Abstract The classical pathway (CP) is a potent mechanism for initiating complement activity and is a driver of pathology in many complement-mediated diseases. The CP is initiated via activation of complement component C1, which consists of the pattern recognition molecule C1q bound to a tetrameric assembly of proteases C1r and C1s. Enzymatically active C1s provides the catalytic basis for cleavage of the downstream CP components, C4 and C2, and is therefore an attractive target for therapeutic intervention in CP-driven diseases. Although an anti-C1s mAb has been Food and Drug Administration approved, identifying small-molecule C1s inhibitors remains a priority. In this study, we describe 6-(4-phenylpiperazin-1-yl)pyridine-3-carboximidamide (A1) as a selective, competitive inhibitor of C1s. A1 was identified through a virtual screen for small molecules that interact with the C1s substrate recognition site. Subsequent functional studies revealed that A1 dose-dependently inhibits CP activation by heparin-induced immune complexes, CP-driven lysis of Ab-sensitized sheep erythrocytes, CP activation in a pathway-specific ELISA, and cleavage of C2 by C1s. Biochemical experiments demonstrated that A1 binds directly to C1s with a Kd of ∼9.8 μM and competitively inhibits its activity with an inhibition constant (Ki) of ∼5.8 μM. A 1.8-Å-resolution crystal structure revealed the physical basis for C1s inhibition by A1 and provided information on the structure–activity relationship of the A1 scaffold, which was supported by evaluating a panel of A1 analogs. Taken together, our work identifies A1 as a new class of small-molecule C1s inhibitor and lays the foundation for development of increasingly potent and selective A1 analogs for both research and therapeutic purposes.

中文翻译：

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6-(4-Phenylpiperazin-1-yl)Pyridine-3-Carboximidamide 和化学类似物对 C1s 蛋白酶和经典补体途径的抑制


摘要经典途径（CP）是启动补体活性的有效机制，并且是许多补体介导的疾病的病理驱动因素。 CP 通过激活补体成分 C1 启动，该成分由与蛋白酶 C1r 和 C1s 的四聚体结合的模式识别分子 C1q 组成。具有酶活性的 C1 为下游 CP 成分 C4 和 C2 的裂解提供了催化基础，因此是 CP 驱动疾病的治疗干预的一个有吸引力的靶标。尽管抗 C1s 单克隆抗体已获得美国食品和药物管理局 (FDA) 的批准，但识别小分子 C1s 抑制剂仍然是当务之急。在这项研究中，我们将 6-(4-苯基哌嗪-1-基)吡啶-3-甲酰亚胺 (A1) 描述为 C1 的选择性竞争性抑制剂。 A1 通过与 C1s 底物识别位点相互作用的小分子虚拟屏幕进行识别。随后的功能研究表明，A1 剂量依赖性地抑制肝素诱导的免疫复合物引起的 CP 激活、Ab 致敏绵羊红细胞的 CP 驱动裂解、途径特异性 ELISA 中的 CP 激活以及 C1 对 C2 的裂解。生化实验表明，A1 直接与 C1s 结合，Kd 为 ∼9.8 μM，并以 ∼5.8 μM 的抑制常数 (Ki) 竞争性抑制其活性。 1.8 Å 分辨率的晶体结构揭示了 A1 抑制 C1s 的物理基础，并提供了有关 A1 支架结构-活性关系的信息，这通过评估一组 A1 类似物得到了支持。 总而言之，我们的工作将 A1 确定为一类新型小分子 C1s 抑制剂，并为开发用于研究和治疗目的的日益有效和选择性的 A1 类似物奠定了基础。