酵母模型在Aurora-B激酶抑制剂筛选中的应用

目的 以酵母细胞为模式菌高通量筛选微生物来源的Aurora-B激酶抑制剂.方法 以野生型酵母菌Y300和ipl1-321温度敏感型突变株为模式菌,筛选微生物来源的Aurora-B激酶抑制剂;体外酶学实验验证候选化合物对Aurora-B激酶的抑制作用;Western Blotting分析候选化合物对肿瘤细胞中histone H3磷酸化的影响.结果 Jadomycin B显示出对ipl1-321温度敏感型突变株的特异性抑制作用;体外酶学实验证实jadomycin B对重组纯化的人Aurora-B激酶具抑制作用;jadomycin B能抑制多种肿瘤细胞中histone H3的磷酸化并呈剂量依赖性.结论 以Y300和ipl1-321酵母细胞为高通量筛选模型筛选到一个新的Aurora-B激酶抑制剂jadomycin B.     

荧光检测技术在蛋白酪氨酸激酶抑制剂高通量筛选中的应用

介绍荧光共振能量转移技术、荧光偏振免疫分析技术、荧光素酶-激酶检测技术和毛细管电泳-激光诱导荧光技术在酪氨酸激酶抑制剂高通量筛选模型中的应用情况。蛋白酪氨酸激酶与多种疾病的发生和发展密切相关,通过高通量筛选寻找其抑制剂已成为新药研发的热点之一。荧光检测技术因具有灵敏、易于检测、适于微量化和安全性高等优势,在药物的高通量筛选中被广泛使用。     

蛋白酪氨酸激酶抑制剂的研究进展

蛋白酪氨酸激酶（PTK）抑制剂是一类作用于细胞信号转导通路的分子靶向药物,针对特定靶点发挥抗肿瘤作用。目前已有十余种蛋白酪氨酸激酶抑制剂上市,且它们在多种实体瘤的治疗中显示出较好的疗效。该文对近年来蛋白酪氨酸激酶抑制剂的研究进展做简要综述。     

蛋白激酶AMPK高效制备及活性检测方法的建立

目的 建立高效的腺苷酸依赖的蛋白激酶（AMPK）的制备方法,并建立1种基于酶联免疫吸附（ELISA）法的AMPK的激酶活性检测方法以用于药物筛选。方法 利用大肠杆菌表达系统,将AMPK的α、β和γ 3个亚基与钙调素依赖性蛋白激酶β(CAMKK β)共表达,重组AMPK被CAMKK β磷酸化修饰并激活,进而纯化得到具有激酶活性的重组AMPK蛋白复合体。根据AMPK底物乙酰辅酶A羧化酶（ACC）的磷酸化修饰位点（Ser79）设计合成生物素标记的多肽（SAMS）作为AMPK激酶的底物。在ATP存在的情况下AMPK对生物素标记的SAMS多肽进行磷酸化修饰,反应产物转移到链霉亲和素包被的酶标板中,SAMS多肽通过链霉亲和素结合到酶标板。以能够识别SAMS磷酸化修饰的抗体作为一抗进行ELISA检测,SAMS的磷酸化修饰水平反应AMPK的激酶活性。结果 利用大肠杆菌表达系统成功得到有激酶活性的AMPK蛋白复合体,建立了ELISA检测AMPK的激酶活性方法。通过对AMPK激活剂A-769662检测,证明该方法可以用于AMPK激活剂/抑制剂的筛选。结论 通过构建大肠杆菌共表达系统,成功获得了AMPK激酶...     

表皮生长因子受体酪氨酸激酶抑制剂BPI-2009的抗肿瘤作用及其机制的研究

目的:探讨一种口服的表皮生长因子受体(epidermalgrowthfactorreceptor ,EGFR)酪氨酸激酶抑制剂(BPI 2 0 0 9)的抗肿瘤作用及其机制。方法:Westernblot方法检测BPI 2 0 0 9对酪氨酸激酶和EGFR自动磷酸化的抑制作用,MTT法检测BPI 2 0 0 9对多种肿瘤细胞的生长抑制作用,使用A4 31肿瘤模型的荷瘤裸鼠进行体内的肿瘤抑制试验。结果:通过对EGFR激酶抑制剂化学文库的筛选,发现BPI 2 0 0 9是一个有效的EGFR激酶抑制剂。BPI 2 0 0 9对EGFR激酶的半数抑制浓度(IC50 )为5nmol·L- 1,当其浓度达到6 2 .5nmol·L- 1的时候可以完全抑制EGFR激酶的活性,但在10 0nmol·L- 1时对Abl、Abl相关基因(Abl relatedgenes ,Arg)以及c- Src酪氨酸激酶都没有明显的抑制作用。BPI 2 0 0 9可以阻断EGFR介导的细胞内蛋白酪氨酸的磷酸化,IC50 为4 5nmol·L- 1。在肿瘤细胞生长抑制试验中,BPI 2 0 0 9对于肿瘤细胞的生长抑制作用与EGFR在细胞中的表达密切相关。人类肿瘤细胞A4 31移植瘤抑制试验的研究表明BPI 2 0 0 9经口给药每天1次在10 0mg·kg- 1,肿瘤抑制率达6 4% ,有明显的剂量效应关系,并没有发现明显的病态和体重下降。结论:BPI 2 0 0 9是一种有效的高选择性的以EGFR酪氨酸激酶为靶点的抗肿瘤药物。     

β-内酰胺酶抑制剂高通量筛选模型的建立

目的 建立β-内酰胺酶抑制剂的高通量筛选模型.方法 从产β-内酰胺酶菌株中提取酶液,以硝噻吩为底物,三唑巴坦作为酶抑制剂,通过检测反应体系吸光度的变化,来判断酶促反应的进行情况以及酶抑制剂的效果,建立384微孔板的高通量筛选模型,优化反应条件并对此模型进行评价.用此模型对7360个化合物组成的随机库进行筛选.结果 确定反应体系条件为:最佳测定波长为482nm,反应温度为30℃,选取15min时间点检测,底物硝噻吩终浓度为0.1mmol/L,评价指标Z’因子在0.8～0.9范围内,表明此模型可以用于高通量筛选.基于此模型初筛出65个有抑制活性的化合物.结论 建立的β-内酰胺酶抑制剂高通量筛选模型具有快速、稳定和微量的特点,是寻找和发现有潜在活性的β-内酰胺酶抑制剂的有效手段.     

苄基哌嗪类衍生物的合成与蛋白酪氨酸激酶抑制活性的研究

目的合成苄基哌嗪类衍生物,考察目标化合物对蛋白酪氨酸激酶(PTK)的抑制活性。方法用呋喃甲醛和丙二酸合成呋喃丙烯酸,以15种取代苄基哌嗪为原料,分别与呋喃丙烯酸反应得到目标化合物。采用酶联免疫吸附法(Elisa)测定所得化合物对PTK的抑制活性,并计算抑制率,筛选出具有抑制PTK活性的化合物。结果所得化合物均对PTK有一定的抑制活性。结论通过优化反应条件,成功合成15种苄基哌嗪类化合物,原料廉价易得,合成方法简单,反应温和。化合物结构经IR、~1H NMR、EA和MS确证。经初步活性筛选发现化合物2h和2k的抑制活性较强。     

配体诱导受体酪氨酸激酶活化的结构基础研究进展

受体酪氨酸激酶(RTK)家族是一类具有内源性蛋白酪氨酸激酶(PTK)活性的单次跨膜膜受体。它们在调控与细胞增殖、分化等相关的信号转导通路中起关键作用。它与配体结合后引起二聚化或结构重排而使胞内区的酪氨酸(Tyr)被自磷酸化、Try的自磷酸化一方面可以激活胞内PTK区的活性，另一方面可以为下游的信号蛋白提供结合位点从而完成活化过程。本文对RTK家族成员的晶体结构及其活性调节机制的研究进行了综述，阐述了由配体诱导RTK活化的结构基础，并简要讨论了RTK抑制剂可能的作用靶点。     

不可逆性酪氨酸激酶抑制剂的研究进展

酪氨酸激酶的过度表达和过度激活在许多肿瘤的发生和发展中具有重要意义,因此,多种酪氨酸激酶成为抗肿瘤药物的靶点。目前已经上市的小分子酪氨酸激酶抑制剂多属于可逆性抑制剂,这些药物具有选择性差、药效不够强烈和持久以及易引发耐药性等缺点。近些年,不可逆性酪氨酸激酶抑制剂的研究正方兴未艾。这一类药物分子以不可逆的共价键与酪氨酸激酶上ATP结合域进行结合,从而使该靶点永久性失活。由于其独特的作用机制,不可逆性酪氨酸激酶抑制剂可以有效地解决可逆性酪氨酸激酶抑制剂的几个缺点。目前,已经有一批不可逆性酪氨酸激酶抑制剂进入市场或临床研究阶段。该篇综述是对不可逆性酪氨酸激酶抑制剂的结构、药理和药化特征及其研究进展等进行总结和阐述。     

多靶点酪氨酸激酶抑制药舒尼替尼

舒尼替尼是一种小分子多靶点酪氨酸激酶抑制药。通过抑制多个受体酪氨酸激酶(RTKs)磷酸化表现出抗肿瘤和抗血管形成活性。相对于更高选择性的激酶抑制剂,它的多靶点协作既具有可以接受的毒性,又大大提高了抗癌活性。2006年1月舒尼替尼获得美国FDA批准用于治疗晚期肾细胞癌(RCC)和服用伊马替尼后疾病出现进展或不耐受伊马替尼的胃肠道间质肿瘤(GIST)。     

酪氨酸激酶参与α1A肾上腺素受体介导的人胚胎肾细胞浆游离？…

AIM: To determine the role of protein-tyrosine kinase (PTK) in alpha 1A-adrenoceptor-mediated increase of [Ca2+]i (intracellular calcium) in human embryo kidney (HEK) 293 cells expressed alpha 1A-adrenoceptor. METHODS: Effects of two PTK inhibitors: genistein and tyrphostin, were investigated on the increase of [Ca2+]i by using Fura-2, The activity of PTK was measured and the accumulation of [3H] InsPs were observed. RESULTS: Norepinephrine stimulated a rapid increase in [Ca2+]i to (371 +/- 31) nmol.L-1 in HEK 293 cells. Norepinephrine-induced increase of [Ca2+]i was inhibited by the tyrosine kinase inhibitors quercetin and tyrphostin by 23.8% and 21.4%, respectively, but the accumulation of [3H]InsPs induced by norepinephrine was not. The activity of the plasma-associated tyrosine kinase was increased to (1.73 +/- 0.72)-fold over the control by norepinephrine 10 mumol.L-1. The norepinephrine-activated PTK was inhibited by calphostin C and depletion of intra- and extra-cellular Ca2+. CONCLUSION: The PTK participates in mobilization of Ca2+ mediated by alpha 1A-adrenoceptors in HEK 293 cell lines.     

蛋白酪氨酸激酶抑制剂的研究进展

目的综述蛋白酪氨酸激酶(protein tyrosine kinase,PTK)抑制剂类抗肿瘤药物的研究进展。方法根据已报道的PTK抑制剂的相关文献,将其分为国外已经上市、国外处于临床研究和我国自主研发的PTK抑制剂进行具体介绍。结果 PTK在细胞内的信号转导中起着重要的作用,与肿瘤细胞的生长、增殖、分化和凋亡密切相关,目前已经有多种结构的PTK抑制剂类抗肿瘤药物上市或进入临床研究。结论随着PTK的作用机制及构效关系研究的不断深入,该类药物终将成为治疗肿瘤的有效药物。     

A shikonin derivative, beta-hydroxyisovalerylshikonin, is an ATP-non-competitive inhibitor of protein tyrosine kinases

Studies of the mechanism of action of a shikonin derivative, beta-hydroxyisovalerylshikonin (beta-HIVS), have revealed that beta-HIVS inhibits the protein tyrosine kinase (PTK) activities of the receptor for epidermal growth factor and v-Src. In this review, we compare the characteristics of the inhibition of PTK activity by beta-HIVS with those of other inhibitors of PTKs. The chemical structure of beta-HIVS is completely different from that of ATP and it does not resemble any of the PTK inhibitors reported to date, except that it includes the benzylidene moiety. In contrast to most PTK inhibitors, the mechanism of inhibition by beta-HIVS is non-competitive with respect to ATP, but competitive with respect to its peptide substrate. This feature of the mechanism of inhibition of PTK by beta-HIVS suggests that it might be useful in a clinical setting with other PTK inhibitors. When Bcr-Abl-positive, human leukemia K562 cells were treated simultaneously with beta-HIVS and STI571 (Gleevec), these compounds had a synergistic effect on both the induction of apoptosis in K562 cells and the inhibition of the phosphorylation activity of PTK, probably because the mechanism of interference with phosphorylation by beta-HIVS and the binding site of beta-HIVS are different from those of STI571.     

Naturally occurring homoisoflavonoids function as potent protein tyrosine kinase inhibitors by c-Src-based high-throughput screening

Protein tyrosine kinase (PTK) inhibitors represent emerging therapeutics for cancer chemoprevention. In our study, hematoxylin (26) was identified as one of the most remarkable c-Src inhibitors in an orthogonal compound-mixing library (32200 compounds) by using an ELISA-based automated high-throughput screening (HTS) strategy. Interestingly, hematoxylin was found to be an ATP competitive broad-spectrum PTK inhibitor in vitro, with IC50 values ranging from nanomolar to micromolar level. Further studies showed that such inhibition was associated with the PTK phosphorylation and subsequent downstream signaling pathways. The structure-activity relationship assessment of the PTK inhibitory potency of hematoxylin analogues isolated from Heamatoxylon campechianum was in good agreement with the result of concurrent molecular docking simulation: the catechol moiety in ring A and the hematoxylin-like three-dimensional structure were essential for c-Src-targeted activities. Hematoxylin and its natural analogues were substantially validated to function as a new class of PTK inhibitors.     

Effects of halogenation on tyrosine phosphorylation and peptide binding to the SRC homology 2 domain of lymphocyte-specific protein tyrosine kinase

Phosphorylation of tyrosine residues by protein tyrosine kinases (PTK) and phosphotyrosine/Src homology 2 (SH2) domain interactions are crucial not only for signal transduction but also for regulation of PTK activity. Tyrosine residues also receive nitration and halogenation under oxidative conditions. It has been reported that nitration of tyrosine residue caused peptides to be a poor substrate for PTK and that nitrotyrosine residues could bind to SH2 domains as a phosphotyrosine mimic to activate Src family kinase. However, the effect of halogenation on tyrosine phosphorylation or SH2 domain binding is not well understood. We examined the phosphorylation of model peptides containing 3-halotyrosine or 3-nitrotyrosine using typical receptor tyrosine kinase, epidermal growth factor receptor (EGFR), and nonreceptor tyrosine kinase, lymphocyte-specific protein tyrosine kinase (Lck). The EGFR- and Lck-mediated phosphorylation was markedly inhibited by tyrosine halogenation. Iodination showed the strongest inhibition of the phosphorylation among four types of halogenation, and its inhibitory effect was stronger than that of nitration. We also examined the effect of iodination and nitration of tyrosine residues on binding to the SH2 domain of Lck, using a model peptide containing the phosphoTyr-Glu-Glu-Ile motif, which has a high affinity for the SH2 domain. The relative affinities of the modified peptides whose phosphotyrosine was substituted with unphosphorylated tyrosine, 3-nitrotyrosine, and 3-iodotyrosine, and of the model peptide were 0.024, 0.26, 1, and 16, respectively. These results suggest that tyrosine iodination may have an effect on the phosphorylation or binding to the SH2 domain similar to nitration. Tyrosine iodination possibly modulates signal transduction, with the potential impairment of cell function.     

酪氨酸激酶抑制剂的进展与评价

目的:探讨酪氨酸激酶抑制剂的进展与临床应用评价。方法:采用国内外专业文献进行综述与评估。结果及结论:蛋白酪氨酸激酶是一类具有酪氨酸激酶活性的蛋白质,近年来其进展迅速,上市和临床应用广泛,其抑制肿瘤细胞的损伤修复、可使肿瘤细胞分裂阻滞。成为研发高效、低毒、特异性强的新型抗癌药的重要方向。     

新型蛋白酪氨酸激酶抑制剂类抗肿瘤药物的研究进展

目的探讨蛋白酪氨酸激酶抑制剂抗肿瘤作用机理及其研究进展。方法综述了最新发现的小分子酪氨酸激酶抑制剂的化学结构、抗肿瘤作用及其作用机制及其发展方向等内容。结果结论蛋白酪氨酸激酶与细胞的增殖、分化、迁移和凋亡有着密切的关系,在细胞生命活动的信号转导途径中扮演着十分关键的角色,筛选酪氨酸激酶抑制剂已经成为开发抗肿瘤药物的新途径。     

Genistein directly inhibits native and recombinant NMDA receptors

The protein tyrosine kinase (PTK) inhibitor genistein has been widely used to examine potential effects of tyrosine phosphorylation on neurotransmitter function. We report here that genistein inhibits N-methyl-d-aspartate (NMDA) receptors through a direct effect. Whole-cell NMDA-activated current was recorded in native receptors from mouse hippocampal slice culture and rat recombinant NR1aNR2A and NR1aNR2B receptors transiently expressed in HEK293 cells. Extracellular application of genistein and NMDA reversibly inhibited NMDA-activated current. The inhibition of NMDA-activated current by genistein applied externally was not affected when genistein was also pre-equilibrated in the intracellular solution. Daidzein, an analog of genistein that does not block PTK, also inhibited NMDA-activated current. Coapplication of lavendustin A, a specific inhibitor of PTK, had no effect on the NMDA response. Moreover, genistein-induced inhibition of NMDA-activated current displayed concentration- and voltage-dependence. Our results demonstrate that genistein has a direct inhibitory effect on NMDA receptors that is not mediated via inhibition of tyrosine kinase. Thus, other PTK inhibitors may be more suitable for studying involvement of PTKs in NMDA receptor-mediated events.     

Protein tyrosine kinase inhibitors: new treatment modalities?

Protein tyrosine kinases (PTKs) have been recognized as attractive cell-signaling targets for drug discovery in the treatment of cancer and other diseases. Most of the PTK inhibitors are small molecules, designed to compete for, or nearby, the ATP-binding site, and are currently in phase I-III clinical trials, mainly for oncological indications. Recent efforts focused on the synthesis of selective PTK inhibitors have generated several promising clinical candidates, which recently culminated in the approval of Gleevec, the first kinase inhibitor registered for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors.