demonstrated the utility of this crosslinking assay to confirm active site hits from a high‐throughput screen (Moynié et al., 2016). Because successful crosslinking between EcACP and FabA is dependent on the proteins engaging in correct PPI, we hypothesized that suramin would block crosslinking by inhibiting the EcACP · FabA interaction as a PPI inhibitor, which could easily be detected by a gel‐based assay.…”

A Review of Fatty Acid Biosynthesis Enzyme Inhibitors as Promising Antimicrobial Drugs

“…FabA structures from E. coli [ 108 ], P. aeruginosa [ 109 ] and Yersinia pestis [ 106 ] have been solved. Several FabZ structures have been reported in E. coli [ 101 ], P. aeruginosa [ 110 ], P. falciparum [ 111 ], Francisella tularensis [ 103 ], Y. pestis [ 103 ] and Helicobacter pylori [ 106 ], for instance.…”

Identification and characterization of potential druggable targets among Essential Hypothetical Proteins of A. baumannii

“…Out of 27, one protein was found to be previously reported as a drug target. 3-Oxo-acyl-acyl carrier protein (ACP) reductase (FabG) is significant in bacterial fatty acid synthesis and has been identified as a target [72,73]. In drug and vaccine design the determination of the 3D structure is important.…”

“…Host non-homology analysis shown that 27 proteins were specific to pathogen, so they are potential to be drug target candidates. 3-oxoacyl-ACP reductase is a known target [72]. The other proteins were identified as 'novel targets,' which require requires additional experimental validation.…”