多肽类ACE抑制剂的设计合成及生物活性

周佳琪; 马春燕; 李晓晖

doi:10.13386/j.issn1002-0306.2022030117

多肽类ACE抑制剂的设计合成及生物活性

doi: 10.13386/j.issn1002-0306.2022030117

1.
大连理工大学生物工程学院，辽宁大连 116024
2.
大连理工大学化工学院，辽宁大连 116024

详细信息

作者简介:
周佳琪（1997−），女，硕士研究生，研究方向：功能性多肽药物的设计与合成，E-mail：jqzhoudut@163.com

通讯作者:
李晓晖（1964−），女，博士，教授，研究方向：多肽药物研究，E-mail：lxhxh@dlut.edu.cn

中图分类号: TS201.1
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出版历程
- 收稿日期: 2022-03-10
- 网络出版日期: 2022-10-20
- 刊出日期: 2022-11-23

Design, Synthesis and Bioactivity of Polypeptide ACE Inhibitors

ZHOU Jiaqi^1
,,
MA Chunyan²,
LI Xiaohui^{1
, ,}

1.
College of Bioengineering, Dalian University of Technology, Dalian 116024, China
2.
College of Chemical Engineering, Dalian University of Technology, Dalian 116024, China

摘要

摘要: 本研究以血管紧张素I转化酶（angiotensin converting enzyme, ACE）抑制肽PHP1和PHP2为研究母肽，通过替换氨基酸残基改变目标多肽的疏水性、带电性等因素，利用生物信息学工具评估多肽潜在的生物活性，设计了19个多肽类似物。采用多肽固相合成法合成目的多肽类似物，并进行体外生物活性检测。结果显示多肽类似物均具有较高的ACE抑制活性，其中，PHP1A-6（IC₅₀=3.87 μmol/L）、PHP2A-3（IC₅₀=3.33 μmol/L）、PHP2A-4（IC₅₀=2.86 μmol/L）和PHP2A-7（IC₅₀=4.58 μmol/L）的ACE抑制活性最高，较母肽有显著提高（P<0.05），PHP1A-3、PHP1A-4、PHP1A-7、PHP2A-1和PHP2A-10具有同母肽相当的抑制活性，IC₅₀<10 μmol/L。绝大部分多肽类似物的α-葡萄糖苷酶抑制活性与母肽相比均有明显提高，PHP1A-3（IC₅₀=3.09 μmol/L）、PHP1A-7（IC₅₀=9.51 μmol/L）、PHP2A-6（IC₅₀=5.58 μmol/L）、PHP2A-11（IC₅₀=2.35 μmol/L）和PHP2A-12（IC₅₀=3.98 μmol/L）的活性最高。其中，PHP1A-3和PHP1A-7具备较强的ACE抑制和α-葡萄糖苷酶抑制双重活性。含Cys的多肽类似物在1 mg/mL浓度下，ABTS⁺ · 清除率均在85%以上，具备潜在的抗氧化活性。分子对接研究了ACE抑制肽与ACE的构效关系，表明抑制肽可以与ACE的氨基酸残基产生多个稳定的氢键、疏水相互作用、π-π堆积及盐桥，增加了对ACE的抑制作用。
- 多肽固相合成 /
- 新型ACE抑制肽类似物 /
- 生物活性 /
- 分子对接
Abstract: In this study, using angiotensin converting enzyme (ACE) inhibitory peptides PHP1 and PHP2 as the parent peptides, the hydrophobicity and electrical properties of target polypeptides were altered by substituting amino acid residues. In addition, the potential bioactivity of these polypeptides was assessed using bioinformatics tools. Following this analysis, 19 polypeptide analogues were designed and synthesized using solid-phase synthesis, and their bioactivities were detected in vitro. The results revealed that the polypeptide analogues showed relatively high ACE inhibitory activities, PHP1A-6 (IC₅₀=3.87 μmol/L), PHP2A-3 (IC₅₀=3.33 μmol/L), PHP2A-4 (IC₅₀=2.86 μmol/L), and PHP2A-7 (IC₅₀=4.58 μmol/L) exhibited the highest ACE inhibitory activity levels, significantly higher than the parent peptides (P<0.05). PHP1A-3, PHP1A-4, PHP1A-7, PHP2A-1 and PHP2A-10 displayed equal levels of inhibitory activity in comparison to the parent peptides (IC₅₀<10 μmol/L). Compared with the parent peptides, the α-glucosidase inhibitory activity levels demonstrated by most of the polypeptide analogues were significantly enhanced, PHP1A-3 (IC₅₀=3.09 μmol/L), PHP1A-7 (IC₅₀=9.51 μmol/L), PHP2A-6 (IC₅₀=5.58 μmol/L), PHP2A-11 (IC₅₀=2.35 μmol/L), and PHP2A-12 (IC₅₀=3.98 μmol/L) exhibited the highest activities. Additionally, PHP1A-3 and PHP1A-7 displayed relatively strong inhibitory activity against both ACE and α-glucosidase. At concentrations of 1 mg/mL, the polypeptides containing Cys displayed an ABTS⁺· scavenging rate of higher than 85%, demonstrating a potential antioxidant activity. The structure-activity relationship between the ACE inhibitory peptides and ACE were explored using molecular docking. The results reflected that inhibitory peptides produced multiple stable hydrogen bonds, hydrophobic interactions, π-π stacking interactions, and salt bridges with ACE amino acid residues, thereby improving the inhibitory effects exerted on ACE.
- solid-phase peptide synthesis /
- novel ACE inhibitory peptide analogue /
- biological activity /
- molecular docking

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图 1 ACE与多肽类似物的复合物模型

Figure 1. The models of ACE in complex with the peptide analogues

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表 1 PHP1、PHP2及其类似物的序列

Table 1. Sequence of PHP1, PHP2 and their analogues

名称	序列	名称	序列
PHP1	Val-Val-Tyr-Pro-Trp-Thr	PHP2	Thr-Lys-Thr-Tyr-Phe-Pro-His-Phe
PHP1A-1	Cys-Val-Tyr-Pro-Trp-Thr	PHP2A-1	Thr-Lys-Leu-Tyr-Phe-Pro-His-Phe
PHP1A-2	Val-Cys-Tyr-Pro-Trp-Thr	PHP2A-2	Ile-Lys-Leu-Tyr-Phe-Pro-His-Phe
PHP1A-3	Val-Val-Tyr-Pro-Trp-Phe	PHP2A-3	Ala-Lys-Leu-Tyr-Phe-Pro-His-Phe
PHP1A-4	Cys-Val-Tyr-Pro-Trp-Phe	PHP2A-4	Ile-Ala-Leu-Tyr-Phe-Pro-His-Phe
PHP1A-5	Val-Cys-Tyr-Pro-Trp-Phe	PHP2A-5	Ala-Ala-Leu-Tyr-Phe-Pro-His-Phe
PHP1A-6	Gly-Val-Tyr-Pro-Trp-Phe	PHP2A-6	Ala-Ala-Leu-Tyr-Phe-Pro-Leu-Phe
PHP1A-7	Val-Ala-Tyr-Pro-Trp-Phe	PHP2A-7	Ala-Lys-Ala-Tyr-Phe-Pro-Arg-Phe
−	−	PHP2A-8	Gly-Lys-Leu-Tyr-Phe-Pro-His-Phe
−	−	PHP2A-9	Ile-Gly-Leu-Tyr-Phe-Pro-His-Phe
−	−	PHP2A-10	Gly-Gly-Leu-Tyr-Phe-Pro-His-Phe
−	−	PHP2A-11	Gly-Gly-Leu-Tyr-Phe-Pro-Leu-Phe
−	−	PHP2A-12	Gly-Lys-Gly-Tyr-Phe-Pro-Arg-Phe
注：PHP1/2A（Analogue of Pig Hemoglobin Peptide 1/2，猪血红蛋白源多肽1/2的类似物）。

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表 2 PHP1、PHP2及其类似物的理化性质

Table 2. Physicochemical properties of PHP1, PHP2 and their analogues

名称	pI	GRAVY	净电荷	脂肪族氨基酸指数	非稳定性指数	半衰期（h）			生物活性预测分数
名称	pI	GRAVY	净电荷	脂肪族氨基酸指数	非稳定性指数	mammalian reticulocytes	Yeast cell	E.coli	生物活性预测分数
PHP1	5.49	0.650	0	96.67	−13.52	100	>20	>10	0.50
PHP1A-1	5.52	0.367	0	48.33	−26.08	1.2	>20	>10	0.82
PHP1A-2	5.49	0.367	0	48.33	−0.95	100	>20	>10	0.82
PHP1A-3	5.49	1.233	0	96.67	11.53	100	>20	>10	0.83
PHP1A-4	5.52	0.950	0	48.33	−1.30	1.2	>20	>10	0.91
PHP1A-5	5.49	0.950	0	48.33	24.10	100	>20	>10	0.98
PHP1A-6	5.52	0.467	0	48.33	11.53	30	>20	>10	0.98
PHP1A-7	5.49	0.833	0	65.00	24.10	100	>20	>10	0.97
PHP2	8.29	−0.725	+1	0.00	19.86	7.2	>20	>10	0.60
PHP2A-1	8.29	−0.163	+1	48.75	9.25	7.2	>20	>10	0.76
PHP2A-2	8.60	0.487	+1	97.50	−1.36	20	0.5	>10	0.87
PHP2A-3	8.64	0.150	+1	61.25	9.25	4.4	>20	>10	0.92
PHP2A-4	6.74	1.200	0	110.00	19.86	20	0.5	>10	0.92
PHP2A-5	6.78	0.863	0	73.75	19.86	4.4	>20	>10	0.95
PHP2A-6	5.57	1.738	0	122.50	32.83	4.4	>20	>10	0.97
PHP2A-7	9.99	−0.263	+2	25.00	23.40	4.4	>20	>10	0.95
PHP2A-8	8.60	−0.125	+1	48.75	−1.36	30	>20	>10	0.94
PHP2A-9	6.74	0.925	0	97.50	19.86	20	0.5	>10	0.93
PHP2A-10	6.74	0.312	0	48.75	35.29	30	>20	>10	0.96
PHP2A-11	5.52	1.188	0	97.50	48.25	30	>20	>10	0.98
PHP2A-12	9.99	−0.812	+2	0.00	−8.44	30	>20	>10	0.96
注：mammalian reticulocytes：哺乳动物的红细胞；Yeast cell：酵母细胞；E.coli：大肠杆菌。

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表 3 合成肽的生物活性

Table 3. Bioactivity of synthetic peptides

名称	（MW m/z）		RP-HPLC		ACE IC₅₀ （μmol/L）	α-Glucosidase IC₅₀ （μmol/L）	ABTS⁺·清除率（%）
名称	Calcd.	[M+H]⁺	Rt（min）	Purity（%）	ACE IC₅₀ （μmol/L）	α-Glucosidase IC₅₀ （μmol/L）	ABTS⁺·清除率（%）
PHP1	763.39	764.4	5.559	98.31	6.34±0.37	8897.09±1.79	5.80±0.51
PHP1A-1	767.33	768.3	5.251	98.67	20.25±5.54^**	116.03±1.09^**	95.89±0.43^**
PHP1A-2	767.33	768.2	5.227	99.73	36.66±7.90^**	88.87±1.74^**	89.76±1.22^**
PHP1A-3	809.41	810.4	7.360	99.67	8.48±0.78	3.09±0.43^**	8.18±0.69
PHP1A-4	813.35	814.5	7.391	97.97	8.64±1.92	104.46±1.79^**	94.00±0.28^**
PHP1A-5	813.35	814.4	7.329	98.95	13.34±2.28	54.49±2.39^**	85.53±4.26^**
PHP1A-6	767.36	768.4	6.984	99.84	3.87±0.21^*	331.95±2.60^**	10.10±0.59^**
PHP1A-7	781.38	782.4	6.483	99.57	9.67±0.42	9.51±0.25^**	12.00±1.21^**
PHP2	1039.51	1040.5	10.169	98.04	8.64±0.14	296.79±2.14	27.73±0.37
PHP2A-1	1051.55	1052.2	6.533	98.14	9.48±1.06	1889.59±1.30^**	23.28±1.11^**
PHP2A-2	1063.59	1064.2	8.474	95.80	40.06±2.84^**	1872.49±1.12^**	25.73±0.50^*
PHP2A-3	1021.54	1022.4	6.572	98.49	3.33±0.34^**	306.32±3.76^**	25.53±1.30^*
PHP2A-4	1006.53	1007.5	9.817	97.43	2.86±0.46^**	278.23±1.71^**	28.54±0.69
PHP2A-5	964.48	965.1	8.370	98.35	58.11±2.30^**	21.24±2.32^**	35.43±1.52^**
PHP2A-6	940.51	941.5	12.752	98.23	33.93±2.87^**	5.58±0.88^**	37.62±0.28^**
PHP2A-7	998.53	999.1	10.995	98.15	4.58±0.57^*	7949.34±0.96^**	18.47±1.47^**
PHP2A-8	1007.52	1008.4	6.272	98.48	15.18±2.72^**	957.91±1.71^**	23.14±0.60^**
PHP2A-9	992.51	993.4	9.866	98.11	25.13±1.86^**	90.82±1.27^**	30.44±1.15^**
PHP2A-10	936.45	937.3	8.192	97.73	8.22±1.02	1022.05±1.97^**	28.60±0.84
PHP2A-11	912.47	913.2	10.339	99.29	11.08±1.47	2.35±0.27^**	29.88±1.32^*
PHP2A-12	970.50	971.3	4.134	99.87	24.94±2.71^**	3.98±0.25^**	21.49±1.73^**
注：Rt为多肽在液相分析图中的保留时间；PHP1系列设计肽与PHP1比较，PHP2系列设计肽与PHP2比较，“”代表差异显著（0.01<P<0.05），“*”代表差异极显著（P<0.01）。

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