Miniaturized weak affinity chromatography for ligand identification of nanodiscs-embedded G-protein coupled receptors,Analytica Chimica Acta

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Miniaturized weak affinity chromatography for ligand identification of nanodiscs-embedded G-protein coupled receptors
Analytica Chimica Acta ( IF 5.7 ) Pub Date : 2020-05-01 , DOI: 10.1016/j.aca.2020.03.062
Lucile Lecas ₁ , Lucie Hartmann ₂ , Lydia Caro ₂ , Sarah Mohamed-Bouteben ₂ , Claire Raingeval ₁ , Isabelle Krimm ₁ , Renaud Wagner ₂ , Vincent Dugas ₁ , Claire Demesmay ₁

Affiliation

Biophysical techniques that enable the screening and identification of weak affinity fragments against a target protein are at the heart of Fragment Based Drug Design approaches. In the case of membrane proteins, the crucial criteria for fragment screening are low protein consumption, unbiased conformational states and rapidity because of the difficulties in obtaining sufficient amounts of stable and functionally folded proteins. Here we show for the first time that lipid-nanodisc systems (membrane-mimicking environment) and miniaturized affinity chromatography can be combined to identify specific small molecule ligands that bind to an integral membrane protein. The approach was exemplified using the AA2AR GPCR. Home-made affinity nano-columns modified with nanodiscs-embedded AA2AR (only about 1 μg of protein per column) were fully characterized by frontal chromatographic experiments. This method allows (i) to distinguish specific and unspecific ligand/receptor interactions, (ii) to assess dissociation constants, (iii) to identify the binding pocket of uncharacterized ligands using a reference compound (whose binding site is known) with competition experiments. Weak affinity ligands with Kd in the low to high micromolar range can be detected. At last, the applicability of this method was demonstrated with 6 fragments recently identified as ligands or non-ligands of AA2AR.

中文翻译：

用于纳米圆盘嵌入 G 蛋白偶联受体配体鉴定的微型弱亲和色谱

能够筛选和鉴定针对靶蛋白的弱亲和力片段的生物物理技术是基于片段的药物设计方法的核心。在膜蛋白的情况下，片段筛选的关键标准是低蛋白质消耗、无偏见的构象状态和快速，因为难以获得足够数量的稳定和功能折叠的蛋白质。在这里，我们首次展示了脂质纳米圆盘系统（膜模拟环境）和小型亲和层析可以结合来识别与完整膜蛋白结合的特定小分子配体。使用 AA2AR GPCR 举例说明了该方法。用纳米圆盘嵌入的 AA2AR 修饰的自制亲和纳米柱（每柱仅约 1 μg 蛋白质）通过正面色谱实验进行了充分表征。该方法允许 (i) 区分特异性和非特异性配体/受体相互作用，(ii) 评估解离常数，(iii) 使用参考化合物（其结合位点已知）通过竞争实验鉴定未表征配体的结合口袋。可以检测到 Kd 在低到高微摩尔范围内的弱亲和性配体。最后，通过最近鉴定为 AA2AR 的配体或非配体的 6 个片段证明了该方法的适用性。(iii) 使用参考化合物（其结合位点已知）通过竞争实验鉴定未表征配体的结合口袋。可以检测到 Kd 在低到高微摩尔范围内的弱亲和性配体。最后，通过最近鉴定为 AA2AR 的配体或非配体的 6 个片段证明了该方法的适用性。(iii) 使用参考化合物（其结合位点已知）通过竞争实验鉴定未表征配体的结合口袋。可以检测到 Kd 在低到高微摩尔范围内的弱亲和性配体。最后，通过最近鉴定为 AA2AR 的配体或非配体的 6 个片段证明了该方法的适用性。

更新日期：2020-05-01

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