Discovery of AHCY as an Off-Target of Doxorubicin by Integrative Analysis of Photoaffinity Labeling Chemoproteomics and Untargeted Metabolomics.
Discovery of AHCY as an Off-Target of Doxorubicin by Integrative Analysis of Photoaffinity Labeling Chemoproteomics and Untargeted Metabolomics.
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DOI：
10.1021/acs.analchem.2c03377
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发表时间：
2022-11
影响因子：
7.4
通讯作者：
S. Qian;Ying Han;Yue Zhang;Yanan Du;Jing Li;Xin Yang;Jingwu Kang
中科院分区：
文献类型：
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作者：
S. Qian;Ying Han;Yue Zhang;Yanan Du;Jing Li;Xin Yang;Jingwu Kang
靶标识别对于理解药物的作用机制至关重要。在这里，我们报告了一种新的策略，以阿霉素（DOX）为模型，结合非靶向代谢组学与光亲和标记化学蛋白质组学的药物靶点（或脱靶）的去卷积。制备了DOX衍生的光亲和探针，并应用于捕获活细胞中的DOX相互作用蛋白。然后通过无标记定量蛋白质组学鉴定捕获的DOX相互作用蛋白。总共鉴定了151种具有高置信度的显著蛋白质（倍数变化>4，p值< 0.005）。基因本体富集分析表明，这些蛋白质主要参与碳代谢、柠檬酸循环、脂肪酸代谢和代谢途径。因此，非靶向代谢组学被应用于定量药物治疗后细胞中显著改变的代谢物。途径富集分析表明，DOX主要干扰嘧啶和嘌呤代谢、碳代谢、蛋氨酸代谢和磷脂酰胆碱生物合成过程。化学蛋白质组学和代谢组学的综合分析表明，腺苷同型半胱氨酸酶（AHCY）是DOX的一个新靶点（脱靶），导致S-腺苷同型半胱氨酸的积累。通过细胞热位移试验、亲和竞争性下拉试验、生物化学试验和siRNA敲除实验证实了所推导的DOX靶点。我们的结果表明，AHCY是DOX的未发现的脱靶区。
Target identification is critically important for understanding the mechanism of action of drugs. Here, we reported a new strategy for deconvolution of drug targets (or off-targets) with photoaffinity labeling chemoproteomics in combination with untargeted metabolomics by using doxorubicin (DOX) as a model. The DOX-derived photoaffinity probes were prepared and applied to capture DOX-interacting proteins in living cells. The captured DOX-interacting proteins were then identified by label-free quantitative proteomics. Totally, 151 significant proteins were identified with high confidence (fold change >4, p-value < 0.005). The gene ontology enrichment analysis suggested that the proteins were mainly involved in carbon metabolism, citrate cycle, fatty acid metabolism, and metabolic pathways. Therefore, untargeted metabolomics was applied to quantify the significantly altered metabolites in cells upon drug treatment. The pathway enrichment analysis suggested that DOX mainly interrupted with the processes of pyrimidine and purine metabolism, carbon metabolism, methionine metabolism, and phosphatidylcholine biosynthesis. Integrative analysis of chemoproteomics and metabolomics indicated that adenosylhomocysteinase (AHCY) is a new target (off-target) of DOX leading to the accumulation of S-adenosyl homocysteine. This deduced DOX target was confirmed by the cellular thermal shift assay, affinity competitive pull-down assay, biochemical assay, and siRNA knock down experiments. Our result suggested that AHCY is the uncovered off-target of DOX.