基于S期激酶相关蛋白2的抗肿瘤多肽药物筛选与应用

Malignant tumors are serious diseases in human being, however, there is no efficient drugs for curing these diseases clinically. Ubiquitin is a small protein, which can specifically modify and degrade its target proteins with the help of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), ubiquitin ligase (E3) and proteasome. Consequently, ubiquitin is involved in regulation of cell cycle, gene expression, transcription modulation, etc. Numerous studies indicated that protein ubiquitination plays an important role in tumors and cardiovascular diseases. Therefore, some proteins involved in ubiquitination process have become targets of new drug development. S-phase kinase-associated protein 2(Skp2) is an E3 ligase which is chosen to be the target of anticancer drug development in this proposal. Peptide aptamers which will specifically bind to Skp2 are screened using yeast two-hybrid systems from a humanized peptide aptamer library which has been prepared in our laboratory. We will clarify the biological function of peptide aptamers through analyses of their effects on cell proliferation, migration, invasion, apoptosis and cell cycle. Meanwhile, we are intent to elucidate the anti-tumor mechanism of peptide aptamers by detection of ubiquitination levels of Skp2 substrates. Moreover, we will further explore the effects of peptide aptamers on planting tumors in xenograft models of nude mice to confirm their anticancer functions. There is no doubt that our study will provide an insight in the study of Skp2-mediated protein ubiquitination and the development of anticancer drugs.

恶性肿瘤是严重危害人类健康的疾病，然而，目前临床上尚缺乏有效的治疗药物。泛素是一类小分子蛋白质，它在泛素激活酶、结合酶、连接酶和降解酶等作用下，对靶蛋白进行特异性修饰并使之降解，从而参与细胞周期、基因表达、转录调节等几乎一切生命活动的调控。研究表明，蛋白泛素化过程在肿瘤、心血管疾病等的发病中起重要作用。因此，参与蛋白泛素化过程的多种蛋白分子已成为新药研发的靶点。本课题将以S期激酶相关蛋白2（Skp2，一种E3泛素连接酶）为靶点，利用酵母双杂交技术在我们已构建好的人源化肽适配体文库中筛选具有抗肿瘤作用的特异性肽适配体，观察其对多种肿瘤细胞增殖、迁移、侵袭等的影响以确定肽适配体的抗肿瘤作用，同时还将检测Skp2底物的蛋白泛素化水平以阐明肽适配体抗肿瘤的作用机制。此外，在异体种植瘤裸鼠模型中研究肽适配体对种植瘤生长的影响，以进一步明确其抗肿瘤作用，从而为抗肿瘤药物研发提供实验依据。

恶性肿瘤是严重危害人类健康的疾病，然而，目前临床上尚缺乏有效的治疗药物。泛素是一类小分子蛋白质，它在泛素激活酶、结合酶、连接酶和降解酶等作用下，对靶蛋白进行特异性修饰并使之降解，从而参与细胞周期、基因表达、转录调节等几乎一切生命活动的调控。研究表明，蛋白泛素化过程在肿瘤、心血管疾病等的发病中起重要作用。因此，参与蛋白泛素化过程的多种蛋白分子已成为新药研发的靶点。原癌基因SKP2，即S期激酶相关蛋白，是一种E3泛素连接酶，通过对多种靶蛋白尤其是抑癌基因的泛素化降解作用参与细胞周期的调控，与肿瘤的发生、发展及预后密切相关。本课题以Skp2为靶点，利用酵母双杂交系统从构建好的人源化肽适配体文库中成功筛选到十一个与之相互作用的肽适配体。GST Pull-down实验证实它们能直接结合Skp2。通过检测不同肿瘤细胞中Skp2及其底物的表达情况，确定A549细胞为最适肽适配体抗肿瘤作用研究的体外细胞系。免疫印迹实验发现肽适配体6-1能显著提高细胞內源p21的表达量，而23-5能提高细胞內源p21和p27的表达量，提示它们可能抑制Skp2与底物结合；细胞周期实验结果进一步显示6-1和23-5确实增加了G2/M期细胞比率，与下调Skp2表达的作用一致。因此，我们在多种肿瘤细胞中深入验证这些肽适配体的抗肿瘤作用，最终证实23-5具有广谱的抗肿瘤作用。后期，我们将该肽适配体进一步改造，促进其穿膜能力，并在异体种植瘤裸鼠模型中进行体内实验，评估肽适配体23-5对种植瘤生长、迁移和侵润的影响。综上所述，本研究成功筛选到能特异性抑制Skp2蛋白与底物结合的人源肽适配体23-5，通过阻断抑癌蛋白p21和p27的降解而显著抑制肿瘤细胞的生长和增殖，有望成为新一代无细胞毒性且特异性针对原癌基因SKP2的多肽抗肿瘤药物，为抗肿瘤药物研发提供新的研究方向，并为肿瘤的临床诊疗奠定良好的实验基础。