以Glut1为靶点非固定化细胞毛细管电泳快速筛选药物新方法研究

The human glucose transporter GLUT1 is an twelve-transmembrane and extremely hydrophobic protein. However, as to extremely hydrophobic proteins, there exists a serious problem, i.e., isolating and purifying these proteins from the cell membrane will typically results in loss of native conformation, and it’s transport mechanism is complicated. Additionally, a number of target protains expressed on cell membranes is limited; the preparation technology of the immobilized cell capillary column is not easy to be mastered and it has a relatively short service life, also, it is difficult to couple the immobilized cell capillary electrophoresis (ICCE) with mass spectrometry. These factors make the method of screening ligands with the cell membrance receptors as targets by ICCE meet new challenges. In the present study, a novel method is proposed for the first time, in which, firstly, the GLUT1-eGFP plasmids will be transferred into HEK293 cells using electrotransfection. Secondly, under the physiological condition or approximately physiological conditions, a series of new technologies about this method will be investigated using DMSO as control sample. Finally, the novel non-immobilized cell capillary electrophoresis (NICCE) will be investigated and established by optimizing a series of parameters of the HPCE, and the interactions of GLUT1 with glucose and some other positive compounds will be studied by qualitative-quantitative analysis metnod and the defferent cells (living HEK293 cells, paraformaldehyde-fixed HEK293 cells, overexpression GLUT1 living HEK293 cells and overexpression GLUT1 paraformaldehyde-fixed HEK293 cells). The kinetic parameters of interaction between GLUT1 and glucose/other positive compounds will be calculated by the model. And the new NICCE screening platform will be established and used to rapidly screen a series of the new synthetic carbohydrate derivatives and the natural products. Furthermore, the efficacy tests of cell growth and cytotoxicity will be carried out for these binding-active compounds. This study will solve the above-mentioned problems effectively, lay the foundation for discovering the source of innovative drugs and the efficacy tests of anti-tumor in vivo, and establish a new method and new technology for screening anti-tumor drugs.

葡萄糖转运体1（GLUT1）是12次跨膜疏水性蛋白质，难以分离纯化并保留活性构象，转运机制复杂；细胞膜表达靶蛋白数量有限；固定化细胞毛细管柱制作技术不易掌握、使用寿命短、与MS联用难等问题，使以膜蛋白为靶点的固定化细胞毛细管电泳（ICCE）筛选方法受到挑战。本项目旨在构建人GLUT1真核表达质粒，建立高表达GLUT1的HEK293细胞，在生理或接近生理条件下，以DMSO为对照样品，进行一系列新技术探索，定性定量研究葡萄糖等阳性物质与活HEK293细胞、固定HEK293细胞、超表达GLUT1的活HEK293细胞、超表达GLUT1的固定HEK293细胞的相互作用，并计算结合动力学参数，建立以GLUT1为靶点NICCE筛选药物新方法。筛选合成糖类衍生物和天然产物，进行细胞增殖和细胞毒性实验。本研究将有效解决上述问题，为体内抗肿瘤药效实验和发现源头创新药物奠定基础、建立新筛选平台并提供筛选新思路。

葡萄糖转运体1（GLUT1）是12次跨膜疏水性蛋白质，难以分离纯化并保留活性构象，转运机制复杂；细胞膜表达靶蛋白数量有限；固定化细胞毛细管柱制作技术不易掌握、使用寿命短、与MS联用难等问题，使以膜蛋白为靶点的固定化细胞毛细管电泳（ICCE）筛选方法受到挑战。对于以此类受体/配体相互作用为靶点药物筛选研究只限于功能实验和放射配基结合实验，但重现性差、毒性大、成本高、耗时长。本项采用前人未曾报道的方法，自制了具筛毛细管柱接口，其方法简便快速、空隙尺度可控、渗透性良好、耐酸碱、耐非水溶剂、耐高温高压、机械强度好、表面性质可控，能够在生理环境或接近生理环境条件下使用，可以控制毛细管柱中细胞运动、而且不影响检测器对样品的检测，细胞可以随时更新，接口方便连接。对具筛毛细管柱接口进行了一系列新的技术探索，本方法完全满足分子间相互作用定性定量研究的要求。以氟他胺和EGCG作为阳性对照，DMSO作为阴性对照，研究了三者与GLUT1之间的相互作用，阳性化合物在GLUT1细胞管中的峰型改变符合非线性色谱理论（NLC）的特征峰型，利用NLC理论对EGCG和氟他胺的动力学参数进行了计算。创建了以GLUT1为靶点非固定化细胞毛细管电泳研究筛选与配体有特异性结合的平台。同时利用新筛选平台筛选合成糖类衍生物和天然产物，进行体外细胞增殖和细胞毒性实验，裸鼠体内抗肿瘤实验，活性位点竞争实验、分子对接实验的研究。通过分子水平相互作用初步筛选，可以大大加快作用靶点明确的抗肿瘤药物的筛选，降低毒副作用和实验成本。在项目执行期间，申请了5项国家发明专利，获得了4项中国发明专利的授权，培养了博士后研究人员1名、博士研究生5名（毕业博士1名）、硕士研究生8名（毕业硕士6名）,发表了科研论文12篇，其中SCI收录论文10篇。