WO2022242672A1 - 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 PDF

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WO2022242672A1
WO2022242672A1 PCT/CN2022/093527 CN2022093527W WO2022242672A1 WO 2022242672 A1 WO2022242672 A1 WO 2022242672A1 CN 2022093527 W CN2022093527 W CN 2022093527W WO 2022242672 A1 WO2022242672 A1 WO 2022242672A1
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polypeptide
ace1
angiotensin
inhibitor
peptide
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杨代常
杜子谦
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易森荟(武汉)生物医药有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

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  • the invention relates to the field of biology, in particular to a polypeptide as an angiotensin-1 conversion enzyme inhibitor.
  • renin-angiotensin system RAS
  • kallikrein-kinin system KS
  • chymosin a biochemical pathway that converts angiotensin I to angiotensin II .
  • RAS renin-angiotensin system
  • RAS is one of the most important blood pressure regulation systems in the human body, because RAS regulates arterial pressure, and it is closely related to the occurrence and development of cardiovascular diseases.
  • angiotensinogen is converted by renin to angiotensin I, followed by cleavage of angiotensin I to angiotensin II by angiotensin-converting enzyme (ACE), which leads to contraction, followed by an increase in blood pressure.
  • Angiotensin II has the ability to bind to angiotensin receptors AT1 and AT2, the two major angiotensin II receptor subtypes that are heterogeneously distributed in peripheral tissues and in the brain.
  • AT1 receptors are ubiquitously expressed and contribute to most of the physiological and pathophysiological effects of angiotensin II.
  • the AT2 receptor is greatly expressed in the developing fetus and very little is expressed in the normal adult cardiovascular system.
  • angiotensin II the physiological effects of angiotensin II are induced by AT2 receptors rather than mediated by AT1 receptors, and activation of AT1 receptors by angiotensin II promotes the construction of vascular beds, AT2 Activation of the receptor results in vasodilation.
  • Angiotensin-I converting enzyme is a peptidyl dipeptidase that catalyzes the conversion of the decapeptide angiotensin I to the octapeptide angiotensin II by removing the carboxy-terminal dipeptide.
  • ACE is a key part of the renin-angiotensin system that regulates blood pressure.
  • Angiotensin-converting enzyme inhibitors are a class of substances that act by competitively inhibiting angiotensin-converting enzyme (ACE). ACE inhibitors inhibit the enzyme activity of ACE1 and reduce the production of angiotensin II, which dilates blood vessels, increasing the amount of blood pumped by the heart and lowering blood pressure.
  • ACEI renin-angiotensin
  • Angiotensin II is converted from angiotensin-converting enzyme (ACE). It is the strongest vasoconstrictor substance in the body and can promote the secretion of aldosterone, leading to water and sodium retention and promoting cell hypertrophy and hyperplasia. It is closely related to the formation of diseases such as hypertrophy.
  • ACEI angiotensin-converting enzyme inhibitors
  • the antihypertensive effect of angiotensin-converting enzyme inhibitors (ACEI) is mainly achieved by inhibiting angiotensin-converting enzymes and preventing the formation of angiotensin II in blood and tissues. Its pharmacological effects are reflected in: 1.
  • Reduce the production of angiotensin II inhibit the production of angiotensin II, reduce the production of angiotensin II, reduce the secretion of aldosterone, reduce the retention of water and sodium, and reduce the amount of venous return to the heart.
  • it also reduces the degradation of bradykinin dilates blood vessels, reduces peripheral resistance, reduces cardiac preload and postload, and increases cardiac output.
  • the left ventricular end-diastolic pressure and volume decrease accordingly, the ventricular wall tension decreases, the renal vascular resistance decreases, and the renal blood flow increases, which is also conducive to the improvement of heart function. 2.
  • Prevent or reverse cardiovascular remodeling inhibit myocardial and vascular hypertrophy and hyperplasia, delay or reverse ventricular and vascular remodeling, improve cardiac and vascular systolic function, and improve myocardial and vascular compliance.
  • the ACEIs commonly used in clinical practice are mainly small powder chemical drugs, such as captopril, enalapril, benazepril, fosinopril, ramipril and so on.
  • Synthetic drugs that inhibit ACE activity although effective in treating hypertension, can also cause undesirable side effects. Common adverse reactions are cough, itchy throat, dry cough, palpitations, tachycardia, and chest pain. Less common are: proteinuria, dizziness, headache, fainting, angioedema, rapid heart rate, etc. Unlike synthetic chemical drugs, antihypertensive peptides did not show any side effects.
  • the first object of the present invention is to provide a polypeptide which acts as an angiotensin-converting enzyme inhibitor.
  • the second object of the present invention is to provide the use of the above-mentioned polypeptide in the preparation of drugs for treating hypertension.
  • a polypeptide is provided, the polypeptide is composed of an antihypertensive peptide and a connecting arm connecting the antihypertensive peptide, the amino acid sequence of the antihypertensive peptide is selected from: KYLCY (SEQ ID NO.6) and FKGKYYP (SEQ ID NO.7), the amino acid sequence of the linking arm is selected from: FKGKYYP (SEQ ID NO.8) and AISGSGGGTYYA (SEQ ID NO.9).
  • the polypeptide described in the present invention preferably comprises 1, 2, 3 or 4 antihypertensive peptides in the composition of the polypeptide, more preferably, the polypeptide described in the present invention has the following SEQ ID NO. 2 or the amino acid sequence shown in SEQ ID NO.5.
  • polypeptide described in the present invention as an ACE inhibitor is provided.
  • the polypeptide of the present invention is used to prepare medicine for treating hypertension.
  • the invention provides a polypeptide as an ACE inhibitor, the polypeptide has a good inhibitory effect on the enzyme activity of ACE1, and can be used as a candidate drug for treating hypertension.
  • Figure 1 is the OD value curve of the five synthetic peptides and the control
  • B1-5 are test peptides 1-5
  • B6, B7, B9, B10 are the results of inhibitor control [IC], enzyme control [EC], background control without enzyme [BC] and solvent control [SC] .
  • Figure 2 is the OD value curve of different concentrations of polypeptides and controls
  • Figure 3 is the IC50 curve of 3% RA.
  • Fig. 4 is the antihypertensive drug effect compared with the model control group before administration
  • A mean blood pressure
  • B diastolic blood pressure
  • C systolic blood pressure
  • Figure 5 shows the blood pressure reduction multiples at each time point after administration and the model control group.
  • the antihypertensive peptide was designed by simulating the high-level conformation of the antihypertensive drug captopril combined with ACE, and the antihypertensive peptide was analyzed simultaneously.
  • the high-level structural features of the antihypertensive peptides, from the antihypertensive peptide database, are based on the known antihypertensive peptides with higher affinity as the reference series.
  • large amounts of new peptide data are generated.
  • Use protein fingerprinting technology to screen out peptides with the same physical and chemical property fingerprints and similar spatial conformational fingerprints. Theoretically, the new antihypertensive peptide should have a better affinity with ACE.
  • Angiotensin-converting enzyme ACE target structure 1. Angiotensin-converting enzyme ACE target structure
  • the peptide synthesis was entrusted to obtain the candidate polypeptides.
  • DCM (appropriate amount means that the resin can be fully agitated), DIEA (diisopropylethylamine), DMF (dimethylformamide), DCM, and nitrogen gas bubbling for 60 minutes. Then add about 5n equivalent methanol, react for half an hour, remove the reaction solution, and wash with DMF and MEOH;
  • the developed process uses high performance liquid chromatography to purify the crude product to the required purity.
  • the purified liquid was concentrated in a lyophilizer, and lyophilized into a white powder.
  • the substrate of ACE1 enzyme has an obvious absorption peak at 345nm, and it will be degraded rapidly in the presence of ACE1 enzyme, and the absorption peak is significantly reduced; we use Captopril (a kind of ACE1 enzyme) specific inhibitor) as a positive control, when captopril exists, the enzymatic activity of ACE1 is greatly reduced, so the substrate will not be released, and the OD value at 345nm is not significantly reduced.
  • Captopril a kind of ACE1 enzyme specific inhibitor
  • ACE1 inhibitor screening kit Bio vision#K719-100, specifically including ACE1 analysis buffer, ACE1 substrate, ACE1 enzyme, ACE1 inhibitor control and 96-well UV transparent plate; microplate reader, 5 A candidate ACE-inhibiting polypeptide.
  • test ACE1 inhibitory polypeptide and ACE1 inhibitor control the test ACE1 inhibitory polypeptide [S] was dissolved in an appropriate solvent at a ratio of 100 times. For each test ACE1 inhibitory polypeptide, dilute to 10 times the desired test concentration with ACE1 assay buffer. To determine the IC50 value of a test ACE1-inhibiting peptide, prepare several dilutions of the test ACE1-inhibiting peptide in ACE1 Assay Buffer. Add 25 ⁇ l of each dilution to the designated wells.
  • the ACE1 inhibitor positive control (10 mM captopril) can be diluted to 100 ⁇ M captopril by adding 5 ⁇ l 10 mM captopril to 495 ⁇ l assay buffer.
  • Prepare a 1 ⁇ M captopril working solution by adding 5 ⁇ l of 100 ⁇ M captopril and 495 ⁇ l of assay buffer.
  • ACE1 enzyme solution Prepare diluted ACE1 enzyme solution by adding 2 ⁇ l of ACE1 enzyme stock solution to 38 ⁇ l of ACE1 assay buffer. Mix thoroughly and place on ice.
  • Candidate inhibitor configuration Prepare wells containing diluted test inhibitor [S], inhibitor control [IC], enzyme control [EC], background control without enzyme [BC] and solvent control [SC]. The specific configuration method is shown in Table 2 below.
  • reaction mixture was prepared according to the experimental number of wells * 50 ⁇ l.
  • the 50 ⁇ l reaction mixture contained: 40 ⁇ l of ACE1 assay buffer + 10 ⁇ l of ACE1 substrate mix. Add 50 ⁇ l of the reaction mixture to the [S], [IC], [EC], [SC], and [BC] wells and mix well.
  • the calculated relative inhibition rate was input into the software for calculation, and the results are shown in Figure 3.
  • the IC50 of the No. 2 candidate polypeptide for ACE1 inhibition was 0.1262 ⁇ M.
  • SHR spontaneously hypertensive rat
  • the systolic blood pressure of the drug group was 5.73 times, 14.48 times, 6.6 times and 4.45 times lower than that of the model control group at 2, 4, 6 and 8 hours after administration; 2, 4, 6, and 8h decreased by 0.51 times, 4.74 times, 1.11 times and 0.98 times respectively; the average blood pressure decreased by 1.28 times, 8.48 times, 2.12 times and 1.04 times respectively at 2, 4, 6 and 8 hours after administration ( Figure 5).
  • Figure 5 Especially in reducing diastolic blood pressure is the most obvious. Wherein 2-4 hours after administration has a better effect than 6-8 hours after administration.

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Abstract

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

血管紧张素转换酶抑制剂 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。
图4为与模型对照组给药前比较的降血压药效；
A:平均血压；B:舒张压；C:收缩压。
图5为给药后与模型对照组的各时间点的血压降低倍数。
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
Figure PCTCN2022093527-appb-000001
【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：测试样品中有2号多肽和5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。同时5号多肽也显示出对ACE1的酶活性具有一定的抑制效果。
【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
Figure PCTCN2022093527-appb-000002
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。
【实施例5】大鼠降压实验
试验方法
采用自发性高血压大鼠(SHR)模型，测试2号多肽的降压效果。选取周龄为11周的雄性SHR大鼠，分为实验组和模型对照组，每组5只；在适应1周后进行给药。试验分为药物组和模型对照组。药物组(2号多肽)给予100μg/kg进行皮下注射；模型对照组给予相同溶媒。在给药前(0h)、给药后2h、4h、6h和8h采用为尾袖法测量大鼠的血压，并记录数据。
试验结果
结果如图4所示，自发性高血压大鼠(SHR)在给予受试药物后，与给药前(0h)比较，在给药后2、4和6小时，收缩压较给药前(0h)持续降低，分别降低了3.14％、14.1和17.98％，在8h后，降低了10.48％。在给药后2、4、6和8h，舒张压较给药前(0h)持续降低，分别降低了21.73％、38.3％、43.67％、48.53％(图4)。与给药后各时间点比较，药物组比模型对照组的收缩压在给药后2、4、6、8h分别降低了5.73倍、14.48倍、6.6倍和4.45倍；舒张压在给药后2、4、6、8h分别降低了0.51倍、4.74倍、1.11倍和0.98倍；平均血压在给药后2、4、6、8h分别降低了1.28倍、8.48倍、2.12倍和1.04倍(图5)。尤以降低舒张压最为明显。其中在给药后2-4小时比给药后6-8小时具有更好的效果。

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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* Cited by examiner, † Cited by third party
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US20060234942A1 (en) * 2002-06-27 2006-10-19 Jerome Tauzin Use of at least one $g(a)62 casein peptide with angiotensin i converting enzyme inhibiting activity for preparing medicines, food products and food complements
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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CN108892710A (zh) * 2018-07-24 2018-11-27 中国科学院海洋研究所 龙须菜降压肽提取物和龙须菜降压肽及其应用
CN111105845A (zh) * 2020-01-13 2020-05-05 深圳职业技术学院 一种基于蛋白折叠指纹条形码设计降血压肽的制备

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US20060234942A1 (en) * 2002-06-27 2006-10-19 Jerome Tauzin Use of at least one $g(a)62 casein peptide with angiotensin i converting enzyme inhibiting activity for preparing medicines, food products and food complements
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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