靶向酸性微环境和己糖激酶II的抗肿瘤药物候选物的合成与生物活性研究

HKII is a promising cancer drug target as it links cancer cell glycolysis with mitochondrial-mediated apoptosis. Many cancer cells exhibit increased glycolysis under abundant oxygen supply and often greatly increased expression of hexokinase II (HKII). HKII is the first rate-limiting enzyme of aerobic glycosis and binds to the voltage dependent anion channel (VDAC) of the outer mitochondrial membrane, having preferential access to ATP produced by oxidative phosphorylation. Binding of HKII to VDAC blocks binding of the pro-apoptotic molecule Bax to VDAC, inhibiting release of cytochrome c release from the mitochondria initiated by Bax binding to VDAC. Several HKII inhibitors are currently being studied as potential anti-cancer drugs, such as analogues for glucose. Acidic environment is another characteristics of cancer. It was induced partly by release of lactate in Warburg effect. Increase of H2CO3 synthesis is another important factor. Such weak acidity could pose effect on the stability of metal complexes, releasing free ligand. Glucose 6- phosphate (G-6-P) is the product of phosphorylation reaction catalyzed by hexokinase II, which can inhibit HKII by negative feedback. In present project, metal complexes of analogues of G-6-P will be designed, synthesized and tested in evaluation of enzyme inhibition activity and anti-cancer effect. The coordination of analogues of G-6-P with metal is aimed to obtain tumor tissue specificity. The ester bond and carbohydrate ring in G-6-P will be changed to stable C-P bond and carbon ring. Such changes will also possibly accompanied by introduction of active SH and/or subsititution of 2-OH by other inert groups. Such structucture changes are hoped to lead tighter HKII binding of analogues than G-6-P, the conformation change of HKII and disassociation of HK from VDAC. The blocking-up of glycosis and apotosis of cancer cell are expected as the results. Besides the anti-cancer drug candidates, understanding of the structure-effect of HKII inhibitor is to deepened by studying the interaction between HKII and small molecule probe like G-6-P analogues through XAFS method.

己糖激酶II（HKII）连接癌细胞有氧糖酵解和线粒体介导的细胞凋亡，在癌细胞中高表达，是一个潜在的抗肿瘤药物靶点。HKII结合在线粒体外膜的VDAC上，既是有氧糖酵解第一个限速酶，也能影响Bax介导的细胞色素C胞浆释放，抑制细胞凋亡。酸性微环境是实体瘤的一个重要特征，类似的酸度能影响金属配合物稳定性使部分配体游离出来。 本项目拟合成具有巯基、稳定的类糖环和C－P键等的6－磷酸葡萄糖（G6P）类似物，并络合成金属配合物。受肿瘤微环境酸性影响，金属配合物解离出G6P类似物，竞争G6P负反馈抑制HKII的结合位点，改变HKII构象使之从线粒体脱离，引起细胞凋亡。项目计划通过同步辐射XAFS法研究HKII与G6P类似物探针分子的作用，研究HKII抑制剂构效关系；通过酶活性抑制实验、细胞和动物水平抗肿瘤活性研究，筛选出2～3个药物候选物。

己糖激酶II（HKII）是连接肿瘤细胞有氧糖酵解和线粒体介导的细胞凋亡信号传导途径的节点，是一个抗肿瘤药物的很可能的靶点。HKII在癌细胞中高表达，结合在线粒体外膜的VDAC上，是有氧糖酵解第一个限速酶，抑制Bax介导的细胞色素C释放到胞浆，抑制细胞凋亡。HKＩＩ能被较高浓度的６－磷酸葡萄糖（Ｇ６Ｐ）负反馈抑制。酸性微环境是实体瘤的一个重要特征，能影响金属配合物稳定性而使部分配体游离出来。.本课题根据研究计划设计了HKII的产物G6P类似物，作为HKII的竞争性抑制剂进行抗肿瘤活性研究。先后合成了3批共约40个化合物，筛选出2个化合物具有较好的肿瘤细胞生长抑制作用，而对快速增长的正常细胞的毒性则相对较低。机制研究表明该类化合物是通过将肿瘤细胞阻滞在G2/M期并引起细胞凋亡。因为这两个化合物的结构特点，不具有两个配位基或者形成螯合物时位阻较大，不能形成稳定的螯合物，因而试图形成金属螯合物而靶向肿瘤酸性微环境的研究内容不能顺利进行。据此，我们根据HKII别构酶的结构特点，综合运用计算机辅助药物设计等方法对其新型抑制剂先导化合物进行虚拟筛选与理性设计，设计并获得45个化合物进行HKII抑制活性评价，并进一步进行对肿瘤的体外活性的研究，从中筛选出两个化合物与多柔吡星的活性相当。以上四个化合物正在进行对HK亚型的选择性的研究。尝试了通过接种细胞进行移植瘤裸鼠的培养，但培养出的裸鼠瘤体尺寸差距太大，因而换用接种肿瘤组织而进行荷瘤裸鼠的培养。选用一系列特征的化合物作为模型化合物，采集并解析了基于同步辐射的XAFS（EXAFS和XANES）谱，初步确立了利用XAFS谱来研究探针小分子与生物大分子之间相互作用的可行性。.本课题发现了4个靶向HKII的抗肿瘤药物候选物，已发表基金标注的SCI论文2篇，申请专利1项，培养研究生3名并顺利毕业，培养博士后1名并出站。