基于小分子探针技术研究协同阿霉素抗乳腺癌并减轻心脏毒性的药物作用靶点

Doxorubicin, sold under the trade name Adriamycin, belongs to a class of anthracyclines. It is still widely used to in the combination chemotherapy for the treatment of various kinds of cancers, particularly breast cancer. However, the clinical use of doxorubicin was limited by its serious cardiotoxicity, including congestive heart failure and cardiomyopathy. In our previous study, we found that a novel small molecule probe named BAA, a danshensu derivative combined with biotin, inhibited the catalytic activity of protein disulfide isomerase ERp57 as well as bound with this protein. Furthermore, we also found this small molecule improved the chemotherapeutic efficacy of doxorubicin in breast cancer while protected against doxorubicin-induced cardiotoxicity both in vivo and in vitro. In this study, small molecule probe was used to identify its unknown binding proteins using chemical proteomic approach under the circumstances. These candidate proteins were analyzed and validated to explore its feasibility as a therapeutic target to regular enhanced anti-breast cancer efficacy and prevented cardiac dysfunction using various experimental methods. The promising candidate lead compound will be found by virtual high-throughput screening based the target protein structure, followed to validate by experimental testing. This study is important because it offers the academic discipline for the development of combined drug with doxorubicin to enhance anti-tumor efficacy and prevent cardiotoxicity. Therefore, our work will highlight and open a new avenue for drug discovery combined used with doxorubicin or other anthracyclines to enhance effect of chemotherapy against breast cancer and reduce cardiotoxicity.

阿霉素是治疗乳腺癌等癌症的蒽环类常用药，多联合其他药物使用以达到理想的疗效，但易诱发心脏毒性。若能发现可协同阿霉素抗乳腺癌并减轻心脏毒性的药物作用靶点，进而基于靶点筛选新型的联合阿霉素等使用的药物是解决其缺陷的有效可行策略。本项目组前期发现：①新型小分子探针BAA与二硫键异构酶ERp57结合并抑制其活性；②该小分子具有联合阿霉素协同抗乳腺癌同时减轻其诱发心脏毒性的功能。本课题拟在此创新性结果的基础上，利用小分子探针联合化学蛋白质组的技术，解析该小分子联合阿霉素协同抗乳腺癌并减轻心脏毒性的靶标蛋白，进一步探讨靶标蛋白作为调控阿霉素抗乳腺癌和心脏毒性治疗靶点的可行性。然后采用计算机高通量虚拟筛选和实验验证相结合的方法，筛选基于该候选靶点的先导化合物。该项目对研究联合阿霉素等蒽环类化疗药协同抗肿瘤同时减轻心脏毒性的规律有重要意义，有望通过找到新的药物靶点而研发出新的与阿霉素等联用的先导化合物。

阿霉素是治疗乳腺癌最有效的化疗药之一，但阿霉素的应产生的心脏毒性，限制了其在临床上的应用。本文旨在探讨协同阿霉素抗乳腺癌并能减轻心脏毒性的药物作用靶点。本研究表明，相同的药物和相同的处理方式，小分子探针BAA在H9c2细胞和MDA-MB-231细胞两种不同的细胞系中显示出了不同的生物活性，在MDA-MB-231细胞中表现出协同阿霉素增强乳腺癌细胞的凋亡的作用，而在H9c2细胞中对阿霉素诱导的心脏毒性有一定的减弱作用。BAA协同阿霉素抗乳腺癌并减轻心脏毒性的作用机制是通过调控阿霉素诱导的内质网应激。在斑马鱼体内实验中发现，BAA对阿霉素诱导的斑马鱼心脏功能减弱具有一定的保护作用。.基于化学结构相似性原理，进行化合物的筛选并结合实验验证，发现咖啡酸苯乙酯也具有协同阿霉素抗乳腺癌同时减轻心脏的作用，且CAPE的药理学活性优于BAA。CAPE单独或联合阿霉素处理两种细胞处理后发现两者内质网应激相关蛋白表达的变化趋势与BAA不一致，可以看出CAPE发挥增效减毒的作用机理与BAA不同。用蛋白质组学的方法分析CAPE协同阿霉素抗乳腺癌并减轻心脏毒性的作用机制，发现其作用机理与内质网中蛋白质加工信号通路有关。同时在动物实验中发现CAPE具有协同阿霉素抑制了小鼠体内乳腺癌移植瘤的生长的作用，且对阿霉素诱导的心脏损伤具有一定的保护作用。.进一步的研究表明，ERp57基因沉默和基因过表达后，在MDA-MB-231细胞和H9c2细胞中加入阿霉素处理一段时间，显示出了不同的作用效果。在MDA-MB-231细胞中ERp57基因过表达后减少了阿霉素诱导的细胞凋亡，促进了乳腺癌细胞存活，细胞毒性减弱；而ERp57基因沉默后却能够进一步降低阿霉素诱导的乳腺癌细胞的细胞存活率。在H9c2细胞中ERp57基因过表达后促进了阿霉素诱导的心肌细胞凋亡，抑制了心肌细胞的存活且细胞毒性增强，而ERp57基因沉默后却能够提高阿霉素诱导的心肌细胞存活率，ERp57可能是体外增强阿霉素抗乳腺癌及减轻心肌细胞毒性的潜在作用靶点。ERp57体外增强阿霉素抗乳腺癌及减轻心肌细胞毒性作用的机制可能与UPR反应和凋亡信号通路密切相关。