核受体PPARγ组装DNA折纸色谱药物筛选新方法的研究

At present, the drug screening methods using nuclear receptor as targets have some problems, such as complicated operation, needing pure ligands of large quantity, and not for complex constituent of natural drugs. By enlightenment from cell membrane chromatography, the nuclear receptor may try to be assembled on the biological template to establish an efficient biochromatography screening of nuclear receptor targeted drugs. The DNA origami makes this idea become possible. The “nano templates” constructed of DNA origami have some advantages as biological macromolecular carriers, such as addressable assembling of protein molecules, high rigidity, good stability, and can be covalently on the surface of silica gel. Based on these, the concept of “DNA origami chromatography” is first proposed in this study. This new biochromatography method can accurately assemble the nuclear receptors on the programmable surfaces of DNA origami. The stationary phase has good mechanical properties, biocompatibility and long service life. Furthermore, the active ingredients of complex natural drugs can be quickly determined. Peroxisome proliferator-activated receptor γ (PPARγ) is an important target in the treatment of various diseases. This study intends to assemble nuclear receptor PPARγ on short tubular DNA origami, and bond it on silica surface. The nuclear receptor PPARγ assembling DNA origami chromatography is established for ginsenosides, which provide a specific, stable, convenient and effective screening method for the development of PPARγ target drugs.

目前以核受体为靶点的药物筛选法存在操作繁琐、纯配体需要量大、无法对复杂成分直接进行筛选等问题。受细胞膜色谱法启示，可尝试将核受体组装在生物模板上，采用高效生物色谱法来对核受体靶点药物进行筛选。DNA折纸术的出现使得这一想法成为可能，其构建的“纳米模板”作为生物大分子载体，能寻址组装蛋白质分子，刚性强、稳定性好。基于此，本研究首次提出“DNA折纸色谱法”的概念。这一新的生物色谱方法，能精确实现核受体在高度有序模板上的定向组装，固定相具有优良的力学性能、生物相容性和较长使用寿命，对于复杂天然产物中活性成分也能快速进行测定。过氧化物酶体增殖物激活受体γ亚型（PPARγ）是多种疾病的重要治疗靶点。本研究拟将核受体PPARγ组装在短管状DNA折纸上，并与硅胶键合，制备核受体PPARγ组装DNA折纸色谱；以人参皂苷为模型药物，为PPARγ靶点药物的开发提供一个特异性强、稳定性好、简便高效的筛选新方法。

以核受体为靶点的传统药物筛选法存在操作繁琐、纯配体需要量大、无法对复杂成分直接进行筛选等问题。本课题尝试将核受体组装在生物模板上，采用高效生物色谱法来对核受体靶点药物进行筛选。DNA折纸能精确组装蛋白质分子，刚性强、稳定性好。因此，本课题构建DNA折纸色谱模型，用于复杂天然产物中药效组分的筛选。本研究以过氧化物酶体增殖物激活受体γ亚型（PPARγ）为治疗靶点，主要研究内容包括三部分。①核受体PPARγ组装DNA折纸生物色谱柱的研制。提取大量M13mp18单链DNA和重组hPPARγ-LBD蛋白，并进行了表征；在PCR仪上，加入M13mp18单链DNA及128条订书钉链进行折叠，得长方形DNA折纸纳米生物模板；将核受体蛋白hPPARγ-LBD组装在DNA折纸上，并键合巯基化硅胶，制得核受体PPARγ组装DNA折纸色谱固定相；采用高压匀浆法，将自制的固定相装填于不锈钢色谱柱中。②核受体PPARγ组装DNA折纸色谱模型的建立及评价。构建以自制生物色谱柱为药物筛选核心的HPLC模型，考察其亲和特性及选择特异性。③核受体PPARγ组装DNA折纸色谱模型筛选中药人参活性成分的研究。采用HPLC法和HPLC/MS法，以人参总皂苷为研究对象，筛选其中与核受体PPARγ蛋白具有亲和作用的组分，从而确定了中药人参中具有药效作用的活性成分。实验结果发现，自制DNA折纸生物色谱柱性能良好，且可专属性地识别PPARγ受体阳性药物——罗格列酮；成功构建核受体PPARγ组装DNA折纸生物色谱药物筛选模型，并筛选出人参皂苷Re为人参皂苷中主要的药效组分。本研究为PPARγ靶点药物的开发提供了一个特异性强、稳定性好、简便高效的筛选新方法，并可为其他胞内受体靶点药物的筛选提供新的思路和方法。