CN111057134B - 一类具有广谱抗菌活性、低毒性且无诱导耐药性的抗菌肽及其应用 - Google Patents
一类具有广谱抗菌活性、低毒性且无诱导耐药性的抗菌肽及其应用 Download PDFInfo
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Abstract
本发明设计合成了一类具有广谱抗菌活性、低毒性且无诱导耐药性的抗菌肽。该抗菌肽是以丙氨酸或/和甘氨酸或/和赖氨酸替换母肽MP‑C的1‑14位的一个或多个氨基酸得到，记为AGK。体外抗菌实验、溶血活性实验和诱导耐药实验表明，抗菌肽AGK具有广谱抗菌活性，低毒性，且无诱导耐药性。其中，AGK‑L1GA5K与Rifampin联合用药后不仅可减小Rifampin耐药性的产生，而且能使Rifampin耐药菌株对Rifampin重新敏感。因此，本发明的抗菌肽AGK有望成为新型抗生素的候选药物，在临床抗菌药物中具有良好的应用前景。
Description
技术领域
本发明属于生物化学技术领域，涉及一类具有广谱抗菌活性、低毒性且无诱导耐药性的抗菌肽，本发明同时还涉及该抗菌肽在制备临床抗菌药物中的应用。
背景技术
细菌的多重耐药性是一个全球性问题，严重威胁人类健康。大多数抗生素通过干扰细胞壁、蛋白质、核酸的生物合成或代谢功能的途径来攻击细菌。细菌很容易通过发展生化溶液来对抗这些特定的过程而进化出对抗生素的耐受能力，从而产生耐药性(ACSApplied Materials and Interfaces,2019,11(38):34609-34620)。抗菌肽(Antimicrobial peptides)构成了大多数多细胞生物中先天防御系统的主要组成部分，并形成了抵御入侵微生物的第一道防线。尽管尚未完全阐明抗菌肽的确切作用机理，但细胞质膜被认为是其主要靶点，抗菌肽通过靶向细胞质膜，使脂质双层结构去极化，增加膜通透性最终杀死微生物，由于这种物理作用方式迅速发生，病原体很难形成有效的抗药性。随着“超级细菌”的出现，新型抗生素的开发已变得至关重要，而抗菌肽代表了这类具有巨大潜力的新型抗生素(Journal of Medicinal Chemistry,2019,62(7):3354-3366)。
天然抗菌肽由于代谢不稳定、生产成本高、容易引起溶血副作用等缺点限制了它们进一步的临床应用。氨基酸替换修饰多肽序列是改善抗菌活性和细胞毒性最简单最常用的一种方法。如：通过用赖氨酸，精氨酸和色氨酸取代myxinidin中的关键氨基酸，表现出高细胞选择性，高耐盐性和抗生物膜特性(Amino Acids,2016,48(2):505-522)；抗菌肽K1K8经氨基酸替换后显示出改善的抗菌谱，更低的溶血活性和增强的血清稳定性(AppliedMicrobiology and Biotechnology,2016,100(11):5069-5077)；多肽Tachyplesin-1具有强抗菌活性和高细胞毒性，通过丙氨酸扫描确定每个氨基酸的结构-功能关系，然后再进行疏水性和电荷修饰，获得了低毒性的同时保留了抗菌活性的类似物(ACS InfectiousDiseases,2017,3(12):917-926)。
Mastoparan C(MP-C)是从欧洲大黄蜂(Vespa crabro)的毒液中鉴定出来的一种典型的α-螺旋两亲性阳离子抗菌肽，具有广谱抗菌活性，但溶血活性高，毒性大的缺点限制了其广泛应用(International Journal of Biological Sciences,2018,14(6):599-607)。
发明内容
本发明的目的是提供一类具有广谱抗菌活性、低毒性且无诱导耐药性的新型抗菌肽。
本发明的另一目的是提供上述抗菌肽在制备临床抗菌药物中的应用。
(一)具有广谱抗菌活性、低毒性且无诱导耐药性的抗菌肽
本发明具有广谱抗菌活性、低毒性且无诱导耐药性的抗菌肽，是以丙氨酸(Ala，A)或/和甘氨酸(Gly，G)或/和赖氨酸(Lys，K)替换抗菌肽MP-C的1-14位的一个或多个氨基酸得到，标记为AGK。
所述抗菌肽MP-C的结构式如图1所示，质谱图如图2所示，MP-C分子量理论计算结果为1507，与质谱鉴定结果一致，证明抗菌肽MP-C结构正确。其氨基酸序列为：Leu-Asn-Leu-Lys-Ala-Leu-Leu-Ala-Val-Ala-Lys-Lys-Ile-Leu-NH2(SEQ ID No.4)。
作为本发明技术方案的优选，上述抗菌肽的代表为AGK-L1G、AGK-L7A、AGK-L1GA5K，其氨基酸序列如下：
AGK-L1G：Gly-Asn-Leu-Lys-Ala-Leu-Leu-Ala-Val-Ala-Lys-Lys-Ile-Leu-NH2(SEQ ID No.1)；
AGK-L7A：Leu-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Val-Ala-Lys-Lys-Ile-Leu-NH2(SEQ ID No.2)；
AGK-L1GA5K：Gly-Asn-Leu-Lys-Lys-Leu-Leu-Ala-Val-Ala-Lys-Lys-Ile-Leu-NH2(SEQ ID No.3)。
更优选地，上述抗菌肽为AGK-L1GA5K，其氨基酸序列为Gly-Asn-Leu-Lys-Lys-Leu-Leu-Ala-Val-Ala-Lys-Lys-Ile-Leu-NH2(SEQ ID No.3)。
上述抗菌肽AGK是采用经典的多肽Fmoc固相合成方法得到的，具体为：以Rink-MBHAResin为原料，在多肽合成过程中以HOBt/HBTU作为缩合剂进行氨基酸耦合，采用茚三酮显色法检定二级胺，按照多肽序列依次耦合得到连有MBHA树脂的多肽，多肽切割及HPLC纯化后即得。
(二)本发明抗菌肽AGK的体外抗菌活性研究
1、体外抑菌实验
采用经典的微量连续二倍稀释法测定抗菌肽对革兰氏阳性菌(金黄色葡萄球菌、枯草芽孢杆菌)及革兰氏阴性菌(大肠杆菌、铜绿假单胞菌、肺炎克雷伯菌)的最小抑菌浓度；以抗生素Gentamicin、Kanamycin、Vancomycin、Rifampin、Polymyxin B作为阳性对照。实验平行重复3次。结果见表1。
表1抗菌肽AGK对常见标准菌株的最低抑菌浓度
表1结果显示，母肽MP-C及抗菌肽AGK-L1G、AGK-L7A、AGK-L1GA5K对金黄色葡萄球菌、枯草芽孢杆菌为代表的革兰氏阳性菌，以大肠杆菌、铜绿假单胞菌、肺炎克雷伯菌为代表的革兰氏阴性菌都具有较强抑制作用，表现为广谱抗菌活性，其抗菌效果与抗生素Polymyxin B较相近。
2、溶血活性实验
为了考察本发明合成的抗菌肽AGK对正常哺乳动物细胞的毒性，我们测定了母肽MP-C及抗菌肽AGK-L1G、AGK-L7A、AGK-L1GA5K与小鼠红细胞孵育1h后的溶血情况，结果见图3。
图3显示，10％溶血率时，母肽MP-C浓度为64μM，抗菌肽AGK-L1G、AGK-L7A、AGK-L1GA5K的浓度大于最高测试浓度256μM，远高于所测其对枯草芽孢杆菌、大肠杆菌的MIC。说明本发明合成的抗菌肽AGK-L1G、AGK-L7A、AGK-L1GA5K的毒性均远低于母肽MP-C，用药更安全。
3、诱导耐药实验
为了考察本发明合成的抗菌肽AGK是否会产生耐药性，我们测定了母肽MP-C及抗菌肽AGK-L1G、AGK-L7A、AGK-L1GA5K与大肠杆菌连续作用15天后的诱导耐药情况，以抗生素Rifampin、Polymyxin B作为对照，结果见图4。
图4显示，母肽MP-C与抗菌肽AGK-L1G、AGK-L7A、AGK-L1GA5K及抗生素Polymyxin B，与大肠杆菌连续作用15天后均没有产生耐药性，而Rifampin迅速产生耐药性，15天后MIC提高了16倍。值得注意的是，抗菌肽AGK-L1GA5K与Rifampin的混合物(等浓度等体积混合)并没有产生耐药性。说明本发明合成的抗菌肽AGK不仅无诱导耐药性，还能减少抗生素耐药性的产生。
为了考察大肠杆菌对Rifampin耐药后是否会导致对抗菌肽的交叉耐药，以及AGK-L1GA5K与Rifampin联合用药后是否会使Rifampin耐药逆转，我们采用经典的微量连续二倍稀释法测定母肽MP-C与抗菌肽AGK-L1G、AGK-L7A、AGK-L1GA5K，以及Rifampin、AGK-L1GA5K与Rifampin的混合物(等浓度等体积混合)对Rifampin连续作用15天后的获得耐药性大肠杆菌的最小抑菌浓度，以对正常大肠杆菌的最小抑菌浓度为对照。实验平行重复3次。结果见表2。
表2抗菌肽AGK对Rifampin连续作用15天后的大肠杆菌的最低抑菌浓度
表2显示，Rifampin诱导耐药后的获得耐药大肠杆菌对母肽MP-C及抗菌肽AGK-L1G、AGK-L7A、AGK-L1GA5K均没有交叉耐药性，更令人惊喜的是，AGK-L1GA5K与Rifampin联合用药后使Rifampin耐药成功逆转。即AGK-L1GA5K无诱导耐药性，与Rifampin联合用药后不仅可减少Rifampin耐药性的产生，而且能使Rifampin耐药菌株对Rifampin重新敏感。
本发明相较于现有技术的有益效果为：
本发明的抗菌肽AGK是在母肽MP-C的基础上进行氨基酸替换得到的，其体外抗菌实验、溶血活性实验和诱导耐药实验表明，抗菌肽AGK具有广谱抗菌活性，低毒性，且无诱导耐药性。其中，AGK-L1GA5K与Rifampin联合用药后不仅可减少Rifampin耐药性的产生，而且能使Rifampin耐药菌株对Rifampin重新敏感。因此，本发明的抗菌肽有望成为新型抗生素的候选药物，在制备临床抗菌药物中具有良好的应用前景。
附图说明
图1为抗菌肽MP-C的结构式；
图2为抗菌肽MP-C的质谱图；
图3为抗菌肽与小鼠红细胞孵育1h后对红细胞的溶血活性结果图；
图4为抗菌肽与大肠杆菌连续作用15天后的诱导耐药结果图；
图5为抗菌肽AGK-L1G的质谱图；
图6为抗菌肽AGK-L7A的质谱图；
图7为抗菌肽AGK-L1GA5K的质谱图。
具体实施方式
下面结合附图和具体实施例对本发明具有广谱抗菌活性、低毒性且无诱导耐药性的抗菌肽AGK的合成作进一步说明。
实施例1：抗菌肽AGK-L1G的合成
(1)树脂的活化及预处理
称取0.7g的MBHA树脂(0.43mmol/g)，加入多肽固相合成仪中，经DCM溶胀30min，DMF洗涤后，茚三酮显色法鉴定树脂，若无色表明树脂正常。
(2)Fmoc-AGK-L1G-MBHA的合成
溶胀后的树脂用含有20％哌啶的DMF溶液洗涤脱去Fmoc保护基团，茚检树脂呈蓝紫色即可。将3倍过量的Leu、3倍过量的HOBt、HBTU，6倍过量的DIEA用重蒸DMF溶解加入到合成仪中搅拌1h，反应到时间后，茚检树脂呈无色透明表明缩合成功，得到Fmoc-Leu-MBHA。
按照上述方法依次缩合Ile、Lys、Lys、Ala、Val、Ala、Leu、Leu、Ala、Lys、Leu、Asn、Gly，得到Fmoc-Gly-Asn-Leu-Lys-Ala-Leu-Leu-Ala-Val-Ala-Lys-Lys-Ile-Leu-MBHA。
(3)多肽切割
将所得Fmoc-Gly-Asn-Leu-Lys-Ala-Leu-Leu-Ala-Val-Ala-Lys-Lys-Ile-Leu-MBHA用含有20％哌啶的DMF溶液洗涤脱去Fmoc保护基团后，依次用DCM、甲醇洗涤，彻底抽干树脂。加入10mL切割试剂(TFA:Tris:水＝9.5:0.25:0.25(v:v:v))反应3h，经乙醚萃取后冷冻干燥。
(4)多肽纯化
RP-HPLC纯化条件为流动相A：0.1％TFA/水，流动相B：0.1％TFA/乙腈，采用线性梯度洗脱，收集目标峰流出液，冻干，得到抗菌肽AGK-L1G。
AGK-L1G的质谱图如图5所示，其分子量理论计算结果为1451，与质谱鉴定结果一致，证明抗菌肽AGK-L1G结构正确。
实施例2：抗菌肽AGK-L7A的合成
(1)树脂的活化及预处理
同实施例1。
(2)Fmoc-AGK-L7A-MBHA的合成
溶胀后的树脂用含有20％哌啶的DMF溶液洗涤脱去Fmoc保护基团，茚检树脂呈蓝紫色即可。将3倍过量的Leu、3倍过量的HOBt、HBTU，6倍过量的DIEA用重蒸DMF溶解加入到合成仪中搅拌1h，反应到时间后，茚检树脂呈无色透明表明缩合成功，得到Fmoc-Leu-MBHA。
按照上述方法依次缩合Ile、Lys、Lys、Ala、Val、Ala、Ala、Leu、Ala、Lys、Leu、Asn、Leu，得到Fmoc-Leu-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Val-Ala-Lys-Lys-Ile-Leu-MBHA。
(3)多肽切割
同实施例1。
(4)多肽纯化
同实施例1。得到抗菌肽AGK-L7A。
AGK-L7A的质谱图如图6所示，其分子量理论计算结果为1465，与质谱鉴定结果一致，证明抗菌肽AGK-L7A结构正确。
实施例3：抗菌肽AGK-L1GA5K的合成
(1)树脂的活化及预处理
同实施例1。
(2)Fmoc-AGK-L1GA5K-MBHA的合成
溶胀后的树脂用含有20％哌啶的DMF溶液洗涤脱去Fmoc保护基团，茚检树脂呈蓝紫色即可。将3倍过量的Leu、3倍过量的HOBt、HBTU，6倍过量的DIEA用重蒸DMF溶解加入到合成仪中搅拌1h，反应到时间后，茚检树脂呈无色透明表明缩合成功，得到Fmoc-Leu-MBHA。
按照上述方法依次缩合Ile、Lys、Lys、Ala、Val、Ala、Leu、Leu、Lys、Lys、Leu、Asn、Gly，得到Fmoc-Gly-Asn-Leu-Lys-Lys-Leu-Leu-Ala-Val-Ala-Lys-Lys-Ile-Leu-MBHA。
(3)多肽切割
同实施例1。
(4)多肽纯化
同实施例1，得到抗菌肽AGK-L1GA5K。
AGK-L1GA5K的质谱图如图7所示，其分子量理论计算结果为1508，与质谱鉴定结果一致，证明抗菌肽AGK-L1GA5K结构正确。
序列表
<110> 倪京满
<120> 一类具有广谱抗菌活性、低毒性且无诱导耐药性的抗菌肽及其应用
<140> 201911303490.1
<141> 2019-12-17
<160> 4
<210> 1
<211> 14
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<400> 1
Gly Asn Leu Lys Ala Leu Leu Ala Val Ala Lys Lys Ile Leu
1 5 10
<210> 2
<211> 14
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<400> 2
Leu Asn Leu Lys Ala Leu Ala Ala Val Ala Lys Lys Ile Leu
1 5 10
<210> 3
<211> 14
<212> PRT
<213> 人工序列（Artificial Sequence）
<220>
<400> 3
Gly Asn Leu Lys Lys Leu Leu Ala Val Ala Lys Lys Ile Leu
1 5 10
<210> 4
<211> 14
<212> PRT
<213> 欧洲大黄蜂（Vespa crabro）
<400> 4
Leu Asn Leu Lys Ala Leu Leu Ala Val Ala Lys Lys Ile Leu
1 5 10
Claims (4)
1.一类广谱低毒的MP-C修饰抗菌肽，其特征在于，所述抗菌肽是以甘氨酸替换抗菌肽MP-C 1位的亮氨酸得到，其氨基酸序列如SEQ ID No.1所示；
或：所述抗菌肽是以丙氨酸替换抗菌肽MP-C 7位的亮氨酸得到，其氨基酸序列如SEQID No.2所示；
或：所述抗菌肽是以甘氨酸替换抗菌肽MP-C 1位的亮氨酸，并以赖氨酸替换抗菌肽MP-C 1位的丙氨酸得到，其氨基酸序列如SEQ ID No.3所示；
所述抗菌肽MP-C的氨基酸序列如SEQ ID No.4所示。
2.如权利要求1所述的一类广谱低毒的MP-C修饰抗菌肽，其特征在于，所述抗菌肽的氨基酸序列如SEQ ID No.3所示。
3.如权利要求1或2所述的一类广谱低毒的MP-C修饰抗菌肽在制备临床抗菌药物中的应用。
4.如权利要求3所述的一类广谱低毒的MP-C修饰抗菌肽在制备临床抗菌药物中的应用，其特征在于，所述抗菌肽用于制备Rifampin联合抗菌药物。
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