WO2024037263A1 - 一种体内低毒性的抑制金葡菌毒素产生的合成肽及其应用 - Google Patents

一种体内低毒性的抑制金葡菌毒素产生的合成肽及其应用 Download PDF

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WO2024037263A1
WO2024037263A1 PCT/CN2023/107416 CN2023107416W WO2024037263A1 WO 2024037263 A1 WO2024037263 A1 WO 2024037263A1 CN 2023107416 W CN2023107416 W CN 2023107416W WO 2024037263 A1 WO2024037263 A1 WO 2024037263A1
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peptide
type
residue
staphylococcus aureus
natural
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French (fr)
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梁小平
王良友
夏文晖
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重程投资管理（上海）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
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 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
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 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
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a synthetic peptide with low toxicity in vivo that inhibits the production of Staphylococcus aureus toxin and its application.
  • Staphylococcus aureus is a common Gram-positive pathogenic bacteria and one of the main microorganisms causing fatal diseases such as burns and war-wound infections, pneumonia, endocarditis, sepsis, and toxic shock. one.
  • clinical treatment of Staphylococcus aureus mostly uses a combination of antibiotics, but the effect is not ideal. Since Staphylococcus aureus is very easy to develop drug resistance and there is no good solution, many commonly used antibiotics are ineffective against it. Controlling Staphylococcus aureus infection is one of the urgent problems in clinical medicine.
  • RNAIII a regulatory RNA molecule
  • RNAIII activates the gene transcription of virulence factors and regulates the translation of virulence factors.
  • the RNAIII level is a protein secreted by Staphylococcus aureus itself, namely RANIII activating protein (RAP).
  • RAP is also called Staphylococcus aureus virulence stimulating factor. Staphylococcus aureus continues to secrete RAP, and only after RAP reaches a certain concentration can it activate the production of virulence factors. S. aureus without RAP production is not pathogenic in itself.
  • Balaban et al. published research results in the journal "Science” showing that the antibodies they prepared to immunize animals with RAP can effectively protect mice from Staphylococcus aureus infection (Balaban N, et al. Autoinducer of virulence as a target for vaccination and therapy against Staphylococcus aureus.Science,1998,280(17):438-440).
  • MRG chemically synthesized modified peptide MRG with the general sequence formula: CH 3 (CH 2 )mXG-(CQHwWHWYC)-(R)nY.
  • MRG is extremely soluble in water and has good anti-Staphylococcus aureus activity.
  • MRG has obtained a U.S. patent (US10905735B2) and a Chinese invention patent (ZL201780021188.X).
  • US10905735B2 US10905735B2
  • ZL201780021188.X Chinese invention patent
  • intravenous administration of MRG in mice is highly toxic. Intravenous administration of more than 15 mg/kg body weight will cause obvious convulsions and even death. Intravenous administration of more than 75 mg/kg body weight will cause all mice to die. cause obvious impact. Therefore, there remains a need for synthetic peptides with low toxicity in vivo that inhibit S. aureus toxin production.
  • the main purpose of the present invention is to improve the in vivo toxicity of modified peptides in the prior art and maintain biological activity.
  • the present invention provides a new synthetic peptide MUS-1 based on MRG sequence modification.
  • the results show that compared with MRG, the modified chemically synthesized peptide MUS-1 is not only highly soluble in water, but also has good anti-Staphylococcus aureus activity.
  • the mice tail vein administration (100mg/kg body weight) did not See obvious toxicity.
  • the chemically synthesized peptide MUS-1 is reconstructed on the basis of the MRG sequence, so the mechanism of action is consistent with MRG, that is, it specifically binds to the autocrine RNAIII activating protein of Staphylococcus aureus and inhibits the toxin production of Staphylococcus aureus. No obvious toxicity was found after administration in mice.
  • one object of the present invention is to provide a chemically synthesized synthetic peptide with low toxicity in vivo that inhibits the production of Staphylococcus aureus toxin.
  • the synthetic peptide can specifically inhibit the production of Staphylococcus aureus toxin.
  • the chemically synthesized synthetic peptide provided by the present invention with low toxicity in vivo and inhibits the production of Staphylococcus aureus toxin is a small molecule polypeptide analogue with the following structural formula: CH 3 (CH 2 ) 10 CO-G-(CQHwWHWYC)-DDD-NH 2
  • G represents: glycine residue
  • C represents: L-type cysteine residue
  • Q represents: L-type glutamine residue
  • H represents: L-type histidine residue
  • W represents: L-type tryptophan Acid residue
  • w represents: D-type tryptophan residue
  • Y represents: L-type tyrosine residue
  • D represents: L-type aspartic acid residue
  • the two cysteine residues represented by C are connected by a disulfide bond.
  • G represents: natural L-glycine residue
  • C represents: natural L-type cysteine residue
  • Q represents: natural L-type glutamine residue
  • H represents: L-type histidine residue
  • W represents: natural L-type Type tryptophan residue
  • w represents: D-isomer of natural tryptophan
  • Y represents: natural L-type tyrosine residue
  • D stands for: natural L-type aspartic acid residue.
  • the chemically synthesized peptide MUS-1 can specifically bind to Staphylococcus aureus virulence stimulating factor RAP.
  • the chemically synthesized peptide MUS-1 can inhibit the production of Staphylococcus aureus toxin.
  • the chemically synthesized peptide MUS-1 is obtained by chemical synthesis.
  • the chemically synthesized peptide MUS-1 has low toxicity in vivo.
  • CH 3 (CH 2 ) 10 CO- is dodecanoyl.
  • Another object of the present invention is to provide the application of the above-mentioned synthetic peptide with low toxicity in vivo that inhibits the production of Staphylococcus aureus toxin in the preparation of drugs against Staphylococcus aureus infection.
  • the present invention provides a method for treating diseases related to Staphylococcus aureus infection, which method includes administering to a patient a therapeutically effective amount of the above-mentioned chemically synthesized peptide MUS-1.
  • the diseases include burns and combat wound infections, pneumonia, endocarditis, sepsis, and toxic shock caused by Staphylococcus aureus infection.
  • the present invention provides a pharmaceutical composition, which contains the above-mentioned chemically synthesized peptide MUS-1, and pharmaceutically acceptable excipients.
  • the main reason for resistance to traditional antibiotic treatments is that bacteria produce inducible enzymes that decompose effective groups in antibiotics under survival pressure after treatment.
  • the present invention uses a polypeptide compound that specifically inhibits RAP activity to establish a treatment plan for Staphylococcus aureus infection, finding a new way out for the treatment of drug-resistant Staphylococcus aureus infection, a common, frequent and fatal disease that has been plaguing clinical practice.
  • the invention is of great significance for the development of new small molecule polypeptide drugs against Staphylococcus aureus infection, and has wide application value and broad market prospects.
  • Figure 1 is a route diagram of the synthetic steps of the synthetic peptide MUS-1 of the present invention.
  • Figure 2 is an HPLC analysis chart of the synthetic peptide MUS-1 of the present invention.
  • Figure 3 is the MS analysis spectrum of the synthetic peptide MUS-1 of the present invention.
  • the amount of Resin (5mmol/0.45mmol/g) is 11.11g. Put the weighed resin into the reaction column and swell it with N,N-dimethylformamide (DMF) for about 30 minutes. Remove the swelling liquid and use DMF Wash 3 times as wash solution.
  • DMF N,N-dimethylformamide
  • Step 3 In step 2, the peptide sequence is coupled to Gly. After the protective group is removed, the last amino acid, dodecanoic acid (lauric acid), needs to be coupled.
  • the condensation reagent used is 1-hydroxybenzotriazole (HOBT), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA), the reaction time is about 1 hour. Use the ninhydrin colorimetric method to detect whether the reaction is complete.
  • HOBT 1-hydroxybenzotriazole
  • HBTU benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate
  • DIPEA N,N-diisopropylethylamine
  • Step 4 Take out the quicksand-like peptide resin in Step 3 and weigh it to obtain 25.1g.
  • First, prepare 200mL of conventional lysis reagent in the ratio of TFA: Anisole: EDT: Anisole 90:5:3:2, and shake well. Then add 8-10mL of cleavage reagent per 1g of peptide resin for lysis. The reaction time is about 2-3 hours. After the reaction is completed, filter off the resin to obtain about 200 mL of filtrate (partial loss during filtration). Slowly add the filtrate to anhydrous ether at a sedimentation ratio of 1:8 (filtrate: methyl tert-butyl ether) and leave it for 30 minutes. After sufficient sedimentation, the linear crude peptide is obtained by centrifugation, washing, and drying:
  • Step 5 Grind the dried linear crude peptide obtained in step 4 to powder, dissolve it in pure water at a concentration of 1mmol/500mL, and obtain a total of 2500mL of linear crude peptide solution. Take out a small sample and perform HPLC analysis to locate the peak time.
  • Step 6 Add the ethanol solution of iodine dropwise to the linear crude peptide solution described in step 5 (dissolve 5g of iodine in 1L of water) until the solution turns light yellow, place it in a 50°C water bath, and stir for 1 hour.
  • Use Vc (take 10g Vc and dissolve it in 1L water) aqueous solution to adjust the solution until it is clear, and obtain the crude solution of the target peptide, namely:
  • Step 7 The crude target peptide after complete cyclization analyzed by HPLC in Step 6 is separated and purified on a C 18 reversed-phase high-performance liquid chromatography column, and after rotary evaporation and freeze-drying, the finished target peptide is obtained.
  • the main steps are as follows:
  • the cyclized crude peptide solution was filtered through a 0.45 ⁇ m filter membrane, and the filtrate was adjusted to pH 4-5 before being purified and prepared by HPLC.
  • HPLC analysis instrument is: DIONEX U3000, analytical column: C 18 , 5 ⁇ m, 4.6 ⁇ 250mm, analysis conditions: mobile phase: Phase A: 1 ⁇ TFA, Phase B: acetonitrile; purification adopts innovative 5cm preparative HPLC, packing C 18 , 10 ⁇ m, 150 ⁇ 250mm.
  • Step 8 Freeze-dry the concentrated liquid packaged in the previous step to obtain white powder, which is the finished product.
  • Pre-freezing First, pre-freeze the concentrated sample solution, that is, place the sample on the partition in the freeze-drying box for pre-freezing. The temperature of the product drops to below -40°C and is maintained for about 120 minutes.
  • Sublimation drying Electric heating is set to 0°C, deviation time is 1 minute, and maintained for about 40 minutes. The electric heating is set to 10°C and the deviation time is 500 minutes. The electric heating is set to 35°C, and the deviation time is 420 minutes.
  • Desorption the temperature rises to about 33°C and is maintained for about 240 minutes.
  • Step 9 Purity identification and structure determination of chemically synthesized MUS-1.
  • Example 2 Observation on the inhibitory effect of cyclic heptapeptide-modified compounds MUS-1 and MRG on Staphylococcus aureus toxin production in vitro
  • Cyclic heptapeptide-modified compound MRG (CH 3 (CH 2 ) 10 CO-G-(CQHwWHWYC)-RRR-NH 2 ) and cyclic heptapeptide-modified compound MUS-1 (CH 3 (CH 2 ) 10 CO-G-(CQHwWHWYC) )-DDD-NH 2 ) were synthesized by Suzhou Tianma Pharmaceutical Group Tianji Biopharmaceutical Co., Ltd., with a purity greater than 98%.
  • Staphylococcus aureus 04018 strain provided by Beijing Institute of Basic Medical Sciences
  • blood plates fresh blood agar culture plates
  • BHI plates were made in our own laboratory
  • imported BHI culture medium (BactoTM Brain Heart Infusin) Purchased from BD Company of the United States
  • DMEM culture medium was purchased from CIBCO Company of the United States
  • imported fetal bovine serum was purchased from PAN BIOTECH Company of the United States
  • 0.22 ⁇ membrane was purchased from PALL Company of the United States
  • MDBK cells were provided by Beijing Institute of Basic Medical Sciences
  • desktop centrifuge Germany EPPDORF Company
  • enzyme linker from MICROPLATE Company of the United States
  • cell culture bottles were purchased from CORNING Company of the United States.
  • the method for determining the inhibition level of Staphylococcus aureus toxin production refers to the literature (Yang G, et al. A novel peptide screened by phage display can mimic TRAP antigen epitope against Staphylococcus aureus infections. J Biol. Chem. 2005, 280: 27431-27435), for details as follows:
  • Example 3 Observation on the survival of animals after cyclic heptapeptide modified compound MUS-1 and MRG were administered to the tail vein of mice
  • mice Different concentrations of MRG and MUS-1 were injected into the tail veins of mice. The activity status of the mice after injection was observed, and the survival of the mice within one week was recorded.
  • mice have good activity status and no obvious adverse conditions. All mice survive and grow normally after one week of observation.
  • MRG was injected into the tail vein of mice at a dose greater than 15 mg/kg body weight, the mice showed obvious convulsions, limited movement, and shortness of breath.
  • Intravenous administration of greater than 75 mg/kg body weight would cause the death of all mice (Table 3).

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Abstract

提供一种体内低毒性的抑制金葡菌毒素产生的合成肽及其应用。所述合成肽的结构为：CH 3(CH 2) 10CO-G-(CQHwWHWYC)-DDD-NH 2。该合成肽通过与金葡菌自分泌的RNAIII激活蛋白特异性结合并抑制金葡菌的毒素产生，并经小鼠静脉给药后未见明显毒性。还提供该合成肽在制备抗金葡菌感染药物的应用。

Description

一种体内低毒性的抑制金葡菌毒素产生的合成肽及其应用 技术领域
本发明涉及一种体内低毒性的抑制金葡菌毒素产生的合成肽及其应用。
背景技术
金黄色葡萄球菌(金葡菌)是一类常见的革兰氏阳性致病菌，是引起烧伤及战伤感染、肺炎、心内膜炎、败血症、中毒性休克等致命性疾病的主要微生物之一。每年仅医院内感染金葡菌的人数就超过数百万，目前临床上对金葡菌的治疗多采用联合使用抗生素的办法，但是效果并不理想。由于金葡菌极易产生耐药性且无好的解决方法，常用的许多抗生素对之无效，控制金葡菌感染是临床医学亟待解决的问题之一。
金葡菌的主要致病物质是毒素，包括溶血毒素、杀白细胞素、肠毒素等。最新研究表明，金葡菌这些毒力因子的合成是受一种可调节RNA分子，即RNAIII控制的，RNAIII激活毒力因子的基因转录，调节毒力因子的翻译。在细菌生长的对数早期其RNAIII水平低，但到对数晚期RNAIII水平会增加40倍，而RNAIII的水平是由金葡菌自身分泌的蛋白，即RANIII激活蛋白(RNA III activating protein，RAP)调节的，故RAP又称为金葡菌毒力刺激因子。金葡菌持续分泌RAP，在RAP达到一定浓度后才有激活毒力因子产生的作用。没有RAP产生的金葡菌本身并不致病。1998年Balaban等在“Science”杂志发表研究结果表明，用他们制备的RAP免疫动物，其抗体可以有效地保护小鼠免受金葡菌的感染(Balaban N,et al.Autoinducer of virulence as a target for vaccine and therapy against Staphylococcus aureus.Science,1998,280(17):438-440)。
本发明人前期经过反复的筛选实验及结构改造，制备了序列通式为：CH3(CH2)m-X-G-(CQHwWHWYC)-(R)n-Y的化学合成修饰肽MRG。MRG能极好地溶于水，而且具有良好的抗金葡菌毒素活性。MRG已经获得美国专利(US10905735B2)和中国发明专利(ZL201780021188.X)。随着深入研究，发现MRG小鼠体内静脉给药毒性较大，大于15mg/kg体重静脉给药会引起明显抽搐甚至死亡，大于75mg/kg体重静脉给药会引起小鼠全部死亡，对成药性造成明显影响。因此，对于体内低毒性的抑制金葡菌毒素产生的合成肽仍存在需求。
发明内容
针对现有技术中的缺陷，本发明的主要目的在于改善现有技术中修饰肽的体内毒性并保持生物活性。
由此，本发明提供一种基于MRG序列改构的新的合成肽MUS-1。结果表明，改造后的化学合成肽MUS-1与MRG相比，不但能极好地溶于水，还具有良好的抗金葡菌毒素活性，小鼠尾静脉给药(100mg/kg体重)未见明显毒性。
所述化学合成肽MUS-1是在MRG序列基础上改构的，因此作用机制与MRG一致，即通过与金葡菌自分泌的RNAIII激活蛋白特异结合并抑制金葡菌的毒素产生，并经小鼠体内给药后未见明显毒性。
所述化学合成肽MUS-1无论从来源还是从结构上完全是新的，未见任何文献报道。
因此，本发明的一个目的是提供一种化学合成的体内低毒性的抑制金葡菌毒素产生的合成肽。该合成肽能特异抑制金葡菌毒素的产生。
本发明提供的化学合成的体内低毒性的抑制金葡菌毒素产生的合成肽，是小分子多肽类似物，结构式如下：
CH3(CH2)10CO-G-(CQHwWHWYC)-DDD-NH2
其中：
G代表：甘氨酸残基；
(CQHwWHWYC)中：C代表：L-型半胱氨酸残基；Q代表：L-型谷氨酰胺残基；H代表：L-型组氨酸残基；W代表：L-型色氨酸残基；w代表：D-型色氨酸残基；Y代表：L-型酪氨酸残基；
D代表：L-型天冬氨酸残基；
C代表的两个半胱氨酸残基通过二硫键相连。
优选地，
G代表：天然L-型甘氨酸残基；
(CQHwWHWYC)中：C代表：天然L-型半胱氨酸残基；Q代表：天然L-型谷氨酰胺残基；H代表：L-型组氨酸残基；W代表：天然L-型色氨酸残基；w代表：天然色氨酸的D-型异构体；Y代表：天然L-型酪氨酸残基；
D代表：天然L-型天冬氨酸残基。
优选地，所述化学合成肽MUS-1能够与金葡菌毒力刺激因子RAP特异结合。
优选地，所述化学合成肽MUS-1能够抑制金葡菌毒素产生。
优选地，所述化学合成肽MUS-1通过化学合成获得。
优选地，所述化学合成肽MUS-1体内低毒性。
CH3(CH2)10CO-为十二烷酰基。
本发明的另一目的是提供上述体内低毒性的抑制金葡菌毒素产生的合成肽在制备抗金葡菌感染的药物中的应用。
此外，本发明提供一种治疗与金葡菌感染相关的疾病的方法，所述方法包括给予患者治疗有效量的上述化学合成肽MUS-1。
优选地，所述疾病包括金葡菌感染引起的烧伤及战伤感染、肺炎、心内膜炎、败血症、中毒性休克。
另外，本发明提供一种药物组合物，其包含上述的化学合成肽MUS-1，以及药学上可接受的辅料。
传统抗生素治疗产生抗药性的原因主要为用药后细菌在生存压力下产生分解抗生素中有效基团的诱导酶。本发明利用特异抑制RAP活性的多肽化合物建立的治疗金葡菌感染的方案，为一直困扰临床的抗药性金葡菌感染这一常见、多发且有致命性的疾病的治疗找到新的出路。
本发明对研制新型抗金葡菌感染的小分子多肽药物具有重要意义，并具有广泛的应用价值及广阔的市场前景。
本申请中，“合成肽”、“化学合成肽”、“环七肽修饰化合物MUS-1”、“MUS-1”具有相同的含义。
附图说明
图1是本发明合成肽MUS-1的合成步骤路线图。
图2是本发明合成肽MUS-1的HPLC分析图谱。
图3是本发明合成肽MUS-1的MS分析图谱。
具体实施方式
下面结合实施例对本发明作进一步详细说明。
MUS-1：CH3(CH2)10CO-G-(CQHwWHWYC)-DDD-NH2
分子式：C91H112N22O22S2
分子量：1930.13
实施例1：环七肽修饰化合物MUS-1的制备
1、合成肽序列：CH3(CH2)10CO-G-(CQHwWHWYC)-DDD-NH2(分子量：1930.13)
2、合成步骤(见图1，为本发明合成肽MUS-1的合成步骤路线图)：
步骤一：合成5mmol MUS-1，选取Rink Amide-AM Resin替代度为0.45mmol/g，根据公式称取树脂的量＝合成的摩尔量/树脂的替代度即需要称取的树脂Rink Amide-AM Resin的量(5mmol/0.45mmol/g)11.11g，将称量好的树脂投入到反应柱中用N,N-二甲基甲酰胺(DMF)进行溶胀约30min，抽掉溶胀液后用DMF作为洗涤溶液洗涤3遍。
步骤二：分两次将脱保护试剂(V哌啶:VDMF＝1:4)加入到步骤一中溶胀好的树脂中鼓氮气反应，分别反应5min和15min，脱保护洗涤后采用茚检试剂：(a)5％茚三酮的无水乙醇溶液(w/v)；(b)苯酚：无水乙醇溶液(4:1，w/v)；(c)吡啶。每个检测试剂滴加二滴，105℃条件下加热3-5min观察其颜色，若显蓝色可以抽掉保护液，若不显色说明脱保护不彻底立刻查明原因重新脱除。去保护完毕后DMF洗涤六遍即可投入Fmoc-Asp(OtBu)-OH和缩合试剂1-羟基苯并三唑(HOBt)及N,N'-二异丙基碳二亚胺(DIC)进行偶联反应(偶联顺序根据肽序从C端至N端，逐个偶联Fmoc保护的氨基酸)，反应时间2-3h。反应完毕后仍用茚三酮显色反应实验来检测反 应是否完全，即取少量树脂于检测管中将上述三种检测液每个溶液滴加二滴105℃下加热3-5min观察其颜色，若无色说明反应完全，反之说明反应不完全，需补投料或重复投料至反应完全。按上述所述方法以此类推将肽序中的氨基酸依次偶联完毕得到：
Gly-Cys(Trt)-Gln(Trt)-His(Trt)-D-Trp(Boc)-Trp(Boc)-His(Trt)-Trp(Boc)-Tyr(tBu)-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Rink Amide-AM Resin
步骤三：在步骤二中肽序偶联至Gly在脱完保护基的情况下需偶联最后一个氨基酸即十二烷酸(月桂酸)，采用的缩合试剂为1-羟基苯并三唑(HOBT)、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)和N,N-二异丙基乙胺(DIPEA)，反应时间约1h。采用茚三酮显色法检测反应是否完全，反应完全后用DMF洗涤树脂3-4遍，甲醇(MeOH)收缩树脂三遍，每遍收缩时间为10min,再将树脂真空抽干至流沙状即得到目的肽的线性肽树脂：
CH3(CH2)10CO-Gly-Cys(Trt)-Gln(Trt)-His(Trt)-D-Trp(Boc)-Trp(Boc)-His(Trt)-Trp(Boc)-Tyr(tBu)-Cys(Trt)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Rink Amide-AM Resin
步骤四：取出步骤三中流沙状的肽树脂称量得到25.1g，首先按TFA:苯甲硫醚:EDT:苯甲醚＝90:5:3:2比例配制200mL的常规裂解试剂，摇匀后按每1g肽树脂加8-10mL的裂解试剂进行裂解，反应时间约2-3h。反应完毕后滤除树脂得到滤液约200mL(在过滤中有部分损失)，按沉降比例1:8(滤液:甲基叔丁基醚)的比例将滤液缓慢加入到无水乙醚中，放置30min，充分沉降后通过离心、洗涤、干燥获得线性粗肽：
CH3(CH2)10CO-Gly-Cys-Gln-His-D-Trp-Trp-His-Trp-Tyr-Cys-Asp-Asp-Asp-NH2，干燥后得到粗肽8.71g。
步骤五：将步骤四中获得干燥后的线性粗肽研磨至粉末状，用纯水溶解，浓度为1mmol/500mL，从而获得线性粗肽溶液共2500mL，取出小样HPLC分析定位出峰时间。
步骤六：向步骤五中所述线性粗肽溶液中滴加碘的乙醇溶液(取5g碘放入1L水中溶解)，直至溶液变成浅黄色，放入50℃水浴中，搅拌1h。用Vc(取10g Vc放入1L水中溶解)水溶液将溶液调至澄清，得到目的肽粗品溶液，即：
CH3(CH2)10CO-Gly-Cyclo(Cys-Gln-His-D-Trp-Trp-His-Trp-Tyr-Cys)-Asp-Asp-Asp-NH2
步骤七：将步骤六中HPLC分析环化完全后的目的肽粗品经过C18反相高效液相色谱柱分离纯化，旋蒸冻干后，得目的肽成品。主要步骤如下：
将环化粗肽溶液经0.45μm滤膜进行过滤，将滤液调至pH4-5后进行HPLC纯化制备。
HPLC分析仪器为：DIONEX U3000，分析柱：C18、5μm、4.6×250mm，分析条件：流动相：A相：1‰TFA，B相：乙腈；纯化采用创新5cm制备型HPLC，填料C18、10μm、150×250mm。
将上述过滤后的粗品溶液使用5cm制备型HPLC纯化，流动相：A相：TFA(取1mL的TFA 加入1000mL水中)，B相：100％乙腈；检测波长：λ＝230nm；柱温：室温；用干净的三角烧瓶收集样品，收集样品纯度大于98％单杂小于1％的纯化溶液为合格品，不合格样品重复上面步骤，将溶液全部处理完，合并合格纯化溶液后进行减压旋蒸浓缩。
步骤八：将上步分装好的浓缩液冷冻干燥得白色粉末，即得成品。
预冻：首先将浓缩好的样品溶液先预冻，即将样品置于冻干箱内隔板上进行预冻，制品温度下降至-40℃以下，维持约120min。
升华干燥：电加热设置为0℃，偏差时间1min，维持约40min。电加热设置为10℃，偏差时间500min。电加热设置为35℃，偏差时间420min。
解吸附：温度升至33℃左右，保持约240min。
步骤九：化学合成MUS-1的纯度鉴定及结构确定。
将合成制备得到的环七肽修饰化合物MUS-1经HPLC分析，纯度大于98％(见图2，为合成肽MUS-1的HPLC分析图谱)；MUS-1理论分子量1930.13，质谱测定分子量为1930.1([M+2H]2+966.0，[M+H]+1931.1。图3为合成肽MUS-1的MS分析图谱)。
表1合成肽MUS-1的HPLC分析数据
实施例2：环七肽修饰化合物MUS-1和MRG体外对金葡菌毒素产生的抑制作用观察
1、实验试剂、耗材及仪器
环七肽修饰化合物MRG(CH3(CH2)10CO-G-(CQHwWHWYC)-RRR-NH2)和环七肽修饰化合物MUS-1(CH3(CH2)10CO-G-(CQHwWHWYC)-DDD-NH2)均由苏州天马医药集团天吉生物制药有限公司合成，纯度大于98％。金葡菌04018菌株：北京基础医学研究所提供；血平板(新鲜血琼脂培养板)购自北京澳博星生物技术有限责任公司、BHI平板本室自制；进口BHI培养基(BactoTM Brain Heart Infusin)购自美国BD公司；DMEM培养基购自美国CIBCO公司；进口胎牛血清购自美国PAN BIOTECH公司；0.22μ膜购自美国PALL公司；MDBK细胞由北京基础医学研究所提供；台式离心机：德国EPPDORF公司；酶联仪：来自美国MICROPLATE公司；细胞培养瓶购自美国CORNING公司。
2、实验方法
金葡菌毒素产生抑制水平测定方法参照文献(Yang G,et al.A novel peptide screened by phage display can mimic TRAP antigen epitope against Staphylococcus aureus infections.J Biol.Chem.2005,280:27431-27435)，具体如下：
(1)取-70℃冰箱中冻存的金葡菌菌株04018，划线至血平板或者BHI平板内；37℃培养箱中培养16h后，观察板内长出单克隆，放置4℃冰箱中备用；
(2)8小时后在超净台内随机挑取平板内的单克隆菌落，接入5mL BHI培养基试管中，共挑取2管；37℃摇床，200rpm振摇，16h后收菌，将2管菌液均匀混和，备用；
(3)化学合成肽MRG和MUS-1及阳性对照品(金葡菌毒素抑制蛋白TP)分别用生理盐水溶解，稀释成不同浓度，加入细菌BHI培养基中。阴性对照组为金葡菌BHI培养基；另设正常细胞加BHI培养基对照组。
(4)将不同浓度的化学合成肽与金葡菌04018共孵育，培养6h后收菌，将细菌悬液按照编号分别取700μL加入至EP管中，离心，8000rpm，5min，取上清，100℃煮沸7min后，14000rpm离心10min；取10μL上清加入预先接种1×105/mL MDBK细胞的96孔细胞培养板内；继续培养18-20h后，加入MTT液(5mg/mL)5μL/孔，3h后加入10％SDS+0.01M HCl液100μL/孔终止。37℃继续孵箱培养20h后，酶联仪595nm处检测并读数。
3、实验结果与结论
实验结果表明，本发明环七肽修饰化合物MUS-1能很好溶于生理盐水；该化学合成肽能够较好地抑制金葡菌毒素产生，对金葡菌毒素引起的MDBK细胞的增殖抑制具有明显保护作用，呈一定的剂量-效应关系。MUS-1的体外抑制金葡菌毒素活性与MRG相当(表2)。
表2 MUS-1和MRG体外对金葡菌毒素产生的抑制作用观察
实施例3：环七肽修饰化合物MUS-1和MRG小鼠尾静脉给药动物存活数观察
1、实验试剂、耗材及仪器
环七肽修饰化合物MRG(CH3(CH2)10CO-G-(CQHwWHWYC)-RRR-NH2)和环七肽修饰化合 物MUS-1(CH3(CH2)10CO-G-(CQHwWHWYC)-DDD-NH2)均由苏州天马医药集团天吉生物制药有限公司化学合成，纯度大于95％。MRG和MUS-1分别用生理盐水溶解，稀释成不同浓度。BALB/c小鼠(35只，雌性，8周)购自维通利华公司。
2、实验方法
小鼠尾静脉分别注射不同浓度的MRG和MUS-1，观察注射药物后小鼠活动状态，记录一周内小鼠存活情况。
3、实验结果
实验结果发现，本发明环七肽修饰化合物MUS-1在静脉注射剂量达100mg/kg体重时小鼠活动状态良好，无明显不良状态，观察一周皆存活，生长正常。小鼠尾静脉注射MRG剂量大于15mg/kg体重时，小鼠出现明显抽搐，活动受限，呼吸急促，大于75mg/kg体重静脉给药会引起小鼠全部死亡(表3)。
表3 MUS-1和MRG小鼠尾静脉给药动物存活数观察
基于以上发明内容的描述，本领域技术人员能够全面地应用本发明，所有相同原理或类似的改动均应视为包括在本发明的范围之内。

Claims (10)

  1. 一种肽，结构式如下：
    CH3(CH2)10CO-G-(CQHwWHWYC)-DDD-NH2
    其中：
    G代表：甘氨酸残基；
    (CQHwWHWYC)中：C代表：L-型半胱氨酸残基；Q代表：L-型谷氨酰胺残基；H代表：L-型组氨酸残基；W代表：L-型色氨酸残基；w代表：D-型色氨酸残基；Y代表：L-型酪氨酸残基；
    D代表：L-型天冬氨酸残基；
    C代表的两个半胱氨酸残基通过二硫键相连。
  2. 根据权利要求1所述的肽，其中，
    G代表：天然L-型甘氨酸残基；
    (CQHwWHWYC)中：C代表：天然L-型半胱氨酸残基；Q代表：天然L-型谷氨酰胺残基；H代表：L-型组氨酸残基；W代表：天然L-型色氨酸残基；w代表：天然色氨酸的D-型异构体；Y代表：天然L-型酪氨酸残基；
    D代表：天然L-型天冬氨酸残基。
  3. 根据权利要求1所述的肽，其中，所述肽能够与金葡菌毒力刺激因子RAP特异结合。
  4. 根据权利要求1所述的肽，其中，所述肽能够抑制金葡菌毒素产生。
  5. 根据权利要求1所述的肽，其中，所述肽通过化学合成获得。
  6. 根据权利要求1所述的肽，其中，所述肽体内低毒性。
  7. 权利要求1所述的肽在制备抗金葡菌感染的药物中的应用。
  8. 一种治疗与金葡菌感染相关的疾病的方法，所述方法包括给予患者治疗有效量的权利要求1的肽。
  9. 根据权利要求8所述的方法，其中，所述疾病包括金葡菌感染引起的烧伤及战伤感染、肺炎、心内膜炎、败血症、中毒性休克。
  10. 一种药物组合物，其包含权利要求1所述的肽，以及药学上可接受的辅料。
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