WO2016195336A1 - Peptide antibactérien ciblant un système toxine-antitoxine de mycobacterium tuberculosis et utilisation correspondante - Google Patents

Peptide antibactérien ciblant un système toxine-antitoxine de mycobacterium tuberculosis et utilisation correspondante Download PDF

Info

Publication number
WO2016195336A1
WO2016195336A1 PCT/KR2016/005657 KR2016005657W WO2016195336A1 WO 2016195336 A1 WO2016195336 A1 WO 2016195336A1 KR 2016005657 W KR2016005657 W KR 2016005657W WO 2016195336 A1 WO2016195336 A1 WO 2016195336A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
toxin
antibiotic
mycobacterium tuberculosis
protein
Prior art date
Application number
PCT/KR2016/005657
Other languages
English (en)
Korean (ko)
Inventor
이봉진
이인균
이상재
이기영
Original Assignee
서울대학교산학협력단
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 서울대학교산학협력단 filed Critical 서울대학교산학협력단
Publication of WO2016195336A1 publication Critical patent/WO2016195336A1/fr

Links

Images

Classifications

    • 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
    • A61K38/08—Peptides having 5 to 11 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
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • 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
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/48—Medical, disinfecting agents, disinfecting, antibacterial, germicidal or antimicrobial compositions
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D9/00—Compositions of detergents based essentially on soap
    • C11D9/04—Compositions of detergents based essentially on soap containing compounding ingredients other than soaps
    • C11D9/22—Organic compounds, e.g. vitamins
    • C11D9/40—Proteins

Definitions

  • the present invention relates to antibiotic peptides and their use for targeting the toxin-antitoxin system of Mycobacterium tuberculosis.
  • Tuberculosis is a serious and infectious acute and chronic disease that can occur anywhere in the body and can even lead to death. About 85% of the lungs develop in the lungs and the bloodstream It can spread and affect any organ in the body along the lymph nodes. Tuberculosis is transmitted from the patient's cough, runny nose and sputum through the air, killing approximately 9 million people in 2013, killing 1.5 million people. In addition, due to the emergence of multidrug-resistant tuberculosis, and more fully resistant tuberculosis bacteria, new antimicrobial agents for treating tuberculosis bacteria are needed.
  • the toxin-antitoxin gene was first known to play a role in maintaining the plasmid of Escherichia coli. When the plasmid containing the toxin-antitoxin gene is lost, the toxin having a stable structure remains but the toxin protein having an unstable structure It breaks down and eventually E. coli dies. Since the discovery of the first toxin-antitoxin gene, it has been found that the toxin-antitoxin gene is present not only in the plasmid but also in the chromosome of E. coli, and is known to be involved in multidrug resistance, biofilm formation and growth inhibition under stress.
  • Toxin-antitoxin systems can be broadly divided into three types (Type I, II, and III).
  • Type I RNA-type toxins attach to the RNA form of toxins and remove the toxicity.
  • type II protein-type antitoxins also attach to the protein form of the toxin and remove the toxicity.
  • type III systems RNA-type antitoxins attach to protein-form toxins and eliminate their toxicity.
  • Type II The most researched of these three types is the Type II system, in which the genes of toxins and antitoxins are encoded as operons.
  • a difficult external condition is encountered by bacteria, such as a rise in temperature or depletion of nutrients, unstable antitoxins are degraded by stress-inducing proteolytic enzymes and the cells are killed because they do not neutralize the toxin's toxicity.
  • the largest part of the type II is the VapBC family, and to date, the toxin (VapC) of the VapBC family is known to inhibit cell growth based on RNA resolution.
  • Toxin-antitoxin systems can be an attractive target for the development of new antibiotics because the artificial separation of the toxin-antitoxin complex will not neutralize the toxic toxins and eventually kill the cells.
  • Mycobacterium tuberculosis more than half of the toxin-antitoxin systems currently belong to the VapBC family, which is known to be involved in the extreme incubation and drug resistance of Mycobacterium tuberculosis. To date, many efforts have been made to develop tuberculosis therapeutics targeting the toxin-antitoxin complex, but so far no successful cases have been developed.
  • VapC30 or toxin
  • Mycobacterium tuberculosis regulates cell growth based on manganese ion-dependent RNA resolution.
  • the structure of the VapBC30 complex was identified, and based on this structure, a peptide was designed to interfere with the binding of the toxin-antitoxin complex, and the peptide successfully inhibited the binding of the toxin-antitoxin complex in vitro.
  • the present invention was completed by revealing that the peptide can be used as an active ingredient of an antibiotic pharmaceutical composition for treating tuberculosis.
  • the present invention provides an antibiotic peptide that inhibits the binding of toxin-antitoxin.
  • the present invention also provides a pharmaceutical composition for antibiotics containing the antibiotic peptide of the present invention as an active ingredient.
  • the present invention also provides an antibiotic quasi-drug containing the antibiotic peptide of the present invention as an active ingredient.
  • the present invention also provides an antibiotic external preparation containing the antibiotic peptide of the present invention as an active ingredient.
  • the present invention provides antibiotic peptides that inhibit toxin-antitoxin binding.
  • the peptide is preferably composed of any one amino acid sequence selected from the group consisting of SEQ ID NO: 4 to 6, but is not limited thereto.
  • the peptide is characterized in that it has an antimicrobial activity against Mycobacterium tuberculosis.
  • the peptide is characterized in that it inhibits the binding of toxin ⁇ 2, ⁇ 4 and antitoxin ⁇ 1, and does not affect toxin activity.
  • the peptide may also be used an amino acid in which one or several amino acids are added, substituted or deleted.
  • a method for synthesizing the peptide it is preferable to synthesize by conventional chemical synthesis method (WH Freeman and Co., Proteins; structures and molecular principles, 1983), specifically, liquid phase peptide synthesis method (Solution Phase Peptide synthesis) , It is more preferably synthesized by solid-phase peptide syntheses, fragment condensation and F-moc or T-BOC chemistry, and more specifically, it is most preferably synthesized by solid-phase peptide synthesis. It doesn't work.
  • the peptide of the present invention can be prepared by the following genetic engineering method.
  • a DNA sequence encoding the peptide is constructed according to a conventional method.
  • DNA sequences can be prepared by PCR amplification using appropriate primers.
  • DNA sequences may be synthesized by standard methods known in the art, such as using automated DNA synthesizers (eg, products from Biosearch or Applied Biosystems).
  • the DNA sequence is inserted into a vector comprising one or more expression control sequences (e.g., a promoter, an enhancer, etc.) operably linked to regulate expression of the DNA sequence, and the host cell is inserted into a recombinant expression vector formed therefrom.
  • expression control sequences e.g., a promoter, an enhancer, etc.
  • substantially pure peptide it is meant that the peptide according to the invention is substantially free of any other protein derived from the host.
  • Genetic engineering methods for peptide synthesis of the present invention reference may be made to Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual et al.
  • the inventors have isolated and purified the VapBC30 complex protein of Mycobacterium tuberculosis (Strain H37Rv) for VapBC30 structural analysis to synthesize an antibiotic peptide that may interfere with toxin-antitoxin binding,
  • the structure was determined through diffraction analysis experiments (see FIG. 2).
  • three peptides designed to affect only the binding site without touching the active site of the toxin SEQ ID NOS: 4 to 6).
  • the antibiotic peptide of the present invention prevents the binding of the toxin-antitoxin complex of Mycobacterium tuberculosis without affecting the active site of the toxin, and as a result, the initiating tRNA is degraded by the isolated toxin to induce the death of Mycobacterium tuberculosis. It can be usefully used as an antibiotic composition for treatment.
  • the present invention also provides an antibiotic composition containing the antibiotic peptide of the present invention as an active ingredient.
  • the peptide is preferably composed of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 6, but is not limited thereto.
  • composition is characterized by having an antimicrobial activity against Mycobacterium tuberculosis.
  • the peptide may also be used an amino acid in which one or several amino acids are added, substituted or deleted.
  • the antibiotic peptide of the present invention prevents the binding of the toxin-antitoxin complex of Mycobacterium tuberculosis without affecting the active site of the toxin (see FIG. 9). As a result, the initiating tRNA is degraded by the isolated toxin to induce the killing of Mycobacterium tuberculosis. Since it will be useful as an antibiotic composition for the treatment of Mycobacterium tuberculosis.
  • composition comprising the antibiotic peptide of the present invention preferably comprises 0.1 to 50% by weight of the antibiotic peptide relative to the total weight of the composition, but is not limited thereto.
  • composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of a medicament.
  • compositions according to the invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. have.
  • Carriers, excipients and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the composition of the present invention, for example, starch, calcium carbonate, sucrose (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared.
  • lubricants such as magnesium stearate and talc are also used.
  • Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • the non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used.
  • As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • composition of the present invention can be administered orally or parenterally, any parenteral administration method can be used, systemic or topical administration is possible, but systemic administration is more preferred, and intravenous administration is most preferred.
  • Preferred dosages of the compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art.
  • the effective dose of the antimicrobial peptide of the present invention is 1 to 2 mg / kg, preferably 0.5 to 1 mg / kg, and can be administered 1 to 3 times a day.
  • the dosage does not limit the scope of the invention in any aspect.
  • the pharmaceutical composition comprising the antibiotic peptide of the present invention as an active ingredient may be administered to a patient in a single dose by bolus form or by infusion for a relatively short period of time. Multiple doses may be administered by a fractionated treatment protocol with long term administration. Since the concentration is determined by taking into consideration various factors such as the age and health condition of the patient as well as the route of administration and the number of treatments of the drug, in view of the above, the present invention is a person having ordinary skill in the art. Appropriate effective dosages for the particular use of the novel peptides as pharmaceutical compositions may be determined.
  • the present invention also provides an antibiotic quasi-drug containing the antibiotic peptide of the present invention as an active ingredient.
  • the antibiotic peptide of the present invention prevents the binding of the toxin-antitoxin complex of Mycobacterium tuberculosis without affecting the active site of the toxin (see FIG. 9). As a result, the initiating tRNA is degraded by the isolated toxin to induce the killing of Mycobacterium tuberculosis. Therefore, it can be usefully used as an antibiotic pharmaceutical composition for treating Mycobacterium tuberculosis, antibiotic quasi-drugs and antibiotic antibiotics.
  • the peptide When the composition of the present invention is used as an quasi-drug additive, the peptide may be added as it is or used together with other quasi-drug or quasi-drug components, and may be appropriately used according to a conventional method.
  • the mixing amount of the active ingredient may be appropriately determined depending on the intended use.
  • the quasi-drug composition of the present invention is not limited thereto, but may preferably be a disinfectant cleaner, a shower foam, a gagreen, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment, a patch, or a filter filler.
  • the present invention also provides an antibiotic external preparation containing the antibiotic peptide of the present invention as an active ingredient.
  • the external preparation is preferably an antibiotic peptide of the present invention based on the total weight of 0.1 to 50 parts by weight, but is not limited thereto.
  • the external preparations may further comprise fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic Emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, any other ingredients commonly used in external preparations or commonly It may contain an adjuvant used.
  • the antibiotic peptide of the present invention prevents the binding of the toxin-antitoxin complex of Mycobacterium tuberculosis without affecting the active site of the toxin (see FIG. 9). As a result, the initiating tRNA is degraded by the isolated toxin to induce the death of Mycobacterium tuberculosis. Therefore, it can be usefully used as an antibiotic pharmaceutical composition for treating Mycobacterium tuberculosis, antibiotic quasi-drugs and antibiotic antibiotics.
  • the antibiotic peptide of the present invention prevents the binding of the toxin-antitoxin complex of Mycobacterium tuberculosis without affecting the active site of the toxin. Since it will induce the death of can be useful as an antibiotic composition for the treatment of Mycobacterium tuberculosis.
  • 1 is a diagram showing the overall structure of the VapBC30 complex.
  • Figure 2 is a diagram showing the binding structure of VapB30 and VapC30 in the VapBC30 complex.
  • Figure 3 is a diagram showing the overall structure of the VapC30 toxin protein.
  • FIG. 4 is a diagram showing the structure of the active site of the VapC30 toxin protein.
  • Figure 5 shows the VapB protein structure of each species.
  • FIG. 6 is a diagram showing the growth curve of E. coli expressing VapC30 protein or VapBC30 complex through OD 600 measurement.
  • Figure 7 is a diagram showing the results of acrylamide modified gel electrophoresis for the experiment in which VapC30 or VapBC30 protein to the tRNA fMet of Mycobacterium tuberculosis (Strain H37Rv).
  • FIG. 8 is a diagram showing the results of fluorescence quenching assay for the manganese ion dependence of the VapC30 protein.
  • Figure 9 is a diagram showing the results of fluorescence disappearance analysis for the experiment in which the peptide (SEQ ID NO: 4 to 6) to the VapBC30 complex.
  • VapB30 gene (SEQ ID NO: 7) of Mycobacterium tuberculosis (Strain H37Rv) was recombined into a pET28a (Novagen) expression vector, the VapC30 gene (SEQ ID NO: 8) to a pET21a (Novagen) expression vector, and the VapB30 protein was N-terminally. It is designed to contain histidine.
  • the recombinant vector thus prepared was transformed into Escherichia coli Rosetta2 (DE3) cells, which were incubated at 37 ° C. until the optical density was 0.5 at 600 nm in 600 LB (Luria Bertani) medium containing ampicillin and kanamycin. . Then 0.5 mM isopropyl 1-thio-beta-di-galactopyranoside was added to induce overexpression, and after 4 hours the cultured cells were 5,600 Centrifuged at g.
  • Example ⁇ 1-1> The cells obtained in Example ⁇ 1-1> were resuspended in buffer A (50 mM Tris-HCl, pH 8.0 and 500 mM NaCl), and then disrupted by ultrasonication, and the cells were again centrifuged at 17,900 g. The supernatant was filled into a fixed metal adsorption chromatography column (nickel-nitrilotriacetic acid-agarose, Novagen). The protein was eluted with A buffer containing 500 mM imidazole and then purified once again via size exclusion chromatography (HiLoad 16/60 Superdex 200 prep-grade column, GE Healthcare). Thereafter, 10 mg of human thrombin (thrombin, Sigma-Aldrich, USA) was added to the purified protein and left overnight at 20 ° C. to remove histidine labeling, and isolated via size exclusion chromatography.
  • buffer A 50 mM Tris-HCl, pH 8.0 and 500 mM NaCl
  • Protein crystals of VapBC30 substituted with Seleno-Methionine consisted of 0.2 M sodium acetate, 0.1 M sodium citrate pH 5.5, 5% (w / v) PEG 4,000 Obtained using an induction solution, the protein crystals of VapBC30 in the circular state were derived from 0.1 M sodium citrate tribasic dihydrate (pH 5.6), 1.0 M ammonium phosphate monobasic. It obtained using the liquid.
  • the obtained protein crystals were transferred to cryogenic protection liquid containing 20% glycerol (v / v), and then the ADSC Quantum 315r CCD detector system (Area Detector Systems Corporation, Poway, California) was used in the BL-5C beamline of the Pohang Accelerator Center. The result of diffraction analysis was obtained. Each diffraction image was obtained by rotating the crystal 1 degree, using the HKL2000 program. Both Se-Met substituted proteins and circular protein crystals belonged to the P3 1 21 space group, and the unit cell size was as follows:
  • VapBC30 complex dimers (heterodimer) exist in the asymmetric unit in both the Se-Met substituted protein and the protein crystal in the circular state (Tables 1 and 2).
  • the value in a parenthesis is the value of the high resolution shell (highest resolution shell).
  • phase difference problem is based on the Autosol of the PHENIX program, the protein modeling is done using the Coot, Paul Emsley program, and the Refmac5 (CCP4 Software Suite) and the Phoenix (PHENIX). To improve the structure. R free was calculated using 5 percent of the data, and water molecules were modeled using the Coot program. All structures were checked for accuracy using Morprobity (Table 2).
  • Structural comparison and overlapping were performed using the Secondary Structural Comparison Function (SSM), and solvent access site analysis was carried out using PISA.
  • SSM Secondary Structural Comparison Function
  • the improved structure was visualized using PyMOL and the residue overlap was visualized with the ClusterX 2.0 program and with ESPript 3.0.
  • each heterotetramer is connected to two heterodimers connected via the VapC30-VapC30 interface (see FIG. 1A).
  • heterodimer) complexes are linked, each heterodimer consists of VapC30 chain A and VacB30 chain B, or VapC30 chain C and VapB30 chain D (Fig. 1b).
  • size exclusion chromatography confirmed that the structure of the VapBC30 complex was 92.3 kDa (FIG. 1C), indicating that VapBC30 was present in the form of heterooctamer in aqueous solution.
  • the VapBC30 complex inhibits the activity of VapC30 through swap blocking of VapB30 (FIG. 2A).
  • the C-terminal portion of VapB30 binds to the residue of VapC30 on the far side and interferes with the enzymatic activity of VapC30 through a swapped inactivation process
  • the C-terminal portion of VapB30 chain D is VapC30 chain A
  • the C-terminal portion of VapB30 chain B binds to VapC30 chain C.
  • Asn96 residues in chain A of VapC30 are involved in hydrogen bonding in the molecule together with Asp99 and Asp119 residues in the active site, whereas in chain C of VapC30, Asn96 residues are linked by hydrogen bonds to Leu75 and Gly76 residues of VapB30 chain B (FIG. 2b and 2c).
  • VapC30 protein was obtained from the results of Examples ⁇ 1-3> and ⁇ 1-4>.
  • the VapC30 protein is characterized by a PIN-domain motif and consists of four parallel beta-strands and six alpha-helices in the order of ⁇ 4- ⁇ 1- ⁇ 2- ⁇ 3.
  • FIG. 3A the C-terminal residues of VapC30 (residues 115-131) do not form any secondary structure (FIG. 3b) and each complex is 2 because the VapBC30 complex is in the form of heterooctamer in aqueous solution and crystalline state.
  • FIG. 3C the VapC30 protein is characterized by a PIN-domain motif and consists of four parallel beta-strands and six alpha-helices in the order of ⁇ 4- ⁇ 1- ⁇ 2- ⁇ 3.
  • FIG. 3A the C-terminal residues of VapC30 (residues 115-131) do not form any secondary structure (FIG. 3b) and each complex is 2 because the VapBC30 complex is in the form of heterooctamer in aqueous solution and crystalline
  • RNA molecules In addition, positively charged amino acids (Lys88, Arg90, His91, and Arg92) are located in the loop between ⁇ 5 and ⁇ 6 helix, which are expected to bind RNA molecules (FIG. 4A), and active sites on the concave surface of the VapC30 homodimer interface. Located, the active site is formed by a loop between ⁇ 1 , ⁇ 1, ⁇ 3 and ⁇ 6 and a C-terminal loop. The active site contains Asp4, Glu40 and Asp99 residues, which are essential for RNA degrading enzyme activity, and these three residues form a powerful hydrogen bonding network and bind to water molecules. Comparing the structure of the toxin protein of the different species and the active site, it was confirmed that the water molecule can play the same role as the metal ion of the other species (Fig.
  • the structure of the VapB30 protein was obtained from the results of Examples ⁇ 1-3> and ⁇ 1-4>.
  • the N-terminal part showed almost the same structure consisting of alpha-helix and loop, while the C-terminal part showed various structures. It was confirmed.
  • the N-terminal alpha-helix (residues 49-62) structure of the VapB30 protein was similar to that of other known VapB proteins, but the overall structure was similar (Fig. 5).
  • VapC30 alone or pET28a (+)-His 6 -VapB30 and pCOLD1-His 6 -VapC30 expression vectors were inserted into E. coli BL21 cells and cultured in the same manner as in Example ⁇ 1-1>. Expression was induced and OD 600 was measured at 30 minute intervals for 4 and a half hours.
  • DNA fragments containing the H37Rv tRNA fMet moiety were amplified by a polymerase chain reaction (PCR), and a recognition site of the T7 RNA synthase was attached to the top.
  • PCR polymerase chain reaction
  • tRNA fMet was transcribed and purified using a Megaclear kit (MEGAclear kit, Ambion).
  • VapC30 protein was mixed with tRNA fMet (3 ⁇ m) of Example ⁇ 10-1> at different concentrations (3, 6, 9 ⁇ m) [10 ul reaction solution-0.1 M NaCl, 20 mM Tris-HCL buffer (pH). 8.0) and 40 units of RiboLock TM RNase inhibitor (Thermo scientific)] After 1 hour at 37 ° C, the cut RNA fragments were identified by 15% acrylamide modified gel electrophoresis with 8M urea.
  • the structure of the VapBC30 complex shows that the binding between the toxin-antitoxin complexes is the ⁇ 1-helix of the toxin (amino acid residues 49-62, SEQ ID NO: 1), the ⁇ 2-helix of the toxin (amino acid residues 17-27, SEQ ID NO: 2) and It consists of three structural elements of the ⁇ 4-helix (amino acid residues 52-65, SEQ ID NO: 3). Therefore, we designed three peptides that affect only the binding site without touching the active site of the toxin.
  • Peptide I mimics the ⁇ 1-helix of the antitoxin
  • peptide II mimics the ⁇ 2-helix of the toxin
  • peptide III mimics the ⁇ 4-helix of the toxin:
  • Peptide I (SEQ ID NO: 4): Glu-Leu-Ala-Ala-Ile-Arg-His-Arg;
  • Peptide II (SEQ ID NO: 5): Asp-Glu-Pro-Asp-Ala-Glu-Arg-Phe-Glu-Ala-Ala-Val-Glu-Ala-Asp-His-Ile; And
  • Peptide III (SEQ ID NO: 6): Arg-Phe-Gly-Glu-Pro-Gly-Gly-Arg-Glu.
  • VapC30 (20 ⁇ m) or VapBC30 complex (20 ⁇ m) was mixed with tRNA fMet [40 ul reaction solution—0.1 M NaCl, 20 mM Tris-HCL buffer (pH 8.0) and 40 units of RiboLock TM RNase inhibitor (Thermo. scientific)] and left at 37 ° C. for 60 minutes, and treated with peptide I (SEQ ID NO: 4), peptide II (SEQ ID NO: 5), and peptide III (SEQ ID NO: 6) at 20, 50 and 200 uM concentrations, respectively, at 37 ° C. It was left for 2 hours.
  • tRNA fMet 40 ul reaction solution—0.1 M NaCl, 20 mM Tris-HCL buffer (pH 8.0) and 40 units of RiboLock TM RNase inhibitor (Thermo. scientific)] and left at 37 ° C. for 60 minutes, and treated with peptide I (SEQ ID NO: 4), peptide II (SEQ ID NO
  • SEQ ID NO: 4 Glu Leu Ala Ala Ile Arg His Arg
  • SEQ ID NO: 5 Asp Glu Pro Asp Ala Glu Arg Phe Glu Ala Ala Val Glu Ala Asp His

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Wood Science & Technology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un peptide antibactérien ciblant un système toxine-antitoxine de Mycobacterium tuberculosis et une utilisation correspondante. Plus particulièrement, un peptide présentant la séquence d'acides aminés représentée par les séquences SEQ ID NO : 4-6, inhibant la liaison toxine-antitoxine de Mycobacterium tuberculosis, inhibe la liaison d'un complexe toxine-antitoxine de Mycobacterium tuberculosis tout en n'affectant pas le site actif des toxines, ce qui permet de décomposer l'initiation d'ARNt par des toxines isolées de manière à induire la mort de Mycobacterium tuberculosis et, par conséquent, le peptide inhibant la liaison toxine-antitoxine de Mycobacterium tuberculosis peut être utile en tant que composition antibactérienne.
PCT/KR2016/005657 2015-05-29 2016-05-27 Peptide antibactérien ciblant un système toxine-antitoxine de mycobacterium tuberculosis et utilisation correspondante WO2016195336A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2015-0076355 2015-05-29
KR1020150076355A KR101746160B1 (ko) 2015-05-29 2015-05-29 결핵균 독소-항독소 체계를 표적으로 하는 항생 펩타이드 및 이의 용도

Publications (1)

Publication Number Publication Date
WO2016195336A1 true WO2016195336A1 (fr) 2016-12-08

Family

ID=57441486

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2016/005657 WO2016195336A1 (fr) 2015-05-29 2016-05-27 Peptide antibactérien ciblant un système toxine-antitoxine de mycobacterium tuberculosis et utilisation correspondante

Country Status (2)

Country Link
KR (1) KR101746160B1 (fr)
WO (1) WO2016195336A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101919364B1 (ko) * 2016-12-07 2018-11-19 한양대학교 산학협력단 결핵균 ＣｌｐＣ１ 결합용 펩타이드
KR101849347B1 (ko) 2017-07-31 2018-04-16 서울대학교 산학협력단 결핵균 독소-항독소 체계를 표적으로 하는 펩타이드 및 이의 용도
KR102083398B1 (ko) * 2018-07-18 2020-03-02 서울대학교산학협력단 결핵균 내인성 독소의 독성을 증가시키며, 독소-항독소 체계를 표적으로 하는 항결핵 펩타이드 및 이의 용도
KR102097040B1 (ko) * 2018-08-10 2020-04-03 서울대학교산학협력단 폐렴구균의 독소-항독소 체계를 표적으로 하는 항생 펩타이드 및 이의 용도
KR102521182B1 (ko) * 2020-11-06 2023-04-12 서울대학교산학협력단 결핵균 독소-항독소 시스템을 표적으로 하는 항균 스테이플 펩타이드 및 이의 용도
KR102551038B1 (ko) * 2020-12-30 2023-07-05 서울대학교산학협력단 폐렴구균 내인성 독소의 독성을 증가시키며, HigBA 독소-항독소 체계를 표적으로 하는 항균 펩타이드 및 이의 용도
KR102581500B1 (ko) * 2021-01-20 2023-09-20 서울대학교산학협력단 폐렴간균 독소-항독소 시스템을 표적으로 하는 항균 펩타이드 및 화합물, 및 이들의 용도

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007039398A (ja) * 2005-08-04 2007-02-15 Institute Of Physical & Chemical Research トキシン−アンチトキシン複合体の形成を指標とする抗生物質のスクリーニング方法
US20070298043A1 (en) * 2003-10-02 2007-12-27 Ehud Gazit Novel Antibacterial Agents and Methods of Identifying and Utilizing Same
KR20090013762A (ko) * 2006-03-22 2009-02-05 유니버시티 오브 메디신 앤드 덴티스트리 오브 뉴 저지 신규 항생제 개발을 위한 박테리아 자멸 경로의 표적화
US20110028697A1 (en) * 2008-03-27 2011-02-03 Takara Bio Inc. Prophylactic/therapeutic agent for infectious disease

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070298043A1 (en) * 2003-10-02 2007-12-27 Ehud Gazit Novel Antibacterial Agents and Methods of Identifying and Utilizing Same
JP2007039398A (ja) * 2005-08-04 2007-02-15 Institute Of Physical & Chemical Research トキシン−アンチトキシン複合体の形成を指標とする抗生物質のスクリーニング方法
KR20090013762A (ko) * 2006-03-22 2009-02-05 유니버시티 오브 메디신 앤드 덴티스트리 오브 뉴 저지 신규 항생제 개발을 위한 박테리아 자멸 경로의 표적화
US20110028697A1 (en) * 2008-03-27 2011-02-03 Takara Bio Inc. Prophylactic/therapeutic agent for infectious disease

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Genbank [O] 29 April 2014 (2014-04-29), XP055332822, Database accession no. KAY40319.1 *
LEE ET AL.: "Structural and Functional Studies of the Mycobacterium Tuberculosis VapBC30 Toxin-antitoxin System: Implications for the Design of Novel Antimicrobial Peptides", NUCLEIC ACIDS RESEARCH, vol. 43, no. 15, 6 July 2015 (2015-07-06), pages 7624 - 7637, XP055332825 *

Also Published As

Publication number Publication date
KR20160140137A (ko) 2016-12-07
KR101746160B1 (ko) 2017-06-12

Similar Documents

Publication Publication Date Title
WO2016195336A1 (fr) Peptide antibactérien ciblant un système toxine-antitoxine de mycobacterium tuberculosis et utilisation correspondante
WO2013169077A1 (fr) Composition pour la prévention ou le traitement de la cachexie
TW200950800A (en) Use of defensins against tuberculosis
WO2012050402A2 (fr) Protéine parkin recombinante à perméation cellulaire et composition pharmaceutique de traitement des maladies dégénératives du cerveau l'incluant
AU2015372767B2 (en) Glucagon derivatives
WO2011132830A1 (fr) Nouveau composé de kanamycine, espèces de bactérie streptomyces produisant de la kanamycine, et procédé de production de kanamycine
WO2019027224A2 (fr) Peptide ciblant un système toxine-antitoxine de mycobacterium tuberculosis et son utilisation
Bowers et al. Isolation, sequencing and expression of the gene encoding a major protein from the backteriophage associated with Bartonella henselae
WO2016108394A1 (fr) Composition comprenant un néo-agaro-oligosaccharide en tant que substance active, pour la prévention ou le traitement de la septicémie ou d'un choc septique
WO2020032324A1 (fr) Peptide antibiotique ciblant un système toxine-antitoxine de streptococcus pneumoniae, et utilisation associée
WO2019004674A1 (fr) Peptide se liant aux récepteurs d'acétylcholine
Gibson et al. Functional characterisation of the regulatory subunit of cyclic AMP-dependent protein kinase A homologue of Giardia lamblia: Differential expression of the regulatory and catalytic subunits during encystation
WO2020171274A1 (fr) Peptide ayant une activité prophylactique ou thérapeutique contre une maladie inflammatoire ou auto-immune et utilisation associée
WO2022158875A1 (fr) Peptide antibactérien et composé ciblant le système toxine-antitoxine de klebsiella pneumoniae et utilisations associées
WO2015072750A1 (fr) Peptide à activité anti-inflammatoire et composition le comprenant
WO2019022469A1 (fr) Utilisation du nicotinamide adénine dinucléotide phosphate (nadph) ou d'un dérivé de celui-ci comme inhibiteur de bêta-lactamase
WO2020139031A1 (fr) Composition à base de crispr-cas pour correction génique
WO2018016814A1 (fr) Variant de facteur de croissance des fibroblastes hautement stable ayant une région d'acides aminés n-terminale modifiée, et son utilisation
WO2021242046A1 (fr) Nouveau peptide anti-inflammatoire et utilisation associée
WO2018182301A1 (fr) Composition pharmaceutique de prévention ou traitement de maladie respiratoire inflammatoire, à protéine de fusion de peptide perméable aux cellules et de peptide ctctla4, contenue en tant que principe actif dans cette composition
WO2021080400A1 (fr) Polypeptide dérivé de l'oenanthe javanica et composition pharmaceutique le comprenant
Tsai et al. Effect of site directed mutagenesis on the activity of recombinant trimucrotoxin, a neurotoxic phospholipase from Trimeresurus mucrosquamatus venom
WO2020263062A1 (fr) Composition vaccinale contre le virus de la fièvre aphteuse
WO2012077849A1 (fr) Copolymère multi-bloc peptidique antimicrobien s'exprimant sur la surface des cellules
WO2011090345A2 (fr) Nouvelle composition antibiotique comprenant de l'acide flufénamique comme principe actif

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16803691

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16803691

Country of ref document: EP

Kind code of ref document: A1