Development of an In Situ G Protein-Coupled Receptor Fragment Molecule Screening Approach with High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance.

IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY ACS Chemical Biology Pub Date : 2025-02-21 Epub Date: 2025-01-21 DOI:10.1021/acschembio.4c00686
Enzo Petracco, Guillaume Ferré, Ivo Kabelka, Flavio Ballante, Jens Carlsson, Emma Mulry, Arka P Ray, James Collins, Florent Allais, Matthew T Eddy
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Abstract

Small molecules are essential for investigating the pharmacology of membrane proteins and remain the most common approach for therapeutically targeting them. However, most experimental small molecule screening methods require ligands containing radiolabels or fluorescent labels and often involve isolating proteins from their cellular environment. Additionally, most conventional screening methods are suited for identifying compounds with moderate to higher affinities (KD < 1 μM) and are less effective at detecting lower affinity compounds, such as weakly binding molecular fragments. To address these limitations, we demonstrated a proof-of-concept application of high-resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) spectroscopy with small molecules that bind the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor. Our approach leverages a streamlined workflow to prepare NMR samples with only milligrams of unpurified cell membranes containing ∼1 μM of A2AAR. Utilizing saturation transfer difference NMR, we identified bound small molecules from spectra recorded within minutes and further derived information on ligand binding poses without the need for detailed structure determination. After establishing optimal criteria for which the HRMAS approach is most sensitive, we leveraged our HRMAS approach to identify and characterize molecular fragments not previously known to be ligands of A2AAR. In molecular docking and simulations, we observed novel binding poses for these fragments, which revealed the potential to grow them into more complex ligands and confirmed HRMAS NMR as a valuable tool for lead compound identification in the context of fragment-based drug discovery.

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高分辨率魔角旋转核磁共振原位G蛋白偶联受体片段分子筛选方法的建立。
小分子对于研究膜蛋白的药理学至关重要,并且仍然是治疗它们的最常用方法。然而,大多数实验性小分子筛选方法需要含有放射性标记或荧光标记的配体,并且通常需要将蛋白质从细胞环境中分离出来。此外,大多数传统的筛选方法适合于鉴定中高亲和度(KD < 1 μM)的化合物,而对低亲和度的化合物(如弱结合的分子片段)的检测效果较差。为了解决这些限制,我们展示了高分辨率魔角旋转核磁共振(HRMAS NMR)光谱的概念验证应用,该小分子结合了人类A2A腺苷受体(A2AAR),这是一种a类G蛋白偶联受体。我们的方法利用简化的工作流程来制备NMR样品,仅使用含有~ 1 μM A2AAR的未纯化细胞膜毫克。利用饱和转移差核磁共振,我们从几分钟内记录的光谱中识别出结合的小分子,并进一步获得配体结合姿势的信息,而无需详细的结构测定。在建立HRMAS方法最敏感的最佳标准后,我们利用HRMAS方法来识别和表征以前不知道是A2AAR配体的分子片段。在分子对接和模拟中,我们观察到这些片段的新结合姿势,这揭示了它们生长成更复杂配体的潜力,并证实了HRMAS NMR在基于片段的药物发现的背景下作为先导化合物鉴定的有价值的工具。
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来源期刊
ACS Chemical Biology
ACS Chemical Biology 生物-生化与分子生物学
CiteScore
7.50
自引率
5.00%
发文量
353
审稿时长
3.3 months
期刊介绍: ACS Chemical Biology provides an international forum for the rapid communication of research that broadly embraces the interface between chemistry and biology. The journal also serves as a forum to facilitate the communication between biologists and chemists that will translate into new research opportunities and discoveries. Results will be published in which molecular reasoning has been used to probe questions through in vitro investigations, cell biological methods, or organismic studies. We welcome mechanistic studies on proteins, nucleic acids, sugars, lipids, and nonbiological polymers. The journal serves a large scientific community, exploring cellular function from both chemical and biological perspectives. It is understood that submitted work is based upon original results and has not been published previously.
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