CN106916205B - Antibacterial hexapeptide and derivatives and application thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了一种抗菌六肽，序列为：Arg‑Arg‑Trp‑Trp‑Arg‑Trp。应用于制备治疗或预防细菌感染的药物中。本发明还提供一种抗菌六肽衍生物，该抗菌六肽衍生物的序列为：Arg‑Arg‑Trp‑Trp‑Arg‑Trp‑β‑苯乙胺。应用于制备治疗或预防细菌感染的药物中。所述细菌为耐药鲍曼不动杆菌。本发明提供的人工设计合成的抗菌六肽及其苯乙胺修饰物，可方便地采用固相合成法获得。该合成抗菌肽及其衍生物对革兰氏阳性菌、革兰氏阴性菌具有广谱杀伤活性，尤其其衍生物对耐药鲍曼不动杆菌表现出更强的抗菌活性，溶血毒性极小，能够应用于治疗或预防因耐药鲍曼不动杆菌而引起的疾病的药物中。

The invention discloses an antibacterial hexapeptide with the sequence: Arg‑Arg‑Trp‑Trp‑Arg‑Trp. Used in the preparation of medicines for treating or preventing bacterial infections. The invention also provides an antibacterial hexapeptide derivative, the sequence of the antibacterial hexapeptide derivative is: Arg‑Arg‑Trp‑Trp‑Arg‑Trp‑β‑phenylethylamine. Used in the preparation of medicines for treating or preventing bacterial infections. The bacterium is drug-resistant Acinetobacter baumannii. The artificially designed and synthesized antibacterial hexapeptide and its phenylethylamine modification provided by the present invention can be easily obtained by solid-phase synthesis. The synthetic antimicrobial peptide and its derivatives have broad-spectrum killing activity against Gram-positive bacteria and Gram-negative bacteria. In particular, its derivatives show stronger antibacterial activity against drug-resistant Acinetobacter baumannii and have minimal hemolytic toxicity. , can be used in drugs to treat or prevent diseases caused by drug-resistant Acinetobacter baumannii.

Description

Translated fromChinese

抗菌六肽及其衍生物和应用Antibacterial hexapeptides and their derivatives and applications

技术领域technical field

本发明涉及一种抗菌六肽及其衍生物及它们的应用，属于生物技术领域。The invention relates to an antibacterial hexapeptide and derivatives thereof and their applications, belonging to the field of biotechnology.

背景技术Background technique

全球每年的细菌感染病患高达15亿人，其中死亡人数460万，特别是多药耐药菌(MDRB)感染导致的高死亡率，严重威胁公众健康。欧洲与美国每年因细菌感染防治的支出高达70亿欧元和65亿美元，而发展中国家则需为此承担更为高昂的代价。MDRB感染在全球的快速蔓延与抗生素滥用及其研发断代密切相关，即1962至2000年间并未开发出新结构类型的抗生素；2000年以来仅有3个新结构类型的抗菌药物进入市场。美国感染病学会(IDSA)自2004年开始呼吁振兴抗生素研发并给予项目资助，拟在2020年前开发10个结构全新的抗菌药物。可以预测，抗MDRB感染药物的研发将获得快速发展。The number of bacterial infections in the world is as high as 1.5 billion every year, and 4.6 million of them die. Especially the high mortality rate caused by multidrug-resistant bacteria (MDRB) infection is a serious threat to public health. Europe and the United States spend up to 7 billion euros and 6.5 billion U.S. dollars a year on the prevention and control of bacterial infections, and developing countries have to pay a higher price for this. The rapid spread of MDRB infection in the world is closely related to the abuse of antibiotics and their research and development, that is, no new structural types of antibiotics were developed from 1962 to 2000; only 3 new structural types of antibiotics have entered the market since 2000. The Infectious Diseases Society of America (IDSA) has called for the revitalization of antibiotic research and development since 2004 and has given project funding to develop 10 new antibacterial drugs by 2020. It can be predicted that the research and development of anti-MDRB infection drugs will develop rapidly.

鲍曼不动杆菌(AB)是一种严重的医源性病原菌，导致呼吸道、血液、尿道、软组织及中枢神经系统感染，在病患之间通过直接或间接接触方式传播。根据2006-2007年国内外院内感染监测数据资料显示，鲍曼不动杆菌在院内感染中的检出率居前四位，在免疫力低下的患者中，可以引起严重的甚至致死性的感染。它可以引起呼吸机相关性肺炎、败血症、脑膜炎、中耳炎、皮肤软组织感染、泌尿系统感染和中枢神经系统感染等严重感染性疾病。由于鲍曼不动杆菌感染引起的发病率和病死率增加，其被称为“革兰阴性的MRSA”。在常规AB感染中，多药耐药鲍曼不动杆菌(MRAB)的发生率从2004年的23％上升到2014年的63％，其平均发生率也显著高于其他革兰氏阴性菌(44％vs 13％)。目前，95％的MRAB对头孢曲松有抗性，90％的MRAB对头孢他啶、左氧氟沙星、美罗培南、哌拉西林-他唑巴坦等药物耐药，是临床耐药菌感染疾病治疗的一大难题。因此，筛选结构新颖的抗菌药物以应对MRAB感染将成为新药研发的热点。Acinetobacter baumannii (AB) is a serious iatrogenic pathogen that causes infections of the respiratory tract, blood, urethra, soft tissue and central nervous system, and is transmitted by direct or indirect contact between patients. According to the domestic and foreign nosocomial infection monitoring data from 2006 to 2007, the detection rate of Acinetobacter baumannii in nosocomial infections ranks among the top four, and it can cause serious and even fatal infections in patients with low immunity. It can cause serious infectious diseases such as ventilator-associated pneumonia, sepsis, meningitis, otitis media, skin and soft tissue infections, urinary system infections and central nervous system infections. Due to the increased morbidity and mortality associated with Acinetobacter baumannii infection, it is known as "Gram-negative MRSA". Among routine AB infections, the incidence of multidrug-resistant Acinetobacter baumannii (MRAB) increased from 23% in 2004 to 63% in 2014, and its average incidence was also significantly higher than other Gram-negative bacteria ( 44% vs 13%). At present, 95% of MRABs are resistant to ceftriaxone, and 90% of MRABs are resistant to ceftazidime, levofloxacin, meropenem, piperacillin-tazobactam and other drugs. problem. Therefore, screening of novel antibacterial drugs to deal with MRAB infection will become a hot spot in new drug development.

抗菌肽(AMP)是存在于所有生物体中的一类短肽，具有阳离子性和两亲性的特点且结构多样，是生物体天然免疫系统对抗病原菌入侵的重要组成部分，在生物进化过程中保持稳定和高效。截至目前，文献已报道约5000条AMP序列，其中74条已进入药物开发阶段。由于AMP主要以细菌细胞膜为作用靶标，与传统抗生素的作用机制存在显著区别，对MDRB具有显著的抗菌效果，是对抗MDRB最具发展潜力的候选结构类型。然而，天然AMP仍然存在氨基酸序列长、生产成本高，耐酶稳定性差、体内易降解，以及细胞溶解毒性等问题，其在临床应用上面临严重困境。通过化学合成方法获得的非天然小分子抗菌肽(sAMP)，其序列仅由3-9氨基酸残基构成，由于非天然氨基酸(如D-型氨基酸或氨基酸残基修饰物)的存在，使其具有活性显著、结构简单、易于合成、稳定性好、安全性高等优势，受到研究人员的广泛关注。因此，通过化学合成方法发现抗菌活性强、耐酶稳定性好、体内安全性高的sAMP及其类似物，是解决MRAB感染问题的有效途径。Antimicrobial peptides (AMPs) are a class of short peptides that exist in all organisms, with cationic and amphiphilic characteristics and diverse structures. They are an important part of the innate immune system of organisms against pathogen invasion. Be stable and efficient. Up to now, about 5000 AMP sequences have been reported in the literature, of which 74 have entered the drug development stage. Because AMP mainly targets bacterial cell membranes, it is significantly different from the action mechanism of traditional antibiotics, and has a significant antibacterial effect on MDRB. It is the most potential candidate structure type against MDRB. However, natural AMP still has problems such as long amino acid sequence, high production cost, poor enzyme resistance and stability, easy degradation in vivo, and cytolytic toxicity, which face serious difficulties in clinical application. The unnatural small molecule antimicrobial peptide (sAMP) obtained by chemical synthesis method, its sequence is only composed of 3-9 amino acid residues, due to the existence of unnatural amino acids (such as D-type amino acids or amino acid residue modifications), making it It has the advantages of remarkable activity, simple structure, easy synthesis, good stability and high safety, and has attracted extensive attention of researchers. Therefore, chemical synthesis to find sAMP and its analogs with strong antibacterial activity, good enzyme resistance and stability, and high in vivo safety is an effective way to solve the problem of MRAB infection.

发明内容SUMMARY OF THE INVENTION

针对现有的问题，本发明的第一目的在于提供一种抗菌六肽。本发明的第二目的在于提供该种抗菌六肽的应用，本发明的第三目的在于提供一种抗菌六肽衍生物，第四目的在于提供该抗菌六肽衍生物的应用。该抗菌六肽及抗菌六肽衍生物对革兰氏细菌有广谱抗菌活性，其中抗菌六肽衍生物对耐药鲍曼不动杆菌的生长表现出高效的抑制作用，并且该抗菌六肽及其衍生物的毒性小。In view of the existing problems, the first object of the present invention is to provide an antibacterial hexapeptide. The second object of the present invention is to provide the application of the antibacterial hexapeptide, the third object of the present invention is to provide an antibacterial hexapeptide derivative, and the fourth object is to provide the application of the antibacterial hexapeptide derivative. The antibacterial hexapeptide and the antibacterial hexapeptide derivative have broad-spectrum antibacterial activity against Gram bacteria, wherein the antibacterial hexapeptide derivative has an efficient inhibitory effect on the growth of drug-resistant Acinetobacter baumannii, and the antibacterial hexapeptide and Its derivatives are less toxic.

为了实现上述第一目的，本发明的技术方案为：一种抗菌六肽，其特征在于：该抗菌六肽的序列为：Arg-Arg-Trp-Trp-Arg-Trp。In order to achieve the above first object, the technical solution of the present invention is: an antibacterial hexapeptide, characterized in that: the sequence of the antibacterial hexapeptide is: Arg-Arg-Trp-Trp-Arg-Trp.

为了实现上述第二目的，本发明的技术方案为：上述抗菌六肽应用于制备治疗或预防细菌感染的药物中。In order to achieve the above-mentioned second purpose, the technical solution of the present invention is that the above-mentioned antibacterial hexapeptide is used in the preparation of a medicine for treating or preventing bacterial infection.

为了实现上述第三目的，本发明的技术方案为：一种抗菌六肽衍生物，其特征在于：该抗菌六肽衍生物的序列为：Arg-Arg-Trp-Trp-Arg-Trp-β-苯乙胺(抗菌肽b)。In order to achieve the above third object, the technical solution of the present invention is: an antibacterial hexapeptide derivative, characterized in that: the sequence of the antibacterial hexapeptide derivative is: Arg-Arg-Trp-Trp-Arg-Trp-β- Phenethylamine (antibacterial peptide b).

为了实现上述第四目的，本发明的技术方案为：上述抗菌六肽衍生物应用于制备治疗或预防细菌感染的药物中。In order to achieve the above fourth object, the technical solution of the present invention is that the above antibacterial hexapeptide derivatives are used in the preparation of medicines for treating or preventing bacterial infections.

进一步的：所述细菌为耐药鲍曼不动杆菌。此处的耐药鲍曼不动杆菌为广义上的耐药鲍曼不动杆菌，包括耐单种药的鲍曼不动杆菌和耐多重药的鲍曼不动杆菌。Further: the bacteria are drug-resistant Acinetobacter baumannii. The drug-resistant Acinetobacter baumannii here is a broad-spectrum drug-resistant Acinetobacter baumannii, including single-drug-resistant Acinetobacter baumannii and multidrug-resistant Acinetobacter baumannii.

本发明思路如下：在天然抗菌肽Lactoferricin B_4-9RRWQWR基础上，结合虚拟组合库设计技术，DNAstar，Bioedit，sequence logo等分析软件和网站，经过多次试验后发现一条具有抗菌活性的全新多肽序列RRWWRW(Arg-Arg-Trp-Trp-Arg-Trp)，在其C端苯乙胺化后仍具有显著的抗菌活性。新发现的抗菌六肽及其衍生物相比LfcinB_4-9杀菌效果更佳，无溶血毒性，尤其是其衍生物对耐药鲍曼不动杆菌表现出更强的杀菌活性，溶血毒性小，有望作为治疗细菌感染尤其是耐药鲍曼不动杆菌的新药研究和开发。The idea of the invention is as follows: on the basis of the natural antibacterial peptide Lactoferricin B_4-9 RRWQWR, combined with the virtual combinatorial library design technology, DNAstar, Bioedit, sequence logo and other analysis software and websites, after many tests, a brand new polypeptide with antibacterial activity was found The sequence RRWWRW (Arg-Arg-Trp-Trp-Arg-Trp) still has significant antibacterial activity after its C-terminal phenethylamination. Compared with LfcinB_4-9 , the newly discovered antibacterial hexapeptide and its derivatives have better bactericidal effect and no hemolytic toxicity, especially its derivatives show stronger bactericidal activity against drug-resistant Acinetobacter baumannii, with less hemolytic toxicity It is expected to be used as a new drug research and development for the treatment of bacterial infections, especially drug-resistant Acinetobacter baumannii.

利用美国应用系统生物公司生产的Pioneer多肽合成仪基于固相合法制备本发明公开的抗菌六肽苯乙胺修饰物(抗菌肽b)和抗菌六肽(抗菌肽a)以及天然抗菌肽Lactoferricin B_4-9RRWQWR。The antibacterial hexapeptide phenethylamine modified product (antibacterial peptide b) and antibacterial hexapeptide (antibacterial peptide a) disclosed in the present invention and the natural antibacterial peptide Lactoferricin B₄ were prepared based on the solid-phase method using the Pioneer peptide synthesizer produced by American Applied Systems Biological Company._-9 RRWQWR.

利用琼脂空板穴扩散法和96孔板法检测本发明抗菌六肽及其衍生物的抑菌和杀菌活性，以预先合成的天然抗菌肽Lactoferricin B_4-9RRWQWR为对照，进行杀菌活性检测，结果表明本发明的抗菌六肽及其衍生物的杀菌活性显著强于对照的天然抗菌肽Lactoferricin B_4-9RRWQWR的杀菌活性。与抗菌肽a相比，本发明的抗菌肽b在低浓度的条件下就能高效抑制耐药鲍曼不动杆菌的生长。能够应用于治疗或预防因耐药鲍曼不动杆菌而引起的疾病的药物中，如应用于治疗因耐药鲍曼不动杆菌而引起的呼吸机相关性肺炎、败血症、脑膜炎、中耳炎、皮肤软组织感染、泌尿系统感染和中枢神经系统感染等严重感染性疾病的药物中。The antibacterial and bactericidal activities of the antibacterial_hexapeptide and its derivatives of the present invention were detected by the agar hole diffusion method and the 96-well plate method. The results show that the bactericidal activity of the antibacterial hexapeptide and its derivatives of the present invention is significantly stronger than that of the control natural antibacterial peptide Lactoferricin B_4-9 RRWQWR. Compared with the antimicrobial peptide a, the antimicrobial peptide b of the present invention can effectively inhibit the growth of drug-resistant Acinetobacter baumannii under the condition of low concentration. It can be used in drugs for the treatment or prevention of diseases caused by drug-resistant Acinetobacter baumannii, such as in the treatment of ventilator-associated pneumonia, sepsis, meningitis, otitis media, It is used in medicines for serious infectious diseases such as skin and soft tissue infections, urinary system infections and central nervous system infections.

抗菌肽在高效杀菌的同时也有可能作用于高等有机体包括人体细胞，因为抗菌肽的作用方式都是在细胞膜上穿孔使得细胞发生渗漏死亡。所以把抗菌肽能否使红细胞发生渗漏作为其是否有毒性的一个标准，如果抗菌肽能使红细胞中的血红蛋白发生渗漏，就可以通过检测其OD₄₉₀值确定毒性的大小。实验表明，在高浓度下本发明的抗菌六肽衍生物溶血率依然非常低，证实本发明抗菌六肽衍生物的溶血毒性极小。Antibacterial peptides may also act on higher organisms including human cells while efficiently sterilizing, because the mode of action of antibacterial peptides is to perforate the cell membrane, causing the cells to leak and die. Therefore, the antibacterial peptide can make red blood cells leak as a standard of its toxicity. If the antibacterial peptide can make the hemoglobin in red blood cells leak, the toxicity can be determined by detecting its OD₄₉₀ value. Experiments show that the hemolysis rate of the antibacterial hexapeptide derivatives of the present invention is still very low at high concentrations, which proves that the hemolytic toxicity of the antibacterial hexapeptide derivatives of the present invention is extremely small.

本发明的有益效果是：本发明提供的人工设计合成的抗菌六肽及其苯乙胺修饰物，可方便地采用固相合成法获得。该合成抗菌肽及其衍生物对革兰氏阳性菌、革兰氏阴性菌具有广谱杀伤活性，尤其其衍生物对耐药鲍曼不动杆菌表现出更强的抗菌活性，溶血毒性极小，能够应用于治疗或预防因耐药鲍曼不动杆菌而引起的疾病的药物中。The beneficial effects of the present invention are as follows: the artificially designed and synthesized antibacterial hexapeptide and its phenethylamine modified product provided by the present invention can be conveniently obtained by a solid phase synthesis method. The synthetic antibacterial peptide and its derivatives have broad-spectrum killing activity against Gram-positive bacteria and Gram-negative bacteria, especially its derivatives have stronger antibacterial activity against drug-resistant Acinetobacter baumannii, and have minimal hemolytic toxicity , which can be used in drugs for the treatment or prevention of diseases caused by drug-resistant Acinetobacter baumannii.

附图说明Description of drawings

图1为本发明抗菌六肽衍生物的质谱图。Figure 1 is the mass spectrum of the antibacterial hexapeptide derivatives of the present invention.

图2为抗菌六肽的质谱图。Figure 2 is the mass spectrum of the antimicrobial hexapeptide.

具体实施方式Detailed ways

下面结合具体实施例对本发明做进一步的描述：The present invention is further described below in conjunction with specific embodiment:

实施例1：Example 1:

化学合成抗菌肽a和抗菌肽b及对照抗菌肽Lactoferricin B4-9。Chemically synthesized antimicrobial peptide a and antimicrobial peptide b and control antimicrobial peptide Lactoferricin B4-9.

抗菌肽a:RRWWRW(Arg-Arg-Trp-Trp-Arg-Trp)Antimicrobial Peptide a:RRWWRW(Arg-Arg-Trp-Trp-Arg-Trp)

抗菌肽b:RRWWRW-PEA(Arg-Arg-Trp-Trp-Arg-Trp-β-苯乙胺)Antimicrobial peptide b: RRWWRW-PEA (Arg-Arg-Trp-Trp-Arg-Trp-β-phenethylamine)

1、抗菌肽b(Arg-Arg-Trp-Trp-Arg-Trp-β-苯乙胺)的制备：1. Preparation of antimicrobial peptide b (Arg-Arg-Trp-Trp-Arg-Trp-β-phenethylamine):

a.用4倍量DMF洗涤树脂并完全干燥之后，加入10mL 20％(哌啶:DMF，质量比)哌啶、DMF混合溶液后振荡1min并干燥，然后再加入10mL 20％(哌啶:DMF，质量比)哌啶、DMF混合溶液振荡30min；干燥反应容器并用4倍量的DMF洗涤树脂，确保没有哌啶残留，用茚三酮检查树脂颗粒应为蓝色。a. After washing the resin with 4 times the amount of DMF and drying it completely, add 10 mL of 20% (piperidine: DMF, mass ratio) mixed solution of piperidine and DMF, shake for 1 min and dry, then add 10 mL of 20% (piperidine: DMF) , mass ratio) piperidine, DMF mixed solution shake for 30min; dry the reaction vessel and wash the resin with 4 times the amount of DMF to ensure that there is no piperidine residue, check the resin particles with ninhydrin should be blue.

b.将Fmoc保护氨基酸(1mmol)、2.1mL 0.45M HBTM/HOBT(1mmol)、248uL DIEA(2mmol)溶液加入树脂并振荡30min；干燥反应容器并用4倍量DMF洗涤树脂，用茚三酮检查如果为无色，则重新准备树脂，如果为蓝色则继续加入氨基酸进行反应；重复上述氨基酸连接反应直到多肽合成结束；偶联完成后，用低浓度三氟乙酸进行裂解，用醚沉淀，沉淀物加入溶剂(四氢呋喃或者其混合溶剂)中，然后再加入苯乙胺反应，反应结束后用高浓度三氟乙酸脱保护基，用醚沉淀。b. Add Fmoc protected amino acid (1mmol), 2.1mL 0.45M HBTM/HOBT (1mmol), 248uL DIEA (2mmol) solution to the resin and shake for 30min; dry the reaction vessel and wash the resin with 4 times the amount of DMF, check with ninhydrin if If it is colorless, prepare the resin again. If it is blue, continue to add amino acids for the reaction; repeat the above amino acid linking reaction until the end of the peptide synthesis; Add the solvent (tetrahydrofuran or its mixed solvent), and then add phenethylamine to react. After the reaction, use high-concentration trifluoroacetic acid to deprotect the protective group, and use ether to precipitate.

c.将多肽-树脂加入至1mL乙酸(AcOH)、2mL三氟乙醇(TFE)和7mL二氯甲烷(DCM)的混合溶液中，在室温搅拌1h；过滤树脂并用2倍量的TFE/DCM(体积比2:8)混合溶液冲洗，确保回收所有产物；蒸馏浓缩产物溶液至5mL以内；向含有100mL上述浓缩溶液的试管中加入乙醚，查看完全保护的多肽是否溶于乙醚；如果不溶，则再次向试管内加入冰乙醚促使产物析出，抽滤获得产物粗品；如果溶解，则向乙醚中加入水促使产物析出，然后再抽滤获得产物粗品。c. The polypeptide-resin was added to a mixed solution of 1 mL of acetic acid (AcOH), 2 mL of trifluoroethanol (TFE) and 7 mL of dichloromethane (DCM), and stirred at room temperature for 1 h; filtered the resin and used 2 times the amount of TFE/DCM ( Volume ratio 2:8) mixed solution rinse to ensure that all products are recovered; distill and concentrate the product solution to less than 5mL; add ether to the test tube containing 100mL of the above concentrated solution to check whether the fully protected polypeptide is soluble in ether; if insoluble, repeat Add glacial ether to the test tube to promote product precipitation, and suction filtration to obtain crude product; if dissolved, add water to diethyl ether to promote product precipitation, and then suction filtration to obtain crude product.

d.称取100mg干燥后的上述固体多肽粗品，溶于10mL 0.1％三氟乙酸(TFA)，样品用0.22μm滤膜过滤之后，在反相高效液相色谱(RP-HPLC)上分离纯化；洗脱液为0.1％TFA水溶液(A)和0.1％的乙腈(ACN)溶液(B)，洗脱条件为0-60％B梯度洗脱30min，收集洗脱峰置于冻干机(-50℃)中冷冻干燥；纯化干燥后的抗菌肽用HPLC测定纯度并用质谱(MS)检测分子质量与理论计算的分子量相符。本文的抗菌肽合成、纯化、鉴定实验在上海紫域生物科技有限公司完成。d. Weigh 100 mg of the dried above-mentioned solid polypeptide crude product, dissolve in 10 mL of 0.1% trifluoroacetic acid (TFA), filter the sample with a 0.22 μm membrane, and separate and purify it on reverse-phase high performance liquid chromatography (RP-HPLC); The eluents were 0.1% TFA aqueous solution (A) and 0.1% acetonitrile (ACN) solution (B), the elution conditions were 0-60% B gradient elution for 30 min, and the elution peaks were collected and placed in a lyophilizer (-50 ℃); the purified and dried antimicrobial peptides were purified by HPLC, and the molecular mass was detected by mass spectrometry (MS), which was consistent with the theoretically calculated molecular weight. The antimicrobial peptide synthesis, purification and identification experiments in this paper were completed in Shanghai Ziyu Biotechnology Co., Ltd.

2、抗菌肽a和抗菌肽Lfcin B_4-9RRWQWR的制备2. Preparation of antimicrobial peptide a and antimicrobial peptide Lfcin B_4-9 RRWQWR

按照标准的Fmoc固相程序人工合成抗菌肽a和抗菌肽Lactoferricin B_4-9，合成肽经反相HPLC(Vydac 218TP1022柱2.2×25cm)纯化，采用乙腈/水/三氟乙酸系统洗脱，MALDI-TOF和EPI质谱分析。抗菌肽合成由上海紫域生物科技有限公司。Antimicrobial peptide a and antibacterial peptide Lactoferricin B_4-9 were synthesized artificially according to the standard Fmoc solid-phase procedure. The synthetic peptides were purified by reverse-phase HPLC (Vydac 218TP1022 column 2.2×25 cm), eluted with acetonitrile/water/trifluoroacetic acid system, MALDI -TOF and EPI mass spectrometry. Antimicrobial peptides were synthesized by Shanghai Ziyu Biotechnology Co., Ltd.

实施例2Example 2

抗菌肽的抗菌活性检测Antibacterial activity detection of antimicrobial peptides

以下所使用的各种标准菌株购于广东省微生物菌种保藏中心，耐药菌由第三军医大学提供。The various standard strains used below were purchased from the Guangdong Provincial Microbial Culture Collection Center, and the drug-resistant bacteria were provided by the Third Military Medical University.

采用琼脂板扩散法对合成抗菌肽b和抗菌肽a的抗菌活性进行检测，并用天然抗菌肽Lfcin B_4-9作为对照，以评价本发明中抗菌肽b和抗菌肽a的杀菌活性。The antibacterial activity of synthetic antibacterial peptide b and antibacterial peptide a was detected by agar plate diffusion method, and natural antibacterial peptide Lfcin B_4-9 was used as a control to evaluate the bactericidal activity of antibacterial peptide b and antibacterial peptide a in the present invention.

按以下步骤进行测定抗菌肽的抗菌活性：The antibacterial activity of antimicrobial peptides was determined by the following steps:

a.菌种复苏：用接种环分别挑取适量大肠杆菌、金黄色葡萄球菌、鲍曼不动杆菌、铜绿假单胞菌、粪肠球菌、MRSA、耐单种药鲍曼不动杆菌(对头孢他啶耐药，下同)、多重耐药鲍曼不动杆菌(对庆大霉素和头孢他啶同时耐药，下同)于各自培养基中划线，标记。37℃恒温培养箱倒置培养(12-16h)。a. Bacterial recovery: Use an inoculation loop to pick appropriate amounts of Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecalis, MRSA, and single drug-resistant Acinetobacter baumannii (for Ceftazidime resistance, the same below) and multidrug-resistant Acinetobacter baumannii (resistance to both gentamicin and ceftazidime, the same below) were streaked and marked in their respective culture media. 37 ℃ constant temperature incubator inverted culture (12-16h).

b.菌种培养：分别挑选单个菌落到100ml液体MHB培养液，置37℃、160转，摇床培养(12-16h)。b. Bacterial culture: select a single colony to 100ml of liquid MHB culture solution, set it at 37°C, 160 rpm, and cultivate on a shaker (12-16h).

c.菌悬液制备：制备菌悬液的浊度在0.5麦氏浊度左右，此时细菌菌落数约为1.5×10⁸cfu/ml，然后再按1:1000稀释成10⁵-10⁶cfu/ml的菌悬液。c. Preparation of bacterial suspension: the turbidity of the prepared bacterial suspension is about 0.5 McFarland turbidity, and the number of bacterial colonies is about 1.5×10⁸ cfu/ml, and then diluted by 1:1000 to 10⁵ -10⁶ cfu/ml of bacterial suspension.

d.抑菌实验：将稀释好的大肠杆菌、金黄色葡萄球菌、鲍曼不动杆菌、铜绿假单胞菌、粪肠球菌、MRSA、耐单种药鲍曼不动杆菌、多重耐药鲍曼不动杆菌菌悬液分别按0.2ml/平板(平板直径为150mm)的量均匀涂于40mL固体LB培养基；待培养基完全凝固后，打孔直径8mm 6个/平板，标记为+/-/CAZ/A/B/C，分别代表阳性对照(庆大霉素)、阴性对照(去离子水)、头孢他啶、三条多肽(A为抗菌肽a；B为抗菌肽b；C为LfcinB64-9)。“+”孔加50ul 1mg/ml庆大霉素溶液；“-”孔加50μl去离子水；“CAZ”孔加50ul 1mg/ml头孢他啶溶液；A/B/C孔分别加50ul 1mg/ml抗菌肽溶液。4℃静置3h后，37℃恒温培养箱培养，12h后观察肉眼抑菌圈大小并测定抑菌圈直径。每条肽在每种菌种下独立进行3次实验。d. Antibacterial test: the diluted Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecalis, MRSA, single drug resistant Acinetobacter baumannii, multidrug resistant The Acinetobacter manis bacteria suspension was evenly coated on 40mL solid LB medium in the amount of 0.2ml/plate (plate diameter is 150mm); -/CAZ/A/B/C, representing positive control (gentamicin), negative control (deionized water), ceftazidime, three polypeptides (A is antimicrobial peptide a; B is antimicrobial peptide b; C is LfcinB64- 9). Add 50ul 1mg/ml gentamicin solution to "+" well; add 50ul deionized water to "-" well; add 50ul 1mg/ml ceftazidime solution to "CAZ" well; add 50ul 1mg/ml antibacterial to well A/B/C Peptide solution. After standing at 4°C for 3 hours, cultured in a constant temperature incubator at 37°C, and after 12 hours, the size of the inhibition zone was observed with the naked eye and the diameter of the inhibition zone was determined. Three experiments were performed independently for each peptide under each strain.

表1 1mg/ml抗菌肽对不同细菌的抗菌活性Table 1 Antibacterial activity of 1mg/ml antimicrobial peptides against different bacteria

注：CAZ-头孢他啶；“–”表示无明显抑菌圈Note: CAZ-ceftazidime; "-" means no obvious inhibition zone

从上表1看出本发明的抗菌肽b和抗菌肽a杀菌能力明显且优于天然抗菌肽。It can be seen from the above table 1 that the antimicrobial peptide b and the antimicrobial peptide a of the present invention have obvious bactericidal ability and are superior to natural antimicrobial peptides.

实施例3合成抗菌肽的抑菌活性检测Example 3 Detection of antibacterial activity of synthetic antimicrobial peptides

以下所使用的各种标准菌株购于广东省微生物菌种保藏中心，耐药菌由第三军医大学提供。The various standard strains used below were purchased from the Guangdong Provincial Microbial Culture Collection Center, and the drug-resistant bacteria were provided by the Third Military Medical University.

采用96孔板法对合成抗菌肽的杀菌活性进行检测，并用天然抗菌肽Lfcin B_4-9作为对照，以评价抗菌肽a、b的抑菌活性。The bactericidal activity of synthetic antimicrobial peptides was detected by 96-well plate method, and the natural antimicrobial peptide Lfcin B_4-9 was used as a control to evaluate the bacteriostatic activity of antimicrobial peptides a and b.

按以下步骤进行测定抗菌肽的抑菌活性：The antibacterial activity of antimicrobial peptides was determined according to the following steps:

a.将大肠杆菌、金黄色葡萄球菌、鲍曼不动杆菌、铜绿假单胞菌、粪肠球菌、MRSA、耐单种药鲍曼不动杆菌、多重耐药鲍曼不动杆菌在灭菌NA培养基平板上过夜培养，挑取单菌落接种于灭菌MHB培养液，37℃170r/min培养18～24h。a. Sterilize Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecalis, MRSA, single-drug-resistant Acinetobacter baumannii, and multidrug-resistant Acinetobacter baumannii Culture on NA medium plate overnight, pick a single colony and inoculate it in sterilized MHB medium, and cultivate at 37°C for 18-24h at 170r/min.

b.将培养后的菌液稀释成10⁵-10⁶cfu/ml的菌悬液。分别将有抑菌活性的待测拟肽溶液按2倍连续稀释后加入96孔微量培养板中，第1孔及第12孔留空不用(避免边际效应致使数据不准确)。除第2孔加入稀释菌液160μl外，3-9各孔均加入100μl，继于第2孔加入抗菌肽原液(浓度1280μg/ml)40μl，混合后吸出100μl加入第3孔中，依次稀释至第9孔，弃去100μl。这样待测抗菌拟肽的终浓度分别为:b. Dilute the cultured bacterial solution into a bacterial suspension of 10⁵ -10⁶ cfu/ml. The peptoid solution with antibacterial activity to be tested was serially diluted by 2 times and added to the 96-well microplate. In addition to adding 160 μl of diluted bacterial solution to the second hole, 100 μl was added to each well of 3-9, followed by adding 40 μl of antimicrobial peptide stock solution (concentration 1280 μg/ml) to the second well, and after mixing, aspirated 100 μl and added it to the third well, and diluted to For the 9th well, discard 100 μl. The final concentrations of the antibacterial peptoids to be tested are as follows:

取浓度为1280μg/ml的待测拟肽稀释为如下浓度(μg/ml)：Take the peptoid to be tested with a concentration of 1280 μg/ml and dilute it to the following concentration (μg/ml):

编号NumberingA2A2A3A3A4A4A5A5A6A6A7A7A8A8A9A9浓度concentration256256128128646432321616884422

第10孔生长对照，第11孔为阳性对照(亚胺培南)，加160μl菌液和40μl同浓度抗生素溶液混合后弃100μl。每个抗菌肽平行做3次。于37℃培养16h后，用ELISA酶标仪分别测OD₆₀₀值，最小的OD₆₀₀值所对应的最小浓度便为该抗菌肽的MIC值。据此计算抗菌肽a、b分别对大肠杆菌、金黄色葡萄球菌、鲍曼不动杆菌、铜绿假单胞菌、粪肠球菌、MRSA、耐单种药鲍曼不动杆菌、多重耐药鲍曼不动杆菌的MIC。The 10th well is a growth control, and the 11th well is a positive control (imipenem), add 160 μl of bacterial solution and 40 μl of antibiotic solution of the same concentration, and then discard 100 μl. Each antimicrobial peptide was done 3 times in parallel. After culturing at 37°C for 16 hours, the OD₆₀₀ values were measured with an ELISA microplate reader, and the minimum concentration corresponding to the minimum OD₆₀₀ value was the MIC value of the antimicrobial peptide. Based on this, the antimicrobial peptides a and b were calculated against Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecalis, MRSA, single-drug-resistant Acinetobacter baumannii, and multidrug-resistant MIC of Acinetobacter mansibacter.

表2抗菌肽对不同细菌的最小抑菌浓度(MIC)Table 2 The minimum inhibitory concentration (MIC) of antimicrobial peptides against different bacteria

上表中的最小抑菌浓度值越小，则代表抗菌能力越强。本发明的合成抗菌肽a和b的MIC比Lfcin B_4-9均要小，合成抗菌肽a和b的抗菌能力大大强于抗菌肽Lfcin B_4-9，但是抗菌肽b特别对耐药细菌鲍曼不动杆菌杀菌效果更佳。The smaller the minimum inhibitory concentration value in the above table, the stronger the antibacterial ability. The MICs of the synthetic antimicrobial peptides a and b of the present invention are smaller than those of Lfcin B_4-9 , and the antibacterial abilities of the synthetic antimicrobial peptides a and b are much stronger than those of the antimicrobial peptide Lfcin B_4-9 , but the antimicrobial peptide b is particularly resistant to drug-resistant bacteria. The bactericidal effect of Acinetobacter baumannii is better.

实施例4体外溶血活性检测Example 4 In vitro hemolytic activity detection

本实施例用于检测合成抗菌肽对人体红细胞是否具有溶血活性，并用固相化学法合成的抗菌肽a和抗菌肽Lfcin B_4-9作为对照。使用的血样取于无菌绵羊血。This example is used to detect whether the synthetic antimicrobial peptide has hemolytic activity on human erythrocytes, and the antimicrobial peptide a and antimicrobial peptide Lfcin B_4-9 synthesized by solid-phase chemical method are used as controls. The blood samples used were obtained from sterile sheep blood.

溶血活性的检测步骤是：The detection steps for hemolytic activity are:

a.将无菌绵羊血离心5min 3000rpm/min，并用PBS缓冲液冲洗3次，重复离心操作，弃去上清液，保留红细胞。a. Centrifuge sterile sheep blood at 3000 rpm/min for 5 min, rinse with PBS buffer three times, repeat the centrifugation operation, discard the supernatant, and retain the red blood cells.

b.0.1ml红细胞液用9.9ml PBS液稀释(红细胞最终浓度为1％)，分别取浓度为512、256、128、64、32、16、8μg/ml的抗菌肽200μl与200μl稀释红细胞液混合后在37℃孵育1小时，然后在4000rpm/min离心5分钟，取悬浮液转移到96孔ELISA板并分别在414nm波长检测悬浮液的吸光值，计算每孔的溶血率。0.01mol/L的PBS作为阴性对照，0.1％Triton-X100(聚乙二醇辛基苯基醚)作为阳性对照。溶血率(％)＝(试验管吸光度-阴性对照管吸光度)/(阳性对照管吸光度-阴性对照管吸光度)×100％。b. 0.1ml of red blood cell solution was diluted with 9.9ml of PBS solution (the final concentration of red blood cells was 1%), and 200μl of antimicrobial peptides with concentrations of 512, 256, 128, 64, 32, 16, and 8μg/ml were mixed with 200μl of diluted red blood cell solution After incubation at 37°C for 1 hour, centrifugation at 4000 rpm/min for 5 minutes, the suspension was transferred to a 96-well ELISA plate and the absorbance of the suspension was detected at a wavelength of 414 nm, and the hemolysis rate of each well was calculated. 0.01mol/L PBS was used as a negative control, and 0.1% Triton-X100 (polyethylene glycol octyl phenyl ether) was used as a positive control. Hemolysis rate (%)=(absorbance of test tube-absorbance of negative control tube)/(absorbance of positive control tube-absorbance of negative control tube)×100%.

c.溶血率实验以抗菌肽a和Lfcin B_4-9为对照，独立三次重复实验。c. Hemolysis rate experiment With antimicrobial peptide a and Lfcin B_4-9 as controls, the experiment was repeated three times independently.

表4三种抗菌肽溶血活血检测结果Table 4 Three kinds of antibacterial peptides hemolysis and blood activation test results

抗菌肽的溶血率值越小，则代表抗菌肽的溶血毒性越小。从表中结果可以看出抗菌肽a、b的溶血毒性都在5％以下，即使在浓度升高的情况下其溶血毒性的增加也不明显。相比于对照天然抗菌肽溶血毒性非常低，特别是在高浓度下未有大幅度的溶血现象发生，是下一步成药的重要基础。The smaller the hemolysis rate value of the antimicrobial peptide, the smaller the hemolytic toxicity of the antimicrobial peptide. From the results in the table, it can be seen that the hemolytic toxicity of antimicrobial peptides a and b are all below 5%, and the increase in hemolytic toxicity is not obvious even when the concentration increases. Compared with the control natural antibacterial peptide, the hemolytic toxicity is very low, especially at high concentrations, there is no significant hemolysis, which is an important basis for the next step.

<210> 1<210> 1

<211>6<211>6

<212> PRT<212> PRT

<213> 人工合成<213> Synthetic

<220><220>

<223> 抗菌肽a<223> Antibacterial peptide a

<400> 1<400> 1

Arg-Arg-Trp-Trp-Arg-TrpArg-Arg-Trp-Trp-Arg-Trp

1 5 61 5 6

<210> 1<210> 1

<211>6<211>6

<212> PRT<212> PRT

<213> 人工合成<213> Synthetic

<220><220>

<223> 抗菌肽b<223> Antimicrobial peptide b

<400>2<400>2

Arg-Arg-Trp-Trp-Arg-Trp-β-苯乙胺Arg-Arg-Trp-Trp-Arg-Trp-β-phenethylamine

1 5 61 5 6

Claims (1)

1. An application of an antibacterial hexapeptide derivative in preparing a medicament for treating or preventing multiple drug-resistant acinetobacter baumannii, single drug-resistant acinetobacter baumannii and pseudomonas aeruginosa infection, wherein the sequence of the antibacterial hexapeptide derivative is as follows: Arg-Arg-Trp-Trp-Arg-Trp-beta-phenylethylamine.

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