Discovery of a small molecule targeting ULK1-modulated cell death of triple negative breast cancer in vitro and in vivo.
Discovery of a small molecule targeting ULK1-modulated cell death of triple negative breast cancer in vitro and in vivo.
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体外和体内发现一种针对 ULK1 调节的三阴性乳腺癌细胞死亡的小分子
DOI：
10.1039/c6sc05368h
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发表时间：
2017-04-01
期刊：
影响因子：
8.4
通讯作者：
Liu B
中科院分区：
文献类型：
--
作者：
Zhang L;Fu L;Zhang S;Zhang J;Zhao Y;Zheng Y;He G;Yang S;Ouyang L;Liu B
ULK 1被鉴定为TNBC中的靶标;因此，通过靶向ULK 1调节的细胞死亡（与自噬和凋亡相关）发现了一种小分子激动剂。众所周知，ULK-51样激酶1（ULK 1）启动自噬，并且在大多数乳腺癌组织中发现ULK 1的下调。因此，激活ULK 1调节的自噬可能是乳腺癌治疗的一个有前途的策略。在这项研究中，我们发现ULK 1在乳腺癌组织样本中显着下调，通过癌症基因组图谱（TCGA）分析和组织芯片（TMA）分析，特别是在三阴性乳腺癌（TNBC）。为了设计ULK 1激动剂，我们整合了计算机筛选和化学合成，以获得一系列小分子候选物。经过多轮激酶和抗增殖活性筛选，我们发现小分子林恩-1604是ULK 1激动剂的最佳候选物。此外，我们通过定点突变和生化分析确定了三个氨基酸残基（LYS 50，LEU 53和TYR 89）是林恩-1604和ULK 1激活位点的关键。随后，我们证明了林恩-1604可以诱导细胞死亡，与MDA-MB-231细胞中ULK复合物（ULK 1-mATG 13-FIP 200-ATG 101）的自噬相关。为了进一步探索林恩-1604诱导的自噬机制，我们通过比较微阵列分析发现了一些潜在的ULK 1相互作用因子，如ATF 3、RAD 21和caspase 3。有趣的是，我们发现林恩-1604诱导的细胞死亡涉及ATF 3、RAD 21和半胱天冬酶3，并伴有自噬和凋亡。此外，我们证明了林恩-1604通过靶向体内ULK 1调节的细胞死亡对TNBC具有良好治疗效果的潜力;因此使这种ULK 1激动剂成为未来TNBC治疗的新型潜在小分子药物候选物。
ULK1 is identified as a target in TNBC; thus a small-molecule agonist is discovered by targeting ULK1-modulated cell death, associated with autophagy and apoptosis. UNC-51-like kinase 1 (ULK1) is well-known to initiate autophagy, and the downregulation of ULK1 has been found in most breast cancer tissues. Thus, the activation of ULK1-modulated autophagy could be a promising strategy for breast cancer therapy. In this study, we found that ULK1 was remarkably downregulated in breast cancer tissue samples by The Cancer Genome Atlas (TCGA) analysis and tissue microarray (TMA) analysis, especially in triple negative breast cancer (TNBC). To design a ULK1 agonist, we integrated in silico screening and chemical synthesis to acquire a series of small molecule candidates. After rounds of kinase and anti-proliferative activity screening, we discovered the small molecule, LYN-1604, to be the best candidate for a ULK1 agonist. Additionally, we identified that three amino acid residues (LYS50, LEU53, and TYR89) were key to the activation site of LYN-1604 and ULK1 by site-directed mutagenesis and biochemical assays. Subsequently, we demonstrated that LYN-1604 could induce cell death, associated with autophagy by the ULK complex (ULK1-mATG13-FIP200-ATG101) in MDA-MB-231 cells. To further explore LYN-1604-induced autophagic mechanisms, we found some potential ULK1 interactors, such as ATF3, RAD21, and caspase3, by performing comparative microarray analysis. Intriguingly, we found that LYN-1604 induced cell death involved in ATF3, RAD21, and caspase3, accompanied by autophagy and apoptosis. Moreover, we demonstrated that LYN-1604 has potential for good therapeutic effects on TNBC by targeting ULK1-modulated cell death in vivo; thus making this ULK1 agonist a novel potential small-molecule drug candidate for future TNBC therapy.