CN110776557A - 多肽及其应用和dpp-ⅳ抑制剂或降血糖药物或保健品 - Google Patents
多肽及其应用和dpp-ⅳ抑制剂或降血糖药物或保健品 Download PDFInfo
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Abstract
本发明涉及抑制二肽基肽酶(DPP‑Ⅳ,dipeptidyl peptidaseⅣ)活性及降血糖的一种多肽化合物Phe‑Pro‑His‑Phe‑Asp‑Leu‑Ser‑His‑Gly‑Ser‑Ala,它的氨基酸序列为Phe‑Pro‑His‑Phe‑Asp‑Leu‑Ser‑His‑Gly‑Ser‑Ala。多肽Phe‑Pro‑His‑Phe‑Asp‑Leu‑Ser‑His‑Gly‑Ser‑Ala具有DPP‑Ⅳ抑制活性及降血糖活性,作为降血糖以及治疗II型糖尿病的保健品和药物先导化合物具有良好的应用前景。
Description
技术领域
本发明涉及多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala在制备抑制二肽基肽酶(DPP-Ⅳ,dipeptidyl peptidaseⅣ)及降血糖药物及保健品中的应用。
背景技术
糖尿病是一种由于体内胰岛素绝对或相对不足而导致的葡萄糖、蛋白质、脂代谢紊乱的综合症。目前,糖尿病已经成为全球性流行病。根据世界卫生组织统计,糖尿病的发病率每年都以一定的幅度在递增。2016年我国糖尿病流行率为9.6%,且随着生活水平的提高,这一数字仍在急剧上升。
二肽基肽酶Ⅳ(DPP-Ⅳ,dipeptidyl peptidaseⅣ)是一种丝氨酸蛋白酶,该酶的底物包括N端第二位是脯氨酸或丙氨酸残基的多肽。它能从肽链N端水解两个氨基酸残基,能快速有效降解胰高血糖素样肽1(GLP-1,glucagon-like peptide-1),GLP-1是胰岛素生成和分泌最有效的刺激剂之一,因此抑制DPP-Ⅳ能增强内源性GLP-1的作用,从而提高血液中胰岛素的水平,进而降低并维持糖尿病人的血糖水平(Bioorg Med Chem.2007,15(7):2715-2735)。并且,GLP-1调节胰岛素分泌具有严格的血糖浓度依赖性,只有在高血糖条件下GLP-1才会提高胰岛素的分泌水平,故DPP-Ⅳ抑制剂不存在因服药而引发低血糖的风险(Best Pract Res Clin Endocrinol Metab.2009,23(4):479-86)
发明内容
本发明的目的是提供多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala在抑制DPP-Ⅳ活性和降血糖中的应用和快速筛选方法;多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala具有DPP-Ⅳ抑制活性及降血糖活性,作为高血糖、II型糖尿病的保健品和药物先导化合物具有良好的应用前景。
为实现上述目的,本发明以所述多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala为抑制DPP-Ⅳ活性和降血糖的有效成份。
其具有序列表SEQ ID NO:1中氨基酸序列;多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala为DPP-Ⅳ抑制剂及降血糖药物及保健品的活性成份,其中可添加药物学上可接受的载体或辅料。
具有抑制DPP-Ⅳ活性和降血糖活性的多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala来源于梅花鹿。由于鹿的蛋白库不够完善,总蛋白数量较少,而牛和鹿的基因同源性超过90%(Sui Z G,Yuan H M,Liang Z,et al.Talanta,2013,107,189-194.),因此本实验选择牛科(bovine)作为蛋白数据库。多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala来源于牛科(bovine)蛋白库的Alpha globin chain蛋白,含11个氨基酸残基,氨基酸序列为Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala,为单链线性结构,白色粉末状,易溶于水,分子量为1214Da;对DPP-Ⅳ活性具有较好的抑制作用,IC50为108±7μM(n=3,Mean±SD)。
多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala具备DPP-Ⅳ抑制剂所要求的特征:
DPP-Ⅳ抑制肽由至少应含有一个Pro残基,特别是当N端第二位为Pro残基时DPP-Ⅳ抑制活性尤为明显。多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala含有Pro残基且其位于N端第二位,因此满足这一影响活性的条件。
本发明与现有技术相比,具有如下有益效果:
本发明首次从鹿茸中获得并确定了活性化合物的结构,化合物具有较好的抑制DPP-Ⅳ的活性,因此作为治疗高血糖、II型糖尿病药物的先导化合物具有良好的潜力和应用前景。
具体实施方式
实施例1多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala的制备
采用LC-MS/MS与Shotgun蛋白质组学技术相结合的方法。以梅花鹿茸为原料,经酶解蛋白,离心,纯化及LC-MS/MS分析,结合构效关系特征,筛选对DPP-Ⅳ具有抑制作用的肽段。
其具体方法如下:
将新鲜梅花鹿茸冷冻干燥后粉碎,加入去离子水使鹿茸浓度为33.3g/L,搅拌均匀后加入鹿茸质量0.5%(W/W)的胰蛋白酶,在40℃下酶解3小时;酶解结束将酶解液过80目筛,残渣同法提取一次。将两次酶解液升温至90℃保温15分钟,分别经8层纱布和200目筛过滤,所得滤液以10000g的速度离心10分钟,收集上清液;上清液经Nanodrop Onec在205nm下测定肽浓度,加水稀释至肽浓度20mg/mL待用;将溶胀后的Sephadex G-25medium填料填装成直径2cm、柱高30cm的凝胶柱。将酶解液加载于凝胶柱上,以去离子水为洗脱液、流速3.5mL/min进行洗脱,以床体积的1/20为一个流份,洗脱2个床体积,收集全部40个流份并经Nanodrop Onec在205nm下测定肽浓度。根据肽浓度合并第16至第25个流份,冷冻干燥,得到鹿茸提取物。
将鹿茸提取物用LTQ Orbitrap Velos进行质谱分析:将鹿茸提取物用0.1%(V/V)甲酸水溶液复溶成0.4mg/mL的溶液,进行LC-MS/MS分析。将毛细管的一端拉成内径约为5μm的尖端,通过气压将C18AQ填料压入柱内,填充柱长度约15cm。将毛细管的尖端与质谱相连。所用的流动相A为0.1%(V/V)甲酸水溶液,流动相B为0.1%甲酸的乙腈溶液,线性梯度洗脱过程为:0%B(0min)—2%B(2min)—25%B(87min)—35%B(97min)—90%B(99min)—90%B(109min)—2%B(110min)—2%B(120min)。流速60μL/min。
设置离子传输毛细管的温度200℃,电喷雾电压1.8KV,归一化碰撞能量35.0%。均使用数据依赖模式(data-dependent mode)对MS和MS/MS进行图谱采集。质谱扫描条件设定为:从每次m/z=400~2000的全扫描中选择10个最高丰度离子峰进行MS/MS扫描,其中动态排除(dynamic exclusion)设置为:重复次数(repeat count)为2,重复容忍时间(repeatduration)30s,动态排除时间(exclusion duration)90s。利用Xcalibur软件(Version2.2,Thermo)进行系统控制和数据收集。
将采集的*.raw文件数据用Thermo Proteome Discoverer Daemon(v1.4)转换成*.mgf格式,再用Mascot(version 2.3.0,Matrix Science,London,UK)于牛科数据库(bovine,蛋白数目17890,下载自http://www.uniprot.org/),进行检索,检索参数如下:不设置酶切位点、最大漏切数和固定修饰;蛋氨酸残基、脯氨酸残基加15.9949Da的可变修饰;母离子的质量容忍度(peptide tolerance)为20ppm,碎片离子的质量容忍度(fragmentions tolerance)为0.8Da。导出肽段结果时设置score>25,调整显著性差异P使肽段假阳性率(FDR,false discovery rate)控制在1%内。鉴定结果见附表一。结合构效关系进行筛选,获得序列为Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala的多肽。
实施例2多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala的DPP-Ⅳ抑制活性检测
原理
对于N端为X-Pro、X-Ala的肽段,DPP-Ⅳ可将该二肽选择性切除,GLP-1具有X-Ala结构,因此DPP-Ⅳ可导致超过95%的GLP-1发生降解。本方法采用Gly-Pro-p-nitroanilide代替GLP-1作为DPP-Ⅳ的模拟底物,DPP-Ⅳ切下Gly-Pro后,生成的对硝基苯胺在405nm有特征吸收峰,由此可计算DPP-Ⅳ的抑制活性。实验方法:
实验所用多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala由南京杰肽生物科技有限公司合成,纯度>95%。将多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala溶解于100mM的Tris-HCl缓冲液(pH=8.0),向96孔板加入25μL的Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala溶液和25μL 1.59mM的Gly-pro-p-nitroanilide溶液(用相同缓冲液配置,下同),混匀,于37℃温育10分钟,再加入50μL 0.01U/mL的DPP-Ⅳ溶液,混匀后于37℃温育60分钟,加入100μL 1M的醋酸-醋酸钠缓冲液(pH 4.0)使反应终止,用酶标仪测定405nm吸光度。Control组用等体积的Tris-HCl缓冲液代替Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala溶液,blank组用等体积的Tris-HCl缓冲液代替Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala溶液和DPP-Ⅳ溶液。
分别配制浓度0.5、0.25、0.1、0.05mM的多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala溶液,按上述方法进行DPP-Ⅳ抑制活性检测,计算Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala抑制率,抑制率计算公式:
其中I%表示抑制率,A多肽表示Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala吸光度值,AControl表示阴性对照组吸光度值,Ablank表示空白组吸光度值。结果如表一所示:
表一不同浓度的Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala对DPP-IV的抑制率
由表一可知,多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala对DPP-IV的半数抑制浓度(IC50)为108±7μM(Mean±SD,n=3)
多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala具有DPP-Ⅳ抑制活性及降血糖活性,作为高血糖、II型糖尿病的保健品和药物先导化合物具有良好的应用前景。
附表:
附表一鹿茸提取物经LC-MS/MS鉴定的肽段
以TGTPGLPGPPGPMGPPGDR为例:肽段的修饰种类为Oxidation(M);3Pro(O)(P),表明该肽段有1处甲硫氨酸氧化修饰和3处脯氨酸羟基修饰。肽段的修饰位置为0.0002002000001002000.0,表示该肽段的3处脯氨酸羟基修饰位于第4、7、16位残基,甲硫氨酸氧化修饰位于第13位残基。
附表一的序列所涉及的氨基酸均为氨基酸的简写,各氨基酸缩写、简写及名称见附表二。
附表二氨基酸名称、缩写与简写
序列表
<110> 中国科学院大连化学物理研究所
<120> 多肽及其应用和DPP-Ⅳ抑制剂或降血糖药物或保健品
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Phe Pro His Phe Asp Leu Ser His Gly Ser Ala
1 5 10
Claims (6)
1.一种多肽,其特征在于:所述多肽为Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala,具有序列表SEQ ID NO:1中氨基酸序列;该多肽的氨基酸序列具体为Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala。
2.一种权利要求1所述在制备DPP-Ⅳ抑制剂、或降血糖药物或保健品、或治疗II型糖尿病的药物或保健品中的应用。
3.按照权利要求2所述的应用,其特征在于:所述DPP-Ⅳ抑制剂和/或降血糖药物是以多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala为活性成份。
4.按照权利要求2所述的应用,其特征在于:所述DPP-Ⅳ抑制剂、或降血糖药物或保健品、或治疗II型糖尿病的药物或保健品中可添加药物学上可接受的载体或辅料。
5.一种DPP-Ⅳ抑制剂、或降血糖药物或保健品、或治疗II型糖尿病的药物或保健品,其特征在于:所述DPP-Ⅳ抑制剂或降血糖药物是以权利要求1所述多肽Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala为活性成份。
6.按照权利要求5所述的DPP-Ⅳ抑制剂、或降血糖药物或保健品、或治疗II型糖尿病的药物或保健品,其特征在于:所述DPP-Ⅳ抑制剂、或降血糖药物或保健品、或治疗II型糖尿病的药物或保健品中可添加药物学上可接受的载体或辅料。
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