The molecular basis of the interactions between synthetic retinoic acid analogues and the retinoic acid receptors
The molecular basis of the interactions between synthetic retinoic acid analogues and the retinoic acid receptors
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DOI：
10.1039/c6md00680a
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发表时间：
2017-03-01
期刊：
影响因子：
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通讯作者：
Whiting, Andrew
中科院分区：
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作者：
Haffez, Hesham;Chisholm, David R.;Whiting, Andrew
全反式视黄酸（ATRA）及其合成类似物EC23和EC 19通过作为核受体蛋白的视黄酸受体（RAR α、β和γ）家族的配体相互作用来指导细胞分化。迄今为止，已经解决了许多天然和合成配体与其靶蛋白复合的晶体结构，提供了配体结合的分子水平快照。然而，需要更深入地了解受体和配体的灵活性和构象自由度，以开发稳定有效的ATRA类似物用于临床。因此，我们已经使用分子建模技术来定义RAR与ATRA和两个合成类似物，EC 19和EC23的相互作用，并将其预测的生化活性与实验测量的相对配体亲和力和辅激活蛋白的募集进行比较。一个全面的分子对接的方法，探索的配体的构象空间表明，全反式维甲酸是能够结合的三个RAR蛋白在一些构象与一个扩展的结构是有利的。与此相反，生物学上不同的异构体，9-顺式-视黄酸（9 CRA），在RAR结合口袋中显示出显着较少的构象灵活性。这些发现被用于通知对接研究的合成类维生素A EC23和EC 19，以及它们各自的甲酯。EC23是ATRA的一个很好的模拟物，并且在所有三种靶RAR蛋白中与ATRA具有相似的结合模式。相比之下，EC 19表现出另一种结合模式，其降低了RAR α/γ中关键极性相互作用的强度，但非常适合于较大的RAR α结合口袋。与此相反，对接的相应的酯揭示了关键的极性相互作用的损失，这可以解释大大降低的生物活性。我们的计算结果使用基于FRET测量的体外结合测定进行了补充，这表明EC23是RAR的强结合泛激动剂，而EC 19对RAR α表现出特异性，如对接研究所预测的。这些发现可以解释EC 23和EC 19在细胞分化测定中的不同行为，并且另外，本文所述的方法可以进一步应用于理解不同类维生素A对RAR α、β和γ的选择性的分子基础。
All-trans-retinoic acid (ATRA) and its synthetic analogues EC23 and EC19 direct cellular differentiation by interacting as ligands for the retinoic acid receptor (RAR alpha, beta and gamma) family of nuclear receptor proteins. To date, a number of crystal structures of natural and synthetic ligands complexed to their target proteins have been solved, providing molecular level snap-shots of ligand binding. However, a deeper understanding of receptor and ligand flexibility and conformational freedom is required to develop stable and effective ATRA analogues for clinical use. Therefore, we have used molecular modelling techniques to define RAR interactions with ATRA and two synthetic analogues, EC19 and EC23, and compared their predicted biochemical activities to experimental measurements of relative ligand affinity and recruitment of coactivator proteins. A comprehensive molecular docking approach that explored the conformational space of the ligands indicated that ATRA is able to bind the three RAR proteins in a number of conformations with one extended structure being favoured. In contrast the biologically-distinct isomer, 9-cis-retinoic acid (9CRA), showed significantly less conformational flexibility in the RAR binding pockets. These findings were used to inform docking studies of the synthetic retinoids EC23 and EC19, and their respective methyl esters. EC23 was found to be an excellent mimic for ATRA, and occupied similar binding modes to ATRA in all three target RAR proteins. In comparison, EC19 exhibited an alternative binding mode which reduces the strength of key polar interactions in RAR alpha/gamma but is well-suited to the larger RARa binding pocket. In contrast, docking of the corresponding esters revealed the loss of key polar interactions which may explain the much reduced biological activity. Our computational results were complemented using an in vitro binding assay based on FRET measurements, which showed that EC23 was a strongly binding, pan-agonist of the RARs, while EC19 exhibited specificity for RARa, as predicted by the docking studies. These findings can account for the distinct behaviour of EC23 and EC19 in cellular differentiation assays, and additionally, the methods described herein can be further applied to the understanding of the molecular basis for the selectivity of different retinoids to RAR alpha, beta and gamma.