CN115368469A - 血管紧张素转换酶抑制剂 - Google Patents
血管紧张素转换酶抑制剂 Download PDFInfo
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- CN115368469A CN115368469A CN202110553352.XA CN202110553352A CN115368469A CN 115368469 A CN115368469 A CN 115368469A CN 202110553352 A CN202110553352 A CN 202110553352A CN 115368469 A CN115368469 A CN 115368469A
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- Prior art keywords
- polypeptide
- ace1
- angiotensin
- inhibitor
- converting enzyme
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Abstract
血管紧张素转换酶抑制剂。本发明提供了一种对ACE抑制作用的多肽，所述多肽对ACE1的酶活性具有较好的抑制效果，可以作为治疗高血压的候选药物。
Description
技术领域
本发明涉及生物学领域，具体地说涉及一种作为血管紧张素-1转换酶抑制剂的多肽。
背景技术
目前，初略的估计全球大约有9亿成人患有高血压，预计到2025年该数字还会增加至大约15亿！因高血压死亡的人数占总死亡人数的13.5％，使得心血管疾病成为人类最危险的死亡因素。人体血压调节涉及不同的生物化学路径，例如肾素-血管紧张素系统(RAS)，激肽释放酶-激肽系统(KKS)，以及将血管紧张素I转化为血管紧张素II的凝乳酶。肾素-血管紧张素系统(RAS)是人体最重要的血压调控系统之一，因为RAS调节动脉压，其与心血管疾病的发生发展有着密切的关系。在该途径中，血管紧张素原被肾素转化为血管紧张素I，接着在血管紧张素转换酶(ACE)的作用下，将血管紧张素I裂解为血管紧张素II，后者导致动脉血管收缩，之后血压升高。血管紧张素II具有结合血管紧张素受体AT1和AT2的能力，这些受体是不均匀地分布在外周组织和脑中的两个主要的血管紧张素II受体亚型。在心血管系统，AT1受体被广泛地表达并促进大部分的血管紧张素II的生理和病理生理作用。AT2受体在发育的胎儿中被极大地表达，并且在正常成人心血管系统中其表达非常少。根据Lemarie and Schiffrin(2010)，血管紧张素II生理的作用是通过AT2受体诱导而不是被AT1受体介导，而由血管紧张素II的AT1受体的激活促进了血管床的构建，AT2受体的激活导致血管舒张。
血管紧张素-I转换酶是一种肽基二肽酶，可通过除去羧基末端的二肽来催化十肽血管紧张素I向八肽血管紧张素II的转化。ACE是调节血压的肾素血管紧张素系统的关键部分。血管紧张素转换酶抑制剂(ACEI)是通过竞争性抑制血管紧张素转换酶(ACE)而发挥作用的一类物质。ACEI可抑制ACE1的酶活性并降低血管紧张素II的产生，其会使血管扩张，从而增加了心脏泵出的血液量并降低血压。ACEI作用于肾素-血管紧张素系统后，能够有效的调节、控制人体血压，治疗充血性心力衰竭，可较好的预防首次心肌梗塞痊愈患者的复发病症，改善预后，显著降低患者的致残率和死亡率。
血管紧张素Ⅱ是由血管紧张素转换酶(ACE)转换而成，是体内最强的缩血管物质，且能促进醛固酮分泌，导致水、钠潴留及促进细胞肥大、增生，与高血压及心肌肥厚等疾病的形成具有密切关系。血管紧张素转换酶抑制剂(ACEI)的降压作用主要通过抑制血管紧张素转换酶，阻止血液及组织中血管紧张素Ⅱ的形成而实现。其药理作用体现在：1.减少血管紧张素Ⅱ的生成，抑制血管紧张素转化酶，使血管紧张素Ⅱ的生成减少，可减少醛固酮分泌，使水钠潴留减轻，静脉回心血量减少，有利于减轻心脏前负荷。同时还减少缓激肽的降解，使血管扩张，外周阻力降低，心脏前后负荷减轻，心输出量增加。左心室舒张末期压力和容积随之减小，心室壁张力降低，肾血管阻力下降，肾血流量增加，也有利于心功能的改善。2.预防或逆转心血管重构，抑制心肌和血管的肥厚、增生，延缓或逆转心室和血管重构，改善心脏和血管的舒缩功能，提高心肌和血管的顺应性。目前，临床上常用的ACEI主要是小粉化学药物，如卡托普利、依那普利、贝那普利、福辛普利、雷米普利等。
抑制ACE活性的合成药物虽然在治疗高血压方面具有较好的效果，但是也会引起不希望的副作用。常见的不良反应为咳嗽，以咽痒、干咳、心悸,心动过速,胸痛。较少见的还有:蛋白尿,眩晕,头痛,昏厥,血管性水肿,心率快等。与合成化学药物不同，抗高血压肽没有显示出任何副作用。
发明内容
本发明的第一个目的是提供一种多肽，其具有血管紧张素转换酶抑制剂的作用。
本发明的第二个目的是提供上述多肽在制备治疗高血压药物中的用途。
根据本发明的一方面，提供一种多肽，所述多肽由降压肽和连接所述降压肽的连接臂构成，所述降压肽的氨基酸序列选自：KYLCY(SEQ ID NO.6)和FKGKYYP(SEQ ID NO.7)，所述连接臂的氨基酸序列选自：FKGKYYP(SEQ ID NO.8)和AISGSGGGTYYA(SEQ ID NO.9)。
本发明所述的多肽，在所述多肽的构成中，优选地包含1个，2个，3个或4个降压肽，更优选的是，本发明所述的多肽具有如SEQ ID NO.2或SEQ ID NO.5所示的氨基酸序列。
根据本发明的另一方面，提供本发明所述的多肽作为ACE抑制剂的用途。将本发明的多肽用于制备治疗高血压的药物。
本发明提供了一种作为ACE抑制剂的多肽，所述多肽对ACE1的酶活性具有较好的抑制效果，可以作为治疗高血压的候选药物。
附图说明
图1为合成的五个多肽以及对照的OD值曲线
B1-5为测试多肽1-5号，B6，B7，B9，B10分别为抑制剂对照[IC]，酶对照[EC]，不含酶的背景对照[BC]和溶剂对照[SC]的结果。
图2为不同浓度多肽以及对照的OD值曲线
E1-3为4.2μM的三次重复结果；E4-6为2.1μM的三次重复结果；E7-9为1.05μM的三次重复结果；E10-12为0.526μM的三次重复结果；F1-3为0.263μM的三次重复结果；F4为抑制剂对照[IC]的结果；F5为酶对照[EC]的结果；F6为不含酶的背景对照[BC]结果。
图3为3％RA的IC50曲线。
具体实施方式
如无特别说明，所有材料均为市售购买。
【实施例1】多肽的设计
根据ACE的晶体结构和构象，在分析研究血管紧张素转化酶ACE-靶标结合位点高级结构的基础上，模拟降压药卡托普利与ACE结合的高级构象设计降压肽，同时分析降压肽的高级结构特征，从降压肽数据库中，依据已知的具有较高亲和力的降压肽作为参考系列。在设计过程中，产生大量新多肽数据。运用蛋白指纹技术筛选出具有相同理化性质指纹，同时具有类似空间构象指纹的多肽。理论上，新降压肽应该和ACE具有较好的亲和力。
一、新降压肽设计步骤
1.血管紧张素转化酶ACE靶标结构；
2.分析可能的多个ACE降压多肽抑制剂结构特征；
3.分析降压肽数据库中大约3000个降压肽实验数据，筛选降压肽作为参考系；
a)筛选出10％左右高亲和力的实验多肽，(IC50(归一化)小于10)；
b)再进一步筛选有重复多个实验支持的多肽序列作为参考，包括5AA，6AA，7AA，8AA和9AA的多肽；
4.提取对ACE抑制多肽的蛋白指纹，用蛋白指纹数据软件对这些多肽进行重新设计，产生大约上万条新多肽作为中间数据；
5.运用蛋白指纹技术筛选出和参考系具有相同理化性质指纹，同时具有类似空间构象指纹的多肽；
6.数据库检索确认设计的多肽不包含在原始数据中。
二、连接臂的设计
1.考虑连接臂的理化特征：以疏水为主，亲水；
2.考虑链接臂的构象特征：标记构象特征；
进行构象分析和亲和力测定后，获得5条候选ACE1抑制多肽，如表1，其中黑体表示降压肽，非黑体为连接臂，多肽的氨基酸序列如SEQ ID NO.1～SEQ ID NO.5所示。
表1候选ACE1抑制多肽序序列以及分子质量
【实施例2】多肽的合成
按照5个候选ACE1抑制多肽的氨基酸序列委托进行多肽合成，获得候选多肽。
1、合成顺序：从序列C端到N端，步骤如下：
a.称取n当量树脂放入反应器，加入DCM(二氯甲院)溶胀半小时，然后抽掉DCM，加入序列中第一个氨基酸2n当量，加2n当量的DIEA，适量的DMF，DCM(适量是指以可使树脂充分鼓动起来为宜)，DIEA(二异丙基乙胺)、DMF(二甲基甲酰胺)、DCM，氮气鼓泡反应60min。然后加入约5n当量甲醇，反应半小时，抽掉反应液，用DMF、MEOH洗净；
b.往反应器中加入序列中第二个氨基酸(也为2n当量)，2n当量H(1-羟基,苯并,三氯四甲基六氯磷酸盐)及DIEA，氮气鼓泡反应半小时，洗掉液体，茚三酮检测，然后用吡啶和乙酸酐封端。最后洗净，加入适量的脱帽液士除Fmoc(9-芴甲氧碳基)保护基，洗净，茚三酮检测；
c.依步骤b的方式依次加入序列中不同的氨基酸并进行各种修饰；
d.将树脂用氮气吹干后从反应柱中取下，倒入烧瓶中，然后往烧瓶中加一定量(切割液和树脂大约以10ml/克的比例)的切割液(组成是95％TFA，2％乙二硫醇，2％三异丙基硅院，1％水)震荡，滤掉树脂；
e.得到滤液，然后向滤液中加入大量乙醚，析出粗产物，然后离心，清洗即可得到序列的粗产物；
2、多肽纯化：
开发工艺用高效液相色谱将粗品提纯至要求纯度。
3、多肽冻干：
纯化好的液体放入冻干机中进行浓缩，冻干成白色粉未。
【实施例3】ACE1抑制多肽筛选
实验原理：ACE1酶的底物在345nm处有着一个明显的吸收峰，在ACE1酶存在的情况下会使其快速降解，吸收峰明显降低；我们以卡托普利(Captopril)(一种ACE1酶的特异性抑制剂)作为阳性对照，当卡托普利存在时，ACE1的酶促活性大大降低，因此底物不会被讲解，在345nm处的OD值没有明显降低。我们通过比较60分钟内的线性345nm处的OD值的下降速率来观察要筛选的设计的候选ACEI抑制多肽对ACE的抑制效果。
1.实验材料及方法：
1.1.实验材料：ACE1抑制剂筛选试剂盒(Bio vision#K719-100)，具体包括ACE1分析缓冲液、ACE1底物、ACE1酶、ACE1抑制剂对照和96孔UV透明板；酶标仪、5种候选ACE抑制多肽。
1.2.实验方法：
1，测试ACE1抑制多肽与ACE1抑制剂对照物的制备：将测试ACE1抑制多肽[S]以100倍的比例溶于适当的溶剂中。对于每种测试ACE1抑制多肽，用ACE1分析缓冲液稀释至所需测试浓度的10倍。要确定测试ACE1抑制多肽的IC50值，请在ACE1分析缓冲液中准备几份测试ACE1抑制多肽的稀释液。将每种稀释液25μl加入指定的孔中。对于抑制剂控制，可通过将5μl 10mM卡托普利添加到495μl分析缓冲液中，将ACE1抑制剂阳性对照(10mM卡托普利)稀释至100μM卡托普利。通过添加5μl 100μM卡托普利和495μl分析缓冲液，制备1μM卡托普利工作溶液。在抑制剂对照孔中加入25μl 1μM卡托普利。
2，ACE1酶溶液的制备：通过将2μl ACE1酶储备液添加到38μl ACE1分析缓冲液中来制备稀释的ACE1酶溶液。彻底混合并放在冰上。
3.候选抑制剂配置：准备含有稀释的测试抑制剂[S]，抑制剂对照[IC]，酶对照[EC]，不含酶的背景对照[BC]和溶剂对照[SC]的孔。具体配置方法见下表2。
表2不同孔之间加入的试剂以及剂量
| [S] | [IC] | [EC] | [BC] | [SC] | |
| 测试抑制剂 | 25μl | —— | —— | —— | —— |
| 对照抑制剂 | —— | 25μl | —— | —— | —— |
| 分析缓冲液 | —— | —— | 25μl | 25μl | —— |
| 溶剂对照 | —— | —— | —— | —— | 25μl |
然后[S]、[IC]、[EC]、[SC]中每孔加入40ulACE1酶溶液，再用ACE1分析缓冲液将每孔的总体积(包括[S]，[IC]，[EC]，[SC]和[BC])调整至200μl/孔。充分混合并在避光下于37℃孵育15-20分钟。
4.反应混合物的制备：按照实验数量的孔数*50μl来制备反应混合物。50μl反应混合物其中包含：ACE1分析缓冲液40μl+ACE1底物混合物10μl。将50μl反应混合物添加到[S]，[IC]，[EC]，[SC]和[BC]孔中，混合均匀。
5.测量：在37℃下以动力学模式立即在OD 345nm下测量吸光度60分钟。在曲线的线性范围内选择任意两个时间点(t1和t2)，并获得相应的吸光度值(OD1和OD2)。6.计算：通过将ΔOD＝(OD1-OD2)值除以Δt(t2-t1)，计算[S]，[EC]，[SC]和[BC]的斜率。从[S]，[EC]和[SC]中减去[BC]的斜率。如果[SC]斜率与[EC]相比有显着差异，请使用[SC]值确定测试ACE1抑制多肽对的效果。得到％RA(相对活性百分比)＝[S]的斜率/[EC]的斜率*100，并计算相对活性的IC50。
2.实验结果：
2.1.候选ACE1抑制多肽初步筛选：
为了测试5个候选ACE1抑制多肽是否对ACE1有抑制效果，首先取每种候选ACE1抑制多肽取1管(2mg)用2ml水溶解，浓度为1mg/ml，再按照上面的实验方法进行操作，345nm处读取60min，3min读取一次OD值，一共21个数值，如图1，表3。
| 时间 | B1 | B2 | B3 | B4 | B5 | B6(IC) | B7(EC) | B9(SC) | B10(BC) |
| 1号多肽 | 2号多肽 | 3号多肽 | 4号多肽 | 5号多肽 | 阳性对照 | 酶对照 | 抑制剂对照 | 空白对照 | |
| 0:00:00 | 0.6752 | 0.6837 | 0.6917 | 0.649 | 0.6952 | 0.6545 | 0.6772 | 0.6577 | 0.6942 |
| 0:03:00 | 0.6676 | 0.6787 | 0.6744 | 0.6492 | 0.6844 | 0.6454 | 0.6636 | 0.6491 | 0.6882 |
| 0:06:00 | 0.6646 | 0.6771 | 0.6678 | 0.6469 | 0.6812 | 0.6433 | 0.6573 | 0.6435 | 0.6883 |
| 0:09:00 | 0.6607 | 0.6767 | 0.6638 | 0.6544 | 0.6782 | 0.6426 | 0.6519 | 0.6386 | 0.6884 |
| 0:12:00 | 0.6562 | 0.6775 | 0.6591 | 0.6413 | 0.6748 | 0.6418 | 0.6466 | 0.6328 | 0.6878 |
| 0:15:00 | 0.651 | 0.6788 | 0.6563 | 0.6454 | 0.6715 | 0.6421 | 0.6431 | 0.6289 | 0.6892 |
| 0:18:00 | 0.6456 | 0.6785 | 0.6493 | 0.6351 | 0.6678 | 0.6417 | 0.639 | 0.6241 | 0.6885 |
| 0:21:00 | 0.6403 | 0.6791 | 0.6461 | 0.6324 | 0.6649 | 0.6421 | 0.6347 | 0.6191 | 0.6891 |
| 0:24:00 | 0.6342 | 0.6779 | 0.6386 | 0.6301 | 0.662 | 0.6418 | 0.6305 | 0.6139 | 0.6887 |
| 0:27:00 | 0.6288 | 0.6761 | 0.6345 | 0.6296 | 0.6576 | 0.6422 | 0.6266 | 0.6103 | 0.6889 |
| 0:30:00 | 0.623 | 0.6802 | 0.6308 | 0.6227 | 0.6541 | 0.6421 | 0.622 | 0.605 | 0.689 |
| 0:33:00 | 0.6175 | 0.6769 | 0.6235 | 0.6413 | 0.6504 | 0.6418 | 0.6186 | 0.6044 | 0.6894 |
| 0:36:00 | 0.6118 | 0.6792 | 0.6189 | 0.6299 | 0.6594 | 0.6416 | 0.6151 | 0.6063 | 0.6897 |
| 0:39:00 | 0.607 | 0.6791 | 0.615 | 0.6138 | 0.645 | 0.6422 | 0.6113 | 0.6056 | 0.6894 |
| 0:42:00 | 0.6026 | 0.6762 | 0.6102 | 0.6107 | 0.641 | 0.6418 | 0.6098 | 0.6029 | 0.6893 |
| 0:45:00 | 0.598 | 0.6778 | 0.6069 | 0.6078 | 0.6376 | 0.6414 | 0.6066 | 0.5977 | 0.6887 |
| 0:48:00 | 0.5921 | 0.6786 | 0.6012 | 0.6055 | 0.6354 | 0.6418 | 0.604 | 0.5955 | 0.6892 |
| 0:51:00 | 0.5879 | 0.6785 | 0.5968 | 0.6035 | 0.6327 | 0.6414 | 0.6008 | 0.5813 | 0.6896 |
| 0:54:00 | 0.583 | 0.679 | 0.5929 | 0.6012 | 0.6302 | 0.6418 | 0.5947 | 0.5793 | 0.6892 |
| 0:57:00 | 0.5782 | 0.6803 | 0.5886 | 0.5987 | 0.6271 | 0.6409 | 0.5918 | 0.5749 | 0.689 |
| 1:00:00 | 0.5735 | 0.6774 | 0.5842 | 0.5961 | 0.6248 | 0.6409 | 0.5891 | 0.5746 | 0.6894 |
| ΔOD | 0.1017 | 0.0063 | 0.1075 | 0.0479 | 0.0704 | 0.0136 | 0.0881 | 0.0831 | 0.0048 |
从图1中和表3的数据能看出：测试样品中只有2号多肽的OD值曲线几乎无变化，进一步查看具体数值并计算60分钟的OD值变化可以看出2号多肽的抑制效果是最佳的，与不加酶的BC组几乎相等，甚至效果好于IC组，可能是由于IC组的抑制剂卡普里托的浓度(1μM)小于2号多肽浓度(2.6μM)，结果显示2号多肽对ACE1的酶活性具有较好的抑制效果。
【实施例4】ACE1抑制多肽筛选
为了进一步测定初步筛选的对ACE1具有较好抑制活性的2号多肽的IC50进行测定，以确定测试抑制剂的IC50值。取2号候选ACE1抑制多肽[S]，以100倍的比例溶于适当的溶剂中溶解，在ACE1分析缓冲液中分别加入待测试抑制剂的稀释液。将每种稀释液25μl加入指定的孔中。然后倍比稀释至1.6mg/ml，0.8mg/ml、0.4mg/ml、0.2mg/ml和0.1mg/ml 5个浓度(见表4)，每个处理3个重复，按照表2方式加入不同浓度的多肽，在345nm处读取60min，2min读取一次。结果如图2，表5。
表4测定IC50的溶液配制
| 对应孔编号 | Stock浓度(uM) | 最终浓度(uM) |
| E1-3 | 1.6mg/ml | 4.2μM |
| E4-6 | 0.8mg/ml | 2.1μM |
| E7-9 | 0.4mg/ml | 1.05μM |
| E10-12 | 0.2mg/ml | 0.526μM |
| F1-3 | 0.1mg/ml | 0.263μM |
| F4 | IC | |
| F5 | EC | |
| F6 | BC |
表5，2号候选ACE1抑制多肽对ACE1的动态抑制的测定值
将计算得到的相对抑制率输入软件进行计算，结果如图3所示，测定结果2号候选多肽对ACE1抑制的IC50为0.1262μM。
SEQUENCE LISTING
<110> 易森荟（武汉）生物医药有限公司
<120> 血管紧张素转换酶抑制剂
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Claims (4)
1.一种多肽，其具有血管紧张素-1转换酶抑制剂的作用，其特征在于所述多肽由降压肽和连接所述降压肽的连接臂构成，所述降压肽的氨基酸序列选自：KYLCY和FKGKYYP，所述连接臂的氨基酸序列选自：FKGKYYP和AISGSGGGTYYA。
2.根据权利要求1所述的多肽，其特征在于在所述多肽所述的多肽具有如SEQ ID NO.2或SEQ ID NO.5所示的氨基酸序列。
3.权利要求1所述的多肽作为ACE抑制剂的用途。
4.权利要求1所述的多肽在制备治疗高血压的药物中的用途。
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| CN101210047A (zh) * | 2006-12-29 | 2008-07-02 | 中国科学院大连化学物理研究所 | 一种活性单肽及其应用 |
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