CN114057835A - 一种抗菌肽类似物及其制备方法与应用 - Google Patents
一种抗菌肽类似物及其制备方法与应用 Download PDFInfo
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- CN114057835A CN114057835A CN202111404945.6A CN202111404945A CN114057835A CN 114057835 A CN114057835 A CN 114057835A CN 202111404945 A CN202111404945 A CN 202111404945A CN 114057835 A CN114057835 A CN 114057835A
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Abstract
本发明属于生物化学中的多肽药物技术领域,公开了一种抗菌肽类似物及其制备方法与应用,基于抗菌肽W3R6,其序列为VWRRWRRFWRR‑NH2,利用二氨基庚二酸Dap对序列中精氨酸或色氨酸进行替换,获得系列抗菌肽类似物。本发明公开了上述抗菌肽类似物不仅能杀死普通病原微生物,而且对临床分离的多药耐药菌表现出十分优异的抗菌活性,且没有溶血活性。其中多药耐药菌包括耐药表皮葡萄球菌、耐药金黄色葡萄球菌、耐药粪肠链球菌等革兰氏阳性细菌,以及耐药大肠杆菌、耐药产气肠杆菌、耐药阴沟肠杆菌、耐药铜绿假单胞菌、耐药鲍曼不动杆菌、耐药肺炎克雷伯菌、耐药荧光杆菌等革兰氏阴性细菌。
Description
技术领域
本发明属于生物化学中的多肽药物技术领域,具体涉及抗菌肽W3R6和该系列改造类似物及其制备方法,以及在制备抗多药耐药细菌感染药物中的用途。
背景技术
抗生素的发现解决了微生物感染的问题,但由于抗生素的过度滥用,导致由耐多药细菌引起的传染病日益增多,给患者带来了极大的危害。耐药细菌细胞膜发生了变化,降低了膜对药物的通透性,阻断了药物的跨膜转运,又或是细菌外排泵的变化显著增加了药物的排出,降低了细菌细胞内药物浓度,此外,作为细菌抵抗外界攻击的屏障的细菌生物膜也会增加细菌的耐药性。传统抗生素对耐药细菌和真菌的作用越来越有限。有报告指出全球每年约有70万人死于耐药细菌感染,并预计这一数字将在未来几年内继续增长,耐药细菌感染将成为世界各地的主要死亡原因之一。因此,迫切需要新型抗菌剂来缓解这一问题。
抗菌肽(Antimicrobial peptide,AMPs),又称宿主防御肽,是广泛存在于植物、动物、昆虫、微生物等生物体内的先天免疫系统的组成部分,具有分子量小、结构简单、杀菌活性强、杀菌机制独特,不易引起耐药性等优点。由于特定的膜的作用机制不同于大多数传统抗生素杀死细菌的作用机制,AMPs不论是对敏感细菌还是耐药细菌都表现出广谱抗菌活性,因此有望替代传统抗生素。此外,AMPs也可与抗生素联合使用,激活耐药细菌对抗生素的敏感性,并通过特定的膜破坏机制和灭活细菌的外排泵系统来逆转耐药。
天然来源的抗菌肽由于抗菌活性低,存在免疫原性,会引起溶血等毒性不明确的特性限制了抗菌肽作为抗菌药物的推广应用,因此,寻找抗菌活性更强,溶血或细胞毒性更低的抗菌肽是解决抗菌肽作为抗菌药物推广的最关键的因素。为了解决这些问题,研究人员设计了一些方法来改善AMPs的临床应用。最常用的方法是通过化学修饰改变其物理和化学性质,以提高其临床应用潜力,如调节其净电荷数、疏水含量、空间结构等。虽然其中一些策略已经取得了实质性的进展,并对AMPs的发展做出了贡献,但由于AMPs的多样性,仍需要进一步的策略,可以普遍应用于AMPs的修改。
发明内容
针对上述不足,本发明提供具有广谱高效的抗菌活性和极低的溶血活性的W3R6系列新型抗菌肽及其应用,基于抗菌肽W3R6,其序列为VWRRWRRFWRR-NH2,利用二氨基庚二酸Dap对序列中精氨酸或色氨酸进行替换,获得系列抗菌肽类似物。
本发明的上述目的是通过以下技术方案实现的:
一种抗菌肽类似物,包括抗菌肽W3R6及其改造类似物的系列抗菌肽,所述系列抗菌肽均由11个氨基酸残基组成,pH=7时均带正电荷,具体序列如下:
本发明进一步提出了上述抗菌肽类似物的制备方法,采用多肽固相合成-Fmoc法合成其全序列,通过RP-HPLC纯化,具体步骤如下:
步骤S1:固相合成,用Fmoc-Linker MBHA Resin S=0.32mmol/g,采用Fmoc/tBu工艺,按上述肽序列,以下表方法从C端向N端,从右到左依次缩合氨基酸链接,最后形成多肽树脂:
按照序列依次偶联各个氨基酸,将多肽树脂洗涤转移出干燥至恒重,待裂解;
步骤S2:多肽树脂裂解,裂解试剂在搅拌下加入步骤S1制备的多肽树脂中,待体系温度稳定后;温控在25~30℃搅拌反应2.5小时,将裂解液滤出,采用5倍液体积量的冰乙醚将其沉淀,滤出沉淀物并采用3倍液体积量的冰乙醚洗涤3次后,室温减压干燥,得固体粗品;
步骤S3:多肽的纯化冻干,将步骤S2固体粗品研细,准备纯化水,在搅拌下缓慢加入研细的固体粗品,同时滴加乙腈水溶液,待固体粗品加完并溶解完全后,用0.45um的微孔滤膜过滤;粗品纯化通过RP-HPLC纯化,采用C-18柱填料制备柱,在常温下分离纯化,收集目标产物,分析检测,归类,杂质纯度要求≥95%,将不合格目标物收集,再次进行分离纯化,将合格主峰减压冷冻干燥,得到粉末状精制多肽。
进一步的,步骤S2裂解试剂按如下方法配制:
按体积为1g肽树脂比10mL±2mL计算裂解试剂用量,TFA:H2O:EDT:TIS=95:1:2:2依次将所需裂解试剂H2O,TFA,EDT,TIS于裂解反应瓶,裂解试剂温度控制在0~10℃。
进一步的,步骤S3RP-HPLC纯化的流动相为A:0.1%TFA/H2O,B:0.1%TFA/CAN。
本发明提出了上述抗菌肽类似物的应用,在制备抗多药耐药细菌感染药物中的用途。
进一步的,多药耐药细菌分为多药耐药革兰氏阳性细菌和多药耐药革兰氏阴性细菌,多药耐药革兰氏阳性细菌包括:耐药表皮葡萄球菌、耐药金黄色葡萄球菌、耐药粪肠链球菌;多药耐药革兰氏阴性细菌包括:耐药大肠杆菌、耐药产气肠杆菌、耐药阴沟肠杆菌、耐药铜绿假单胞菌、耐药鲍曼不动杆菌、耐药肺炎克雷伯菌和耐药荧光杆菌。
本发明与现有技术相比的有益效果是:
本发明中的抗菌肽类似物不仅能杀死普通病原微生物,而且对临床分离的多药耐药菌表现出十分优异的抗菌活性,且没有溶血活性。抗菌肽为人工合成,具有分子量小、人工合成方便,杀菌作用强、抗菌谱广、溶血活性低等优点,具有广泛的应用前景。
附图说明
下面结合附图和实施例对本发明进一步说明。
图1为抗菌肽W3R6及其类似物的溶血活性;
图2为抗菌肽W3R6对耐药金黄色葡萄球菌杀菌动力学曲线图;
图3为抗菌肽W3R6对耐药大肠杆菌杀菌动力学曲线图;
图4类似物W3R6-A1对耐药金黄色葡萄球菌杀菌动力学曲线图;
图5为类似物W3R6-A1对耐药大肠杆菌杀菌动力学曲线图;
图6为类似物W3R6-A4对耐药金黄色葡萄球菌杀菌动力学曲线图;
图7为类似物W3R6-A4对耐药大肠杆菌杀菌动力学曲线图。
具体实施方式
下面通过具体实施例详述本发明,但不限制本发明的保护范围。如无特殊说明,本发明所采用的实验方法均为常规方法,所用实验器材、材料、试剂等均可从商业途径获得。
临床所分离的多药耐药菌株来自大连医科大学附属第一医院。
实施例1抗菌肽W3R6及其类似物的制备
多肽W3R6及其类似物的合成采用固相有机合成法,利用Fmoc-保护氨基酸策略,SPPS固相合成技术,完成多肽合成,裂解,氧化,纯化得到目标产物。具体步骤以序VWR{Dap}WRRFW{Dap}R-NH2为例。
第一步:首先进行固相合成,用Fmoc-Linker MBHA Resin S=0.32mmol/g,采用Fmoc/tBu工艺,按上述肽序列,以表1方法从C端向N端,从右到左依次缩合氨基酸链接,最后形成此多肽树脂:
表1 AA1-AA11
依次偶联下列氨基酸:A-01 Fmoc-Arg(Pbf)-OH,A-02 Fmoc-Dap(Boc)-OH,A-03Fmoc-Trp(Boc)-OH,A-04 Fmoc-Phe-OH,A-05 Fmoc-Arg(Pbf)-OH,A-06 Fmoc-Arg(Pbf)-OH,A-07 Fmoc-Trp(Boc)-OH,A-08 Fmoc-Dap(Boc)-OH,A-09 Fmoc-Arg(Pbf)-OH,A-10Fmoc-Trp(Boc)-OH,A-11 Fmoc-Val-OH
将肽树脂洗涤转移出干燥至恒重,待裂解;
第二步进行肽树脂裂解,裂解试剂的配制:按体积为1g肽树脂比10mL±2mL计算裂解试剂用量:TFA:H2O:EDT:TIS=95:1:2:2依次将所需裂解试剂H2O,TFA,EDT,TIS于裂解反应瓶,裂解试剂温度控制在0~10℃;裂解试剂在搅拌下加入肽树脂中,待体系温度稳定后;再温控在25~30℃搅拌反应2.5小时,将裂解液滤出,采用5倍液体积量的冰乙醚将其沉淀,滤出沉淀物并采用3倍液体积量的冰乙醚洗涤3次后,室温减压干燥,得固体粗品;
第三步进行肽的纯化冻干,将粗品研细,准备纯化水,在搅拌下缓慢加入研细粗品,同时滴加乙腈水溶液,待粗品加完并溶解完全后,用0.45μm的微孔滤膜过滤;粗品纯化采用C-18柱填料制备柱,流动相A:0.1%TFA/H2O,B:0.1%TFA/CAN,在常温下用合适梯度进行分离纯化,收集目标产物,分析检测,归类,杂质纯度要求≥95%,将不合格目标物收集,用合适梯度再次进行分离纯化,将合格主峰减压冷冻干燥,得到粉末状精制多肽。
其他类似物的合成方法与W3R6-A1的合成及纯化方法除序列不同,其余操作方法与W3R6-A1相同。
最终得到的抗菌肽W3R6及其类似物的序列信息如表2所示。
表2抗菌肽W3R6及其类似物的序列信息
实施例2抗菌肽W3R6及其类似物溶血活性测定
配置0.9%的生理盐水,将采集的新鲜人血稀释为2%的人血红细胞悬液,0.9%的生理盐水溶解抗菌肽W3R6及其系列类似物并稀释成终浓度为200μM至12.5μM。将750μL肽溶液稀释液与750μL人血红细胞等体积混合,以0.9%的生理盐水为阴性对照,0.1%TritonX-100为阳性对照。上述各组共同放入37℃水浴锅内孵育1h,取出后于离心机以1500rpm/min离心10min,取上清200μL加入96孔细胞培养板中,酶标仪检测各孔414nm处的吸光度。根据下列公式计算溶血活性:溶血活性={(Fn-F0)/(Ft-F0)}×100%,其中Fn为实验组吸光度,Ft为阳性对照组吸光度,F0为阴性对照组吸光度。
结果如图1所示。
由图1可知,所设计的系列抗菌肽即使在200μM的高浓度下也不会引起人血发生溶血现象。
实施例3抗菌肽W3R6及其类似物的抗菌活性检测
将37℃过夜培养至对数生长期的细菌,稀释成2×105CFU/mL备用。实验组分别为:抗菌肽和LB液体培养基、无菌水和LB液体培养基作为空白组;抗菌肽和菌液作为实验组,抗菌肽的终浓度按半倍稀释方法配置为200μM至0.39μM。
将50μL无菌水、液体培养基、不同浓度抗菌肽及抗生素和50μL菌液加入96孔板中混匀,37℃条件下培养过夜后,通过酶标仪检测各孔600nm处的吸光度。计算抗菌肽与阳性对照抗生素的最小抑菌浓度MIC,抑菌率达到95%及以上为MIC值。
结果如表3所示,抗菌肽W3R6及其类似物对临床分离的革兰氏阳性细菌、革兰氏阴性细菌均表现出很强的抗菌活性。
W3R6-A1和W3R6-A2对多药耐药的表皮葡萄球菌抗菌作用最强,其MIC值为0.78μM,改造肽W3R6-A1对耐药的金黄色葡萄球菌的抗菌活性最强,其MIC值为0.78μM,其对耐药金黄色葡萄球菌的抗菌活性比其母肽W3R6提高了8倍,其他改造肽W3R6-A2-A4对金黄色葡萄球菌的抗菌活性提高了2倍,W3R6-A5对耐药金黄色葡萄球菌的活性与母肽活性相当,说明W3R6系列抗菌肽在极低浓度下就可抑制葡萄球菌的生长。W3R6-A1和W3R6-A3对耐药粪肠链球菌的抗菌活性与其母肽相比提高了2倍,其MIC值为3.13μM。
与母肽相比,W3R6-A1对多药耐药的大肠杆菌杀伤活性提高了2倍,其MIC值为3.13μM。此外W3R6-A1和W3R6-A3均对其他多药耐药菌显示抗菌活性,包括耐药产气肠杆菌、耐药阴沟肠杆菌、耐药铜绿假单胞菌、耐药鲍曼不动杆菌、耐药肺炎克雷伯菌和耐药荧光杆菌。尤其W3R6-A1对耐药铜绿假单胞菌、耐药鲍曼不动杆菌以及耐药荧光杆菌显示较强的抗菌活性,其MIC值分别为25μM、25μM和12.5μM。
表3抗菌肽W3R6及其系列类似物的抗菌活性
实施例4抗菌肽W3R6及其类似物对代表性多药耐药细菌的杀菌动力学
选用两种临床典型的多药耐药细菌,分别为大肠杆菌和金黄色葡萄球菌,经耐药性测试分析,多药耐药的大肠杆菌对以下抗生素:哌拉西林、哌拉西林/他唑巴坦、氨苄西林、氨苄西林/舒巴坦、头孢哌酮/舒巴坦、头孢曲松、头孢他啶、头孢西丁、头孢吡肟、亚胺培南、美罗培南、庆大霉素、妥布霉素、环丙沙星、氧氟沙星、左旋氧氟沙星、头孢呋辛、氨曲南、厄他培南、复方新诺明、头孢噻肟、氟康唑、氯林可霉素、达托霉素耐药;多药耐药的金黄色葡萄球菌对以下抗生素:氨苄西林、氨苄西林/舒巴坦、头孢曲松、庆大霉素、环丙沙星、左旋氧氟沙星、复方新诺明、克林霉素、四环素、红霉素、苯唑西林、莫西沙星、青霉素、阿莫西林/棒酸耐药。
将37℃过夜培养至对数生长期的细菌,稀释至OD600=10-3备用。实验组分别取10μL终浓度为1×MIC、4×MIC和16×MIC的W3R6系列抗菌肽加入1mL稀释好的细菌,于37℃恒温培养箱中孵育,不同时间点孵育完成后取出于4℃离心机以4000r/min离心10min,倒掉上清,用PBS清洗三次,洗掉抗菌肽,稀释至5×103CFU/mL浓度涂板。对照组取1mL菌液,选择与实验组抗菌肽相同的时间点孵育,选择稀释液涂在LB琼脂平板上,平板置于37℃恒温培养箱培养18-24h后计数菌落。
实验结果如图2至图7所示,没有做任何处理的耐药金黄色葡萄球菌和耐药大肠杆菌,随着孵育时间的增加,细菌数目呈一定趋势的增长;实验组中,抗菌肽W3R6在16×MIC、4小时内将多药耐药的金黄色葡萄球菌全部杀死,3小时内将多药耐药的大肠杆菌全部杀死,杀菌作用呈浓度依赖和时间依赖趋势。类似物W3R6-A1和W3R6-A4在16×MIC、8小时内可将多药耐药的金黄色葡萄球菌和多药耐药的大肠杆菌全部杀死,杀菌作用呈浓度依赖和时间依赖趋势。
以上所述实施方式仅为本发明的优选实施例,而并非本发明可行实施的全部实施例。对于本领域一般技术人员而言,在不背离本发明原理和精神的前提下对其所作出的任何显而易见的改动,都应当被认为包含在本发明的权利要求保护范围之内。
Claims (8)
2.一种抗菌肽类似物的制备方法,其特征是,采用多肽固相合成-Fmoc法合成其全序列,通过RP-HPLC纯化,具体步骤如下:
步骤S1:固相合成,用Fmoc-LinkerMBHA Resin S=0.32mmol/g,采用Fmoc/tBu工艺,按上述肽序列,从C端向N端,从右到左依次缩合氨基酸链接,最后形成多肽树脂:
按照序列依次偶联各个氨基酸,将多肽树脂洗涤转移出干燥至恒重,待裂解;
步骤S2:多肽树脂裂解,裂解试剂在搅拌下加入步骤S1制备的多肽树脂中,待体系温度稳定后;温控在25~30℃搅拌反应2.5小时,将裂解液滤出,采用5倍液体积量的冰乙醚将其沉淀,滤出沉淀物并采用3倍液体积量的冰乙醚洗涤3次后,室温减压干燥,得固体粗品;
步骤S3:多肽的纯化冻干,将步骤S2固体粗品研细,准备纯化水,在搅拌下缓慢加入研细的固体粗品,同时滴加乙腈水溶液,待固体粗品加完并溶解完全后,用0.45um的微孔滤膜过滤;粗品纯化通过RP-HPLC纯化,采用C-18柱填料制备柱,在常温下分离纯化,收集目标产物,分析检测,归类,杂质纯度要求≥95%,将不合格目标物收集,再次进行分离纯化,将合格主峰减压冷冻干燥,得到粉末状精制多肽。
3.如权利要求2所述的一种抗菌肽类似物的制备方法,其特征是,所述步骤S2裂解试剂按如下方法配制:
按体积为1g肽树脂比10mL±2mL计算裂解试剂用量,TFA:H2O:EDT:TIS=95:1:2:2依次将所需裂解试剂H2O,TFA,EDT,TIS于裂解反应瓶,裂解试剂温度控制在0~10℃。
4.如权利要求2所述的一种抗菌肽类似物的制备方法,其特征是,所述步骤S3 RP-HPLC纯化的流动相为A:0.1%TFA/H2O,B:0.1%TFA/CAN。
5.一种抗菌肽类似物的应用,其特征是,在制备抗多药耐药细菌感染药物中的用途。
6.如权利要求5所述的一种抗菌肽类似物的应用,其特征是,所述多药耐药细菌分为多药耐药革兰氏阳性细菌和多药耐药革兰氏阴性细菌。
7.如权利要求6所述的一种抗菌肽类似物的应用,其特征是,所述多药耐药革兰氏阳性细菌包括:耐药表皮葡萄球菌、耐药金黄色葡萄球菌、耐药粪肠链球菌。
8.如权利要求6所述的一种抗菌肽类似物的应用,其特征是,所述多药耐药革兰氏阴性细菌包括:耐药大肠杆菌、耐药产气肠杆菌、耐药阴沟肠杆菌、耐药铜绿假单胞菌、耐药鲍曼不动杆菌、耐药肺炎克雷伯菌和耐药荧光杆菌。
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