KR101492720B1 - Antibiotic composition comprising 2-bromoalkanoic acids - Google Patents

Abstract

The present invention relates to an antibiotic composition comprising 2-bromoalkanoic acid as an active ingredient. 2-bromo alkanoic acid (2-bromoalkanoic acids) of the present invention exhibits a strong inhibitory activity on Pseudomonas aeruginosa (P seudomonas aeruginosa) that causes lung diseases such as cystic fibrosis, cystic fibrosis, sepsis, systemic infection or chronic airway Can be usefully used as antibiotic compositions for the treatment of infectious diseases.

Description

An antibiotic composition comprising 2-bromoalkanoic acids as an active ingredient,

The present invention relates to an antibiotic composition comprising 2-bromoalkanoic acid as an active ingredient.

Pseudomonas aeruginosa , an infectious pathogen, is parasitic in the lungs of patients with impaired immunity or cystic fibrosis and causes fatal symptoms. The risk of Pseudomonas aeruginosa infection has been known for a long time, and antibiotic studies to overcome this have long been advocated, and the individual efficacy of antibiotics has been clinically studied (G. Doring et al., 2000, 16, 749). Antibiotics used as antibiotics for Pseudomonas aeruginosa infection are classified into several types depending on the mechanism of action. Penicillin and cephalosporins are beta-lactam antibiotics that inhibit bacterial cell wall formation, among which ceftazidime is widely used as a treatment for pseudomonas aeruginosa. Ceftazidime is injected mainly through blood vessels, and the daily dose is 100 to 250 mg per kg of body weight. Novobiocin and Quinolones react with topoisomerase to interfere with the production of DNA. The most widely used ciprofloxacin is orally administered, and the daily dose is 30 mg / kg of body weight. In addition, Rifampin inhibits transcription from DNA to RNA, while Chloramphenicol and Tetracycline antibiotics bind to the ribosomes of bacteria to inhibit protein formation. These antibiotics may also vary in efficacy, but in the case of chronic infections such as cystic fibrosis, antibiotics resistant strains are produced when a single antibiotic is prescribed for a long period of time. Pseudomonas aeruginosa resistance develops as an antibiotic resistant strain by mutation or by receiving an antibiotic resistance gene from another individual.

In order to inhibit the development of these antibiotic resistant strains, a complex formulation using various antibiotics is used clinically, but a new type of new drug having a novel inhibition target is required without inducing the generation of resistant strains.

Korean Patent Publication No. 2012-0138769

It is an object of the present invention to provide an antibiotic composition comprising 2-bromoalkanoic acids as an active ingredient.

In order to achieve the above object, the present invention provides an antibiotic composition comprising 2-bromoalkanoic acids as an active ingredient.

In one embodiment of the present invention, the 2-bromoalkanoic acid is 2-bromohexanoic acid represented by the following formula (1), 2-bromooctanoic acid represented by the following formula (2) ) Or 2-bromodecanoic acid represented by the following formula (3).

[Chemical Formula 1]

(2)

(3)

.

.

In one embodiment of the invention, the antibiotic composition may exhibit antimicrobial activity by reducing or inhibiting antibiotic resistance.

In one embodiment of the invention, the antimicrobial composition may be an indication of an antimicrobial activity against Pseudomonas aeruginosa (P seudomonas aeruginosa).

In one embodiment of the present invention, the antibiotic composition may be one that inhibits PHA (polyhydroxyalkanoate) or rhamnolipid production.

In one embodiment of the invention, the antibiotic composition may be to reduce or inhibit the swarming motility of the pathogen.

In one embodiment of the present invention, the antibiotic composition may be to reduce or inhibit biofilm formation of pathogenic bacteria.

In one embodiment of the present invention, the antibiotic composition can prevent or treat cystic fibrosis, sepsis, systemic infection or chronic airway infections.

The 2-bromoalkanoic acids of the present invention exhibit strong inhibitory activity against Pseudomonas aeruginosa , which is a cause of lung diseases such as cystic fibrosis, and thus exhibit cystic fibrosis, sepsis, systemic infection or chronic airway infection ≪ RTI ID = 0.0 > antibiotic < / RTI >

Figures 1A, 1B and 1C are cross- sectional views of P. aeruginosa The PA14 strain 2-bromo alkanoic acids (2-BrHA, 2-BrOA ) a result obtained by measuring the growth of Pseudomonas aeruginosa (P. aeruginosa) according to the incubation time after treatment with (A: Control, B: 2-BrHA treatment Group, C: 2-BrOA treated group).
Figures 1D, 1E, and 1F illustrate, in accordance with one embodiment of the present invention, P. aeruginosa The PA01 strain 2-bromo alkanoic acids (2-BrHA, 2-BrOA ) a result obtained by measuring the growth of Pseudomonas aeruginosa (P. aeruginosa) according to the incubation time after treatment with (D: control group, E: 2-BrHA treatment Group, F: 2-BrOA treated group).
Figure 2A is intended for 2-bromo alkanoic acids (2-BrHA, 2-BrOA , 2-BrDA) of the three in accordance with one embodiment of the invention P. aeruginosa Saengsanryangreul shows the results of measurement of PA14 Wild-type and Δ PA14- phaG mutant person Norris and the PHA of the type feed.
Figure 2B is intended for 2-bromo alkanoic acids (2-BrHA, 2-BrOA , 2-BrDA) of the three in accordance with one embodiment of the invention P. aeruginosa PAO1 wild type and the PAO1- ? RhlA mutant type of ranorizide and PHA.
FIG. 3 is a schematic diagram showing the synthesis process of rhamnoside and PHA.
Fig. 4 shows that P. aeruginosa Fungus RT-PCR results were obtained by confirming the expression level of PhaG and RhlA after treating wild-type and mutant forms with 2-bromoalkanoic acids (2-BrHA, 2-BrOA, 2-BrDA).
Figure 5 is a graph showing the effect of P. aeruginosa PAO1 and P. aeruginosa (2-BrHA, 2-BrOA, 2-BrDA) to the wild type and mutant forms of the two strains PA14 and PHA after the treatment with 2-bromoalkanoic acids (2-BrHA, 2-BrOA, 2-BrDA).
FIG. 6A shows the results of confirming the presence or absence of rhamnolipid after treating 2-BrHA compound with PA14 and PAO1 of P. aeruginosa .
FIG. 6B is a photograph of P. aeruginosa The swarming motility of the strains, PAO1 and PA14, was examined after treatment with 2-BrHA compound.
Figure 6C is a map of P. aeruginosa (2-BrHA, 2-BrOA, 2-BrDA) were treated with PAO1 and PA14, and the degree of biofilm formation after 24 hours and 48 hours was measured.
Figure 6D is a photograph of P. aeruginosa (2-BrHA, 2-BrOA, 2-BrDA) to the strains PAO1 and PA14 and the degree of biofilm formation in%.

The present invention is characterized by providing an antibiotic composition comprising 2-bromoalkanoic acids as an active ingredient.

P. aeruginosa is a pathogenic bacterium that causes refractory infections in patients with sepsis, systemic infection, chronic airway infections, and pancreaticobiliary fibrosis. Particularly, patients with lowered resistance due to surgery, burns, trauma, and chemotherapy treatments cause septic shock by Pseudomonas aeruginosa, causing high temperature, blood pressure drop, etc., and ultimately leading to death. In addition, the treatment of Pseudomonas aeruginosa infection is mostly based on antibiotics, and it is not easy to treat because the antibiotic abuse is resistant to conventional antibiotics.

Accordingly, the inventors of the present invention have been studying antibiotics capable of effectively treating P. aeruginosa. The inventors of the present invention have found that the 2-bromoalkanoic acid of the present invention has an activity of inhibiting the formation of rhamnolipids and biofilms, which are pathogens of P. aeruginosa , In the first place.

These results can be confirmed by one embodiment of the present invention. In the culture of P. aeruginosa in a 70 mM fructose medium to which 2 mM 2-bromoalkanoic acid compound (2-BrHA, 2-BrOA, 2-BrDA) As a result, the accumulation of PHA and rhamnolipid was inhibited in both PA14 and PA01 strains as compared to the control without treatment of 2-bromoalkanoic acid compound. This result shows that 2-BrHA (2-bromohexanoic acid ) Was the best effect (see Table 1).

According to another embodiment, P. aeruginosa Measurement of the production of rhamnoside and PHA in PA14 wild-type and PA14- ? PhaG mutants showed that the content of PHA and rhamnolipid was more effectively suppressed in the PA14- ? PhaG mutant than in the wild type , wild-type and Δ PA14- phaG mutant type in both the 2-and 2-BrHA BrOA was inhibited the PHA and people went feed production effectively, 2-BrDA shown in the figure similar to the control group not treated with anything PHA person went It was found that feed generation was hardly suppressed (see FIG. 2A).

Similar to these results, P. aeruginosa In the same experiment with PAO1, it was found that the PHA was more inhibited in the PAO1- ? RhlA mutant type than in the wild type, and PHA and rhamnoside production were effectively inhibited in the 2mM 2-BrHA (see FIG. 2B).

According to another embodiment, P. aeruginosa PAO1 and P. aeruginosa In the PA14 strain, the degree of inhibition of rhamnose-free synthesis, the swarming mobility and the presence or absence of biofilm formation of the three 2-bromoalkanoic acid compounds (2-BrHA, 2-BrOA and 2-BrDA) It was found that the mallow alkanoic acid compound inhibited the pathogenic factors of the Pseudomonas aeruginosa. Among them, 2-BrHA (2-bromohexanoic acid) having the shortest alkyl group showed the best inhibition ).

Therefore, the 2-bromoalkanoic acids of the present invention have an activity of inhibiting the production of rhamnolipid and the biofilm, which are the pathogens of P. aeruginosa , and are useful for the treatment of pulmonary diseases such as cystic respiratory diseases It can be used for applications.

Further, the present invention is characterized in that the 2-BrHA compound having the shortest alkyl group among the 2-bromoalkanoic acids has a superior effect of inhibiting the pathogenic factor of P. aeruginosa.

In the present invention, the term " P. aeruginosa " refers to the fact that four toxic factors exoenzyme S (Exo S), exoenzyme T (Exo T), exoenzyme U (Exo U) and exoenzyme Y Type III secretion system is used for injection.

Strains producing Exo U and Exo T induce caspase (cysteinyl protease family) dependent host cell death more slowly. Exo U is a potent cytolytic toxin with similar activity to phospholipase A2 with broad specificity. Exo S and Exo T are proteins with N-terminal GTPase-activating protein activity and C-terminal adenosine diphosphate (ADP) -ribosyltransferase activity. Exo Y is a host factor-dependent adenylate cyclase.

Pseudomonas aeruginosa is an opportunistic pathogen and rarely infects healthy humans. However, it causes damage to normal tissues by burns, permeable trauma, surgical operations, deterioration of immune function, normal microbial diversity Total: microbial flora) can cause disease due to loss of protection. The infectious process forms colonies on the skin or mucosal surface and then invades the blood vessels through local site infiltration and damage to the underlying tissues.

Pseudomonas vaccine is the first and only vaccine to prevent P. aeruginosa infection in patients with cystic fibrosis. Cystic fibrosis is a hereditary disease that causes abnormal mucus production in the lungs. Thick, sticky mucus blocks the lungs and pancreas and causes life-threatening chest infections and digestive disorders. The disease is mainly exacerbated by P. aeruginosa and is very difficult to treat. This vaccine has been found to protect the lungs of patients with cystic fibrosis by inhibiting P. aeruginosa infection and progressive colony formation. The P. aeruginosa vaccine is the first vaccine approved in Europe for Orphan Drug.

In the present invention, the diseases caused by P. aeruginosa include, but are not limited to, cystic fibrosis, sepsis, systemic infections, and chronic airway infections.

The pharmaceutical composition for the prophylaxis or treatment of the above-mentioned diseases according to the present invention may comprise a pharmaceutically effective amount of 2-bromoalkanoic acid compound alone or in the form of one or more pharmaceutically acceptable salts . The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and the free acid may be an organic acid or an inorganic acid. The organic acids include, but are not limited to, citric, acetic, lactic, tartaric, maleic, fumaric, formic, propionic, oxalic, trifluroacetic, benzoic, gluconic, methosulfonic, glycolic, succinic, Glutamic acid and aspartic acid. The inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

The compound according to the present invention can be isolated from nature or can be prepared by chemical synthesis methods known in the art.

The pharmaceutically effective amount of the 2-bromoalkanoic acid compound or its salt according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight. However, the pharmacologically effective amount may be appropriately changed depending on the severity of symptoms of cystic fibrosis, sepsis, systemic infection and chronic airway infections, the age, body weight, health condition, sex, administration route and treatment period of the patient.

The term " pharmaceutically acceptable " as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

In addition, the antibiotic composition according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or muscular, and the dose of the active ingredient may be appropriately determined depending on the administration route, age, sex, And the antibiotic composition according to the present invention can be administered in combination with a known compound having an effect of preventing, ameliorating or treating cystic fibrosis, sepsis, systemic infection and symptoms of chronic airway infections. have.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

Used strain

Strain used in the experiment of the present invention, Pseudomonas aeruginosa (P. aeruginosa) with, PA14 wild-type and Δ PA14- phaG mutant type was obtained in the NR set PA14, P. aeruginosa PAO1 wild type and mutant type is PAO1- Δ rhlA University of Washington Transposon Were purchased from the Mutant Collection.

The media used in this study were NR (nutrient rich) medium, LB medium, and mineral salt medium. NR medium (1% w / v yeast extract, 1.5% w / v nutrient solution and 0.4% (w / v) ammonium sulfate) were used for the maintenance, storage and cultivation of the strain, 1.5% Bacto agar was added for the medium. For the biosynthesis of PHA and rhamnolipid, M1 mineral salt medium was used, and the concentration of each carbon source and nitrogen source (ammonium sulfate) was changed as needed. In addition, 60 μg / ml tetracycline and 30 μg / ml gentamycin were used for the stability of the introduced recombinant plasmids.

< Example  2>

2- Bromoalkanoic acid  Analysis of the effect on the growth of Pseudomonas aeruginosa

The present inventors conducted the following experiment to investigate the effect of 2-bromoalkanoic acid on growth of P. aeruginosa strains PA14 and PA01.

First, PA14 and PA01 strains were inoculated into a test tube containing NR medium, shake cultured at 30 ° C at 180 rpm for 12 hours, and 500 ml of M1 medium containing 70 mM fructose and 1.0 g of ammonium sulfate , 2 mM 2-BrHA and 2-BrOA were added to the flask, and the cells were cultured until the growth was stopped.

The cultured cells were centrifuged at 10,000 × g for 10 minutes, washed with methanol, and dried in a vacuum dryer for 24 hours. In addition, as a control, the cells were cultured in M1 medium without addition of the 2-bromoalkanoic acids 2-BrHA and 2-BrOA, and the degree of growth with time was measured using dry cell weight. Concentrations of fructose and ammonium ions in the medium were determined by 3,5-dinitrosalicylic acid (DNS) and Nessler 'reagent method, respectively.

As a result, it was observed that there was no difference in dry cell weight between the group treated with 2-BrHA and 2-BrOA compounds and the control group treated with nothing (see Fig. 1). Thus, it was found that the 2-bromoalkanoic acid had no effect on the growth of P. aeruginosa .

[Table 1] P. aeruginosa Analysis of the effect of adding 2-BrHA, 2-BrOA or 2-BrDA compounds to the PA14 and PAO1 strains during the synthesis of rhamnose and PHA

< Example  3>

2- Bromoalkanoic acid  by Randomized PHA Analysis of production inhibition effect

P. aeruginosa is known to synthesize PHA and rhamnoripid using PhaG and RhlA when cultured together with saccharides. In this case, 2-BrOA is known to inhibit the accumulation of PHA when cultured using fructose. The present inventors have found that 2-BrOA and 2-BrDA, which are homologous compounds with 2-BrOA, In order to confirm that the production of rhamnoside can be inhibited, the following experiment was prepared.

<3-1> Rannolipid  analysis( Rhamnolipid assay )

The present inventors have M9 salts medium is 0.2% fructose, 2 mM MgSO _4, 0.0005% methylene blue, 0.02% cetyl trimethyl ammonium bromide was added in order to analyze the change in the production of indigo Norris feed by 2-bromo alkanoic acids 2 mM 2-bromoalkanoic acid was added. On the other hand, glutamate was added to the medium to a concentration of 0.05% as a nitrogen source, and 1.8% agar was added thereto to prepare a rhamnolipid solid medium. Each colony grown in the LB solid medium was inoculated into a rhamnolipid solid medium using a toothpick and incubated at 30 ° C for 24 hours, and then again at room temperature for at least 48 hours until a blue rim was formed around the colonies Lt; / RTI &gt;

<3-2> Orcinol  analysis( Orcinol assay )

Orcinol analysis was performed to determine the amount of ranoripidide secreted during incubation in a M1 mineral-salt medium. For the analysis of orcinol, 300 mu l of the supernatant separated by centrifugation of the M1 medium was dissolved in 600 mu l dimethyl ether And the ether fractions were collected, dried and evaporated, and 100 ㎕ of distilled water was added. 100 μl of a 1.71% solution of orsinol and 800 μl of a 60% (v / v) sulfuric acid solution were added to 100 μl of each sample, followed by heating at 80 ° C for 30 minutes. Then, each sample was cooled for 15 minutes, and the absorbance was measured at a wavelength of 421 nm. The concentration of rhamnoside was calculated from the Ramson standard solution curve between 0 and 50 mg / ml, and the alkyl group of rhamnoside was measured by gas chromatography. Each culture was centrifuged, and the supernatant was collected by freeze-drying and then extracted with chloroform. The filtered chloroform extract was concentrated using a rotary evaporator (Eyela N-1000, Japan). A portion of the extract of chloroform was subjected to GC analysis after methanolysis as described in Example 3-3 below.

<3-3> Intracellular PHA  Quantitative analysis

To analyze the amount of PHA in the cells, 20 mg of the dried cells were reacted with 1 ml of chloroform, 0.85 ml of methanol, 0.15 ml of concentrated sulfuric acid mixture, and cultured at 100 ° C for 3 hours. After separating the organic layer containing the reaction product, MgSO 4 was added thereto to completely remove the residual water, and then analyzed by gas chromatography. Each peak was normalized using standard 3-hydroxy-methyl ester obtained by sulfuric acid methanolysis of purified PHA, a known composition determined by quantitative NMR analysis. Gas chromatograms were obtained using a Hewlett Packard 5890A gas chromatograph equipped with an HP-5 column and a flame ionization detector. The column was heated at 10 [deg.] C / min in the range of 80 to 250 [deg.] C.

As a result of the above experiment, it was confirmed that the production of rhamnoside and PHA increased with the growth of the cells, and the production of rhamnose was confirmed to occur while the nitrogen source was almost exhausted and the PHA was accumulated (see FIGS. 1A and 1D). On the other hand, PHA showed maximum accumulation at the last station where the carbon source is depleted. After all of the carbon source was used, PHA synthesis was reduced, but the synthesis of rhamnoside was continued (see Figures 1A, 1D). These results indicate that the PHA is converted to rhamnolipid by the PHA degrading enzyme (PhaZ) present in the cell under the condition that the carbon source is depleted.

On the other hand, 2 mM 2- bromo-alkanoic acid compound (2-BrHA, 2-BrOA , 2-BrDA) P. aeruginosa in 70 mM fructose media supplemented with The PHA and rhamnose contents of the PA14 strain were found to be 16% and 1.56 g / l, respectively. In the untreated PA14 strain, PHA and rhamnose were the highest yields of 1.56 g / The treatment with 2-BrOA was 5.1 wt% and 0.50 g / l, respectively, and the treatment with 2-BrOA was 10.2 wt% and 1.56 g, respectively. / l &lt; / RTI &gt; (see Table 1 and Figures 1B and 1C).

In a 70 mM fructose culture medium supplemented with 2 mM 2-bromoalkanoic acid compound (2-BrHA, 2-BrOA, 2-BrDA), P. aeruginosa The production of PHA and rhamnorefide of the PAO1 strain was confirmed, and the PHA and rhamnorefeed were 13 wt% and 1.82 g / l, respectively, in the case of no treatment of the PAO1 strain, and 2-BrHA compound The treatment with 2-BrOA compound reduced to 3.8 wt% and 0.55 g / l, respectively, and the treatment with 2-BrDA compound decreased to 9.5 wt% and 1.4 wt%, respectively. , And 1.80 g / l, respectively (see Table 1 and Figures 1E and 1F).

On the other hand, in the PA14 strain, 2-bromoalkanoic acid inhibited the production of PHA and rhamnoside by%, and BrHA, which has the shortest chain length of alkyl group, showed the strongest inhibitory effect. That is, 2-BrHA inhibited 89.3%, 2-BrOA had 67.8%, and 2-BrDA had 35.5% inhibitory effect on PHA production. In the production of rhamnose, 2- It was confirmed that 87.8% of BrHA, 68.2% of 2-BrOA and 0.3% of 2-BrDA were inhibited (FIG. 2A). In addition, it was confirmed that the PAO1 strain had an inhibitory effect in a similar manner (Fig. 2B).

RhlA and PhaG enzymes are known to be involved in the synthesis of rhamnose and PHA in the fatty acid synthesis pathway, respectively. The present inventors conducted experiments using transposon mutants of rhlA and phaG to confirm the mechanism by which 2-bromoalkanoic acid inhibits the production of rhamnose and PHA. The rhlA mutation in the PA14 strain exhibited excessive rhamnolipid expression, and was excluded from the present experiment, and PAO1- [Delta] rhlA Mutant type was used. Meanwhile, phaG The mutant PA14- ? PhaG mutant strains prepared in strain PA14 were used.

PAO1 - ? Rhla The mutant strains showed a 148% increase in PHA production compared to the wild type (see FIG. 2), suggesting RhIA and PhaG are in competition with the same fatty acid precursor. However, when 2 mM of 2-BrHA or 2-BrOA was treated, PHA accumulation was significantly decreased to 6.9% and 9.46%, respectively, as compared with the wild type (see FIG. 2B).

On the other hand, the PA14- △ phaG mutant type strain showed that the person went feed synthesis of 76.1% as compared to wild-type, 3.1% as compared to the addition of 2 mM 2-BrHA or 2-BrOA, people went feed is wild-type, respectively, 7.5% (see Fig. 2A).

This strong inhibition of rhamnoside and PHA synthesis by 2-BrHA or 2-BrOA is believed to be accomplished by double-targeting two enzymes (PhaG, RhlA) linked by PHA to rhamnolipid synthesis from the fatty acid synthesis pathway (See FIG. 3).

< Example  4>

2- Bromoalkanoic acid  by PhaG  And Of Rhla  Inhibitory activity assay

In order to confirm whether the inhibitory effect of PHA and rhamnolipid production by 2-bromoalkanoic acid on the expression of PhaG and RhlA enzymes involved in these biosynthetic pathways was inhibited by the present inventors, -Bromoalkanoic acid was treated and RT-PCR was performed.

To examine the expression of RNA, cells were grown in M1 medium containing 70 mM fructose with or without 2 or 5 mM 2-bromo alkanoic acid at a temperature of 30 DEG C for 72 hours. The cells were collected, RNA was isolated using an RNeasy mini kit according to the manufacturer's protocol, and phaG And rhlA One-step RT-PCR was performed using an oligonucleotide primer and a Roto-Grene SYBR Green RT-PCR kit for gene expression.

As a result, even when 2-bromoalkanoic acid was treated in PAO1 wild type and mutant type, phaG And rhlA (Fig. 4A, Fig. 4B). Therefore, the present inventors determined that the 2-bromoalkanoic acid inhibited the production of PHA and rhamnorefide by inhibiting PhaG and RhlA enzymes themselves.

2-Bromoalkanoic acid was judged to double-target PhaG and RhlA enzymes. The present inventors comparatively analyzed the inhibitory effect of 2-bromoalkanoic acid to inhibit PHA and rhamnolipid in order to clarify this.

As a result, as shown in FIG. 5, 2-BrHA and 2-BrOA both inhibited PHA and rhamnolipid synthesis and showed inhibitory effects of 90% and 70%, respectively, Showed about 25 ~ 35% inhibition effect on the synthesis of PHA and showed almost no inhibitory effect on the synthesis of rhamnose. Thus, the fact that the effect of 2-BrDA treatment is different from that of PHA and rhamnolipid synthesis inhibition indicates that only a specific gene at a higher level in the fatty acid biosynthesis pathway is not the only target of 2-bromoalkanoic acid . That is, the 2-bromoalkanoic acid according to the present invention can be expected to double the effect of each of PhaG and RhlA.

< Example  5>

2- Bromoalkanoic acid  by Rannolipid  Production inhibitory activity Swamming  Analysis of motility inhibitory activity

<5-1> 2- Bromoalkanoic acid  by Rannolipid  Production inhibitory activity

Pseudomonas aeruginosa ( P. aeruginosa ) generally produces a lipid surfactant called ranoripide, which is lipid-bound to rhamnose. The present inventors conducted a rhamnolipid analysis to investigate the activity of inhibiting rhamnoside production by 2-bromoalkanoic acid treatment.

The concentration of Ramanolipid detected in PA14 and PAO1 of P. aeruginosa of the present invention can be visualized by the presence of a blue border around the colony in a plate assay. In the medium without 2-bromoalkanoic acid treatment, PA14 and PAO1 strain (Fig. 6A). However, in the medium treated with 5 mM 2-BrHA or 2-BrOA, no rhamnolipid secretion was observed (Fig. 6A).

Therefore, it was found that the 2-bromoalkanoic acid compound of the present invention had an activity to inhibit rhamnoside.

<5-2> 2- Bromoalkanoic acid  by Swamming  Analysis of motility inhibitory activity

P. aeruginosa is capable of organized, multicellular movement called 'swarming', which is known to be benefited by type IV flies as well as flagella. Furthermore, ranorifide, in conjunction with its precursor HAA (C10-C10), is involved in swarming motility by reducing surface tension to induce surface conditions necessary for efficient colony formation, Mutations are known to lack such swarming motility. Therefore, the inventors of the present invention conducted the following experiment to determine the swarming mobility of 2-bromoalkanoic acid.

The plate medium for Swamming motility measurement was prepared by adding 1 mM MgSO ₄ , 0.2% glucose to M9 salt medium, supplementing with 0.05% glutamate, adding 5 mM 2-bromo compound if necessary, and adding 0.6% agar A hardened medium was used. 2 [mu] L of the strain cultured in LB medium overnight was inoculated at the center of the plate medium for swarming motility measurement, cultured at 37 [deg.] C for 16 hours, and further cultured at 30 [deg.] C for 48 hours.

As a result, as expected, 2-bromoalkanoic acid inhibited the swarming motility of P. aeruginosa (Fig. 6B), and it was judged that this was due to the inhibitory activity of rhamnoside production.

< Example  6>

2- Bromoalkanoic acid  Of biofilm formation

On the other hand, Pseudomonas aeruginosa is known to form biofilms, which are aggregates of cells surrounded by extracellular matrix composed of protein, nucleic acid, exopolysaccharide, DNA and the like.

The inventors of the present invention conducted the following test to see if the 2-BrHA compound of the present invention can inhibit the formation of the above-mentioned biofilm of P. aeruginosa.

First, biofilm formation was carried out by modifying the experimental method of O'Toole and Kolter. The strain was cultured in 4 ml of M1 medium supplemented with 70 mM fructose. When necessary, 5 mM 2-bromo-compound was added and reacted in a glass tube at 30 ° C for 24 to 48 hours without stirring. The biofilm formation was measured by visual observation of the surface of the culture medium in contact with air. When the surface of the culture medium was covered with the cells and the substrate layer, the biofilm was regarded as a biofilm. The test tube was washed with distilled water and stained with a 0.1% crystal violet solution. Then, 4.5 ml of 95% ethanol was added to each tube, and the absorbance was measured at 600 nm to quantitatively analyze the biofilm.

Interestingly, it was confirmed that the treatment of 2-BrHA, 2-BrOA, and 2-BrDA compounds inhibited the formation of biofilm before the production of ranorifide (FIGS. 6C and 6D). On the other hand, the difference in the biofilm formation varied depending on the length of alkyl groups. In the case of PA14 cultured for 48 hours in the medium treated with 5 mM 2-BrHA, 2-BrOA, 2-BrDA, Biopharmaceuticals were reduced to 18%, 26% and 67%, respectively. In the case of PAO1 cultured for 48 hours in media treated with 5 mM 2-BrHA, 2-BrOA, and 2-BrDA, biofilm was 29%, 40%, 69% %, Respectively. From these results, the present inventors found that the degree of inhibition of biofilm was the greatest in 2-BrHA having the shortest alkyl length (see FIG. 6D).

Since the biofilm of Pseudomonas aeruginosa is known to impart antibiotic resistance to Pseudomonas aeruginosa, the 2-Bromoalkanoic acid according to the present invention can inhibit the formation of biofilm of Pseudomonas aeruginosa, and thus can be usefully used as an antibiotic of P. aeruginosa.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (8)

An antibiotic composition comprising 2-bromohexanoic acid represented by the following formula (1) as an active ingredient;
[Chemical Formula 1]

. delete The method according to claim 1,
Wherein said antibiotic composition exhibits antimicrobial activity by reducing or inhibiting antibiotic resistance. The method according to claim 1,
The antibiotic compositions antibiotic composition characterized to exhibit the antibacterial activity against Pseudomonas aeruginosa (P seudomonas aeruginosa). The method according to claim 1,
Wherein said antibiotic composition inhibits PHA (polyhydroxyalkanoate) or rhamnose riboside production. The method according to claim 1,
Wherein the antibiotic composition reduces or inhibits swarming motility of the pathogen. The method according to claim 1,
Wherein said antibiotic composition reduces or inhibits biofilm formation of pathogenic bacteria. The method according to claim 1,
Wherein said antibiotic composition is characterized by preventing or treating cystic fibrosis, sepsis, systemic infection or chronic airway infections.

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