KR101135179B1 - A ANTI-BACTERIAL COMPOSITION COMPRISING FabI-INHIBITORY VINAXANTHONE - Google Patents

Abstract

The present invention relates to a compound having an antimicrobial activity by inhibiting FabI, a new antibiotic target, and specifically, an antimicrobial composition represented by Formula 1, a penicillium sp. Producing the compound, and (a ) Penicillium Culturing the strain, (b) extracting the culture solution with acetone and then extracting with ethyl acetate; And (c) performing chromatography on the extract to obtain a compound of the present invention.

Vinasanson, vinaxanthone, Fab I inhibitor, MRSA, QRSA, antibacterial, antibiotic resistance

Description

Antimicrobial composition containing FAA inhibitor Subasonson {A ANTI-BACTERIAL COMPOSITION COMPRISING FabI-INHIBITORY VINAXANTHONE}

The present invention relates to a new concept antimicrobial compound that inhibits FabI, a novel antibiotic target, specifically, an antimicrobial composition represented by Formula 1, a penicillium strain (Penicillium sp.) Producing the compound, and the compound It is about how to.

Direct or indirect damage by pathogenic microorganisms causes many economic, environmental and medical problems. Especially in the food industry, the loss due to corruption in the food distribution process, the harmful effects of the human body caused by the use of excessive chemical pesticides on agricultural products in the agricultural industry, and the emergence of antibiotic resistant strains due to the abuse of antibiotics Causes many problems. Although rational antibiotic therapy in the treatment of infectious diseases is an important factor in determining the patient's prognosis, the emergence of such antibiotic resistant strains has made it difficult to select effective antibiotics unlike the past.

For example, after the treatment of methicillin-resistant S. aureus (MRSA), which is only treated with vancomycin, the last treatment for staphylococcus, the most common cause of human infection, has been questioned since the 1970s, In 1988, vancomycin resistant Enterococcus (VRE) was first discovered in Europe, and in late 1990, vancomycin intermediate-resistant S. was found in Japan, the United States, France and Korea . aureus , VISA). Furthermore, vancomycin-resistant Staphylococcus aureus (VRSA), a highly resistant to vancomycin, was reported for the first time in the world by the Centers for Disease Control in 2002, so the so-called "super bacteria" could be spread. This is very high. Accordingly, the problem of antibiotic resistance has emerged as a global crisis, and the development of a new concept of antibiotics is urgently required.

Meanwhile, as a result of research into hospital microbial genomes, which began in the mid-1990s, new antibiotic targets were discovered, opening up the possibility of new antibiotic development. Among these new antibiotic targets discovered and validated using microbial genome information, enoyl-ACP reductase is essential for the growth of microorganisms, which are responsible for the end of the elongation phase of the fatty acid synthesis cycle. As an enzyme, it is present in both Gram-positive and negative bacteria and has attracted attention as a new antibiotic target because of its very low homology to humans.

Enoyl reductase contains three isoforms, Fab I, Fab K, and Fab L, of which Fab I is Staphylococcus. aureus ) and E. coli are common to most major pathogenic bacteria. Therefore, new Fab I inhibitors can be developed into a wide range of antimicrobial therapeutics.

Accordingly, the present inventors have made intensive efforts to develop Fab I inhibitors from natural products such as microorganisms and plants, and as a result, the Vinasanson compound isolated from fungi Penicillium sp F131 isolated from domestic soil strongly inhibits Fab I. As well as confirming that it has antibacterial activity, the present invention was completed.

An object of the present invention is to provide a composition for antibacterial of a novel mechanism of action.

Another object of the present invention to provide a composition for inhibiting Fab I activity comprising the antimicrobial composition.

Still another object of the present invention is to provide a penicillium (Penicillium sp.) Strain producing the antimicrobial composition.

Another object of the present invention is to (a) culturing the penicillium strain (Penicillium), (b) extracting the culture solution obtained from the step (a) with acetone, followed by extraction with ethyl acetate; And (c) performing chromatography on the extract obtained from step (b) to obtain a compound of the present invention.

As one embodiment, the present invention relates to an antimicrobial composition comprising a compound represented by the following formula (1), an isomer thereof, a derivative thereof, or a pharmaceutically acceptable salt thereof.

The IUPAC name of the compound represented by Formula 1 is 5,7-diacetyl-6- (5-carboxy-6,7-dihydroxy-4-oxochromen-3-yl) -2,3-dihydroxy -9-oxoxanthene-1-carboxylic acid (5,7-diacetyl-6- (5-carboxy-6,7-dihydroxy-4-oxochromen-3-yl) -2,3-dihydroxy-9-oxoxanthene- 1-carboxylic acid, collectively referred to as vinaxanthone, and is a yellow powdery compound having a molecular weight of 576.418240 g / ml and a molecular formula C ₂₈ H ₁₆ O ₁₄ . The compound, 'vinaxanthone', can be synthesized from vanillin, a kind of aromatic hydrocarbon compound, and is known as a phospholipase C inhibitor, but the antimicrobial activity of the compound is still unknown. Has not been reported.

Preferably the compounds of the present invention include isomers, derivatives or pharmaceutically acceptable salts thereof. In the present invention, the 'isomer' refers to a relationship between compounds having the same chemical formula but not identical, and the kind of such isomers includes structural isomers, geometric isomers, optical isomers, and geometric isomers. A stereoisomer means a compound having the same chemical composition but different in terms of the arrangement of atoms or groups in space, and an optical isomer (enantiomer) means two stereoisomers of a compound having mirror images that do not overlap each other, and diastereomers Isomers refer to stereoisomers that have two or more asymmetric centers and whose molecules are not mirror images of each other.

The term "pharmaceutically acceptable salts" in the present invention means the relatively non-toxic inorganic and organic acid addition salts of the compounds.

In the present invention, "antibacterial" means a property that resists microorganisms, such as bacteria and fungi, more specifically, antibiotics and the like to inhibit the growth of bacteria. When using the antimicrobial composition containing the binasanson of the present invention, it was confirmed that the antimicrobial activity in the individual by inhibiting the growth and growth of bacteria.

In addition, the compounds of the present invention may exhibit an antimicrobial effect against antibiotic resistant bacteria. As used herein, the term 'antibiotic resistance' refers to resistance of antibiotics to antibiotics as a result of continuous use of drugs to treat or prevent any disease and its complications, or any bacterial disorders and complications thereof. Means that. Examples of such antibiotics include cephalosporins, quinolones and fluoroquinolones, penicillins, beta lactamase inhibitors, carbepenems, monobactams, macrolides and lincosamines, glycopeptides, rifampins, oxazolidinones, tetracyclines, aminos Glycosides, streptogramine and sulfonamides, and the like. The antibiotic resistant bacteria exhibit resistance even when treated with the antibiotics listed above, so that the disease is maintained continuously in the individual.

Preferably, when using the composition of the present invention, it can exhibit excellent antimicrobial therapeutic effect against antibiotic resistant bacteria as well as general antibacterial activity.

The term "treatment" in the present invention refers to local or systemic effects in animals, in particular mammals, and more particularly in humans, caused by pharmacologically active substances, and can be used to diagnose, treat, ameliorate, treat or prevent a disease or Any substance intended to be used in the enhancement of the desired physical or mental development and / or condition of an animal or human.

From a preferred embodiment of the present invention, in vitro antibacterial experiments on the compound of the present invention, vinaxanthone, the vinaxanthone compound of the present invention is Staphylococcus aureus, MRSA (methicillin-resistant staphylococcus aureus) or QRSA (Quinolone-resistant Staphylococcus aureus) strains were confirmed to exhibit antimicrobial activity (Example 5).

In the present invention, 'Staphylococcus aureus' is one of the bacteria widely distributed in the natural world, there are many kinds, and not only food poisoning, but also causing the purulent diseases such as purulent, otitis media and cystitis of the skin. In Korea, after the Salmonella and enteritis Vibrio bacteria, the bacteria causing a lot of food poisoning, among them, Staphylococcus aureus is known as the major bacteria causing food poisoning.

'MRSA (methicillin-resistant staphylococcus aureus)' of the present invention refers to a bacterium that has become resistant to antibiotics (methicillin) as a type of Staphylococcus aureus, and 'Qinolone-resistant Staphylococcus aureus' is a quinolone. Refers to bacteria that show resistance.

The compounds of the present invention can be synthesized according to methods commonly used in the art, and preferably natural compounds produced from strains can also be obtained. Although it can be obtained from the penicillium sp. (Penicllium sp.), By the above strains are not limited to the type of strain from which the compound of the present invention can be obtained. Preferably it can be produced from Penicillium F131, more preferably the compound of the present invention can be obtained from Accession No. KCTC 11462BP strain.

As another aspect, the present invention relates to a composition for inhibiting Fab I activity comprising a compound represented by the formula (1), an isomer thereof, a derivative thereof or a pharmaceutically acceptable salt thereof. Preferably, the composition of the present invention can inhibit Fab I enzyme activity present in Staphylococcus aureus MRSA (methicillin-resistant staphylococcus aureus) or QRSA (Quinolone-resistant Staphylococcus aureus).

In the present invention, the term 'Fab I (acyl-carrier protein) reductase' refers to an enoyl-acyl carrier at the end of four reactions included in each cycle of fatty acid biosynthesis of bacteria. Refers to a bacterial enzyme believed to function as a protein (ACP) reductase. The enzyme is widely distributed in bacteria and plants and is a protein enzyme necessary for synthesizing the lipids constituting the bacterial biofilm. Inhibiting Fab I enzymes can produce antibiotic and antimicrobial effects and can be used as a 'target protein' for the treatment of resistant bacteria. Triclosan '. This 'Fab I' is known to be present in the strain of superbacterial resistant bacteria, such as superbacterial staphylococcus (methicillin-resistant S. aureus , MRSA). In a preferred embodiment of the present invention it was confirmed that the compound has an inhibitory activity against Fab I (Example 4).

As a preferred embodiment, the present invention may further include a pharmaceutically acceptable carrier in the compound of the present invention.

The term "pharmaceutically acceptable carrier" in the present invention refers to liquid or solid fillers involved in the transport or transport of any subject composition or component from one organ or part of the body to another organ or part of the body. Refers to a pharmaceutically acceptable material, composition or vehicle, such as a diluent, excipient, solvent or encapsulating material, wherein the composition of the present invention comprises a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredients described above for administration. It may further include. The carrier, excipient and diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, Polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the compositions of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral dosage forms, external preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. have. Specifically, when formulated, it may be prepared using conventional diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like with the compound of Formula 1. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include, but are not limited to, suspending agents, solvents, emulsions, syrups, and the like, and various excipients, such as wetting agents, sweeteners, fragrances, It can be prepared by adding a preservative or the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the compositions of the present invention may be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage may be based on the condition and weight of the patient, disease Depending on the degree, drug form, route of administration, and duration, it may be appropriately selected by those skilled in the art. It may be administered once or several times daily as needed, and used alone or in combination with methods using surgery, hormone therapy, drug treatment and biological response modifiers for the prevention or treatment of pathogenic bacteria and resistant bacteria. Can be. Preferably the composition of the present invention may be administered at 0.1 to 50 mg / kg, more preferably at 5 to 30 mg / kg. The most preferred dosage of the composition of the present invention is about 30 mg / kg. As described above, the route of administration of the invention is also not limited, but may preferably be administered parenterally, and more preferably by subcutaneous injection method.

As another aspect, the present invention relates to a method for preparing a compound of the present invention, preferably (a) Penicillium sp Culturing the strain; (b) extracting the culture solution obtained from step (a) with acetone, followed by extraction with ethyl acetate; And (c) chromatographing the extract obtained from step (b).

In the present invention, penicillium strain culture for producing a compound of the present invention may be cultured in a medium containing nutrients that can be used by conventional microorganisms. As a nutrient source of the strain can be used without limitation, a nutrient source commonly used in the art, preferably a known nutrient source used in the conventional culture of the fungus can be used. For example, as a carbon source, glucose, starch syrup, dextrin, starch, molasses, animal oil, vegetable oil, etc. may be used, and as nitrogen sources, bran, soybean meal, wheat, malt, cottonseed gourd, fishmeal, corn stew ricker, gravy, yeast extract, sulfuric acid Ammonium, sodium nitrate, urea and the like can be used. If necessary, it is very effective to add salt, potassium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other inorganic salts that promote the formation of ions. As a culture method, shaking culture or political culture is possible under aerobic conditions.

The culture temperature is slightly different depending on the conditions when the culture in each of the above conditions, it is usually suitable to incubate at 20 to 37 ℃, preferably can be cultured at 26 to 30 ℃. In addition, the culture period can also be cultured for a known period used in the art, the period can be adjusted as necessary. Preferably, shaking and stationary cultures can be cultured for 4 days to 7 days, and the yield of the compound of the present invention was confirmed to reach the highest value during the culture period.

From one preferred embodiment of the present invention, culturing the strain under the conditions as described above yields the compound of the present invention (Examples 1 and 2), and the compound of the present invention is not only in the culture medium but also in the cells. Since it may exist, it can be efficiently separated and purified according to the following method for effective extraction.

Method of extraction and purification of the compound can be used without limitation the methods commonly used in the art, it is obvious that the amount and yield can be adjusted by changing the type of medium, culture conditions, extraction purification method, etc. as necessary Do. In a preferred embodiment of the present invention, the extraction was performed using acetone and ethyl acetate, the specific method is as follows.

After adding an organic solvent such as acetone to the culture solution and mycelium and stirring to extract the active ingredient from the cell, the acetone was evaporated and the solvent extracted with ethyl acetate, and then the ethyl acetate solvent layer containing the active ingredient was concentrated under reduced pressure. After removal of ethyl acetate, acetonitrile: water was subjected to ODS (ODS) chromatography using a solvent of 4: 6, 5: 5, 6: 4, 7: 3. The obtained active fractions were subjected to sephadex LH-20 chromatography again with methanol as a solvent, and finally purified by HPLC to obtain the pure material of the present invention (Example 3). The present inventors have found that using such a method, it is possible to effectively extract and purify vinasanson from penicillium F131 strain.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

The compounds of the present invention can be usefully used for the treatment of superbacteria by strongly inhibiting Fab I enzyme, having strong antimicrobial activity against antibiotic resistant bacteria, and particularly showing antimicrobial activity against MRSA.

Example 1.  Penicillium F131  Isolation of strain

The present inventors isolated bacteria, fungi, actinomycetes from the soil of the country, and screened strain F131 strain having strong Fab I inhibitory antimicrobial activity in order to screen strains producing substances having Fab I inhibitory activity. The F131 strain producing vinaxanthone was identified as penicillium sp. As a result of comparing an ITS sequence with an NCI sequence database. The present inventors deposited the strain to the Genetic Resource Center of Korea Biotechnology Research Institute on February 03, 2009 (Accession Number: KCTC 11462BP).

Example 2.  Penicillium sp.  Culture of F131 Strains

In order to cultivate the F131 strain, a medium containing 0.3% yeast extract, malt extract 0.3%, tryptone 0.5%, and glucose 1% before sterile sterilization was used as a seed medium. The pH was adjusted to 5.5 and used. A 100 ml Erlenmeyer flask containing 20 ml of the seed medium was sterilized at 121 ° C. for 20 minutes, inoculated with platinum from a slope culture tube of F131 strain, shake-cultured at 28 ° C. for 3 days, and then the primary seed culture solution. Used as. Then a 500 ml Erlenmeyer flask containing sterile medium containing 0.3% yeast extract, 0.3% malt extract, 0.5% tryptone and 1% glucose. (48) were inoculated with the seed culture solution and shaken at 28 ° C for 7 days.

Example 3. Isolation and Purification of Compounds of the Invention

The acetone extract of the culture medium and mycelium cultured in Example 1 was solvent extracted three times with ethyl acetate. The ethyl acetate solvent layer containing the active ingredient thus obtained was concentrated under reduced pressure to remove ethyl acetate, followed by acetonitrile: water using a solvent of 4: 6, 5: 5, 6: 4, 7: 3. SOD chromatography was performed. The active fraction thus obtained was subjected to Sephadex LH-20 chromatography again using methanol as a solvent, and finally purified by HPLC to obtain the pure material of the present invention. The active fraction was subjected to HPLC (YMC ODS 150 × 20 mm, Flow rate: 7 mL / min, UV 300 nm) using 25% acetonitrile containing 0.01% TFA as a mobile phase to obtain a vinoxanthone compound. .

Physicochemical properties and chemical structure of the compound of the present invention purified as described above are as follows (Formula 1).

1) Appearance of material: Yellow powder

2) Mass Spectrometry Spectrum (ESI-MS): 577.5 (M + H) ⁺

3) Molecular formula: C ₂₈ H ₁₆ O ₁₄

5) Nuclear Magnetic Resonance (NMR) Absorption Spectrum: Hydrogen nuclear magnetic resonance spectrum data measured using DMSO-d6 as a solvent and tetramethylsilane (TMS) as reference materials are as follows.

¹ H-NMR (300 MHz, DMSO- d ₆ ): 12.75 (2H, brs, 11,9'-OH), 11.45 (2H, brs, 2,6'-OH), 9.44 (2H, brs, 3, 7'-OH), 8.53 (1H, s, H-8), 8.17 (1H, s, H-2 '), 6.96 (1H, s, H-8'), 6.94 (1H, s, H-4 ), 2.55 (3H, s, H-13), 2.53 (3H, s, H-15)

¹³ C-NMR (75 MHz, DMSO- d ₆ ): 201.1 (C-12), 199.2 (C-14), 172.9 (C-4 '), 172.6 (C-9), 167.4 (C-11), 167.3 (C-9 '), 154.2 (C-3), 152.6 (C-2'), 152.8 (C-7 '), 152.1 (C-10 _a ), 150.7 (C-8' _a ), 150.3 ( C-4a), 141.7 (C-6 '), 141.1 (C-2), 136.3 (C-7), 133.4 (C-5), 132.6 (C-6), 126.3 (C-8), 120.9 ( C-3 '), 120.5 (C-8a), 119.8 (C-5'), 119.6 (C-1), 112.4 (C-4a), 110.0 (C-9a), 102.3 (C-4), 102.4 (C-8 '), 32.1 (C-13), 29.1 (C-15).

Example 4. Fab I Inhibitory Activity of Compounds of the Invention

In order to confirm Fab I inhibitory activity of the compounds according to the present invention, enzyme titer assays were performed as follows. Ward's method (Biochemistry 38, 12514 (1999)) was modified and used to measure Fab I titers. Staphylococcus aureus Fab I prepared by genetic recombination technology was used. The titer was measured in 50 mM sodium phosphate buffer (pH 7.5), and 400 μM of trans-2-octennoyl N-acetylcysteamine thioester and 200 μM of NADPH were used as a substrate. And Fab n was used 150 nM. After adding the enzyme and reacting at room temperature for 60 minutes, the absorbance decrease of NADPH was measured at 340 nm using a UV-spectrometer, and the results are shown in FIG. 2.

Example 5 In vitro Antimicrobial Activity of Compounds of the Invention

When culturing the test strains were cultured in MHB (Mueller Hinton broth) and the antimicrobial activity was measured by broth microdilution. After diluting the test culture incubated overnight to 2 x 100,000 / ㎖ and dispense 100 μl per well in a 96 well plate, and then gradually fold the compound from a concentration of up to 128 ㎍ / ㎖ Diluted treatment. The compound was diluted in DMSO (dimethylsulxoside) and the experiment was carried out with the concentration of DMSO adjusted to about 1/100. After incubation for 20 hours, the growth of bacteria was examined by measuring the OD value at 650 nm. The minimum concentration of compound that completely inhibited the growth of bacteria was determined by MIC.

Antimicrobial Activity of Vinaxanthone Strain vinaxanthone Oxacillin Norfloxacin One S.aureus 503 32 0.25 One 2 S. aureus KCTC 1916 (209) 32 0.25 0.25 3 S.aureus RN 4220 32 0.25 One 4 S.aureus MRSA CCARM 3167 32 500 8 5 S.aureus MRSA CCARM 3506 32 500 One 6 S.aureus QRSA CCARM 3505 32 0.5 250 7 S.aureus QRSA CCARM 3519 32 0.5 125 8 Bacillus subtilis KCTC 1021 32 0.25 0.25 9 Bacillus cereus KCTC 1661 4 0.125 0.25

As shown in Table 1, the vinaxanthone compound of the present invention showed antimicrobial activity against Staphylococcus aureus, and MIC was 32 µg / ml. In particular, it was confirmed that oxacillin or nofloxacin had no antimicrobial activity against the superbacterial MRSA strain and the quinolone resistant Staphylococcus aureus strain, but vinasone showed the same antimicrobial activity.

The vinaxanthone compound according to the present invention exhibits excellent Fab I inhibitory activity and exhibits antimicrobial activity against MRSA, and thus can be usefully used for the treatment of superbacteria.

1 is a diagram showing the chemical formula of vinaxanthone.

Figure 2 is a graph showing the inhibitory activity against Fab I of vinaxanthone.

Claims (7)

An antimicrobial composition comprising a compound represented by Formula 1, an isomer thereof, or a pharmaceutically acceptable salt thereof. [Formula 1]

According to claim 1, wherein the composition is antimicrobial composition having antimicrobial activity against at least one bacteria selected from the group consisting of MRSA (methicillin-resistant staphylococcus aureus) and QRSA (Quinolone-resistant Staphylococcus aureus). The antimicrobial composition of claim 1 wherein the composition further comprises a pharmaceutically acceptable carrier. Fab I (enoyl-ACP [acyl-carrier protein] reductase) activity comprising a compound represented by the formula (1). [Formula 1]

The Fab I according to claim 4, wherein Fab I inhibits Fab I enzyme activity of at least one bacterium selected from the group consisting of MRSA (methicillin-resistant staphylococcus aureus) and QRSA (Quinolone-resistant Staphylococcus aureus). Composition for inhibiting activity. (a) culturing Penicillium sp. F131 strain having accession number KCTC 11462 BP; (b) extracting the culture solution obtained from step (a) with acetone, followed by extraction with ethyl acetate; And (c) performing chromatography on the extract obtained from step (b) to obtain a compound represented by the following Chemical Formula 1; Method of producing a composition for antimicrobial comprising a. [Formula 1]

delete

Images