混合物中低丰度高亲和力配体的指数富集技术及应用

The integration of competitive binding, magnetic separation of bound ligands with HPLC-MS analysis is a validated new technique for screening ligands in compound mix, however its utilization is limited when samples are from solution-phase combinatorial synthesis, or extracts of plants, or traditional chinese medicine, wherein compounds with biological activities greatly vary in abundance. To address this problem, a strategy of iterative enrichment of low abundance and high affinity ligands is proposed by using all bound ligands from the previous competitive binding systems as the sample for the next competitive binding system, and a proportion-down series of quantities of a magnetic immobilized tagret to competitively binding ligands with their averaged binding ratios below 10%, ultimately resulting in an Affinity-Based-Selective-Exponential-Enrichment (ABSEE) of ligands of higher affinity. This ABSEE-based screen technique will facilitat discovering higher-affinity ligands from complex mixtures.We choose human cyclic nucleotide phosphodiesterase 4 (PDE4) and glutathione-S-transferase isozyme mu (GSTM) as enzyme targets. Comlex mixtures of known inhibitors will be synthesized via solution-phase combinatorial synthesis and a simulate natural product fractions spiking with known inhibitors will be applied to test the effectiveness of ABSEE and search for the optimal conditions for ABSEE. Based on ABSEE, libraries of compounds mixtures prepared by solution-phased combinatirail syntheses, and preliminary fractionations of natural products, will be utilized for the screening of high-affinity inhibitors of PDE4, and GSTM. Phamarcological actions of hits will be evaluated. Through those studies, a new mixture-based library technique will be developed for rapid discovery of high-affinity ligands in trace abundances in complex mixture samples based on ABSEE.

磁分离竞争结合配体联用HPLC-MS分析可同步测定混合物中多种配体亲和力，但不适于组合合成复杂混合物及粗分级天然产物混合物中低丰度高亲和力先导配体发现。将结合配体提取混合物作为样品再迭代竞争结合并磁分离结合配体，用等比例衰减的磁珠固定化靶蛋白量使每轮配体平均结合率尽可能低于10%，可产生亲和力驱动的选择性指数富集 (ABSEE)，以快速发现复杂混合物中痕量高亲和力配体。用人磷酸二酯酶同工酶4、谷胱甘肽-S 转硫酶同工酶mu为可溶靶蛋白模型并用磁珠固定化；用液相组合合成已知亲和力抑制剂混合物及含已知亲和力抑制剂的粗分级天然产物，验证ABSEE和所需靶蛋白量递减序列；基于ABSEE，从液相组合合成未知亲和力抑制剂混合物及粗分级天然产物混合物中，快速发现两种靶蛋白的高亲和力抑制剂，初步考察新抑制剂的药理活性。据此建立混合物中低丰度高亲和力配体快速发现新技术，促进混合组合库技术在药物发现中的应用。

基于磁分离竞争结合配体联用HPLC-MS同步测定混合物中多种配体亲和力方法，建立亲和力驱动的选择性指数富集技术(ABSEE)，以快速发现复杂混合物中痕量高亲和力配体。以人磷酸二酯酶同工酶4(PDE4B2)和谷胱甘肽S-转硫酶mu(GSTM)及其抑制剂混合物为模型，将结合配体提取混合物作为样品再迭代竞争结合并磁分离结合配体，用等比例衰减的磁珠固定化靶蛋白量使每轮配体平均结合率尽可能低于10%，考察ABSEE和磁珠固定化靶蛋白递减序列。首先完成ABSEE所需关键原材料兼性粒子表面亲水磁珠的制备与表征；系统比较和优化人磷酸二酯酶同工酶4(PDE4B2)全长及截短突变体表达及固定化，且因全长表达困难，用双对数模型证明截短突变体为替代靶筛选可行，合成PDE4抑制剂并筛选及优化炎症细胞模型；完成GST同工酶的表达及固定化，设计合成GSTM选择性二价潜抑制剂、体内外实验证实BDEA、EDEA、ADEA对耐顺铂乳腺癌有显著增敏活性，而GSTA在耐顺铂肺癌A549为主导亚型。完成用结核分支杆菌二氢叶酸还原酶为靶的表达固定化，以合成甲氨蝶呤衍生物纯品人工混合模拟混合物验证ABSEE效应和靶蛋白递减序列，以甲氨蝶呤衍生物组合合成小库(>20个成分)、添加目标配体的姜黄醇提物为样品，证明ABSEE可快速筛选混合库中亚纳摩尔级但丰度0.1%的高亲和力配体，初步建立了混合物中低丰度高亲和力配体快速发现新技术。进一步完成甲硫氨酸脱甲酰基酶、甲硫氨酸氨肽酶的表达固定化并设计三个酶的抑制剂组合合成大库初步筛选，为最终常规应用ABSEE 筛选复杂混合库发现先导配体奠定基础。