基于亲合整体柱色谱的药物靶标鉴定新技术

Thus far, the knowledge on the action mechanism and targets of currently used small molecular drugs remains limited. With the development of pharmacology, it was realized that the small molecular drugs may have several action targets in the body. Identification of these drug targets is very helpful to reveal the action mechanism of the drugs, and subsequently to improve their efficacy by the following structure-activity relationship study. Moreover, it is also very valuable to predict the possible side-effect of the lead compounds prior to the clinic trial hence reduction of the failure risk of the drug develop project. The aim of the present proposal is to develop a new technique which can be used to effectively enrich the drug targets from the cell lysate. The proposed new technique is based on affinity chromatography with the monolithic capillary column. The small molecular drugs will be chemically modified so as to introduce a linker with a double bond terminal. The modified drug derivative can be copolymerized with other monomers and cross linker to form the monolith bed in the fused silica capillary. It can be predicted that the high separation performance of the monolith column will improve the efficiency of the drug target enrichment from the cell lysate. In combination with the mass spectrometry technology, the enriched proteins can be identified. Finally, such identified proteins will be validated with the biochemical analysis, such as western blotting. The effect of the chemical modification of the model drugs and the conditions used for monolith preparation will be systematically investigated so as to find a way to improve the affinity specific interaction of the affinity matrix to the drug target proteins, and to reduce the nonspecific adsorption. Our research will produce very useful tool for drug discovery.

目前对大多数药物的作用机理和靶标知之甚少。随着药理学的进步，人们已经认识到小分子药物在体内往往具有多个作用靶标。明确药物的作用靶标，可以阐明药物的作用机理，通过构效关系优化药物的结构并提高药效；还能够预测可能出现的副作用，降低药物研发的风险。本项目拟开展高效率富集药物靶标蛋白的亲合色谱整体柱技术。对研究的小分子药物进行结构修饰后，作为功能化单体与交联剂在石英毛细管内同聚，就可以形成亲合整体柱。利用有机整体柱比表面大、传质阻力小，分离纯化步骤简单，对蛋白分离效率高的特点，提高对细胞中的药物靶标蛋白的富集效率。采用蛋白组学的质谱分析技术，鉴定被亲和整体柱富集的药物靶标；再用生化分析和分子生物学的方法对靶标蛋白进行验证。我们还将研究小分子药物的化学修饰和固定化方式对亲合基质富集靶标蛋白效率的影响，从而提高靶标蛋白亲合选择性并降低非特异性吸附。此项目将为新药发现的研究提供非常有用的技术。

鉴定小分子药物在体内的靶标蛋白谱是药理学研究和新药发现的关键步骤。但是，小分子药物靶标的鉴定工作是一件令人生畏的挑战性的工作。直到目前为止，仍没有通用、可靠的小分子药物靶标的鉴定方法。因此，发展可普遍适用的药物靶标蛋白鉴定技术对于化学生物学研究和新药物研发都具有重要的意义。.本项目旨在建立一个研究小分子药物在细胞内的靶标谱的技术平台。 利用这一平台，我们研究了大环内酯类免疫抑制剂在细胞内的靶标蛋白谱的鉴定，发现了FK506，环孢素A，雷帕霉素的一个新靶标PGAM5。用生物化学和细胞生物学和分子生物学的方法进一步验证的结果。 将这两种免疫抑制剂经化学修饰引入双键，作为功能单体参与聚合，一步法制备亲和整体柱。利用该方法，我们共鉴定33种FK506的结合蛋白，32种环孢素A的结合蛋白，其中包括这两种药物已知的靶标蛋白。这一结果说明，亲和整体柱技术可以高效的富集小分子药物的靶标蛋白谱。我们发现线粒体丝氨酸/苏氨酸磷酸酶PGAM5是环孢素A，FK506的结合蛋白。采用液相色谱质谱联用和毛细管电泳激光诱导荧光分析的生化测试方法，定量考察了环孢素A，FK506及雷帕霉素对PGAM5活性的抑制。细胞活性测试以及RNAi实验证实了PGAM5是免疫抑制剂环孢素A，FK506及雷帕霉素的一个新的靶标蛋白。我们进一步用细胞实验发现这三种小分子化合物能够抑制细胞的坏死。 这一发现为发现治疗与细胞坏死相关疾病如心肌梗死，中风等疾病的药物提供了新的依据。基于这一技术，我们还进行了一系列的小分子药物的靶标鉴定。