摘要
肠促胰岛素分泌肽胰高血糖素样肽-1(GLP-1)和葡萄糖依赖性促胰岛素释放多肽(GIP)能通过血糖依赖机制促进胰岛素分泌,其特异性结合受体GLP-1R和GIPR是治疗2型糖尿病的良好靶点。以本实验室前期设计的口服降糖多肽OHP2为基础,设计了可口服的新型降糖多肽——ODA。ODA较OHP2的亲脂性提高,在Caco-2细胞中的胞吞能力及跨细胞转运能力更强。ODA保留了OHP2对GLP-1R的激活能力,增强了与GIPR的结合能力。口服低剂量ODA(0.53 mg/kg)即可达到与口服OHP2(1.06 mg/kg)相当的降糖水平。研究结果显示,ODA是治疗2型糖尿病极具潜力的口服药物。
GLP-1由小肠L细胞分泌产生,经GLP-1R介导,以葡萄糖依赖的方式作用于胰岛β细胞,促进胰岛素分
在目前的糖尿病治疗中,肠促肽类降糖药物占据了重要地位,其中以GLP-1R激动剂为
本实验室前期基于GLP-1R激动剂Exendin-4的结构特征,利用计算机辅助设计得到了具有降糖活性的口服GLP-1R激动剂OHP
DMEM高糖培养基、DMEM/F-12培养基(美国Gibco公司);胰蛋白酶、G418(美国Sigma-Aldrich公司);胎牛血清(FBS,美国CLARK Bioscience公司);Exendin-4(上海吉尔生化有限公司);OHP2(南京金斯瑞生物科技有限公司);ODA(合肥森尔生物科技有限公司);异硫氰酸荧光素(异构体Ⅰ,FITC,上海阿拉丁生化科技股份有限公司);Pierce BCA法蛋白测定试剂盒(美国Thermo Scientific公司);牛血清白蛋白(BSA,广州赛国生物科技有限责任公司);氨苄青霉素、链霉素、潮霉素(上海生工生物工程有限公司);Steady-Gl
基于OHP2的结构,引入GIP 活性位点,同时OHP2耐酶解位点保持稳定,得到增强GIP激动活性的ODA序列(由合肥森尔生物科技合成)。从RCSB PDB蛋白质数据库(https://www.rcsb.org)获取Exendin-4和GLP-1受体胞外结构域的PDB文件(5OTT)、GIP和GIP受体胞外结构域的PDB文件(2QKH),利用MOE(Molecular Operating Environment)分子操作系统和Rosetta Design在线设计服务系统(http://rosettadesign.med.unc.edu)进行亲和力打分计算。
使用去离子水溶解样品至0.5 mg/mL,用圆二色光谱仪Jasco J-810,在室温下以1 nm分辨率,50 nm/min的扫描速度在190 ~ 250 nm范围内对样品进行检测。
利用蛋白质结构服务器SWISS-MODEL(http://swissmodel.expasy.org)进行同源建模,ODA和OHP2均以Exendin-4为模板序列,选择全球性模型质量估测(GMQE)值最高的建模结果。
细胞铺板:Caco-2细胞密度为每毫升1 × 1
细胞铺板:Caco-2细胞密度为每毫升1 × 1
细胞培养:使用含10%胎牛血清、潮霉素B、G418的DMEM/F12培养基培养A11A细胞。细胞铺板:A11A细胞密度为每毫升2 × 1
靶标分子预处理:从RCSB PDB蛋白质数据库(https://www.rcsb.org)下载GIP和GIP受体胞外结构域的PDB文件(2QKH),选择Quick Prepare对分子进行加氢去水并赋予电荷,修正PDB结构缺陷。定义活性位点(site)以确定配体分子结合部位。配体分子预处理:通过MOE同源建模获得配体分子的PDB文件,加氢去水并赋予电荷。分子对接:选择蛋白-蛋白对接以半柔性对接方法计算结果。
3%碳酸氢钠溶液,Exendin-4溶液(0.02 mg/mL),葡萄糖溶液(0.2 g/mL),OHP2溶液(0.424 mg/mL),ODA溶液(0.848 mg/mL,0.424 mg/mL,0.212 mg/mL)。
基于GLP-1(HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG)、GIP(YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ)、Exendin-4(HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS)和OHP2(HGEGTFTSDLSSQMEEEAVKEFIEWLVNGGPSSGAPPPSC)的分子结构,在保留OHP2良好的抗酶解活性以及口服GLP-1R激动活性的前提下,通过进一步引入GIP的活性位点,本实验设计了一条新型口服降糖多肽——ODA。通过Rosetta Design评估其对GLP-1和GIP两个受体的亲和力,亲和力强度与打分绝对值呈正相关。打分结果显示,与OHP2相比,ODA保留了GLP结合能力,同时提高了GIP受体亲和力。(
Peptides | Score | |
---|---|---|
GLP-1R | GIPR | |
Exendin-4 | -21.6 | N/A |
GIP (1-42) | N/A | -16.8 |
OHP2 | -21.6 | -15.3 |
ODA | -22.5 | -15.8 |
N/A: Not applicable; OHP2: Oral hypoglycemic peptide 2; ODA: Oral dual agonist
为了得到ODA的二级结构信息,本实验对ODA的CD谱进行分析。如

Figure 1 Determination of Exendin-4, OHP2 and ODA
A: Analysis of the secondary structure of ODA by CD spectroscopy; B: Simulation of the tertiary structure of ODA based on SWISS-MODEL
本实验通过SWISS-MODEL模拟预测ODA的三级结构,由于序列的同源相似性,ODA和OHP2的三级结构模拟均以Exendin-4为模板序列。Exendin-4,OHP2和ODA的GMQE分别为0.71,0.73和0.68。如
为了评价ODA的胞吞能力,对ODA进行了流式细胞术和荧光酶标仪法分析。如

Figure 2 Endocytosis of Exendin-4, OHP2 and ODA in Caco-2 cells
A: Percentages of FIT
本实验通过Transwell法考察ODA在单层细胞模型上的跨膜转运水平。结果如

Figure 3 Transcytosis of Exendin-4, OHP2 and ODA in Caco-2 cells ()
A: Concentration-time profiles of FITC-labelled drugs at the basolateral side of the Transwell model during 8 hours incubation; B: Concentration of FITC-labelled drugs at the basolateral side of Transwell model in the 8th hour
本实验采用荧光素酶报告基因法评估了ODA对GLP-1R的激活能力,结果如

Figure 4 ODA binding ability to GLP-1R and GIPR in vitro
A: Evaluation of the ability of Exendin-4, OHP2 and ODA to activate GLP-1R by A11A cellular luciferase reporter gene assays (); B: Binding of ODA to GIPR, simulated by MOE molecular docking
选取健康C57BL/6J小鼠,通过腹腔注射葡萄糖耐量试验(IPGTT)考察口服不同剂量ODA对血糖的控制作用,以皮下注射Exendin-4组为阳性对照,ODA单次口服给药的糖耐量试验结果如

Figure 5 IPGTT of ODA in healthy mice ()
A: Fasting blood glucose versus time curve of IPGTT in healthy mice with Exendin-4 (sc, 0.10 mg/kg), OHP2 (ig, 1.06 mg/kg), ODA-H (ig, 2.12 mg/kg), ODA-M (ig, 1.06 mg/kg) and ODA-L (ig, 0.53 mg/kg) respectively; B: The area under the glucose levels curve (AUC) of IPGTT
口服索马鲁肽是目前市场上唯一一款可口服的GLP-1R激动剂,依赖促吸收剂N-[8-(2-羟基苯甲酰基)-氨基]辛酸钠(SNAC)才能实现口服吸收。SNAC可在胃部升高局部区域的pH,通过短期局部缓冲作用保护索马鲁肽防止胃蛋白酶的降
综合代谢性疾病如肥胖和糖尿病等通常具有异质性和并发性等特点,单一靶点药物治疗有一定局限
综上所述,本研究设计得到的口服ODA保留了OHP2对GLP-1R的激活能力,增强了与GIPR的结合能力,从而服用小剂量ODA即可达到降糖疗效。此外,简单的分子结构不仅更易于工业化生产,还能避免过度修饰带来的生物安全隐患。未来,口服ODA将为糖尿病患者提供更安全、更便利、更有效的用药选择。
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