Institute for Chemical Biology & Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400, USA.
J Antimicrob Chemother. 2011 Mar;66(3):564-73. doi: 10.1093/jac/dkq509. Epub 2011 Jan 22.
As an initial step in developing novel antibacterials against Burkholderia pseudomallei, we have characterized the FabI enoyl-ACP reductase homologues in the type II fatty acid biosynthesis pathway from this organism and performed an initial enzyme inhibition study.
A BLAST analysis identified two FabI enoyl-ACP reductase homologues, bpmFabI-1 and bpmFabI-2, in the B. pseudomallei genome, which were cloned, overexpressed in Escherichia coli and purified. Steady-state kinetics was used to determine the reaction mechanism and the sensitivity of bpmFabI-1 to four diphenyl ether FabI inhibitors. The antibacterial activity of the inhibitors was assessed using a wild-type strain of Burkholderia thailandensis (E264) and an efflux pump mutant (Bt38).
Consistent with its annotation as an enoyl-ACP reductase, bpmFabI-1 catalysed the NADH-dependent reduction of 2-trans-dodecenoyl-CoA via a sequential Bi Bi mechanism. In contrast, bpmFabI-2 was inactive with all substrates tested and only bpmfabI-1 was transcriptionally active under the growth conditions employed. The sensitivity of bpmFabI-1 to four diphenyl ethers was evaluated and in each case the compounds were slow-onset inhibitors with K(i) values of 0.5-2 nM. In addition, triclosan and PT01 had MIC values of 30 and 70 mg/L for B. pseudomallei as well as a wild-type strain of B. thailandensis (E264), but MIC values of <1 mg/L for the efflux pump mutant Bt38. A reduction in MIC values was also observed for the pump mutant strain with the other diphenyl ethers.
Provided that efflux can be circumvented, bpmFabI-1 is a suitable target for drug discovery.
作为开发针对伯克霍尔德氏菌的新型抗菌药物的初始步骤，我们对该菌的 II 型脂肪酸生物合成途径中的 FabI 烯酰-ACP 还原酶同源物进行了特征描述，并进行了初步的酶抑制研究。
通过 BLAST 分析在伯克霍尔德氏菌基因组中鉴定出两个 FabI 烯酰-ACP 还原酶同源物 bpmFabI-1 和 bpmFabI-2，将其克隆、在大肠杆菌中过表达并纯化。使用稳态动力学测定来确定 bpmFabI-1 的反应机制和对四种二苯醚 FabI 抑制剂的敏感性。使用野生型伯克霍尔德氏菌泰国变种（E264）和外排泵突变株（Bt38）评估抑制剂的抗菌活性。
与它被注释为烯酰-ACP 还原酶一致，bpmFabI-1 通过顺序 Bi Bi 机制催化 2-反式-十二烯酰-CoA 的 NADH 依赖性还原。相比之下，bpmFabI-2 对所有测试的底物均无活性，只有在使用的生长条件下 bpmfabI-1 转录活跃。评估了 bpmFabI-1 对四种二苯醚的敏感性，在每种情况下，化合物均为缓慢作用抑制剂，K(i) 值为 0.5-2 nM。此外，三氯生和 PT01 对伯克霍尔德氏菌以及野生型伯克霍尔德氏菌泰国变种（E264）的 MIC 值分别为 30 和 70 mg/L，但对外排泵突变株 Bt38 的 MIC 值<1 mg/L。泵突变株的 MIC 值也随着其他二苯醚的使用而降低。
只要可以规避外排，bpmFabI-1 就是药物发现的合适靶标。
