一种新的MDM2-p53信号通路抑制剂的研究

本研究以研发新型小分子MDM2抑制剂为目的,建立了以分子对接为基础的虚拟筛选流程.利用虚拟筛选流程对SPECS化合物库的分子进行类药性筛选、分子对接粗筛、二次筛选以及排序挑选,并通过细胞实验验证这些分子激活p53并抑制肿瘤细胞生长的活性.结果表明M12能够激活p53及其下游信号通路,抑制肿瘤细胞周期并促进肿瘤细胞凋亡.M12与已知MDM2-p53抑制剂结构完全不同,是一种潜在的癌症治疗候选药物.     

筛选p53靶向药物的细胞模型的构建

为了筛选可以恢复肿瘤细胞中p53功能的小分子,作者用表达野生型p53的人类直肠癌细胞HCT116建立了一株能够应答激活p53信号通路的荧光素酶报告基因的稳定细胞系,同时用表达野生型p53的人类骨肉瘤细胞U2-OS建立了一株能够应答激活p53信号通路的mCherry红色荧光蛋白报告基因的稳定细胞系.为了检测筛选p53靶向药物的有效性,利用三种已知的以p53为靶点的小分子药物(cisplatin,doxorubicin以及Nutlin-3)处理这两种稳定细胞系,结果显示p53信号通路在这两个稳定细胞系中均能够被激活.为了探索小分子RNA作为恢复p53功能的靶标药物,并进一步验证这两种细胞模型用于药物筛选的可行性,分别检测了MDM2和MDMX的5个不同shRNA.通过比较HCT116稳定细胞的荧光素酶活性和U2-OS稳定细胞中荧光蛋白的荧光强度,我们筛选出了有效沉默MDM2或MDMX的shRNA.数据表明,这两种细胞模型不仅可用作筛选激活p53的小分子化合物的平台,而且可用于筛选激活p53信号通路的小分子RNA.     

6-氮杂甾醇类Ⅱ型5-α还原酶抑制剂的三维定量构效关系研究和虚拟筛选

采用Topomer CoMFA对37个6-氮杂甾醇类抑制剂进行三维定量构效关系分析,新建模型的交互验证和拟合相关系数为q2=0.774,r2=0.965,结果表明模型具有良好的可信度和预测能力.将基于配体的Topomer search虚拟筛选、基于受体的分子对接虚拟筛选和基于3D-QSAR模型的分子活性预测等方法运用到新抑制剂的分级筛选,最终获得4个高活性的新抑制剂分子.新抑制剂的Surflex-dock结果显示6-氮杂甾醇类抑制剂与5AR-Ⅱ靶点的作用模式主要是氢键作用.MTT法生物学活性测试结果显示新抑制剂能显著抑制BPH-1细胞增殖,且抑制程度呈浓度依赖性.通过分子对接机理解释和细胞学实验的抑制前列腺增生活性测试,这两种研究方式相互验证,证明此虚拟筛选方法能够为治疗良性前列腺增生的新药设计提供有效候选化合物.     

COX-2/DHODH双重抑制剂存在可行性的初步对接虚拟筛选研究

首次开展了探索环氧合酶-2(COX-2)和二氢乳清酸脱氢酶(DHODH)双重抑制剂存在的可行性研究工作。通过自建COX-2抑制剂数据库,采用虚拟筛选方法,以DHODH的晶体结构为靶标,对建的COX-2抑制剂数据库进行对接虚拟筛选,筛选出多个对DHODH可能有抑制作用并且具有COX-抑制活性的分子。研究表明,现有数据库中的25000系列、7000系列、26000系列以及33000系列的抑制剂最有可能成为DHODH和COX-2的双抑制剂,特别是其中的2500、7004和7006这三个抑制剂,具有进一步实验研究的价值。     

MDM2蛋白与抑制剂IMZ相互作用的研究

癌症是最难攻克的疾病之一,它被认为是基因疾病.P53是一种癌抑制蛋白,而MDM2是一种绑定在P53上致使其失去活性的一种癌蛋白,P53失去活性意味着癌症的发生.本文论述的是抑制剂IMZ（药物）与MDM2蛋白的相互作用,作用的目的是打断MDM2与P53的结合,使P53保持活性,这样就可以用来抑制癌症.     

白桦脂酸体外抗肿瘤的活性和机制

研究白桦脂醇（Betulin）和白桦脂酸（Betulinic Acid, BA）对3种癌细胞株的增殖抑制作用以及白桦脂酸诱导MCF 7细胞毒的分子机制. 应用结晶紫染色方法对白桦脂醇和白桦脂酸对肿瘤细胞的生长抑制作用进行筛选； 应用RT-PCR技术检测MCF 7细胞p21 mRNA, p53 mRNA, Bcl 2 mRNA和Bax mRNA的表达. 结果表明, 白桦脂醇和白桦脂酸有显著抑制肿瘤细胞生长的作用, 且有浓度依赖性; 白桦脂酸能够诱导MCF 7细胞凋亡, 并诱导其p21 mRNA和p53 mRNA表达增高, 但对其Bcl 2 mRNA和Bax mRNA 的表达无影响.     

抑癌基因p53与肿瘤发生及基因治疗的研究进展

概述了抑癌基因p53的结构和特性,以及p53的抑癌作用机理、基因治疗的研究进展.抑癌基因p53是人类细胞中非常重要的基因,野生型p53基因产物具有抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡的作用.在p53基因发生突变或缺失时,可引起多种肿瘤.当人工将野生型p53基因与载体结合成重组DNA分子导入肿瘤患者体内后,有可能达到治疗恶性肿瘤的目的.     

肿瘤抑制因子p53蛋白在毕赤酵母中的克隆表达

TP53基因在调控细胞信号通路和抑制肿瘤细胞中发挥着重要作用.本研究利用毕赤酵母表达系统以获得大量重组p53蛋白,为此,本文将人TP53基因克隆至表达载体p PIC3.5K,电转化进入毕赤酵母GS115,通过组氨酸筛选和G418筛选,获得高拷贝稳定转化子.SDS-PAGE和Western Blot检测结果表明p53蛋白可以在毕赤酵母中表达.本研究所获酵母菌株为今后大规模生产重组p53蛋白奠定了基础.     

HIV辅助受体CCR5常见小分子抑制剂抑制效果的综合评价

通过系统比较CCR5常见小分子抑制剂的抑制效果,寻找CCR5的优化对接靶点,筛选最优抑制剂,为新型抑制剂的研发提供理论依据.基于Material Studio软件,我们构建了7类29种小分子抑制剂的三维结构,全面模拟了各种抑制剂与CCR5对接的效果;利用分子对接绝对自由能、相对自由能、对接姿态百分比和LibDock综合得分等参数评估小分子抑制剂的抑制效果.研究初步发现,CCR5小分子抑制剂的最优对接位点是第二个胞外环与N末端之间的site4.小分子抑制剂的抑制效果由强到弱排序依次为:抑制剂27(吡咯烷类)、抑制剂8(苯并环庚烯类)、抑制剂12(哌啶类)、抑制剂14(螺环二酮哌啶类)、抑制剂21(4-哌啶-1-丁胺类)、抑制剂5(天然小分子类)和抑制剂17(托品烷类);其研究表明,吡咯烷类抑制剂27可能成为CCR5最优抑制剂候选对象.     

MdmX在Axin-p53信号通路中的调控机制

鼠双微体基因X(MdmX)是肿瘤抑制因子p53信号通路中重要的负调控蛋白,它能与p53直接结合,抑制p53的转录活性.体轴发育抑制因子Axin作为构架蛋白与p53及同源结构域互作蛋白激酶2(HIPK2)等相互作用形成复合物,诱导p53的转录活化,共同促进细胞的凋亡.本研究将MdmX引入Axin-p53信号通路的调控中,发现MdmX通过竞争Axin与p53的结合,抑制Axin诱导p53的转录激活.p53结合区域缺失的MdmX突变体MdmXΔp53则不能抑制Axin对p53的激活.这些实验结果为进一步深入研究Axin-p53信号通路在细胞凋亡及肿瘤发生发展中的作用提供了重要的理论依据.     

Amplification of a gene encoding a p53-associated protein in human sarcomas.

Despite extensive data linking mutations in the p53 gene to human tumorigenesis, little is known about the cellular regulators and mediators of p53 function. MDM2 is a strong candidate for one such cellular protein; the MDM2 gene was originally identified by virtue of its amplification in a spontaneously transformed derivative of mouse BALB/c cells and the MDM2 protein subsequently shown to bind to p53 in rat cells transfected with p53 genes. To determine whether MDM2 plays a role in human cancer, we have cloned the human MDM2 gene. Here we show that recombinant-derived human MDM2 protein binds human p53 in vitro, and we use MDM2 clones to localize the human MDM2 gene to chromosome 12q13-14. Because this chromosomal position appears to be altered in many sarcomas, we looked for changes in human MDM2 in such cancers. The gene was amplified in over a third of 47 sarcomas, including common bone and soft tissue forms. These results are consistent with the hypothesis that MDM2 binds to p53, and that amplification of MDM2 in sarcomas leads to escape from p53-regulated growth control. This mechanism of tumorigenesis parallels that for virally-induced tumours, in which viral oncogene products bind to and functionally inactivate p53.     

DNA damage stress induces the dissociation of Smurf1/2 from MDM2 in a slow manner

The tumor suppressor p53 locates at the key point of cell growth or apoptosis balance, and the expression level of p53 is tightly controlled by ubiquitin ligases including MDM2. Upon DNA damage stresses, p53 was accumulated and activated, leading to cell cycle arrest or apoptosis. We previously showed that Smad ubiquitylation regulatory factor 1/2 (Smurf1/2) promotes p53 degradation by interacting with and stabilizing MDM2, and consequently enhancing MDM2-mediated ubiquitylation of p53. However, it is unclear how the Smurf1-MDM2 interaction is regulated in response to DNA damage stress. Here, we show that in response to etoposide treatment Smurf1 dissociates from MDM2, resulting in MDM2 destabilization and p53 accumulation. The negative regulation of Smurf1 on apoptosis is released. Notably, this dissociation is a slow process rather than a rapid response, implicating high expression of Smurf1 might confer the resistance against p53 activation. Consistent with this notion, we observed that Smurf1/2 ligases are highly expressed in colon cancer, esophageal squamous cell carcinoma and pancreatic cancer tissues, suggesting the oncogenic tendency of Smurf1/2.     

Inactivation of the p53 pathway in retinoblastoma

Most human tumours have genetic mutations in their Rb and p53 pathways, but retinoblastoma is thought to be an exception. Studies suggest that retinoblastomas, which initiate with mutations in the gene retinoblastoma 1 (RB1), bypass the p53 pathway because they arise from intrinsically death-resistant cells during retinal development. In contrast to this prevailing theory, here we show that the tumour surveillance pathway mediated by Arf, MDM2, MDMX and p53 is activated after loss of RB1 during retinogenesis. RB1-deficient retinoblasts undergo p53-mediated apoptosis and exit the cell cycle. Subsequently, amplification of the MDMX gene and increased expression of MDMX protein are strongly selected for during tumour progression as a mechanism to suppress the p53 response in RB1-deficient retinal cells. Our data provide evidence that the p53 pathway is inactivated in retinoblastoma and that this cancer does not originate from intrinsically death-resistant cells as previously thought. In addition, they support the idea that MDMX is a specific chemotherapeutic target for treating retinoblastoma.     

中医药治疗便秘的用药规律及网络药理学探析

基于数据挖掘及网络药理学方法,探究中药治疗便秘的用药规律并分析核心中药的作用机制。通过检索中国知网、维普数据库、万方数据库中有关中药复方治疗便秘的文献,筛选符合要求的文献并提取数据,对中药复方的药物频次、关联规则及聚类进行分析。基于中药系统药理学数据库与分析平台获取核心药物的化学成分及靶点,通过GeneCards、OMIM、TTD等数据库获取疾病靶点,借助Venny平台获取潜在靶点,采用STRING数据库、DAVID数据库进行相关机制分析。基于AutoDock软件进行分子对接。结果表明:筛选出复方85个;使用频次在10以上的有25味药,其中白术的使用频率最高;关联规则挖掘出白术-黄芪、白术-黄芪-当归分别是支持度最高的组合;聚类分析可将药物分为5类;挖掘出核心中药白术、当归、枳实和火麻仁。网络药理分析显示,核心中药治疗便秘的机制是化学成分作用于AKT1等靶点,进而调节癌症通路、磷脂酰肌醇3激酶-蛋白激酶B信号通路、p53信号通路等发挥治疗作用。该研究为中医药治疗便秘的用药规律提供了理论基础,为治疗便秘的中药复方的开发与发展提供了思路。     

P53和MDM2蛋白表达对肝细胞肝癌预后判断的意义

目的 :研究肝细胞肝癌中突变型P5 3和MDM2蛋白的表达对临床预后判断的意义。方法 :应用免疫组织化学方法 ,检测 72例原发性肝细胞肝癌手术切除标本突变型P5 3、MDM2蛋白的表达 ;与临床病理学指标和术后生存期进行分析比较。结果 :突变型P5 3蛋白阳性 2 8例(38 89% ) ,MDM2蛋白阳性 2 3例 (31 94 % ) ,二者阳性表达有相关性 (r =0 2 4 8,P <0 0 5 )。突变型P5 3、MDM2蛋白阳性表达病例生存率明显低于突变型P5 3、MDM2蛋白阴性表达病例 (P <0 0 1)。突变型P5 3和MDM2蛋白表达均阳性病例 13例 (18 0 6 % ) ,中位生存期的生存率最低。单因素及多因素分析显示 ,突变型P5 3蛋白表达、MDM2蛋白表达、肿瘤大小与中位生存期的生存率有关 ,MDM2是统计学上最有意义的独立预后指标 (P <0 0 0 0 1)。结论 :应用免疫组织化学方法检测突变型P5 3和MDM2蛋白的表达可作为原发性肝细胞肝癌预后判断的指标。     

DNA damage stress induces the dissociation of Smurf1/2 from MDM2 in a slow manner

The tumor suppressor p53 locates at the key point of cell growth or apoptosis balance, and the expression level of p53 is tightly controlled by ubiquitin ligases including MDM2. Upon DNA damage stresses, p53 was accumulated and activated, leading to cell cycle arrest or apoptosis. We previously showed that Smad ubiquitylation regulatory factor 1/2 (Smurf1/2) promotes p53 degradation by interacting with and stabilizing MDM2, and consequently enhancing MDM2-mediated ubiquitylation of p53. However, it is uncle...     

NUMB controls p53 tumour suppressor activity

NUMB is a cell fate determinant, which, by asymmetrically partitioning at mitosis, controls cell fate choices by antagonising the activity of the plasma membrane receptor of the NOTCH family. NUMB is also an endocytic protein, and the NOTCH-NUMB counteraction has been linked to this function. There might be, however, additional functions of NUMB, as witnessed by its proposed role as a tumour suppressor in breast cancer. Here we describe a previously unknown function for human NUMB as a regulator of tumour protein p53 (also known as TP53). NUMB enters in a tricomplex with p53 and the E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing ubiquitination and degradation of p53. This results in increased p53 protein levels and activity, and in regulation of p53-dependent phenotypes. In breast cancers there is frequent loss of NUMB expression. We show that, in primary breast tumour cells, this event causes decreased p53 levels and increased chemoresistance. In breast cancers, loss of NUMB expression causes increased activity of the receptor NOTCH. Thus, in these cancers, a single event-loss of NUMB expression-determines activation of an oncogene (NOTCH) and attenuation of the p53 tumour suppressor pathway. Biologically, this results in an aggressive tumour phenotype, as witnessed by findings that NUMB-defective breast tumours display poor prognosis. Our results uncover a previously unknown tumour suppressor circuitry.