KR102521182B1 - Antibacterial Stapled Peptides Targeting Toxin-antitoxin System of Mycobacterium tuberculosis and Use Thereof - Google Patents

Abstract

The present invention relates to antibacterial staple peptides or uses thereof that target the Mycobacterium tuberculosis VapBC30 toxin-antitoxin system.

Description

Antibacterial Stapled Peptides Targeting Toxin-antitoxin System of Mycobacterium tuberculosis and Use Thereof

The present invention relates to antimicrobial staple peptides targeting the toxin-antitoxin system of Mycobacterium tuberculosis .

The bacterial toxin-antitoxin (TA) system is considered essential for bacterial survival. Bacterial toxin-antitoxin systems play an essential role in cell survival as binding regulators. Typically toxins act as negative regulators and are involved in various functions such as RNA cleavage, acetylation, phosphorylation, and inhibition of ATP synthesis. Throughout this control action, the toxin inhibits growth and ultimately kills the bacterial cell. Unlike toxins, antitoxins act as positive regulators in cell survival through neutralization of RNase (ribonuclease) activity by binding to cognate toxins at the protein or genetic level. Under various stress conditions, such as malnutrition or antibiotic treatment, bacteria use mechanisms related to the balance between toxin and antitoxin expression levels to maintain homeostasis. Since the toxin-antitoxin system is closely related to bacterial survival, the toxin-antitoxin system is recognized as a promising antibacterial target for the development of novel antibacterial agents.

There is an abundant toxin-antitoxin system in the entire genome of M. tuberculosis . Surprisingly, proteins belonging to the toxin-antitoxin system account for more than 5% of all proteins in the H37Rv strain. Since the treatment of tuberculosis infection still requires long-term combination therapy, a novel mechanism of action different from that of typical antibiotics is required, especially in the case of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). . Continuing studies indicate that the toxin-antitoxin system is likely involved in the infection and pathogenicity of bacteria, so the functional and regulatory role of the toxin-antitoxin system in their bacterial survival has been extensively discussed in the past decade.

Specifically, the structure-function relationship study of the toxin-antitoxin system from Mycobacterium tuberculosis provides information on the protein-protein interaction (PPI) between toxin and antitoxin, which can be a promising therapeutic target in the development of novel antibacterial agents. In particular, the toxin-antitoxin system, despite its frequent prevalence in bacterial pathogens, is not present in eukaryotic cells, making it a more attractive target. Accordingly, the present inventors conceived of hindering the interaction between toxin and antitoxin by small molecules, which can induce RNase activity of toxin protein by isolating the antitoxin protein.

According to previous studies, three helical peptide segments designed utilizing the crystal structure of the Mycobacterium tuberculosis VapBC30 complex ( Rv0623 - Rv0624 , PDB ID: 4XGQ) mimic the binding interface between VapC30 and VapB30 and bind to the corresponding toxin-antitoxin complex in vitro. It was confirmed that it exhibits inhibitory activity against However, in general, linear peptides have disadvantages of poor cell permeability and proteolytic stability. In addition, high conformational flexibility contributes to a low binding force to a target protein, resulting in a decrease in bioactivity.

Schafmeister, C. E., Po, J., and Verdine, G. L. (2000), J. Am. Chem. Soc. 122, 5891-5892.

It is an object of the present invention to provide antibacterial staple peptides targeting the Mycobacterium tuberculosis toxin-antitoxin system.

In order to achieve the above object, the present invention provides an antibacterial staple peptide that inhibits binding within VapBC30, a toxin-antitoxin conjugate of Mycobacterium tuberculosis . Specifically, the present invention inhibits the binding of VapB30 and VapC30 in the toxin-antitoxin conjugate VapBC30 of Mycobacterium tuberculosis, and provides an antibacterial staple peptide characterized in that two or more amino acids constituting the peptide are linked to each other.

In addition, the present invention provides an antibacterial composition against Mycobacterium tuberculosis, particularly a composition for treating tuberculosis, containing the antimicrobial staple peptide as an active ingredient.

The antibacterial staple peptide of the present invention mimics the binding of the toxin-antitoxin complex VapBC30 of Mycobacterium tuberculosis, but inhibits the binding of the toxin-antitoxin complex without affecting the activity of the toxin. induces the death of Therefore, the antibacterial staple peptide of the present invention can be used as an antibiotic composition that can provide a novel antibacterial strategy against Mycobacterium tuberculosis.

Figure 1 shows the design of staple peptides based on alanine scanning. (a) Alanine scan results of three VapBC30-derived linear peptides (VapB30 α1 _{52 -59} , VapC30 α2 _{14 -30} , and VapC30 α4 _{48 -56} ). Activity was expressed as 100% of the activity of the original peptide. (b) non-natural amino acids with olefins used for peptide stapling. (c) Positioning of stapled moieties. (d) synthetic peptides.
Figure 2 shows the HPLC chromatogram of the purified peptide. All spectra were measured on an Agilent HP 1260 system (Agilent Technologies, Santa Clara, CA, USA). A Zorbax C18 column (2.7 μm, 120 Å, 4.6 Х 50 mm) was used as the stationary phase. For the mobile phase, solvent A (water with 0.1% v/v trifluoroacetic acid (TFA)) and solvent B (acetonitrile) were used as a gradient. Gradients were given as follows: i) a linear gradient of B from 5 to 50% (0 to 5 min); ii) a linear gradient of B from 50 to 100% (5 to 7 min); iii) isocratic hold (B, 100%) (7-15 min); and iv) a linear gradient of B from 100 to 5% (15 to 17 minutes). The flow rate was set at 1.0 mL/min.
Figure 3 shows the high resolution (HR) mass spectrum of the peptide. All spectra were obtained with a super-resolution ESI Q-TOF mass spectrometer (Bruker, USA) (V30-SP-1 to V30-SP-11) and a high-resolution LC/MSMS spectrometer (AB Sciex, USA) (V30-SP-8-FITC to V30-SP-9-FITC) was used.
4 presents in vitro assay data for staple peptides. (a) Schematic diagram of the in vitro RNase (ribonuclease) activity assay. (b) In vitro RNase activity assay results of synthetic staple peptides. (c) Concentration-dependent RNase activity of V30-SP-8 (left) and V30-SP-9 (right). (d) CD spectra of V30-SP-8 and V30-SP-9.
Figure 5 shows the CD spectroscopic analysis of linear peptides with successive increases in TFE.
6 is a confocal laser scanning microscope image (left) of smegma bacteria treated with fluorescein isothiocyanate (FITC)-labeled peptide (10 μM), and a bright-field image of smegma bacteria ( middle) and merged images (right). (a) Empty control. (b) linear peptides. (c) V30-SP-8. d) V30-SP-9.
7 shows confocal laser scanning microscopy images (left), brightfield images (middle) and merged images (right) of smegma bacteria treated with FITC-labeled peptide (10 μM). (a) V30-SP-8. (b) V30-SP-9.
8 shows flow cytometry data. (a) Results of flow cytometric analysis of smegma cells obtained in the untreated (negative control) group and upon addition of FITC-labeled peptides (linear form, V30-SP-8 and V30-SP-9). (b) Bar graph showing the amount of cellular uptake of each peptide. Activity was normalized based on that of V30-SP-9.
Figure 9 shows the antibacterial activity of V30-SP-8 and V30-SP-9 (a) Smegma bacteria were treated with various concentrations of V30-SP-8 and V30-SP-9. Cells were labeled with LIVE/DEAD BacLight staining (Syto 9; propidium iodide (PI)) and analyzed by fluorescence activated cell sorting (FACS). The antibacterial activity of 70% isopropyl alcohol represents a positive control. A LIVE cell/DEAD cell area was set using this control. (b) Histogram for FACS data. Histograms represent three sets of independent experiments.

The present invention provides antimicrobial staple peptides targeting the toxin-antitoxin system of Mycobacterium tuberculosis.

In one embodiment, the antibacterial staple peptide of the present invention inhibits the binding of VapB30 and VapC30 in the toxin-antitoxin conjugate VapBC30 of Mycobacterium tuberculosis, and two or more amino acids constituting the peptide may be linked to each other. At this time, the antimicrobial staple peptide may consist of 5 to 20, preferably 8 to 17, particularly 8, 9, 13, 15, or 17 amino acids.

Staple peptides may be formed, for example, by linking two amino acids together (stapled) or by linking three amino acids together (stitching). Staple peptides in which three amino acids are linked together are also referred to as stitch peptides.

As used herein, the term "stapling" refers to a technique capable of stabilizing the α-helix structure of a peptide by introducing a hydrocarbon cross-link or the like into the peptide. "Peptide stapling" refers to covalently linked ("stapled"), e.g., two olefin-containing side chains present in a polypeptide using a ring-closing metathesis (RCM) reaction to form a bridge. ) refers to the synthesis method. For example, for peptide stapling, a hydrocarbon (eg, a compound containing a carbon-carbon double bond) at any position (i) in the peptide sequence and the 4th position (i+4) toward the C-terminus therefrom After introducing any amino acid functionalized with , the residues of these two amino acids are cross-linked by a metathesis reaction using a Grubbs catalyst.

As used herein, the term "amino acid" refers to a molecule comprising an amino group and a carboxyl group. Amino acids include alpha-amino acids and beta-amino acids. Amino acids can be unnatural amino acids or natural amino acids. Exemplary amino acids include natural alpha-amino acids such as the D- and L-isomers of the 20 common naturally occurring alpha amino acids found in peptides, unnatural alpha-amino acids, natural beta-amino acids (e.g., beta-alanine), and including, but not limited to, unnatural beta-amino acids. The amino acids used in the construction of the peptides of the present invention may be prepared by organic synthesis or obtained by other routes, such as isolation or degradation from natural sources. Amino acids are commercially available or can be synthesized.

The term "peptide" as used herein includes polymers of amino acid residues linked together by peptide (amide) bonds, and refers to proteins, polypeptides, and peptides of any size, structure, or function. A peptide or polypeptide may refer to an individual protein or a collection of proteins.

When two or more amino acids of the staple peptide are linked to each other, the corresponding amino acids may be linked through cross link or bridge formation. For example, the bridge may be a hydrocarbon bridge, and specifically, may be formed of a double hydrocarbon bond or a single hydrocarbon bond. In another example, the amino acids linked to each other may be linked through a disulfide bond, a carbon-carbon double bond, or an amide bond.

In one embodiment, the antimicrobial staple peptides of the invention may include two or more non-natural amino acids. Preferably, amino acids linked to each other in the peptides of the present invention include non-natural amino acids, examples of which are shown in Table 1 below. Preferably, the non-natural amino acid is a non-natural amino acid having an alkene group, for example, the alkene group is selected from the group consisting of pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, and dodecenyl groups. It may be one or more selected species. Most preferred unnatural amino acids are (S)-2-(4′-pentenyl)-alanine (S ₅ ), (S)-2-(7′-octenyl)-alanine (S ₈ ), (R)- 2-(4′-pentenyl)-alanine (R ₅ ), (R)-2-(7′-octenyl)-alanine (R ₈ ), 2,2-bis-(4′-pentenyl)- It may be at least one selected from the group consisting of glycine (B ₅ ) and 2,2-bis-(7'-octenyl)-glycine (B ₈ ).

Looking at the structure of the VapBC30 complex, the binding between the toxin-antitoxin complex involves the α1-helix of the antitoxin VapB30 (amino acid residues 52-59 of VapB30, SEQ ID NO: 23) and the α2-helix of the toxin VapC30 (amino acid residues 14-30, SEQ ID NO: 24). ) and the α4-helix of the toxin VapC30 (amino acid residues 48-56, SEQ ID NO: 25). Accordingly, the present inventors tried to prepare a new stapled peptide while imitating the peptide of the corresponding part.

Accordingly, the antimicrobial staple peptide of the present invention may have any one of the amino acid sequences of SEQ ID NOs: 1 to 11 below.

EXAAXRHR (SEQ ID NO: 1);

EXAAIXHR (SEQ ID NO: 2);

ELAAXRHX (SEQ ID NO: 3);

RXGEXGGRE (SEQ ID NO: 4);

RXGEPGGRX (SEQ ID NO: 5);

AERFXAAVEADXI (SEQ ID NO: 6);

DEPDAERFXAAVEADXI (SEQ ID NO: 7);

XLAAXRHX (SEQ ID NO: 8);

XLAAIRHX (SEQ ID NO: 9);

RXGEXGGRX (SEQ ID NO: 10); and

XPDAERFXAAVEADX (SEQ ID NO: 11);

In the above formula, X is (S)-2-(4'-pentenyl)-alanine (S ₅ ), (S)-2-(7'-octenyl)-alanine (S ₈ ), (R)-2 -(4′-pentenyl)-alanine (R ₅ ), (R)-2-(7′-octenyl)-alanine (R ₈ ), 2,2-bis-(4′-pentenyl)-glycine (B ₅ ) or 2,2-bis-(7′-octenyl)-glycine (B ₈ ).

Another preferred antibacterial staple peptide of the present invention may have an amino acid sequence of any one of SEQ ID NOs: 12 to 22.

ER ₅ AAS ₅ RHR (SEQ ID NO: 12);

ES ₅ AAIS ₅ HR (SEQ ID NO: 13);

ELAAR ₅ RHS ₅ (SEQ ID NO: 14);

RR ₅ GES ₅ GGRE (SEQ ID NO: 15);

RR ₈ GEPGGRS ₅ (SEQ ID NO: 16);

AERFR ₈ AAVEADS ₅ I (SEQ ID NO: 17);

DEPDAERFR ₈ AAVEADS ₅ I (SEQ ID NO: 18);

S ₅ LAAB ₅ RHS ₅ (SEQ ID NO: 19);

R ₈ LAAIRHS ₅ (SEQ ID NO: 20);

RR ₅ GEB ₅ GGRR ₅ (SEQ ID NO: 21); and

R ₈ PDAERFB ₅ AAVEADS ₈ (SEQ ID NO: 22).

The most preferred antibacterial staple peptide of the present invention may have the amino acid sequence of SEQ ID NO: 19 or 20.

S ₅ LAAB ₅ RHS ₅ (SEQ ID NO: 19); or

R ₈ LAAIRHS ₅ (SEQ ID NO: 20).

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

In a drug development program targeting tuberculosis (TB), the inventors have discovered certain peptides that mimic the binding of the VapBC30 complex, but inhibit bacterial cell growth and result in cell death. In the present invention, candidate peptides based on hydrocarbon stapling strategy were optimized and in vitro RNA activity evaluation was performed. Among _the staple peptides modified from the VapB30 α1 _52-59 peptide of the present invention, the staple peptide of SEQ ID NO: 19 (V30-SP-8) successfully penetrates the cell membrane of Mycobacterium smegmatis and inhibits 50% of the isolates. It was confirmed that the antibacterial activity was exerted at an inhibitory concentration (MIC ₅₀ ) < 6.25 μM.

Accordingly, the present invention provides an antimicrobial staple peptide characterized by having a MIC50 of less than 6.25 μM.

Therefore, the staple peptide according to the present invention can be used for various purposes and uses requiring antibacterial activity, such as compositions for preventing, suppressing, improving or treating infectious diseases caused by pathogenic bacteria, viruses, yeasts or fungi, compositions for animal feed , It can be used for purposes such as additives for animal feed, functional food compositions, food additives, food preservatives, disinfectants, disinfectants and detergents, but is not limited thereto.

Accordingly, the present invention provides an antimicrobial composition comprising the staple peptide. Preferably, the composition is a pharmaceutical composition for preventing or treating tuberculosis.

The term "tuberculosis" used in the present invention is directly infected by fine droplets or droplets from a patient infected with Mycobacterium tuberculosis through the respiratory tract and, by its definition, is a disease caused by infection with Mycobacterium tuberculosis. Currently, because tuberculosis bacilli are transmitted through the air, tuberculosis often occurs in lung tissue. Therefore, tuberculosis usually means pulmonary tuberculosis, but tuberculosis bacteria can invade most tissues or organs, including the lungs, pleura, lymph glands, spine, brain, nerves, kidneys, gastrointestinal tract, and bones, and cause symptoms. [0026] Depending on the organs infected with Mycobacterium tuberculosis, various infectious diseases such as bone joint tuberculosis such as hip tuberculosis, bone tuberculosis, and pelvic tuberculosis, tuberculosis of the urogenital system such as testicular tuberculosis, bladder tuberculosis, and kidney tuberculosis, tuberculosis of the digestive system such as intestinal tuberculosis, and tuberculosis of the central nervous system such as tuberculous meningitis can be divided Mycobacterium tuberculosis multiplies slowly using nutrients in the host's body, and when infected with tuberculosis, symptoms such as feeling weak, easily fatigued, and losing weight usually appear. In addition, symptoms appear differently depending on the organs infected with Mycobacterium tuberculosis. In the case of pulmonary tuberculosis, symptoms such as coughing, sputum, and chest pain occur. Lymphatic tuberculosis causes enlarged lymph nodes throughout the body, especially in the upper neck or armpit, causing pain and pressure. In addition, symptoms such as back pain in the case of spinal tuberculosis and headache and vomiting in the case of tuberculosis of the central nervous system appear. When a person's body is infected with Mycobacterium tuberculosis, normal tissues of the body are destroyed at a very slow rate by an inflammatory reaction with immune cells, and cheese-like pus is formed, and granulomas (lump lumps) develop around it. do.

Tuberculosis that can be prevented or treated with the pharmaceutical composition of the present invention is not limited thereto, but may preferably be pulmonary tuberculosis, and more preferably may be infectious tuberculosis caused by Mycobacterium tuberculosis.

The term " Mycobacterium tuberculosis " used in the present invention may be multi drug resistance tuberculosis (MDR-TB) or extensively drug resistant tuberculosis (XDR-TB).

As used herein, the term "prevention" refers to any activity that inhibits or delays the onset of the disease by ingestion or administration of the composition, and "treatment" means that the symptoms of the disease are improved or improved by ingestion or administration of the composition. It means any action that benefits.

As used herein, the term "improvement" refers to an effect of alleviating the onset or symptoms of the disease or inhibiting or reducing the growth and division of Mycobacterium tuberculosis by ingesting or administering the composition.

The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable excipient. At this time, pharmaceutically acceptable excipients are commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto.

In addition to the above components, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like may be further included.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically applied) depending on the desired method, and the dosage depends on the condition and weight of the patient and the severity of the disease. , Depending on the drug form, administration route and time, it can be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and activity of the drug of the patient's disease , sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, body weight, absorption rate, inactivation rate and excretion rate of the active ingredient in the body, type of disease, and concomitant drugs, generally 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight may be administered daily or every other day or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of obesity, gender, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

<Experiment method>

The present invention can be carried out by the following experimental methods.

1. Protein Preparation

The toxin-antitoxin complex proteins VapBC30 and VapC30 proteins alone were overexpressed in E. coli cells and purified according to (Lee et al., Nucleic Acids Res. Volume 43, Issue 15, 3 September 2015, 7624-7637).

2. peptide synthesis

Peptides were prepared using Fmoc chemistry on Rink Amide MBHA Resin (BeadTech) with a loading capacity of 0.41 mmol/g. All syntheses were performed on the 82 μmol scale. The dry resin was swollen with N,N -dimethylformamide (DMF) for 2 hours prior to use. The Fmoc protecting group was removed by treatment with 20% piperidine in DMF (2 Х 10 min). After Fmoc deprotection, the resin was washed with DMF (Х3), MeOH (Х3), dichloromethane (DCM, Х3) and N -methylpyrrolidone (NMP, Х3). 4 equivalents (eq) of 1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU) as an activator for both natural and unnatural amino acids; Coupling was performed in NMP for 2 hours using 4 equivalents of Fmoc protected amino acid and 8 equivalents of diisopropylethylamine (DIPEA). After complete washing with DMF (Х9), completion of the coupling reaction was confirmed by a Kaiser test.

3. Metathesis and Purification

Ring-closure metathesis of resin-linked N -Fmoc, a side chain-protected peptide, was performed using 20 mol% Grubbs catalyst in 1,2-dichloroethane (DCE) for 2 hours at room temperature under an Ar atmosphere. After washing with DCM (Х2), MeOH (Х2) and DCM (Х2), the reaction was repeated two more times. The reaction was monitored using an Agilent HP 1260 system (Agilent Technologies, Santa Clara, Calif., USA) after cleavage of the peptides from the resin aliquots. After Fmoc removal, N-terminal modification of the peptides with acetyl groups or fluorescein isothiocyanate (FITC) was performed according to the following conditions. i) Acetylation: acetic anhydride (30 equiv.) and diisopropylethylamine (60 equiv.) in NMP at room temperature for 45 min; ii) FITC labeling: FITC (1.5 eq) in pyridine/DMF/DCM (v/v/v = 12/7/5) overnight at room temperature. The resin was washed with DMF (Х3), MeOH (Х3) and DCM (Х3) and vacuum dried overnight. Peptides were deprotected and cleaved from the resin by treatment with a mixture of trifluoroacetic acid/triisopropylsilane/water (v/v/v = 95/2.5/2.5) for 2 hours. The volatile components were then removed under reduced pressure. The crude mixture was dissolved in a 1:1 mixture of acetonitrile and water and filtered through a 0.45 μm syringe filter. The desired product was purified by reverse-phase high performance liquid chromatography (HPLC) using a Zorbax C18 column (Agilent, 5 μm, 80 Å, 21.2 X 150 mm) and analyzed by liquid chromatography-mass spectrometry (LC-MS) and ultra-resolution Electrospray ionization quadrupole time-of-flight (ESI Q-TOF) mass spectrometry (Brooker, USA) was characterized.

4. In vitro ribonuclease assay

The ribonuclease activity of VapC30 released from VapBC30 by the peptide was confirmed using the RNase Alert Kit (IDT, Coralville, Iowa, USA). In this assay, a fluorophore is covalently attached to one end of a synthetic RNA strand and quenched by a quenching group at the other end of the synthetic RNA. When synthetic RNA containing a fluorophore-cheer pair interacts with a ribonuclease, the synthetic RNA is digested and fluorescence is detected. The emitted fluorophore emitted fluorescence at 520 nm upon excitation at 490 nm. The generated fluorescence (relative fluorescence units, RFU) was observed in a Spectramax Gemini XPS microplate reader (Molecular Devices, San Jose, CA, USA). Various concentrations of peptide (1.25, 2.5, 5 and 10 μM), 10 μM VapC30 toxin, and 10 μM VapBC30 complex were used.

5. CD spectroscopy

CD measurements were performed using a Chirascan-Plus spectropolarimeter (Applied Photophysics, Leatherhead, UK) at 20 °C in a cell with a 1 mm optical path. Peptide samples were prepared at a concentration of 50 μM in a mixture of water and acetonitrile (9:1, v/v). To analyze only the linear peptides, TFE was successively added to the solvent (1, 10, 20, 50, 100%). CD scans were performed from 180 to 260 nm with a 1 nm bandwidth and a scan rate of 100 nm/min. Three scans were averaged and the solvent background was subtracted from the averaged scan. The degree of helix of each peptide was calculated with CDNN software.

6. Cloning and expression of smegma bacteria ( M. smegmatis )

The genes encoding Mycobacterium tuberculosis (strain H37Rv) VapB30 ( rv0623 ) and VapC30 ( rv0624 ) were amplified using primers Rv0623-BHI_F, Rv0623-Hid_rN, Rv0624-Ndel_F and Rv0624-ERV-R (Table 1) and the expression vector pYUBDuet ( Addgene, Watertown, MA) to prepare VapB30 and VapC30 with an N-terminal hexa-histidine tag. Vectors carrying rv0623 and rv0624 were transformed into E. coli Top10 competent cells (Thermo Fisher Scientific, Waltham, MA, USA). The plasmid extracted from the transformed Top10 cells was electroporated into Smegma mc ² 155. Preparation and electroporation of smegma competent cells were performed according to a reported protocol (Bio-Rad, Hercules, CA, USA, www.Bio-Rad.com). Smegma mc ² 155 strains carrying rv0623 and rv0624 were cultured in Middlebrook 7H9 liquid culture medium supplemented with 10% albumin-dextrose-saline (ADS), 0.5% glycerol, and 0.1% Tween 80 at 37°C and 100 rpm. grown up Hygromycin (Hyg, 50 μg/mL) was used for culturing smegma bacteria. When the OD ₆₀₀ of the culture reached 0.5, 0.5 mM isopropyl 1-thio-D-galactopyranoside was added to induce expression and the culture was grown at 37°C for 24 hours.

7. Confocal Microscopy

The permeability of the peptide to smegma bacteria was evaluated using the FITC-labeled peptide using a confocal microscope (Leica Micro-systems, Wetzlar, Germany). Cultures of Smegma grown to mid-log phase (OD ₆₀₀ 0.3-0.5) in complete 7H9 medium were diluted 5-fold in Mueller-Hinton broth (BD Bioscience). Diluted cell suspensions aliquoted into Eppendorf tubes were treated with an antimicrobial peptide preparation at 10 μM. After incubation at 37° C. and 100 rpm for 1 hour, the cell suspension was washed with phosphate buffered saline (PBS). Images were acquired on a TCS8 confocal microscope (Leica Microsystems) with a 63x oil immersion objective.

8. Antibacterial activity test ( MIC test)

The minimal inhibitory concentrations (MICs) of several antibacterial peptide preparations against smegma bacteria harboring VapBC30 were determined by a two-fold serial dilution method in 7H9 medium. Log-phase cultures of smegma bacteria were diluted to an OD ₆₀₀ value of 0.01. 200 μL of the diluted culture was inoculated into a 96-well plate (polystyrene round square well, SPL, Korea) and 100 μL of a peptide solution at a concentration of 100 μM to 3.125 μM was added to each well. Visible growth was scored after 1 day incubation at 37 °C. Wells containing bacterial pellets were labeled positive wells and wells containing clear broth were labeled negative wells. MIC was defined as the lowest peptide concentration that completely inhibited bacterial growth. Each test was performed in duplicate.

9. flow cell analyze

Both permeability and viability of the peptides to smegma were evaluated using flow cytometry. Sample preparation for permeability evaluation followed the same protocol as sample preparation for confocal images. For viability assays, smegma bacteria harboring VapBC30 were grown in complete 7H9 medium to mid-log phase (OD ₆₀₀ 0.3-0.5) and treated with antimicrobial peptide preparations at 10 μM. After culturing for 24 hours at 37° C. and 100 rpm, cells were stained with LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen) containing PI-phycoerythrin (PE) and Syto 9-FITC. Both flow cytometry analyzes were performed on a FACSLyric (BD Bioscience, USA) and data were analyzed using FlowJo software (TreeStar).

Example 1: linear of peptides alanine scanning and staple of peptides design

A helical fragment at the binding interface of VapC30 or VapB30 acts as an inhibitor of protein-protein interactions between the glycolytic toxin and the antitoxin. Alanine scanning was performed to determine the contribution of each residue to the inhibitory activity and to identify the appropriate location of unnatural amino acids for peptide stapling (Fig. 1a).

Each peptide having an alanine substitution was added to the VapBC30 complex, and the expression of VapC30 RNase activity was evaluated through an in vitro RNase assay.

In the case of the VapB30 α1 _{52 -59} peptide, none of the mutant peptides exhibited a significantly appreciable change in inhibitory activity (less than 10% reduction compared to the wild-type peptide). Therefore, the positions of the olefin-containing residues were selected according to the following criteria (FIG. 1b): (i) less than 5% change in inhibitory activity when alanine substituted and (ii) i and i+3 to align the residues in the same helix plane. Pick two positions from , 4, or 7. For the double staple (stitched) peptide, the B ₅ residue was incorporated in the middle of the two unnatural amino acids at i and i+7 (Fig. 1c).

For the other two linear peptides (VapC30 α2 _{14 -30} and VapC30 α4 _{48 - 56} ), some mutations (E6A, R7A, F8A, E9A, V12A and E13A for the former, R1A, E4A, G7A, R8A and E13A for the latter) E9) inhibited more than 10% of the inhibitory activity against the toxin-antitoxin complex compared to the corresponding original peptide (Fig. 1a).

Based on these results, we determined suitable positions for stapled residues to generate appropriate synthetic targets (Fig. 1d). Entries 1 to 11 of FIG. 1d correspond to SEQ ID NOs: 12 to 22, respectively. For some VapC30 α2 _14-30 derivatives, those that did not contribute to the inhibitory activity were truncated (Entry 6 and ₁₁ in Fig. 1d).

Example 2: staple to the peptide for in vitro RNase analyze

Based on the alanine scanning results, a total of 11 staple peptides shown in FIG. 1d were prepared through solid-phase peptide synthesis with a purity greater than 95% (refer to Experimental Methods “2. Peptide Synthesis” and “3. Metathesis and Purification”). After purification by HPLC, it was confirmed through LC-MS analysis that the target peptide was produced (FIG. 2, Table 4). In addition, the desired peptide was characterized by high-resolution mass spectrometry (FIG. 3).

An in vitro RNase assay was performed to test the ability to disrupt interactions within the VapBC30 complex (see experimental methods section "4. In vitro ribonuclease assay"). As a result, it was confirmed that the inhibitor peptide of the present invention induces the recovery of VapC30 RNase activity by releasing the VapC30 toxin protein from the VapB30 antitoxin protein. Subsequently, the fluorescence intensity increased in proportion to the number of free VapC30 toxin proteins as the substrate RNA serving as a linker between the fluorophore and the quencher was cleaved (FIG. 4a).

In an initial evaluation, comparison between staple peptides was performed by treating equal amounts of the peptide (10 μM) with a mixture of toxin-antitoxin complex and substrate RNA. As a result, the five staple peptides modified by the addition of the VapB30 α1 _52-59 peptide were found to be superior to other staple peptides in terms of disruptive activity (Entries 1 to 3, 8, and 9 in FIG _. 1d). Among them, V30-SP-8 and V30-SP-9 were found to be the most effective peptides (FIG. 4b). These peptides were designed to mimic the toxin binding region of VapB30 and bind to the inactive site of VapC30. Through the above experiments, it was confirmed that the staple peptides of the present invention, particularly V30-SP-8 and V30-SP-9, compete with VapB30 when binding to VapC30, thereby expressing the active site of VapC30 and inducing digestion of bacterial RNA. . From this, it can be seen that the staple peptide of the present invention can act as an antibacterial agent against VapBC30-expressing pathogens such as Mycobacterium tuberculosis.

In addition, the concentration dependence of the peptide of the present invention was confirmed (FIG. 4c). As a result of treatment with V30-SP-8 and V30-SP-9 at concentrations of 1.25, 2.5, 5, and 10 μM, respectively, activity was shown even at less than 10 μM. V30-SP-8 and V30-SP-9 were modified from the same linear peptide, but the stapled linkers used for their synthesis were different. Cell inhibitory properties have a compound effect based on peptide helix and cell permeability, which contributes to the dissociation of VapC from VapBC30. Therefore, even if similar in vitro RNase activity is observed, the two staple peptides may differ in helix and corresponding cell membrane permeability.

Example 3: linear peptide and staple of peptides CD spectroscopy

The helical propensity of two staple peptides, V30-SP-8 and V30-SP-9, and their linear counterparts were analyzed by circular dichroism (CD) spectroscopy (see experimental method "5. CD spectroscopy").

The linear peptide showed a strong negative peak at about 195 nm, indicating a typical random coil pattern, while the staple peptide showed two negative peaks at 208 and 222 nm, indicating a helical structure in aqueous media ( Fig. 4d). Surprisingly, contrary to the expectation that the stitch peptide V30-SP-8 would be more helical than the single-staple peptide V30-SP-9, the single-staple strategy was more effective in inducing helix (helix diagram: for V30-SP-8 26.1% and 33.8% for V30-SP-9). This result may indicate that there is further room for structural optimization of stitch peptides.

For linear peptides, 2,2,2-trifluoroethanol (TFE) was added to the solvent to induce a helical structure. Even though the solvent was completely exchanged with TFE, the TFE-free solvent did not increase the helicity to the same level as the staple peptide (FIG. 5). These results indicate that peptide stapling is efficient.

Example 4: Evaluation of cell permeability

To determine whether the staple and linear peptides cross the smegma cell membrane, fluorescein isothiocyanate (FITC)-labeled V30-SP-8 and V30-SP-9 and the linear forms of these two staple peptides were examined. Confocal microscopy studies were performed on VapBC30-bearing smegma bacteria (refer to experimental method "7. Confocal microscopy" section).

The results are shown in Figures 6a to 6d.

In the case of the linear peptide, almost no fluorescence signal was observed in the cell, which means that the ability to pass through the bacterial membrane was poor (FIG. 6b). This is considered to be because, according to CD spectroscopic analysis, the linear peptide has low cell permeability and does not form an α-helical structure in an aqueous medium. In contrast, V30-SP-8-FITC and FITC-V30-SP-9-FITC were uptaken by smegma bacteria (Figs. 6c and 6d, Fig. 7), which correlated with the helix propensity.

Cellular uptake of the staple peptide was about 2-fold higher than that of the linear peptide (FIG. 8). Therefore, it can be seen that the stapling technique improves cell membrane penetration and intracellular peptide localization.

Example 5: Measurement of antibacterial activity

Previous studies have confirmed that expression of VapC30 alone without VapB30 results in bacterial cell death. Based on the above results, the present inventors hypothesized that a chemical tool for separating VapB30 and VapC30 would exhibit antibacterial activity. To support this hypothesis, an antibacterial activity test was performed by treating smegma mc ² 155 cells with a staple peptide and its linear counterpart (see Experimental Method “8. Antimicrobial Activity Test (MIC Test)”). As a result, cell growth was inhibited at 6.25 μM V30-SP-8 and V30-SP-9.

Additionally, the extent of inhibition at concentrations of 6.25 μM, 12.5 μM, 25 μM and 50 μM (above 6.25 μM) was evaluated via flow cytometry. The effect of adding V30-SP-8 and V30-SP-9 to Smegma mc ² 155 cells was tested by flow cytometry of cells labeled with LIVE/DEAD BacLight staining (Experimental Method "9. Flow Cytometry" section). reference). The results are shown in FIG. 9 .

Smegma mc ² 155 cells treated with 70% isopropyl alcohol and DMSO showed 96% and 25% apoptotic cells, respectively (cells are permeable to propidium iodide (PI)). When smegma mc ² 155 cells were treated with the staple peptide of the present invention, the ratio of PI-permeable cells increased according to the peptide concentration, indicating the bactericidal activity of the peptide of the present invention.

As successful internalization of V30-SP-8 was demonstrated by confocal images (Figs. 6b and 6c), the results indicate that the antibacterial effect was indeed exerted inside the bacterial cells. V30-SP-8 inhibiting 50% of the isolates when the antibacterial activities of V30-SP-8 and V30-SP-9 were normalized to 70% isopropyl alcohol and DMSO as 100% and 0%, respectively. The minimum inhibitory concentration (MIC ₅₀ ) values of were between 12.5 μM and 25.0 μM (FIG. 9B). This shows an efficacy similar to that of vancomycin (MIC ₅₀ = 20 μM against smegma), which is already known to have antibacterial activity.

Since helix is one of the important factors affecting cell permeability, the difference in helix between the tested peptides could explain the lower effect of the linear peptide at 50 μM compared to that of the staple peptide at 6.25 μM. These results of the present invention indicate significant potential for V30-SP-8 in applications in antibacterial drug discovery.

conclusion

From the above example, 11 novel stapled peptides targeting the VapBC30 toxin-antitoxin protein complex were identified. Among them, the design of peptide sequences by alanine scanning and subsequent screening through in vitro RNase analysis showed that V30-SP-8 and V30-SP-9 could be effective as peptide hits. CD spectroscopy analysis and confocal laser scanning microscopy demonstrate the effectiveness of our approach by showing a correlation between α-helicality and cell permeability. As a result of treating the staple peptide with smegma bacteria, V30-SP-8 showed antibacterial activity with a MIC50 of less than 6.25 μM, which was superior to vancomycin.

Taken together, these results indicate that the novel staple peptides of the present invention, particularly V30-SP-8, exhibit great antibacterial activity by interfering with the interaction within the VapBC30 protein complex. The mechanism of action of the novel staple peptides of the present invention is distinct from typical antibacterial agents that generally block DNA, RNA, protein or cell wall synthesis. This feature indicates that the novel staple peptide of the present invention can be a promising candidate for solving drug resistance.

<110> SNU R&DB Foundation <120> Antibacterial Stapled Peptides Targeting Toxin-antitoxin System of Mycobacterium tuberculosis and Use Thereof <130> S20P-0021-KR <160> 25 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (1)..(8) <223> X is (S) -2- (4'-pentenyl) -alanine, (S) -2- (7'-octenyl) -alanine, (R)-2-(4'-pentenyl)-alanine, (R)-2-(7'-octenyl)-alanine, 2,2-bis-(4'-pentenyl)-glycine or 2,2-bis-(7'-octenyl)-glycine <400> 1 Glu Xaa Ala Ala Xaa Arg His Arg 1 5 <210> 2 <211> 8 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (1)..(8) <223> X is (S) -2- (4'-pentenyl) -alanine, (S) -2- (7'-octenyl) -alanine, (R)-2-(4'-pentenyl)-alanine, (R)-2-(7'-octenyl)-alanine, 2,2-bis-(4'-pentenyl)-glycine or 2,2-bis-(7'-octenyl)-glycine <400> 2 Glu Xaa Ala Ala Ile Xaa His Arg 1 5 <210> 3 <211> 8 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (1)..(8) <223> X is (S) -2- (4'-pentenyl) -alanine, (S) -2- (7'-octenyl) -alanine, (R)-2-(4'-pentenyl)-alanine, (R)-2-(7'-octenyl)-alanine, 2,2-bis-(4'-pentenyl)-glycine or 2,2-bis-(7'-octenyl)-glycine <400> 3 Glu Leu Ala Ala Xaa Arg His Xaa 1 5 <210> 4 <211> 9 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (1)..(9) <223> X is (S) -2- (4'-pentenyl) -alanine, (S) -2- (7'-octenyl) -alanine, (R)-2-(4'-pentenyl)-alanine, (R)-2-(7'-octenyl)-alanine, 2,2-bis-(4'-pentenyl)-glycine or 2,2-bis-(7'-octenyl)-glycine <400> 4 Arg Xaa Gly Glu Xaa Gly Gly Arg Glu 1 5 <210> 5 <211> 9 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (1)..(9) <223> X is (S) -2- (4'-pentenyl) -alanine, (S) -2- (7'-octenyl) -alanine, (R)-2-(4'-pentenyl)-alanine, (R)-2-(7'-octenyl)-alanine, 2,2-bis-(4'-pentenyl)-glycine or 2,2-bis-(7'-octenyl)-glycine <400> 5 Arg Xaa Gly Glu Pro Gly Gly Arg Xaa 1 5 <210> 6 <211> 13 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (1)..(13) <223> X is (S) -2- (4'-pentenyl) -alanine, (S) -2- (7'-octenyl) -alanine, (R)-2-(4'-pentenyl)-alanine, (R)-2-(7'-octenyl)-alanine, 2,2-bis-(4'-pentenyl)-glycine or 2,2-bis-(7'-octenyl)-glycine <400> 6 Ala Glu Arg Phe Xaa Ala Ala Val Glu Ala Asp Xaa Ile 1 5 10 <210> 7 <211> 17 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (1)..(17) <223> X is (S) -2- (4'-pentenyl) -alanine, (S) -2- (7'-octenyl) -alanine, (R)-2-(4'-pentenyl)-alanine, (R)-2-(7'-octenyl)-alanine, 2,2-bis-(4'-pentenyl)-glycine or 2,2-bis-(7'-octenyl)-glycine <400> 7 Asp Glu Pro Asp Ala Glu Arg Phe Xaa Ala Ala Val Glu Ala Asp Xaa 1 5 10 15 Ile <210> 8 <211> 8 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (1)..(8) <223> X is (S) -2- (4'-pentenyl) -alanine, (S) -2- (7'-octenyl) -alanine, (R)-2-(4'-pentenyl)-alanine, (R)-2-(7'-octenyl)-alanine, 2,2-bis-(4'-pentenyl)-glycine or 2,2-bis-(7'-octenyl)-glycine <400> 8 Xaa Leu Ala Ala Xaa Arg His Xaa 1 5 <210> 9 <211> 8 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (1)..(8) <223> X is (S) -2- (4'-pentenyl) -alanine, (S) -2- (7'-octenyl) -alanine, (R)-2-(4'-pentenyl)-alanine, (R)-2-(7'-octenyl)-alanine, 2,2-bis-(4'-pentenyl)-glycine or 2,2-bis-(7'-octenyl)-glycine <400> 9 Xaa Leu Ala Ala Ile Arg His Xaa 1 5 <210> 10 <211> 9 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (1)..(9) <223> X is (S) -2- (4'-pentenyl) -alanine, (S) -2- (7'-octenyl) -alanine, (R)-2-(4'-pentenyl)-alanine, (R)-2-(7'-octenyl)-alanine, 2,2-bis-(4'-pentenyl)-glycine or 2,2-bis-(7'-octenyl)-glycine <400> 10 Arg Xaa Gly Glu Xaa Gly Gly Arg Xaa 1 5 <210> 11 <211> 15 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (1)..(15) <223> X is (S) -2- (4'-pentenyl) -alanine, (S) -2- (7'-octenyl) -alanine, (R)-2-(4'-pentenyl)-alanine, (R)-2-(7'-octenyl)-alanine, 2,2-bis-(4'-pentenyl)-glycine or 2,2-bis-(7'-octenyl)-glycine <400> 11 Xaa Pro Asp Ala Glu Arg Phe Xaa Ala Ala Val Glu Ala Asp Xaa 1 5 10 15 <210> 12 <211> 8 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (2) <223> X is (R) -2- (4'-pentenyl) -alanine <220> <221> MISC_FEATURE <222> (5) <223> X is (S) -2- (4'-pentenyl) -alanine <400> 12 Glu Xaa Ala Ala Xaa Arg His Arg 1 5 <210> 13 <211> 8 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (2)..(6) <223> X is (S) -2- (4'-pentenyl) -alanine <400> 13 Glu Xaa Ala Ala Ile Xaa His Arg 1 5 <210> 14 <211> 8 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (5) <223> X is (R) -2- (4'-pentenyl) -alanine <220> <221> MISC_FEATURE <222> (8) <223> X is (S) -2- (4'-pentenyl) -alanine <400> 14 Glu Leu Ala Ala Xaa Arg His Xaa 1 5 <210> 15 <211> 9 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (2) <223> X is (R) -2- (4'-pentenyl) -alanine <220> <221> MISC_FEATURE <222> (5) <223> X is (S) -2- (4'-pentenyl) -alanine <400> 15 Arg Xaa Gly Glu Xaa Gly Gly Arg Glu 1 5 <210> 16 <211> 9 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (2) <223> X is (R) -2- (7'-octenyl) -alanine <220> <221> MISC_FEATURE <222> (9) <223> X is (S) -2- (4'-pentenyl) -alanine <400> 16 Arg Xaa Gly Glu Pro Gly Gly Arg Xaa 1 5 <210> 17 <211> 13 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (5) <223> X is (R) -2- (7'-octenyl) -alanine <220> <221> MISC_FEATURE <222> (12) <223> X is (S) -2- (4'-pentenyl) -alanine <400> 17 Ala Glu Arg Phe Xaa Ala Ala Val Glu Ala Asp Xaa Ile 1 5 10 <210> 18 <211> 17 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (9) <223> X is (R) -2- (7'-octenyl) -alanine <220> <221> MISC_FEATURE <222> (16) <223> X is (S) -2- (4'-pentenyl) -alanine <400> 18 Asp Glu Pro Asp Ala Glu Arg Phe Xaa Ala Ala Val Glu Ala Asp Xaa 1 5 10 15 Ile <210> 19 <211> 8 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (1) <223> X is (S) -2- (4'-pentenyl) -alanine <220> <221> MISC_FEATURE <222> (5) <223> X is 2,2-bis-(4'-pentenyl)-glycine <220> <221> MISC_FEATURE <222> (8) <223> X is (S) -2- (4'-pentenyl) -alanine <400> 19 Xaa Leu Ala Ala Xaa Arg His Xaa 1 5 <210> 20 <211> 8 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (1) <223> X is (R) -2- (7'-octenyl) -alanine <220> <221> MISC_FEATURE <222> (8) <223> X is (S) -2- (4'-pentenyl) -alanine <400> 20 Xaa Leu Ala Ala Ile Arg His Xaa 1 5 <210> 21 <211> 9 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (2) <223> X is (R) -2- (4'-pentenyl) -alanine <220> <221> MISC_FEATURE <222> (5) <223> X is 2,2-bis-(4'-pentenyl)-glycine <220> <221> MISC_FEATURE <222> (9) <223> X is (R) -2- (4'-pentenyl) -alanine <400> 21 Arg Xaa Gly Glu Xaa Gly Gly Arg Gly 1 5 <210> 22 <211> 15 <212> PRT <213> artificial sequence <220> <223> synthsized peptide <220> <221> MISC_FEATURE <222> (1) <223> X is (R) -2- (7'-octenyl) -alanine <220> <221> MISC_FEATURE <222> (8) <223> X is 2,2-bis-(4'-pentenyl)-glycine <220> <221> MISC_FEATURE <222> (15) <223> X is (S) -2- (7'-octenyl) -alanine <400> 22 Xaa Pro Asp Ala Glu Arg Phe Xaa Ala Ala Val Glu Ala Asp Xaa 1 5 10 15 <210> 23 <211> 8 <212> PRT <213> Mycobacterium tuberculosis <400> 23 Glu Leu Ala Ala Ile Arg His Arg 1 5 <210> 24 <211> 17 <212> PRT <213> Mycobacterium tuberculosis <400> 24 Asp Glu Pro Asp Ala Glu Arg Phe Glu Ala Ala Val Glu Ala Asp His 1 5 10 15 Ile <210> 25 <211> 9 <212> PRT <213> Mycobacterium tuberculosis <400> 25 Arg Phe Gly Glu Pro Gly Gly Arg Glu 1 5

Claims (13)

An antibacterial staple peptide that inhibits the binding of VapB30 and VapC30 in VapBC30, a toxin-antitoxin conjugate of Mycobacterium tuberculosis , and consists of the amino acid sequence of SEQ ID NO: 19 or 20:
S ₅ LAAB ₅ RHS ₅ (SEQ ID NO: 19); or
R ₈ LAAIRHS ₅ (SEQ ID NO: 20).
delete delete delete delete delete delete delete delete delete The antimicrobial staple peptide according to claim 1 , characterized in that it has a MIC50 of less than 6.25 μM.
A pharmaceutical composition for preventing or treating tuberculosis, comprising the antimicrobial staple peptide of claim 1 or 11.
delete

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