计算机辅助药物设计抗TNF-α青蛙缓激肽类似物及其机制研究

Tumor necrosis factor -α (TNF-α) plays a key role in autoimmune diseases process such as rheumatoid arthritis disease. The TNF-α monoclonal antibody makes a great improvement in the treatment of autoimmune diseases. However, the shortages of the existing monoclonal antibody drug are obvious, which could cause various adverse reactions making serious side effects on the patients. Therefore, the development of novel anti- TNF-α drugs attracts more and more scientists’ attention. This project is based on applier’s previous research found that some special Bradykinin analogs isolated from frog skin secretions are able to inhibit the activity of TNF-α. Moreover, the applier is involved in a novel anti-TNF-α monoclonal antibody research project, where the professional knowledge and experimental skills make sure this project is available. This project is going to focus on anti-TNF-α frog bioactive peptides, which have not been reported yet. Use computer-aided drug design platform to optimize the amino acid structure of Bradykinin analogs, combined with the pre-established anti-TNF-α screening platform to screen activity of Bradykinin analogs synthesized by solid-phase peptide synthesis. Then their related anti-TNF-α mechanism in vivo and in vitro is going to be studied in order to supply novel high efficacy candidates for the development of anti- TNF-α peptide drug.

肿瘤坏死因子-α（TNF-α）在类风湿性关节炎等自身免疫型疾病病变过程中起主要作用，以TNF-α为靶点的单克隆抗体药物用于治疗自身免疫性疾病效果明显；但现有的单克隆抗体药物存在结构复杂，免疫原性强等缺点；研究开发新型的抗TNF-α药物是当今科学研究的热点。本项目基于申请人前期研究发现的实验现象：青蛙表皮分泌物中含有的某种Bradykinin类似物对TNF-α活性具有抑制作用；结合目前课题组进行的抗TNF-α单克隆抗体研究，开展目前尚未见报道的抗TNF-α青蛙活性多肽的研究工作；采用计算机辅助药物设计技术指导Bradykinin类似物氨基酸结构改造优化，借助前期所建立的以TNF-α为靶点的活性筛选平台，对运用固相肽合成技术化学合成的Bradykinin类似物进行活性筛选，在体内和体外对其作用机制进行探究；以期获得药效高、特异性强的抗TNF-α多肽类药物候选物。

自从传统的TNF-α抗体在治疗风湿性关节炎和其他自身免疫疾病中取得显著成效后，研究者们一直致力于寻找新型抗TNF-α免疫治疗蛋白药物。本项目旨在开展以TNF-α为靶点，运用计算机技术对天然活性多肽进行改造和筛选；在体外研究活性多肽生物活性及作用机制；构建动物模型探讨抗TNF-α活性多肽体内的药理功能，阐明其作用机制。从而获得具有抗TNF-α活性多肽，为抗TNF-α多肽药物的研发提供重要的候选物和理论依据。经过3年努力，课题组运用分子生物学技术并结合高效液相色谱及LCQ等质谱技术成功构建青蛙活性多肽分离提取及鉴定平台，获得多种新型活性多肽；运用生物信息学和结构生物学等相关知识，选择结构简单便于改造的纳米抗体为基础结构，运用计算机软件设计对具有抗TNF-α生物活性的多肽进行氨基酸结构改造和修饰，获得新型抗TNF-α生物活性蛋白。运用固相肽合成技术或构建活性蛋白原核表达系统获得活性蛋白，以TNF-α为靶点进行活性筛选。rhTNFα剂量依赖性的抑制L929细胞的生长，12h时IC50值为1.961ng/ml,活性蛋白的EC50为2.487pM。高内涵分析亦表明活性蛋白具有拮抗hTNFα诱导的L929细胞凋亡作用。右旋半乳糖胺（D-GalN）的给药剂量为1000mg/kg，致敏时间为20min，rhTNFα最小致死剂量为65ug/kg。当给药剂量为4.2mg/kg且给药间隔60min时，活性蛋白能较大效率的抵抗rhTNFα引起的小鼠死亡。佐剂诱导大鼠关节炎模型研究结果表明活性蛋白对AA大鼠关节炎症具有改善作用，能一定程度上降低关节炎大鼠的关节肿胀、关节炎指数，改善关节组织的病变，影响相关组织中炎症信号通路的蛋白表达水平，具体的药理机制还需我们进一步研究。