A Substrate Mimic Allows High-Throughput Assay of the FabA Protein and Consequently the Identification of a Novel Inhibitor of Pseudomonas aeruginosa FabA.
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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DOI：
10.1016/j.jmb.2015.10.027
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发表时间：
2016-01-16
影响因子：
5.6
通讯作者：
Naismith JH
中科院分区：
文献类型：
--
作者：
Moynié L;Hope AG;Finzel K;Schmidberger J;Leckie SM;Schneider G;Burkart MD;Smith AD;Gray DW;Naismith JH
真核生物和原核生物具有脂肪酸合成酶（FAS）生物合成途径，其包括反复的链延长、还原和脱水反应。细菌FASII途径与人FAS途径显著不同，并且由于与人FAS的差异，细菌FASII途径是针对革兰氏阴性菌的抗生素开发的长期目标，并且已知几种现有的抗菌剂抑制FASII酶。N-乙酰基半胱胺（NAC）脂肪酸硫酯已被用作天然酰基载体蛋白途径中间体的模拟物，以测定FASII酶，我们现在报告使用（E）-2-癸烯酰基-NAC测定来自铜绿假单胞菌的FabV。此外，我们已经将FabA（双功能脱水/差向异构化酶和FASII途径中的关键靶标）的现有UV吸光度测定转换为高通量酶偶联荧光测定，该测定已用于筛选不同小分子的文库。通过这种方法，发现N-（4-氯苄基）-3-（2-呋喃基）-1H-1，2，4-三唑-5-胺（N42 FTA）竞争性抑制（pIC 50 = 5.7 ± 0.2）铜绿假单胞菌FabA对3-羟基癸酰基-NAC的加工。N42 FTA被证明是有效的阻断大肠杆菌酰基载体蛋白和FabA的交联，直接模拟的生物过程。N42FTA与铜绿假单胞菌FabA蛋白的共复合物结构使亲和力合理化并提示未来的设计机会。采用NAC脂肪酸模拟物来开发FASII途径中单个酶的进一步高通量测定，应有助于发现新的抗菌剂。FabA是革兰氏阴性菌中一个很有前途的药物靶点。已经开发了使用底物类似物的FabA的高通量偶联测定。已使用该测定法鉴定了一类新的非共价抑制剂。该化合物抑制FabA的生物活性。已确定与FabA结合的化合物的晶体结构。
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. 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.