喹啉衍生物拮抗VB1诱导TD性状的分子机制研究

Quinoline derivatives are widely used in medicine. However, they cause various side effects such as neuritis, retinal damadge etc. The molecular mechanism remains obscure. Our previous studies have shown that quinoline derivatives inhibit the growth of yeast（S. cerevisiae）by specifically targeting thaimine transporters. Is this mode of action correlated with their side effects? In this project, the cellular mechanism of clioquinol (CQ) will be firstly sutdied by determination of effects of CQ on thaimine status and related.physiological metabolism in animal cells. Next, interaction sites, functional motifs and binding capacity between CQ and targeting proteins (thaimine related THI7p、SLC19A etc.) will be investigated by using computer modeling analysis combining with Surface Plasmon Resonance (SPR) technique, and sentivive genes to CQ associated with thaimine metabolism will be analyzed using functional variomics and genetic screening techniques as well. Additionally, thiamine deficiency (TD) animal model will be set up to study the effect of CQ on thiamine metabolism, the correlation between CQ and TD phenotype, and possible approaches of its side effects attenuation in vivo. These results will be confirmed by other quinoline derivatives. The work is expected to explain the medicinal molecular mechanism of quinoline derivatives,and provide significant therapeutic strategy for the side-effect attenuation of quinoline derivatives. It is meaningful to combine quinoline derivatives with vitamines to treat some diseases in the future.

喹啉类药临床应用广泛，但药物引起的神经炎、视网膜损伤等副作用，机理不明晰。我们前期应用化学遗传学方法研究发现：对硫胺素(VB1)转运子竞争性抑制是含喹啉环衍生物发挥生物学活性的一个普遍规律。但与副作用是否相关？项目拟从细胞、分子和在体三个层次开展：首先，研究氯碘喹啉(CQ)处理细胞效应，检测对细胞内硫胺素稳态及其参与生理功能的影响，阐明CQ拮抗VB1细胞机制，检测CQ衍生物机制保守性。其次，研究CQ拮抗VB1生化分子机制，计算机模建分析结合表面等离子共振等检测化合物与靶蛋白离体拮抗结合能力、构效关系及关键结构域；分子遗传分析响应等位基因对CQ及其衍生药物细胞敏感性遗传贡献，阐明CQ拮抗VB1分子基础。最后，通过食物及食物联合CQ诱导建立TD(VB1缺乏)动物模型，进一步研究喹啉在体对VB1代谢的影响及副作用消除策略。结果将拓展喹啉类活性机制认识，为联合用药预防副作用有重要的临床意义。

喹啉衍生物临床应用广泛,但长期服用引起的神经炎、视网膜损伤等副作用的机理不明晰。本课题组前期研究发现：喹啉环衍生物在酵母细胞中会对硫胺素(VB1)转运子竞争性抑制。本项目从动物细胞、分子和在体三个层次研究了氯碘羟喹（CQ）与VB1的拮抗竞争机制：首先，我们发现CQ对SH-SY5Y，HeLa，SMMC7721，QSG7701有生长抑制作用，可引起细胞自吞噬；通过高效液相色谱检测CQ对细胞内VB1和焦磷酸硫胺素含量的影响，发现CQ明显减少细胞内VB1的含量，而焦磷酸硫胺素含量出现了增加。通过检测以焦磷酸硫胺素为辅酶的转酮醇酶酶活，表明CQ显著减弱该酶活性，而对转酮醇酶的mRNA和蛋白表达水平的研究发现，虽然CQ对转酮醇酶的mRNA水平无明显影响，但下调了其蛋白的表达，表明CQ参与的VB1拮抗效应具有真核细胞保守性。其次，利用实时荧光定量PCR检测CQ对硫胺素转运子mRNA水平的影响，发现SLC19A2的mRNA表达量无明显差异，已知CQ作用下细胞内VB1的含量明显下降，由此推测CQ与VB1竞争结合位点,计算机建模分子对接证实它们之间存在竞争结合位点，分别是：arg-29, tyr-63, trp-284,和gln-418；而且我们的研究发现CQ通过引起胞内Ca2+积累来诱导SH-SY5Y细胞的周期停滞；而CQ介导的VB1缺乏和Ca2+累积之间的相关性研究有待未来的探讨；最后，我们建立VB1缺乏小鼠模型，检测CQ对体内VB1和焦磷酸硫胺素含量的影响，发现CQ明显减少细胞内VB1的含量，并显著减弱转酮醇酶酶的活性，VB1的添加可以回补CQ的效应。从而在在体水平再次证实CQ参与VB1吸收、转运和代谢表现VB1拮抗效应。本项目的研究结果对回答喹啉类化合物药用分子机制，临床如何联合VB1使用喹啉类药物消除副作用具有重要指导作用; 也为以VB1信号系统为靶标的新药研发提供理论依据。