应用质谱技术筛选激酶抑制剂
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摘要
蛋白激酶是一类磷酸转移酶,可将ATP的γ磷酸基转移到底物特定的氨基酸残基上,使蛋白质磷酸化。蛋白激酶在调节细胞的多种生命活动和疾病的进程中发挥着关键作用。异常的磷酸化与许多疾病相关,如癌症、炎症或免疫系统紊乱等。以蛋白激酶作为药物靶点,寻找与之相互作用的小分子化合物,并进一步优化用于新药研发是当今蛋白激酶研究领域的一个重要方向。传统的激酶抑制剂筛选方法虽然可以采用高通量的方式进行,但仍然存在以下缺点:偶联反应易导致非特异性结果、放射性同位素或荧光标记底物价格昂贵并可能产生同位素污染、筛选操作过程复杂易出现假阴性或假阳性结果等。因此,结合高通量的特点,寻求补充的激酶抑制剂的筛选方法非常有意义。
随着质谱技术的不断改进和完善,质谱的应用范围已扩展到生命科学研究的许多领域。质谱用于酶活性的测定及酶动力学研究也越来越广泛。本文应用质谱技术建立了激酶抑制剂的筛选方法,具体如下:
(一)以新功能蛋白激酶RX218为研究对象,建立了激酶抑制剂高通量筛选方法。采用经典的Fmoc固相合成策略,以及平行合成及“混-分”编码合成等组合化学技术合成了含有86条多肽的肽库,并结合Kinase-Glo~(?)发光检测法筛选到了RX218激酶底物(Pep8)。以Pep8为底物,经过优化激酶反应体系中的ATP浓度、激酶用量、底物Pep8浓度等首次建立了激酶RX218的Kinase-Glo~(?)发光检测筛选模型。应用该模型对我课题组的640个单一纯化合物进行筛选后,发现8个活性化合物。其中活性较高的2个化合物的IC_(50)值分别为38.7μM(P3-A7)和43.2μM(V8807)。
(二)以Pep8为激酶RX218的多肽底物,采用HPLC-MS联用技术中的质谱阀切换功能进行样品脱盐前处理,通过优化液相梯度、质谱检测条件以及激酶反应体系等,建立了激酶抑制剂的色谱-质谱联机筛选方法。研究中对LC-MS筛选体系进行了系统的方法学考察,并对筛选模型的稳定性进行了评价。结果表明,该方法线性、重复性良好。Z′因子评价表明该方法可用于高通量筛选。
(三)应用已建立的模型对20个联苯类化合物进行了初步构效关系研究,并发现化合物V8804活性高于V8807。利用UHP氧化法合成了内标物Pep8(O_2)-P,在存在内标及无内标的条件下,应用质谱法测定其IC_(50)值分别为19.1μM及14.4μM。同时应用Kinase-Glo~(?)发光检测法测得其IC_(50)值为15.4μM,证明两种方法检测结果一致。
(四)应用已建立的模型,筛选了我课题组现有的1,2,7-三取代-1H-咪唑并[4,5-g]喹喔啉6-酮类混合物化学库(1850个化合物)。发现了4个对RX218有抑制作用的化合物,其IC_(50)值分别为38.3μM(F3-E),98μM(E5-A),110μM(D6-D),72μM(D6-E)。全文总共发现了13个活性化合物,其中活性较高的4个化合物为P3-A7,V8807,V8804,F3-E。
综上所述,本论文创新点如下:
1.建立了LC-MS激酶抑制剂筛选新方法。该法特点是:均质显著减少了假性结果、不需要标记底物节约了筛选成本、无同位素标记免除了放射性对实验者以及放射性同位素垃圾对环境的危害、不需要偶联反应可以直接对抑制作用进行定量。
2.将组合化学混合物合成化学与LC-MS高通量筛选相结合进行混合物化学库筛选,显著提高了筛选效率。
3.应用HPLC-MS联用技术及质谱的阀切换功能进行样品的脱盐处理。
4.LC-MS激酶抑制剂筛选新方法与传统Kinase-Glo~(?)发光检测法结果一致。
5.筛选并首次确定了激酶RX218的多肽底物Pep8。
6.发现了4个抑制RX218的较高活性化合物(IC_(50)＜50μM)。
Phosphorylation by protein kinase is believed to be the most common protein post-translational covalent modification.Protein kinases serve important regulatory functions in essential of all cell processes including cell growth,migration, differentiation and death.The protein kinases catalyzed abnormal phosphorylation has been linked to many diseases,such as cancer,inflammation,as well as immune system disorder.Very recently,a serine/threonine kinase(RX218) was found to be highly expressed in lung and esophagus tumor tissues,which might be a new drug target. Traditional kinase assays for lead compounds identification suffer from the drawbacks including enzymatic link-coupling reactions frequently offering non specific observations or false results,requirement of radio or fluorescent labeling substrates linking with the exposure of dangerous materials to researchers and the potentials of wasting radio-activating material,in addition of high cost and time consuming.Therefore, development of high throughput screening alternative methods is highly anticipated.
In the last decade with the significant achievements,mass spectrometry(MS) technologies have had a significant impact on many research areas of life science.In this thesis,a MS-based kinase inhibitor screening method is developed.The successes in this thesis include:
1.A method based on the LC-MS technology is developed for high throughput screening of RX218 Ser/Thr kinase inhibitors.By employing parallel and "split-mix" combinatorial chemistry strategies and standard Fmoc peptide chemistry,total 86 peptides were synthesized and purified by preparative HPLC.Together with Kinase-Glo~(?) Luminescence assay,a peptide substrate(Pep8) of RX218 is identified for the first time.By using Pep8 as substrate,kinase RX218 Kinase-Glo~(?) assay is then established for the first time after systematic optimization of ATP concentration, amount of kinase and substrate.Furthermore,640 individual pure compounds were screened and 8 compounds were hitted.Two of them are giving their values of IC_(50) at38.7μM(P3-A7) and 43.2μM(V8807) respectively.
2.MS-based auto assay was developed for Kinase RX218 inhibitor identification by using pep8 as substrate.This includes establishment of desalting method by using Valve switch function of mass spectrometry,obtaining its linearity and repeatability through optimization of kinase reaction system,gradient of liquid chromatography, and mass spectrometric detection parameters.The linearity,repeatability were good. The obtained Z'factor in thesis valuated the method for HTS.
3.Twenty compounds with biphenyl scaffold were evaluated by the new MS-based assay.A novel compound(V8804) was found with improved IC_(50) value of 14.4μM and 19.1μM,respectively.To validation of the method,Kinase-Glo~(?) assay analyzed V8804 that give IC_(50) of 15.4μM.These results demonstrate that our MS-based assay is sensitive and comparable.
4.By using this MS-based high-throughput screening technology,one thousand eight hundred and fifty compounds from a chemical library of 1,2,7-trialky-1H-imidazo[4, 5-g]quinoxalin-6-one scaffold were screened.Four compounds inhibiting RX218 were identified with IC_(50) values of 38.3μM(F3-E),98μM(E5-A),110μM (D6-D) and 72μM(D6-E),respectively.
Over all,this thesis devotes significant contributions:
1.A MS-based kinase inhibitor screening method was established with the advantages of homologous assay that could reduce the false positive and/or negative results,labeling-free save the cost,radio-free eliminated the risk of exposure and waste of the radio activating materials,and precise quantization of phosphorylated product.
2.Integration of mixture chemical library and high throughput MS technology significantly enhanced the screening throughput.
3.The desalting method is developed by using the function of valve switch function of mass spectrometry.
4.The MS-based assay is coincident with the one of Kinase-Glo~(?) luminescence assay.
5.A peptide substrate(Pep8) of Kinase RX218 is identified for the first time.
6.Total 13 compounds were identified to be active against RX218.Among them,4 were less than 50μM of their IC_(50) values.
随着质谱技术的不断改进和完善,质谱的应用范围已扩展到生命科学研究的许多领域。质谱用于酶活性的测定及酶动力学研究也越来越广泛。本文应用质谱技术建立了激酶抑制剂的筛选方法,具体如下:
(一)以新功能蛋白激酶RX218为研究对象,建立了激酶抑制剂高通量筛选方法。采用经典的Fmoc固相合成策略,以及平行合成及“混-分”编码合成等组合化学技术合成了含有86条多肽的肽库,并结合Kinase-Glo~(?)发光检测法筛选到了RX218激酶底物(Pep8)。以Pep8为底物,经过优化激酶反应体系中的ATP浓度、激酶用量、底物Pep8浓度等首次建立了激酶RX218的Kinase-Glo~(?)发光检测筛选模型。应用该模型对我课题组的640个单一纯化合物进行筛选后,发现8个活性化合物。其中活性较高的2个化合物的IC_(50)值分别为38.7μM(P3-A7)和43.2μM(V8807)。
(二)以Pep8为激酶RX218的多肽底物,采用HPLC-MS联用技术中的质谱阀切换功能进行样品脱盐前处理,通过优化液相梯度、质谱检测条件以及激酶反应体系等,建立了激酶抑制剂的色谱-质谱联机筛选方法。研究中对LC-MS筛选体系进行了系统的方法学考察,并对筛选模型的稳定性进行了评价。结果表明,该方法线性、重复性良好。Z′因子评价表明该方法可用于高通量筛选。
(三)应用已建立的模型对20个联苯类化合物进行了初步构效关系研究,并发现化合物V8804活性高于V8807。利用UHP氧化法合成了内标物Pep8(O_2)-P,在存在内标及无内标的条件下,应用质谱法测定其IC_(50)值分别为19.1μM及14.4μM。同时应用Kinase-Glo~(?)发光检测法测得其IC_(50)值为15.4μM,证明两种方法检测结果一致。
(四)应用已建立的模型,筛选了我课题组现有的1,2,7-三取代-1H-咪唑并[4,5-g]喹喔啉6-酮类混合物化学库(1850个化合物)。发现了4个对RX218有抑制作用的化合物,其IC_(50)值分别为38.3μM(F3-E),98μM(E5-A),110μM(D6-D),72μM(D6-E)。全文总共发现了13个活性化合物,其中活性较高的4个化合物为P3-A7,V8807,V8804,F3-E。
综上所述,本论文创新点如下:
1.建立了LC-MS激酶抑制剂筛选新方法。该法特点是:均质显著减少了假性结果、不需要标记底物节约了筛选成本、无同位素标记免除了放射性对实验者以及放射性同位素垃圾对环境的危害、不需要偶联反应可以直接对抑制作用进行定量。
2.将组合化学混合物合成化学与LC-MS高通量筛选相结合进行混合物化学库筛选,显著提高了筛选效率。
3.应用HPLC-MS联用技术及质谱的阀切换功能进行样品的脱盐处理。
4.LC-MS激酶抑制剂筛选新方法与传统Kinase-Glo~(?)发光检测法结果一致。
5.筛选并首次确定了激酶RX218的多肽底物Pep8。
6.发现了4个抑制RX218的较高活性化合物(IC_(50)＜50μM)。
Phosphorylation by protein kinase is believed to be the most common protein post-translational covalent modification.Protein kinases serve important regulatory functions in essential of all cell processes including cell growth,migration, differentiation and death.The protein kinases catalyzed abnormal phosphorylation has been linked to many diseases,such as cancer,inflammation,as well as immune system disorder.Very recently,a serine/threonine kinase(RX218) was found to be highly expressed in lung and esophagus tumor tissues,which might be a new drug target. Traditional kinase assays for lead compounds identification suffer from the drawbacks including enzymatic link-coupling reactions frequently offering non specific observations or false results,requirement of radio or fluorescent labeling substrates linking with the exposure of dangerous materials to researchers and the potentials of wasting radio-activating material,in addition of high cost and time consuming.Therefore, development of high throughput screening alternative methods is highly anticipated.
In the last decade with the significant achievements,mass spectrometry(MS) technologies have had a significant impact on many research areas of life science.In this thesis,a MS-based kinase inhibitor screening method is developed.The successes in this thesis include:
1.A method based on the LC-MS technology is developed for high throughput screening of RX218 Ser/Thr kinase inhibitors.By employing parallel and "split-mix" combinatorial chemistry strategies and standard Fmoc peptide chemistry,total 86 peptides were synthesized and purified by preparative HPLC.Together with Kinase-Glo~(?) Luminescence assay,a peptide substrate(Pep8) of RX218 is identified for the first time.By using Pep8 as substrate,kinase RX218 Kinase-Glo~(?) assay is then established for the first time after systematic optimization of ATP concentration, amount of kinase and substrate.Furthermore,640 individual pure compounds were screened and 8 compounds were hitted.Two of them are giving their values of IC_(50) at38.7μM(P3-A7) and 43.2μM(V8807) respectively.
2.MS-based auto assay was developed for Kinase RX218 inhibitor identification by using pep8 as substrate.This includes establishment of desalting method by using Valve switch function of mass spectrometry,obtaining its linearity and repeatability through optimization of kinase reaction system,gradient of liquid chromatography, and mass spectrometric detection parameters.The linearity,repeatability were good. The obtained Z'factor in thesis valuated the method for HTS.
3.Twenty compounds with biphenyl scaffold were evaluated by the new MS-based assay.A novel compound(V8804) was found with improved IC_(50) value of 14.4μM and 19.1μM,respectively.To validation of the method,Kinase-Glo~(?) assay analyzed V8804 that give IC_(50) of 15.4μM.These results demonstrate that our MS-based assay is sensitive and comparable.
4.By using this MS-based high-throughput screening technology,one thousand eight hundred and fifty compounds from a chemical library of 1,2,7-trialky-1H-imidazo[4, 5-g]quinoxalin-6-one scaffold were screened.Four compounds inhibiting RX218 were identified with IC_(50) values of 38.3μM(F3-E),98μM(E5-A),110μM (D6-D) and 72μM(D6-E),respectively.
Over all,this thesis devotes significant contributions:
1.A MS-based kinase inhibitor screening method was established with the advantages of homologous assay that could reduce the false positive and/or negative results,labeling-free save the cost,radio-free eliminated the risk of exposure and waste of the radio activating materials,and precise quantization of phosphorylated product.
2.Integration of mixture chemical library and high throughput MS technology significantly enhanced the screening throughput.
3.The desalting method is developed by using the function of valve switch function of mass spectrometry.
4.The MS-based assay is coincident with the one of Kinase-Glo~(?) luminescence assay.
5.A peptide substrate(Pep8) of Kinase RX218 is identified for the first time.
6.Total 13 compounds were identified to be active against RX218.Among them,4 were less than 50μM of their IC_(50) values.
引文
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