线粒体靶向抗肿瘤药物的合成和生物活性及其机理

发布时间：2018-06-01 03:55

本文选题：线粒体靶向 + 抗肿瘤药物　；参考：《武汉大学》2014年博士论文

【摘要】：在最近的一系列抗肿瘤治疗研究报道中,发现肿瘤在基因表达水平上存在明显差异,同一类型的肿瘤在不同患者身上都有着不同的突变类型,甚至在同一肿瘤上也出现了不同的突变体。这些研究报道均表明,仅仅针对某一单独的基因或者单独的代谢途径的抗肿瘤治疗,是无法解决问题的。因此,寻找一种稳定不易突变且具有肿瘤病理学广泛适用性的靶标,就成为了抗肿瘤治疗研究亟待解决的问题。因为在肿瘤形成过程中线粒体功能经常发生改变,而且线粒体是细胞死亡的关键调控因素,所以,线粒体靶向药物代表了一种抗肿瘤治疗(特别是针对耐化疗的细胞系)的新方向。目前,这类线粒体靶向抗肿瘤药物生物活性及其机理的研究方法,主要集中在细胞水平上的实验。但是,关于线粒体靶向抗肿瘤药物影响线粒体功能导致的线粒体功能变化的直接结果,则很少见报道。为了克服这些局限性,我们把线粒体从细胞或组织中提取出来,再利用仪器直接检测离体线粒体的功能,从而实时动态观察线粒体靶向抗肿瘤药物对离体线粒体具体的生理参数的影响。这些结果有助于人们从亚细胞(线粒体)水平上理解线粒体靶向抗肿瘤药物的生物活性及其机理。另外,对于某些有特异靶向作用的药物来说,只有通过生物体的吸收、生物大分子对药物的结合、运载和释放,才能使药物小分子到达生物体内的靶向目标,最终实现靶向给药。线粒体靶向抗肿瘤药物对血清蛋白、DNA等生物大分子的相互作用情况,对于理解药物在分子水平、基因水平的作用机理,以及探索药物前期研发过程中的ADME (absorption, distribution, metabolism and excretion)指标都有积极的指导意义。本论文共分为六章：第一章：本章对线粒体靶向抗肿瘤药物的分类及研究进展做了较全面的介绍。第二章：选取大鼠离体线粒体、人血清白蛋白(HSA)和小牛胸腺DNA(ctDNA)为研究对象,系统研究了α-TOS对离体线粒体和生物大分子的生物功能和活性的影响。通过实验研究,我们发现α-TOS不仅会抑制线粒体的呼吸作用、引发线粒体的膜渗透性转换(MPT),还会对线粒体的生物膜与超微结构造成强烈扰动。此外,我们还发现α-TOS与人体内重要的运输蛋白HSA以一种适中的方式在位点Ⅰ结合形成复合物,并且此结合过程不会对HSA本身的结构及功能造成负面影响。α-TOS与遗传物质DNA的结合方式,为一种典型的沟槽作用,并且结合位点极有可能在DNA的大沟区。第三章：本章选取大鼠离体线粒体和人血清白蛋白(HSA)为研究对象,系统研究了F16两种异构体(o-F16,m-F16)对离体线粒体和生物大分子的生物功能和活性的影响。研究发现,o-F16对离体线粒体的影响与F16比较类似,都能够引起线粒体膜电位的坍塌,进而引发线粒体的膜渗透转换(MPT),对线粒体的生物膜与超微结构造成强烈的破坏,同时抑制线粒体的呼吸作用。而m-F16对离体线粒体的毒性明显小于F16和o-F16,虽然能引发线粒体的膜渗透转换(MPT),并对线粒体的膜结构与超微结构造成破坏,但与F16和o-F16不同,这种破坏不是通过引起线粒体膜电位的坍塌来实现的。对o-F16和m-F16与HSA相互作用的研究发现,o-F16对HSA荧光猝灭的机理与F16相同,均为动态猝灭,而m-F16对HSA荧光猝灭机理为静态猝灭。我们根据上述实验现象,提出了一种可能的构效关系理论模型。第四章：本章选取人血清白蛋白(HSA)为研究对象,通过荧光光谱法、紫外光谱法、圆二色光谱法和分子对接模拟计算,系统研究了桦木酸(BA)和HSA的相互作用。通过实验研究,我们发现BA与人体内重要的运输蛋白HSA以范德华力和氢键的方式在位点I结合形成复合物,并且此结合过程不会对HSA本身的结构及功能造成负面影响。第五章：本课题组曾经尝试通过柔性连接基团将线粒体靶向抗肿瘤小分子F16与其他小分子连接,但线粒体靶向效果不甚理想。基于此,本章尝试将连接基团更换为共轭结构,设计并合成了一种通过苯乙炔键将F16与BODIPY连接的连接物,并初步测定了其具有较为明显的线粒体靶向效果。希望通过更换连接基团的方法,解决前期设计方案中出现的瓶颈问题(无法定位到线粒体),并为以后的功能性小分子设计(线粒体靶向抗肿瘤)提供一定参考。第六章：本章设计并合成了一种pH敏感的双功能(抗肿瘤、抗氧化)药物维生素E-5-氟尿嘧啶连接物,此化合物可以在酸性环境中释放并表现出抗肿瘤和抗氧化的双重活性,而在中性和碱性环境中无此活性。此研究为基于抗氧化的胃部化疗研究提供了一种潜在的思路。
[Abstract]:In a recent series of anti-cancer studies, there was a significant difference in the level of gene expression. The same type of tumor had different types of mutations in different patients and even different mutants on the same tumor. It is impossible to solve the problem of antitumor therapy for individual metabolic pathways. Therefore, finding a target that is stable and unmutable and with extensive application of tumor pathology has become an urgent problem to be solved in the study of antitumor treatment. The mitochondrial function often changes during the process of tumor formation, and the mitochondria are cells. Therefore, mitochondrial targeting drugs represent a new direction for anticancer therapy, especially for chemotherapy resistant cell lines.
At present, the research method of this kind of mitochondria targeting the bioactivity and its mechanism of antitumor drugs is mainly concentrated on the cell level. However, the direct results about mitochondrial function changes caused by mitochondrial target to the function of mitochondria are rarely reported. In order to overcome these limitations, we put mitochondria in the mitochondria. The function of the isolated mitochondria was detected directly from the cells or tissues, and the effect of the mitochondria targeted antitumor drugs on the specific physiological parameters of isolated mitochondria was observed in real time. These results help people to understand the biological activity of mitochondria targeting antitumor drugs at the level of subcellular (mitochondria). And its mechanism.
In addition, for some specific targeted drugs, only through the absorption of organisms, the combination of biological macromolecules to the drug, carrier and release, can the small molecules reach the target target in the organism, and finally achieve the target drug delivery. Mitochondria target anti swelling drugs on the interaction of serum proteins, DNA and other biological macromolecules. The use of ADME (absorption, distribution, metabolism and excretion) has a positive guiding significance in understanding the molecular level of the drug at the molecular level and the mechanism of the gene level, as well as in the exploration of the early stage of drug development.
This paper is divided into six chapters.
Chapter 1: this chapter gives a comprehensive introduction to the classification and research progress of mitochondrial targeted antitumor drugs.
In the second chapter, we selected the isolated mitochondria, human serum albumin (HSA) and calf thymus DNA (ctDNA) as the research object. The effects of alpha -TOS on the biological function and activity of isolated mitochondria and biological macromolecules were systematically studied. Through experimental study, we found that the alpha -TOS not only inhibits mitochondrial respiration and induces membrane permeation of mitochondria. MPT also causes strong disturbance to the membrane and ultrastructure of mitochondria. In addition, we also found that alpha -TOS combined with important transporter HSA in the human body to form complex in a moderate way of site I, and this binding process does not have a negative impact on the structure and function of HSA itself. Alpha -TOS and genetic material D The binding mode of NA is a typical groove action, and the binding sites are likely to be located in the groove area of DNA.
In the third chapter, in this chapter, the effects of F16 two isomers (o-F16, m-F16) on the biological function and activity of isolated mitochondria and biological macromolecules were studied in vitro, and the effect of o-F16 on isolated mitochondria was similar to that of F16, which could cause mitochondrial membrane electricity. The collapse of the site leads to the membrane osmotic transformation of mitochondria (MPT), which destroys the membrane and ultrastructure of mitochondria and inhibits the mitochondrial respiration. The toxicity of m-F16 to mitochondria is significantly less than that of F16 and o-F16, although it can lead to mitochondrial membrane osmotic transformation (MPT), and the membrane structure and ultrastructure of mitochondria. Structural damage is caused, but unlike F16 and o-F16, this failure is not caused by the collapse of the mitochondrial membrane potential. The study of the interaction of o-F16 and m-F16 with HSA shows that the mechanism of o-F16 quenching of HSA fluorescence is the same as F16, and the quenching mechanism of m-F16 to HSA is static quenching. We are based on the above experiments. A possible theoretical model of structure-activity relationship is proposed.
The fourth chapter: in this chapter, human serum albumin (HSA) is selected as the research object. The interaction between Betula acid (BA) and HSA is systematically studied by fluorescence spectrometry, ultraviolet spectroscopy, circular two color spectroscopy and molecular docking simulation. Through experimental study, we found that BA and the important transport protein HSA in human body are in the mode of van Edward force and hydrogen bond The site I combines to form a complex, and this binding process does not negatively affect the structure and function of HSA itself.
In the fifth chapter, we tried to connect the mitochondria targeting the small molecule F16 with other small molecules through the flexible connection group, but the mitochondrial targeting effect was not ideal. Based on this, this chapter tried to replace the connecting group as conjugated structure, and designed and synthesized a connection of F16 and BODIPY via a benzyl acetylene bond. It has been preliminarily determined that it has a more obvious mitochondrial targeting effect. It is hoped that the replacement of the connecting group can solve the bottleneck problem in the early design scheme (unable to locate the mitochondrion), and provide some reference for the later functional small molecule design (mitochondria targeting anti-tumor).
The sixth chapter: this chapter designs and synthesizes a pH sensitive bifunctional (antitumor, antioxidant) drug vitamin E-5- fluorouracil junction, which can be released in the acid environment and exhibit dual activity of antitumor and antioxidation, and no activity in neutral and alkaline environments. This study is based on antioxidant stomach. The study of therapy provides a potential way of thinking.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R979.1

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