以紫草素及二甲基紫草素肟衍生物为小分子探针探索其抗肿瘤作用的新靶点和新机制

Shikonin, a naturally occurring naphthoquinone, exhibited outstanding anticancer activity both in vitro and in vivo. The covalent bonding of shikonin with cellular nucleophiles such as DNA and proteins and the toxicity of active oxygen produced by the redox cycling of the naphthazarin moiety have been considered as molecular mechanisms for its anticancer effects. Therefore shikonin was characterized as a non-specific anticancer agent. Taking shikonin as a lead, we had prepared a series of 5,8-O-dimethoxy shikonin 1,4-dioxime derivatives and evaluated their anti-proliferate activity against certain cancer cell lines. The results from both in vitro and in vivo studies implied that compared with the lead, these dioxime derivatives exhibited more potent inhibitory effects on the growth of cancer cells but with low toxicity towards normal ones. The difference observed in the cytotoxic profile between these dioxime derivatives and the lead compound suggested that these dioximes shared different biological targets and mechanism with shikonin. The forthcoming studies will be conducted to examine the molecular mechanism of these novel dioximes as promising anticancer agents. The mian strategies in our research contain the following steps. First of all, using these shikonin dioxime derivatives as molecular probes, the targeted protein will be discovered by the purification procedure and further structural characterization after the binding of these probes with it. Then the relationship between the targeted protein and tumor cell growth could be well elucidated by the bioinformatics analysis of tumors. At last, the plausible mechanism for the significant anticancer effects of these dioximes will be obtained. All of these studies will be helpful in understanding the procedure of the development and apoptosis of cancer cells and will certainly make great contributions for the discovery of new molecularly targeted anticancer agents with natural naphthoquinones as lead compounds.

紫草素是天然萘醌类化合物，其抗肿瘤作用已被大量研究证实。作用的分子机制是源于紫草素萘醌结构氧化还原产生的活性氧及烷基化对DNA和蛋白等生物大分子的破坏，是一种非特异性的细胞毒作用。我们近年来对紫草素进行了一系列结构改造研究，通过对紫草素萘茜母核的羟基甲基化醌羰基肟化得到一类二甲基紫草素肟（DMSKO）衍生物，这些化合物的氧化还原产生活性氧及烷基化作用消失，但体内外试验对肿瘤细胞生长抑制作用选择性地增强，毒性远低于紫草素。很显然DMSKO与紫草素有着完全不同的作用机制。本研究以DMSKO为小分子探针，通过蛋白结合、分离和结构分析确定其作用靶点，并结合肿瘤生物信息学分析研究靶蛋白与肿瘤细胞生长的关系，在分子层面明确紫草素衍生物抗肿瘤作用机制。为开发以萘醌结构为先导的分子靶向抗肿瘤新药奠定基础，同时也为揭示肿瘤细胞的发生、发展、凋亡的生物学规律，阐明小分子的调节机制提供理论依据。

首先，我们制备了小分子探针，测试了体外活性，筛选出活性最好的探针分子进行了与靶蛋白结合试验，但由于DMAKO 为前药，在细胞内被代谢为活性化合物，且探针与靶蛋白的结合可能是可逆不稳定性结合，通过该方法没能钓到其结合蛋白。进而我们通过蛋白质组学方法，提取收集 HCT-15 细胞与 DMAKO-20 孵育前和孵育3 h 后的蛋白，通过双向电泳技术将蛋白有效地分离，在选择两者的差异蛋白后通过质谱鉴定肽段结构，利用 Sequest 软件搜索比对相应数据库，最终鉴定差异蛋白的结构。结果显示DMAKO影响最大的蛋白为电压依赖性离子通道蛋白-2（VDAC2），它是维持线粒体膜稳定的关键蛋白，对VDAC2的抑制导致线粒体膜被破坏，从而导致细胞凋亡。这可能是紫草素衍生物抗肿瘤作用的机制之一，VDAC2是该机制的重要靶蛋白。随后为解决紫草素和阿卡宁衍生物作为抗肿瘤先导化合物的毒性和选择性问题，我们设计了5,8-O-二甲基阿卡宁-1,4-萘醌肟衍生物（DMAKO-20），体外对多种肿瘤细胞抑制试验显示与其先导化合物比较他们具有较强的抗肿瘤活性和良好的选择性。DMAKO-20对HCT-15、HCT-116及K562有很强的选择性抑制活性IC50<1μM，对MCF-7、DU-145、MDA-MB-231、HepG2及PANC具有中等强度抑制的作用IC50<10μM。而对正常人皮肤成纤维细胞HSF几乎没有毒性。动物体内人结肠癌细胞HCT-15裸鼠体内移植瘤抑制试验显示，剂量为10mg/kg/d，抑制率为59.3%，与阳性对照5-FU 25 mg/kg/d的抑制率为57.9%相当，但给药组没有出现5-Fu组同样的毒副反应。DMAKO-20与阿卡宁不同，由于萘醌结构被掩蔽，失去产生活性氧和烷基化能力，已不是传统意义上的细胞毒药物。它可能是肿瘤细胞特异性高表达的CYP1B1酶的良好底物，能被该酶代谢释放出一氧化氮和萘醌单肟衍生物，二者发挥多靶点协同抗肿瘤作用，我们把它定义为由肿瘤细胞特异性酶活化的多靶点协同抗肿瘤前药，其发现及其机制的深入研究有望为解决困扰肿瘤化疗的毒性、选择性、耐药、复发和转移等世界性难题提供新思路。