细胞分裂过程中正确的染色体分离依赖于染色体内聚力的及时溶解。分离酶 (EC3.4.22.49) 是一种半胱氨酸蛋白酶，通过裂解粘连蛋白的 kleisin 亚基，在有丝分裂中发挥关键作用，从而为癌症治疗提供了一个有希望的靶点。然而，分离适合高通量筛选的活性人分离酶的挑战限制了有效抑制剂的鉴定。在这里，我们利用嗜热毛壳菌分离酶（ctSPD）的蛋白酶结构域对小分子抑制剂进行了高通量筛选，该结构域不仅与人类分离酶具有显着的序列相似性，而且易于获得。在对包含 9,172 种化合物的文库进行初步筛选并随后使用人分离酶进行验证后，我们确定了 walrycin B 及其类似物、毒黄素、3-甲基毒黄素和 3-苯基毒黄素是人分离酶的有效抑制剂。随后的微尺度热泳分析和分子动力学模拟表明，walrycin B 与分离酶的活性位点结合，并与底物竞争结合。此外，基于细胞的研究表明，walrycin B 及其类似物可有效诱导细胞周期停滞在 M 期，激活细胞凋亡，并最终导致有丝分裂中的细胞死亡。最后，在小鼠异种移植模型中，walrycin B 表现出显着的抗肿瘤功效，且副作用极小。总之，这些发现凸显了 walrycin B 在癌症治疗中的治疗潜力，及其在未来涉及 separase 的研究中作为化学工具的实用性。版权所有 © 2024。由 Elsevier Inc. 出版。

Proper chromosome segregation during cell division relies on the timely dissolution of chromosome cohesion. Separase (EC3.4.22.49), a cysteine protease, plays a critical role in mitosis by cleaving the kleisin subunit of cohesin, thereby presenting a promising target for cancer therapy. However, challenges in isolating active human separase suitable for high-throughput screening have limited the identification of effective inhibitors. Here, we conducted a high-throughput screening of small-molecule inhibitors using the protease domain of Chaetomium thermophilum separase (ctSPD), which not only shares significant sequence similarity with human separase but is also readily available. After conducting a primary screening of a library containing 9,172 compounds and subsequent validation using human separase, we identified walrycin B and its analogs, toxoflavin, 3-methyltoxoflavin, and 3-phenyltoxoflavin, as potent inhibitors of human separase. Subsequent microscale thermophoresis assays and molecular dynamics simulations revealed that walrycin B binds to the active site of separase and competes with substrates for binding. Additionally, cell-based studies showed that walrycin B and its analogs effectively induce cell cycle arrest at the M phase, activate apoptosis, and ultimately lead to cell death in mitosis. Finally, in a mouse xenograft model, walrycin B exhibited significant antitumor efficacy with minimal side effects. Together, these findings highlight the therapeutic potential of walrycin B for cancer treatment and its utility as a chemical tool in future studies involving separase.Copyright © 2024. Published by Elsevier Inc.