KR101746160B1 - Antibiotic peptides targeting toxin-antitoxin system of Mycobacterium tuberculosis and use thereof - Google Patents

Abstract

The present invention relates to an antimicrobial peptide targeting the Mycobacterium tuberculosis toxin-antitoxin system and its use. More particularly, the present invention relates to an antimicrobial peptide that inhibits toxin-antitoxin binding of Mycobacterium tuberculosis and has an amino acid sequence as shown in SEQ ID NOS: Antitoxin complex of the Mycobacterium tuberculosis without affecting it, and as a result, the initiation tRNA is degraded by the separated toxin to induce the death of the Mycobacterium tuberculosis. Therefore, the peptide that inhibits the toxin-antitoxin binding of the Mycobacterium tuberculosis It can be usefully used as an antibiotic composition.

Description

Antibiotic peptides targeted against Mycobacterium tuberculosis toxin-antitoxin system and its use {Antibiotic peptides targeting toxin-antitoxin system of Mycobacterium tuberculosis and use thereof}

The present invention relates to antibiotic peptides that target the toxin-antitoxin system of Mycobacterium tuberculosis and uses thereof.

Tuberculosis is a contagious and infectious acute and chronic disease that can occur anywhere in the body. It is a terrible disease that may even lead to death. It affects about 85% of the lungs. About 85% It can be propagated to any organ of the body along the lymph nodes to influence. Tuberculosis spreads from the cough, runny nose and sputum of the patient through the air. Approximately 9 million people are infected with tuberculosis in 2013 and 1.5 million people have died. In addition, multidrug - resistant tuberculosis, and furthermore, complete - resistant tubercle bacilli have emerged, and new antibacterial agents are needed to treat tuberculosis.

The toxin-antitoxin gene was first known to play a role in maintaining the plasmid of Escherichia coli. When the plasmid having the toxin-antitoxin gene is lost, the toxin having the stable structure is maintained, but the antitoxin protein having the unstable structure is maintained And E. coli is finally killed. Since the discovery of the toxin-antitoxin gene for the first time, it has been found that there is a toxin-antitoxin gene in the chromosome of E. coli as well as in the plasmid, and it is known to be involved in multidrug resistance, biofilm formation and inhibition of growth under stress conditions.

 The toxin-antitoxin system can be roughly classified into three types (Type I, II, III). In the type I system, the RNA-type antitoxin is attached to the RNA-type toxin to eliminate its toxicity. In the type II system, the protein-type antitoxin is also attached to the protein-type toxin to eliminate its toxicity. In the Type III system, the RNA-type antitoxin is attached to a toxin in the form of a protein to eliminate its toxicity.

Among the three types, the most studied type is the type II system. In the type II system, the toxin and antitoxin genes are encoded as operons. When a hard external condition such as a temperature rise or depletion of nutrients is encountered, the unstable antitoxin is degraded by the stress-inducing protease, and the cell is destroyed by failing to neutralize the toxin toxicity. It is known that the VapBC family of toxins (VapC), which occupies the most part of type II, is the cell line that inhibits cell growth based on RNA degradation.

The toxin-antitoxin system can be a fascinating target for the development of new antibiotics because the toxin-antitoxin complex can be artificially detoxified and the toxin-containing toxin can not neutralize and eventually kill the cell. In Mycobacterium tuberculosis, more than half of the toxin-antitoxin systems are now found to belong to the VapBC family, and this VapBC family is known to be associated with extreme latency and drug resistance of Mycobacterium tuberculosis. To date, much effort has been made to develop a therapeutic agent for tuberculosis targeting the toxin-antitoxin complex, but so far there has been no successful example.

Therefore, We have begun to study the toxin-antitoxin system that is encoded in the gene. Through this study, we found that VAPC30, a toxin of Mycobacterium tuberculosis, regulates cell growth based on manganese ion-dependent RNA resolution. In addition, the structure of VapBC30 complex was elucidated. Based on this structure, a peptide capable of interfering with the binding of the toxin-antitoxin complex was designed, and the peptide successfully inhibited the binding of the toxin-antitoxin complex in vitro And that the peptide can be used as an active ingredient of an antibiotic pharmaceutical composition for the treatment of tuberculosis, thereby completing the present invention.

It is an object of the present invention to provide an antibiotic peptide targeting the toxin-antitoxin system of Mycobacterium tuberculosis and a pharmaceutical composition containing it as an active ingredient

In order to achieve the above object, the present invention provides an antibiotic peptide which inhibits binding of toxin-antitoxin.

In addition, the present invention provides a pharmaceutical composition for antibiotics containing the antibiotic peptide of the present invention as an active ingredient.

In addition, the present invention provides an antibiotic quasi-drug containing the antibiotic peptide of the present invention as an active ingredient.

The present invention also provides an external preparation for antibiotics comprising the antibiotic peptide of the present invention as an active ingredient.

The antibiotic peptides of the present invention inhibit the binding of the toxin-antitoxin complex of Mycobacterium tuberculosis without affecting the active site of the toxin, and as a result, the initiation tRNA is degraded by the separated toxin, And thus can be effectively used as an antibiotic composition for the treatment of Mycobacterium tuberculosis.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the overall structure of a Vap BC30 complex.
2 is a view showing the binding structure of VapB30 and VapC30 in the Vap BC30 complex.
Fig. 3 shows the overall structure of the VapC30 toxin protein. Fig.
Figure 4 shows the structure of the active site of the VapC30 toxin protein.
5 is a diagram showing 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.
FIG. 7 is a graph showing the results of acrylamide-modified gel electrophoresis on an experiment in which tRNA ^fMet of Mycobacterium tuberculosis (Strain H37Rv) was treated with VapC30 or VapBC30 protein.
FIG. 8 is a graph showing a fluorescence quenching assay result of manganese ion dependence of VapC30 protein. FIG.
9 is a graph showing the results of fluorescence extinction analysis for an experiment in which a peptide (SEQ ID NOS: 4 to 6) was treated with a Vap BC30 complex.

Hereinafter, the present invention will be described in detail.

The present invention provides antibiotic peptides that inhibit toxin-antitoxin binding.

The peptide is preferably composed of any amino acid sequence selected from the group consisting of SEQ ID NOS: 4 to 6, but is not limited thereto.

The peptide is characterized by having an antibacterial activity against Mycobacterium tuberculosis.

The peptide is characterized by inhibiting the binding of alpha 2, alpha 4, and alpha 1 of the toxin toxin, and does not affect the activity of the toxin.

Amino acids in which one or several amino acids are added, substituted or deleted can also be used as the peptide.

As a method for synthesizing the peptide, it is preferable to synthesize it by a conventional chemical synthesis method (WH Freeman and Co., Proteins; structures and molecular principles, 1983) in the art. Specifically, Solution Phase Peptide synthesis Solid phase peptide syntheses, fractional condensation, and F-moc or T-BOC chemical methods. More preferably, the solid phase peptide syntheses are most preferably synthesized by a solid phase peptide synthesis method. It does not.

In addition, the peptides of the present invention can be produced by a genetic engineering method as described below. First, a DNA sequence encoding the peptide is constructed according to a conventional method. DNA sequences can be prepared by PCR amplification using appropriate primers. Alternatively, DNA sequences can be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer (e. G., Biosearch or Applied Biosystems). The DNA sequence is operatively linked to insert into a vector comprising one or more expression control sequences (e.g., promoters, enhancers, etc.) that regulate the expression of the DNA sequence, and the host cell is transformed with the recombinant expression vector formed therefrom Subsequently, the resulting transformant is cultured under conditions and under conditions suitable for expression of the DNA sequence, and the substantially pure peptide encoded by the DNA sequence is isolated from the culture by a method known in the art , Chromatography). By "substantially pure peptide" is meant that the peptide according to the invention is substantially free of any other proteins derived from the host. Genetic engineering methods for peptide synthesis of the present invention can be found in the following references: Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor Laboratory, 1982; Sambrook et al., Molecular Cloning: A Laboratory Manual et al.

In a specific example of the present invention, the present inventors isolated and purified a Vap BC30 complex protein of Mycobacterium tuberculosis (strain H37Rv) for analysis of VapBC30 structure for synthesizing an antibiotic peptide capable of interfering with toxin-antitoxin binding, The structure was determined through a diffraction analysis experiment (see FIG. 2). As a result, three peptides were designed (SEQ ID NOS: 4 to 6) that affect only the binding site without touching the active site of the toxin.

In addition, when the synthetic peptide was treated with VapBC30 complex, it was confirmed that the fluorescence increases in proportion to the concentration of the peptide, which means that the complex formation between VapBC is inhibited by the peptide and RNA is degraded by VapC30. Particularly, when peptide III mimicking the α-4 helices of the toxin was added, it was confirmed that RNA was most decomposed (see FIG. 9).

In addition, in order to confirm the activity of VapC30 protein, it was confirmed that the expression of VapC30 alone in Escherichia coli inhibited the growth of E. coli compared with the expression of VapBC30 complex (see FIG. 6) (See FIG. 7), and in the presence of manganese ion (Mn ²⁺ ), the activity of VAPC30 RNAase was increased (see FIG. 8).

Accordingly, the antibiotic peptide of the present invention inhibits the binding of the toxin-antitoxin complex of Mycobacterium tuberculosis without affecting the active site of the toxin, and as a result, the initiation tRNA is degraded by the separated toxin to induce the death of Mycobacterium tuberculosis, Can be usefully used as an antibiotic composition for treatment.

The present invention also provides an antibiotic composition comprising the antibiotic peptide of the present invention as an active ingredient.

The peptide is preferably composed of any amino acid sequence selected from the group consisting of SEQ ID NOS: 4 to 6, but is not limited thereto.

The composition is characterized by having antibacterial activity against Mycobacterium tuberculosis.

Amino acids in which one or several amino acids are added, substituted or deleted can also be used as the peptide.

The antibiotic peptides of the present invention inhibit the binding of the toxin-antitoxin complex of Mycobacterium tuberculosis without affecting the active site of the toxin (see FIG. 9), and as a result, the initiation tRNA is degraded by the isolated toxin, Therefore, it can be effectively used as an antibiotic composition for the treatment of Mycobacterium tuberculosis.

The composition comprising the antibiotic peptide of the present invention preferably comprises 0.1 to 50% by weight of the antibiotic peptide based on the total weight of the composition, but is not limited thereto.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of medicaments.

The composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral preparations, suppositories and sterilized injection solutions according to a conventional method have. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition of the present invention may be administered orally or parenterally, and any parenteral administration method may be used, and systemic administration or topical administration is possible, but systemic administration is more preferable, and intravenous administration is most preferable.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, 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 amounts do not in any way limit the scope of the invention

A pharmaceutical composition comprising the antibiotic peptide of the present invention as an active ingredient can be administered to a patient in a single dose by bolus injection or by infusion for a relatively short period of time, or by a fractionated treatment protocol in which multiple doses are administered over a prolonged period of time. Since the concentration of the effective dose of the patient is determined in consideration of various factors such as the route of administration and the number of treatments as well as the age and health condition of the patient, in view of this point, Lt; RTI ID = 0.0 > of < / RTI > novel peptide as a pharmaceutical composition.

In addition, the present invention provides an antibiotic quasi-drug containing the antibiotic peptide of the present invention as an active ingredient.

The antibiotic peptides of the present invention inhibit the binding of the toxin-antitoxin complex of Mycobacterium tuberculosis without affecting the active site of the toxin (see FIG. 9), and as a result, the initiation tRNA is degraded by the isolated toxin, Therefore, it can be usefully used as an antibiotic pharmaceutical composition for treatment of Mycobacterium tuberculosis, an antibiotic quasi drug, and an external medicine for antibiotics.

When the composition of the present invention is used as a quasi-drug additive, the peptide may be added as it is or may be used together with other quasi-drugs or quasi-drugs, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

The quasi-drug composition of the present invention may be a disinfectant cleaner, a shower foam, a gagrin, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment agent, a patch or a filter filler.

The present invention also provides an external preparation for antibiotics comprising the antibiotic peptide of the present invention as an active ingredient.

The external preparation preferably contains the antibiotic peptide of the present invention in an amount of 0.1 to 50 parts by weight based on the total weight, but is not limited thereto.

The external preparation may further contain at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickening and gelling agents, softening agents, antioxidants, suspending agents, stabilizing agents, foaming agents, fragrances, surfactants, water, And may contain other optional components commonly used in emulsifying agents, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, And may contain auxiliary agents used.

The antibiotic peptides of the present invention inhibit the binding of the toxin-antitoxin complex of Mycobacterium tuberculosis without affecting the active site of the toxin (see FIG. 9), and as a result, the initiation tRNA is degraded by the isolated toxin, Therefore, it can be usefully used as an antibiotic pharmaceutical composition for treatment of Mycobacterium tuberculosis, an antibiotic quasi drug, and an external medicine for antibiotics.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

Example 1: Determination of the structure of the Vap BC30 complex of Mycobacterium tuberculosis

<1-1> Production of recombinant vector of toxin (VapC30) and antitoxin (VapB30) and induction of expression

To determine the structure of toxin and antitoxin complexes of Mycobacterium tuberculosis, recombinant vectors containing toxin and antitoxin genes were prepared and expressed in Escherichia coli.

Specifically, the VapB30 gene (SEQ ID NO: 7) of the Mycobacterium tuberculosis strain (SEQ ID NO: 7) was recombined with the pET28a (Novagen) expression vector and the VapC30 gene (SEQ ID NO: 8) was replaced with pET21a Was prepared to contain histidine. The prepared recombinant vector was transformed into Escherichia coli Rosetta2 (DE3) cells and cultured in LB (Luria Bertani) medium containing ampicillin and kanamycin at 370C until the optical density reached 0.5 at UV 600 nm . Thereafter, 0.5 mM isopropyl 1-thio-beta -D-galactopyranoside was added to induce excessive expression, and after 4 hours, the cultured cells were incubated for 5,600 g.

Recombinant pCOLD1 (Takara) expression vector was inserted into E. coli BL21 cells to obtain VapC30 alone protein, which was then expressed together with GroES and GroEL chaperon proteins and expressed and separated in the same manner as above.

Proteins in which methionine was replaced with selenomethionine (Se-Met) for phase difference resolution were expressed and separated in the same manner as described above in M9 medium containing selenomethionine.

All the separated proteins were concentrated using a centrifugal filter (Amicon) for crystal formation.

<1-2> Isolation and purification of expressed toxin and antitoxin protein

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. The disrupted cells were centrifuged again at 17,900 g , And the supernatant was loaded onto a fixed metal adsorption chromatography column (nickel-nitrilotriacetic acid-agarose, Novagen). Protein was eluted with A buffer containing 500 mM imidazole and purified once again by size exclusion chromatography (HiLoad 16/60 Superdex 200 prep-grade column, GE Healthcare). Then, 10 mg of human thrombin (Sigma-Aldrich, USA) was added to the purified protein to remove the histidine label, left overnight at 20 ° C, and separated by size exclusion chromatography.

<1-3> Structure analysis of toxin-antitoxin protein

In order to confirm the structure of the protein purified in <1-2>, protein crystals were formed by a sitting drop vapor diffusion method to analyze the structure.

Concretely, 0.5 μl of the purified protein solution and the crystal inducing solution for inducing crystal formation were mixed with each other to make a sitting drop, and then left at 20 ° C. Protein crystals of VapBC30 substituted with Seleno-Methionine were prepared from crystals consisting of 0.2 M sodium acetate (sodiuim acetate), 0.1 M sodium citrate pH 5.5, 5% (w / v) PEG 4,000 The protein crystals of VapBC30 in the circular state were prepared using 0.1 M sodium citrate tribasic dihydrate (pH 5.6), 1.0 M ammonium phosphate monobasic crystal induction Solution.

The obtained protein crystals were transferred to a cryoprotectant containing 20% glycerol (v / v), and then the ADSC Quantum 315r CCD detector system (Area Detector Systems Corporation, Poway, Calif.) Was used in a BL- And the diffraction analysis results were obtained. Each diffraction image was obtained by rotating the crystal by 1 degree, and the HKL2000 program was used. Both the Se-Met substituted protein and the circular protein crystals belong to the P3 ₁ 21 space group, the size of the unit lattice is as follows:

Se-Met substituted protein: a = 96.31 A, b = 96.31 A, and c = 233.03 A; And

Circular protein: a = 96.38 A, b = 96.38 A, and c = 232.79 A.

As a result, it was confirmed that four VapBC30 complex heterodimers were present in the asymmetric unit in both the Se-Met substituted protein and the circular protein crystal (Tables 1 and 2).

Data collection SAD (Se peak) Protein crystal I (Se-Met) Protein crystals II (circular) X-ray wavelength (Å) 0.9791 0.9791 1.0000 Space group P3 ₁ 21 P3 ₁ 21 P3 ₁ 21 Unit cell length (Å) a = b = 96.44 a = b = 96.31 a = b = 96.38 c = 233.24 c = 233.03 c = 232.79 Unit cell angle (°) alpha = beta = 90, gamma = 120 alpha = beta = 90, gamma = 120 alpha = beta = 90, gamma = 120 Resolution range (Å) 50.0-2.80 (2.75-2.70) ^a 50.0-2.70 (2.75-2.70) ^a 50.0-2.70 (2.75-2.70) ^a Total / unique reflections 1,093,614 / 31,502 233,858 / 34,963 272, 621 / 35,181 Completeness (%) 99.9 (100) ^a 98.9 (99.9) ^a 99.3 (99.0) ^a &Lt; I /? (I) 83.4 (12.4) ^a 37.0 (4.68) ^a 41.0 (5.39) ^a R _merge ^b 0.173 (0.531) ^a 0.087 (0.613) ^a 0.088 (0.693) ^a

^a The value in the insertion hole is the value of the highest resolution shell.

^b R _merge = Σ _h Σ _i | _{I (h) i -> I} (h) | / Σ h Σ i I (h) i, I (h) is a value showing the intensity of reflection h, Σ _h is the sum of the reflection h, Σ _i is a reflection h I &lt; / RTI &gt;

<1-4> Determination of protein structure and improvement of structure

The phase difference problem uses Autosol of the PHENIX program and protein model formation using the Coot and Paul Emsley program and the Refmac5 and CCP4 Software Suite and PHENIX To improve the structure. Five percent of the data were used to calculate R _free , and water molecules were modeled using the Coot program. All structures were confirmed using Molprobity (Table 2).

Protein residues of 94.79% and 93.67% for the Se-Met replacement protein and the circular protein were distributed in the favored region of the Ramachandran plot and there was no outlier. Molprobity found that the two proteins belonged to 100 ^th and 99 ^th percentiles with a score of 1.64 and 1.79, and 100 ^th percentages had the highest structural accuracy at similar resolution structures (Table 2).

The structure comparison and overlap were performed using the secondary structure comparison function (SSM) and the solvent access site analysis was performed using PISA. The improved structure was visualized using PyMOL. Residual overlaps were visualized using the ClustalX 2.0 program and Esprit 3.0 (ESPript 3.0).

Model structure improvement data Protein crystal I (Se-Met) Protein crystals II (circular) R _work / R _free ^c 0.242 / 0.273 0.205 / 0.238 No. of nonhydrogen atoms / average B-factor (Å ² ) Protein atoms 4,713 / 59.8 4,944 / 61.1 Water oxygen atoms 57 / 48.73 106 / 48.29 Wilson B-factor (Å ² ) 59.92 54.2  R.m.s. deviations from ideal geometry Bond lengths (Å) / bond angles (˚) 0.0113 / 1.287 0.0112 / 1.2053 Rms Z-scores ^d Bond lengths / bond angles 0.565 / 0.585 0.563 / 0.567  Ramachandran plot (%) Favored 94.79 ^e 93.67 ^e Outliers 0.0 ^e 0.0 ^e Rotamer outliers (%) 2.77 ^e 2.03 ^e

^c R = Σ || F _obs | - | F _calc || / Σ | F _obs |, R _free values were calculated by randomly choosing 5% unused for reflection in reflection, and R _work values were calculated from the remaining reflections.

^d REFMAC. &lt; / RTI &gt;

^e mol Pro obtained using Bt (MolProbity).

<1-5> Structural features of VapBC30 complex

The structures of VapBC30 complexes were determined at the resolution of 2.7 Å using the results of <1-3> and <1-4>.

As shown in Figure 1, there are two VapBC30 complex heterotetramers in each asymmetric unit of crystal form I and II (Figure 1a), and each heterotetramer has two heterodimers connected via the VapC30-VapC30 interface heterodimer complexes were connected. Each heterodimer was composed of VapC30 chain A and VacB30 chain B, or VapC30 chain C and VapB30 chain D (FIG. 1B). In addition, size exclusion chromatography confirmed the structure of the VapBC30 complex to be 92.3 kDa (Fig. 1c), indicating that VapBC30 is present in the form of a heterooctamer in aqueous solution.

Also as shown in Figure 2, the Vap BC30 complex inhibits the activity of VapC30 through swapped blocking of VapB30 (Figure 2a). Specifically, the C-terminal region of VapB30 binds to the residue of VapC30 at the far site and interferes with the enzymatic activity of VapC30 through a swapped inactivation process. The C-terminal region of VapB30 chain D interferes with the VapC30 chain A And the C-terminal region of the VapB30 chain B binds to the VapC30 chain C. Asn96 residues in the chain A of VapC30 are involved in hydrogen bonding in molecules with Asp99 and Asp119 residues of the active site while in the chain C of VapC30 the Asn96 residues are hydrogen bonded to the Leu75 and Gly76 residues of the VapB30 chain B 2b and 2c).

<1-6> Structural features of VapC30 protein

The structure of VapC30 protein was determined through the results of Examples 1-3 and 1-4.

As shown in FIG. 3, 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 beta 4 - beta 1 - beta 2 - beta 3 (Fig. 3A). In addition, the C-terminal residues (residues 115-131) of VapC30 do not form any secondary structure (FIG. 3b), and because the VapBC30 complex is present in the form of hetero- Homo dimer is formed by hydrogen bonding and hydrophobic bonding by moieties of? 3,? 4,? 5 and? 6 helix (FIG. 3c).

It is also expected that the positive charge amino acids (Lys88, Arg90, His91 and Arg92) are located in the loop between the a5 and the a6 helix and are expected to bind to RNA molecules (Fig. 4a) and that the active site on the concave surface of the VapC30 homodimer interface , And active sites are formed by loops and alpha-end loops between alpha 1 , beta 1, alpha 3 and alpha 6. The active site contains conserved Asp4, Glu40, and Asp99 residues essential for RNAase activity, and these residues form a strong hydrogen bonding network and associate with water molecules. Comparing the structure of the active site with the toxin protein of other species, it was confirmed that the water molecule could act like the metal ion of other species (FIG. 4b).

<1-7> Structural features of VapB30 protein

The structure of VapB30 protein was determined through the results of Examples 1-3 and 1-4.

As shown in FIG. 5, when comparing the eight VapB30 monomers of the asymmetric unit of crystalline form I and II, the N-terminal region showed almost the same structure consisting of alpha-helix and loop, but the C- Respectively. In addition, when compared to other known VapB proteins, the N-terminal alpha-helix (residues 49-62) of the VapB30 protein is similar but the overall structure is not similar (FIG. 5).

Example 2 Confirmation of VapC30 Protein Inhibitory Effect on E. coli Growth

In order to confirm the effect of VapC30 protein on the growth of E. coli, the following experiment was conducted.

VapC30 alone or in pET28a (+) - His ₆ -VapB30 and cultured pCOLD1-His ₆ -VapC30 then inserted an expression vector, as for E. coli (E.coli BL21) cells, Example <1-1> in the same manner, and Expression was induced and OD ₆₀₀ was measured at 30 minute intervals for 4 and a half hours.

As a result, when VapC30 alone was expressed alone, the growth of E. coli was inhibited and the growth inhibitory effect of VapB30 was neutralized when the VapBC30 complex was expressed and compared with the control (Fig. 6).

Example 3 Confirmation of Initiation tRNA (initiator tRNA) Degradability of VapC30 Protein

The following experiments were carried out to investigate the activity of VspC30 protein RNAase.

<3-1> Synthesis of tRNA used as a substrate

The DNA fragment containing the ^fMet portion of the Mycobacterium tuberculosis (H37Rv) tRNA was amplified by polymerase chain reaction (PCR) and the recognition site of the T7 RNA synthetase was attached to the upper portion thereof. Using T7 RNA synthetase, tRNA ^fMet was transcribed and purified using a MegaClear kit (Ambion).

<3-2> Identification of initiation tRNAase activity of VapC30

After VapC30 to the different protein concentrations (3, 6, 9 ㎛) mixture as in Example <10-1> of ^{tRNA fMet (3 ㎛) [10} ul reaction solution - 0.1 M NaCl, 20 mM Tris -HCL buffer (pH 8.0) and 40 units of RiboLock ^™ RNase inhibitor (Thermo scientific)]. The sections were allowed to stand at 37 ° C for 1 hour, and the cleaved RNA fragments were confirmed by 15% acrylamide denaturing gel electrophoresis containing 8M Urea.

As a result, when the VapBC30 complex (30 mu m) was treated, the RNA was present intact, whereas when the VapC30 protein was treated, RNA was completely decomposed in a concentration-dependent manner (FIG.

<3-3> The manganese ion of the VapC30 protein (Mn ²⁺ ) Identification of dependent RNA degradation ability

In addition, a fluorescence quenching assay was performed to confirm the effect of manganese ions on initiation tRNA degradation of VapC30 protein.

Specifically, VAPC30 or VapBC30 protein and tRNA ^fMet were mixed and treated with 10 uM MgCl ₂ , 10 uM MnCl ₂ and 10 uM EDTA, respectively, and fluorescence (SPECTRAmax GEMINI XS spectrofluorometer) was measured over time.

As a result, it was confirmed that when magnesium ion (Mg ²⁺ ) was treated, the activity of VapC30 was hardly increased, but that the activity of VapC30 was increased by three times when manganese ion was present (FIG. 8).

<Example 4> Peptide design for inhibiting toxin-antitoxin binding

In the structure of VapBC30 complex, the binding between the toxin-antitoxin complexes is determined by binding of α1-helices (amino acid residues 49-62, SEQ ID No. 1), toxin α2-helices (amino acid residues 17-27, (amino acid residues 52-65, SEQ ID NO: 3). Therefore, three peptides were designed that affect only the binding site without touching the active site of the toxin. Peptide I mimics the α1-helices of the antitoxin, peptide II mimics the α2-helices of the toxin, and peptide III mimics the α4-helices of the toxins:

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.

EXPERIMENTAL EXAMPLE 1 Confirmation of Inhibitory Effect on Complex Formation of Synthetic Peptides

The following experiment and fluorescence extinction analysis were performed to confirm the inhibitory effect of the peptide synthesized in Example 9 on the formation of VapBC30 complex.

Specifically, VapC30 (20 ㎛) or VapBC30 complex (20 ㎛) and then mixed with tRNA ^fMet [40 ul reaction solution - 0.1 M NaCl, 20 mM Tris -HCL buffer (pH 8.0) and 40 units RiboLock ^TM RNase inhibitor (Thermo The peptide I (SEQ ID NO: 4), peptide II (SEQ ID NO: 5) and peptide III (SEQ ID NO: 6) were treated at 20, 50 and 200 uM concentrations, respectively, at 37 DEG C And allowed to stand for 2 hours.

As a result, when the fluorescence value of 20 uM Vap BC30 protein was taken as 100% and the fluorescence value of 20 uM Vap BC30 protein was taken as 0%, it was confirmed that fluorescence increased depending on the concentration when the peptide was treated . This means that complex formation between VapBCs is inhibited by peptides and RNA is degraded by VapC30. Particularly, when peptide III mimicking the α-4 helix of toxin was added, it was confirmed that RNA was most decomposed (FIG. 9).

<110> SNU R & DB Foundation <120> Peptides targeting toxin-antitoxin system of Mycobacterium          tuberculosis and therof <130> 15P-02-029 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 14 <212> PRT <213> Mycobacterium tuberculosis <400> 1 Leu Arg Asp Glu Leu Ala Ala Ile Arg His Arg Cys Ala Ala   1 5 10 <210> 2 <211> 11 <212> PRT <213> Mycobacterium tuberculosis <400> 2 Asp Ala Glu Arg Phe Glu Ala Ala Val Glu Ala   1 5 10 <210> 3 <211> 14 <212> PRT <213> Mycobacterium tuberculosis <400> 3 Pro Gly Gly Arg Glu Leu Asp Leu Trp Leu His Arg Ala Ala   1 5 10 <210> 4 <211> 8 <212> PRT <213> Mycobacterium tuberculosis <400> 4 Glu Leu Ala Ala Ile Arg His Arg   1 5 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthesized peptides <400> 5 Asp Glu Pro Asp Ala Glu Arg Phe Glu Ala Ala Val Glu Ala Asp His   1 5 10 15 Ile     <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthesized peptides <400> 6 Arg Phe Gly Glu Pro Gly Gly Arg Glu   1 5 <210> 7 <211> 255 <212> DNA <213> Mycobacterium tuberculosis <400> 7 atggcgctga gtatcaagca cccggaagcc gaccggctcg cgcgagcgct tgcggcgcgc 60 accggcgaga cgttgaccga ggcagtggtt accgcgttgc gcgagcggct cgctcgtgag 120 actgggcgtg cccgtgttgt cccgttgcgc gacgagcttg ccgcgattcg gcaccggtgc 180 gcagcgttgc cggtggtcga caaccggtcc gctgaggcga ttctcggcta tgacgagcgc 240 ggattgccgg cctga 255 <210> 8 <211> 396 <212> DNA <213> Mycobacterium tuberculosis <400> 8 atggtgatcg acacgtccgc gctcgttgcg atgctcagcg acgagccaga cgcagagcgg 60 ttcgaggccg ccgtcgaagc cgaccacatc cggctgatgt cgacggcgtc ttacctggaa 120 acggcactcg tgatagaagc ccgcttcggt gaaccgggcg gacgtgagct ggatctgtgg 180 cttcatcgcg ccgcggtcga ccttgttgcc gtgcatgccg accaagcgga tgccgcgcgc 240 gccgcctacc gcacgtacgg caagggaagg catcgtgcgg ggctcaacta cggcgactgc 300 ttctcatacg gcctcgccaa gatcagcggc cagccactcc tgttcaaggg cgaagatttc 360 caacacaccg acatcgccac ggtcgcgctg ccctaa 396

Claims (10)

An antibiotic peptide which inhibits binding of a Vap BC30 complex, which is a toxin-antitoxin conjugate of Mycobacterium tuberculosis, which is composed of the amino acid sequence of any one of SEQ ID NOS: 4 to 6.
The antibiotic peptide according to claim 1, wherein the antisense peptide consisting of the amino acid sequence of SEQ ID NO: 4 is characterized by inhibiting the binding of? 1 composed of the amino acid sequence of SEQ ID NO: 1 corresponding to amino acid residues 49-62 of VapB30, Antimicrobial peptide.
2. The antibiotic peptide according to claim 1, wherein the antisense peptide consisting of the amino acid sequence of SEQ ID NO: 5 is characterized by inhibiting the binding of? 2 consisting of the amino acid sequence of SEQ ID NO: 2 corresponding to amino acid residues 17-27 of VapC30, Antimicrobial peptide.
The antibiotic peptide according to claim 1, wherein the antisense peptide consisting of the amino acid sequence of SEQ ID NO: 6 inhibits the binding of? 4 consisting of the amino acid sequence of SEQ ID NO: 3 corresponding to amino acid residue 52-65 of VapC30, Antibiotic peptides.
The antibiotic peptide of claim 1, wherein the peptide does not affect the activity of the toxin.
An antibiotic composition for mycobacterium tuberculosis containing the antibiotic peptide of claim 1 as an active ingredient.
delete delete An antibiotic quasi-drug for tubercle bacilli containing the antibiotic peptide of claim 1 as an active ingredient.
A tuberculosis antibiotic external preparation containing the antibiotic peptide of claim 1 as an active ingredient.

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