摘要
WHO研究报告表明,全球恶性肿瘤患者越来越多,发病情况日趋严重。所有的数据均表明,抗癌治疗已成为促进人类健康,提高人们生活质量的全球性课题。目前临床上常用的抗肿瘤药物如烷化剂、抗代谢物、抗肿瘤抗生素、生物碱类、激素类,起到缓解癌症症状、降低癌症患者死亡率、提高癌症患难者生活质量、延长患者生命等作用,然而,这类抗癌药具有难以避免的选择性差、毒副作用强、易产生耐药等缺点。发现选择性作用于特定靶点的高效、低毒、特异性强的分子靶点抗癌药物已成为当今抗肿瘤药物研究开发的重要方向。蛋白酪氨酸激酶(protein tyrosine kinase, PTK)是近年来发现的抗肿瘤药物作用的重要分子靶点。蛋白酪氨酸激酶是一类催化ATP上γ-磷酸转移到蛋白酪氨酸残基上的激酶,能催化多种底物蛋白质酪氨酸残基磷酸化,在细胞生长、增殖、分化中具有重要调节作用。蛋白酪氨酸激酶功能的失调则会引发生物体内的一系列疾病。已有的资料表明,超过50%的原癌基因和癌基因产物都具有蛋白酪氨酸激酶活性,它们的异常表达将导致细胞增殖调节发生紊乱,进而导致肿瘤发生。此外,酪氨酸激酶的异常表达还与肿瘤的侵袭和转移,肿瘤新生血管的生成,肿瘤的化疗抗性密切相关。因此,以酪氨酸激酶为靶点进行药物研发成为国际上抗肿瘤药物研究的热点,根据PTK是否存在于细胞膜受体可将其分成非受体型和受体型。
非受体酪氨酸激酶可以分为十一个大家族：SRC、ABL、JAK、FAK等。在信号转导通路中起着重要作用,近来发现非受体型蛋白酪氨酸激酶与人类癌症密切。已经鉴定的SRC家族成员包括Fgr、Fyn、Sre、Yes、BIK、Hck、Lck、Lyn。SRC家族蛋白的过度表达与相当多肿瘤疾病,如乳腺癌、肝癌和结肠癌密切相关,主要是由于它具有癌基因活性,与生长因子共同调节肿瘤细胞的生长,在肿瘤细胞的增殖、存活、粘连和迁移中起着重要作用。Sre家族激酶的结构在很大程度上具有相似性,他们含有一个或者多个膜定位所需的氨基端的酰基化位点,SH3结构域、SH2结构域、催化结构域和羧基未端Tyr529抑制位点。ABL家族包括两个成员：Abl和Arg。JAK与少数几种人类白血病密切相关,在细胞因子信号转导的初始步骤中起着至关紧要的作用。JAK家族包括四个成员：TYK2、JAD1、JAK2、JAK3。FAK家族包括FAK, Pyk2, CAKβ, RAFTK, cadTK, FAK2。FAK在不同肿瘤组织中的表达是不同的,在前列腺癌、乳腺癌、结肠癌、卵巢癌、口腔癌和甲状腺癌的发生过程中,FAK的表达均有提高。尤其是在乳腺癌和结肠癌组织中,FAK表达极高。
受体型酪氨酸激酶迄今为止,已有大约60种受体酪氨酸激酶被发现,根据受体或配体的类型可归入到20个亚家族中。许多肿瘤的发生、发展都与受体型酪氨酸激酶的异常表达有着极其密切的联系。几个主要的受体型酪氨酸激酶有以下几种：1、表皮生长因子受体家族,包括EGFR、ErbB2、ErbB4等成员,其酪氨酸激酶活性在调节细胞增殖及分化中起着至关重要的作用。EGFR在许多肿瘤中的过表达和/或突变,借助信号转导至细胞生长失控和恶性化,所参与的过程与新生血管生成,肿瘤的侵袭和转移,肿瘤的化疗抗性及预后密切相关。2、血小板衍生生长因子受体家族成员包括PDGFRa, PDGFRβ,还包括集落刺激因子1受体、干细胞生长因子受体、FLK2/FLT3。PDGFR主要存在于成纤维细胞、平滑肌细胞中,但也同时在肾、睾丸、脑中表达。PDGFR与胃肠道基质瘤、肥大细胞白血病发生发生有密切的关系。3、成纤维细胞生长因子受体(Fibroblast growth factor receptor, FGFR),家族成员包括FGFR1 (flg)、FGFR2 (bek)、FGF3R、FGFR4。FGFRs主要由中胚层来源的细胞产生,有强烈促增殖和分化作用,对内皮细胞、血管平滑肌细胞、成纤维细胞等均有很强的促有丝分裂作用。它具有促血管生成的作用。4、血管内皮细胞生长因子受体(Vascular endothelial growth factor receptor, VEGFR)家族,成员包括：VEGFR1 (Flt-1)、VEGFR2 (KDR/Flk-1)、VEGFR3 (Flt-4),通过与相应的配体结合,VEGFR在实体瘤的恶性生长和转移中,肿瘤的新生血管生成起着非常重要的作用,为肿瘤的生长提供了所必需的营养和氧气。
目前已上市及正在进行临床试验的酪氨酸激酶抑制剂可以分为以下三类：
1、EGF受体酪氨酸激酶抑制剂许多癌细胞有高水平EGF受体高表达,小分子EGF受体酪氨酸激酶抑制剂是近年来的抗癌药研究热点之一,包括Gefitinib、Elotinib均是EGF受体酪氨酸激酶抑制剂,能明显改善癌症的症状,耐受良好,不良反应轻微。
2、VEGF受体酪氨酸激酶抑制剂VEGF是一种可扩散的血管内皮特异性有丝分裂素和血管生长因子,在生理性和病理性血管形成过程中起关键作用。目前已有数个疗效较好的VEGF受体酪氨酸激酶的小分子抑制剂如Zactima,Vatalanib等进入临床。
3、PDGF受体酪氨酸激酶抑制剂研究发现,PDGF及其受体可刺激血管生成而促进癌症发生,如PDGF受体之一flt-3在急性粒细胞白血病中过度表达,引发白血病,而PDGF受体激酶抑制剂如CP-547632、SU11248等具有抑制内皮细胞增殖,缩小癌肿的临床效用。
基于已知VEGFR2抑制剂与VEGFR2形成复合物的晶体结构和活性数据,我们运用药物设计软件Syby17.3的GALAHAD、CoMFA、Surflex-DOCK等模块进行药物设计,在设计的38个化合物中,我们发现AW1与VEGFR2的晶体结构上的ATP结合位点对接评分最高。因此我们首先合成了AW1,然后采用均相时间分辨荧光免疫分析方法检测AW1对酪氨酸激酶VEGFR2活性抑制作用,接下来检测AW1对脐静脉血管内皮细胞增殖的作用以及对GTL-16细胞裸鼠移植瘤的抑制作用。
研究的主要方法及结果如下：
一、酪氨酸激酶抑制剂AW1的合成及其结构鉴定。
以间溴苯胺为起始原料,与水合氯醛及盐酸羟胺反应生成间溴异亚硝基乙酰苯胺,然后在浓硫酸作用下经环合、水解得到6—溴靛红,再经Wolff-Kishner黄鸣龙反应得到6-溴吲哚酮。6-溴吲哚酮与2-氯-5-[(5-呋喃甲醛)-2-基]苯甲酸在三乙胺作用下缩合,生成目标化合物AW1。
AW1的1H-NMR图谱、红外图谱及质谱图结果一致：1H-NMR (500 MHz) delta 7.04 (s,1H),7.27(d,1H, J=8.0 Hz),7.38(s,1H),7.47 (dd,2H, J,=4.0Hz),7.72 (d,1H, J=8.5Hz),8.01(1d,1H, J=8.5 Hz),8.36 (d,2H, J=8.5 Hz),10.71 (s,1H); IR (KBr)cm-13037.1、1690.8.1592.6、1543.1、1398.6、1231.9、1304.5、684.4;MS (m/z), ESI-1,444 (M-H).
二、均相时间分辨荧光免疫方法的建立以及AW1对酪氨酸激酶VEGFR2活性评价
我们根据国外在酪氨酸激酶抑制剂高通量筛选领域的最新研究进展,建立了一种用于蛋白酪氨酸激酶抑制剂高通量筛选的时间分辨荧光免疫分析方法：以黑色96孔板为实验容器,每孔加入20μl缓冲液(或酪氨酸激酶抑制剂溶液),10μl反应底物,10μl VEGFR-2激酶,10μl ATP。在37℃的孵箱中孵育30 min。然后,依次加入25μl的链激酶素标记的XL-665及25μl EuK标记的抗磷酸化的酪氨酸激酶抗体,室温反应60 min。用Tecan Genios Pro检测荧光信号,参数设置如下：Em=670 nm, Ex1=670 nm, Ex2=612 nm, lag time=150 s, integration time= 500 s, gain= 150。反应设3复孔,检测时每孔读数10次,设阴性对照及空白对照。反应体系中,在EuK和XL-665过量前提下测得的VEGFR-2、ATP和多肽底物的最适浓度分别为5 ng/μl,100μM和1μM。用本体系评价所合成的化合物AW1对VEGFR-2的酶活性抑制作用。0.16μg/ml-100μg/ml的AW1明显抑制多肽底物的磷酸化,表明AW1对VEGFR-2激酶活性具有显著的抑制作用。
三、AW1抑制脐静脉血管内皮细胞增殖
从临床收集新生儿脐带,进行脐静脉血管内皮细胞的体外培养并进行鉴定。用MTT法检测AW1对人脐静脉血管内皮细胞的增殖活性抑制作用。将AW1的母液进行2倍比稀释,使在培养液中的最终浓度为：0.625μg/ml,1.25μg/ml,2.5gg/ml,5μg/ml,10μg/ml,在37℃,5%CO2培养箱中培养48小时,然后用MTT法检测HUVEC的活性。以抑制率表示AW1对HUVEC活性的影响。在浓度分别为0.625μg/ml,1.25μg/ml,2.5μg/ml,5μg/ml,10μg/ml的AW1作用下,HUVEC生存的抑制率分别为0.130±0.012,0.230±0.013,0.340±0.025,0.355±0.029,0.423±0.030。结果表明,AW1明显抑制HUVEC的生长,且抑制作用呈剂量依赖性。
四、AW1对人胃癌GTL-16细胞裸鼠移植瘤的生长抑制作用
收集培养瓶中的肿瘤细胞,用PBS悬浮,调整细胞浓度为2×107,用等体积的BD Matrigel混合。混合好的细胞液皮下注射于裸鼠的左前肢内侧,0.1ml/只。每天观察肿瘤生长情况,每三天测一次裸鼠的重量及体积。用游标卡尺测定其体积大小。肿瘤体积计算方法如下：V=axb2/2(a：肿瘤最大直径值；b：与a垂直的直径值)。当肿瘤体积≥100mm3时,可开始给药。按裸鼠体重灌胃给药。
AW1以5mg/kg,15mg/kg,45mg/kg,每天给药2次,连续12天。低、中、高三个剂量对GTL-16细胞裸鼠移植瘤的抑制率分别为24.6%,48.1%,63%；三个剂量对GTL-16细胞裸鼠移植瘤的生长抑制作用呈明显的剂量依赖性。
根据上述实验结果,本论文的结论如下：
一、基于已知VEGFR2抑制剂与VEGFR2形成复合物的晶体结构和活性数据和药物设计,我们成功合成了化合物AW1。
二、本实验室在国内首先成功建立了一种用于蛋白酪氨酸激酶抑制剂高通量筛选的时间分辨荧光免疫分析方法,并用该方法证实了AW1对酪氨酸激酶VEGFR-2具有明显的抑制作用。
三、AW1能够明显抑制原代脐静脉血管内皮细胞的增殖。
四、AW1对裸鼠GTL-16在体移植瘤具有明显的抑制作用。
综上所述,我们成功地合成了一种VEGFR-2酪氨酸激酶抑制剂AWl,并发现它具有明显的抑制VEGFR-2活性、抑制脐静脉血管内皮细胞的增殖及明显地抑制在体移植瘤的生长。为开发以酪氨酸激酶为靶点的抗肿瘤药物提供了实验依据。
Reports from WHO indicate that the number of people suffering from malignant tumor is getting larger along with the situation tougher and tougher. Tumor incidence is estimated to increase by 50% by the year 2020 with number of newly increased cancer sufferers rising from 10,000,000 to 15,000,000 per year. Epidemiologic survey in China revealed that cancer mortality was higher than that of any other disease causing death, both in country and city,104.01/10,000 for the country and 94.71/10,000for city. All data indicated that tumor therapy had become a global project to promote human health and raise life quality and level.
Drug therapy has developed quickly in recent years for tumor suffers and already played a critical role in the area of chemotherapy. Regular chemical drugs used in chemotherapy including alkylating agents, antimetabolites, antitumor antibiotics, hormones, etc. did ease cancer syndrome, decrease death rate, enhance patients' quality life and prolong life-expectancy. However, as a result of the popular and frequent use of them, traditional anti-tumor drugs inevitably show disadvantages: low-selectivity, intensive toxicity, and drug resistance.
Along with the rapid progress on life science research, human beings gained rich knowledge about malignant tumor, especially cellular signal transduction, regulation of cell cycle, induction of cell apoptosis, angiogenesis and basic interaction mechanism intra and outra cellularlly. As a result, targeting therapy advocated in recent years by targeting specific molecule within correlative signal channel in tumors, is a new therapy method and proved available.
1. Protein tyrosine kianse
Protein tyrosine kianse are enzymes that catalyze the transfer of the yphosphate group from adenosine triphosphate to target proteins. They play an important role in diverse normal cellular regulatory processes. Disturbance of PTK may induce a series disease. It's reported that over 50% of oncogene and oncogenic product display PTK activity. Dysfunction of them will cause disorders during cell proliferation, thus tumor occurs. People all over the world invest a lot of money on the research and development of PTK. Tyrosine kinases can be classified as receptor protein kinases (RPK) and nonreceptor protein kinases (NRPK) according to whether they locate on cell membrane or not.
1) nonreceptor protein tyrosine kinases
Nonreceptor protein tyrosine kinases can be ascribed to 11 families:SRC, ABL, JAK, FAK, etc. NRPK plays an important role in cell signal transduction and correlates with a lot of human cancers.
1.1 SRC
Within SRC members include Fgr、Fyn、Sre、Yes、BIK、Hck、Lck、Lyn. Overexpression of SRC protein indicates tumor diseases such as breast carcinoma, liver cancer and colon cancer. SRC play an important role in proliferation, survival, adhesion and metastasis by showing oncogene activity and coaccomandate growth of tumor cells. High similarity is observed within Sre family, including acylating position in amino terminal needed for membrane allocation, SH2 domain, SH3 domain, catalyting domain and inhibitory position Tyr529 in carboxyl domain.
1.2 ABL
There are 2 members in ABL family:Abl and Arg. Abl protein structure includes SH3,SH2,PTK,DNA binding domain, actin binding domain, etc, exerting cell inhibitory activity.
1.3 JAK
JAK is highly correlated with several kind of human leukemia and plays a critical role in initial process of cellular signal transduction.4 members are included in JAK: TYK2, JAK1T, JAK2, JAK3.TYK2, JAK1T, JAK2 are expressed commonly while JAK3 is limitedly expressed in hematopoietic cell.7 conserved sequence exit in amino sequence of JAK.JH1,JH2 domain in carboxyl terminal show kinase activity. No homogeneity is observed in domain JH7-JH3 in amino terminal compared with other NRPK families members.
1.4 FAK
FAK family includes FAK, Pyk2, CAKβ, RAFTK, cadTK, FAK2. Different cancer express different amount of FAK. FAK is expressed more during the process of prostatic cancer, breast cancer, colon cancer, ovarian cancer, oral carcinoma and thyroid cancer. FAK is a special cytoplasm kinase, without SH2 and SH3.
2) Receptor protein tyrosine kinases
Receptor tyrosine kinases are membrane-spanning cell surface proteins that play critical roles in the transduction of extracellular signals to the cytoplasm. They are characterized by immunoglobulin-like sequences in their amino-terminal extracellular domains, alipophilic transmembrane segment, and an intracellular carboxyl-terminal domain that includes the tyrosine kinase catalytic site. Ligand binding induces dimerization of these receptor tyrosine kinases, resulting in autophosphorylation of their cytoplasmic domains and activation of tyrosine kinase activity. Multiple cytoplasmic signaling pathways, including the Ras/Raf mitogen-activated protein kinase pathway, the phosphoinositol3-kinase/Akt pathway, the signal transducer and activator of transcription 3 pathway, the protein kinase C pathway, and scaffolding proteins may then be activated. Intracellular mediators in these pathways transduce signals from membrane receptors through the cytosol and into the nucleus, culminating in altered DNA synthesis and cell division as well as effects on a variety of biological processes, including cell growth, migration, differentiation, and death
2.1 EGFR (Epidermal growth factory receptor)
The EGFR family comprises four transmembrane tyrosine kinase growth factor receptors:EGFR itself (ErbB1) (EGFR/HER1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4(HER4). Binding of a specific set of ligands to the receptor promotes EGFR dimerization and the autophosphorylation of the receptors on tyrosine residues. Upon autophosphorylation of the receptor, several signal transduction pathways downstream of EGFR become activated. The Ras/Raf mitogen-activated protein kinase pathway and the phosphoinositol3-kinase/Akt pathway are two major signaling routes for the HER family. The EGFR signal transduction pathways have been implicated in the regulation of various neoplastic processes, includingcell cycle progression, and inhibition of apoptosis, tumor cell motility, invasion, and metastasis. EGFR activation also stimulates vascular endothelial growth factor, which is the primary inducer of angiogenesis
2.2 PDGFR (platelet-derived growth factor receptor)
Platelet-derived growth factor (PDGF) signals through a cell surface tyrosine kinase receptor (PDGFR) to stimulate various cellular functions, including growth, proliferation, and differentiation. Two distinct PDGFR types have been identified:a andβ. Intracellular activation of this receptor can lead to cell transformation and generation of a mitotic signal. Both receptor types are over expressedin several solid tumors as well as in the surrounding stroma.
2.3 VEGFR (vascular endothelial growth factor receptor)
Angiogenesis is a complex process that occurs in a variety of physiologic and pathophysiologic states and is a remodeling of an established primitive network of blood vessels. VEGF is secreted by all almost all solid tumors and tumor-associated stroma in response to hypoxia. It is highly specific for vascular endothelium and regulates both vascular proliferation and permeability. Excessive expression of VEGF levels correlate with increased microvascular density, cancer recurrence, and decreased survival. There are six different ligands for the VEGFR, VEGF-A through -E and placenta growth factor. Ligands bind to specific receptors on endothelial cells, mostly VEGFR-2 (FLK-1/KDR), but it will also bind to VEGFR-1 (Flt-1) and-3. The binding of VEGF-A to VEGFR-1 induces endothelial cell migration. VEGFR-2 induces endothelial cell proliferation, permeability, and survival. VEGFR-3 is thought to mediate lymphangiogenesis. Binding of VEGF to VEGFR-2 receptors results in activation and autophosphorylation of intracellular tyrosine kinase domains, with triggering of intracellular signaling cascade.
Reviewing data and the crystal structure of compound formed by VEGFR-2 and its inhibitors, we designed 38 chemicals using Sybyl 7. Also we calculated the dock score and found that AW1, with a furan ring binding to I molindole at the same time a benzene ring binding to the molindole get a high score. After the synthesis of it, we do the researches including in vitro enzyme activity, cell growth inhibitory activity and in vivo activity.
The research methods and results are below:
1. Synthesis of protein tyrosine kinase inhibitor AW 1 and its structure identification
6-bromine-2-indolinone was synthesized from 2-Broaniline via condensation, cyclization, hydrolysis and reduction, after which AW1 was synthesized from 6-bromine-2-indolinone and 2-chloro benzoic acid.
'H-NMR, IR, MS spectrum proved the compound to be AW1.1H-NMR (500 MHz)57.04 (s,1H),7.27(d,1H, J=8.0 Hz),7.38(s,1H),7.47 (dd,2H, J,=4.0Hz),7.72 (d,1H, J=8.5Hz),8.01(1d,1H, J=8.5 Hz),8.36 (d,2H, J=8.5 Hz),10.71 (s,1H); IR (KBr) cm -1 3037.1、1690.8、1592.6、1543.1、1398.6、1231.9、1304.5、684.4; MS (m/z),ESI-1,444(M-H)。
2. Establishment of homogeneous time-resolved fluorescence immunoassay and activity evaluation of compound AWI
The most popular methods for screening tyrosine kinase inhibitors are in-plate binding assays and radiometric assays. The in-plate binding assays include enzyme- catalyzed colorimetric and luminescent read-outs and time-resolved fluorescence4. These methods require plate coating and multiple wash and incubation steps, which limits throughput. The radiometric assays using 33P or 32P, although extremely sensitive, can require filtration, which is labor intensive and relatively slow. A higher throughput radioisotopic method is SPA (scintillation proximity assay, Amersham International) as it does not require separation steps. However, because the amount of radioactivity used in the assay is kept to a minimum for cost and safety reasons, the measurement times can be the rate-limiting step in throughput.
Specific fluorescence signals atλ=670 nm andλ,=612 nm are measured by multifunctional microplate reader when fluorescence is emitted through resonance energy transfer between fluorescent materials (EuK and XL-665). The inhibitory activity of AW Ion VEGFR-2 (Vascular Endothelia Growth Factor Receptor 2) kinase activity was investigated. In this system, the concentrations of VEGFR-2, adenosine triphosphate (ATP) and poly-peptide substrate were 5 ng/μl,100μM and 1μM, respectively. AW1 significantly inhibited VEGFR-2 kinase activity.
3. Inhibitory proliferation of AW1 on human umbilical vein endothelial cells
HUVECs were collected from umbilical cord of infants. After the identification of HUVECs, the inhibitory activity of AW1 on HUVECs was assayed. The result showed that AW1 significantly inhibited the growth of HUVECs in a dose-dependent manner.
4. Inhibitory effect of AW1 on GTL-16 cells xenograft tumor growth in nude mouse
Tumor cells GTL-16 were implanted in nude mouse. The mice with tumor size over 100 mm3 were selected for the study. Four dose groups were set:Omg/kg, 5mg/kg,15mg/kg,45mg/kg, bid, weight and tumor size were recorded every three days in order to observe the inhibitory activity of AW1.
We observed that AW1 inhibited the growth GTL-16 in nude mouse. Significant difference (P<0.05) was showed between the drug-giving groups and the blank group, in a dose-dependent manner.
Conclusion
1.1H-NMR, IR, MS spectrum proved the compound synthesized to be AW1: 1H-NMR (500 MHz)δ7.04 (s,1H),7.27(d,1H, J=8.0 Hz),7.38(s,1H),7.47 (dd,2H, J1=4.0Hz),7.72 (d,1H, J=8.5Hz),8.01(1d,1H, J=8.5 Hz),8.36 (d,2H, J=8.5 Hz), 10.71 (s,1H);IR(KBr) cm-1 3037.1、1690.8、1592.6、1543.1、1398.6、1231.9、1304.5、684.4; MS (m/z), ESI-1,444 (M-H)。
2. Homogeneous time-resolved fluorescence for high throughput screening of receptor protein tyrosine kinase was established by our lab, which leads all over the country. Three contents including VEGFR-2, adenosine triphosphate (ATP) and poly-peptide substrate. of the reaction were optimized to the most appropriate concentration,5 ng/μl,100μM and 1μM, respectively. Inhibitory activity of AW1 on VEGFR-2 was identified through homogeneous time-resolved fluorescence.
3. HUVEC grows in a single layer, shaped short fusiform or pebble, with applanatus, polygon, clear boundary and abundant cytoplasm. After correlative antigen immunohistochemistry staining, the cells were shaped spherical, fusiform or polygon, with buffy particles, expecially intensive around the nucleus, while no staining was observed in the control group. AW1 significantly inhibited the proliferation of HUVEC.
4. AW1 inhibited the growth GTL-16 in nude mouse. Significant difference was showed between the drug-giving groups and the blank group, in a dose-dependent manner.
引文
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