基于生物信息学对耐药结核分枝杆菌潜在药物靶点的挖掘分析

发布时间：2018-08-31 13:09

【摘要】：结核病是全球性的重大健康问题,近年来,由于艾滋病病毒与结核分枝杆菌双重感染的流行,加之抗生素的滥用,使结核分枝杆菌多重耐药问题日趋严峻,已成为结核病防治的重大难题,也是结核病全球蔓延、疫情恶化的重要原因。自1963年发现利福平、异烟肼等一线抗结核药物以来,尚未研发出新型的、高效的抗结核药物用以替代传统的抗结核药物。究其原因,主要是由于结核分枝杆菌有效的药物作用靶分子数量不足,远远不能满足当前结核病防治的需要,尤其是多重耐药结核分枝杆菌治疗的需要。因此结核分枝杆菌药物作用靶标的高通量筛选是新型抗结核药物研发及结核病防治的第一步,也是关键的一步。结核分枝杆菌细胞壁主要由荚膜、分支菌酸、肽聚糖、阿拉伯糖、内膜组成。这些结构对于维持结核分枝杆菌细胞形态的完整性、抵御外界化学物质的侵蚀、逃脱免疫以及产生耐药性和致病性都起着重要的作用,因此结核分枝杆菌细胞壁相关物质代谢及合成途径可以作为抗结核药物作用靶点。传统的抗结核菌一线药物如异烟肼等,其作用靶点也正是抑制结核分枝杆菌细胞壁中的分枝菌酸的合成。但是结核分枝杆菌的基因型极其复杂和多变,传统的药物作用靶点如inhA、ahpC、nadh、katG、KasA等都出现了耐药性突变,新的细胞壁相关基因的筛选迫在眉睫。此外,结核分枝杆菌细胞壁的组成成分复杂、功能繁多,传统的结核分枝杆菌细胞壁相关基因的筛选主要通过基因或敲除或RNA沉默等方法对潜在的细胞壁合成及代谢靶分子进行逐一的筛选,实验成本高,效率低,而且由于实验技术的限制,目前尚无任何一种实验方法可以实现全基因组的细胞壁合成相关分子的系统性筛选,因此,亟待建立以结核分枝杆菌信号传导动态网络为基础的细胞壁合成相关基因全景扫描及筛选新模式。鉴于此,本研究收集了2013年5月之前的所有结核分枝杆菌H37Rv的基因芯片共计43个系列2861张芯片。通过整合聚类,建立结核分枝杆菌细胞壁相关基因、未知基因和一般基因的三元素网络图,通过聚类基因簇的模体分析,高通量注释结核分枝杆菌细胞壁相关基因,为研发有效的、敏感的、副作用低的抗结核药物奠定分子基础。到目前为止人们关于异烟肼对结核分枝杆菌的影响研究结果仍然表浅,公认的结论仅仅停留在其对结核分枝杆菌的KatG基因和对细胞壁的结构产生影响这一层面。根据人们对该基因的功能研究和细菌形态学发现其主要功能都集中于细菌耐药的相关研究,但对于异烟肼如何杀伤细菌的机制研究甚少。异烟肼一定对分枝杆菌产生了更致命的作用,但这些作用由于观察和证实的困难性还没有被揭示。而这往往才是最主要的作用。对于如何揭示这些药物作用靶点,我们考虑利用药物的副作用作为切入点来探索药物对分枝杆菌的作用靶点。药物作用于人体后产生一系列的生物化学反应,人们利用药物主要反应治疗疾病,并且尽量规避众多副作用。但是就是这些副作用为确定药物作用靶点提供了可能。Monica Campillos等人通过不同药物相似的副反应推断他们有相同的作用靶点。这个理论为我们探索药物作用靶点提供了可能。异烟肼是一线抗结核药物,在治疗结核病的同时也会对人体的肝脏、维生素B6的代谢和氧化应激过程产生影响。这也证明这些组织和生物过程中共同存在着异烟肼的结合靶点或作用靶点。而且异烟肼又是可以作用于结核的药物,那么人体和结核分枝杆菌中一定存在着一个或一些异烟肼共同作用的靶点。联合分析异烟肼作用于结核分枝杆菌和人体的相关基因,通过大量交互性基因序列比对找到共同的基因序列,从生物学和统计学的角度进一步分析其成为药物作用靶点的可能性。这种方法突破了传统的细胞学或者分子学方法筛选药物作用靶点的瓶颈,极大的提高筛选靶点的速度和效率。为研究异烟肼的药理及对结核分枝杆菌细菌的致死作用提供了理论基础。
[Abstract]:Tuberculosis is a major global health problem. In recent years, due to the epidemic of double infection of HIV and Mycobacterium tuberculosis, coupled with the abuse of antibiotics, the problem of multiple drug resistance of Mycobacterium tuberculosis has become increasingly serious. It has become a major problem in tuberculosis control and prevention. It is also an important reason for the global spread of tuberculosis and the deterioration of the epidemic situation. Since the discovery of rifampicin, isoniazid and other first-line anti-tuberculosis drugs in 1997, no new, highly effective anti-tuberculosis drugs have been developed to replace traditional anti-tuberculosis drugs. The main reason is that the number of effective drug targets of Mycobacterium tuberculosis is insufficient, far from meeting the needs of current tuberculosis control, especially multiple anti-tuberculosis drugs. High-throughput screening of targets for drug-resistant Mycobacterium tuberculosis is therefore the first and key step in the development of new anti-tuberculosis drugs and in the prevention and treatment of tuberculosis. Mycobacterium tuberculosis cell morphological integrity, resistance to external chemical erosion, escape from immunity and the production of drug resistance and pathogenicity play an important role, so the metabolism and synthesis of cell wall-related substances of Mycobacterium tuberculosis can be used as a target of anti-tuberculosis drugs. Traditional first-line anti-tuberculosis drugs such as isoniazid However, the genotype of Mycobacterium tuberculosis is extremely complex and changeable. The traditional drug targets such as inhA, ahpC, nadh, katG, KasA, etc. all have drug resistance mutations, and the screening of new cell wall-related genes is imminent. The components of cell wall of Mycobacterium tuberculosis are complex and have many functions. The traditional screening methods of genes related to cell wall of Mycobacterium tuberculosis mainly through gene knockout or RNA silencing to screen the potential target molecules of cell wall synthesis and metabolism one by one. The experimental cost is high and the efficiency is low. Therefore, it is urgent to establish a new panoramic scanning and screening model for genes related to cell wall synthesis based on the dynamic signal transduction network of Mycobacterium tuberculosis. Bacillus H37Rv gene chips consist of 43 series of 2861 chips. Through integrated clustering, a three-element network diagram of cell wall-related genes, unknown genes and general genes of Mycobacterium tuberculosis was established. Through the analysis of clustering gene clusters, high-throughput annotation of cell wall-related genes of Mycobacterium tuberculosis was made, which is an effective, sensitive and side-effect for research and development. The molecular basis is laid by low levels of anti-tuberculosis drugs. Up to now, studies on the effect of isoniazid on Mycobacterium tuberculosis are still superficial, and the accepted conclusion is only that it affects the KatG gene of Mycobacterium tuberculosis and the structure of the cell wall. Morphological studies have found that the main functions of isoniazid are related to bacterial resistance, but little is known about how isoniazid kills bacteria. Isoniazid must have a more lethal effect on Mycobacterium, but these effects have not been revealed due to the difficulty of observation and confirmation. This is often the most important role. To uncover these drug targets, we consider using the side effects of drugs as a starting point to explore the targets of drugs on Mycobacterium. Drugs act on the human body and produce a series of biochemical reactions. People use the main reactions of drugs to treat diseases and try to avoid many side effects. But these side effects are Monica Campillos et al. inferred that different drugs had the same target through similar side effects. This theory provides a possibility for us to explore drug targets. Isoniazid is a first-line anti-tuberculosis drug that treats tuberculosis but also generates vitamin B6 in the human liver. It also proves that these tissues and biological processes have the binding or action targets of isoniazid, and isoniazid is a drug that can act on tuberculosis, so there must be one or some targets of isoniazid in human and Mycobacterium tuberculosis. Nicotinic hydrazine acts on the related genes of Mycobacterium tuberculosis and human body, finds the common gene sequence through a large number of interactive gene sequence alignment, and further analyzes the possibility of nicotinic hydrazine as a drug target from the biological and statistical point of view. This study provides a theoretical basis for studying the pharmacology of isoniazid and its lethal effect on Mycobacterium tuberculosis.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

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