WO2011069280A1 - Analogue de pexiganan antimicrobien et son procédé de préparation - Google Patents

Analogue de pexiganan antimicrobien et son procédé de préparation Download PDF

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Publication number
WO2011069280A1
WO2011069280A1 PCT/CN2009/001423 CN2009001423W WO2011069280A1 WO 2011069280 A1 WO2011069280 A1 WO 2011069280A1 CN 2009001423 W CN2009001423 W CN 2009001423W WO 2011069280 A1 WO2011069280 A1 WO 2011069280A1
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WO
WIPO (PCT)
Prior art keywords
pexiganan
lys
analogue
analog
ttt
Prior art date
Application number
PCT/CN2009/001423
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English (en)
Chinese (zh)
Inventor
吴晓琰
孙玉琨
龚铁军
Original Assignee
Wu Xiaoyan
Sun Yukun
Gong Tiejun
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wu Xiaoyan, Sun Yukun, Gong Tiejun filed Critical Wu Xiaoyan
Priority to PCT/CN2009/001423 priority Critical patent/WO2011069280A1/fr
Priority to CN2009801620719A priority patent/CN102656184A/zh
Publication of WO2011069280A1 publication Critical patent/WO2011069280A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
    • 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
    • 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 present invention relates to a Pexiganan analog (1-23) which has an antibacterial action and can be used as an antibacterial polypeptide drug.
  • the present invention also provides a method of producing the Pexiganan analog (1-23) by chemical synthesis or genetic engineering.
  • Insects of organisms invading the outside microbes produce antibacterial peptides that protect the body from pathogens through natural immunity. More than 800 antimicrobial peptides have been isolated from various organisms in the past 20 years, and more than 200 antimicrobial peptides have been isolated from various frog skins. This peptide, which has a relatively low molecular weight (20-60 amino acids), kills bacteria, fungi, protozoa and certain viruses. It is effective against some resistant microorganisms and is a new broad-spectrum antibacterial drug. "New antibiotics”. The antibacterial peptide itself is not toxic, and is amino acid after metabolism, which has been widely noted and studied at home and abroad.
  • the high price of chemical synthetic products limits its wide application.
  • the difficulty in preparing antimicrobial peptides by genetic engineering techniques is that the antibacterial peptides expressed by the fermentation will kill the engineered bacteria, and the fermentation is difficult to carry out.
  • Fermented with Pichia Pastori s yeast the small peptide secreted into the fermentation broth has a small molecular weight, a small amount, and is difficult to recover.
  • it is expressed by fusion protein, which forms insoluble inclusion bodies and avoids damage to engineering bacteria.
  • the fusion protein carrier has a small molecular weight and a high expression level to increase the yield of the polypeptide of interest.
  • the object of the present invention is to overcome the defects of the prior art and to successfully produce by using genetic engineering technology.
  • Pexiganan analogues (1-23) can reduce costs by up to 500 mg per liter of fermentation broth.
  • the Pexiganan analog (1-23) of the present invention is characterized in that the amino acid sequence of the Pexiganan analog (1-23) consists of 23 amino acids, and the Pexiganan analog (1-23) has the structural formula:
  • the 22 amino acid sequence is Pexiganan
  • the C terminus is Arg
  • the C terminus Arg is -OH or NH2.
  • the present invention also provides a method of producing the Pexiganan analog (1-23) by genetic engineering or chemical synthesis.
  • the Pexiganan analog (1-23) of the present invention is an analog of the antibacterial peptide Magainin, which has a strong antibacterial action and can directly kill bacteria, fungi, protozoa and certain viruses without causing drug resistance.
  • the use of the Pexiganan analog (1-23) of the present invention is useful as an antimicrobial polypeptide drug.
  • antibacterial peptides such as Pexiganan used so far are chemically synthesized products, which are expensive and hinder their wide application. Genetic engineering using Pichia Pastoris yeast to secrete the expression of antimicrobial peptides to fermentation In the liquid, since the molecular weight is small and the amount of expression is also low, it is very difficult to recover from the fermentation broth. Antibacterial peptide
  • Pexigarmn analogues (1-23) are expressed in the form of fusion proteins, which are simple to prepare and have industrial production significance.
  • Figure 1 is an HPLC detection map of Pexiganan analog (1-23)
  • Figure 2 is a diagram showing the bactericidal effect of Pexiganan analogues (1-23);
  • Figure 3 is a diagram showing the synthesis process of a Pexiganan analogue (1-23) synthesized by solid phase synthesis. detailed description
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • the solid phase resin is Wang Resin, which performs solid phase synthesis. After the synthesis, the obtained peptide resin with side chain protecting group is cleaved, that is, the connection between the polypeptide and the resin is cut off, and the deprotection step is completed in one step.
  • the C-terminus is a free carboxyl group.
  • the reagents used were of analytical grade or peptide synthesis grade: N-methylpyrrolidone, chloroform, hexahydrobipyridyl, 2.0 M DIEA NMP, DMAP/DMF, HBTU 100 mmole/0.5M HOBT in F, and the like.
  • Electromagnetic stirring at a constant temperature of 30 ° C for 6 hours removing the resin by filtration, collecting the supernatant, and washing the resin once with a small amount of trifluoroacetic acid, combining the collected liquid, adding diethyl ether (about 20-30 ml) to precipitate, and filtering with diethyl ether. The precipitate was washed once and immediately placed in a desiccator.
  • the crude Pexiganan analog (1-23) was dissolved in 0.5% acetic acid at a concentration of 5 mg/ml, and subjected to HPLC.
  • the main peak of Pexiganan analog (1-23) was purified by preparative HPLC using Dalian elestat.
  • the separation and purification conditions were as follows: Gradient separation was carried out by using 0.1% trifluoroacetic acid and 80% acetonitrile containing 0.1% trifluoroacetic acid, and the obtained Pexiganan analog (1-23) was concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain a product.
  • the fusion protein is cleaved by Clostripain.
  • GCA CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG CCG TTT CGC AAG CAA
  • Lys lie Leu Lys Lys Arg
  • the Arg-containing codon was amplified by a conventional PCR method, cloned into a Lac-containing promoter plasmid to form a fusion protein gene, and an expression plasmid was constructed and transfected into Escherichia coli JM109.
  • Pexiganan analogue (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, added with ampicillin (Ampici ll in ) to a final concentration of 50 ⁇ g/ml, 37 ° C, and shaken overnight. 150rpm). On the second day, transfer to 20L New Brunswik fermenter.
  • the culture medium is composed of M9 medium, with glucose concentration of 1%, ampicillin (Ampici ll in ) concentration of 50 ⁇ g/ml, aeration of 20L/min, dissolved oxygen. Maintained at more than 20%, the antifoaming agent is domestically produced, the pH is maintained at 7 ⁇ 8, and it is adjusted with ammonia water.
  • adding of IPTG at a concentration of 0. 5mM, continue to ferment 4 ⁇ 6 hours, bacterial cells were collected by centrifugation wet weight 30 ⁇ 50g / L o
  • the collected cells were homogenized with pH 8.0 Tri s buffer 20 mM, then lysozyme lg/L was added, incubated at 37 ° C for 1 hour, frozen at 20 ° C overnight, sonicated 3 times, each time 1 minute or so.
  • the precipitate was collected by centrifugation, washed 2 times with 2M Urea, and washed once with water. Separation and purification of inclusion bodies: The precipitate is dissolved in 8 M urea. Sephadex G-100 was chromatographed at A280 nm, and the collected solution was dialyzed overnight, lysed with Clostripain protease, and traced by HPLC.
  • the Met-containing codon was amplified by a conventional PCR method, cloned into a Lac-containing promoter plasmid to form a fusion protein gene, and an expression plasmid was constructed and transfected into Escherichia coli JM109.
  • Pexiganan analogue (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, and added with ampicillin (Ampici ll in) to a final concentration of 50 ⁇ g/ml, 37 ° C, and shaken overnight. 150rpm). On the second day, transfer to 20L New Brunswik fermenter.
  • the culture medium is composed of M9 medium, with glucose concentration of 1%, ampicillin (Ampici ll in ) concentration of 50 ⁇ g/ml, aeration of 20L/min, dissolved oxygen. Maintained at more than 20%, the antifoaming agent is domestically produced, the pH is maintained at 7 ⁇ 8, and it is adjusted with ammonia water.
  • Lys lie Leu Lys Lys Arg
  • the codons containing Asn-Gly were amplified by conventional PCR and cloned into the Lac-containing promoter plasmid to form a fusion protein gene, and the expression plasmid was constructed and transfected into Escherichia coli JM109.
  • Pexiganan analog (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, added with ampicillin (Ampici ll in ) to a final concentration of 50 ⁇ g/ml, and 37 shaker overnight (150 rpm). On the second day, transfer to 20L New Brunswik fermenter.
  • the culture medium is composed of M9 medium and added with glucose. Degree 1%, ampicillin (Ampici llin) concentration of 50 ⁇ g / ml, ventilation capacity of 20L / min, dissolved oxygen maintained above 20%, antifoaming agent is domestic foaming enemy, pH is maintained at 7 ⁇ 8, with Ammonia adjustment.
  • IPTG was added, and the concentration was 0.5 mM.
  • the fermentation was continued for 4 to 6 hours, and the cells were collected by centrifugation to a wet weight of 30 to 50 g/L.
  • the fusion protein of Pexiganan analogue (1-23) was lysed with 2M NH20H/6M guanidine Guanidin ⁇ HC1 at pH 9. 0, 45 °C for 8 hours, followed by HPLC, cleavage and ultrafiltration with molecular weight of 1000
  • the salt was purified by HPLC to give the Pexiganan analog (1-23) product.
  • Trp Gly lie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val 4 ⁇ TGG GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC GGC AAA GCG TTC GTT
  • Lys lie Leu Lys Lys Arg
  • Trp-containing codon was amplified by a conventional PCR method, cloned into a Lac-containing promoter plasmid to form a fusion protein gene, and an expression plasmid was constructed and transfected into Escherichia coli JM109.
  • Pexiganan analogue (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, and added with ampicillin (Ampici ll in ) to a final concentration of 50 ⁇ g/ml, 37 ° C, and shaken overnight. 150rpm). On the second day, transfer to 20L New Brunswik fermenter.
  • the culture medium is composed of M9 medium, with glucose concentration of 1%, ampicillin (Ampici ll in ) concentration of 50 ⁇ g/ml, aeration of 20L/min, dissolved oxygen. Maintained at more than 20%, the antifoaming agent is domestically produced, the pH is maintained at 7 ⁇ 8, and it is adjusted with ammonia water.
  • IPTG IPTG
  • the concentration is 0.5 mM
  • reaction was carried out for 48 hours (or 5 hours at 37 ° C), and the reaction was stopped by adding a 10-fold amount of a reducing agent (such as mercaptoethanol) under nitrogen in the dark.
  • a reducing agent such as mercaptoethanol
  • the reaction solution was extracted with ethyl acetate to remove BNPS-Skatole, and the aqueous phase was stored in lyophilized, dissolved and then purified by HPLC to obtain Pexiganan analog (1-23) product.
  • a reducing agent such as mercaptoethanol
  • the Pexiganan analog (1-23) of the present invention has an antibacterial action as an antibacterial polypeptide drug, and can directly kill bacteria, fungi, protozoa and certain viruses without causing drug resistance.
  • the left dish is a Pexiganan analogue (1-23) and the E.Coli resistant bacteria are pre-mixed in a saline solution for half an hour after plating, and the right dish is the premixed physiology of E. coli resistant bacteria.
  • the plate was incubated for half an hour in a saline liquid.
  • the plate was incubated overnight at 37 ° C.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Communicable Diseases (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un analogue de Pexiganan antimicrobien, dont la séquence d'acides aminés est GIGKFLKKAKKFGKAFVKILKKR. En outre, la présente invention concerne également un procédé de préparation de l'analogue.
PCT/CN2009/001423 2009-12-11 2009-12-11 Analogue de pexiganan antimicrobien et son procédé de préparation WO2011069280A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2009/001423 WO2011069280A1 (fr) 2009-12-11 2009-12-11 Analogue de pexiganan antimicrobien et son procédé de préparation
CN2009801620719A CN102656184A (zh) 2009-12-11 2009-12-11 抗菌肽Pexiganan类似物及其制备方法

Applications Claiming Priority (1)

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PCT/CN2009/001423 WO2011069280A1 (fr) 2009-12-11 2009-12-11 Analogue de pexiganan antimicrobien et son procédé de préparation

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104356221A (zh) * 2014-10-24 2015-02-18 杭州阿德莱诺泰制药技术有限公司 一种制备培西加南的方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111925430B (zh) * 2020-08-18 2022-05-24 中国海洋大学 抗菌肽及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5424290A (en) * 1992-10-26 1995-06-13 Magainin Pharmaceuticals Inc. Biologically active peptides and uses therefor
CN1920020A (zh) * 2005-08-24 2007-02-28 天津天士力制药股份有限公司 抗菌肽Pexiganan的基因工程生产方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5424290A (en) * 1992-10-26 1995-06-13 Magainin Pharmaceuticals Inc. Biologically active peptides and uses therefor
CN1920020A (zh) * 2005-08-24 2007-02-28 天津天士力制药股份有限公司 抗菌肽Pexiganan的基因工程生产方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104356221A (zh) * 2014-10-24 2015-02-18 杭州阿德莱诺泰制药技术有限公司 一种制备培西加南的方法

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