Department of Early Discovery Biochemistry, Genentech, South San Francisco, California, USA.
Department of Biochemical and Cellular Pharmacology, Genentech, South San Francisco, California, USA.
J Biol Chem. 2024 Jul;300(7):107467. doi: 10.1016/j.jbc.2024.107467. Epub 2024 Jun 12.
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 in vitro 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.
补体系统在先天免疫反应中起着至关重要的作用，是抵御入侵病原体的第一道防线。然而，补体系统的失调与许多疾病的发病机制有关，从阿尔茨海默病到年龄相关性黄斑变性和罕见的血液疾病等。因此，补体抑制剂具有减轻疾病负担的巨大潜力。虽然有几种补体抑制剂已在临床应用，但仍有很大的未满足的医疗需求，需要发现和开发新型抑制剂来治疗患有补体系统疾病的患者。补体抑制剂发展的一个关键障碍是确定其作用机制。补体级联的进展涉及形成许多多聚体蛋白复合物，从而为抑制剂在途径中的多个节点发挥作用创造了潜力。对于靶向中央成分 C3 及其片段 C3b 的分子来说尤其如此，C3b 既作为 C3 转化酶的底物，又作为 C3 和 C5 转化酶中的支架蛋白发挥双重作用。在这里，我们使用生物层干涉法报告了补体替代途径的逐步体外重建。通过物理分离途径中的每一步，我们能够确定作用于途径中单个步骤的抑制剂的动力学特征，并阐明已知和新型 C3 抑制剂的完整作用机制。该方法在药物发现和进一步阐明补体系统的生物化学方面可能具有实用价值。
