KR102125888B1 - Novel cyclic peptide, manufacturing method of the same anc use of the same - Google Patents

Abstract

The present invention provides a novel cyclic peptide having anti-tuberculosis activity produced by a biological method using a Split intein platform. The novel cyclic peptide according to the present invention has a very different framework amino acid sequence from the cyclic peptide having conventional anti-tuberculosis activity. The novel cyclic peptide according to the present invention can be applied to various resistant tuberculosis, and is very useful as a pharmaceutical material.

Description

Novel cyclic peptide, manufacturing method of the same anc use of the same}

The present invention relates to novel cyclic peptides and the like, and more particularly, to a novel cyclic peptide having anti-tuberculosis activity, to a method for preparing it using Split intein, and to a pharmaceutical use thereof.

It is known that peptides and proteins have very low toxicity compared to conventional synthetic compounds because their constituents are amino acids, and have a very high economic value as a pharmaceutical material. However, in the case of a linear peptide or a non-cyclic protein pharmaceutical material, there is a problem in that medicinal effects are not exerted in the human body due to low structural stability such as degeneration or decomposition while passing through the human digestive system.

On the other hand, cyclic peptides are widely recognized in the industry as a next-generation pharmaceutical material capable of overcoming structural stability problems of linear peptides or non-cyclic protein pharmaceutical materials. Techniques for producing cyclic peptides include chemical synthesis methods, separation and purification from microorganisms producing cyclic peptides, and phage display methods. Among them, the chemical method has a disadvantage that it is difficult to cyclize a peptide composed of amino acids having high hydrophobicity. The phage display method can theoretically produce almost all types of cyclic peptides, but it is difficult to secure structural stability because disulfide bonds for generating cyclic structures are easily degraded in vivo. In addition, the method of separation and purification from microorganisms has limitations in producing cyclic peptides of new structures because the structure of the cyclic peptides produced by the microorganisms itself is determined.

Mycobacterium tuberculosis is a large and complex bacterium that causes TB disease in humans and other mammals. Tuberculosis is a highly contagious disease that is transmitted to people by the human respiratory route. There were 9.4 million new cases of tuberculosis in 2009, including 300,000 MDR-TB carriers and 1.1 million of HIV carriers [WHO Action for Life-Toward a World Without Tuberculosis; World Health Organization; Geneva, Switzerland, 2006]. MDR-TB (multi-drug resistant tuberculosis) requires an extended treatment period of at least 6 months to 2 years. The global emergence of XDR-TB (extensively drug resistant tuberculosis) is estimated at 6.6% of the MDR M. tuberculosis strains [behavior for WHO life-towards a world without tuberculosis; World Health Organization; Geneva, Switzerland, 2006]. Rufomycin, also known as Ilamycin, is produced by Streptomyces atratus ATCC 14046. Rufomycin (Rufomycin) is a cyclic peptide with a 7 amino acid backbone, and has anti-tuberculosis activity. However, lupomycin is very insoluble in water, so it is very limited in application to the human body as a therapeutic agent. To solve this, the development of lupomycin derivatives that are both water-soluble and have high anti-tuberculosis activity has continued. For the development of such a lupomycin derivative, a method in which various salts are mainly added to lupomycin (U.S. Patent No. 3330725) or a method in which various substituents are introduced into an amino acid constituting a skeleton (International Patent Publication WO 00/78797), etc. Was used. In addition, rifampicin is an antibiotic used to treat bacterial infections, including tuberculosis. Rifampicin was introduced in 1967. It is used in combination with other antibiotics such as isoniazid, ethambutol, and pyrazineamides to treat tuberculosis and latent meningitis. In the case, resistance to the drug is greatly increased. Rifampicin can cause some side effects when taken with other drugs, and the most serious side effects are related to liver toxicity of rifampicin.

The present invention has been derived under the conventional technical background, the object of the present invention is to provide a novel cyclic peptide having anti-tuberculosis activity.

In addition, an object of the present invention is to provide a method for biologically producing a novel cyclic peptide using a split intein (Split intein).

It is also an object of the present invention to provide a medicinal use of the novel cyclic peptide.

In order to solve the above object, an example of the present invention provides a novel cyclic peptide having the structure of formula (I): or a pharmaceutically acceptable salt thereof.

[Formula Ⅰ]

In order to solve the above object, an example of the present invention is a polynucleotide corresponding to the C-terminal domain of the split intein, a polynucleotide encoding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and the N-terminal of the split intein A polynucleotide corresponding to a domain is sequentially connected and configured, and provides a recombinant polynucleotide used for the production of a recombinant vector for cloning or expressing a cyclic peptide or for producing a transformant for producing a cyclic peptide.

In order to solve the above object, an example of the present invention is a polynucleotide corresponding to the C-terminal domain of the split intein, a polynucleotide encoding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and the N-terminal of the split intein Provided is a recombinant vector for cloning or expressing a cyclic peptide comprising a recombinant polynucleotide composed of sequentially linked polynucleotides corresponding to a domain.

In order to solve the above object, an example of the present invention is a polynucleotide corresponding to the C-terminal domain of the split intein, a polynucleotide encoding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and N- of the split intein. A recombinant polynucleotide consisting of sequentially linked polynucleotides corresponding to terminal domains; Or, the polynucleotide corresponding to the C-terminal domain of the split intein, the polynucleotide encoding the peptide consisting of the amino acid sequence of SEQ ID NO: 1, and the polynucleotide corresponding to the N-terminal domain of the split intein are sequentially connected. Provided is a transformant characterized in that a recombinant vector for expression of a cyclic peptide comprising a constructed recombinant polynucleotide is introduced.

In order to solve the above object, an example of the present invention comprises the steps of expressing a recombinant polynucleotide by culturing the aforementioned transformant; And crushing the transformant expressing the recombinant polynucleotide, and separating the cyclic peptide from the lysate.

In order to solve the above object, an example of the present invention provides a pharmaceutical composition for preventing or treating tuberculosis, which includes a cyclic peptide having the structure of Formula (I): or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula Ⅰ]

The novel cyclic peptide according to the present invention has a very different framework amino acid sequence from the cyclic peptide having conventional anti-tuberculosis activity. The novel cyclic peptide according to the present invention can be applied to various resistant tuberculosis, and is very useful as a pharmaceutical material.

1 shows a map of a pET29b-His-TEV vector custom-made in the present invention and a multi-cloning site.
2 is a result of analyzing various products generated in the process of inserting Npu_DnaE_C-CLLMWLY-Npu_DnaE_N into the pET29b-His-TEV vector by Southern blot.
3 is a schematic cleavage map of the pET29-CYC_RUF-Intein recombinant vector produced in the present invention.
Figure 4 shows the DNA base sequence of the pET29-CYC_RUF-Intein recombinant vector produced in the present invention.
5 is pET29-CYC_RUF Intein-derived traits peptide expressed by culturing the conversion of E. coli Rosetta2 (DE3) cells with the recombinant vectors, and the resultant through a crushing and centrifugation of the cell culture supernatant Ni ^{+ 2} affinity After purification by chromatography column, the purified product was analyzed by SDS-PAGE(a) and Western blot(b).
6 is a spectral result of cyclic peptide obtained by culturing E. coli Rosetta2 (DE3) cells transformed with pET29-CYC_RUF-Intein recombinant vector by LC/MS/MS.
7 is a peak spectrum result when the CYC_RUF extract and Control extract obtained in the present invention were fractionated by Preparative HPLC.
8 is a result of re-analyzing CYC_RUF extract and Control extract obtained in the present invention by Preparative HPLC, and fractions of peaks specific to CYC_RUF extract by Analytical HPLC.

Hereinafter, terms used in the present invention will be described.

The terms "protein", "polypeptide" and "peptide" used in the present invention may be used as interchangeable concepts, unless otherwise specified.

As used herein, the term "polynucleotide" refers to any non-modified or modified polyribonucleotide (RNA) or polydeoxyribonucleotide (DNA). The polynucleotide can be single- or double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is a mixture of single- and double-stranded regions, single- or double-stranded, Or hybrid molecules comprising DNA and RNA, which may be mixtures of single- and double-stranded regions. Also, in the present invention, polynucleotides may be used interchangeably with nucleic acid molecules or oligonucleotides.

The term "transformation", which is used in the present invention, introduces DNA into a host, so that the DNA can be cloned as a factor of a chromosome or by completion of chromosomal integration, and introduces external DNA into cells to cause artificial genetic changes. It means a phenomenon.

As used in the present invention, the term "transformant" is a polynucleotide encoding one or more target proteins or an expression vector containing the same is introduced into a host such as a host cell, a host microorganism, a host plant, a host insect, or a host animal and transformed. It means converted life.

The term "pharmaceutically acceptable" used in the present invention means that it does not significantly stimulate the organism and does not inhibit the biological activity and properties of the active substance administered.

The term "prevention" as used in the present invention means all actions to suppress symptoms or delay progression of a specific disease by administration of the composition of the present invention.

As used herein, the term "treatment" refers to any act that ameliorates or beneficially alters the symptoms of a particular disease by administration of a composition of the invention.

As used herein, the term “administration” means providing a subject composition of the present invention to an individual in any suitable way. At this time, the individual refers to all animals, such as humans, monkeys, dogs, goats, pigs, or rats, who have a disease in which symptoms of a specific disease can be improved by administering the composition of the present invention.

The term "pharmaceutically effective amount" used in the present invention means an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, which is the type, severity, drug activity, drug of the individual. Sensitivity to, time of administration, route of administration and rate of discharge, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field can be determined.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention relates to a novel cyclic peptide having anti-tuberculosis activity or a pharmaceutically acceptable salt thereof. The novel cyclic peptide according to an example of the present invention has the structure of Formula I below. The novel cyclic peptide according to an embodiment of the present invention has a very different skeleton amino acid sequence from the cyclic peptide having a conventional anti-tuberculosis activity, specifically, cysteine-leucine-leucine-methionine-tryptophan-leucine-tyrosine amino acid residue sequence Have

[Formula Ⅰ]

In the present invention, as the pharmaceutically acceptable salt, a variety of known salts commonly used in the pharmaceutical field can be selected, and an acid addition salt formed by free acid is useful. Acid addition salts are prepared by conventional methods, for example, by dissolving the compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equivalent amounts of the compound and acid or alcohol in water (eg, glycol monomethyl ether) can be dried by evaporation of the mixture and then the precipitated salt can be suction filtered. At this time, an organic acid and an inorganic acid may be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and tartaric acid may be used as the inorganic acid, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, and trifluoro may be used as the organic acid. Acetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, Galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanic acid, hydroiodic acid and the like can be used. In addition, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the inexpensive compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salts, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

One aspect of the present invention relates to a biological technology for preparing a novel cyclic peptide, and is used in the construction of a recombinant vector for cloning or expressing a cyclic peptide into a specific category, or in the production of a transformant for producing a cyclic peptide. It includes recombinant polynucleotides, recombinant vectors for cloning or expressing cyclic peptides, transformants for producing cyclic peptides, and methods for producing cyclic peptides using transformants. Recombinant polynucleotide according to an embodiment of the present invention is a polynucleotide corresponding to the C-terminal domain of the split intein, a polynucleotide encoding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and the N-terminal domain of the split intein The polynucleotides corresponding to are sequentially connected. In addition, the recombinant vector for cyclic peptide expression according to an embodiment of the present invention is a polynucleotide and a split-in encoding a peptide consisting of the polynucleotide corresponding to the C-terminal domain of the split intein, the amino acid sequence of SEQ ID NO: 1 It comprises a recombinant polynucleotide consisting of a polynucleotide corresponding to the N-terminal domain of the sequential linkage. In addition, the transformant for producing a cyclic peptide according to an embodiment of the present invention is a polynucleotide corresponding to the C-terminal domain of the split intein, a polynucleotide encoding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and a split A recombinant polynucleotide composed of polynucleotides corresponding to the N-terminal domain of the intein sequentially connected; Or, the polynucleotide corresponding to the C-terminal domain of the split intein, the polynucleotide encoding the peptide consisting of the amino acid sequence of SEQ ID NO: 1, and the polynucleotide corresponding to the N-terminal domain of the split intein are sequentially connected. A recombinant vector for expressing a cyclic peptide containing the constructed recombinant polynucleotide is introduced. In addition, the method for preparing a cyclic peptide according to an embodiment of the present invention comprises the steps of expressing a recombinant polynucleotide by culturing the transformant for producing the cyclic peptide; And crushing the transformant expressing the recombinant polynucleotide, and isolating the cyclic peptide from the lysate.

The amino acid sequence of SEQ ID NO: 1 corresponds to the arrangement of amino acid residues constituting the novel cyclic peptide of the present invention. Polynucleotides encoding peptides in the present invention may or may not include untranslated sequences (eg, introns) (eg, cDNA). The information to which the peptide is coded is specified using codons. Typically, amino acid sequences are encoded by polynucleotides using a universal genetic code. “Codon” refers to a triplet of nucleotides that determines the amino acid sequence in a polypeptide chain. Most organisms use 20 or 21 amino acids to prepare their polypeptides, which are proteins or protein precursors. Since there are four possible nucleotides in DNA, adenine (A), guanine (G), cytosine (C) and thymine (T), there are 20 possible amino acids and 64 possible triplets capable of encoding the terminal signal. Due to this redundancy, most amino acids are encoded by one or more triplets. Accordingly, a change in the nucleotide sequence can be tolerated without affecting the amino acid sequence of the encoded polypeptide, and this is referred to as "silent variation" by "codon degeneracy." In the present invention, the polynucleotide encoding the peptide consisting of the amino acid sequence of SEQ ID NO: 1 preferably consists of the nucleotide sequence of SEQ ID NO: 2.

The biological technique for preparing the novel cyclic peptide according to the present invention is based on the cyclization of amino acid residues by Split intein. Intein (intein) is a protein splicing (protein splicing) is a segment of the protein that can cut itself and bond the rest of the extein (Extein) through peptide bonds. Inteins are largely classified into large inteins and mini inteins, while large inteins contain a homing endonuclease domain (HEG), while mini inteins have a homing There is no endonuclease domain. Further, the intein may exist as two fragments called a C-terminal domain and an N-terminal domain, and the two intein fragments are separated into two and encoded by a gene that is transcribed and translated. This intein is called Split intein and has been identified in a variety of cyanobacteria and archaea [Caspi et al, Mol Microbiol. 50: 1569-1577 (2003); Choi J. et al, J Mol Biol. 556: 1093-1106 (2006.); Dassa B. et al, Biochemistry. 46:322-330 (2007.); Liu X. and Yang J., J Biol Chem. 275:26315-26318 (2003); Wu H. et al, Proc Natl Acad Sci USA. Zech. 5:9226-9231 (1998.); and Zettler J. et al, FEBS Letters. 553:909-914 (2009)]. About 600 inteins are listed worldwide, of which less than 5% are split inteins, and most of the split inteins belong to the family of cyanobacteria known as the split DnaE intein. The kind of split inteins used in the present invention is not greatly limited, and may be selected from various known split inteins. The present invention relates to the available split intein International Patent Publication No. WO 2013/045632, International Patent Publication No. WO 2014/004336, International Patent Publication No. WO 2017/132580, Nucleic Acids Research, 2015, Vol. 43, No. 13, 6450-6458, and the like. In the present invention, in relation to the split intein, the polynucleotide corresponding to the C-terminal domain of the split intein consists of the nucleotide sequence of SEQ ID NO: 3 and corresponds to the N-terminal domain of the split intein It is preferable that the nucleotide consists of the nucleotide sequence of SEQ ID NO:4. The polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 and the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 are genes encoding the C-terminal domain and N-terminal domain of the split intein Npu DnaE, respectively.

As used herein, the term "vector" refers to any medium for cloning and/or transfer of a base to a host cell. In the present invention, the vector may be a replication unit (replicon) capable of bringing replication of foreign DNA fragments. “Replication unit” refers to any genetic unit (eg, plasmid, phage, cosmid, chromosome, virus) that functions as an autologous unit of DNA replication in vivo, ie is capable of replication by self-regulation. . In addition, the term "vector" as used in the present invention includes viral and non-viral mediators for introducing a base into a host cell in vitro, ex vivo or in vivo. In addition, the term "vector" used in the present invention may also include mini-spherical DNA. For example, the vector can be a plasmid that does not have a bacterial DNA sequence. Removal of rich bacterial DNA sequences in the CpG region has been done to reduce transgene expression silencing and to bring more sustained expression from plasmid DNA vectors. In addition, the term "vector" used in the present invention is a transposon such as Sleeping Beauty [Izsvak et al. J. MoI. Biol. 302:93-102 (2000)], or artificial chromosomes. In the present invention, the recombinant vector may be a cloning vector or an expression vector. The term "cloning vector" as used in the present invention is defined as a substance capable of transporting and reproducing a DNA fragment into a host cell. In the present invention, the cloning vector may further include a polyadenylation signal, a transcription termination sequence, and multiple cloning sites. At this time, the multiple cloning site (multiple cloning site) includes at least one endonuclease (endonuclease) restriction enzyme restriction site (restriction site). In addition, the cloning vector may further include a promoter. For example, in the present invention, a polynucleotide encoding a framework amino acid sequence of a cyclic peptide may be located upstream of a polyadenylation signal and a transcription termination sequence, and at least one The endonuclease restriction site may be located upstream of the polyadenylation signal and transcription termination sequence. As used herein, the term "expression vector" refers to a gene construct comprising essential regulatory elements operably linked to an insert such that the insert is expressed when present in the individual's cells. The expression vector can be prepared and purified using standard recombinant DNA technology. The type of the expression vector is not particularly limited as long as it functions to express a desired gene in various host cells of prokaryotic and eukaryotic cells and to produce a desired protein, but retains a promoter exhibiting strong activity and strong expression, and is in a natural state. Vectors capable of producing a large amount of foreign protein in a similar form are preferred. It is preferable that the expression vector contains at least a promoter, an initiation codon, a gene encoding the desired protein, and a termination codon terminator. In addition, DNA encoding a signal peptide, additional expression control sequences, non-translated regions on the 5'and 3'sides of the desired gene, a selectable marker region, or a replicable unit may be appropriately included. "Promoter" means a minimal sequence sufficient to direct transcription. In addition, sufficient promoter constructs can be included to allow expression of a cell type specific or external signal or modulator-driven gene that is induced by the agent, and these constructs may be located in the 5'or 3'portion of the gene. . The expression vector according to the present invention may contain both conservative promoters and inducible promoters. Promoter sequences can be derived from prokaryotic, eukaryotic, or viral. The term “operably linked” means that one function is regulated by another with the polynucleotide sequence association on a single polynucleotide. For example, if the promoter is capable of controlling the expression of the coding sequence (i.e., when the coding sequence is under the transcriptional control of the promoter), the promoter is operated in conjunction with the coding sequence, or the ribosome binding site can facilitate translation. If located, the ribosome binding site is linked to the coding sequence and operates. The coding sequence can be operably linked to a regulatory sequence in either the sense direction or the antisense direction. Expression vectors used in the present invention include, for example, E. coli-derived plasmids (pYG601BR322, pBR325, pUC118 and pUC119); Bacillus subtilis -derived plasmids (pUB110 and pTP5); Yeast-derived plasmids (YEp13, YEp24 and YCp50); λ-phages (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11 and λZAP); Animal viruses such as retrovirus, adenovirus or vaccinia virus; And insect viruses such as Baculovirus. In addition, in the present invention, the expression vector may include GST, GFP, His-tag, Myc-tag, etc., if necessary, to confirm whether the target protein is expressed or to facilitate purification of the target protein. The expression vector of the present invention is not limited by this. In one embodiment of the present invention, a pET29b vector was used when constructing an expression vector comprising a polynucleotide encoding a framework amino acid sequence of a cyclic peptide. In addition, when the target proteins are expressed by the expression vector containing the tag sequence, the expressed protein can be detected or purified by affinity chromatography. For example, when a glutathione-S-transferase is included in an expression vector as a tag, glutathione, which is the substrate of this enzyme, can be used for detection or purification of proteins, and hexahistidine (Hisx6) is used as an expression vector in the tag. When included, a desired target protein can be easily detected or recovered using a Ni-NTA His-binding resin column (Novagen, USA). Polynucleotide encoding the peptide consisting of the amino acid sequence of SEQ ID NO: 1 in the recombinant vector according to an embodiment of the present invention, polynucleotide corresponding to the C-terminal domain of the split intein and N-terminal domain of the split intein For technical characteristics of the polynucleotide corresponding to, refer to the description of the recombinant polynucleotide. Recombinant vector according to an embodiment of the present invention preferably has a cleavage map of Figure 3, more preferably consists of the nucleotide sequence of SEQ ID NO: 5.

The transformant according to an embodiment of the present invention is characterized in that the above-described recombinant polynucleotide or the above-described recombinant vector is introduced. In the present invention, the recombinant polynucleotide is preferably introduced into the host through a recombinant expression vector, and the host is transformed. As a method for preparing a transformant by introducing the recombinant expression vector into a host cell, transient transfection, micro-injection, transduction, cell fusion, calcium phosphate precipitation method, liposome-mediated transfection (liposemmediated transfection), DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, electroinjection, PEG, etc. There is a chemical treatment method, a method using a gene gun, and the like, but is not limited thereto. When the transformant into which the recombinant expression vector has been introduced is cultured in a nutrient medium, a target protein can be prepared in large quantities, and separation is possible. Medium and culture conditions may be appropriately selected and used depending on the host cell. During culture, conditions such as temperature, medium pH, and culture time should be appropriately adjusted to be suitable for cell growth and mass production of proteins. As a host cell that can be transformed with the expression vector according to the present invention, if it is known in the art, such as a prokaryotic cell, a plant cell, an insect cell, an animal cell, the type is not greatly limited, and preferably the efficiency of introducing DNA is A host having a high expression efficiency of the introduced DNA is usually used. For example, well-known prokaryotic hosts such as Escherichia, Pseudomonas, Bacillus, and Streptomyces can be used as host cells, and preferably E. coli. The expression of the protein by the host cell can be induced using the inducing factor IPTG (isopropyl-1-thio-β-D-galactopyranoside), the induction time can be controlled to maximize the amount of protein. The target protein recombinantly produced in the present invention can be recovered from cell lysates. When the target protein is membrane-bound, it can be liberated from the membrane using a suitable surfactant solution (eg, Triton-X 100) or by enzymatic cleavage. Cells used for protein expression can be destroyed by various physical or chemical means such as freeze-thaw repetition, sonication, mechanical destruction, or cell degraders, and can be isolated or purified by conventional biochemical separation techniques (Sambrook et al., Molecular Cloning: A laborarory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989; Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182.Academic Press.Inc., San Diego, CA, 1990). For example, as a method for isolating or purifying proteins expressed by host cells, solvent extraction, electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunosorbent affinity) Chromatography, reverse phase HPLC, gel permeation HPLC), isoelectric focus and various changes or complex methods thereof. For example, the novel cyclic peptides according to the present invention are cultivated with transformants, the culture medium is centrifuged to obtain a supernatant, an organic solvent such as methyl chloride is added to the supernatant, and the organic solvent distribution layer is separated. After that, it can be obtained by removing the organic solvent.

Polynucleotide encoding the peptide consisting of the amino acid sequence of SEQ ID NO: 1 in the transformant according to an embodiment of the present invention, the polynucleotide corresponding to the C-terminal domain of the split intein and the N-terminal of the split intein For technical characteristics of the polynucleotide corresponding to the domain, refer to the description of the recombinant polynucleotide. Recombinant vector introduced into the host cell in the transformant according to an embodiment of the present invention has a cleavage map of Figure 3, more preferably consists of the nucleotide sequence of SEQ ID NO: 5.

One aspect of the present invention relates to the pharmaceutical use of the novel cyclic peptide. An example of the present invention provides a pharmaceutical composition for preventing or treating tuberculosis, comprising a cyclic peptide having the structure of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula Ⅰ]

The type of tuberculosis to which the composition for preventing or treating tuberculosis according to an embodiment of the present invention can be applied is not particularly limited, for example, MDR tuberculosis or silver XDR tuberculosis. MDR tuberculosis (Multidrug-resistant tuberculosis) refers to tuberculosis resistant to both isoniazid and rifampicin, the most important drugs for the treatment of tuberculosis. XDR tuberculosis (extensively drug-resistant tuberculosis) is a tuberculosis with additional resistance to at least one of antibiotics against fluoroquinolone and at least one of anti-tuberculosis injections (amikacin, kanamycin, capreomycin). Speak. The novel cyclic peptide, which is an active ingredient of the composition for preventing or treating tuberculosis in one example of the present invention, has a very different framework amino acid sequence from the cyclic peptide having anti-tuberculosis activity, and thus can be applied to various resistant tuberculosis.

In the composition for preventing or treating tuberculosis according to an embodiment of the present invention, the content of the novel cyclic peptide as an active ingredient may be adjusted in various ranges depending on the specific form of the composition, purpose of use, and aspect.

The content of the novel cyclic peptide as an active ingredient in the pharmaceutical composition according to the present invention is not significantly limited, for example, 0.01 to 99% by weight, preferably 0.5 to 50% by weight, more preferably, based on the total weight of the composition It may be 1 to 30% by weight. In addition, the pharmaceutical composition according to the present invention may further include an additive such as a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient. Carriers, excipients and diluents that may be included in the pharmaceutical 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. In addition, the pharmaceutical composition of the present invention may further contain one or more known active ingredients having anti-tuberculosis activity in addition to the novel cyclic peptide. The pharmaceutical composition of the present invention may be formulated into a formulation for oral administration or a formulation for parenteral administration by conventional methods, and when formulated, a filler, a bulking agent, a binder, a wetting agent, a disintegrant, a surfactant, etc. It can be prepared using diluents or excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient in the active ingredient, for example, starch, calcium carbonate, sucrose ), can be prepared by mixing Lactose or gelatin. In addition, lubricants such as magnesium stearate talc may be used in addition to simple excipients. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions and syrups, but may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used. Furthermore, it can be preferably formulated in accordance with each disease or ingredient using methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA or by appropriate methods in the art. The pharmaceutical composition of the present invention may be administered orally or parenterally to mammals, including humans, depending on the desired method, and the parenteral administration methods include external application to the skin, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, and muscle Intra-injection or intra-thoracic injection. The dosage of the pharmaceutical composition of the present invention is not particularly limited as long as it is a pharmaceutically effective amount, and the range thereof depends on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and disease severity. Varies. The typical daily dosage of the pharmaceutical composition of the present invention is not particularly limited, but is preferably 0.1 to 3000 mg/kg, more preferably 1 to 2000 mg/kg, once a day, based on the active ingredient. Or it may be divided into several times and administered. In addition, the conventional single dose of the pharmaceutical composition of the present invention can be adjusted so that the concentration of the active ingredient in plasma is 1 to 50 μg/ml, preferably 5 to 25 μg/ml.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are only for clearly illustrating the technical features of the present invention, and are not intended to limit the protection scope of the present invention.

One. Split Intein (Split intein ) Recombinant vector for expression of base cyclic peptide and production of new cyclic peptide using the same

(1) Nostoc Synthesis of split α subunit of DNA polymerase III (DnaE) intein gene from punctiforme PCC73102 (Npu)

Commissioned by Bioneer Company (Korea), Nostoc The full-length Split DnaE intein gene (Npu_DnaE) was synthesized from punctiforme PCC73102 (Npu). Npu_DnaE consists of an N-terminal split intein with 102 amino acid residues (Npu_DnaE_N) and a C-terminal split intein with 36 amino acid residues (Npu_DnaE_C). To produce a cyclic peptide, two parts of the split Npu_DnaE gene consisting of Npu_DnaE_N and Npu_DnaE_C were arranged as (Npu_DnaE_C)-(target peptide sequence to be cyclized)-(Npu_DnaE_N).

[Full length amino acid sequence of Npu_DnaE]

* Npu_DnaE_N part: normal letter

* Npu _ DnaE _C part: bold letter

[Npu_DnaE full-length DNA sequence]

* Npu_DnaE_N part: normal letter

* Npu _ DnaE _C part: bold letter

(2) Construction of a split Npu DnaE intein with a target peptide using overlap extension PCR

The target peptide to be cyclized consists of 7 amino acid residues as follows. Using the overlap extension PCR (overlap extension PCR) method, a DNA nucleotide sequence encoding a target peptide was inserted between the Npu_DnaE_C nucleotide sequence and the Npu_DnaE_N nucleotide sequence. Full length Npu_DnaE_C-CLLMWLY-Npu_DnaE_N DNA fragments were obtained by overlap extension PCR of two previously produced PCR products, Npu_DnaE_C-CLLMWLY and CLLMWLY-Npu_DnaE_N.

* Amino acid sequence of the desired peptide: CLLMWLY (1-letter abbreviation)

* Target peptide DNA base sequence: TGC CTG TTG ATG TGG CTG TAT

Types of primers and sequence information used in performing overlap extension PCR are shown in Table 1 below.

Primer classification Primer sequence (5' ---> 3') Npu_DnaE_C_F CCCCCC CATATG ATC AAA ATA GC C ACA CGT Npu_DnaE_C_R_Peptide ATA CAG CCA CAT CAA CAG GCA ATT AGA AGC TAT GAA GCC Npu_DnaE_N_F_Peptide TGC CTG TTG ATG TGG CTG TAT TGT TTA AGC T AT GAA ACG G Npu_DnaE_N_R CCCCCC CTCGAG ATT CGG CAA ATT ATC AAC

For cloning into the pET29b-His-TEV recombinant vector described below, NdeI (CATATG) and XhoI (CTCGAG) restriction sites (indicated in bod) are shown in Table 1 above for Npu_DnaE_C_F primer and Npu_DnaE_N_R primer. Was inserted.

(3) Construction of pET29b-His-TEV vector

The pET29b vector purchased from Novagen was modified to remove the S tag and thrombin site, insert the TEV protease cleavage site at the N-terminus of the 7xHis tag and the protein to be expressed, and insert the pET29b-His-TEV. Vectors were prepared. 1 shows a map of a pET29b-His-TEV vector custom-made in the present invention and a multi-cloning site.

(4) Construction of pET29-CYC_RUF-Intein recombinant vector

The amplified DNA products of Npu_DnaE_C-CLLMWLY-Npu_DnaE_N and pET29b-His-TEV vector were completely digested by incubating for 2 hr at 37° C. with restriction enzymes NdeI and XhoI (New England Biolabs) in CutSmart buffer. Thereafter, the restriction enzyme-treated Npu_DnaE_C-CLLMWLY-Npu_DnaE_N and pET29b-His-TEV were mixed at a ratio of 1:1, and ligated with T4 ligase at 4°C overnight to prepare a pET29-CYC_RUF-Intein recombinant vector. The pET29-CYC_RUF-Intein recombinant vector consists of the nucleotide sequence of SEQ ID NO: 5. Thereafter, 5 μl of the ligation mixture was transformed into NEB Turbo competent cells.

2 is a result of analyzing various products generated in the process of inserting Npu_DnaE_C-CLLMWLY-Npu_DnaE_N into the pET29b-His-TEV vector by Southern blot. Figure 2 (a) is a result of analyzing the PCT products of Npu_DnaE_C-CLLMWLY-Npu_DnaE_N. In (a) of FIG. 2, lane 1 represents Npu_DnaE_C-CLLMWLY, lane 2 represents CLLMWLY-Npu_DnaE_N, and lane 3 represents Npu_DnaE_C-CLLMWLY-Npu_DnaE_N. Figure 2 (b) is a result of analyzing the Npu_DnaE_C-CLLMWLY-Npu_DnaE_N and pET29b-His-TEV vectors treated with restriction enzymes NdeI and XhoI. In (b) of FIG. 2,'Vectors' represents a pET29b-His-TEV vector, and'Inserts' represents Npu_DnaE_C-CLLMWLY-Npu_DnaE_N.

Thereafter, NEB Turbo competent cells were cultured at 37° C. for 12 hr to select transformed colonies, and cloned plasmids were extracted using a plasmid extraction kit. Subsequently, the DNA nucleotide sequence of the extracted plasmid was measured by requesting Macrogen (Korea). As a result, it was confirmed that the Npu_DnaE_C-CLLMWLY-Npu_DnaE_N DNA fragment was correctly inserted into the pET29b-His-TEV vector. 3 is a schematic cleavage map of the pET29-CYC_RUF-Intein recombinant vector produced in the present invention. Figure 4 shows the DNA base sequence of the pET29-CYC_RUF-Intein recombinant vector produced in the present invention.

(5) Production of novel cyclic peptides using pET29-CYC_RUF-Intein recombinant vector

The cloned pET29-CYC_RUF-Intein recombinant vector was transformed into E. coli Rosetta2 (DE3) cells. Thereafter, the transformed cells were inoculated into 5 ml LB medium to which kanamycin was added, and cultured with shaking at 37°C. Thereafter, 5 ml of the culture medium was inoculated into 500 ml LB medium to which kanamycin was added, followed by shaking culture at 37° C. until the value at OD ₆₀₀ became 0.6. Thereafter, IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to the culture solution to a concentration of 1 mM to induce peptide expression, and cultured at 16° C. for 15 hr. Thereafter, the cell culture solution was centrifuged to collect the cell pellet, and the collected cell pellet was dissolved in Lysis buffer (30mM Tris pH 8.0, 150mM NaCl, 15mM Imidazole, 5mM β-mercaptoethanol), crushed with ultrasound, and centrifuged to remove the supernatant. Was collected. Then, the collected supernatant was passed through a Ni ^{2 +} affinity chromatography column, and the expression product was eluted with an elution buffer (30 mM Tris pH 8.0, 150 mM NaCl, 250 mM Imidazole, 5 mM β-mercaptoethanol). Thereafter, the eluted expression products were analyzed by SDS-PAGE and Western blot to confirm the presence of 7xHis_TEV-Npu_DnaE_C-CLLMWLY-Npu_DnaE_N-6xHis protein, split Npu_DnaE C-terminal domain and split Npu_DnaE N-terminal domain.

5 is pET29-CYC_RUF Intein-derived traits peptide expressed by culturing the conversion of E. coli Rosetta2 (DE3) cells with the recombinant vectors, and the resultant through a crushing and centrifugation of the cell culture supernatant Ni ^{+ 2} affinity After purification by chromatography column, the purified product was analyzed by SDS-PAGE(a) and Western blot(b). As shown in Figure 5, the purified product had full length 7xHis_TEV_Npu_DnaE_C-CLLMWLY-Npu_DnaE_N-6xHis(~20kDa) protein, as well as Npu_DnaE_N-6xHis(13kDa) and 7xH_D_E_D_E_D_E_D_E_D_E_D_E_D_E_D_E_D_E_D_E_D_E_D_E_D_E_D_E_D_E_D_E_D(E) These results show that the full-length 7xHis_TEV_Npu_DnaE_C-CLLMWLY-Npu_DnaE_N-6xHis(~20kDa) protein expressed by transformed BL21(DE3) cells undergoes a splicing process, resulting in three protein fragments: 7xHis_TEV_Npu_DnaE_C(Nu_DnaE_CN) It means cleavage into a cyclic peptide consisting of -6xHis (13kDa) and CLLMWLY amino acid sequence.

2. Structure analysis of novel cyclic peptide

E. coli Rosetta2 (DE3) cells transformed with the pET29-CYC_RUF-Intein recombinant vector were cultured to induce peptide expression, and supernatant was obtained through crushing and centrifugation of the cultured cells. Subsequently, an equal volume of methylene chloride was added to the obtained supernatant, and extraction by organic solvent distribution was performed. Thereafter, the methylene chloride layer was fractionated, concentrated through evaporation under reduced pressure, and the concentrate was dissolved in methanol to a solid concentration of 50 mg/ml and filtered through a 0.2 μm syringe filter. Then, the final concentration of the solid content of the filtrate was adjusted to 0.1 mg/ml and analyzed by loading 10 μg in LC/MS/MS.

In addition, E. coli Rosetta2 (DE3) cells transformed with the pET29b-His-TEV vector were cultured and a sample obtained through the same process was used as a control.

<Analysis condition>

* Flow rate: 0.8 ml/min

* Development solvent: 0 to 2 min is 5% acetonitrile, 2 to 13 min is 5 to 100% gradient acetonitrile, and 13 to 15 min is 100% acetonitrile concentration gradient

6 is a spectral result of analyzing the cyclic peptide obtained by culturing E. coli Rosetta2 (DE3) cells transformed with pET29-CYC_RUF-Intein recombinant vector by LC/MS/MS. In Figure 6, the control (Control) is a sample obtained by culturing E. coli Rosetta2 (DE3) cells transformed with a pET29b-His-TEV vector.

The amino acid structure was calculated through spectral results of FIG. 6 and fragment reanalysis by collision induced fragmentation, and as a result, the final expressed cyclic peptide was predicted to have the structure of Formula I below.

[Formula Ⅰ]

3. Anti-tuberculosis activity of novel cyclic peptide

(1) Preparation of analysis sample

E. coli Rosetta2 (DE3) cells transformed with pET29-CYC_RUF-Intein recombinant vector were inoculated in 500 ml LB medium and shake cultured at 37° C. until the value at OD ₆₀₀ reached 0.6. Thereafter, IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to the culture solution to a concentration of 1 mM to induce peptide expression, and cultured at 16° C. for 15 hr. Thereafter, the cell culture solution was centrifuged to collect the cell pellet, and the collected cell pellet was dissolved in Lysis buffer (30mM Tris pH 8.0, 150mM NaCl, 15mM Imidazole, 5mM β-mercaptoethanol), crushed with ultrasound, and centrifuged to remove the supernatant. Was collected. Subsequently, an equal volume of methylene chloride was added to the obtained supernatant, and extraction by organic solvent distribution was performed. Thereafter, the methylene chloride layer was fractionated and concentrated through evaporation under reduced pressure to prepare an analytical sample CYC_RUF extract.

In addition, E. coli Rosetta2 (DE3) cells transformed with the pET29b-His-TEV vector were cultured, and the control extract was prepared through the same procedure.

In addition, the CYC_RUF extract was fractionated by Preparative HPLC to obtain a peak corresponding to the separated and purified CYC_RUF. 7 is a peak spectrum result when the CYC_RUF extract and Control extract obtained in the present invention were fractionated by Preparative HPLC. The scientific basis for securing the peak corresponding to CYC_RUF was the first, and the peak corresponding to CYC_RUF was eluted in the section where the acetonitrile concentration was 55-65% through the LC/MS/MS experiment results, and the second was actually Preparative HPLC It is to select only the peaks that are not measured in the control extract but only in the CYC_RUF extract.

CYC_RUF separation and purification conditions through preparative HPLC are as follows.

<Analysis condition>

Column: YMC-Pack R&D C18 (250 mm×20 mm I.D., S-5 μm, 12 nm)

<development conditions>

0-20 minutes: 5% acetonitrile with 0.1% formic acid

20-70 minutes: Concentration gradient of 5-95% acetonitrile with 0.1% formic acid

70-90 minutes: 95% acetonitrile with 0.1% formic acid

Flow rate: 14 ml/min

As shown in FIG. 7, peaks corresponding to the 55-65% acetonitrile concentration section were secured, and 3 peaks different from the control extract were observed in the CYC_RUF extract. Thereafter, the obtained fraction was re-analyzed CYC_RUF through analytical HPLC. 8 is a result of re-analyzing CYC_RUF extract and Control extract obtained in the present invention by Preparative HPLC, and fractions of peaks specific to CYC_RUF extract by Analytical HPLC.

Analytical HPLC analysis conditions are as follows.

<Analysis condition>

Column: YMC-Triart C18 (250 mm×4.6 mm I.D., S-5 μm, 12 nm)

<development conditions>

0-90 min: gradient of 5-95% acetonitrile with 0.1% formic acid

Flow rate: 0.8 ml/min

As a result, as shown in FIG. 8, a single peak eluting at 50 minutes was finally confirmed, and LC/MS/MS analysis results showed the same results as before, and finally confirmed that it was the desired CYC_RUF.

(2) Method for measuring anti-tuberculosis activity

Samples for measuring anti-tuberculosis activity are CYC_RUF extract and Control extract mill-purified CYC-RUF prepared above, and each sample was dissolved in dimethyl sulfoxide (DMSO) for treatment with anti-tuberculosis activity.

In order to evaluate the anti-tuberculosis activity of the sample, MIC (Minimum Inhibitory Concentration) for Mycobacterium tuberculosis was measured using Alamar Blue Assay. Specifically, the antimicrobial susceptibility test was performed in a 96-well microplate with a black transparent bottom (black view plate, Packard Instrument Company, Meriden, Conn.) to minimize background fluorescence. In addition, the outer circumferential well was filled with sterile water to prevent dehydration in the well during the experiment. Initial drug dilutions were prepared with dimethylsulfoxide (DMSO) or distilled water, and then a 2-fold dilution was performed with 0.1 ml of 7H9GC (without Tween 80) in a microplate. BACTEC 12B-passaged inoculum was initially diluted 1:2 in 7H9GC and 0.1 ml was added to the wells. It was then determined to have bacterial titers of 1×10 ⁶ , 2.5×10 ⁶ and 3.25×10 ⁵ CFU/ml in plate wells for H37Rv, H37Ra and M. avium, respectively. Frozen inoculum was initially diluted 1:20 in BACTEC 12B medium and then 1:50 in 7H9GC. Thereafter, 0.1 ml of the diluent was added to the wells so that the final bacterial titers for H37Rv and H37Ra were 2.0×10 ⁵ and 5×10 ⁴ CFU/mL, respectively. Wells containing only the drug were used to detect the autofluorescence of the compound. Additional controls consisted of only bacteria (B) or medium (M). The plate was incubated at 37°C, and on the 4th day of culture, 20 μl of 10×alamarBlue solution (Alamar Biosciences / Accumed, Westlake, Ohio) and 12.5 μl of 20% Tween 80 were added to one B well and one M well and the plate It was cultivated at 37°C. Wells were observed at 12 hr and 24 hr to read the color change from blue to pink and over 50,000 fluorescence units (FU). Fluorescence was measured with a Cytofluor II microplate fluorimeter (PerSeptive Biosystems, Framingham, Mass.) in a bottom reading mode excitation at 530 nm and emitting at 590 nm. Reagents were added to the entire plate when B wells were pink to 24 hr. When wells remain blue or when fluorescence units (FU) of 50,000 or less are measured, additionally M wells and B wells are tested daily until color changes occur and reagents are added to all remaining wells when color changes occur. The plate was incubated at 37° C. and the results recorded 24 hr after administration of the reagents. Visual MIC (Minimum Inhibitory Concentration) was defined as the lowest drug concentration to prevent color change. Background fluorescence was performed for all wells with an average of 3 M wells for fluorescence MIC. Percent inhibition was defined as follows. The lowest drug concentration exhibiting 90% or more inhibition was considered MIC.

(3) Measurement results of anti-tuberculosis activity

Table 2 below shows the results of measuring anti-tuberculosis activity of CYC_RUF extract, control extract, isolated purified CYC_RUF and rufomycin.

Analytical sample MIC (μg/ml) Control extract > 10 CYC_RUF extract 7.27 Separately purified CYC_RUF 0.92 Rufomycin 0.019

As described above, the present invention has been described through examples, but the present invention is not necessarily limited thereto, and various modifications can be made without departing from the scope and spirit of the present invention. Accordingly, the protection scope of the present invention should be construed to include all embodiments falling within the scope of the claims appended to the present invention.

<110> Myongji University Industry and Academia Cooperation Foundation <120> Novel cyclic peptide, manufacturing method of the same anc use of the same <130> ZDP-18-0349 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Back bone amino acid of novel cyclic peptide <400> 1 Cys Leu Leu Met Trp Leu Tyr 1 5 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Back bone gene of novel cyclic peptide <400> 2 tgcctgttga tgtggctgta t 21 <210> 3 <211> 108 <212> DNA <213> Artificial Sequence <220> <223> C-termini domain gene of split intein Npu_DnaE <400> 3 atgatcaaaa tagccacacg taaatattta ggcaaacaaa atgtctatga cattggagtt 60 gagcgcgacc ataattttgc actcaaaaat ggcttcatag cttctaat 108 <210> 4 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> N-termini domain gene of split intein Npu_DnaE <400> 4 tgtttaagct atgaaacgga aatattgaca gtagaatatg gattattacc gattggtaaa 60 attgtagaaa agcgcatcga atgtactgtt tatagcgttg ataataatgg aaatatttat 120 acacaacctg tagcacaatg gcacgatcgc ggagaacaag aggtgtttga gtattgtttg 180 gaagatggtt cattgattcg ggcaacaaaa gaccataagt ttatgactgt tgatggtcaa 240 atgttgccaa ttgatgaaat atttgaacgt gaattggatt tgatgcgggt tgataatttg 300 ccgaattaa 309 <210> 5 <211> 1081 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of recombinant vector pET29-CYC_RUF-Intein <400> 5 atgggccatc atcatcatca tcatcacagc agcgaaaacc tgtattttca gggacatatg 60 atcaaaatag ccacacgtaa atatttaggc aaacaaaatg tctatgacat tggagttgag 120 cgcgaccata attttgcact caaaaatggc ttcatagctt ctaattgcct gttgatgtgg 180 ctgtattgtt taagctatga aacggaaata ttgacagtag aatatggatt attaccgatt 240 ggtaaaattg tagaaaagcg catcgaatgt actgtttata gcgttgataa taatggaaat 300 atttatacac aacctgtagc acaatggcac gatcgcggag aacaagaggt gtttgagtat 360 tgtttggaag atggttcatt gattcgggca acaaaagacc ataagtttat gactgttgat 420 ggtcaaatgt tgccaattga tgaaatattt gaacgtgaat tggatttgat gcgggttgat 480 aatttgccga attaactcga gcaccaccac caccaccact gagatccggc tgctaacaaa 540 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt 600 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg 660 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 720 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 780 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 840 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 900 gtagtgggcc atcgccctga tagacggatt ttcgcccttt gacgttggag tccacgttct 960 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 1020 ttgatttata agggattttg ccgatttccg cctattggtt aaaaaatggg cttgatttaa 1080 c 1081

Claims (16)

A cyclic peptide having the structure of formula (I): or a pharmaceutically acceptable salt thereof.
[Formula Ⅰ]

The polynucleotide corresponding to the C-terminal domain of the split intein, the polynucleotide encoding the peptide consisting of the amino acid sequence of SEQ ID NO: 1, and the polynucleotide corresponding to the N-terminal domain of the split intein are sequentially connected and configured Recombinant polynucleotide.
The method of claim 2, wherein the polynucleotide encoding a peptide consisting of the amino acid sequence of SEQ ID NO: 1 is a recombinant polynucleotide, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2.
The method according to claim 2, wherein the polynucleotide corresponding to the C-terminal domain of the split intein consists of the nucleotide sequence of SEQ ID NO: 3 and the polynucleotide corresponding to the N-terminal domain of the split intein is SEQ ID NO: 4 Recombinant polynucleotide, characterized in that consisting of a nucleotide sequence.
The polynucleotide corresponding to the C-terminal domain of the split intein, the polynucleotide encoding the peptide consisting of the amino acid sequence of SEQ ID NO: 1, and the polynucleotide corresponding to the N-terminal domain of the split intein are sequentially connected and configured Recombinant vector comprising a recombinant polynucleotide.
The method of claim 5, wherein the polynucleotide encoding the peptide consisting of the amino acid sequence of SEQ ID NO: 1 is a recombinant vector, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2.
The method according to claim 5, wherein the polynucleotide corresponding to the C-terminal domain of the split intein consists of the nucleotide sequence of SEQ ID NO: 3 and the polynucleotide corresponding to the N-terminal domain of the split intein is SEQ ID NO: 4 Recombinant vector, characterized in that consisting of a nucleotide sequence.
The recombinant vector according to claim 5, comprising the nucleotide sequence of SEQ ID NO: 5.
The polynucleotide corresponding to the C-terminal domain of the split intein, the polynucleotide encoding the peptide consisting of the amino acid sequence of SEQ ID NO: 1, and the polynucleotide corresponding to the N-terminal domain of the split intein are sequentially connected and configured Recombinant polynucleotides; Or, the polynucleotide corresponding to the C-terminal domain of the split intein, the polynucleotide encoding the peptide consisting of the amino acid sequence of SEQ ID NO: 1, and the polynucleotide corresponding to the N-terminal domain of the split intein are sequentially connected. A transformant, wherein a recombinant vector comprising the constructed recombinant polynucleotide has been introduced into a non-human organism or cell.
The transformant according to claim 9, wherein the polynucleotide encoding the peptide consisting of the amino acid sequence of SEQ ID NO: 1 is composed of the nucleotide sequence of SEQ ID NO: 2.
The polynucleotide corresponding to the C-terminal domain of the split intein is composed of the nucleotide sequence of SEQ ID NO: 3, and the polynucleotide corresponding to the N-terminal domain of the split intein is SEQ ID NO: 4 Characterized in that it comprises a nucleotide sequence of the transformant.
10. The method of claim 9, wherein the recombinant vector for expression of the cyclic peptide is composed of a nucleotide sequence of SEQ ID NO: 5 transformant.
A method for expressing a recombinant polynucleotide by culturing the transformant of any one of claims 9 to 12; And
A method for producing a cyclic peptide comprising crushing the transformant expressing the recombinant polynucleotide and separating the cyclic peptide from the lysate.
A pharmaceutical composition for preventing or treating tuberculosis, comprising a cyclic peptide having the structure of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula Ⅰ]

15. The pharmaceutical composition of claim 14, further comprising a pharmaceutically acceptable carrier.
15. The pharmaceutical composition of claim 14, wherein the tuberculosis is MDR tuberculosis or XDR tuberculosis.

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