Screening for small-molecule fragments that can lead to potent inhibitors of protein–protein interactions (PPIs) is often a laborious step as the fragments cannot dissociate the targeted PPI due to their low μM–mM affinities. Here, we describe an NMR competition assay called w-AIDA-NMR (weak-antagonist induced dissociation assay-NMR), which is sensitive to weak μM–mM ligand–protein interactions and which can be used in initial fragment screening campaigns. By introducing point mutations in the complex’s protein that is not targeted by the inhibitor, we lower the effective affinity of the complex, allowing for short fragments to dissociate the complex. We illustrate the method with the compounds that block the Mdm2/X-p53 and PD-1/PD-L1 oncogenic interactions. Targeting the PD-/PD-L1 PPI has profoundly advanced the treatment of different types of cancers.

中文翻译：

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用于检测蛋白质-蛋白质相互作用的 Hit/Lead 抑制剂的竞争 NMR

筛选可导致蛋白质-蛋白质相互作用 (PPI) 强效抑制剂的小分子片段通常是一个费力的步骤，因为这些片段由于其低 μM-mM 亲和力而无法解离目标 PPI。在这里，我们描述了一种称为 w-AIDA-NMR（弱拮抗剂诱导解离分析-NMR）的 NMR 竞争分析，它对弱 μM-mM 配体-蛋白质相互作用敏感，可用于初始片段筛选活动。通过在抑制剂未靶向的复合物蛋白质中引入点突变，我们降低了复合物的有效亲和力，允许短片段解离复合物。我们用阻断 Mdm2/X-p53 和 PD-1/PD-L1 致癌相互作用的化合物来说明该方法。