Skp2是泛素连接酶的重要组分，可介导多种抑癌蛋白（如p27）的降解。因Skp2基因表达在恶性肿瘤中常异常增高，开发靶向Skp2的药物已成为抗肿瘤药物研究的热点。现有研究多集中在抑制Skp2与连接酶组分的相互作用，尚未见药物能直接抑制Skp2基因在肿瘤中表达，产生这一空白的主要原因在于缺乏可靠的基于Skp2表达的高通量药物筛选平台。本项目拟利用最新的CRISPR/Cas9基因编辑技术建立一个高效可靠的平台来筛选Skp2表达抑制剂，并在细胞水平评价候选化合物对肿瘤细胞生长、凋亡等的影响，及这些影响与Skp2抑制的相关性；进而在动物水平评估Skp2抑制剂单独使用以及与抗肿瘤药物联合使用的抗肿瘤活性；进一步通过研究候选化合物调控Skp2表达的作用机制来探讨Skp2在肿瘤中异常表达的机理及其调控网络。二者结合相互促进，为开发抗肿瘤药物提供新的研究策略及先导化合物。

Skp2 (S-phase kinase associated protein 2) is a major component of the SCF E3 ubiquitinligase complex, and has been shown to play an essential role in promoting degradation of many tumor suppressors (e.g, p27) via the ubiquitin-proteasome system. As the Skp2 gene is frequently over-expressed in common human cancers, Skp2 has been widely accepted as an effective anti-cancer drug target. Although.small molecules targeting Skp2 interactions with other SCF complex proteins have been discovered, anti-cancer efficacies of these compounds are often limited because of the complexity of the oncogenic network involved by Skp2. While small molecules that can decrease Skp2 expression are expected to be more effective in antagonizing the Skp2 activity in cancer cells, such anti-cancer agents are.currently not available owing to the lack of an effective strategy for developing reliable high-throughput assays (HTS) that can be used for screening for transcription-targeted agents. The main objective of this application is to utilize the cutting-edge CRISPR/Cas9 genome editing tool to develop a bicistronic reporter-based HTS platform for the discovery of anti-cancer Skp2 inhibitors that can decrease Skp2 expression in cancer cells. It has been recently demonstrated by us that bicistronic reporter-based assays are reliable and powerful in HTS screening for transcription inhibitors. We will further determine effects of identified Skp2 transcription inhibitors on cancer cell growth, apoptosis, cell.cycle progression, and cross-talks with other oncogenic signaling while assessing their anti-cancer activities when administrated alone or combined with other therapeutic agents using xenograft models. We will also elucidate how the Skp2 inhibitors suppress Skp2 expression, and utilize these agents as chemical probes for chemical genetics studies to explore the mechanisms by which Skp2 is overexpressed in cancer cells. These in-vitro and in-vivo studies together would not only yield lead compounds targeting Skp2 for cancer treatments, but result in a novel strategy for the discovery of effective anti-cancer agents.