Table_1_Comprehensive Target Screening and Cellular Profiling of the Cancer-Active Compound b-AP15 Indicate Abrogation of Protein Homeostasis and Organelle Dysfunction as the Primary Mechanism of Action.xlsx

Dienone compounds have been demonstrated to display tumor-selective anti-cancer activity independently of the mutational status of TP53. Previous studies have shown that cell death elicited by this class of compounds is associated with inhibition of the ubiquitin-proteasome system (UPS). Here we extend previous findings by showing that the dienone compound b-AP15 inhibits proteasomal degradation of long-lived proteins. We show that exposure to b-AP15 results in increased association of the chaperones VCP/p97/Cdc48 and BAG6 with proteasomes. Comparisons between the gene expression profile generated by b-AP15 to those elicited by siRNA showed that knock-down of the proteasome-associated deubiquitinase (DUB) USP14 is the closest related to drug response. USP14 is a validated target for b-AP15 and we show that b-AP15 binds covalently to two cysteines, Cys203 and Cys257, in the ubiquitin-binding pocket of the enzyme. Consistent with this, deletion of USP14 resulted in decreased sensitivity to b-AP15. Targeting of USP14 was, however, found to not fully account for the observed proteasome inhibition. In search for additional targets, we utilized genome-wide CRISPR/Cas9 library screening and Proteome Integral Solubility Alteration (PISA) to identify mechanistically essential genes and b-AP15 interacting proteins respectively. Deletion of genes encoding mitochondrial proteins decreased the sensitivity to b-AP15, suggesting that mitochondrial dysfunction is coupled to cell death induced by b-AP15. Enzymes known to be involved in Phase II detoxification such as aldo-ketoreductases and glutathione-S-transferases were identified as b-AP15-targets using PISA. The finding that different exploratory approaches yielded different results may be explained in terms of a “target” not necessarily connected to the “mechanism of action” thus highlighting the importance of a holistic approach in the identification of drug targets. We conclude that b-AP15, and likely also other dienone compounds of the same class, affect protein degradation and proteasome function at more than one level.

研究证实，二烯酮类化合物（dienone compounds）可展现肿瘤选择性抗癌活性，且该活性不依赖于TP53的突变状态。既往研究表明，该类化合物诱导的细胞死亡与泛素-蛋白酶体系统（ubiquitin-proteasome system, UPS）的抑制作用密切相关。本研究拓展了既往研究结论，证实二烯酮类化合物b-AP15可抑制长寿命蛋白质的蛋白酶体降解过程。研究发现，经b-AP15处理后，分子伴侣VCP/p97/Cdc48和BAG6与蛋白酶体的结合水平显著升高。通过对比b-AP15与小干扰RNA（small interfering RNA, siRNA）诱导的基因表达谱，结果显示，靶向蛋白酶体相关去泛素化酶（deubiquitinase, DUB）USP14的敲低与该药物的应答相关性最高。USP14是b-AP15的已验证靶点，本研究证实b-AP15可与该酶泛素结合口袋内的两个半胱氨酸残基Cys203与Cys257发生共价结合。与此一致的是，USP14基因敲除会使细胞对b-AP15的敏感性显著降低。但研究发现，仅靶向USP14并不能完全解释所观测到的蛋白酶体抑制现象。为探寻其他潜在靶点，本研究分别采用全基因组CRISPR/Cas9文库筛选技术与蛋白质组整体溶解度改变（Proteome Integral Solubility Alteration, PISA）分析法，鉴定出机制必需基因与b-AP15的互作蛋白。编码线粒体蛋白的基因敲除会降低细胞对b-AP15的敏感性，这表明线粒体功能异常与b-AP15诱导的细胞死亡存在紧密关联。通过PISA分析法，本研究鉴定出醛酮还原酶、谷胱甘肽S-转移酶等已知参与II相解毒过程的酶类为b-AP15的靶点。不同探索性研究方法得到不同结果这一现象，可通过「靶点未必与「作用机制」直接相关」这一视角加以解释，这也凸显了采用整体性策略鉴定药物靶点的重要性。综上，b-AP15很可能与同类其他二烯酮类化合物一样，可从多个层面调控蛋白质降解过程与蛋白酶体功能。