JAK-STAT6信号传导通路抑制剂的高通量筛选及作用机理的初步探讨
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摘要
目的
构建可用于高通量筛选JAK-STAT6信号传导通路抑制剂的工程细胞株,建立稳定可靠的筛选方法。在此基础上对1600种结构多样性的小分子化合物进行了筛选,并对筛选得到的可有效抑制JAK-STAT6通路的2种化合物进行了相关抑制机理的初步探讨,为最终获得针对性治疗哮喘等过敏性疾病的新药研发奠定基础。
方法
1.利用脂质体法将带有STAT6特异性识别启动子IgE基因序列(其后连有虫荧光素酶报告基因)的质粒IgE-luc和表达潮红霉素B的质粒共转染至HeLa细胞,经潮红霉素B抗性筛选及报告基因检测,得到稳定表达虫荧光素酶的工程细胞株。
2.通过优化溶剂DMSO浓度, IL-4作用浓度及孵育时间等筛选条件,建立可靠的筛选方法,并在此基础上对1600种化合物进行了筛选。
3.应用MTT法和流式细胞检测技术,观察高通量筛选得到的抑制效果较理想两种化合物NC000383, NC000438(化合物A,B)在不同剂量和不同作用间隔时间下对HeLa细胞代谢活力,增值及细胞周期的影响；观察化合物对IL-4诱导的BJAB细胞CD23分子表达的影响；利用磷酸钙瞬时转染技术,建立在IFN-Υ或IL-4刺激后可表达虫荧光素酶的细胞。通过报告基因的检测,探讨化合物对JAK-STAT6通路作用的特异性。
结果
1.通过对构建的多个细胞克隆的荧光素酶活性的检测,我们获得了依赖于STAT6活性的稳定表达细胞株,该细胞株可作为后期高通量筛选的细胞模型。
2.通过对筛选条件的优化,结合操作的可行性,确定了高通量筛选时IL-4作用时间为8小时,终浓度为20ng/ml;化合物溶剂DMSO浓度体积百分比低于1。在此条件下,系统Z’因子达到0.64。通过对1600种化合物进行筛选,得到3种抑制效果较理想的化合物,选中率为1.85%o,其IC50值分别为：12.1μmol/L,16.5μmol/L,7.5μmol/L。
3.MTT检测结果显示,在一定的剂量下,两种化合物对HeLa细胞代谢活力和增殖均有一定抑制作用,这种抑制作用具有剂量依赖性和时间依赖性,并与细胞接种密度相关。其中化合物A剂量为1.0ug/ml时,对细胞代谢活力抑制率高于50%,化合物B则在25%以内。流式细胞仪检测结果显示两种化合物均可引起HeLa细胞周期的延迟,表现为G2-M期阻滞及S期蓄积(P<0.05),各剂量组之间变化也具有显著性(P<0.05)。同时能降低由IL-4介导的BJAB细胞CD23分子的表达。在所测试的三个剂量组中(A:0.25μg/ml,0.5μg/ml,1.0μg/ml; B:1.0μg/ml,1.5μg/ml,2.0μg/ml),随着化合物剂量的增加,CD23分子的表达水平逐渐降低。细胞瞬时转染结果显示两种化合物均能降低相应细胞因子刺激引起的分别依赖于STAT1和STAT6信号传导通路活性的荧光素酶的表达,统计学分析无显著差异,显示两种化合物对STAT6通路的抑制不具有特异性。
结论
1、建立了基于JAK-STAT6信号传导通路的工程细胞株和稳定可靠的高通量筛选系统,并利用该系统,得到3种可有效抑制该传导通路的化合物。
2、通过对其中两种化合物作用机理的初步探讨,推测化合物可能由于某些因素使细胞周期延长,细胞代谢活力下降,细胞增殖迟缓,进而引起相关基因(如IgE)的表达降低,其受体CD23表达亦降低,从而对IgE-luc细胞荧光素酶表达产生抑制作用。这种抑制作用对STAT6和STAT1通路均有影响。我们将在后期的实验中继续探讨化合物引起的细胞周期的延长以及CD23分子表达降低的机理。同时将对其它可能作用位点进行检测以及相关动物水平的研究。并期望以此深入,进一步探讨STAT6激活基因转录调控的分子机制,为治疗与该信号传导通路相关的哮喘等过敏性疾病的新药研发奠定基础。
Objective:Asthma is the most common chronic disease in children and young adults. The most effective treatment for asthma is glucocorticoids. These drugs are effective in controlling symptoms attributed to airway inflammation, but also have potentially serious side-effects. One of the principal goals in the development of new methods for treating asthma is therefore to identify non-steroidal drugs that can effectively control the inflammatory response underlying asthma symptoms. Many cytokines are involved in asthma, for example, interleukins (IL) IL-4and IL-13, which are derived from T-helper2(Th2) cells. It has been demonstrated that IL-4/JAK/STAT6signal pathway is highly active in asthma patients. The goal of our research is to establish a cell model which can be applied to identify potential new hits that selectively inhibit IL-4/JAK/STAT6signal pathway.
Methods:The plasmid was cut with pCMV-luc and inserted into the synthetic STAT6DNA binding oligonucleotides sequences (IgE promotor sequences) by gene recombination. Using lipofectin method, the plasmid contains STAT6DNA binding oligonucleotides sequences (IgE promotor sequences) followed by luciferase DNA, and the plasmid expressing Hygromycine B were cotransfected into HeLa cell. After Hygromycine B selection, the luciferase activities of different cell clones were measured under IL-4stimulation. The suitable cell clone was selected to perform optimal conditions for high throughput screening. Some factors, such as final concentration of DMSO, working concentration of IL-4and incubation time were measured to optimize the assay condition.1,600compounds were screened by this reliable method.
Furthermore, the potential molecular mechanisms of these inhibitors were investigated. HeLa cells were treated with different doses and intervals by NC00383or NC00438(compound A, compound B), which were obtained from high-throughput screening and had suppressive effect. The effects on cellular metabolic activity, cell proliferation and cell cycle distribution of HeLa were analyzed by MTT method and flow cytometer. STAT1or STAT6-dependent cell lines were established by the calcium-phosphate co-precipitant method, which can be activated by IFN-y and IL-4.
Results:Measured by luciferase activity, The stable cell lines, which can express luciferase activity in a specific JAK-STAT6activation-dependent manner, were established to select inhibitors of JAK-STAT pathway via high-throughput screen. Considering optimization and feasibility of the assay condition, the stable cells were treated with IL-4for8hours,20ng/ml; the concentration of DMSO was lower than1. Under this condition,Z'-factor value of the system was near0.64. Three compounds were screened to have suppressive effect on JAK/STAT6pathway, with the hit rate at1.85%o, and the IC50value are showed respectively:12.1μmol/L,16.5μmol/L,7.5μmol/L.
The MTT assay revealed that, at the given doses, the effects of both compounds on cellular metabolic activity and proliferation of HeLa cells were suppressively, with dose-dependent and time-dependent, which were also related to the different density of inoculability. The inhibitory rate of cellular metabolic activity of HeLa cells, which treated with compound A (1.0μg/ml) was higher than50%; while the rate of which treated with compound B was lower than25%. Results of flow cytometer assay showed that both compound A and B could prolong the HeLa cells cycle distribution, represented the blockage at G2-M phase and accumulation at S phase (P<0.05). The differences between each group are statistically significant (P<0.05). Besides that, IL-4-induced CD23expression was inhibited by compound A and B in BJAB cells. As the dosage increasing (A:0.25μg/ml,0.5μg/ml,1.0μg/ml, B:1.0μg/ml,1.5μg/ml,2.0μg/ml), the expression of CD23was lower down. Results of precipitate transfection experiment showed that both of these compounds could induce the expression of STAT1or STAT6-dependent luciferase. Since there's no statistically significant differences, these two kinds of compounds didn't have specific suppressive effect to STAT6pathway.
Conclusion:The engineering cell line and high-throughput system depending on JAK/STAT6signal transdution pathway were established. This system can be used to screen compounds that inhibit JAK-STAT6signal transduction pathway. In our study, three effective compounds have been screened.
According to the research of mechanism about these two compounds, we speculate that for some reasons they could prolong the cell cycle of HeLa cells, then reduce the cellular metabolic activity and slow down the proliferation, which induce the lower expression of related gene (e.g. IgE) and its receptor CD23, leading to suppressive effect to expression of IgE-luc luciferase, and affect the STAT6and STAT1pathway. We'll study more about if the prolonged S phase induced by the compounds was originally affected by expression of related genes or protein, and also what is the mechanism of decreased expression of CD23, the detection of other possible acting sites, which is important to the animal level research. More studies need to be done, for molecular mechanism of STAT6activating gene transcription and regulation, which will establish the basis for treatment of allergic diseases related to this signal transduction pathway.
构建可用于高通量筛选JAK-STAT6信号传导通路抑制剂的工程细胞株,建立稳定可靠的筛选方法。在此基础上对1600种结构多样性的小分子化合物进行了筛选,并对筛选得到的可有效抑制JAK-STAT6通路的2种化合物进行了相关抑制机理的初步探讨,为最终获得针对性治疗哮喘等过敏性疾病的新药研发奠定基础。
方法
1.利用脂质体法将带有STAT6特异性识别启动子IgE基因序列(其后连有虫荧光素酶报告基因)的质粒IgE-luc和表达潮红霉素B的质粒共转染至HeLa细胞,经潮红霉素B抗性筛选及报告基因检测,得到稳定表达虫荧光素酶的工程细胞株。
2.通过优化溶剂DMSO浓度, IL-4作用浓度及孵育时间等筛选条件,建立可靠的筛选方法,并在此基础上对1600种化合物进行了筛选。
3.应用MTT法和流式细胞检测技术,观察高通量筛选得到的抑制效果较理想两种化合物NC000383, NC000438(化合物A,B)在不同剂量和不同作用间隔时间下对HeLa细胞代谢活力,增值及细胞周期的影响；观察化合物对IL-4诱导的BJAB细胞CD23分子表达的影响；利用磷酸钙瞬时转染技术,建立在IFN-Υ或IL-4刺激后可表达虫荧光素酶的细胞。通过报告基因的检测,探讨化合物对JAK-STAT6通路作用的特异性。
结果
1.通过对构建的多个细胞克隆的荧光素酶活性的检测,我们获得了依赖于STAT6活性的稳定表达细胞株,该细胞株可作为后期高通量筛选的细胞模型。
2.通过对筛选条件的优化,结合操作的可行性,确定了高通量筛选时IL-4作用时间为8小时,终浓度为20ng/ml;化合物溶剂DMSO浓度体积百分比低于1。在此条件下,系统Z’因子达到0.64。通过对1600种化合物进行筛选,得到3种抑制效果较理想的化合物,选中率为1.85%o,其IC50值分别为：12.1μmol/L,16.5μmol/L,7.5μmol/L。
3.MTT检测结果显示,在一定的剂量下,两种化合物对HeLa细胞代谢活力和增殖均有一定抑制作用,这种抑制作用具有剂量依赖性和时间依赖性,并与细胞接种密度相关。其中化合物A剂量为1.0ug/ml时,对细胞代谢活力抑制率高于50%,化合物B则在25%以内。流式细胞仪检测结果显示两种化合物均可引起HeLa细胞周期的延迟,表现为G2-M期阻滞及S期蓄积(P<0.05),各剂量组之间变化也具有显著性(P<0.05)。同时能降低由IL-4介导的BJAB细胞CD23分子的表达。在所测试的三个剂量组中(A:0.25μg/ml,0.5μg/ml,1.0μg/ml; B:1.0μg/ml,1.5μg/ml,2.0μg/ml),随着化合物剂量的增加,CD23分子的表达水平逐渐降低。细胞瞬时转染结果显示两种化合物均能降低相应细胞因子刺激引起的分别依赖于STAT1和STAT6信号传导通路活性的荧光素酶的表达,统计学分析无显著差异,显示两种化合物对STAT6通路的抑制不具有特异性。
结论
1、建立了基于JAK-STAT6信号传导通路的工程细胞株和稳定可靠的高通量筛选系统,并利用该系统,得到3种可有效抑制该传导通路的化合物。
2、通过对其中两种化合物作用机理的初步探讨,推测化合物可能由于某些因素使细胞周期延长,细胞代谢活力下降,细胞增殖迟缓,进而引起相关基因(如IgE)的表达降低,其受体CD23表达亦降低,从而对IgE-luc细胞荧光素酶表达产生抑制作用。这种抑制作用对STAT6和STAT1通路均有影响。我们将在后期的实验中继续探讨化合物引起的细胞周期的延长以及CD23分子表达降低的机理。同时将对其它可能作用位点进行检测以及相关动物水平的研究。并期望以此深入,进一步探讨STAT6激活基因转录调控的分子机制,为治疗与该信号传导通路相关的哮喘等过敏性疾病的新药研发奠定基础。
Objective:Asthma is the most common chronic disease in children and young adults. The most effective treatment for asthma is glucocorticoids. These drugs are effective in controlling symptoms attributed to airway inflammation, but also have potentially serious side-effects. One of the principal goals in the development of new methods for treating asthma is therefore to identify non-steroidal drugs that can effectively control the inflammatory response underlying asthma symptoms. Many cytokines are involved in asthma, for example, interleukins (IL) IL-4and IL-13, which are derived from T-helper2(Th2) cells. It has been demonstrated that IL-4/JAK/STAT6signal pathway is highly active in asthma patients. The goal of our research is to establish a cell model which can be applied to identify potential new hits that selectively inhibit IL-4/JAK/STAT6signal pathway.
Methods:The plasmid was cut with pCMV-luc and inserted into the synthetic STAT6DNA binding oligonucleotides sequences (IgE promotor sequences) by gene recombination. Using lipofectin method, the plasmid contains STAT6DNA binding oligonucleotides sequences (IgE promotor sequences) followed by luciferase DNA, and the plasmid expressing Hygromycine B were cotransfected into HeLa cell. After Hygromycine B selection, the luciferase activities of different cell clones were measured under IL-4stimulation. The suitable cell clone was selected to perform optimal conditions for high throughput screening. Some factors, such as final concentration of DMSO, working concentration of IL-4and incubation time were measured to optimize the assay condition.1,600compounds were screened by this reliable method.
Furthermore, the potential molecular mechanisms of these inhibitors were investigated. HeLa cells were treated with different doses and intervals by NC00383or NC00438(compound A, compound B), which were obtained from high-throughput screening and had suppressive effect. The effects on cellular metabolic activity, cell proliferation and cell cycle distribution of HeLa were analyzed by MTT method and flow cytometer. STAT1or STAT6-dependent cell lines were established by the calcium-phosphate co-precipitant method, which can be activated by IFN-y and IL-4.
Results:Measured by luciferase activity, The stable cell lines, which can express luciferase activity in a specific JAK-STAT6activation-dependent manner, were established to select inhibitors of JAK-STAT pathway via high-throughput screen. Considering optimization and feasibility of the assay condition, the stable cells were treated with IL-4for8hours,20ng/ml; the concentration of DMSO was lower than1. Under this condition,Z'-factor value of the system was near0.64. Three compounds were screened to have suppressive effect on JAK/STAT6pathway, with the hit rate at1.85%o, and the IC50value are showed respectively:12.1μmol/L,16.5μmol/L,7.5μmol/L.
The MTT assay revealed that, at the given doses, the effects of both compounds on cellular metabolic activity and proliferation of HeLa cells were suppressively, with dose-dependent and time-dependent, which were also related to the different density of inoculability. The inhibitory rate of cellular metabolic activity of HeLa cells, which treated with compound A (1.0μg/ml) was higher than50%; while the rate of which treated with compound B was lower than25%. Results of flow cytometer assay showed that both compound A and B could prolong the HeLa cells cycle distribution, represented the blockage at G2-M phase and accumulation at S phase (P<0.05). The differences between each group are statistically significant (P<0.05). Besides that, IL-4-induced CD23expression was inhibited by compound A and B in BJAB cells. As the dosage increasing (A:0.25μg/ml,0.5μg/ml,1.0μg/ml, B:1.0μg/ml,1.5μg/ml,2.0μg/ml), the expression of CD23was lower down. Results of precipitate transfection experiment showed that both of these compounds could induce the expression of STAT1or STAT6-dependent luciferase. Since there's no statistically significant differences, these two kinds of compounds didn't have specific suppressive effect to STAT6pathway.
Conclusion:The engineering cell line and high-throughput system depending on JAK/STAT6signal transdution pathway were established. This system can be used to screen compounds that inhibit JAK-STAT6signal transduction pathway. In our study, three effective compounds have been screened.
According to the research of mechanism about these two compounds, we speculate that for some reasons they could prolong the cell cycle of HeLa cells, then reduce the cellular metabolic activity and slow down the proliferation, which induce the lower expression of related gene (e.g. IgE) and its receptor CD23, leading to suppressive effect to expression of IgE-luc luciferase, and affect the STAT6and STAT1pathway. We'll study more about if the prolonged S phase induced by the compounds was originally affected by expression of related genes or protein, and also what is the mechanism of decreased expression of CD23, the detection of other possible acting sites, which is important to the animal level research. More studies need to be done, for molecular mechanism of STAT6activating gene transcription and regulation, which will establish the basis for treatment of allergic diseases related to this signal transduction pathway.
引文
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