AccA3作为新型抗结核候选物IMB-SD62作用靶点的研究

With MDR-TB and XDR-TB infections being reported worldwide, the development of new anti-tuberculosis (anti-TB) drugs is very urgent. In our previous study, an active anti-TB compound IMB-SD62 with a new skeleton structure was identified. IMB-SD62 had good in vitro activity against both standard and resistant strains of tuberculosis, and had significant anti-tuberculosis activity in vivo. At the same time, it had low toxicity and the prospect of medicine. By proteome chip and surface plasmon resonance (SPR), we found that IMB-SD62 had a strong binding to acetyl-CoA carboxylase α subunit (AccA3) which was necessary for tuberculosis survival, and preliminarily verified the interaction between IMB-SD62 and AccA3. Therefore, this study would confirm the interaction between IMB-SD62 and AccA3 at the molecular level by isothermal titration calorimetry (ITC) and fluorescence quenching assay, verify the inhibitory activity of IMB-SD62 through the enzyme level and bacterial level study, and clarify the mechanism and target of IMB-SD62 by key site mutation and X ray diffraction. Furthermore, a screening model using the target was constructed to screen new anti-tuberculosis drugs, which proved that the target could be used for drug discovery. The significance of this research is that, the mechanism study of IMB-SD62 will be helpful to obtain new targets for anti-tuberculosis drugs, and provide new theoretical and experimental basis for creating new anti-tuberculosis drugs with independent intellectual property rights.

由于耐药结核广泛传播，新型抗结核药物的研发迫在眉睫。前期我们发现了具有全新骨架结构的抗结核候选物IMB-SD62，其对结核标准株和耐药株均具有良好体外活性，体内抗结核活性显著，毒性低且具有成药前景。深入研究，通过蛋白质组芯片发现其对结核生存所必需的乙酰辅酶A羧化酶α亚基（AccA3）具有较强结合作用，表面等离子共振实验初步验证IMB-SD62与AccA3存在相互作用。故本研究拟在此基础上，通过等温滴定量热等技术在分子水平确认其与AccA3的相互作用，通过酶及细菌水平的研究验证化合物对AccA3的抑制活性，通过点突变、X射线衍射等明确IMB-SD62的作用机制及靶标。后续将以该靶标构建药物筛选模型，筛选新型抗结核药物，证明该靶标可用于药物发现。本研究的意义在于：阐释抗结核候选物IMB-SD62的作用机制，有助于获得抗结核药物新靶标，为创制具有自主知识产权的抗结核新药提供新的理论和实验依据。

结核病是由结核分枝杆菌（Mycobacterium tuberculosis，MTB）感染引起的一种严重危害人类健康的慢性传染病。随着耐药结核的出现和蔓延，临床主要的抗结核药物已经不能有效应对这一严峻形势，因此新型抗结核药物的研发迫在眉睫。前期我们发现了具有全新骨架结构的抗结核候选物IMB-SD62，其对结核标准株和耐药株均具有良好活性，体内抗结核活性显著，毒性低且具有成药前景。深入研究，通过蛋白质组芯片发现其对结核生存所必需的乙辅酶A羧化酶α亚基（AccA3）具有较强结合作用。本项目在此基础上，利用表面等离子共振和荧光淬灭实验，在分子水平上证明了化合物与AccA3蛋白的相互作用；通过检测IMB-SD62在对照菌株及AccA3过表达菌株上的敏感性差异（相差4倍），在细菌水平上证明了IMB-SD62对AccA3蛋白的抑制作用，并且通过构建酶活检测体系，在酶水平上进一步验证了化合物对AccA3的抑制作用；分子对接结果显示，AccA3蛋白的Y208和K168位点对于维持化合物与蛋白结合的稳定性较为重要，将两个位点进行点突变后，SPR结果显示IMB-SD62与蛋白的结合力降低，进一步验证了分子对接结果；后续我们利用构建的海分枝杆菌AccA3过表达菌株，建立了靶向AccA3的抗结核药物筛选模型，通过筛选获得了2个靶向AccA3的具有抗结核活性的化合物，证明该靶标可用于抗结核先导物的发现。综上所述，本研究阐释了新型抗结核先导物IMB-SD62的作用机制，获得了潜在的新型抗结核药物靶标AccA3，为开发具有自主知识产权的抗结核药物奠定基础。