Human biology is regulated by a complex network of protein–protein interactions (PPIs), and disruption of this network has been implicated in many diseases. However, the targeting of PPIs remains a challenging area for chemical probe and drug discovery. Although many methodologies have been put forth to facilitate these efforts, new technologies are still needed. Current biochemical assays for PPIs are typically limited to motif–domain and domain–domain interactions, and assays that will enable the screening of full-length protein systems, which are more biologically relevant, are sparse. To overcome this barrier, we have developed a new assay technology, “PPI catalytic enzyme-linked click chemistry assay” or PPI cat-ELCCA, which utilizes click chemistry to afford catalytic signal amplification. To validate this approach, we have applied PPI cat-ELCCA to the eIF4E–4E-BP1 and eIF4E–eIF4G PPIs, key regulators of cap-dependent mRNA translation. Using these examples, we have demonstrated that PPI cat-ELCCA is amenable to full-length proteins, large (>200 kDa) and small (∼12 kDa), and is readily adaptable to automated high-throughput screening. Thus, PPI cat-ELCCA represents a powerful new tool in the toolbox of assays available to scientists interested in the targeting of disease-relevant PPIs.

中文翻译：

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全长蛋白质-蛋白质相互作用的高通量化学探测

人类生物学受到蛋白质-蛋白质相互作用 (PPI) 的复杂网络的调节，该网络的破坏与许多疾病有关。然而，质子泵抑制剂的靶向仍然是化学探针和药物发现的一个具有挑战性的领域。尽管已经提出了许多方法来促进这些努力，但仍然需要新技术。目前 PPI 的生化测定通常仅限于基序-结构域和结构域-结构域相互作用，而能够筛选生物学上更相关的全长蛋白质系统的测定很少。为了克服这一障碍，我们开发了一种新的检测技术，“PPI催化酶联点击化学检测”或PPI cat-ELCCA，它利用点击化学来提供催化信号放大。为了验证这种方法，我们将 PPI cat-ELCCA 应用于 eIF4E–4E-BP1 和 eIF4E–eIF4G PPI，它们是帽依赖性 mRNA 翻译的关键调节因子。使用这些例子，我们证明了 PPI cat-ELCCA 适合全长蛋白质、大蛋白 (>200 kDa) 和小蛋白 (∼12 kDa)，并且很容易适应自动化高通量筛选。因此，PPI cat-ELCCA 代表了对疾病相关 PPI 靶向感兴趣的科学家可用的检测工具箱中的强大新工具。