基于生物信息学手段开口箭中甾体类抗白血病多药耐药活性成分的发现与优化

Tupistra chinensis is commonly used in adjuvant treatment of leukemia in TCM clinic. Our previous study showed that its total steroidal fraction could significantly inhibit the tumor growth of sensitive leukemia cell K562 and multi-drug resistance (MDR) cell K562/Adr in nude mice transplanted tumors, with the inhibition rates as high as 86% and 87%, respectively. The steroidal fraction and some purified steroids could show high potent inhibitory activity towards cell proliferation of leukemia MDR cell lines K562/Adr, HL-60/Adr, and reverse the multi-drug resistance to daunorubicin..This project will be focused on isolation and purification of the steroidal components from Tupistra chinensis by a variety of chemical and chromatographic methods. The chemical structure of steroids will be identified through the integrated spectroscopic and chemical methods. Then evaluate the anti-leukemic MDR activity of purified steroid by in vitro cell assay. Guided by structural bioinformatics, we conducted rational structure modification of the bioactive compounds and prepared their derivatives to optimize the anti-leukemic MDR candidates through microbial transformation and chemical modification..Anti-leukemic MDR targets and mechanisms of optimized leading compounds could be predicted by network models of bioinformatics, and then verified by molecular pharmacological methods. The anti-leukemic MDR mechanisms would be verified and clarified from the aspects of transporter expression, activity of GSTs enzymes, repair of the drug targets, and induction apoptosis of the leukemia MDR cells. Thereby, the selected candidates for anti-leukemic MDR were obtained..Subsequently, experiments on both in vivo anti-leukemic MDR activity and mechanisms of selected candidates were conducted in K562, K562/Adr bearing nude mice, and preliminary experiments in pharmacokinetics (ADME/T) were also carried out to the candidates with potent in vivo anti-leukemic MDR activity through Pipeline Pilot software. Thus, these data may together help us investigate druggability and prospects as novel anti-leukemic MDR drugs.

开口箭（Tupistra chinensis）在中医临床上常用于白血病辅助治疗，我们前期研究发现其总甾体部位能显著抑制荷白血病多药耐药（MDR）细胞K562/Adr裸鼠移植瘤的生长，抑瘤率达87%，总甾体部位及其部分甾体类能显著抑制白血病耐药细胞增殖并有效逆转对柔红霉素的耐药作用。本项目拟对开口箭中甾体类进行分离鉴定，筛选出抗白血病MDR活性化合物。以结构生物信息学为指导，通过生物转化和化学手段等对活性化合物进行修饰，优选出活性先导化合物。利用生物信息学网络模型及分子药理学手段预测和验证先导化合物在抑制耐药细胞内转运蛋白表达、谷胱甘肽转移酶活性、靶点修复及诱导耐药细胞发生凋亡等方面抗白血病MDR作用机制，筛选出抗白血病MDR候选化合物。利用荷白血病MDR细胞裸鼠模型研究候选化合物的体内活性，并采用Pipeline Pilot软件对其成药性进行研究，筛选出抗白血病MDR候选药物。

开口箭（Tupistra chinensis Bak.）是百合科开口箭属植物，具有清热解毒、散淤止痛之功效，现代中医临床上主要用于咽喉炎、恶性肿瘤等疾病的治疗，药用部位为根茎。本项目通过多种色谱学分离手段对开口箭根茎中甾体皂苷类成分进行分离，并利用理化性质、现代波谱学以及化学等手段鉴定了它们的结构。共分离鉴定的72个化合物中，包括5个变形螺甾皂苷类化合物，43个螺甾类化合物，24个呋甾皂苷类化合物化合物，其中37个新化合物。利用体外筛选模型，系统筛选了甾体皂苷的抗肿瘤和抗炎活性，部分化合物活性显著。通过微生物转化手段，对部分活性化合物进行了微生物转化，制备了一批新的活性衍生物。进一步，对活性显著的两个甾体皂苷类化合物（SPD和T17）开展了抗肿瘤作用机制研究。SPD能诱导HL-60细胞和HepG2细胞发生凋亡。SPD还能降低细胞线粒体膜电位，且促进caspase-3和caspase-9的活化，增大Bax与Bcl-2的比值，促进PARP剪切化，能通过线粒体通路诱导HL-60细胞和HepG2细胞发生凋亡。SPD还能诱导HL-60细胞和HepG2细胞发生自噬。T-17可首先将SGC-7901和AGS胃癌细胞阻滞在G0/G1期，并通过内源性线粒体途径时间和剂量依赖的诱导细胞凋亡。T-17还能够诱导SGC-7901和AGS细胞的保护性自噬。在体外研究的基础上，采用裸鼠移植瘤模型，研究了开口箭总甾体皂苷（TCS）的体内抗肿瘤活性。体内实验结果显示，TCS可显著抑制移植瘤的增长，能有效减小裸鼠移植瘤的体积，给药组裸鼠体重与模型组相比无明显差异，脏器指数也没有变化，显示TCS没有毒副作用。免疫组化显示，TCS能显著的抑制裸鼠体内移植瘤的增殖。因此，开口箭总甾体皂苷能显著的抑制肿瘤生长作用，具有良好的研发前景。.通过本项研究，阐明了开口箭抗肿瘤药效物质和作用机制，为开口箭进一步开发奠定了基础。本项目已发表SCI论文5篇，获国家发明专利3项。