The complement system plays a critical role in the innate immune response, acting as a first line of defense against invading pathogens. However, dysregulation of the complement system is implicated in the pathogenesis of numerous diseases, ranging from Alzheimer’s to age-related macular degeneration and rare blood disorders. As such, complement inhibitors have enormous potential to alleviate disease burden. While a few complement inhibitors are in clinical use, there is still a significant unmet medical need for the discovery and development of novel inhibitors to treat patients suffering from disorders of the complement system. A key hurdle in the development of complement inhibitors has been the determination of their mechanism of action. Progression along the complement cascade involves the formation of numerous multimeric protein complexes, creating the potential for inhibitors to act at multiple nodes in the pathway. This is especially true for molecules that target the central component C3 and its fragment C3b, which serve a dual role as a substrate for the C3 convertases and as a scaffolding protein in both the C3 and C5 convertases. Here, we report a step-by-step reconstitution of the complement alternative pathway using bio-layer interferometry. By physically uncoupling each step in the pathway, we were able to determine the kinetic signature of inhibitors that act at single steps in the pathway and delineate the full mechanism of action of known and novel C3 inhibitors. The method could have utility in drug discovery and further elucidating the biochemistry of the complement system.

中文翻译：

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补体系统替代途径的重建能够快速描述新型抑制剂的作用机制


补体系统在先天免疫反应中起着至关重要的作用，是抵御病原体入侵的第一道防线。然而，补体系统失调与许多疾病的发病机制有关，包括阿尔茨海默病、年龄相关性黄斑变性和罕见的血液疾病。因此，补体抑制剂具有减轻疾病负担的巨大潜力。虽然一些补体抑制剂已在临床使用，但对于发现和开发新型抑制剂来治疗患有补体系统疾病的患者，仍然存在显着的未满足的医疗需求。补体抑制剂开发的一个关键障碍是确定其作用机制。补体级联的进展涉及大量多聚蛋白复合物的形成，从而使抑制剂有可能在途径的多个节点发挥作用。对于靶向中心成分 C3 及其片段 C3b 的分子来说尤其如此，它们具有作为 C3 转化酶的底物以及作为 C3 和 C5 转化酶中的支架蛋白的双重作用。在这里，我们报告了使用生物层干涉测量法逐步重建补体替代途径。通过物理解偶联途径中的每个步骤，我们能够确定在途径中的单个步骤中起作用的抑制剂的动力学特征，并描述已知和新型 C3 抑制剂的完整作用机制。该方法可用于药物发现并进一步阐明补体系统的生物化学。