人源糖原合成酶激酶-3β（Human Glycogen Synthase Kinase-3β）的性质研究及其抑制剂的高通量筛选模型建立
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摘要
糖原合成酶激酶-3(GSK-3)是一个多功能的丝氨酸/苏氨酸类激酶，在真核生物中普遍存在。在哺乳动物中包括两个亚型，即GSK-3α和GSK-3β。GSK-3至少在三条细胞通路上有作用：Wnt/wingless,PI-3'Kinase以及Hedgehog信号通路，该酶的作用主要包括调节糖原的合成代谢，参与细胞的分化与增殖等。研究发现，GSK-3在某些疾病，例如阿尔茨海默尔氏病症以及非胰岛素依赖型糖尿病(NIDDM)中，其活性会异常升高。现已发现了几种针对该酶的抑制剂，例如aloisine,paullones和马来酰胺类化合物等。这些抑制剂的确在分子水平特异性地抑制GSK-3的活性，而对其他激酶几乎没有作用；关于这些抑制剂的研究工作也已经在细胞水平和动物模型上开展起来，为开发以GSK-3为靶点的新的治疗药物创造了良好的基础。
本论文的目的是克隆表达纯化重组人源GSK-3β(HsGSK-3β)并研究其酶学性质；在此基础上，建立HsGSK-3β抑制剂的高通量分子筛选模型。并通过对国家新药筛选中心化合物库的筛选，寻找高亲和力的HsGSK-3β抑制剂，期望能进一步成为治疗二型糖尿病以及神经退行性疾病的先导化合物。
本论文的研究内容主要包括通过PCR方法获得HsGSK-3β的编码序列，克隆至原核表达载体pGEX-KG上；将重组质粒pGEX-KG/HsGSK-3β转化大肠杆菌BL21(DE3)/codon plus后，经IPTG诱导表达，蛋白以可溶形式存在。GST融合重组蛋白经过谷胱甘肽亲和层析纯化，凝血酶切割，SP阳离子交换层析去处GST蛋白，超滤离心，Q-Sepharose阴离子交换层析纯化，得到较纯净的HsGSK-3β。
随后，我们确定了包括同位素的用量，酶反应的线性范围以及反应时间。对HsGSK-3β进行了一系列的酶学性质的考查，对测活条件进行了优化。考察了包括Mg~(2+)、DTT、金属螯合剂、离子强度等因素对酶活性的影响，确定了测活反应的体系为30ul反应体系，在酶反应体系中含有50mM Tris-HCl pH7.5，10mM MgCl_2，2mM DTT，50uM ATP，0.2uCiγ-~(33)PATP，50uM CREB，100nMHsGSK-3β，反应时间为15分钟。随后，进行了pH依赖性、酶动力学分析、酶抑制作用分析以及酶的稳定性分析。pH依赖性实验表明HsGSK-3β属于中性蛋白激酶。分析了HsGSK-3β特异性多肽底物CREB以及激酶共同底物ATP的酶动力学性质，以及阳性抑制剂Aloisine A对HsGSK-3β的抑制作用，结果表明重组HsGSK-3β具有天然HsGSK-3β的激酶活性。酶稳定性分析说明酶能够长期保存在-80℃，贮存溶液为50mM Hepes pH7.2，100mM NaCl，1mM DTT，1mM
华东师范大学硕士毕业论文
EDI人的条件下。
考察了DMso以及测活体系改变(体积改变而导致ATP:Y一，3P ATP掺入比
的改变)对活性的影响，确定进行分子水平高通量抑制剂筛选的50ul酶反应体
系为50 mM Tris一HCI pH 7.5，10 mM MgC12，2 mMD盯，25 uMATp，0.15uCi
竹3，P灯P，50 uMe咫B，100咖价osK一邓，4%DMSO和40 ug加l样品。建立
了HsGsK一3p抑制剂高通量筛选的标准化操作流程。对国家新药筛选中心化合物
库中的2720个纯化合物样品进行筛选，筛选在2天内完成，其中有145个样品
的抑制率在50%以上。挑选这些样品进行了两孔原浓度复筛以及稀释5倍后的两
孔筛选，对稀释5倍的两孔的抑制率都在50%以上的化合物进行多浓度的抑制测
定，计算ICS。值。
筛选得到ICS。大于10 uM而小于20 tlM的化合物共有6个，IC50小于10 uM
的化合物有3个。其中抑制效果最好的化合物编号为SH00011738，分子量330.36，
IC50为2.24u加1，是结构新颖的化合物。筛选到这些化合物，为进一步进行细胞
水平检测其抑制凋亡，降低血糖等作用以及化合物结构改造打下了良好的基础。
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase, highly conserved and has been identified in every eukaryote investigated to date. It is encoded by two isoforms in mammals, termed GSK-3 and GSK-3 . GSK-3 plays an important role in at least three signal transductory systems, namely, the Wnt/wingless, Hedgehog and PI' 3 -kinase pathways, which influence proliferation and cell survival, respectively, highlighted by the wide array of substrates controlled by this enzyme that includes cytoplasmic proteins and nuclear transcription factors. Several diease such as in Non-insulin dependent diabetes metillus (NIDDM), Alzheimer's disease, neurological disorders, and cancer have been related to GSK-3, as a potential therapeutic target. Several new GSK-3 inhibitors, such as the aloisines, the paullones and the maleimides, have been developed. Although they are just starting to be characterized in cell culture and animal model experiments, these new inhibitors hold promise as therapeutic agents.
Here we reported the cloning, expression, purification and characterization of molecule-based assay for high-throughput inhibitor screening. By screening the compound library of National Center for Drug Screening, we found some hits with novel structure skeleton which should be further studied in cell functions.
The coding region of HsGSK-3 was amplified by PCR method from an EST clone containing the HsGSK-3 gene and cloned into the vector pGEX-KG. The recombinant plasmid pGEX-KG/HsGSK-3 was then transformed into E.coli BL21(DE3)/codon plus cells. The GST-HsGSK-3P was overexpressed in the supernatant after IPTG induced. After sonicating, it was first purified by the Glutathione Chromatophy (5 ml) and thrombin cleavage overnight. Then the protein was loaded onto the SP column (5 ml) and eluted by 50 mM - 1 M NaCl gradient. The eluent was hyperfiltered with centrifuge tube and load onto the Q column (1ml). The flow through collected was the purified enzyme.
First we ensured the dose-depedent and time-depedent of the enzyme, then optimized the reaction system by Mg2+, DTT dependence, metal chelating agents, ion strength. By these, the opitimized reaction system containes 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 2 mMDTT, SOuMATP, 0.2uCi -33PATP, SOuMCREB, 100 nM /&GSK-3P in 30 ul as the activity assay system. The characterization of the HyGSK-3(3 includes, pH dependence, the kinetics of enzyme reaction and inhibition analysis and enzyme stability. The pH profile data shows that #sGSK-3p is a neutral kinase while the analyses of kinetic constants for synthesized substrates CREB and ATP, inhibition by Aloisnine A indicate that recombinant /M}SK-3p activity retains the enzyme characters of natural /ftGSK-3p\ Enzyme stability analysis shows that the enzyme can be stored at -80 掳C for long time with the solution 50 mM Hepes pH7.2, 100 mM NaCl,. 1 mM DTT and 1 mM EDTA as the storage buffer.
According to characterization of HsGSK-3 , the model of high-throughput screening was developed. The 50 ul reaction system included 50 mM Tris-HCl pH7.5, 10 mM MgCl2, 2 mM DTT, 50 uM CREB, 25 uM ATP and 0.15 uCi y-33P ATP, 4% DMSO and 2 ul compound (the final concentration is 40 ug/ml). We established the standard operation procedure of high-throughput screening for GSK-3 inhibitors. 2720 samples of National Center for Drug Screening were screened in 2days and there were 145 samples are higher than 50% inhibition. They were selected to do the second screening with the same compound concentration and 5-fold dilution. While one's inhibitories are both higher than 50%, we calculated 50% inhibitory concentration (IC50) by 3-fold compound dilution up to 0.549ug/ml.
From this screening we obtained 6 compounds whose IC50 values are between 10uM and 20uM while 3 compounds whose IC50 values are lower than 10uM. The best inhibitor of HsGSK-3p is named SH00011738, whose IC50 value is 2.24uM and whose molecular mass is 330.36. As the result showed that the screening model is reliable and effective, offering clues for further research on drug discovery and d
本论文的目的是克隆表达纯化重组人源GSK-3β(HsGSK-3β)并研究其酶学性质；在此基础上，建立HsGSK-3β抑制剂的高通量分子筛选模型。并通过对国家新药筛选中心化合物库的筛选，寻找高亲和力的HsGSK-3β抑制剂，期望能进一步成为治疗二型糖尿病以及神经退行性疾病的先导化合物。
本论文的研究内容主要包括通过PCR方法获得HsGSK-3β的编码序列，克隆至原核表达载体pGEX-KG上；将重组质粒pGEX-KG/HsGSK-3β转化大肠杆菌BL21(DE3)/codon plus后，经IPTG诱导表达，蛋白以可溶形式存在。GST融合重组蛋白经过谷胱甘肽亲和层析纯化，凝血酶切割，SP阳离子交换层析去处GST蛋白，超滤离心，Q-Sepharose阴离子交换层析纯化，得到较纯净的HsGSK-3β。
随后，我们确定了包括同位素的用量，酶反应的线性范围以及反应时间。对HsGSK-3β进行了一系列的酶学性质的考查，对测活条件进行了优化。考察了包括Mg~(2+)、DTT、金属螯合剂、离子强度等因素对酶活性的影响，确定了测活反应的体系为30ul反应体系，在酶反应体系中含有50mM Tris-HCl pH7.5，10mM MgCl_2，2mM DTT，50uM ATP，0.2uCiγ-~(33)PATP，50uM CREB，100nMHsGSK-3β，反应时间为15分钟。随后，进行了pH依赖性、酶动力学分析、酶抑制作用分析以及酶的稳定性分析。pH依赖性实验表明HsGSK-3β属于中性蛋白激酶。分析了HsGSK-3β特异性多肽底物CREB以及激酶共同底物ATP的酶动力学性质，以及阳性抑制剂Aloisine A对HsGSK-3β的抑制作用，结果表明重组HsGSK-3β具有天然HsGSK-3β的激酶活性。酶稳定性分析说明酶能够长期保存在-80℃，贮存溶液为50mM Hepes pH7.2，100mM NaCl，1mM DTT，1mM
华东师范大学硕士毕业论文
EDI人的条件下。
考察了DMso以及测活体系改变(体积改变而导致ATP:Y一，3P ATP掺入比
的改变)对活性的影响，确定进行分子水平高通量抑制剂筛选的50ul酶反应体
系为50 mM Tris一HCI pH 7.5，10 mM MgC12，2 mMD盯，25 uMATp，0.15uCi
竹3，P灯P，50 uMe咫B，100咖价osK一邓，4%DMSO和40 ug加l样品。建立
了HsGsK一3p抑制剂高通量筛选的标准化操作流程。对国家新药筛选中心化合物
库中的2720个纯化合物样品进行筛选，筛选在2天内完成，其中有145个样品
的抑制率在50%以上。挑选这些样品进行了两孔原浓度复筛以及稀释5倍后的两
孔筛选，对稀释5倍的两孔的抑制率都在50%以上的化合物进行多浓度的抑制测
定，计算ICS。值。
筛选得到ICS。大于10 uM而小于20 tlM的化合物共有6个，IC50小于10 uM
的化合物有3个。其中抑制效果最好的化合物编号为SH00011738，分子量330.36，
IC50为2.24u加1，是结构新颖的化合物。筛选到这些化合物，为进一步进行细胞
水平检测其抑制凋亡，降低血糖等作用以及化合物结构改造打下了良好的基础。
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase, highly conserved and has been identified in every eukaryote investigated to date. It is encoded by two isoforms in mammals, termed GSK-3 and GSK-3 . GSK-3 plays an important role in at least three signal transductory systems, namely, the Wnt/wingless, Hedgehog and PI' 3 -kinase pathways, which influence proliferation and cell survival, respectively, highlighted by the wide array of substrates controlled by this enzyme that includes cytoplasmic proteins and nuclear transcription factors. Several diease such as in Non-insulin dependent diabetes metillus (NIDDM), Alzheimer's disease, neurological disorders, and cancer have been related to GSK-3, as a potential therapeutic target. Several new GSK-3 inhibitors, such as the aloisines, the paullones and the maleimides, have been developed. Although they are just starting to be characterized in cell culture and animal model experiments, these new inhibitors hold promise as therapeutic agents.
Here we reported the cloning, expression, purification and characterization of molecule-based assay for high-throughput inhibitor screening. By screening the compound library of National Center for Drug Screening, we found some hits with novel structure skeleton which should be further studied in cell functions.
The coding region of HsGSK-3 was amplified by PCR method from an EST clone containing the HsGSK-3 gene and cloned into the vector pGEX-KG. The recombinant plasmid pGEX-KG/HsGSK-3 was then transformed into E.coli BL21(DE3)/codon plus cells. The GST-HsGSK-3P was overexpressed in the supernatant after IPTG induced. After sonicating, it was first purified by the Glutathione Chromatophy (5 ml) and thrombin cleavage overnight. Then the protein was loaded onto the SP column (5 ml) and eluted by 50 mM - 1 M NaCl gradient. The eluent was hyperfiltered with centrifuge tube and load onto the Q column (1ml). The flow through collected was the purified enzyme.
First we ensured the dose-depedent and time-depedent of the enzyme, then optimized the reaction system by Mg2+, DTT dependence, metal chelating agents, ion strength. By these, the opitimized reaction system containes 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 2 mMDTT, SOuMATP, 0.2uCi -33PATP, SOuMCREB, 100 nM /&GSK-3P in 30 ul as the activity assay system. The characterization of the HyGSK-3(3 includes, pH dependence, the kinetics of enzyme reaction and inhibition analysis and enzyme stability. The pH profile data shows that #sGSK-3p is a neutral kinase while the analyses of kinetic constants for synthesized substrates CREB and ATP, inhibition by Aloisnine A indicate that recombinant /M}SK-3p activity retains the enzyme characters of natural /ftGSK-3p\ Enzyme stability analysis shows that the enzyme can be stored at -80 掳C for long time with the solution 50 mM Hepes pH7.2, 100 mM NaCl,. 1 mM DTT and 1 mM EDTA as the storage buffer.
According to characterization of HsGSK-3 , the model of high-throughput screening was developed. The 50 ul reaction system included 50 mM Tris-HCl pH7.5, 10 mM MgCl2, 2 mM DTT, 50 uM CREB, 25 uM ATP and 0.15 uCi y-33P ATP, 4% DMSO and 2 ul compound (the final concentration is 40 ug/ml). We established the standard operation procedure of high-throughput screening for GSK-3 inhibitors. 2720 samples of National Center for Drug Screening were screened in 2days and there were 145 samples are higher than 50% inhibition. They were selected to do the second screening with the same compound concentration and 5-fold dilution. While one's inhibitories are both higher than 50%, we calculated 50% inhibitory concentration (IC50) by 3-fold compound dilution up to 0.549ug/ml.
From this screening we obtained 6 compounds whose IC50 values are between 10uM and 20uM while 3 compounds whose IC50 values are lower than 10uM. The best inhibitor of HsGSK-3p is named SH00011738, whose IC50 value is 2.24uM and whose molecular mass is 330.36. As the result showed that the screening model is reliable and effective, offering clues for further research on drug discovery and d
引文
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