靶向RANKL的抗骨质疏松活性多肽的设计、合成及生物活性研究

发布时间：2018-01-17 02:29

本文关键词：靶向RANKL的抗骨质疏松活性多肽的设计、合成及生物活性研究　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

更多相关文章： 抗骨质疏松 多肽 RANKL OPG模拟物

【摘要】：随着人口老龄化的不断加剧,骨质疏松已成为全球范围内越来越严重的公共健康问题。目前临床上应用的以双膦酸盐为代表的抗骨质疏松药物都存在着一定的不足之处,寻找新型、高效、低毒的抗骨质疏松药物是当前骨科领域的主要目标之一。近些年来,破骨细胞病理生理学研究发现,RANK-RANKL-OPG骨调节轴共同参与调节破骨前体细胞向破骨细胞分化成熟过程,被认为是开发新型抗骨质疏松药物的热点靶标。RANKL与其靶蛋白RANK的结合可以激活下游NF-κB信号通路,造成包括骨质疏松在内的多种病症发生。而OPG作为RANKL的天然诱饵受体,能够有效抑制RANKL诱导的破骨细胞分化成熟。通过比较OPG/RANKL与RANKL/RANK复合物晶体衍射结构发现,OPG的90s loop序列是OPG与RANKL结合的关键位点。OPG通过90s loop占领RANKL,使得RANKL丧失与RANK结合的能力,发挥其对骨骼的保护作用。本课题根据OPG 90s loop序列,合理设计并合成了包括直链肽、二硫键肽、二硫键糖肽、二硒键衍生物以及三氮唑键衍生物等不同类型的OPG模拟物多肽共6条。经文献检索,所有化合物均为首次报道。目标化合物均经过HR-MS确证,所有中间体都经过ESI-MS,1H-NMR,13C-NMR确证。细胞水平活性实验中,我们提取了ICR小鼠破骨前体细胞,经过目标化合物处理后,进行了TRAP染色。测试结果初步显示,在30μM化合物浓度下,除OPG-1外,目标化合物均表现出不同程度的破骨前体细胞分化成熟抑制作用。其中,糖基化衍生物OPG-4显示出最佳的抑制效果,略优于阳性对照OP3-4。我们选取OPG-4再次进行了浓度梯度的TRAP染色实验,化合物显示出剂量依赖性的抑制作用,测得其半数抑制浓度(IC50)为28.51μM。表面等离子共振实验中,我们通过Biacore T200生物传感仪测定了OPG-4与靶标蛋白RANKL的结合能力。实验结果表明,OPG-4与RANKL的结合常数在微摩尔数量级,与阳性对照相当。体内活性测试中,我们选择了雌性C57BL/6小鼠,通过卵巢切除造模,成功获得OVX骨质疏松小鼠模型。腹腔注射给予小鼠OPG-4(20 mg/kg/day)处理6周后,取股骨进行HE染色和Micro-CT扫描。我们发现处理组(OVX+OPG)小鼠骨密度以及骨小梁数目较去卵巢组(OVX)组有明显增多,具有有统计学意义的明显差异(Student’s t test,p0.05)。本课题设计并合成的糖基化OPG模拟物OPG-4在体内外活性实验中都表现出了良好的破骨细胞抑制活性,可以作为候选化合物开展深入研究。通过对其进行氨基酸残基随机突变或更换糖基化类型以及位点等尝试,我们有理由相信能够得到体内外活性更优异的化合物。在后续研究中,我们还将对OPG-4/RANKL复合物继续进行单晶衍射研究,力图阐明两者相互作用的分子机制。本研究对开发新型抗骨质疏松药物有着重大意义。
[Abstract]:With the aging of the population. Osteoporosis has become a more and more serious public health problem in the world. At present, the anti-osteoporosis drugs, such as bisphosphonates, which are used in clinic, have some shortcomings. Low-toxicity anti-osteoporosis drugs are one of the main targets in the field of orthopedics. In recent years, the pathophysiology of osteoclasts has been discovered. The RANK-RANKL-OPG bone regulatory axis is involved in the process of regulating the differentiation and maturation of osteoclasts from osteoclasts to osteoclasts. The binding of RANKL to its target protein RANK can activate the downstream NF- 魏 B signaling pathway. Causes many diseases, including osteoporosis, and OPG acts as a natural bait receptor for RANKL. It can effectively inhibit the differentiation and maturation of osteoclasts induced by RANKL. It was found by comparing the crystal diffraction structure of OPG/RANKL and RANKL/RANK complex. The 90s loop sequence of OPG is the key site of OPG binding to RANKL. OPG occupies RANKL through 90 s loop. The ability of RANKL to bind to RANK was lost and its protective effect on bone was exerted. According to the sequence of OPG 90 s loop, the straight chain peptide was reasonably designed and synthesized. There were 6 polypeptides of disulfide bond peptide, disulfide glycopeptide, diselenide bond derivative and triazolium bond derivative. All the compounds were reported for the first time. The target compounds were confirmed by HR-MS and all intermediates were confirmed by ESI-MSX 1H-NMR-13C-NMR. The osteoclast precursor cells of ICR mice were extracted and treated with the target compounds. The results of TRAP staining showed that at the concentration of 30 渭 M compounds, except OPG-1. The target compounds all showed different degrees of inhibition of osteoclast differentiation and maturation. Among them, glycosylation derivative OPG-4 showed the best inhibitory effect. A little better than the positive control OP3-4. We selected OPG-4 again for the concentration gradient of TRAP staining experiment, the compound showed a dose-dependent inhibitory effect. The half inhibitory concentration (IC50) was determined to be 28.51 渭 M. in the surface plasmon resonance experiment. The binding ability of OPG-4 to target protein RANKL was determined by Biacore T200 biosensor. The binding constant of OPG-4 and RANKL was in the order of micromolarity, which was similar to that of the positive control. In vivo activity test, we selected female C57BL / 6 mice and made the model by ovariectomy. The model of OVX osteoporosis mice was successfully obtained. The mice were treated by intraperitoneal injection of OPG-4(20 mg / kg / day for 6 weeks. The bone mineral density and the number of trabeculae in the treated group were significantly higher than those in the ovariectomized group. There was a significant difference in t test between the two groups. In this study, the glycosylated OPG mimics OPG-4 showed good osteoclast inhibitory activity in vitro and in vivo. It can be used as a candidate compound for further research, by random mutation of amino acid residues or by changing glycosylation types and sites. We have reason to believe that we can obtain more active compounds in vivo and in vitro. In the further study, we will continue to study the single crystal diffraction of OPG-4/RANKL complex. This study is of great significance to the development of new anti-osteoporosis drugs.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91

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