【作者】 李慧；

【导师】 杨光富； 吴嘉炜；

【作者基本信息】 华中师范大学 ， 农药学， 2012， 博士

【摘要】 线粒体作为真核细胞的重要“能量工厂”,是细胞进行呼吸作用的场所。线粒体呼吸链(又称氧化磷酸化电子传递链)位于线粒体内膜上,由四个相对分子质量很大的跨膜蛋白复合体(Ⅰ、Ⅱ、Ⅲ、和Ⅳ)、介于Ⅰ/Ⅱ与Ⅲ之间的泛醌以及介于Ⅲ与Ⅳ之间的细胞色素c共同组成。线粒体呼吸链的功能是进行生物氧化,并与称之为复合体V的ATP合成酶相偶联,共同完成氧化磷酸化过程,并生产能量分子ATP。琥珀酸-细胞色素c氧化还原酶(SCR)是由复合体Ⅱ和复合体Ⅲ所组成。复合体ⅠI (SQR,EC1.3.5.1)可以催化电子由succinate至Q的传递。而复合体Ⅲ(QCR)又称为细胞色素bc1复合体(Cytochrome bc1complex, EC1.10.2.2)是呼吸作用能量转换的中心成分,它主要催化电子由氢醌(QH2)到细胞色素c(cyt c)的传递并促使质子跨膜。研究表明复合体Ⅱ和复合体Ⅲ均可作为理想的农用杀菌剂作用靶标,具有重要的农学意义和应用价值。本文希望能从分子水平上研究复合体Ⅱ、复合体Ⅲ以及它们的复合酶(SCR)分别与底物,抑制剂的相互作用,为抑制剂的科学合理设计提供理论指导,分别设计开发出以复合体Ⅱ和复合体Ⅲ为靶点的新型杀菌剂。在本论文中我们主要进行了以下四方面的工作：一、建立了琥珀酸-细胞色素c氧化还原酶提取与纯化,以及纯度和活性鉴定的方法。试图进行SCR的结构生物学研究,获得了一些微晶,但没有衍射。二、建立了适合于本课题的研究体系,对其进行拓展,发展了适合高通量筛选和动力学研究的紫外法酶活检测体系,并针对本课题建立了等温微量热酶活检测的新方法。三、对复合体Ⅱ抑制剂进行了筛选及抑制动力学研究。1、分析了复合体Ⅱ的十种商品化杀菌剂,确定它们活性的优劣次序,并通过动力学的手段进一步确定它们的作用方式,我们发现它们与succinate以及DCIP是非竞争性的,而与底物Q是竞争性或混合性的,说明这些抑制剂与底物succinate以及DCIP在复合体Ⅱ上的结合位点不同,但与底物Q在复合体Ⅱ上的结合位点相近。2、对我们课题组以复合体Ⅱ为靶点合成的新化合物进行分析,发现部分化合物的离体活性与商品化抑制剂相当,但活体活性较差,研究表明它们不能将复合体Ⅱ的催化活性抑制到底,均有30%-40%左右的剩余活力,基于此我们提出用部分非竞争机制来解释不能抑制到底的原因,并通过动力学实验进行了验证。活性最好的化合物Ki值可达0.4464±0.0624μM。四、对复合体Ⅲ抑制剂进行了筛选及抑制动力学研究。1、分析了复合体Ⅲ商品化的杀菌剂,详细测定了其中六种杀菌剂的抑制动力学。2、详细研究了噁唑菌酮类抑制剂的动力学。发现新合成的该类化合物中活性最好的化合物为8d,对8d和对照样品商品化噁唑菌酮的动力学研究表明它们均与底物细胞色素c非竞争,而与底物DBH2竞争的与酶结合,且指出之前的相关动力学报道失当的原因。另外,我们还发现化合物8d和噁唑菌酮对SCR的抑制能力比对QCR的抑制能力分别强35和15倍,这与嘧菌酯表现出的对SCR的抑制能力与对QCR的抑制能力相当明显不同。该结果表明噁唑菌酮类抑制剂不仅能抑制复合体Ⅲ的催化活力而且能干扰复合体Ⅱ和复合体Ⅲ之间的相互作用。3、对课题组根据PFVS方法发现的新化合物进行了生物活性测试,发现含二苯甲酮结构和含喹喔啉结构的Strobilurins类衍生物具有进一步的研发价值。1)含二苯甲酮结构的Strobilurins类衍生物中活性最好的化合物为Ⅲ1b(Ki=4.28nM),它对SCR和QCR体系的动力学结果一致。该系列部分化合物还具有抗疟疾活性。另外,如果将二苯甲酮的两个苯环做了一个并环形成三个环的结构,可得到活性更好的Ⅲ4a(K=1.8987±0.0432nM),会表现出慢结合的动力学特征。2)含喹喔啉结构的Strobilurins类衍生物中发现大量高活性的化合物,如化合物9f(Ki=0.04300nM)。这些化合物大多表现出慢结合的动力学特征,它们与底物细胞色素c非竞争,而与底物DBH2竞争性的与酶结合。我们还通过合作研究,得到了其中代表性化合物9e与鸡心来源的复合体Ⅲ的共结晶结构,该结构证实了化合物9e的确结合在底物DBH2的同一结合位点,从而验证了我们动力学结果的正确性,另外发现该化合物与复合体Ⅲ形成了若干个C-F-H键,这可能是该化合物之所以与酶具有高亲和力以及出现慢结合动力学行为的主要原因。4、发现同结构类型的化合物表现出不同的动力学特征。因此提出对具有同样结构类型的甲氧基丙烯酸酯类复合体Ⅲ抑制剂进行新的分类。按照抑制剂与酶结合解离速度的不同分为经典型(快结合,解离常数>0.01s-1)抑制剂与非经典型(慢结合,解离常数≤0.01s-1)抑制剂,并特别指出对于非经典型抑制剂进行动力学参数拟合时,传统慢结合经验公式适用于实验中所用[I]>10[E]0且0.001s-1<K-0≤0.01s-1,其优势在于使用简单方便；而不化简的慢紧结合公式适用于实验中所用[I]与[E]o相当甚至更小,此时K_o≤0.001s-1,其优势在于拟合的更准确。综上所述,本文建立起了琥珀酸-细胞色素c氧化还原酶的提取、纯化、活性及纯度鉴定体系,并分别针对以复合体Ⅱ和复合体Ⅲ为靶点的抑制剂进行筛选及抑制动力学研究,对出现的部分非竞争机制和时间依赖型(慢结合)机制进行了详细的阐述,并针对同类型的抑制剂会呈现出不同的动力学行为提出了新的分类标准,为设计高活性、开发新类型抑制剂提供重要的理论指导和新思路。更多还原

【Abstract】 As the important "energy factory" of eukaryotic cells, mitochondrion is the place for cellular respiration. The mitochondrial respiration chain (OXPHOS electron transfer chain) is located on the inner membrane of mitochondrion and comprising of four large trans-membrane protein complexes (mitochondrial respiratory Complex Ⅰ, Ⅱ, Ⅲ and Ⅳ) as well as ubiquinone between Complex Ⅰ/Ⅱ and III and cytochrome c between Complex Ⅲ and Ⅳ. The function of mitochondrial respiration chain is biological oxidation which is coupled with phosphorylation by ATP synthesis enzyme (ATPase, called Complex V) and then complete OXPHOS producing energy molecule ATP.Succinate-ubiquinone reductase (SCR) composed by complex Ⅱ and III. Complex Ⅱ (SQR, EC1.3.5.1) catalyzes electron transfer from succinate to Q, and complex III (also known as cytochrome bcl complex, EC1.10.2.2) is a key component of the respiratory chain, in which it catalyzes electron transfer from ubiquinol (QH2) to cytochrome c (cyt c) while translocating protons across the inner mitochondrial membrane. Both complex Ⅱ and III can used to target for fungicides.First, we established the extraction and purification of succinate-ubiquinone reductase, and evaluated its purity and activity. We want to research the structural biology of SCR, obtain some of crystals, but diffraction not good and need to further optimization.Second, we developed a UV assay for SCR, SQR and QCR enzyme assay for highthroughput screening and a new enzyme assay for SCR through isothermal titration calorimetry.Third, we studied the inhibitor kinetics of complex Ⅱ inhibitors.1、Determined the activity and mode of action for ten commercial inhibitors of complex Ⅱ. We found they are non-competitive inhibitors with respect to the substrate succinate and DCIP, but competitive inhibitors with respect to substrate Q. that indicated the bind site of these inhibitors different with succinate and DCIP, but near to Q.2、We have biologically evaluated to new compounds which synthesized by our group. Partial compounds can inhibit the activity of complex Ⅱ nearly with commercial inhibitors, but the complex Ⅱ activity can not disappear by these, about30%-40%activity were remained. So we proposed and confirmed a partial noncompetitive inhibition mechanism. The Ki value of best compound was0.4464±0.0624μM.Fourth, we studied the inhibitor kinetics of complex III inhibitors.1、 Determined in detail for six commercial inhibitors of complex Ⅲ.2、 New oxazolidinedione derivatives were biologically evaluated. The further inhibitory kinetics studies against SCR revealed that the representative compound8d and famoxadone are both non-competitive inhibitors with respect to the cytochrome c, but competitive inhibitors with respect to DBH2. In addition, compound8d and famoxadone showed, respectively,35-fold and15-fold greater inhibitory activity against the porcine SCR than complex Ⅲ, indicating that these two inhibitors not only inhibited the activity of complex Ⅲ, but possibly affect the interaction between the complex Ⅱ and the complex Ⅲ.3、 Based on the method of pharmacophore-linked fragment virtual screening, we found the strobilurin derivatives which containing functional groups benzophenone and quinoline oxime morpholine were worth further research.1) ⅢIb (Ki=4.28nM) was best and partial compounds have antimalarial activity. Furthermore, if we connect two benzene ring, get Ⅲ4a (Ki=1.8987±0.0432nM) which activity will be better and show slow-binding.2) we found lots of high-activity compounds in new quinoline oxime morpholine strobilurin derivatives such as9f (Ki=0.04300nM). These high-activity compounds were slow-binding inhibitors and noncompetitive with cytochrome c but competitive with DBH2. Additionally, we determined the crystal structure of compound9e bound to the chicken complex III at2.70A resolution, providing a molecular basis for understanding its ultrapotency and confirm our results about kinetics were correct.4、We found strobilurin inhibitors have similar structure but different kinetic feature. According to the dissociation constant, we proposed a new classification which classical (Ko>0.01s-1) and non-classical (Ko≤0.01s-1) inhibitors of strobilurin. Furthermore, we proposed in the inhibition experiment,[I]> IO[E]o traditional formula can be used, but [I] comparable with [E]o, we need to use no simplification formula if we want to obtain more accurate data.In summary, this paper established the systems of extraction, purification, enzyme assay and purity evaluation of succinate-ubiquinone reductase. Furthermore, we studied the inhibitor kinetics and designed the inhibitors to target to complex Ⅱ and complex Ⅲ, elaborated in detail the partial noncompetitive and time-dependent (slow-binding) inhibition mechanism, and proposed a new classification. All these results would shed light on the research of inhibitor with high activity and new structure.更多还原