靶向肿瘤相关重要蛋白BRD4和RhoA小分子抑制剂的发现及作用机制的研究

发布时间：2018-04-09 22:01

本文选题：表观遗传相关蛋白　切入点：细胞骨架类蛋白　出处：《南昌大学》2017年硕士论文

【摘要】：近年来,随着生物医药领域的迅猛快速发展,靶向特定蛋白筛选其特异性的小分子抑制剂已成为该领域的重点。本文中着眼于表观遗传相关蛋白及细胞骨架类蛋白这两个不同的领域中的重要蛋白进行小分子抑制剂的筛选。组蛋白乙酰化作为表观遗传学中重要的一类翻译后修饰,在调控基因转录与染色体构成等多个领域中发挥着重要的作用,并且与癌症的发生发展密切相关,因此,组蛋白乙酰化已成为国内外的研究热点。BRD4蛋白作为组蛋白乙酰化识别因子"reader",是属于BET家族的一员,它能够特异性结合乙酰化修饰的赖氨酸,并招募其他转录因子调控BCL2, CDK6, c-Myc等下游致癌基因。目前研究表明,BRD4的异常表达与MLL, AML等白血病以及睾丸中腺癌等众多疾病相关,因此靶向BRD4蛋白筛选小分子抑制剂是十分有必要的,在本文中,我们利用BRD4蛋白可以识别乙酰化的赖氨酸这一原理,结合AlphaScreen技术灵敏均相无放射性等优点,我们建立了 BRD4的“HITs”筛选平台,初步筛选得到了一类骨架新颖,活性较好的小分子抑制剂,该小分子在体外可以在0.81±0.03μM左右显著抑制BRD4蛋白识别乙酰化的组蛋白,不仅如此该化合物在细胞水平还可以抑制血液病细胞的增殖等,因此其是一个潜在的成药的小分子。RhoA作为GTP酶的一种,是重要的细胞信号通路转导因子,并影响细胞骨架的形态等,属于小G蛋白家族的一员。RhoA与上游的三种蛋白相互作用,并受这三种蛋白的调控,从而在RhoA-GTP活性状态及RhoA-GDP非活性状态之间相互转换。目前研究表明,RhoA的高表达与多种肿瘤的发生发展密切相关,例如乳腺癌,前列腺癌,睾丸癌,肺癌等。因此,RhoA作为重要的抗癌靶点,寻找针对RhoA的小分子抑制剂是必不可少的。而现有RhoA的小分子抑制剂存在活性差选择性差等重要的问题,因此我们基于蛋白热迁移实验建立了靶向RhoA的高通量筛选平台,筛选得到了抑制RhoA酶活性及与GEF相互作用的共价先导化合物,并以此化合物为分子探针,解释了 RhoA蛋白变构调控的新机制。
[Abstract]:In recent years, with the rapid development of biomedical field, the screening of specific small molecular inhibitors targeting specific proteins has become the focus in this field.In this paper, small molecular inhibitors were screened for two different domains, epigenetic related proteins and cytoskeletal proteins.Histone acetylation, as an important post translational modification in epigenetics, plays an important role in the regulation of gene transcription and chromosome formation, and is closely related to the development of cancer.Histone acetylation has become a hot topic at home and abroad. BRD4 protein is a member of the BET family as the recognition factor of histone acetylation, and it can specifically bind to the acetylated lysine.Other transcription factors were recruited to regulate BCL2, CDK6, c-Myc and other downstream oncogenes.Current studies have shown that the abnormal expression of BRD4 is associated with many diseases, such as MLL, AML and adenocarcinoma of testis, so it is necessary to screen small molecular inhibitors for targeting BRD4 protein.Using the principle that BRD4 protein can recognize acetylated lysine, and combining the advantages of AlphaScreen technique, such as sensitive homogeneity and non-radioactivity, we have established a "HITs" screening platform for BRD4.The small molecule inhibitor with good activity could significantly inhibit the acetylated histone recognized by BRD4 protein at 0.81 卤0.03 渭 M in vitro, and not only that the compound could inhibit the proliferation of hematological disease cells at the cell level, etc.Therefore, it is a potential proprietary drug molecule. RhoA, as one of the GTP enzymes, is an important cellular signal transduction factor and affects the morphology of the cytoskeleton. It belongs to the small G protein family member. RhoA interacts with three upstream proteins.The three proteins are regulated by these proteins, thus switching between the active state of RhoA-GTP and the inactive state of RhoA-GDP.Current studies have shown that the high expression of RhoA is closely related to the occurrence and development of many tumors, such as breast cancer, prostate cancer, testicular cancer, lung cancer and so on.Therefore, as an important anticancer target, it is necessary to find small molecular inhibitors for RhoA.However, the existing small molecular inhibitors of RhoA have some important problems, such as poor selectivity, etc. Therefore, we have established a high-throughput screening platform for targeting RhoA based on protein heat transfer experiments.The covalent lead compounds which inhibit the activity of RhoA enzyme and interact with GEF were obtained and used as molecular probes to explain the new mechanism of RhoA protein structure regulation.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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