三阴性乳腺癌（TNBC）是乳腺癌中治疗最困难的亚型，而针对TNBC的异质性已成为一种令人着迷的治疗策略。在本研究中，我们首次提出双靶向PAK1和HDAC6是TNBC治疗的一种有前景的新策略，因为它们在能量代谢和表观遗传修饰的调节中起着重要作用。我们发现了一种新型的双靶向PAK1/HDAC6抑制剂，6-（2-（环丙胺基）-6-（2,4-二氯苯基）-7-氧基吡啶[2,3-d]嘧啶-8（7H）-基）-N-羟基己酰胺（ZMF-23），对MDA-MB-231细胞的PAK1和HDAC6表现出显著的抑制活性和强大的抗增殖潜力。此外，SPR和CETSA实验证明了ZMF-23与PAK1/HDAC6的靶向结合。在机械方面，ZMF-23强烈抑制了细胞的PAK1和HDAC6活性，阻碍了PAK1和HDAC6调控的有氧糖酵解和细胞迁移。通过RNA测序分析，发现ZMF-23能诱导TNF-α调控的程序化坏死，进一步增强了细胞凋亡。此外，ZMF-23触发了PAK1-微管蛋白/HDAC6-Stathmin调控的微管结构变化，进而诱导了G2/M周期阻滞。此外，通过抑制PAK1和HDAC6，ZMF-23在TNBC裸鼠和小鱼模型中显示出显著的抗增殖作用。总之，ZMF-23是一种具有TNBC治疗潜力的双重PAK1/HDAC6抑制剂。版权所有 © 2023 Elsevier B.V. 发布。

Triple-negative breast cancer (TNBC) is the most poorly treated subtype of breast cancer, and targeting the heterogeneity of TNBC has emerged as a fascinating therapeutic strategy. In this study, we propose for the first time that dual-targeting PAK1 and HDAC6 is a promising novel strategy for TNBC treatment due to their essential roles in the regulation of energy metabolism and epigenetic modification. We discovered a novel dual-targeting PAK1/HDAC6 inhibitor, 6 - (2-(cyclopropylamino) - 6 - (2,4-dichlorophenyl) - 7 - oxopyrido [2,3-d] pyrimidin - 8 (7H) -yl) - N-hydroxyhexanamide (ZMF-23), which presented profound inhibitory activity against PAK1 and HDAC6 and robust antiproliferative potency in MDA-MB-231 cells. In addition, SPR and CETSA assay demonstrated the targeted binding of ZMF-23 with PAK1/HDAC6. Mechanically, ZMF-23 strongly inhibited the cellular PAK1 and HDAC6 activity, impeded PAK1 and HDAC6 regulated aerobic glycolysis and migration. By RNA-seq analysis, ZMF-23 was found to induce TNF-α-regulated necroptosis, which further enhanced apoptosis. Additionally, ZMF-23 triggered PAK1-tubulin/HDAC6-Stathmin regulated microtubule structure changes, which further induce the G2/M cycle arrest. Moreover, prominent anti-proliferative effect of ZMF-23 was confirmed in the TNBC xenograft zebrafish and mice model via PAK1 and HDAC6 inhibition. Collectively, ZMF-23 is a novel dual PAK1/HDAC6 inhibitor with TNBC treatment potential.Copyright © 2023. Published by Elsevier B.V.