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首页> 外文期刊>Journal of Molecular Biology >A Substrate Mimic Allows High-Throughput Assay of the FabA Protein and Consequently the Identification of a Novel Inhibitor of Pseudomonas aeruginosa FabA
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A Substrate Mimic Allows High-Throughput Assay of the FabA Protein and Consequently the Identification of a Novel Inhibitor of Pseudomonas aeruginosa FabA

机译：底物模拟物允许高通量测定FabA蛋白，因此可鉴定铜绿假单胞菌FabA的新型抑制剂

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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-chlorobenzyI)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC(50) = 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. (C) 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
机译：真核生物和原核生物具有脂肪酸合酶（FAS）生物合成途径，包括迭代链延长，还原和脱水反应。细菌FASII途径与人FAS途径有显着不同，由于与人FAS的差异，细菌FASII途径是针对革兰氏阴性细菌的抗生素开发的长期目标，并且已知几种现有的抗菌剂可抑制FASII酶。 N-乙酰半胱胺（NAC）脂肪酸硫酯已被用作模拟天然酰基载体蛋白途径中间体的方法来测定FASII酶，现在我们报道使用（E）-2-癸烯酰-NAC对铜绿假单胞菌的FabV进行测定。此外，我们已将FabA，双功能脱水/表位化酶和FASII途径中的关键靶点的现有UV吸收测定法转换为高通量酶联荧光测定法，该方法已用于筛选各种小分子文库。通过这种方法，发现N-（4-氯苯甲酰基）-3-（2-呋喃基）-1H-1,2,4-三唑-5-胺（N42FTA）具有竞争性抑制作用（pIC（50）= 5.7 + / -0.2）铜绿假单胞菌FabA对3-羟基癸酰基-NAC的加工。 N42FTA被证明可有效阻止大肠杆菌酰基载体蛋白和FabA的交联，这是生物学过程的直接模拟。 N42FTA与铜绿假单胞菌FabA蛋白的共复杂结构合理化了亲和力，并提出了未来的设计机会。利用NAC脂肪酸模拟物为FASII途径中的单个酶开发进一步的高通量测定方法，应有助于发现新的抗菌药物。 （C）2015作者。由Elsevier Ltd.发行。这是CC BY许可下的开放访问文章（http://creativecommons.org/licenses/by/4.0/）。

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