明确传统抗癌药物作用靶标是恶性肿瘤精准化治疗亟待解决的关键问题。蟾毒灵（BF）是临床常用抗癌药物华蟾素的主要有效活性成分之一，但其确切的抗癌作用靶标尚未阐明。前期研究表明，传统定量蛋白质组学方法不能准确鉴定BF抗癌作用靶蛋白。本项目拟引入能够精准挖掘BF作用靶标的基于活性的蛋白质组学分析（ABPP）策略：利用前期已合成的既保留了BF抗癌活性又可进行点击化学反应及光交联的BF活性分子探针，钓取与其特异性共价结合的功能活性蛋白质组，进而采用生物大分子质谱鉴定并结合生物信息学分析及分子对接软件预测，发现BF抗癌作用的潜在靶标蛋白。在此基础上，借助细胞生物学和分子生物学手段，确证潜在靶蛋白在BF对抗肿瘤过程的生物学功能和调控机制，最终阐明BF抗癌作用靶标及分子机制。本项目的开展可为临床精准使用华蟾素及BF提供理论依据，同时为小分子药物靶标发现和确证提供一条切实可行的途径。

Defining the targets of traditional anticancer drugs is a critical issue to be addressed urgently for the precise chemotherapy of malignant tumor. Bufalin (BF) is one of the main effective active ingredients of the commonly used anticancer drug cinobufacine, however, its exact anti-cancer targets have not yet been elucidated. Our previous studies found that traditional quantitative proteomics methods could not accurately identify the targets of BF. Therefore, this project intends to introduce a powerful strategy of activity-based protein profiling (ABPP) to precisely explore the target proteins of BF. Recently, we have obtained the excellent BF active probes with photoaffinity groups and click-chemical groups as well as similar anticancer activities of BF. In this study, we will use the BF active probes to capture the functional active proteome with specific covalent binding, followed by identification using biomolecular mass spectrometry, analysis with bioinformatics and prediction by molecular docking software, and to discover the potential target proteins of BF. Furthermore, we will explore the biological functions as well as regulatory mechanisms of the potential targets in BF-treated cells by the means of cell biology and molecular biology, and ultimately clarify the anticancer targets and molecular mechanisms of BF. Collectively, our study will provide theoretical basis for the clinical use of cinobufacine and BF, and also provide a practical way for target discovery and confirmation of other small molecule drugs.