【作者】 杨胜乾；

【导师】 王莉莉；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药理学， 2011， 硕士

【摘要】 蛋白激酶是细胞内一类最重要的功能蛋白,在细胞信号通路中发挥着重要的作用。酪氨酸蛋白激酶(PTKs)是重要的蛋白激酶,其介导或者参与的细胞内信号通路在调节细胞增殖、分泌、分化和凋亡过程中占重要地位。其功能的失调与恶性肿瘤、免疫疾患、动脉粥样硬化、糖尿病等多种疾病的发生密切相关。PTKs抑制剂可有效调节PTKs活性,治疗PTKs参与的“细胞信号紊乱”相关疾病。研发活性高、选择性强的新型PTKs抑制剂具有重要意义。巨噬细胞集落刺激因子受体激酶(c-Fms),是由原癌基因c-fms编码的一种受体酪氨酸激酶,与血小板衍生生长因子α、β受体(PDGFRα,β)、干细胞生长因子受体(SCFR/c-Kit)、FMS样酪氨酸激酶3(FLT-3)等同属于Ⅲ型受体酪氨酸激酶家族。c-Fms与其配体巨噬细胞集落刺激因子(M-CSF)结合后,形成二聚体,进而表现出酪氨酸激酶的催化活性,激活c-Fms介导的胞内信号转导途径,促进单核巨噬细胞系的存活、增殖和分化。c-fms基因序列缺失、重复复制、突变、染色体易位等因素导致c-Fms的激酶活性异常增强,与骨髓增生异常综合征(MDS)、急性髓系白血病(AML)、急性巨核细胞白血病(AMKL)、动脉粥样硬化、乳腺癌、子宫颈癌、类风湿性关节炎等疾病密切相关。鉴于c-Fms独特的生物学作用与病理生理学特点,研发靶向该受体激酶的抑制剂可能发现新的靶向治疗药物。目前处于临床前研究阶段发展较好的c-Fms抑制剂有GW2580、Ki20227和JNJ-28312141,它们可有效抑制c-Fms激酶的磷酸化,改善小鼠肿瘤细胞转移引起的骨质溶解和糜烂;JNJ-28312141还可抑制大鼠移植肿瘤细胞的生长。因此,新型c-Fms抑制剂的发现是目前国际新药研发的一个重点和热点。鉴于高效筛选模型是新药发现的关键和核心的环节。因此,确定本课题研究的目的是:(1)建立c-Fms抑制剂的高效筛选模型和方法;(2)进行筛选化合物,以期发现活性强的新型c-Fms抑制剂。本研究工作由两部分组成:第一部分,c-Fms抑制剂筛选模型的建立,包括两个方面:(1)基于高内涵分析技术c-Fms抑制剂筛选模型的建立;(2)基于激酶盘不依赖M-CSF的c-Fms抑制剂细胞水平筛选模型的建立。第二部分,利用M-CSF依赖的细胞株和所建立的基于高内涵分析技术的c-Fms激酶抑制剂筛选模型进行化合物的筛选。第一部分,c-Fms抑制剂筛选模型的建立。基于高内涵分析技术c-Fms抑制剂筛选模型的建立:采用分子克隆和细胞转染技术,将构建的含有人c-Fms cDNA全长序列的真核表达载体,转染已稳定表达GFP-STAT1融合蛋白的人骨肉瘤细胞(U2OS),采用有限稀释法和小玻片培养法获得同时稳定表达人c-Fms和GFP-STAT1融合蛋白的单克隆细胞株(c-Fms/GFP-STAT1_U2OS)。以重组人巨噬细胞集落刺激因子(rhM-CSF)处理该细胞,诱导GFP-STAT1核转位,采用高内涵分析系统获取细胞图像,并用细胞核转位分析模块(Nuclear Trafficking Analysis Module)定量分析GFP-STAT1的核转位程度。在该细胞模型上对rhM-CSF的有效浓度、处理时间和细胞接种密度等实验条件进行优化,通过Z′因子评估该细胞模型的可靠性,并采用已知的c-Fms抑制剂GW2580等进一步确证模型的有效性。结果表明,实验中细胞接种密度0.2~1.0×104个/孔,rhM-CSF处理细胞的时间在30min较为适宜;rhM-CSF诱发细胞内GFP-STAT1发生核转位的EC50为43.50±3.68 ng/mL;rhM-CSF终浓度为200ng/mL时,Z′因子值为0.618。c-Fms选择性抑制剂GW2580抑制GFP-STAT1核转位的IC50为24.86±1.50nM,完全抑制c-Fms介导GFP-STAT1核转位的浓度为0.3μM。多靶标的受体酪氨酸激酶抑制剂Sutent抑制GFP-STAT1核转位的IC50为14.85±0.55nM;Imatinib抑制GFP-STAT1发生核转位的IC50为196.14±21.69nM。综上表明,本研究成功建立了能够用于c-Fms抑制剂筛选的基于高内涵分析的新细胞模型。基于激酶盘不依赖M-CSF的c-Fms抑制剂细胞水平筛选模型的建立:采用DNA重组技术,将人的c-Src豆蔻酰化多肽、TEL(Translocation E26 transforming-specific Leukaemia)转录因子的螺旋-环-螺旋结构域(HLH domain)、人c-Fms激酶结构域(c-Fms kinase domain,cFmskd)以及c-Myc多肽标签的DNA序列克隆到pCORON/neo载体上,构建pCORON/neo-HcSrc-Tel-cfmskd-Myc激酶盘真核表达载体。通过脂质体转染方法将该载体分别转染已稳定表达GFP-STAT1融合蛋白的人骨肉瘤细胞和稳定表达EGFP-STAT3融合蛋白的幼仓鼠肾细胞(EGFP-STAT3_BHK),在细胞中表达mTEL-cFmskd豆蔻酰化融合蛋白,诱导GFP-STAT1或EGFP-STAT3发生核转位。采用IN Cell Analyzer1000获取细胞图像,并以细胞核转位分析模块定量分析GFP-STAT1和EGFP-STAT3融合蛋白的核转位程度。用0.03μM、0.3μM的c-Fms抑制剂GW2580、Sutent和Imatinib分别处理转染激酶盘真核表达载体12h后的EGFP-STAT3_BHK细胞,考察c-Fms抑制剂对表达的mTEL-cFmskd豆蔻酰化融合蛋白诱导EGFP-STAT3发生核转位的影响。结果,成功构建的pCORON/neo-HcSrc-Tel-cfmskd-Myc激酶盘真核表达载体转染细胞24h后,可在U2OS或BHK细胞中有效诱导GFP-STAT1或EGFP-STAT3发生核转位现象。在黑色底透96孔培养板中,100、200、400、600和800ng/孔的载体转染BHK细胞,对细胞内EGFP-STAT3的核转位程度无明显影响,所对应的Z′因子值分别为0.600、0.701、0.574、0.812和0.637。0.3μM的c-Fms抑制剂GW2580(P<0.05)和Sutent(P<0.01)能有效抑制mTEL-cFmskd诱导EGFP-STAT3核转位,也进一步证实该筛选模型的可行性。以上结果表明该体系可作为c-Fms抑制剂筛选的新方法。第二部分,利用M-CSF依赖的小鼠骨髓性白血病细胞(M-NFS-60)和基于高内涵分析技术的依赖M-CSF的c-Fms抑制剂筛选模型,对靶向c-Fms设计及合成的34个新化合物进行活性筛选。在M-NFS-60细胞模型上,有10个化合物对M-NFS-60细胞的增殖具有较好的抑制活性,其中化合物LK-A051、LK-B030和LK-B023的I C50分别为0.64±0.03μM、0.63±0.02μM和1.41±0.20μM。在c-Fms/GFP-STAT1_U2OS细胞模型上,阳性化合物GW2580抑制c-Fms介导的GFP-STAT1核转位的IC50为24.86±1.50nM。在已筛选的新化合物中,LK-A051和LK-B023对GFP-STAT1核转位具有较好的抑制活性,其IC50分别为3.88±0.49nM和1.62±0.04μM。化合物LK-A051的活性明显高于三个已知c-Fms抑制剂。综上所述,本课题主要结论如下:1)基于c-Fms介导的细胞内信号转导通路,成功建立基于高内涵分析技术依赖rhM-CSF的c-Fms抑制剂筛选模型。已知的c-Fms抑制剂GW2580、Sutent和Imatinib在该模型上的抑制活性是文献已报道其他细胞模型的3～10倍。因此,与其他模型相比,该模型具有灵敏、高效等优点。2)成功构建以人c-Fms为代表的pCORON/neo-HcSrc-Tel-cfmskd-Myc激酶盘真核表达载体。该载体在GFP-STAT1_U2OS和EGFP-STAT3_BHK细胞中表达能有效诱导胞内GFP-STAT1和EGFP-STAT3发生核转位。100～800ng/孔的载体转染细胞诱导EGFP-STAT3发生核转位的Z′因子值均大于0.5,且c-Fms激酶抑制剂能显著抑制EGFP-STAT3核转位的发生,提示该模型可用于c-Fms抑制剂的筛选。该模型为更广泛的激酶抑制剂筛选模型的建立提供了一种通式,为激酶抑制剂的筛选提供了一个新的思路。3)采用M-NFS-60细胞和c-Fms/GFP-STAT1_U2OS细胞筛选模型,完成了34个新化合物的筛选,发现化合物LK-A051的活性明显高于阳性化合物。更多还原

【Abstract】 Kinases, one of the most important classes of protein in cells, play a key role in cellular signaling pathways. Protein tyrosine kinases (PTKs) are one of main member of kinases, and the signal transduction pathways mediated by PTKs are implicated in the control of a variety of biological processes, including cell proliferation, secretion, differentiation, and apoptosis. Disorder of PTKs may lead to many diseases, including malignant tumor, immunizing disease, atherosclerosis and diabetes. However, protein tyrosine kinases inhibitors can effectively block tyrosine kinases phosphorylation and subsequently block the cellular signals that are involved in the development of disease. Therefore, developing novel PTKs inhibitors with higher potencies and selectivity possess a great prospect.Macrophage-colony stimulating factor receptor(c-Fms), encoded by the c-fms proto-oncogene, is a member of type III receptor tyrosine kinase family which include platelet-derived growth factor receptorα,β(PDGFRα,β), stem cell growth factor receptor(SCFR/c-Kit) and FMS-like tyrosine kinase-3(FLT-3). After binding of its ligand (M-CSF or CSF-1) to the extracellular domain of c-Fms, dimerization was formed, phosphorylation of the intracellular FMS domain was induced, and then the catalytic activity of tyrosine kinases was exhibited. M-CSF/c-Fms complex with the catalytic activity of tyrosine kinases activates downstream cytoplasmic signaling, which lead to the survival, proliferation and differentiation of the monocyte- macrophage lineage. Deletion, repeat replication, mutation and chromosome translocation of c-fms gene are closely associated with many diseases, including myelodysplastic syndrome (MDS), acute myelocytic leukemia (AML), acute megakaryoblastic leukemia (AMKL), atherosclerosis, mammary cancer, cervical carcinoma and rheumatoid arthritis. These have made c-Fms as an attractive therapeutic target for c-Fms-associated diseases. Currently reported c-Fms inhibitors such as GW2580, Ki20227 and JNJ-28312141, are all in pre-clinical status. Therefore, researches have been focusing on the study of novel c-Fms inhibitors in past few years. However, efficient screening models and methods for PTKs inhibitors are the key to get potent candidate compounds from a great many compounds. Therefore, the purposes of the present study are, (1) to establish screening assays for human c-Fms inhibitors, and (2) then screening new compounds that were designed based on the structure of c-Fms and synthesized in our laboratory.According to the purposes of the study, the present work is composed of two parts.In the first part, to construct a eukaryotic expression vector including the cDNA for full-length human c-Fms, and then the vector was transfected into human osteosarcoma cell (U2OS) in which GFP-fused STAT1 (GFP-STAT1) protein had been stably expressed. By limiting dilution analysis and small slide culture, the cells (c-Fms /GFP-STAT1_U2OS) expressing human c-Fms and GFP-fused STAT1 protein were sorted. By using IN Cell Analyzer, GFP-STAT1 fusion protein nuclear translocation mediated by recombinant human macrophage colony-stimulating factor (rhM-CSF) in cells was observed, and then the data were analyzed with Nuclear Trafficking Analysis Module. The half-effective concentration (EC50) of rhM-CSF in GFP-STAT1 nuclear translocation assay was 43.50±3.68ng/ml. The performance of high-content screening assay based on the c-Fms /GFP-STAT1_U2OS cells that respond to 200ng/ml rhM-CSF could be assessed by calculating the Z′-factor of the model. The value of Z′-factor was 0.618, which means the screening assay was an excellent for screening of c-Fms inhibitors. Time course analysis of the GFP-STAT1 nuclear translocation indicated that the maximal translocation occurred approximately at 30 minutes after stimulation with 200ng/ml rhM-CSF. However, the seeding densities ranging from 0.2×104 to 1.0×104 cells per well in a black, clear-bottom 96-well plate showed no significant effect on GFP-STAT1 nuclear translocation. In addition, human c-Fms inhibitor GW2580 completely inhibited GFP-STAT1 nuclear translocation mediated by 200ng/ml rhM-CSF at 0.3μM, and IC50 was 24.86±1.50nM. Sutent and Imatinib, two Multitargeted tyrosine kinase inhibitors, also displayed antagonism activity against GFP-STAT1 nuclear translocation /GFP-STAT1_U2OS cells at submicromolar concentrations (ICin c-Fms 50 was 14.85±0.55nM and 196.14±21.69nM, respectively).By recombinant DNA technology, the DNA sequence of polypeptide for myristoylation of human c-Src, helix-loop-helix domain of human TEL, kinase domain of macrophage colony-stimulating factor receptor and c-Myc tag were inserted into pCORON/neo plasmid to generate kinase panel eukaryotic expression vector pCORON/neo-HcSrc-Tel-cfmskd-Myc. mTEL-cFmskd expression vector was transfected into GFP-STAT1_U2OS cells and EGFP-STAT3_BHK cells in a black, clear-bottom 96-well plate by Lipofection. Using IN Cell Analyzer 1000, the GFP-STAT1 or EGFP-STAT3 fusion protein nuclear translocation induced by mTEL-cFmskd myristoylated fusion protein was observed 24 hours after transfection. 100, 200, 400, 600 and 800ng/well of kinase panel eukaryotic expression vector showed no obvious effect on EGFP-STAT3 fusion protein nuclear translocation, and the value of Z′-factor were calculated to be 0.600, 0.701, 0.574, 0.812 and 0.637, respectively. In addition, GW2580 (P<0.05) and Sutent (P<0.01) could markedly inhibit EGFP-STAT3 nuclear translocation at 0.3μM. But the nuclear translocation was resistant to Imatinib at the same concentration. These results suggested that M-CSF-independent high-content screening assay was available.In the second part, 34 novel compounds were evaluated in M-CSF-dependent M-NFS-60cells proliferation assay and rhM-CSF-dependent GFP-STAT1 translocation assay. In M-NFS-60 cells proliferation assay, GW2580 inhibited the growth of M-CSF-dependent M-NFS-60 cells at micromolar concentrations (IC50 was 0.69±0.32μM) as reported. LK-A051, LK-B030 and LK-B023 also inhibited the growth of the cells (IC50 was 0.64±0.03μM, 0.63±0.02μM, and 1.41±0.20μM, respectively). Yet, in M-CSF-dependent high-content screening assay, only LK-B023 and LK-A051 effectivelly inhibit GFP-STAT1 nuclear translocation (IC50 was 1.62±0.04μM and 3.88±0.49nM).In summary, major conclusions of the present work are as follows:1) The cells (c-Fms/GFP-STAT1_U2OS) expressing human c-Fms and GFP-fused STAT1 protein were successfully sorted. GFP-STAT1 protein nuclear translocation mediated by rhM-CSF in c-Fms/GFP-STAT1_U2OS cells was observed by IN Cell Analyzer. M-CSF-dependent high-content screening assay for human c-Fms inhibitors were established, and reported c-Fms inhibitors all dose related inhibited GFP-STAT1 nuclear translocation mediated by rhM-CSF in the c-Fms/GFP-STAT1_U2OS cells. And the activity of kown c-Fms inhibitors in high-content screening assay was 3 to 10 times than other cell assay. Thus, indicating the established high-content screening assay was more sensitive and efficient.2) pCORON/neo-HcSrc-Tel-cfmskd-Myc kinase panel eukaryotic expression vector was transfected into GFP-STAT1_U2OS cells and EGFP-STAT3_BHK cells and successfully expressed mTEL-cFmskd. The GFP-STAT1 and EGFP-STAT3 fusion protein nuclear translocation induced by mTEL-cFmskd myristoylated fusion protein was observed 24 hours after transfection. GW2580 and Sutent obviously inhibited EGFP-STAT3 nuclear translocation at 0.3μM. But the nuclear translocation was resistant to Imatinib at the same concentration. The model could be used in screening of c-Fms kinase inhibitors, and the present assay also provides a general formula for establishing assays for other kinase inhibitors. 3) 34 new compounds had been evaluated in vitro, and compound LK-A051 was demonstrated to be a more powerful human c-Fms inhibitor than the reported inhibitors.更多还原