治疗自身免疫性疾病的低毒高效天然产物 Hirterllanines 构效关系的研究

Hirtellanine N is an novel isoflavanol isolated from TCM herb Campylotropis hirtellae.The bioassay indicated that Hirteallanine N possess very potent immune suppressive activities. The IC50 of Hirtellanine N for T-cell inhibition is 0.13 µM while the IC50 for B-cell inhibition is 0.26 µM, Compared with cyclosporine, Hirtellanine N had similar activity in B-cell inhibition although it is less active to inhibit the T-cell. However, Hiratellanine N is much less toxic than Cyclosproine, hence, it could be an ideal lead for stracture- activity relation study. This project aim to have an in depth understanding about the SAR of Hirtellanine N. We plan to modify the substitutes on the A ring and B ring of the isoflavane backbone. To examine how the substitutions affect the biological activities. The modification on 3-OH group of Hirtellanine N will be one of the focus of the project, since the 3-OH group is the major cause of the instability of the compound. The T-cell inhibition model will be used as the primary indicator of the activities while the toxicity examined with MTT method will be used as the secondary indicator during the structure modification. The first milestone of the project is to identify the bioactive core of the compound. The further STR studies will be carried out to improve the activity of T-cell inhibition, improve stability and the solubility. After the best compound selected, the early safety assessment and the validation of the effectiveness on animal model will be carried out to identify the probability as the drug candidate for the treatment of Rheumatoid Arthritis.

类风湿性关节炎是一种以关节和关节周围组织的非感染性炎症为主的全身性自身免疫性疾病，其致残 率高且治疗棘手，已成为世界公认的难治性疾病。我们在前期的研究中，从中药大红袍中分离 得到50余个新化合物，生物试验中显示出良好的免疫抑制活性和低细胞毒性，其中最新发现的 “Hirtellanine N” 活性优而毒性低。其B细胞抑制活性与环孢菌素相近，而T细胞抑制活性是Hirtellanine A 的五倍，其毒性是环孢菌素的三分之一，因此可作为候选药物进行进一步研究。但前期研究中也发现该化合物具有水溶性低, 稳定性差等缺点, 本课题拟通过对其化学结构的修饰，确定药效基团，建立其构效关系，提高其对T-淋巴细胞的抑制活性、改善水溶性及稳定性；并在动物模型上对其药效学进行验证，为研发拥有自主知识产权的治疗类风湿性关节炎的创新药物提供重要的依据和基础。

本研究一共合成具有免疫抑制潜力的化合物117个，通过体外活性的测试发现一个化合物BYIS-01 具有极强的免疫抑制活性，其抑制Con A 和LPS诱导的小鼠脾细胞增殖的IC50 分别为6 nM和1 nM, 强于对照药环孢菌素。尽管该化合物的毒性也大于环孢菌素，但是充分体现了该类化合物免疫抑制活性的潜力。我们采用绵羊红细胞引起的小鼠跖肿胀迟发超敏反应模型，和DNFB诱导的小鼠耳肿胀模型上检测该化合物在体内的免疫抑制活性，我们发现在口服剂量0.5-12MPK（QD）的剂量下，BYIS-01显著抑制绵羊红细胞引起小鼠趾肿胀，和和DNFB诱导的小鼠耳肿胀。遗憾的是在胶原蛋白诱导的大鼠关节炎模型BYIS-01未能显示出显著的效果。该化合物在MRL/MpJ-Faslpr系统性红斑狼疮模型小鼠上也未能显示出具有剂量相关性的疗效。仅在中剂量组的动物中发现该化合物一定程度上缓解红斑狼疮疾病的严重程度，表现在其显著地降低了抗双链DNA IgG的浓度。这两组体内药效学实验的结果相互矛盾，提示我们需要详尽了解该化合物的作用机制，并结合作用机制进一步寻找最合适的给药方式和适应症。我们采用蛋白芯片与蛋白质谱相结合的办法，以MST，western blot 和biacore 验证，发现BYIS-01可能结合的蛋白靶点为：CAT、IMPDH1、HSPA8。