Many human cancers over-express B cell lymphoma 2 (Bcl-2) or X-linked inhibitor of apoptosis (IAP) proteins to evade cell death. The pro-apoptotic ARTS (Sept4_i2) protein binds directly to both Bcl-2 and XIAP and promotes apoptosis by stimulating their degradation via the ubiquitin-proteasome system (UPS). Here we describe a small molecule, A4, that mimics the function of ARTS. Microscale thermophoresis assays showed that A4 binds XIAP, but not cellular inhibitor of apoptosis protein 1 (cIAP1). A4 binds to a distinct ARTS binding pocket in the XIAP-BIR3 (baculoviral IAP repeat 3) domain. Like ARTS, A4 stimulated poly-ubiquitylation and UPS-mediated degradation of XIAP and Bcl-2, but not cIAP1, resulting in caspase-9 and -3 activation and apoptosis. In addition, over-expression of XIAP rescued HeLa cells from A4-induced apoptosis, consistent with the idea that A4 kills by antagonizing XIAP. On the other hand, treatment with the SMAC-mimetic Birinapant induced secretion of tumour necrosis factor-α (TNFα) and killed ~50% of SKOV-3 cells, and addition of A4 to Birinapant-treated cells significantly reduced secretion of TNFα and blocked Birinapant-induced apoptosis. This suggests that A4 acts by specifically targeting XIAP. The effect of A4 was selective as peripheral blood mononuclear cells and normal human breast epithelial cells were unaffected. Furthermore, proteome analysis revealed that cancer cell lines with high levels of XIAP were particularly sensitive to the killing effect of A4. These results provide proof of concept that the ARTS binding site in XIAP is “druggable”. A4 represents a novel class of dual-targeting compounds stimulating apoptosis by UPS-mediated degradation of important anti-apoptotic oncogenes.

许多人类癌症过度表达 B 细胞淋巴瘤 2 (Bcl-2) 或 X 连锁凋亡抑制剂 (IAP) 蛋白以逃避细胞死亡。促凋亡 ARTS (Sept4_i2) 蛋白直接与 Bcl-2 和 XIAP 结合，并通过泛素-蛋白酶体系统 (UPS) 刺激它们的降解来促进细胞凋亡。在这里，我们描述了一种模拟 ARTS 功能的小分子 A4。微量热泳分析显示 A4 结合 XIAP，但不结合细胞凋亡蛋白 1 (cIAP1)。A4 与 XIAP-BIR3（杆状病毒 IAP 重复 3）结构域中的一个独特的 ARTS 结合口袋结合。与 ARTS 一样，A4 刺激多泛素化和 UPS 介导的 XIAP 和 Bcl-2 降解，但不刺激 cIAP1，导致 caspase-9 和 -3 活化和细胞凋亡。此外，XIAP 的过表达使 HeLa 细胞免于 A4 诱导的细胞凋亡，与 A4 通过对抗 XIAP 杀死的想法一致。另一方面，用模拟 SMAC 的 Birinapant 处理诱导肿瘤坏死因子-α (TNFα) 的分泌并杀死约 50% 的 SKOV-3 细胞，向 Birinapant 处理的细胞中添加 A4 显着减少 TNFα 的分泌并阻断Birinapant 诱导的细胞凋亡。这表明 A4 通过专门针对 XIAP 起作用。A4 的作用是选择性的，因为外周血单个核细胞和正常人乳腺上皮细胞不受影响。此外，蛋白质组分析显示，具有高水平 XIAP 的癌细胞系对 A4 的杀伤作用特别敏感。这些结果证明了 XIAP 中的 ARTS 结合位点是“可药用的”。