PDF(Pubmed)
Abstract：
Miniaturized weak affinity chromatography is emerging as an interesting alternative to conventional biophysical tools for performing fragment-screening studies in the context of fragment-based drug discovery. In order to push back the analytical limits, it is necessary not only to control non-specific interactions with chromatographic support, but also to adapt this methodology by comparing the results obtained on an affinity column to a control column. The work presented in this study focused on fragment screening that targets a model membrane protein, the adenosine A2A receptor, embedded in nanodiscs (NDs) as biomimetic membranes. By studying the retention behavior of test fragment mixtures on supports modified with different types of NDs, we were able to determine the contribution of ND-related non-specific interactions, in particular the electrostatic effect of anionic phospholipids and the hydrophobic effect of neutral phospholipids. Different strategies for the preparation of control columns (empty NDs, orthosteric site blocking) were investigated and are presented for the first time. With these two types of control columns, the screening enabled the identification of two new fragments of AA2AR, which were confirmed by competition experiments and whose Kd values, estimated directly during the screening or after the competition experiments in frontal mode, were in good agreement.
摘要：
在基于片段的药物发现的背景下，微型弱亲和色谱正在成为常规生物物理工具的有趣替代方法，用于进行片段筛选研究。为了推回分析极限，不仅需要控制与色谱载体的非特异性相互作用，但也通过比较在亲和柱与对照柱上获得的结果来调整这种方法。这项研究中提出的工作集中在靶向模型膜蛋白的片段筛选上，腺苷A2A受体，嵌入纳米圆盘（ND）作为仿生膜。通过研究测试片段混合物在用不同类型的ND修饰的载体上的保留行为，我们能够确定与ND相关的非特异性相互作用的贡献，特别是阴离子磷脂的静电效应和中性磷脂的疏水效应。制备控制柱的不同策略(空ND，正构位点阻断）进行了研究，并首次提出。对于这两种类型的控制柱，筛选能够鉴定出两个新的AA2AR片段，通过竞争实验证实了这一点，其Kd值，在筛选过程中或在正面模式的竞赛实验之后直接估计，达成了很好的协议。
