KR101209344B1 - Peptide Compound Having an Inhibition Activity for Orientia tsutsugamushi Infection or Proliferation and Pharmaceutical Composition for Treating Scrub Typhus Using the Same - Google Patents

Abstract

The present invention was made on the basis of the C-terminal sequence of TSA56 of Tsutsugamu strain known to be involved in the infection of host cells, and has binding activity with fibronectin of the host cells interacting with the TSA56, and the HeLa cell of the cervical cancer cell line. Disclosed are a peptide compound having an infection inhibitory activity and a pharmaceutical composition for improving Tsutsugamusosis using the same.

Description

Peptide Compound Having An Inhibition Activity for Orientia tsutsugamushi Infection or Proliferation and Pharmaceutical Composition for Treating Scrub Typhus Using the Same}

The present invention relates to a peptide compound having an activity of inhibiting infection or proliferation of Tsutsugamushi bacteria, and a pharmaceutical composition for improving Tsutsugamushi disease using the same.

Tsutsugamosis (scrub typhus) is caused by Orientia tsutsugamushi ( hereinafter referred to as "Tsutsugamushi" bacterium), which is an absolute intracellular parasitic bacterium.

When a tick infected with Tsutsugamu bacteria touches a rodent or a person and bites them, the Tsutsugamu bacteria invade the human body and develop the disease.

In Eastern Russia, Japan, China, Malaysia, Thailand, Vietnam, Northern Australia, Pakistan, and Afghanistan, it is known that 100 million people in the region are at risk, with 1 million people per year.

Infection with Tsutsugamu bacteria leads to incubation periods of 1 to 2 weeks, leading to clinical symptoms such as eschar, high fever, chills, headache, spotty rash, and lymphadenopathy.

The diagnosis of Tsutsuga's disease is made by clinical diagnosis by clinical symptoms, bacteriological diagnosis by isolates of causative organisms, and serological diagnosis by measuring the presence of antibodies in serum (Kim et al., 20: 105-116, 1988; Et al., Korean Journal of Microbiology 24: 281-289, 1989). Antibacterial drugs such as doxycycline or chloramphenicol have been used in the treatment, but recently, resistant bacteria have been reported in India and Southeast Asia.

The present invention discloses a peptide compound that has an infection or proliferation inhibitory activity of Tsutsugamu strain, and thus can be used for the treatment of Tsutsugamu strain.

It is an object of the present invention to provide a peptide compound having an activity of inhibiting infection or proliferation of Tsutsugamu bacteria.

Another object of the present invention is to provide a pharmaceutical composition for improving Tsutsugamus disease using the compound.

Other and specific objects of the present invention will be presented below.

In one aspect, the present invention relates to a peptide having an activity capable of inhibiting the infection or proliferation of Tsutsugamu bacteria of the present invention.

The inventors of the present invention have previously shown that infection of host cells of Tsutsugashimi strains includes 56-kDa type-specific antigen (hereinafter, abbreviated as “TSA56”), which is an outer membrane protein of Tsutsugamus strain, and fibronectin, an extracellular matrix glycoprotein of host cells. It has been shown through interactions with (Lee et al., (2008) J Infect Dis . 198 : 250-257).

Based on the results of these previous studies, the inventors anticipated antagonistic inhibition and designed and fabricated a total of 10 peptides based on the C-terminal sequence of TSA56, which binds to the fibronectin of these peptides. After confirming the activity, as a result of measuring the inhibitory activity of the host cell infection by the synthesized peptides, it was confirmed that some of the peptides inhibit the cell infection of the Tsutsugamushi bacteria at a significant level, in particular SEQ ID NO: 4 and 5 Burn peptides most effectively inhibited bacterial infection in host cells.

Therefore, these peptides are expected to be able to inhibit the biological infection of Tsutsugamushi bacteria, and may be used as a therapeutic agent for infection control by inhibiting proliferation by infecting other cells after the biological infection of Tsutsugamushi bacteria.

The present invention has been completed based on these experimental results, and the peptide compound having an activity capable of inhibiting the infection or proliferation of the Tsutsugamu bacterium of the present invention is a peptide compound selected from (1) to (9) below or a pharmaceutical thereof As an acceptable salt.

 A peptide compound comprising the amino acid sequence of SEQ ID NO: 1 or a peptide compound having a sequence substantially similar to the amino acid sequence of SEQ ID NO: 1;

 A peptide compound comprising the amino acid sequence of SEQ ID NO: 2 or a peptide compound having a sequence substantially similar to the amino acid sequence of SEQ ID NO: 2;

 A peptide compound comprising the amino acid sequence of SEQ ID NO: 3 or a peptide compound having a sequence substantially similar to the amino acid sequence of SEQ ID NO: 3;

 A peptide compound comprising the amino acid sequence of SEQ ID NO: 4 or a peptide compound having a sequence substantially similar to the amino acid sequence of SEQ ID NO: 4;

 A peptide compound comprising the amino acid sequence of SEQ ID NO: 5 or a peptide compound having a sequence substantially similar to the amino acid sequence of SEQ ID NO: 5;

A peptide compound comprising the amino acid sequence of SEQ ID NO: 8 or a peptide compound having a sequence substantially similar to the amino acid sequence of SEQ ID NO: 8;

 A peptide compound comprising the amino acid sequence of SEQ ID NO: 10 or a peptide compound having a sequence substantially similar to the amino acid sequence of SEQ ID NO: 10;

As used herein, the term "peptide compound" refers to a compound consisting of a series of amino acid residues by which amino acids are linked by peptide bonds. Generally it consists of 10 or more and 100 or less amino acids, preferably 15 or more and 50 or less amino acids. The peptide compound is a concept that includes peptide derivatives and peptide analogs that have a low degree of activity and still have activity that can inhibit the infection or proliferation of Tsutsugamushi bacteria, so that some amino acids in the peptide are non-natural amino acids. Or a peptide replaced with an amino acid analog (such as when substituted with L-amino acid and methionine is substituted with a methionine analog), and at the N-terminus and / or C-terminus of the peptide, methyl, ethyl, n-propyl, It is a concept including the case where the alkyl group, such as isopropyl, the cycloalkyl group, such as cyclopentyl and cyclohexyl, and the aryl group, such as phenyl, (alpha)-naphthyl, etc. is couple | bonded. Furthermore, compounds as illustrated above are bonded to a substituent on the side chain of an amino acid (eg, -OH, -SH, an amino group, an imidazolyl group, an indolyl group, a guanidino group, etc.) in a peptide compound molecule, or a sugar chain is bound. Includes cases. Verification and screening of such peptide derivatives or analogs having the ability to inhibit infection or proliferation of Tsutsugamu bacteria is possible within the range of ordinary skill of those skilled in the art according to the methods disclosed in the experimental examples below.

In addition, in the present specification, each "peptide compound comprising the amino acid sequence of SEQ ID NO:" is a peptide compound having an activity capable of inhibiting the infection or proliferation of Tsutsugamus spp even though the degree of activity is low, and the N-terminal and And / or a compound in which a few amino acids are added at the C-terminus. It is generally known in the art that even if a few amino acids are added to the N-terminus and / or C-terminus of a peptide or polypeptide, the original activity of the peptide or polypeptide is often maintained. The number of amino acids added is 30 or less, preferably 10 or less, more preferably 5 or less. Verification and screening of such peptide compounds for inhibiting the proliferation or the proliferation of Tsutsugamu bacteria in vivo is also within the scope of ordinary skill of those skilled in the art as long as the disclosure is referred to.

In addition, in the present specification, each of "peptide compounds having sequences substantially similar to the amino acid sequence of SEQ ID NO" means a peptide compound having an activity capable of inhibiting the infection or proliferation of Tsutsugamu bacteria even though the degree of activity is low. It refers to the case where an amino acid is substituted by the other amino acid similar in structure and / or polarity to the amino acid. Groups of amino acids of similar structure and / or polarity are known in the art, where lysine, arginine, histidine and the like can be grouped into basic amino acids, aspart, glutamine and the like can be grouped into acidic amino acids and glycine, glutamine, serine , Threonine, tyrosine, cysteine and the like can be grouped into non-charged polar amino acids. In addition, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan and the like can be grouped into non-polar amino acids, phenylalanine, tryptophan and the like can be grouped into aromatic amino acids. Preferred degrees of substitution here are 10% or less (i.e., substituted for at least 90% homology of the amino acid sequence), particularly when substituted with 5% or less (i.e., replaced with 95% or more homologous of the amino acid sequence). If). Validation and selection of peptide compounds having sequences substantially similar to those of the amino acid sequences of the respective SEQ ID NOs has the ability to inhibit the biological infection or proliferation of Tsutsugamushi bacteria. Do.

In addition, in the present specification, "pharmaceutically acceptable salt" means a salt that does not inhibit the activity of the peptide compound of the present invention, organic salts commonly used in the formation of metal salts or acid addition salts (for example acetate, citrate, Oleate, oxalate, glutonate, etc.) or inorganic salts (such as chloride, antiacid, borate, carbonate, etc.) may be used.

Peptide compounds of the invention can be prepared genetically using synthetic DNA techniques or prepared by chemical synthesis methods.

Genetic engineering is a technique for transforming and expressing recombinant DNA containing a gene encoding a peptide in a host cell, such a recombinant DNA promoter, activator, enhancer, operation Factor, ribosome binding site, initiation signal, stop signal, cap signal, polyadenylation signal, and the like, and host cells include bacteria such as E. coli , Saccharomyces sp. Genus yeast, insect cells, mammalian cells and the like can be used. Techniques for making recombinant DNA, transformation techniques, appropriate culture techniques for host cells, and the like are all known in the art.

Chemical synthesis methods are also well known in the art, such as solid phase synthesis techniques, solution phase synthesis techniques, enzyme synthesis methods, etc. may be used and specific examples are described in Stewart JM and Young JD (1984) “Solid Phase Peptide Synthesis, 2nd edition”. ], Bodanzsky M. and Bodanzsky A. (1984) “The practice of Peptide Synthesis”, Lloyd-Williams, P., Albericio, F. and Giralt, E. (1997) “Chemical Approaches to the Synthesis of Peptides and Proteins ”, Kullmann W. (1980)“ Proteases as catalysts for enzymic syntheses of opioid Peptides ”J. Biol . Chem . 255, 8234-8238, and the like. Such chemical synthesis techniques would be particularly useful for the production of small peptides with a low number of amino acids.

In another aspect, the present invention relates to a pharmaceutical composition for improving Tsutsugamussis comprising the peptide compound as described above or a pharmaceutically acceptable salt thereof as an active ingredient.

As used herein, the term "tsutsugamushi" means a clinical symptom according to the bioinfection of Tsutsugamushi, proliferation in vivo of Tsutsugamushi, and / or the bioinfection and proliferation of Tsutsugamushi.

As used herein, the term "active ingredient" alone refers to a component that can exhibit the desired activity or can itself exhibit activity with a carrier which is inactive.

As used herein, “improvement” is meant to include suppressing / delaying the onset of Tsutsugamusosis, preventing Tsutsugamusosis, and treating Tsutsugamus.

In the composition of the present invention, the active ingredient may be included in any amount (effective amount) as long as it can exhibit the improvement activity of Tsutsugamussis according to the use, formulation, formulation purpose, etc., and the conventional effective amount may be based on the total weight of the composition. When determined to be within the range 0.001% to 15% by weight. The term "effective amount" herein refers to a mammal, such as a rodent or the like, to which it is applied, to inhibit, alleviate, or treat the clinical symptoms of Tsutsugamushi bacteria, their proliferation, their biological infection, and their growth. The amount of active ingredient that can be used. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.

Subjects to which the compositions of the present invention can be applied (prescribed) are mammals and humans, such as rodents, and especially humans.

The composition of the present invention may further include an antibiotic that is already used for the treatment of Tsutsugamus symptom in order to increase or enhance the effect of improving Tsutsugashiosis. Representative examples of such antibiotics include doxycycline and chloramphenicol.

Pharmaceutical compositions of the present invention, in addition to its active ingredients, include pharmaceutically acceptable carriers, excipients, and the like, oral formulations (tablets, suspensions, granules, emulsions, capsules, syrups, liposome capsules, protenoid capsules, etc.) And parenteral formulations (such as aqueous or oily suspensions for sterile injection) and the like. Regarding formulation of liposome encapsulation or proteoid encapsulation, reference may be made in particular to US Pat. No. 4,925,673, US Pat. No. 5,013,556 and the like.

As used herein, "pharmaceutically acceptable" means that the subject of application (prescription) does not have more toxicity (sufficiently low toxicity) to which the subject of application (prescription) is adaptable without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (such as corn starch, potato starch, etc.), cellulose, derivatives thereof (such as sodium carboxymethyl cellulose, ethylcellulose, etc.) malt, gelatin, talc, solids Lubricants (e.g. stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oils (e.g. peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerin, etc.), alginic acid, emulsifiers (e.g. TWEENS), wetting agents (e.g. Sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, water, saline, phosphate buffer solutions and the like. The carrier may be selected from one or more of suitable pharmaceutical formulations according to the formulation of the pharmaceutical composition of the present invention.

Excipients may be selected and used according to the formulation of the pharmaceutical composition of the present invention, for example, when the pharmaceutical composition of the present invention is prepared with an aqueous suspending agent, suitable excipients are sodium carboxymethyl cellulose, methyl cellulose, hydropropylmethylcellulose And suspending agents and dispersing agents such as sodium alginate and polyvinylpyrrolidone. Suitable excipients when prepared from injection solutions include Ringer's solution, isotonic sodium chloride, and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally.

The daily dose of the pharmaceutical composition of the present invention is usually 0.001 to 150 mg / kg body weight, and may be administered once or several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as route of administration, age, sex, weight, and patient's severity of the patient, the dose is limited in any aspect to the scope of the present invention Should not be understood to be.

As described above, according to the present invention, it is possible to provide a peptide compound having an activity of inhibiting infection or proliferation of Tsutsugamu bacteria. This peptide compound can be commercialized and used as a pharmaceutical composition for the purpose of preventing, treating, and treating Tsutsuga's disease.

FIG. 1A is a diagram showing the structure of the C-terminal region of TSA56 based on the peptide compound design of the present invention and a diagram showing the site from which each peptide sequence was derived.
1B is a result showing the binding activity (red dots) of the peptide compound of the present invention to fibronectin and the infection inhibitory activity of HeLa cells, a cervical cancer cell line of Tsutsugashimi bacteria.
FIG. 1C is a photograph taken by immunofluorescence of the degree of proliferation of Tsutsugamus in HeLa cell host cells when the peptide compound of SEQ ID NO: 5 and the peptide peptide compound of SEQ ID NO: 9 are treated.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experiments.

<Examples> Preparation of Peptide Compounds

As shown in FIG. 1A, a TSA56 peptide (> 90% purity) consisting of 20 to 35 amino acids based on the C-terminal amino acid sequence (aa 232-442) of TSA56 was synthesized using a peptide synthesizer (PeptrEX ^™ , Peptron Inc., Daejeon, Korea).

The prepared peptide sequence is shown in Table 1 below.

SEQ ID NO. Amino acids no. Residues One
2
3
4
5
6
7
8
9
10 AA 232-261
AA 252-281
AA 282-301
AA 292-321
AA 312-341
AA 334-363
AA 353-382
AA 373-402
AA 393-422
AA 413-442 FAIHNHEQWRSLVVGLAALSNANKPSASPV
NANKPSASPVKVLSDKIIQIYSDIKPFADI
AGINVPDTGLPNSASIEQIQ
PNSASIEQIQSKIQELGDTLEELRDSFDGY
EELRDSFDGYINNAFVNQIHLNFVMPPQAQ
FVMPPQAQQQQGQGQQQQAQATAQEAVAAA
QATAQEAVAAAAVRLLNGSDQIAQLYKDLV
QIAQLYKDLVKLQRHAGIRKAMEKLAAQQE
AMEKLAAQQEEDAKNQGKGDCKQQQGASEK
CKQQQGASEKSKEGKVKETEFDLSMVVGQV

<Experimental Example> Tsutsugamusosis Improvement Activity of TSA56 Peptides

Experimental Example 1 Cell Culture

DMEM medium containing 100 units / ml penicillin-streptomycin and 10% fetal bovine serum (FBS) from cervical cancer cell line HeLa cells (ATCC CCL-2, American Type Culture Collection) and mouse fibroblast line L929 cells (ATCC NCTC929) Incubated in a 37 ° C., 5% CO ₂ incubator.

Experimental Example 2 Preparation of Tsutsugamu Strain

Tsutsugamu was isolated by modifying the Percoll gradient purification method (Tamura A, et. Al., Microbiol Immunol 1982; 26: 321-8).

The Boryeong strain of Tsutsugashi strain was infected by incubating for 90 minutes in the cultured mouse fibrinase L929 in a carbon dioxide incubator at 34 ° C. The extent of infection was determined using indirect immunofluorescence technique (Cho NH, et. Al., Infect Immun 2000; 68: 594-602.).

Cells were harvested by centrifugation at 6000 g for 20 minutes when infection rate was greater than 90% and pellets suspended in 6.5 mL of Tris-sucrose buffer (33 mM Tris-Cl (pH 7.4) and 250 mM sucrose). I was. The suspended cells are homogenized (100 stokes) using a Polytron homogenizer (Wheaton, USA), centrifuged for 200 g for 5 minutes to obtain a supernatant, and the supernatant is mixed with 40% Percoll medium (Pharmacia, Sweden) using TS buffer. 25,000 g, and centrifuged for 30 minutes to collect the Tsutsugamu bacteria layer and centrifuged again for 77,000 g, 30 minutes. The Tsutsugamushi layer was collected again, washed three times with TS buffer and then resuspended in DMEM medium and stored in liquid nitrogen until use.

<Experiment 3> Tsutsugamusosis improvement activity experiment of TSA56 peptide

Experimental Example 3-1 Binding Activity Measurement Experiment of TSA56 Peptide with Fibronectin

Fibronectin binding activity of the TSA peptide was evaluated using ELISA according to the method of Konkel et al. (Mon Micro et al., Mol Microbiol . 57 : 1022-1035).

Specifically, 1.0 μg of peptide was coated on a 96 well plate, and BSA (bovine serum albumin) was coated as a negative control. The wells were then blocked with 1% BSA for 1 hour at 25 ^° C. and washed with PBS containing 0.1% w / v BSA and 0.01% v / v Tween 20. Fibronectin (1.0 μg, Sigma-Aldrich) was diluted with PBS containing 0.02% w / v BSA, added to each well and allowed to react for 3 hours.

The wells were then washed three times and combined with primary antibodies (mouse anti-fibronectin antibody, Chemicon, Temecular, CA, USA) and secondary antibodies (anti-mouse IgG-HRP, Sigma chemicals, St. Louis, MO, USA) I was. Finally, the reaction was performed by adding a substrate solution (TMB Substrate Solution; Endogen, Woburn, MA) and quantified by measuring the degree of binding at 492 nm.

The results are shown in B (red dots) of FIG. Referring to FIG. 1, the binding activity of the peptide of SEQ ID NO: 4 and the peptide of SEQ ID NO: 5 to fibronectin is the highest.

Experimental Example 3-2 Experiment for Measuring Inhibitory Activity of TSA56 Peptide Against Cellular Infection of Tsutsugamu Strain

In order to evaluate the inhibitory activity of TSA56 peptide against cellular infection of Tsutsugamushi bacteria, HeLa cells were treated with 50 nM TSA56, and only DMEM medium was used as a negative control. Next, Tsutsugamushi was added and reacted for 3 hours. The infected cells were then harvested using trypsin, washed three times with PBS to remove surface adherent cells, re-plated in a 12 mm cover slip on a 24 well plate and left for 24 hours, followed by immunofluorescence in Tsutsugamusi Bacteria were visualized and Tsutsugamushi bacteria grown in 100 randomly selected host cells were counted.

The results are shown in B of FIG. 1. Referring to B of FIG. 1, the higher the binding activity of the fibronectin of the TSA56 peptide, the higher the inhibitory activity of the infection of the Tsutsugamushi bacterium. SEQ ID NOs: 1, 2, 3, 4, Peptides 5, 8, and 10 showed statistically significant activity of inhibiting cellular infection of Tsutsugamu bacteria. Particularly, peptides of SEQ ID NOs. 4 and 5 showed the most effective inhibition of bacterial infection.

For reference, a photograph taken by immunofluorescence of the degree of proliferation of Tsutsugashi bacteria in host cells is shown in FIG. The photo representatively shows the peptide of SEQ ID NO: 9 and the negative control, which was found to be insignificant with the peptide of SEQ ID NO: 5 having the highest activity.

Statistical processing

Statistical analysis was performed using s t -test (SigmaPlot, Jandel, San Rafael, CA, USA) and the results were presented as mean ± standard deviation. Statistical significance was verified at P <0.05 level.

Attach an electronic file to a sequence list

Claims (10)

delete Peptide compound consisting of the amino acid sequence of SEQ ID NO: 2 having the activity of inhibiting the biological infection or proliferation of Tsutsugamushi bacteria in vivo or a pharmaceutically acceptable salt thereof.
delete Peptide compound consisting of the amino acid sequence of SEQ ID NO: 4 having the activity of inhibiting the biological infection or proliferation of Tsutsugamushi bacteria in vivo or a pharmaceutically acceptable salt thereof.
Peptide compound consisting of the amino acid sequence of SEQ ID NO: 5 or the pharmaceutically acceptable salt thereof having the activity of inhibiting the biological infection or proliferation of Tsutsugamushi bacteria in vivo.
delete delete Claims 2, 4 and 5, wherein any one of the peptide compound or a pharmaceutically acceptable salt thereof as an active ingredient Tsutsugamushi improvement pharmaceutical composition.
9. The method of claim 8,
The composition is a pharmaceutical composition for improving Tsutsugamus syrup, characterized in that it further comprises an antibiotic.

9. The method of claim 8,
Wherein the antibiotic is doxycycline or chloramphenicol Tsutsugamushi disease improvement pharmaceutical composition, characterized in that.

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