College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China.
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China.
Talanta. 2017 Apr 1;165:508-515. doi: 10.1016/j.talanta.2016.12.089. Epub 2016 Dec 30.
Screening agonists of peroxisome proliferator-activated receptor-γ (PPARγ) from natural products is particularly motivated by the need for effective anti-diabetic agents. However, method for direct identification of PPARγ ligands from a complex sample is rarely reported. Here we propose a novel immobilized fusion protein affinity chromatography (IFPAC) to achieve rapid multicomponent screening. First, functional human PPARγ ligand binding domain (hPPARγLBD) was bacterially produced by fusion to glutathione S-transferase (GST). The unpurified GST-hPPARγLBD was directly applied to a 96-well filter plate prepacked with glutathione sepharose. Due to the strong affinity between GST and glutathione, the fusion protein could selectively attach to the glutathione matrix with an oriented immobilization, which was rapidly purified under non-denaturing conditions. Experimental results indicated that the prepared 96-affinity column array exhibited excellent selectivity and sensitivity for fishing novel interacting compounds. The proposed approach was applied in the high-throughput screening of PPARγ ligands from natural products, followed by rapid characterization of active compounds using HPLC-ESI-Q-TOF-MS/MS. Isochlorogenic acid A in Dendranthema indicum flowers was found to be a PPARγ ligand. Our findings suggested the IFPAC could be a powerful tool for drug discovery from natural products.
从天然产物中筛选过氧化物酶体增殖物激活受体-γ (PPARγ) 的激动剂特别需要有效的抗糖尿病药物。然而，从复杂样品中直接鉴定 PPARγ 配体的方法很少有报道。在这里，我们提出了一种新的固定化融合蛋白亲和层析（IFPAC）方法，以实现快速的多组分筛选。首先，通过与谷胱甘肽 S-转移酶（GST）融合，在细菌中产生功能性人 PPARγ 配体结合域（hPPARγLBD）。未纯化的 GST-hPPARγLBD 直接应用于 96 孔过滤板，该过滤板预先填充有谷胱甘肽琼脂糖。由于 GST 和谷胱甘肽之间的强亲和力，融合蛋白可以选择性地附着在谷胱甘肽基质上，实现定向固定化，在非变性条件下可快速纯化。实验结果表明，制备的 96 个亲和柱阵列对钓取新型相互作用化合物具有优异的选择性和灵敏度。该方法已应用于从天然产物中高通量筛选 PPARγ 配体，并使用 HPLC-ESI-Q-TOF-MS/MS 快速表征活性化合物。发现菊花中的绿原酸 A 是一种 PPARγ 配体。我们的研究结果表明，IFPAC 可以成为从天然产物中发现药物的有力工具。
