Abstract：
Breast cancer is one of the most common female malignant tumors, with triple-negative breast cancer (TNBC) being the most specific, highly invasive, metastatic and associated with a poor prognosis. Our previous study showed that the natural product ganoderic acid A (GAA) has a certain affinity for MDM2. In this study, two series of novel GAA PROTACs C1-C10 and V1-V10 were designed and synthesized for the treatment of breast cancer. The antitumor activity of these compounds was evaluated against four human tumor cell lines (MCF-7, MDA-MB-231, SJSA-1, and HepG2). Among them, V9 and V10 showed stronger anti-proliferative effects against breast cancer cells, and V10 showed the best selectivity in MDA-MB-231 cells (TNBC), which was 5-fold higher than that of the lead compound GAA. Preliminary structure-activity analysis revealed that V-series GAA PROTACs had better effects than C-series, and the introduction of 2O-4O PEG linkers could significantly improve the antitumor activity. Molecular docking, surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), and Western blot researches showed that both V9 and V10 could bind with MDM2, and degrade the protein through the ubiquitin-proteasome system. Molecular dynamics simulation (MD) revealed that V10 is a bifunctional molecule that can bind to von Hippel-Lindau (VHL) at one end and target MDM2 at the other. In addition, V10 promoted the upregulation of p21 in p53-mutant MDA-MB-231 cells, and induced apoptosis via down-regulation of the bcl-2/bax ratio and the expression of cyclin B1. Finally, in vivo experiments showed that, V10 also exhibited good tumor inhibitory activity in xenografted TNBC zebrafish models, with an inhibition rate of 27.2% at 50 μg/mL. In conclusion, our results suggested that V10 has anti-tumor effects on p53-mutant breast cancer in vitro and in vivo, and may be used as a novel lead compound for the future development of TNBC.
摘要：
乳腺癌是女性最常见的恶性肿瘤之一，三阴性乳腺癌（TNBC）是最具体的，高度侵入性,转移并与不良预后相关。我们先前的研究表明，天然产物灵芝酸A（GAA）对MDM2具有一定的亲和力。在这项研究中,设计并合成了两个系列的新型GAAPROTACsC1-C10和V1-V10，用于治疗乳腺癌。评价这些化合物对四种人肿瘤细胞系(MCF-7、MDA-MB-231、SJSA-1和HepG2)的抗肿瘤活性。其中,V9和V10对乳腺癌细胞具有更强的抗增殖作用，V10在MDA-MB-231细胞（TNBC）中显示出最佳的选择性，比先导化合物GAA高5倍。初步的结构活性分析表明，V系列GAAPROTACs比C系列具有更好的效果，2O-4OPEG接头的引入可以显着提高抗肿瘤活性。分子对接,表面等离子体共振（SPR），细胞热转移测定（CETSA），Westernblot研究表明，V9和V10均可与MDM2结合，并通过泛素-蛋白酶体系统降解蛋白质。分子动力学模拟（MD）表明，V10是一种双功能分子，可以一端与vonHippel-Lindau（VHL）结合，另一端与MDM2结合。此外,V10促进p53突变型MDA-MB-231细胞中p21的上调,并通过下调bcl-2/bax比值和cyclinB1的表达诱导细胞凋亡。最后,体内实验表明,V10在异种移植TNBC斑马鱼模型中也表现出良好的肿瘤抑制活性，在50μg/mL时抑制率为27.2%。总之,我们的结果表明，V10在体外和体内对p53突变的乳腺癌具有抗肿瘤作用，并可能作为一种新型的先导化合物用于TNBC的未来发展。
