CN113461785B - Glp-1受体激动剂及其应用 - Google Patents
Glp-1受体激动剂及其应用 Download PDFInfo
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- CN113461785B CN113461785B CN202110392318.9A CN202110392318A CN113461785B CN 113461785 B CN113461785 B CN 113461785B CN 202110392318 A CN202110392318 A CN 202110392318A CN 113461785 B CN113461785 B CN 113461785B
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Abstract
本发明公开了一种GLP‑1受体激动剂，属于生物医药领域。本发明应用高通量药物筛选技术，从大型环肽库中筛选到名为100‑44(PL00201001)的多肽，能够激活GLP‑1受体，引起细胞信号通路的激活。PL00201001含有80个氨基酸环肽，通过解压缩技术得到一系列衍生的小环肽和线性肽，都具有GLP‑1受体激动剂的作用。PL00201001及其一系列多肽能够作为GLP‑1受体激动剂，进一步开发出用于糖尿病、肥胖症及其相关疾病的多肽类新药。
Description
技术领域
本发明属于生物医学技术领域，具体涉及GLP-1受体激动剂及其应用。
背景技术
蛋白偶联受体（G protein- coupled receptors, GPCRs）是一类7次跨膜受体，属于细胞膜表面受体，约占编码人类基因组编码蛋白的4%。FDA批准的药物的靶点有大约475个，其中GPCRs约有108个，占据34%。GPCRs受体药物每年全球销售额超过1800亿美元，约占全球药物市场的27%以上，可见GPCRs受体药物开发具有广阔的前景。
胰高血糖素样肽-1受体（glucagon-like peptide 1 receptor，GLP-1R）是有463个氨基酸组成的B类GPCRs。GLP-1R受到内源性GLP-1多肽包括（GLP-1(1-36)NH2、GLP-1(7–36)NH2、GLP-1(1-37)和GLP-1(7-37) 和外源性多肽exendin-4的调节。
胰高血糖素样肽-1（1-37）（glucagon-like peptide 1，GLP-1），是由37个氨基酸组成的多肽，其氨基酸序列为HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG，是一种由回肠内分泌细胞分泌的多肽激素，它通过作用于 GLP-1 受体，可以调节胰岛素分泌、抑制胰高血糖素分泌以及延缓胃肠道排空、抑制食欲，从而达到控制糖的摄入，进而调控血糖浓度。
目前上市的GLP-1受体激动剂类药物有艾塞拉肽、利西拉来、利拉鲁肽、阿必鲁肽、度拉鲁肽和索马鲁肽，这些药物都是以GLP-1序列为基础开发出的一系列GLP-1类似物药物。2018年全球GLP-1受体激动剂市场规模约达88.27亿美元，仍保持较快增长。
多肽类药物分子量上介于小分子药物和生物制品之间，有着独特的特性，因为多肽在机体内作为信号分子参与众多生理功能，所以多肽药物往往充当替代疗法的角色，以弥补内源性多肽激素水平的缺乏，2019年，多肽类药物，占全球医药市场的5%，全球销售额超过500亿美元。
现有GLP-1受体激动剂都是基于内源性GLP-1序列改造而来，目前没有全新多肽序列，且都为线性GLP-1类似物，在体内容易降解，半衰期短。本发明发现全新序列GLP-1受体激动剂多肽，再成环条件下仍然具有较高的活性，预测可以提高多肽在体内的半衰期。
多肽药物多以天然活性多肽为基础进行改造，本发明应用自主知识产权的大型多肽库，采用高通量筛选技术，获得GLP-1受体激动剂的编号为PL00201001多肽及系列不同氨基酸数量的线性和环状多肽。
发明内容
发明目的：本发明要解决的技术问题是提供一种GLP-1受体激动剂及其应用。
为解决上述技术问题，本发明采用如下技术方案：
GLP-1受体激动剂，其氨基酸序列如SEQ ID NO.1~6所示。
其中，SEQ ID NO.1所示的氨基酸序列的第1个氨基酸和第80个氨基酸通过肽键连接，形成蛋白质为环肽；
SEQ ID NO.2所示的氨基酸序列形成的蛋白质为为线性肽；
SEQ ID NO.3所示的氨基酸序列的第1个氨基酸和第58个氨基酸通过肽键连接，形成蛋白质为环肽；
SEQ ID NO.4所示的氨基酸序列形成的蛋白质为为线性肽；
SEQ ID NO.5所示的氨基酸序列的第1个氨基酸和第43个氨基酸通过肽键连接，形成蛋白质为环肽；
SEQ ID NO.6所示的氨基酸序列的第1个氨基酸和第17个氨基酸通过肽键连接，形成蛋白质为环肽。
上述GLP-1受体激动剂在制备治疗糖尿病、肥胖症药物中的应用。
一种药物组合物，包括上述GLP-1受体激动剂或者其药学上可接受的载体。
采用检测细胞内钙离子浓度的方法，从大型多肽库中筛选到一种80环肽及系列不同氨基酸数量的线性和环状多肽的氨基酸序列及其作为GLP-1受体激动剂药物的应用。
本发明提供的GLP-1受体激动剂PL00201001 80氨基酸环肽，是从自主知识产权多肽库中，通过高通量筛选技术，检测细胞内钙离子浓度变化得到的80环肽，及一系列解压缩的小环肽和线性肽。
PL00201001多肽的发现包括细胞培养过程，细胞铺板，多肽库溶解及稀释，GLP-1受体激动的检测。
有益效果：
本发明公开了一种GLP-1受体激动剂PL00201001多肽及系列解压缩多肽，本发明对GLP-1受体有非常显著的激动作用，是一种GLP-1受体激动剂。
附图说明
图1：确认PL00201001的激动GLP-1R受体的作用。
图2：PL00201001激动GLP-1受体浓度响应曲线。
图3：PL00201001对应80个氨基酸线性肽（PL00201245)对GLP-1R受体的作用。
图4：PL00201001解压缩后得到58个氨基酸数量的环肽（PL00201063）对GLP-1R受体的作用。
图5：PL00201001解压缩后得到48个氨基酸数量的线性肽（PL00201071）对GLP-1R受体的作用。
图6：PL00201001解压缩后得到43个氨基酸数量的环肽（PL00201050）对GLP-1R受体的作用。
图7：PL00201050、PL00201063、PL00201071、PL00201245、PL002011372激动GLP-1受体浓度响应曲线。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本实用新型，而不应当也不会限制权利要求书中所详细描述的本发明。
实施例1：
1. 关键试剂：多肽库（自制）、过表达GLP-1R的CHO细胞（Chinese hamster ovarycell，中国仓鼠暖巢细胞）系，简写为CHO-K1/GLP-1R/Gα15细胞（Genscript）、Calcium 6Assay Kit（Molecular Devices）。
2. 高通量筛选过程
2.1. CHO-K1/GLP1/Gα15细胞的培养
2.1.1. 细胞复苏：从液氮罐中取出细胞，在37℃水浴锅中快速解冻细胞。将细胞移至15 ml离心管中，缓慢加入9mL预热的解冻培养基，800转离心5分钟，移除上清培养基。用5ml解冻培养基重悬细胞，转移至T25培养瓶中，放于37℃，5%CO2的培养箱中培养。细胞复苏第二天更换培养液为生长培养基。
2.1.2. 细胞传代：当细胞长满培养瓶80-90%，先用DPBS润洗细胞，再用0.25%胰酶消化细胞；收集细胞悬液至离心管中，800转离心5分钟，移除上清培养基；加入6-8mL新鲜生长培养基，重悬细胞，按照1：3~1：10的比列进行传代，放于37℃，5%CO2的培养箱中培养。传代后每2-3天进行换液。
2.1.3. 细胞冻存：当细胞长满培养皿80-90%，先用DPBS润洗细胞，再用0.25%胰酶消化细胞；收集细胞悬液至离心管中，800转离心5分钟，移除上清培养基；用冻存培养基重悬细胞，进行细胞计数，将细胞稀释到2~3×106/mL。每个冻存管分装1mL细胞冻存悬液。将分装好细胞的冻存管放入冻存盒中，将冻存盒放入-80°C冰箱过夜保存后，将冻存管转移到液氮罐中。
2.2. CHO-K1/GLP1/Gα15细胞铺板
测试前24小时，将培养瓶中的CHO-K1/GLP1/Gα15细胞经过0.25%胰酶消化处理并悬浮于细胞培养液中，以每孔10000个细胞的密度使用分液仪加至黑色透明底384孔板中培养，每孔25μl，37℃, 5% CO2过夜培养。
2.3. Calcium 6 assay kit找工作液准备
测试当日，将Calcium 6试剂盒中的组分A溶解于钙染料缓冲液(Loading buffer)并加入250 mM丙磺舒配制成含有5 mM 丙磺舒的钙染料溶液。
在细胞培养板中每孔加入25 μl钙染料溶液，放置于37℃，5% CO2条件下继续培养2小时。
2.4. 多肽库溶解及稀释
2.4.1. 多肽库溶解
2.4.2. 将多肽库96孔深孔板放于离心机4000 rpm离心2~3 分钟。用自动分液仪5.1.2 向96孔深孔板中加入250μL/孔超纯水中。用硅胶盖密封，放置95℃水浴5 分钟。注：此时多肽浓度约为：200 µM。
2.4.3. 溶解后的96深孔板多肽放于离心机4000 rpm离心2~3 分钟。
2.5. 多肽库稀释
将溶解后用工作站转移至384孔板中，用loading buffer稀释至10uM。
2.6. FLIPR检测
细胞加入Calcium 6染料2小时后，将细胞培养板取出并避光放置于室温10分钟，然后和多肽溶液板一起放入FLIPR仪器，进行检测。设置GLP-1为阳性对照。
3. PL00201001确认
3.1. 按照步骤2的过程对初筛到的多肽进行确认。
3.2. 按照步骤2的过程检测活性多肽的EC50值
检测当天，将将活性多肽母液用1×Loading buffer(含20mMHEPES)（pH:7.4）稀释到50μM（5×浓度）， 再以3倍稀释8个梯度，每个浓度做3个复孔，测试活性多肽的EC50值。
4. PL00201001衍生多肽筛选及确认
对80环肽PL00201001进行氨基酸的解压缩，设计出10~80不同氨基酸序列的环肽或者线性肽。
本发明得到的多肽序列如下：
SEQ ID NO.1
PL00201001：第1个和第80个氨基酸通过肽键成环
HHHHHHFIHECFVRCCWTKWHRPNCNTESQCVWWFHCIWAAHKEHDANHKQCQDKDEPYEQQHHKWKMFLLYMIAFLYCL
SEQ ID NO.2
PL00201245：PL00201001线性肽
HHHHHHFIHECFVRCCWTKWHRPNCNTESQCVWWFHCIWAAHKEHDANHKQCQDKDEPYEQQHHKWKMFLLYMIAFLYCL
SEQ ID NO.3
PL00201063：第1个和第58个氨基酸通过肽键成环
RCCWTKWHRPNCNTESQCVWWFHCIWAAHKEHDANHKQCQDKDEPYEQQHHKWKMFLL
SEQ ID NO.4
PL00201071：48个氨基酸线性肽
CWTKWHRPNCNTESQCVWWFHCIWAAHKEHDANHKQCQDKDEPYEQQH
SEQ ID NO.5
PL00201050：第1个和第43个氨基酸通过肽键成环
CWTKWHRPNCNTESQCVWWFHCIWAAHKEHDANHKQCQDKDEP
SEQ ID NO.6
PL00201372：第1个和第17个氨基酸通过肽键成环
FHCIWAAHKEHDANHKQC
按照步骤2筛选过程进行筛选，并根据步骤3.2对筛选到的解压缩活性多肽进行EC50验证。
5. 实验结果：
1. 通过高通量筛选，从近8万条80环肽中找到PL00201001编号多肽，能够在细胞水平激动GLP-1R受体，并进行再次确认实验，如图1所示。
2. 在确认PL00201001激动作用后，进行PL00201001对GLP-1R受体浓度响应曲线，得到EC50值为0.6μM，如图2所示。
3. 对80环肽PL00201001进行解压缩筛选，结果如图3至图6所示。
4. 对80环肽PL00201001进行解压缩后，活性多肽进行对GLP-1R受体浓度响应曲线验证，如图7所示。
序列表
<110> 湖南中晟全肽生化有限公司
<120> GLP-1受体激动剂及其应用
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<170> SIPOSequenceListing 1.0
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<211> 80
<212> PRT
<213> 人工序列(Artificial Sequence)
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Trp Trp Phe His Cys Ile Trp Ala Ala His Lys Glu His Asp Ala Asn
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Lys Trp Lys Met Phe Leu Leu Tyr Met Ile Ala Phe Leu Tyr Cys Leu
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His His His His His His Phe Ile His Glu Cys Phe Val Arg Cys Cys
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Trp Thr Lys Trp His Arg Pro Asn Cys Asn Thr Glu Ser Gln Cys Val
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Trp Trp Phe His Cys Ile Trp Ala Ala His Lys Glu His Asp Ala Asn
35 40 45
His Lys Gln Cys Gln Asp Lys Asp Glu Pro Tyr Glu Gln Gln His His
50 55 60
Lys Trp Lys Met Phe Leu Leu Tyr Met Ile Ala Phe Leu Tyr Cys Leu
65 70 75 80
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<211> 58
<212> PRT
<213> 人工序列(Artificial Sequence)
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Arg Cys Cys Trp Thr Lys Trp His Arg Pro Asn Cys Asn Thr Glu Ser
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Gln Cys Val Trp Trp Phe His Cys Ile Trp Ala Ala His Lys Glu His
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Asp Ala Asn His Lys Gln Cys Gln Asp Lys Asp Glu Pro Tyr Glu Gln
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Gln His His Lys Trp Lys Met Phe Leu Leu
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<210> 4
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<213> 人工序列(Artificial Sequence)
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Cys Trp Thr Lys Trp His Arg Pro Asn Cys Asn Thr Glu Ser Gln Cys
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Val Trp Trp Phe His Cys Ile Trp Ala Ala His Lys Glu His Asp Ala
20 25 30
Asn His Lys Gln Cys Gln Asp Lys Asp Glu Pro Tyr Glu Gln Gln His
35 40 45
<210> 5
<211> 43
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Cys Trp Thr Lys Trp His Arg Pro Asn Cys Asn Thr Glu Ser Gln Cys
1 5 10 15
Val Trp Trp Phe His Cys Ile Trp Ala Ala His Lys Glu His Asp Ala
20 25 30
Asn His Lys Gln Cys Gln Asp Lys Asp Glu Pro
35 40
<210> 6
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Phe His Cys Ile Trp Ala Ala His Lys Glu His Asp Ala Asn His Lys
1 5 10 15
Gln Cys
Claims (4)
1.GLP-1受体激动剂，其特征在于，其氨基酸序列如SEQ ID NO.1所示;
其中，所述SEQ ID NO.1所示的氨基酸序列的第1个氨基酸和第80个氨基酸通过肽键连接，形成蛋白质为环肽。
2.权利要求1所述的GLP-1受体激动剂在制备治疗糖尿病药物中的应用。
3.权利要求1所述的GLP-1受体激动剂在制备治疗肥胖症药物中的应用。
4.一种药物组合物，其特征在于，包括权利要求1所述的GLP-1受体激动剂或者其药学上可接受的载体。
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