KR102002172B1 - Novel use of compound having anti-virulence activity and anti-biofilm formation activity - Google Patents

Abstract

The present invention relates to a novel use of a compound exhibiting an anti-virulence activity and a biofilm formation inhibitory effect, and more particularly, to a novel use of a compound having an outer membrane protein A membrane protein A, OmpA) by inhibiting the gene promoter of the compound of formula (I).
The compound of formula (I) of the present invention inhibits the expression of the gene and protein of the outer membrane protein A, which is a pathogenic protein, without showing a direct bactericidal effect against the pathogenic bacteria, and thus the anti-virulence agent . In addition, it can inhibit the formation of a biofilm closely related to the expression of OmpA, and thus can be effectively used for the prevention and treatment of infectious diseases caused by infection with pathogenic bacteria.

Description

[0001] The present invention relates to novel compounds having anti-virulence activity and anti-biofilm formation activity,

The present invention relates to a novel use of a compound exhibiting an anti-virulence activity and a biofilm formation inhibitory effect, and more particularly, to a novel use of a compound having an outer membrane protein A membrane protein A, OmpA) by inhibiting the gene promoter of the compound of formula (I).

Antibiotics is a metabolite produced by microorganisms. It is a drug that inhibits or kills the growth of other microorganisms in a small amount and treats infectious diseases caused by pathogens. It is used worldwide because it shows excellent efficacy against infection. Antibiotics are antimicrobial agents extracted from fungi. Antibiotics such as penicillin and cephalosporin, antibiotics such as griseofulvin and fucidin, and macrolide, aminoglycoside, tetracycline and chloramphenicol are examples of antibiotics extracted from fungi. Penicillin has fewer side effects than other antibiotics, but some bacteria produce enzymes that break down penicillin, and artificially synthesized methicillin and oxacillin are used together.

One of the most powerful antibiotics in the world, vancomycin, was developed in the 1950s as a result of the spread of Staphylococcus aureus resistant to methicillin, an alternative to penicillin, and a serious infection of Staphylococcus aureus Have been used to treat.

However, due to the abuse of antibiotics, there has been a problem in the effective use of antibiotics due to the emergence of super bacteria, which are able to resist the pathogens themselves and become resistant to any antibiotics due to their strong resistance. Bacteria can become natural mutants that can overcome antibiotics, and these resistant bacteria can transmit the resistance gene to other bacteria, and the spread of resistant bacteria can be done quickly. Thus, the abuse of antibiotics causes bacterial antibiotic resistance, and chronic diseases caused by bacterial infections are becoming global issues.

On the other hand, unlike antibiotics, which are intended to inhibit and kill bacteria, anti-virulence agents inhibit only the virulence of bacterial growth, so they are more resistant to bacterial antifungal agents than general antibiotics Less induced. An important example of an antimicrobial agent is a biofilm formation inhibitor that causes antibiotic resistance and also an antinfectious agent that inhibits the production of pathogenic proteins without affecting cell growth.

At the present time when antibiotic resistance is a problem, there is no antibiotic resistance problem because it does not affect the growth of infectious bacteria at the present time, and at the same time, the production of biofilm and / Which is the most important pathogen.

Accordingly, the inventors of the present invention have found that a compound having the structure of the following formula (I) inhibits the OmpA gene promoter and inhibits the expression of the OmpA gene and the expression of the protein while screening for a compound inhibiting the gene promoter of OmpA which is a pathogenic protein in infective bacteria The present inventors have found that there is an effect of inhibiting the formation of a biofilm and have completed the present invention.

Accordingly, an object of the present invention is to provide an anti-virulence composition for a pathogenic bacterium which comprises, as an active ingredient, a compound of the following general formula (I) or a pharmaceutically acceptable salt thereof.

(I)

It is another object of the present invention to provide a pharmaceutical composition for the prevention and treatment of infectious diseases caused by pathogenic bacteria including the above antiviral composition.

Another object of the present invention is to provide a composition for inhibiting biofilm of a pathogenic bacterium comprising the compound of the general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

(I)

In order to accomplish the above object of the present invention, the present invention provides an anti-virulence composition for a pathogenic bacterium comprising the compound of the general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

(I)

In order to accomplish another object of the present invention, the present invention provides a pharmaceutical composition for the prevention and treatment of infectious diseases caused by a pathogenic bacterium comprising the above antiviral composition.

In order to accomplish still another object of the present invention, there is provided a composition for inhibiting biofilm of a pathogenic bacterium comprising, as an active ingredient, a compound of the following general formula (I) or a pharmaceutically acceptable salt thereof.

(I)

Hereinafter, the present invention will be described in detail.

The present invention provides an anti-virulence composition for pathogenic bacteria comprising, as an active ingredient, a compound of the formula (I) or a pharmaceutically acceptable salt thereof:

(I)

In the present invention, the pathogenic bacterium may be a bacterium that expresses outer membrane protein A (OmpA) and exhibits pathogenicity by the protein.

More specifically, as the bacteria are preferred in the present invention include Acinetobacter baumannii (Acinetobacter baumannii), Acinetobacter knife core Shetty kusu (Acinetobacter calcoaceticus), Acinetobacter H. Emory Tea Syracuse (Acinetobacter haemolyticus), Acinetobacter Junior (Acinetobacter junii), Acinetobacter Jones you (Acinetobacter johnsonii), Acinetobacter Lee wopi (Acinetobacter lwoffii), Acinetobacter radio Recife stance (Acinetobacter radioresistens), Acinetobacter Ur destination (Acinetobacter ursingii , Acinetobacter schindleri), Acinetobacter Parque booth (Acinetobacter parvus), Acinetobacter Lee Bay (Acinetobacter baylyi , Acinetobacter bouvetii), Acinetobacter tow Nourishing (Acinetobacter towneri), Acinetobacter tandoyi (Acinetobacter tandoii), Acinetobacter so Montego Bay (Acinetobacter grimontii), Acinetobacter Syed Zia reunbeo (Acinetobacter tjernbergiae), and Acinetobacter Nourishing Stavanger (Acinetobacter gerneri), Tino liquid Bacillus play right pneumoniae in (Actinobacillus pleuropneumoniae , Actinobacillus < RTI ID = 0.0 > suis ), Aggregatibacter actinomycetemcomitans , Chlamydia trachomatis ( Chlamydia spp. ), trachomatis , Chlamydia caviae , Chlamydia muridarum), Carol shown in Pillars pneumoniae (Chlamydophila pneumoniae), climb the pillar shown Sita key (Chlamydophila psittaci), Chrono bakteo sakajaki (Cronobacter sakazakii), E. coli (Escherichia coli ), Flavobacterium psychrophilum , Haemophilus influenzae ( Haemophilus < RTI ID = 0.0 > influenzae , Haemophilus parasuis , Histophilus < RTI ID = 0.0 > somni), Klebsiella pneumoniae (Klebsiella in pneumoniae ), leptospira ( Leptospira interrogans , Mannheimia haemolytica , Neisseria gonorrhoeae , Neisseria meningitidis meningitidis , Pasteurella multocida , Pseudomonas aeruginosa, aeruginosa), Liege Mercure Relais Anna Tee Festiva flops (Riemerella anatipestifer), Salmonella (Salmonella spp) or yeosi California (may be Yersinia spp), most preferably Acinetobacter baumannii (Acinetobacter be baumannii), but not limited to, bacteria that express the OmpA can all be included thereto.

OmpA protein is a protein that exists in the outer membrane of bacteria and is mainly found on the cell membrane surface of Gram-negative bacteria. In many pathogenic bacteria, the OmpA protein is closely related to bacterial attachment, penetration, intracellular survival, avoidance of host cell immune system, resistance to antimicrobial peptides or the production of inflammatory cytokines, thus playing an important role in pathology . The pathological features of these OmpA proteins are more closely related to infection of the central nervous system, respiratory and genitourinary tracts.

In addition, the OmpA protein is closely involved in various pathological phenomena because it is expressed on a wide range of frequencies with a high frequency on the surface of pathogenic bacteria. OmpA, which is expressed on the outer surface of pathogenic bacteria, has been reported as a pathogenic protein in many infectious diseases such as pneumonia, meningitis, gingivitis, urethritis, cystitis, ulcerative colitis and encephalitis (Veterinary Microbiology 163 (2013) 207222).

According to one embodiment of the present invention, the inventors of the present invention have confirmed that the compound of the formula (I) inhibits the gene promoter of OmpA in Asminobacter baumanni to inhibit the expression of OmpA gene and protein 2 to 4). At the same time, it has been confirmed that the compound of formula (I) can be used as an anti-virulence agent since it does not exhibit a direct proliferation inhibitory effect or bactericidal effect on bacteria.

Therefore, the compound of formula (I) is characterized by inhibiting the expression of the OmpA gene and protein by inhibiting the OmpA gene promoter, which is a pathogenic substance, and exhibiting an antiviral activity.

The present invention also provides a pharmaceutical composition for the prevention and treatment of infectious diseases caused by a pathogenic bacterium comprising an anti-pathogenic composition comprising the compound of the formula (I) as an active ingredient.

In the present invention, the pathogenic bacterium may be a bacterium that expresses outer membrane protein A (OmpA) and exhibits pathogenicity by the protein.

More specifically, as the bacteria are preferred in the present invention include Acinetobacter baumannii (Acinetobacter baumannii), Acinetobacter knife core Shetty kusu (Acinetobacter calcoaceticus), Acinetobacter H. Emory Tea Syracuse (Acinetobacter haemolyticus), Acinetobacter Junior (Acinetobacter junii), Acinetobacter Jones you (Acinetobacter johnsonii), Acinetobacter Lee wopi (Acinetobacter lwoffii), Acinetobacter radio Recife stance (Acinetobacter radioresistens), Acinetobacter Ur destination (Acinetobacter ursingii , Acinetobacter schindleri), Acinetobacter Parque booth (Acinetobacter parvus), Acinetobacter Lee Bay (Acinetobacter baylyi , Acinetobacter bouvetii), Acinetobacter tow Nourishing (Acinetobacter towneri), Acinetobacter tandoyi (Acinetobacter tandoii), Acinetobacter so Montego Bay (Acinetobacter grimontii), Acinetobacter Syed Zia reunbeo (Acinetobacter tjernbergiae), and Acinetobacter Nourishing Stavanger (Acinetobacter gerneri), Tino liquid Bacillus play right pneumoniae in (Actinobacillus pleuropneumoniae , Actinobacillus < RTI ID = 0.0 > suis ), Aggregatibacter actinomycetemcomitans , Chlamydia trachomatis ( Chlamydia spp. ), trachomatis , Chlamydia caviae , Chlamydia muridarum), Carol shown in Pillars pneumoniae (Chlamydophila pneumoniae), climb the pillar shown Sita key (Chlamydophila psittaci), Chrono bakteo sakajaki (Cronobacter sakazakii), E. coli (Escherichia coli ), Flavobacterium psychrophilum , Haemophilus influenzae ( Haemophilus < RTI ID = 0.0 > influenzae , Haemophilus parasuis , Histophilus < RTI ID = 0.0 > somni), Klebsiella pneumoniae (Klebsiella in pneumoniae ), leptospira ( Leptospira interrogans , Mannheimia haemolytica , Neisseria gonorrhoeae , Neisseria meningitidis meningitidis , Pasteurella multocida , Pseudomonas aeruginosa, aeruginosa), Liege Mercure Relais Anna Tee Festiva flops (Riemerella anatipestifer), Salmonella (Salmonella spp) or yeosi California (may be Yersinia spp), most preferably Acinetobacter baumannii (Acinetobacter be baumannii), but not limited to, bacteria that express the OmpA can all be included thereto.

In the present invention, the infectious diseases are selected from the group consisting of dermatitis, dermatitis such as dermatitis, dermatitis and rash, dermatitis, pneumonia, mastitis, phlebitis, cystitis, meningitis, enteritis, urinary tract infection, osteomyelitis, keratitis, meningitis, prostatitis, endocarditis, but is not limited to, wound wounds, food poisoning, sepsis, bacteremia, or toxic shock syndrome, including, but not limited to, any disease that can be caused by the infectious bacterium.

The composition of the present invention may further comprise a pharmaceutically acceptable additive. Examples of the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, calcium stearate, magnesium stearate, White sugar, dextrose, sorbitol and talc may be used. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

In addition, the composition of the present invention can be administered in various formulations of oral and parenteral administration at the time of actual clinical administration. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, . ≪ / RTI >

Solid formulations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid formulations are prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose or gelatin, . In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used .

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

On the other hand, injecting agents may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives and the like.

In addition, the therapeutic compositions of the present invention may be formulated with any physiologically acceptable carrier, excipient or stabilizer (Remington: The Science and Practice of Pharmacy, l9th Edition, Alfonso, R., ed., Mack Publishing Co. : 1995)). Acceptable carriers, excipients or stabilizers are nontoxic to the recipient at the dosages and concentrations employed and include buffer solutions such as phosphoric acid, citric acid and other organic acids; Antioxidants including ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides; Proteins, such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrin; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt-forming counterions such as sodium; And / or non-ionic surfactants such as tween, pluronics or polyethylene glycol (PEG).

 The dosage of the present invention may vary depending on the patient's age, weight, sex, dosage form, health condition and disease severity, and is generally 0.01 to 100 mg / kg / day, preferably 0.1 to 20 mg / / kg / day, and more preferably 5 - 10 mg / kg / day. It may also be administered at a fixed interval according to the judgment of a doctor or pharmacist.

The administration route of the composition of the present invention may be administered orally or parenterally by a known method such as injection or infusion by intravenous, intraperitoneal, intracerebral, subcutaneous, intramuscular, intravenous, intraarterial, intracerebral, or intralesional routes, Injection or injection by a sustained release system. In addition, the pharmaceutical composition of the present invention can be used alone or in combination with methods for using surgery, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of infectious diseases.

The pharmaceutical composition of the present invention may also be included in the composition of the present invention, depending on the specific condition or disease to be treated or prevented, and further therapeutic agents normally administered to treat or prevent the condition. An additional therapeutic agent that is normally administered to treat or prevent a disease or condition is known to be "suitable for the disease or condition being treated ". Such agents include, without limitation, antibiotics, antiinflammatory agents, substrate metalloprotease inhibitors, lipoxygenase inhibitors, cytokine antagonists, immunosuppressants, anticancer agents, antiviral agents, cytokines, growth factors, immunomodulators, prostaglandins, , Or agents that increase the susceptibility of bacterial microorganisms to antibiotics.

Examples of antibiotics suitable for administration of the compounds of the invention and compositions thereof include, but are not limited to, quinolones,? -Lactams, macrolides, glycopeptides and lipopeptides.

For example, U.S. Patent No. 5,523,288, U.S. Patent No. 5,783,561, and U.S. Patent No. 6,140,306 disclose agents that increase the susceptibility of bacteria to the antibiotic in the present invention in order to increase the susceptibility of Gram-positive and Gram- Discloses a method using a sterilizing / permeability-enhancing protein (BPI). Drugs that increase the permeability of the outer membrane of bacteria are described in Vaara, M., Microbiological Reviews (1992), pp. 395-411, and the sensitization of Gram-negative bacteria is described in Tsubery, J. Med. Chem. (2000) 3085-3092], and these drugs may be included in an agent that increases the susceptibility of the bacteria of the present invention.

The present invention also provides a composition for inhibiting biofilm of a pathogenic bacterium comprising, as an active ingredient, a compound of the formula (I) or a pharmaceutically acceptable salt thereof.

(I)

In the present invention, the biofilm refers to a mucilaginous bacterium complex formed by bacteria, and is a complex composed of bacterial colonies, which are solid biological surfaces, and an outer membrane, which is an inanimate surface. Accordingly, in the present invention, the biofilm means the entire film including the outer membrane and bacterial colonies contained therein. Bacteria in which biofilms are formed are more than 1,000 times more resistant to antibiotics than others, and the formation of biofilms is known to play an important role in the development of infectious diseases (Rasmussen and Givskov 2006).

In addition, the biofilm is commonly found in living, medical and industrial environments that are often encountered around. In the case of pathogenic microorganisms which can live in the living body, various kinds of tissues / organs including the host's epithelial cells, bones, teeth, and inner wall of vessels, various artificial implants (catheter, implant) A film can be formed. When biofilms are formed, the microorganisms are resistant to harsh environments, and are resistant to antibiotics and immune cells. Therefore, they are very difficult to remove and cause chronic inflammatory diseases. Recent reports indicate that about 65% of all infectious diseases are caused by biofilm formation (Ymele-Leki and Ross, 2007, Applied and Environmental Microbiology 73 (6): 1834-41). Therefore, the inhibition of biofilm by the compounds of formula (I) of the present invention may be very important in the prevention / treatment of infection by pathogenic microorganisms.

In one embodiment of the present invention, the inventors have found that the compound of formula I effectively inhibits the biofilm formation of Asnithobacter baumannii (Examples 5 and 9).

On the other hand, OmpA is known to be overexpressed during the formation of biofilms in bacteria (Proteomics 2006, 6, 42694277). Therefore, inhibition of the expression of OmpA can inhibit the formation of biofilm, which can be effective in the prevention and treatment of pathogenic infection. As described above, the compound of formula (I) of the present invention inhibits the OmpA gene promoter, thereby inhibiting the expression of OmpA gene and protein, and consequently inhibiting the formation of biofilm induced by the expression of OmpA.

Accordingly, the present invention provides a composition, wherein the compound of formula I inhibits biofilm formation by inhibiting the expression of the OmpA gene.

In one embodiment of the invention, the result of infection A. baumannii strains in immunocompetent mice and number removes mouse neutrophils and confirm the viability and in vivo distribution of the bacteria after treatment of the compound of formula (I) mouse, the compounds of formula (I) A . baumannii It was confirmed that the bacterium was effective in both the infected normal immune state and the neutrophil-removed mouse, and it was confirmed that the bacterium was removed from the mouse group treated with the compound of the formula I compared to the mouse group not treated with the compound of the formula (Example 7).

The compound of formula (I) of the present invention inhibits the expression of the gene and protein of the outer membrane protein A, which is a pathogenic protein, without showing a direct bactericidal effect against the pathogenic bacteria, and thus the anti-virulence agent . In addition, it can inhibit the formation of a biofilm closely related to the expression of OmpA, and thus can be effectively used for the prevention and treatment of infectious diseases caused by infection with pathogenic bacteria.

Fig. 1 is a schematic diagram of a reporter strain prepared by cloning nptl, a kanamycin antibiotic resistance gene, after removing the OmpA structural protein gene following the promoter DNA of OmpA.
Figure 2 is a graphical representation of an experimental method for screening compounds that inhibit the OmpA promoter.
FIG. 3 is a graph showing the results of inhibition of proliferation and reproductive inhibition against reporter strain of the wild-type strain of the compound of formula I. FIG.
FIG. 4 shows the result of real time PCR for the change in the expression level of the OmpA gene after treating the compound of formula I with Asnithobacter baumannii.
FIG. 5 shows the results of SDS-PAGE (FIG. 5A) and Western blot (FIG. 5B) of changes in the expression level of OmpA protein after treating the compound of formula I with Asnithobacter baumanni.
Fig. 6 shows the results of evaluating the biofilm formation inhibiting ability after treating the compound of formula I with Asnithobacter baumannii.
FIG. 7 is a graph showing the results of evaluation of the cytotoxicity of the compound of formula I through the WST-1 assay. FIG.
Figure 8 shows A. baumannii ATCC 17878 strain and a lux A. baumannii with gene insertion The result of confirming the expression of fluorescence in the fluorescent expression apparatus (IVIS) of the ATCC 17878 strain.
9 is lux A. baumannii with gene insertion ATCC 17878 strain was treated with a compound of the formula (I), and the survival rate of the mice and the distribution of the infectious bacteria after 1 hour, 24 hours and 48 hours were confirmed in mice.
FIG. 10 shows the results of real time PCR for the change in the expression level of the OmpA gene after treatment of the compound of formula (I) with 8 strains of Asnithobacter baumanni clinical isolates.
FIG. 11 shows the results of evaluating the biofilm formation inhibiting ability after treating the compound of formula I with 8 strains of Ashinobacter baumanni ATCC 19606 strain and clinical isolates.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Test compound

In order to search for a substance that inhibits the expression of the outer membrane protein A (OmpA) gene, which is a structural protein expressed in the outer membrane of pathogenic bacteria, the present inventors purchased the compounds shown in the following Table 1 from Korean Compound Bank, Respectively.

Acinetobacter Structures of compounds used for screening substances that inhibit the promoter of outer membrane protein A of baumannii compound
number rescue compound
number rescue I

VII

II

VIII

III

IX

IV

X

V

XI

VI

≪ Example 2 >

Selection of Compounds Inhibiting OmpA Gene Promoter

<2-1> Experimental strain

The strain A. baumannii ATCC 17978 was purchased from the American Type Culture Collection (ATCC), and the strain was cultured on a MacConkey agar plate or Luria-Bertani (LB) medium.

<2-2> Preparation of Reporter strain

In order to search for the compound, the reporter strain was constructed by transforming the pFL02 plasmid constructed by cloning the kanamycin antibiotic resistance gene, nptI, into the A. baumannii ATCC 17978 by removing the OmpA structural protein gene following the promoter DNA of OmpA, Is shown in Fig.

Specifically, ompA Reporter plasmids pFL01 and pFL02 plasmid vectors were constructed in order to develop a reporter strain that can inhibit gene expression. Reporter strains were constructed by transforming pFL02 plasmid into A. baumannii ATCC 17978.

&Lt; 1 > Development of pFL01 plasmid vector (Fig. 1)

OmpA using antibiotic resistance gene We constructed pFL01 plasmid vector for construction of reporter plasmid. Antibiotic markers other than nptI ( kanamycin resistance gene), which are used as reporter genes in reporter plasmid construction, are indispensable. Therefore, a novel cloning vector pFL01 was constructed by inserting a specific antibiotic marker erythromycin resistant gene ( ermAM ) for A. baumannii in the tetracycline resistance gene region of pBR322.

1) A pair of primer sets (E03 / E04) capable of amplifying ermAM ( a gene causing erythromycin resistance) and ermAM PCR was carried out using the pIL252 plasmid containing the gene as a template. PrimerSTAR GXL Taq DNA polymerase (Takara) was used to make the PCR products blunt-ended.

② The amplified ermAM ( 1,040 bp) DNA fragment was purified using a gel extraction kit.

③ The backbone plasmid pBR322 was treated with restriction enzymes EcoRI and SphI, and then both ends of the vector were smoothed using DNA polymerase I (Large [Klenow] Fragment).

④ The ermAM amplified by PCR reaction in the pBR322 vector in which both ends of the DNA were smoothed The gene was cloned by ligation, and the plasmid vector thus constructed was named pFL01.

&Lt; 2 > Development of pFL02 plasmid vector (Fig. 1)

A. baumannii 's ompA reporter plasmid system was constructed in which the expression of antibiotic resistance gene ( nptI ) was regulated by the promoter. The preliminary experimental outline consists of primer sets of three pairs (O05 / O06, K03 / K04, R01 / R02) and two-step PCR reaction to A. baumannii The open reading frame (ORF) of the promoter site of ompA , nptI ( kanamycin antibiotic resistance gene) and A. In the baumannii , the cloning site that causes replication of the plasmid is sequentially connected to one DNA fragment. Sequentially linked DNA fragments are cloned into the pFL01 vector to construct a reporter plasmid in which the expression of the antibiotic resistance gene is regulated by the ompA promoter.

A pair of primer sets (O05 / O06) used in the first PCR reaction is a primer capable of amplifying the promoter region of the ompA gene on the chromosome of A. baumannii . The K03 / K04 primer set is a primer capable of amplifying the ORF of nptI , and the remaining pair of primer sets (R01 / R02) is a primer capable of amplifying the replication starting site that causes plasmid replication in A. baumannii to be.

② The Tm value of all the primers used is designed to be 54 ° C to 56 ° C. Especially, the reverse primer (O06, K04) used for amplifying the ORF of promoter and nptI of ompA has nptI And 25 nucleotides that are homologous to the origin of replication. This is to sequentially bind the promoter region of ompA , the ORF of nptI , and the initiation site of plasmid replication through overlap extension PCR.

③ In order to easily clone the amplified PCR product into the pFL01 plasmid vector, the base sequence CTGCAG , which recognizes the restriction enzyme PstI, was added to the 5 'end of the forward primer (O05) used to amplify the ompA promoter site, The base sequence GACGTC , which is recognized by the restriction enzyme AatII, was added to the 5 'end of the reverse primer (R02) used to amplify the starting site.

④ and the pUC4K plasmid pWH1266 were each used as a template for DNA was used as a genomic DNA of A. baumannii template to amplify the ompA promoter region in the first step PCR reaction, to amplify the ORF with the origin of replication region of nptI . Each DNA amplified by PCR was purified by gel extraction kit. Each of the purified DNAs was used as template DNA in the overlap extension PCR reaction, which is the second PCR step. The DNA concentrations used in PCR reactions were 100 ng each. PCR was performed using the O05 and R02 primers used in the first PCR reaction to sequentially bind the amplified three different DNA fragments ( ompA promoter, ORF of nptI , and the cloning start site) to one smooth end DNA. Respectively. In the second step PCR, the amplified DNA fragments were purified using a gel extraction kit.

(5) The purified DNA fragments were ligated to pFL01 plasmid treated with the restriction enzymes PstI and AatII and then cloned. The newly constructed vector was confirmed by restriction enzyme treatment and DNA sequencing. The constructed vector was named pFL02.

<3> Reporter strain development (Fig. 2)

The constructed recombinant plasmid pFL02 was transformed into A. baumannii cells using a helper plasmid (pRK2013). Transformed A. baumannii ( pFL02 plasmid) was screened on Luria-Bertani (LB) solid medium supplemented with ampicillin (100 μg / ml) and erythromycin (30 μg / ml).

( 2 ) To confirm that the kanamycin antibiotic resistance gene ( nptI ) possessed by the constructed reporter strain is regulated by the ompA promoter, bacteria were cultured in LB solid medium supplemented with kanamycin (50 μg / ml). A. baumannii , which does not have the pFL02 plasmid, and A. baumannii , which contains the plasmid pFL02, were inoculated into LB broth and LB broth supplemented with erythromycin (30 μg / ml), and cultured at 37 ° C for 12 hours with shaking. Each shake culture was diluted with LB broth to an optical density of 600 nm [OD ₆₀₀ ] of 0.1. Each diluted bacterial strain (10 μl) was plated on LB solid medium supplemented with kanamycin (50 μg / ml) to confirm the growth of the bacteria.

③ As a result, A. baumannii not containing pFL02 could not grow in medium supplemented with kanamycin (50 μg / ml). However, A. baumannii with pFL02 The fungus was found to grow by sprinkling on medium supplemented with kanamycin (50 μg / ml). This fact implies that the kanamycin resistance gene ( nptI ) present in the pFL02 plasmid is regulated by the ompA gene expression regulatory system of A. baumannii .

The characteristics of the strains and plasmids used for the above reporter plasmid and reporter strain and the primers used are as follows.

&Lt; 2-3 > Search for compounds inhibiting OmpA promoter

Anti-virulent agents are substances that inhibit specific pathogenic factors without inhibiting the growth of bacteria, unlike antibiotics. Therefore, we sought to select substances that only inhibit reporter strain growth without inhibiting growth of wild-type strains.

The principle of selecting a substance that inhibits the OmpA promoter using the reporter strain is shown in FIG.

Whether the compound inhibits growth on the reporter strain can be determined by adding kanamycin-containing culture media to each well of a 96-well plate, inoculating the reporter strain and the compound to be searched, and culturing. After 24 hours, the bacterial growth of reporter strain without compound treatment and reporter strain with compound was evaluated by comparing optical density measurement with ELISA reader.

Specifically, 100 μl of Mueller-Hinton broth containing 50 μg / ml of kanamycin was added to each well of a 96-well plate, and A. baumannii with pFL02 plasmid cultured overnight in LB broth The reporter strain, ATCC 17978, was placed at 6.0 × 10 ⁵ . Each compound was added to each well at a concentration of 2.5 μM, and the mixture was incubated at 37 ° C. for 24 hours in a final volume of 150 μl. As control, 100 μl of Mueller-Hinton broth containing no kanamycin in 3 or more wells per plate and A. baumannii with pFL02 plasmid cultured overnight in LB broth ATCC 17978 reporter strain was added at a final volume of 150 μl in a final volume of 6.0 × 10 ^5, and then cultured in a 37 ° C. incubator for 24 hours. The growth of bacteria was measured at OD600 using an ELISA reader for 24 hours. A. baumannii with pFL02 plasmid used as control We compared the bacterial growth of ATCC 17978 reporter strain and the bacterial growth of reporter strain containing compound. In order to ensure the reliability of the first experimental results, the first experiment and the procedure were the same, and the second experiment was performed once again with the concentration of the compound being only 5 μM.

The results of the growth inhibition effect on the reporter strain are shown in Table 4 below. In the table below,% inhibition is the inhibition of bacterial growth of the reporter strain containing the compound compared to the control. For example, if the% inhibition value is 73%, it means that the compound inhibited the growth of the reporter strain by 73% as compared with the control.

Growth inhibition effect on reporter strain of compounds compound
number rescue At a concentration of 2.5 [mu] M
% inhibition At a concentration of 5.0 μM
% inhibition I

73.92 93.60 II

-7.0 2.94 III

-4.26 15.03 IV

19.81 33.06 V

-22.80 6.20 VI

9.61 24.86 VII

-6.25 11.86 VIII

-10.64 2.74 IX

-18.61 17.87 X

-17.45 7.90 XI

-14.00 -6.37

As can be seen in Table 4 above, it has been found that the compound of formula I significantly inhibits the growth of the reporter strain. On the other hand, all of the other compounds having a chemical structure similar to that of the compound of the formula (I) were found to have no or only insufficient effect of inhibiting bacterial growth of the reporter strain. That is, all of the above compounds can be used in combination with 1-isobenzofuranmethanol group or 5-fluoro-2,4 (1H, 3H) -pyrimidinedione ) Group, it was found that only the compound of the formula (I) containing both of the above two groups is superior in the effect of inhibiting the bacterial growth of the reporter strain.

That is, since the compound having the structure of the following formula (I) inhibits the OmpA promoter with excellent effect, the nptI which is resistant to kanamycin is not expressed, and consequently the reporter strain is resistant to the kanamycin-containing medium And the growth was inhibited.

(I)

On the other hand, since anti-virulence should not inhibit the growth of wild-type strain, the growth inhibition of the compound of formula (I) against the wild-type strain was evaluated according to the following method.

Specifically, wild-type A. baumannii ATCC17978 and reporter strain were inoculated into 10 ml of Luria-Bertani (LB) broth and cultured for 16 hours at 37 ° C with shaking at 150 rpm. The culture broth was centrifuged at 8,000 rpm for 5 minutes, and then the supernatant was removed. The supernatant was resuspended in phosphate-buffered saline (PBS) and diluted to a value of 1.0 at an absorbance of 600 nm. In a 96-well microplate, 50 μl of a dilution of Mueller-Hinton broth (MHB) was added to allow 6.0 × 10 ⁵ bacteria per well. The compound of formula (I) was then stepwise diluted in MHB in wells at a maximum concentration of 30 [mu] M, twice, and 50 [mu] l of the compound was added. At this time, wells containing no compound were added as a control group. 50 μl of MHB without kanamycin was added to the wells containing Wild-type A. baumannii ATCC17978, and 50 μl of MHB prepared so that the final concentration of kanamycin was 50 μg / ml in the well containing the reporter strain was added . Therefore, the total solution is 150 μl in each well. Absorbance values at 600 nm and 0 h were measured immediately using a VersaMax microplate reader. The 96-well plate was incubated at 37 ° C for 24 hours, and the absorbance was measured. The absorbance values were obtained from 24 hours to 0 hours to exclude the effect of the absorbance by the compound itself, and the values were obtained by comparing the respective bacteria with the group not treated with the drug.

The results are shown in Fig.

As shown in FIG. 3, the compound of formula (I) does not inhibit growth of wild-type strain but inhibits growth only to reporter strain, and thus it can be used as anti-virulence agent.

&Lt; Example 3 >

OmpA gene expression inactivation (real time PCR)

Since the compound of the formula (I) inhibits the OmpA gene promoter, expression of the OmpA gene will also be lowered, so that it is desired to confirm this by real-time PCR.

Specifically, the reporter strain cultured overnight in Luria-Bertani (LB) agar plate medium was diluted in PBS until absorbance at 600 nm was 1.0. Ten microliters of the compound solution was added to 980 microliters of Mueller-Hinton broth (MHB) so that the final concentration of the compound of formula I was between 0.625 μM and 10 μM. As a control, 10 μl of DMSO used as a solvent for the compound of formula (I) was added instead of the compound of formula (I). They were cultured at 37 DEG C for 24 hours while shaking at 150 rpm. The total RNA of the bacteria was then extracted using the RNeasy Protect Bacteria Mini Kit from Quiagen. When extracting RNA, a process for DNase treatment by Quiagen was added to prevent gDNA contamination. Total RNA was quantitated using a Bio-Rad nanodrop, and 1 μg of RNA was used as a template and cDNA was prepared using Fermentas oligo dT primer and M-MLV reverse transcriptase. The reverse transcription reaction was performed using a Biorad thermal cycler. For quantitative real-time PCR analysis, primers were designed using Biosystems' Primer Express Software (version 3.0). The primer sequences for OmpA and 16S rRNA PCR were as follows. OmpA (sense primer: 5'-TAT TGC ACT TGC TAC TAT GCT TGT TG-3 ', anti-sense primer: 5'-GTT ACC ATC TTT ACC GCC ATT GT-3'); 16S rRNA (sense primer: 5'-ACT CCT ACG GGA GGC AGC AGT-3 ', anti-sense primer: 5'-TAT TAC CGC GGC TGC TGG C-3'). Real-time PCR was performed using ABI PRISM 7500 Real-Time System instrument using Biosystems Power SYBR Green PCR Master Mix reagent. The polymerization reaction was carried out at 95 ° C for 15 minutes, followed by 15 cycles of 95 ° C for 15 seconds and 60 ° C for 1 minute, followed by 15 cycles at 95 ° C, 1 minute at 60 ° C, and 15 cycles at 95 ° C It took a melting process for a second. The amplification specificity was determined by analyzing the melting curve. OmpA mRNA was quantified using 16S rRNA mRNA levels, and fold change values were calculated using ΔΔCt values.

The results are shown in Fig.

As shown in FIG. 4, the compound of formula I inhibited the expression of the OmpA gene by 38% at a concentration of 2.5 μM and by 61% at a concentration of 10 μM as compared to an untreated control.

<Example 4>

Inhibition of OmpA protein expression

After the OmpA gene is expressed, it is made into the OmpA protein in the cytoplasm and travels to the bacterial outer membrane. Therefore, the compound of formula (I) was treated to isolate only the outer membrane protein of the bacterium, and the amount of OmpA protein was reduced.

Specifically, wild-type A. baumannii ATCC17978 was inoculated into 10 ml of Luria-Bertani (LB) broth and cultured for 16 hours at 37 ° C with shaking at 150 rpm. The culture broth was centrifuged at 8,000 rpm for 5 minutes, and then the supernatant was removed. The supernatant was resuspended in phosphate-buffered saline (PBS) and diluted to a value of 1.0 at an absorbance of 600 nm. 20 μl of the diluted bacterium and 10 μl of the compound solution of 2.5 μM final concentration of the compound of formula (I) were inoculated into Mueller-Hinton broth (MHB) to make a final 20 ml broth. As a control, 10 μl of DMSO used as a solvent for the compound of formula (I) was added instead of the compound of formula (I). They were cultured at 37 DEG C for 24 hours while shaking at 150 rpm. The culture broth was centrifuged at 8,000 rpm for 5 minutes, and then the supernatant was removed. Then, the supernatant was resuspended in 10 mM HEPES buffer (pH 7.4), the cells were disrupted by an ultrasonic disrupter, and centrifuged at 8000 rpm for 5 minutes. And large lumps were removed and the supernatant was obtained. The supernatant was centrifuged at 100,000 xg at 100,000 xg for 1 hour, and the precipitate was suspended in HEPES buffer containing 2% sodium lauryl sarcosinate. The supernatant was allowed to stand at room temperature for 30 minutes to remove endogenous proteins, and then 100,000 xg to obtain pure outer membrane protein. The outer membrane protein thus obtained was stained with Coomasie Brilliant Blue at 10% gel and subjected to sodium-dodecyl sulfate-gel electrophoresis (SDS-PAGE). In the same manner as above, SDS-PAGE protein was electrotransferred to nitrocellulose membrane at 10% gel and reacted with polyclonal anti-mouse OmpA antibody and horseradish peroxidase-conjugated secondary antibody. Proteins that react with antibodies were developed using an enhanced chemiluminescence system. Differences in expression of OmpA protein in bacteria treated with the control and the compound of formula (I) were measured using a densitometer.

The results are shown in Fig.

As shown in FIG. 5, the compound of formula I was found to inhibit the expression of the OmpA protein by about 29% as compared to the untreated control.

&Lt; Example 5 >

Inhibition of biofilm formation

OmpA protein is a protein that has various functions such as bacterial formation of pathogenic bacteria, cell attachment and invasion, and cell death induction. Therefore, if the OmpA protein is not properly expressed, this pathogenicity should be reduced so that it can be used as an anti-virulent agent. Therefore, the present inventors evaluated whether the compound of formula (I) inhibiting the promoter of OmpA inhibited the biofilm formation of bacteria.

Specifically, reporter strain cultured overnight in Luria-Bertani (LB) broth was adjusted to 2.0 at optical density (OD) at 600 nm and diluted 200-fold with LB broth. 297 μl of the diluted bacterial suspension was added to 5 ml of polystyrene tube, and 3 μl of the compound of formula (I) was added thereto so as to have a final concentration of 0.625 μM to 5 μM. As a negative control, 3 μl of DMSO used as a solvent of the compound of formula (I) was added instead of the compound of formula (I), and 100 μM of virstatin was added as a positive control. The bacterial solution was subjected to stationary culture at 30 DEG C for 24 hours. After incubation, the absorbance at 600 nm was measured to confirm the growth of airborne bacteria. Airborne bacteria were removed and the tubes were washed once with 300 μl of PBS. Bacteria forming the biofilm were stained with 0.1% crystal violet solution for 15 minutes at room temperature, washed twice with distilled water for 3 minutes, extracted with 95% ethanol from the tubes, And the absorbance was measured at 600 nm to measure biofilm formation ability.

The results are shown in Fig.

As shown in FIG. 6, it was confirmed that the compound of formula I significantly inhibited the formation of biofilm (bacterium) of Asnithobacter baumannii at a concentration of 1.25 μM. On the other hand, at the concentration of 5 μM, the effect of inhibiting biofilm formation was better than that of virtatin, which was used as a positive control.

&Lt; Example 6 >

Cytotoxicity Assessment

As shown in the above examples, the compound of formula (I) of the present invention exhibited excellent anti-virulence effect. However, even if it is a compound showing excellent activity, it is difficult to develop it as a drug if it is cytotoxic. Therefore, The cytotoxicity of compounds of formula I was evaluated.

Specifically, in order to confirm the cytotoxicity of the compound of formula (I) in human cells, the final concentration of the compound of formula (I) was treated to 1, 10, 100, 1000, 10000 nM U937 cells as human monocytic cell line. First, 4.0 x 10 ⁴ U937 cells per well were added to a 96-well plate, and the compound of formula (I) was treated by concentration. Cell cultures were incubated in a CO ₂ incubator at 37 ° C for 24 hours. After incubation for 3 hours with WST1 solution (Takara), the cell viability was measured by absorbance at 450 nm, and the absorbance was compared with the untreated group.

The results are shown in Fig.

As shown in Fig. 7, it was confirmed that no toxicity was shown even at 10 [mu] M.

< Example  7>

A. baumannii  Influence on the mouse model infected with the bacteria (in vivo)

Based on the embodiments of the compound [formula I] A. baumannii In order to confirm the effect on the mice infected with the bacteria, the following experiment was carried out.

<7-1> Test strains

A. baumannii the mouse in vivo distribution of the pathogen-infected bacteria, such as to confirm the killing of bacteria by medication A. fluorescence gene into the chromosome of baumannii type strain (ATCC 17978) lux gene was introduced. Fluorescence expression of the fluorescently expressed strain was confirmed by using InVivo Imaging System (IVIS) (Caliper Life Sciences, Massachusetts, USA). The results are shown in Fig.

&Lt; 7-2 > Mouse model of immune status in which normal immunity mouse and callus neutrophils are removed

8-week-old female Balb / c mice (Hyochang Science Co., Korea) were purchased and stabilized for one week before the experiment. The mice were then treated with cyclophosphamide (Sigma, USA) diluted in phosphate-buffered saline (PBS) at a dose of 150 mg / kg (3 mg for 20 g mice) And 1 day prior to intravenous injection twice to remove neutrophil to create a neutrophil-null mouse model. The mice were then infected with A. baumannii in a mouse under normal normal conditions and in a neutrophilic condition with no neutrophils removed. These results suggest that A. baumannii infections in hospitals mainly occur in immunocompromised patients such as intensive care units and patients with underlying diseases.

<7-3> In the mouse model A. baumannii  Fungal infection

The normal immunized mouse of Example 7-2 and the neutrophil-removed mouse were infected with the fluorescence-expressing A. baumannii bacteria prepared in Example 7-1. Bacteria were inoculated into LB medium (Luria-Bertani broth) one day before the mice were infected with the bacteria and cultured for 16 hours at 37 ° C. After centrifuging the bacterial culture, the supernatant was discarded and only germs were obtained. The bacteria were diluted with 100 ml of phosphate-buffered saline so that the absorbance was 1.0 at a wavelength of 600 nm. The diluted strains were centrifuged again and the supernatant was discarded. The bacterial strain was concentrated to 5 times and 20 ml of phosphate buffered saline Respectively. The mice were then injected intraperitoneally with 200 μl of 2.0 × 10 ⁹ cells in the normal and neutrophil-free conditions. After 1 hour of infection, the cells were injected intraperitoneally with 200 [mu] l of phosphate buffered saline (0.625, 1.25, 2.5, 5, 10, 20, 40 [ 200 μl of saline was intraperitoneally injected.

<7-4> Effect of compound of formula I on survival rate in mouse model

The normal immunized mouse of Example 7-2 and the mouse having the neutrophil-removed state were subjected to fluorescence expression A. baumannii After infecting the bacterium 1 hour later, the compound of formula I was injected into the abdominal cavity at a dose of 200 μl by calculating the drug concentration according to the weight of the mouse. The concentration of the compound in the normal immune state mouse was set to 10 μM, and the mice were injected once with 0.625, 1.25, 2.5, 5, and 10 μM of the compound concentration in the neutrophil-removed mouse. The survival rate of the mice was observed up to 72 hours per day.

The results are shown in Table 5 and Table 6.

As shown in the following Tables 5 and 6, the survival rate of the mice was 50% when the mice were infected with the normal immune status mouse and the compound was not treated, whereas when the compound of the formula I was treated, the survival rate was increased to 76.9% . In addition, the survival rate of the mice infected with the neutrophil-removed mice and treated with the compound of the formula I was 23.5%, while the survival rate of the mice treated with 10 μM of the compound of the formula I increased to 84.6% the compound of I A. baumannii The mice were found to be effective in both normal immune and neutrophil-infected mice.

&Lt; 7-5 > Effect of Compound (I) on In vivo Bacterial Distribution in Mouse Model

Fluorescent expression of A. baumannii was infused into the peritoneal cavity and 1 hour later, 10 μM of the compound of formula I was intraperitoneally injected. The concentrations of the compounds were injected with 10 μM of A. baumannii infected mice. The in vivo distribution of the infecting bacteria was confirmed by IVIS imaging system 1 hour and 24 hours after the drug treatment.

The results are shown in Fig.

As shown in Fig. 9, fluorescence expression A. baumannii In the group of mice (infected group) in which the bacteria were infected with peritoneal fluid and the compound of the formula (I) was not treated, most of the bacteria died after 24 hours of peritoneal infection and 3 mice died after 24 hours, In the mouse group treated with the compound of the present invention, the fluorescence was not expressed in 2 mice after 24 hours of the fungal infection, and only one mouse resulted in pneumonia and died.

This allows the compound of formula I to be reacted with A. baumannii It was confirmed that there was an effect of suppressing the pathogenicity of the bacteria.

&Lt; Example 8 >

Evaluation of Mouse Toxicity of Compound (I)

To determine whether high concentrations of the compounds of formula (I) cause toxicity in vivo in mice, high single dose toxicity studies were conducted.

Female Balb / c mice at 8 weeks of normal immunosuppressive state and callus decontamination state were intraperitoneally injected with a compound of formula I at a concentration of 250, 500 μM. As a control, since the compound of formula (I) was dissolved in DMSO, the same volume of DMSO entered when dissolved at a concentration of 500 μM was administered once per abdominal cavity. Three mice were used for each group, and the mice injected with the compound of the formula (I) at a high concentration were observed for 10 days to confirm the survival rate of the mice.

As a result, all the mice in the experimental group including the control group were found to survive for 10 days, and it was confirmed that the compound of the formula (I) was not toxic to the mice.

&Lt; Example 9 >

Inhibition of OmpA gene expression and inhibition of biofilm formation

The following experiment was conducted to examine whether the compounds of formula (I) inhibit OmpA gene expression and inhibit biofilm formation when tested for clinical isolates.

As a clinical isolate, eight strains of A. baumannii were isolated from Kyungpook National University Hospital between 2011 and 2014, and strains were purchased from Kyungpook National University Hospital. The strains were grown on MacConkey agar plate or Luria-Bertani (LB) medium.

<9-1> ompA gene expression inactivation (real time PCR)

Real-time PCR was performed in the same manner as described in Example 3 above.

Specifically, clinical isolates cultured overnight in Luria-Bertani (LB) agar plate medium were diluted in PBS until absorbance at 600 nm was 1.0. Ten microliters of the diluted solution and 10 microliters of the compound solution to give a final concentration of 10 microliters of the compound of formula I were inoculated into 980 microliters of Mueller-Hinton broth (MHB). As a control, 10 μl of DMSO used as a solvent for the compound of formula (I) was added instead of the compound of formula (I). They were cultured at 37 DEG C for 24 hours while shaking at 150 rpm. The total RNA of the bacteria was then extracted using the RNeasy rotect Bacteria Mini Kit from Quiagen. Total RNA was quantitated using nanodrop and cDNA was prepared using oligo dT primer and MMLV reverse transcriptase using 1 μg of RNA as template. Quantitative real-time PCR analysis was performed and the same primers as described in Example 3 above were used. In the same manner as in Example 3, the polymerization reaction was carried out at 95 ° C. for 15 minutes, followed by repeating a cycle of 95 ° C. for 15 seconds and 60 ° C. for 1 minute for 40 cycles, followed by 95 ° C. for 15 seconds, 1 min and 95 ° C for 15 sec. The amplification specificity was determined by analyzing the melting curve. OmpA mRNA was quantified using 16S rRNA mRNA levels, and fold change values were calculated using ΔΔCt values.

The results are shown in Fig.

Compounds of the formula I (62520) decreased statistically in 6 out of 8 weeks (1321, 1325, 1606, 2140, 2204, 2300) of OmpA gene expression compared to the untreated control, , 1539 strain), the amount of OmpA gene expression was increased or increased more than the control group.

<9-2> Ability to inhibit biofilm formation

The ability to form biofilm was measured in the same manner as in Example 5 above.

Specifically, ATCC 19606 cultured overnight in LB medium and clinical isolates of 8 weeks were adjusted to 2.0 in optical density (OD) at 600 nm and diluted 200-fold in LB medium. 297 μl of the diluted bacterial suspension was added to 5 ml of a polystyrene tube, and 3 μl of the compound of the formula (I) was added to a final concentration of 10 μM. As a negative control, 3 [mu] l of DMSO used as a solvent for the compound of formula (I) was added instead of the compound of formula (I). The bacterial solution was subjected to stationary culture at 30 DEG C for 24 hours. After incubation, the absorbance at 600 nm was measured to confirm the growth of airborne bacteria. Airborne bacteria were removed and the tubes were washed once with 300 μl of PBS. Bacteria forming the biofilm were stained with 0.1% crystal violet solution for 15 minutes at room temperature, washed twice with distilled water for 3 minutes, extracted with 95% ethanol from the tubes, And the absorbance was measured at 600 nm to measure biofilm formation ability.

The results are shown in Fig.

The compound of formula I (62520) was isolated from a clinical isolate of A. baumannii at a concentration of 10 μM for 6 weeks (1321, 1325, 1606, 2140, 1472, 1539 strains) (ATCC 19606) and 2 weeks (2204, 2300 strains) were not statistically significant, but the compound of formula I inhibited biofilm formation ability Respectively. Also, it was confirmed that the compound of formula (I) inhibits the biofilm-forming ability of the strain of 2 weeks (1472 strains, 1539 strains) in which OmpA gene expression was not inhibited by treatment with the compound of formula (I).

The compound of formula (I) of the present invention inhibits the expression of the gene and protein of the outer membrane protein A, which is a pathogenic protein, without showing a direct bactericidal effect against the pathogenic bacteria, and thus the anti-virulence agent . In addition, it is possible to inhibit the formation of a biofilm closely related to the overexpression of OmpA, and thus it can be effectively used for the prevention and treatment of infectious diseases caused by infection with pathogenic bacteria.

Claims (9)

An anti-virulence composition for Acinetobacter baumannii comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.
(I)

delete The composition according to claim 1, wherein the compound inhibits the expression of the outer membrane protein A (OmpA) gene, which is a virulence substance, to exhibit an antinuclear activity.
A pharmaceutical composition for the prevention and treatment of infectious diseases caused by Acinetobacter baumannii comprising the composition according to claim 1 or 3 as an active ingredient.
The pharmaceutical composition according to claim 4, wherein the infectious disease is selected from the group consisting of pneumonia, urinary tract infection, meningitis, endocarditis, war wound and bacteremia.
delete A composition for inhibiting biofilm of Acinetobacter baumannii comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.
(I)

delete The composition according to claim 7, wherein the compound inhibits biofilm formation by inhibiting expression of an outer membrane protein A (OmpA) gene.

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