KR20130029952A - Novel compound and antibiotic composition comprising the same - Google Patents

Abstract

PURPOSE: A compound is provided to have an excellent antibiotic activity to various organic materials which have resistance to β-lactam antibiotics. CONSTITUTION: A compound or a pharmaceutically acceptable salt thereof is indicated in chemical formula 3. The compound decreases an ATP-dependent-D-alanyl-D-alanine ligase activity. The pharmaceutical composition for preventing an infection comprises the compound or a pharmaceutically acceptable support thereof. The composition has an activity decreasing the cell wall synthesis of bacteria.

Description

Novel compound and Antibiotic Composition comprising the same

The present invention relates to novel anti-bioactive compounds and antibiotic compositions comprising the compounds, and more particularly, to compounds that inhibit bacterial cell growth and the use of the compounds.

The use of antibiotics to treat infectious diseases worldwide has increased significantly over the last few decades. In 1954, two million pounds of antibiotics were produced in the United States alone. Today, the figure is over 50 million pounds. According to the Central Center for Disease Control (CDC), people consume 235 million antibiotics per year.

The widespread abuse or overuse of antibiotics has raised antibiotic resistance, which contributes to serious public health problems. Antibiotic resistance occurs when the bacteria causing the infection are not rescued by the antibiotics used to stop the infection. Bacteria survive and multiply and become more harmful. For example, Staphylococcus aureus is the main cause of infection from hospitals, which, epidemiologically, satisfactorily responds to vancomycin antibiotics. However, many Staphylococcus aureus strains have recently been shown to be resistant to vancomycin. Moreover, partly because of bacterial resistance to antibiotics, the mortality rate of some infectious diseases such as tuberculosis is increasing again.

Antibiotics are used therapeutically to treat bacterial infections. Many types of antibiotics, classified according to their mechanism of action, are currently used. Known types of antibiotics include, for example, cell wall synthesis inhibitors, cell membrane inhibitors, protein synthesis inhibitors, and inhibitors that affect or bind to DNA or RNA synthesis.

Inhibitors of cell wall synthesis, such as beta lactam antibiotics, generally inhibit some of the synthetic steps of bacterial peptidoglycan. Penicillin is generally effective against non-resistant streptococcus, gonococcus and staphylococcus. Amoxicillin and ampicillin show a broad spectrum in Gram-negative bacteria. Cephalosporins are commonly used as surgical precautions and penicillin replacements for Gram-negative bacteria. Monobactam is generally useful for treating allergic subjects.

Many antibiotics suitable for use in the treatment of bacterial-related diseases and disorders are described, for example, in The Physician's Desk Reference (PDR), Medical Economics Company (Montvale, NJ), (53rd Ed.), 1999; Mayo Medical Center Formulary, Unabridged Version, Mayo Clinic (Rochester, MN), January 1998; Merck Index: An Encyclopedia of Chemicals, Drugs and Biologicals, (11th Ed.), Merck & Co., Inc. (Rahway, NJ), 1989; University of Wisconsin Antimicrobial Use Guide, http: //www.medsch. wisc.edu/clinsci/5amcg/amcg.html: Introduction on the Use of the Antibiotics Guideline, of Specific Antibiotics Classes, Thomas Jefferson University, http://jeffiine.tju.edu/CWIS/OAC/antibiotics guide / intro.html And known in the art.

Peptidoglycans have been recognized as an attractive target for antibiotic development because peptidoglycans are an essential component of bacterial cell walls and similar structures and biosynthetic pathways do not exist in plants or mammals (Spratt, BG (1994) Science 264, 388-393.). D-alanine in the peptidoglycan structure is one of the central molecules of the cross-linking step of the peptidoglycan assembly to prevent proteolysis and increase cell wall stability. D-alanine branches in peptidoglycan synthesis consist of three catalysts: pyridoxal phosphate-dependent D-alanine racemase, ATP-dependent-D-alanyl-D-alanine ligase (ligase; Ddl), and ATP-dependent D-alanyl-D-alanine-adding enzymes. Ddl catalyzes the formation of D-alanyl-D-alanine dipeptide, a precursor of peptidoglycan. Dimerization of D-alanine begins with the attack of adenosine triphosphate (ATP) to the first D-alanine by gamma-phosphate to attach acyl phosphate to the first D-alanine. This product is accompanied by an attack by the amino group of the second D-alanine, finally removing phosphoric acid and producing a D-alanyl-D-alanine product (Walsh CT (1989) J Biol Chem . 264 , 2393- 6).

As a related prior patent, Korean Patent Publication No. 10-1996-0030942 is a novel antibiotic WAP-8294A, A1, A2, A4, AX, AX-8, AX-9 produced from a strain belonging to the genus Lysobacter And AX-13 or a pharmaceutically acceptable salt thereof; The antibiotic WAP consisting of culturing a microorganism having a production capacity of antibiotic WAP-8294A belonging to the genus Lysobacter to produce an antibiotic and propagating it in a culture medium, and recovering the antibiotic. Production method of -8294A; And it relates to an antimicrobial composition containing the antibiotic or pharmaceutically acceptable salt,

As another related prior patent, Korean Patent Publication No. 10-2002-7003755

The present invention relates to novel (7R) -7-acylamino-3-heteroarylthio-3-cepem-4-carboxylic acids or pharmacologically acceptable salts and prodrugs thereof, which are directed to β-lactam antibiotics. Novel intermediates and novel methods for producing new compounds and intermediate compounds that exhibit antibiotic activity against a wide range of organisms, including organisms resistant to, are useful as antibiotics, and are useful for preparing such new compounds. It is described.

The present invention solves the above problems, and it is an object of the present invention to provide a compound having an anti-living property.

Another object of the present invention is to provide a composition comprising the compound.

The above object of the present invention provides a compound of Formula 3 or a pharmaceutically acceptable salt thereof.

[Formula 1]

[Formula 2]

(3)

In one embodiment of the invention, the compound preferably inhibits ATP-dependent-D-alanyl-D-alanine ligase activity, but is not limited thereto.

The present invention also provides a pharmaceutical composition for preventing or treating infection by a pathogen comprising the compound of the present invention and a pharmaceutically acceptable carrier.

The present invention also provides a feed composition for preventing or treating infection by a pathogen comprising the compound of the present invention and a feed acceptable carrier.

In another aspect, the present invention provides a pesticide composition for preventing or treating an infection comprising the compound of the present invention and a pharmaceutically acceptable carrier.

In one embodiment of the present invention, the composition preferably has bacterial cell wall synthesis inhibitory activity, but is not limited thereto.

In addition, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable excipient, carrier, diluent and the like.

Carriers usable in the present invention include macromolecules that are slowly metabolized, such as liposomes, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers. Salts of inorganic acids such as, for example, hydrochloride, hydrobromide, phosphate and sulfate; Pharmaceutically acceptable salts such as salts of organic acids such as acetate, propionate, malonate and benzoate; Liquids such as water, saline, glycerol and ethanol, and auxiliary materials such as wetting agents, emulsifying agents or pH buffering substances may be used.

Pharmaceutically acceptable carriers are described in Remingtion's Pharmaceutical Sciences, Mack Publishing Company, 1991.

In addition, the composition is formulated in a unit dosage form suitable for intrabody administration of a patient, preferably in the form of a formulation useful for the administration of a medicament of the invention, according to conventional methods in the pharmaceutical art and commonly used in the art. Oral, or skin, intravenous, intramuscular, intraarterial, intramedullary, intramedural, intraventricular, lung, transdermal, subcutaneous, intraperitoneal, intranasal, intragastrointestinal, topical, sublingual, intravaginal Or by parenteral administration routes including the rectal route, but are not limited thereto.

Compound doses effective for the treatment of bacteria are about 1-50 mg or 1-20 mg per kg body weight of the receptor per day, more generally 0.1 to about 100 mg per kg body weight of the receptor per day. Effective dose ranges of pharmaceutically acceptable salts and prodrugs can be calculated based on the weight of the parent compound to be delivered. If the salt or prodrug exhibits activity by itself, the effective dose can be estimated using the weight of the salt or prodrug, as mentioned above, or by other means known to those skilled in the art.

For oral therapeutic administration, the active compounds can be used in the form of tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, in combination with one or more excipients.

Tablets, troches, pills, capsules, and the like may contain one or more of the following ingredients: binders such as microcrystalline cellulose, tragacanth gum, acacia, corn starch or gelatin; Excipients such as dicalcium phosphate, starch or lactose; Disintegrants such as corn starch, potato starch, alginic acid, Primogel and the like; Lubricants, eg magnesium stearate or Steotes; Glidants such as colloidal silicon dioxide; Sweeteners such as sucrose, fructose, lactose, saccharin or aspartame; Flavoring agents such as peppermint, methylsalicylate, wintergreen oil or cherry flavor; And peptide antibacterial agents such as Fuzeon ^™ .

If the unit dosage form is a capsule, it may contain, in addition to substances of this type, a liquid carrier such as vegetable oil or polyethylene glycol. Various other materials may be present as coatings or to alter the physical form of the solid unit dosage form.

The pharmaceutical composition of the present invention may be prepared, packaged or sold in a formulation suitable for rectal administration. Such compositions may be, for example, in the form of suppositories, retention enema preparations and solutions for rectal or colonic washing. The pharmaceutical compositions of the invention may also be prepared, packaged or sold in formulations suitable for vaginal administration. Such compositions may be, for example, in the form of suppositories, embedded or coated vaginal intercalating substances such as tampons, cleaning formulations, vaginal cleaning solutions.

The pharmaceutical compositions of the present invention may be prepared, packaged or sold in a formulation suitable for ophthalmic administration. For topical administration, the compounds of the present invention may be applied in pure form, ie in liquids. Typically, however, the compound may be administered to the skin as a composition or formulation in combination with a dermatologically acceptable carrier. Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols, glycols, and two or more of these mixtures, wherein the compounds of the present invention may optionally be dissolved or dispersed at effective levels using non-toxic surfactants.

The pesticide composition according to the invention may further comprise other additional ingredients, such as agriculturally acceptable supports, carriers or fillers.

As used herein, the term "support" refers to a natural or synthetic organic or inorganic material with which the active material is bound, in particular for ease of application to parts of the plant. Therefore, this support should generally be inert and agriculturally acceptable.

The retard can be a solid or a liquid. Examples of suitable supports include clays, natural or synthetic silicates, silicas, resins, waxes, solid fertilizers, water, alcohols, in particular butanol, organic solvents, mineral oils and vegetable oils and derivatives thereof. Mixtures of such supports can also be used.

The pesticide composition may also contain other additional ingredients. In particular, the pesticide composition may further contain a surfactant. The surfactant may be an ionic or nonionic emulsifier, dispersant or wetting agent or a mixture of these surfactants. These include, for example, polyacrylates, lignosulfonates, phenolsulfonates or naphthalenesulfonates, polycondensates of ethylene oxide with fatty alcohols or fatty acids or fatty amines, substituted phenols (particularly alkylphenols or arylphenols) , Sulfosuccinic acid ester salts, taurine derivatives (particularly alkyl taurates), phosphoric acid esters of polyoxyethylated alcohols or phenols, fatty acid esters of polyols, and derivatives of these compounds having sulfate, sulfonate and phosphate functional groups. If the active substance and / or the inert support is water insoluble and the vector reagent for application is water, it is generally necessary to have at least one surfactant present. Preferably the surfactant content may be 5 to 40% by weight of the composition.

Additional components may also include, for example, protective colloids, tackifiers, thickeners, thixotropic agents, penetrants, stabilizers, sequestrants. More generally, the active material may be combined with any solid or liquid additive according to conventional formulation techniques.

In general, the pesticide composition according to the present invention may contain 0.05 to 99% by weight, preferably 10 to 70% by weight of active substance.

The composition according to the invention is an aerosol dispenser, capsule suspension, cold mist concentrate, dustable powder, emulsifiable concentrate, oil-in-water emulsion, water-in-oil emulsion, encapsulated granules, fines, fluid concentrate for seed treatment, gas (under pressure) ), Gas-generating products, granules, radish concentrates, macrogranules, microgranules, oil dispersible powders, oil miscible fluid concentrates, oil miscible liquids, pastes, plant rodlets, powders for dry seed treatment Seeds coated with pesticides, soluble concentrates, soluble powders, solutions for seed treatment, suspension concentrates (fluid concentrates), ultra low volume (ulv) solutions, ultra volume suspensions, water dispersible granules or tablets, It can be used in various forms such as water dispersible powders for slurry treatment, water soluble granules or tablets, soluble powders for seed treatment and hydrating agents.

These compositions include commercially available concentrated compositions that must be diluted before being applied to the crop, as well as compositions that are in direct application to the plant or seed to be treated using a suitable device such as a spraying or spreading device.

The pesticide composition of the present invention can be used for the therapeutic or prophylactic control of insects as well as for the therapeutic or prophylactic control of phytopathogenic bacteria of crops.

Thus, according to a further aspect of the present invention, an effective non-phytotoxic amount of a composition as defined above is seed treatment, leaf application, stem application, or the seed, plant and / or fruit of the plant or the plant is growing or growing therefrom. Soil and / or inert substrates (e.g., organic substrates (e.g. sand, rock wool, glass wool, expandable minerals (e.g. pearlite, vermiculite, zeolites, expandable clays)), pumice, coal rock material / tuff, synthetic organic Substrates (e.g. polyurethane), organic substrates (e.g. peat, compost, wood waste (e.g. coir, wood fibers / chips, bark)) and / or liquid substrates (e.g. suspended hydroponic systems, Nutrient Film Technique, A method for treating or preventing insects as well as therapeutically or prophylactically controlling insect phytopathogenic bacteria of crops, which is applied through irrigation / drip application (irrigation) to aeroponics). This is provided.

The expression "effective non-phytotoxic amount" is sufficient to control or eradicate pests and / or diseases present in or prone to crops, while the crops do not involve any significant phytotoxic symptoms. Means quantity. Such amounts can vary widely depending on the pests and diseases to be eradicated or controlled, the type of crop, the climatic conditions and the compounds included in the composition according to the invention. The amount can be determined by those skilled in the art and can be determined by systematic field experiments.

The treatment method according to the invention may be useful for the treatment of propagation materials, such as tubers or rhizomes, as well as for the treatment of seeds, seedlings or transplanted seedlings and plants or transplanted plants. The treatment method may also be useful for the treatment of roots. The treatment method according to the invention may also be useful for treating the ground parts of plants, such as the body, stems or sacks, leaves, flowers and fruits of related plants.

Plants that may be protected by the method of the present invention include cotton; maybe; Vine plants; Fruit or vegetable crops, seedlings, major crops such as Graminae sp. (E.g. corn, grass or wheat, rice, barley and wheat), flowers, horticulture and forest crops; As well as genetically modified homologs of these crops may be mentioned.

In addition, the feed composition comprising the compound of the present invention or a salt thereof is used as a feed for animals such as mammals, birds, reptiles, amphibians, fish, preferably mammals. For example, the feed of the present invention is used as feed for pets such as dogs, cats, rats, cattle feed such as cows and pigs, feed for poultry such as chickens and turkeys, feed for breeding fish such as sea breams and defenses , Preferably used as feed for pets or feed for livestock.

The feed of the present invention can be prepared by appropriately compounding the compound of the present invention or a salt thereof in feed materials. Examples of feed materials include cereal grains, crude steel, vegetable oils, animal feedstuffs, other feedstuffs, and refined products.

Examples of cereals include maize, wheat, barley, oats, rye, brown rice, buckwheat, buckwheat, sorghum, blood, corn, soybeans and the like.

Examples of the crude steel include rice bran, defatted rice bran, wheat bran, horse meal, wheat, embryo, barley bran, screening, pellets, corn bran, and corn embryo.

Examples of vegetable oils include soybean meal, soybean meal, flaxseed meal, cottonseed meal, groundnut meal meal, safflower meal, palm oil palm, palm oil, fomarak, sunflower oil, rapeseed meal, cowpea oil and mustard oil.

As animal feed materials, for example, fish meal (North American wheat, imported wheat, whole wheat, coastal wheat), fish bulrush, meat meal, fish bone meal, blood meal, decomposing hair, bone meal, animal by-products, feather mill, silkworm, skim milk powder, Casein, dried whey, and the like.

Other feedstocks include plant foliage (alfalfa, haycube, alfalfa leaf powder, pseudo acacia powder, etc.), corn processing industrial by-products (corn gluten, wheat, corn gluten feed, corn staple, etc.), starch processed products, sugar, fermentation. Industrial products (yeast, beer foil, malt sprouts, alcohol foil, walnuts, etc.), agricultural production by-products (citrus fruits, tofu, coffee, cocoa, etc.), others (cassava, bean, guam, seaweed, Krill, spirulina, chlorella, minerals, etc.) etc. are mentioned.

Typical products include proteins (casein, albumin, etc.), amino acids, sugars (starch, cellulose, sucrose, glucose, etc.), minerals, vitamins, and the like.

As can be seen from the present invention, we found two lead compounds that inhibit bacterial cell growth in the ˜ug / ml range and confirmed their direct binding to target proteins using NMR and DPI results. .

1 is a SDS-PAGE photograph of purified DdlA (Xoo0352), and 2 is a crystal photograph grown at 0.2 M MgCl ₂ , 15% (w / v) PEG 4000, and 0.1 M Tris pH 8.5.
3 is the overall structure of DdlA and 4 is a detailed view of its active site with ATP molecules.
FIG. 5 is a diagram illustrating a VLS experiment to search for antagonist drugs for specific target proteins.
6 is a diagram showing the process of bacterial cell growth inhibition assay with selected compounds derived from VLS.
Figure 7 shows the 1D ¹ H-NMR spectrum for compound T5401603. This spectrum shows the degree of affinity for the compound for DdlA.
Figure 8 shows different concentrations (2- 3.3% DMSO, 3-0.025 mM, 4-0.05 mM, 5- 0.075 mM, 6-0.1 mM, 7-0.125 mM, 8-0.15 mM, 9- 0.175 mM, 10-0.2 mM, 11-0.25 mM) is used to show DPI profiles of Ddl-His and drug (T0506-1741) interactions.

The present invention will now be described in more detail by way of non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention, and the scope of the invention is not to be construed as limited by the following examples.

Preparation Example: Preparation of Compound of the Invention

The compound used in the present invention was purchased from ENAMINE Ltd. of Ukraine and used. The company web site: http://www.enamine.net/

The company address is 23 Alexandra Matrosova Street 01103 Kiev, Ukraine.

Example 1: Xanthomonas oryzae pv . Cloning of DdlA from oryzae (Xoo), expression, purification, crystallization and structure determination

Cloning

Gene Coding to DdlA is Xanthomonas oryzae pv. The genome of oryzae (Xoo KACC10331 strain) was cloned through polymerase chain reaction (PCR). Oligonucleotide primer sequences were designed based on previously reported gene sequences (Lee, BM, et al. (2005) Nucleic Acids Res 33 , 577-86.) The forward and reverse primers were 5'GGG GGG CAT ATG CGC, respectively. AAG ATC CGG GTC GGC CTC A-3 '(SEQ ID NO: 1) and 5'GGG GG G GAT CC T CAG TGT AGC TCG ACC GCG CTG C-3' (SEQ ID NO: 2). Nde I and Bam HI restriction sites are shown in bold. The PCR amplicon was double-treated with Nde I and Bam HI and the residues of the tobacco etch virus (TEV) protease cleavage site and 6xHis in front of the Nde I site in the pET11a vector (Novagen) to facilitate purification of the expressed protein. Was ligated with a modified pET11a vector (His-TEV-pET11a) designed to have.

Overexpression and Purification

Expression of DdlA was performed using E. coli strain BL21 (DE3) in Luria-Bertani medium containing 50 μg / ml ampicillin. Induction was performed by adding 0.5 mM Isopropyl-beta-D-thiogalactopyranoside to the culture at 310K when OD ₆₀₀ reached 0.6. After induction, the E. coli were incubated for an additional 20 hours at 288 K. Cultured Escherichia coli were collected by centrifugation (Vision VS24-SMTi V5006A rotor) for 10 minutes at 6,000 rpm at 277K. The collected E. coli was then resuspended in cold cell A buffer buffer [25 mM Tris-HCl pH 7.5, 300 mM NaCl, 15 mM Imidazole, 3 mM beta-mercaptoethanol] and sonicated on ice (Sonomasher, S & T). Science, Korea). The lysate was then centrifuged at 277K, 13,000 rpm for 30 minutes to obtain a cell extract (Vision VS24-SMTi V508A rotor). About 25% of the total expressed DdlA was water soluble (not shown). Supernatant containing water-soluble DdlA was loaded into Ni-NTA His.Bind resin (Novagen) previously equilibrated with lysis buffer A to use affinity purification. Affinity purification was performed at 277 K according to the manufacturer's protocol. Fragmentation buffer A containing 30 mM imidazole was used to wash non-specifically bound proteins. Fragmentation buffer A containing 200 mM imidazole was applied to elute the DdlA protein with 6 × His-tag. The obtained protein solution was dialyzed at 277K for 12 hours using Buffer B [25 mM Tris-HCl pH 7.5, 3 mM beta-mercaptoethanol]. Further purification was performed using a UNO6 Q column (Bio-Rad). At this time, buffer E [25 mM Tris-HCl, pH 7.5, 1 M NaCl, 3 mM beta-mercaptoethanol] was used as the elution reagent. Purity of the purified protein was examined using SDS-PAGE during each step. After two steps of purification, only one protein band of about 43 kDa size was observed on the SDS-PAGE gel (FIG. 1). The protein was then concentrated to 6 mg ml ⁻¹ in crushing buffer containing 25 mM NaCl using Centri-Prep (Millipore).

Crystallization and X- Lay diffraction  Data collection

Crystallization of the protein was performed via Hydra II e-drop (Matrix), an automated pipetting system, using several screening kits such as Crystal screen HT, Index HT, and Salt Rx HT (Hampton Research), and high efficiency at 287K. High throughput (HT) crystal screening has begun. We obtained several different crystallization conditions, in particular 0.3 M Dimethylethyl- (3-Sulfopropyl) -ammonium Determination of diffraction quality was obtained using a protein concentration of 6 mg / ml under conditions of 0.2 M MgCl ₂ with (additive), 15% (w / v) PEG 4000, 0.1 M Tris pH 8.5. The antifreeze 20% ( v / v ) glycerol was added to the crystallization conditions and then frozen to 100K in liquid nitrogen.

X-ray diffraction data was collected at a beamline 17A in the Japanese Photon Factory using the ADSC Quantum 270 CCD detector up to 2.3 Å resolution. Hayeoseo integrating data sets was calculated using the DENZO and SCALEPACK (Otwinowski, Z. and Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276 , 307-326. The auto-indexing process inferred the decision space group of P4 ₃ 2 ₁ 2 and the unit cell parameter a = b = 83.0 c = 97.6 Å.

As shown in FIG. 3, the final model includes 360 amino acid residues having 142 water molecules and their ATP binding activation site. The molecule was divided into three domains based on the alpha / beta structure: N-terminal domain (R2-G131), central domain (V132-A225) and C-terminal domain (G226-L360). The active site is located at the interface between the N and C terminal domains in an open structure to facilitate the binding of the substrate to the wide-hole space.

Example  2: Target  enzyme DdlA For Virtual  Library screening ( VLS ) process

Since Xoo0352 (DdlA) is one of the potential target genes for white leaf blight according to the GlaxoSmithKline (GSK) report, antagonism of the substrate binding site of the DdlA enzyme via virtual library screening (VLS), a computer simulation using ICM Molsoft. Red drug lead compounds were searched (FIG. 5).

VLS was performed using a 18 million compound database in the ICM Molsoft modeling suit for drug screening. Substrates of each flexible structure present in the compound database found through the Lipinski formula in VLS were docked into the substrate binding pocket grid of the receptor using ICM and assigned a score that reflects the quality of the complex. Three checks were performed in parallel at the same time to check for regeneration of each substrate binding result. 703 medicinal leads with top scores were selected and each complex thereof examined visually (numerically) with coefficients such as shape complementarity, hydrogen bonding network, and ligand flexibility. 500 compounds were obtained based on this final selection step for characterization through experimental analysis.

Example 3: Bacterial cell growth inhibition assay of synthesized chemical compounds against Xoo , E. coli , and pseudomonas , and determination of minimum inhibitory concentration ( MIC ) of each effective screened compound

3 ml of Xoo incubated overnight were inoculated into 100 ml of NB medium. The bacterial cultures were incubated until the OD ₆₀₀ reached 0.6. 150 μl of bacterial culture was transferred to a 96 well ELISA plate containing 1 mM screened compound retrieved from VLS. DMSO was used to dissolve the compound into stock compound at 30 mM concentration. DMSO concentrations were kept below 5% for all bacterial growth assays and the negative control used only the same concentration of DMSO. The plates were incubated at 28 with continuous shaking and measured at regular intervals of 0, 3, 6, 9, and 12 hours at absorbance 600 nm. Compounds that inhibit Xoo growth of at least 50% were selected to determine the MIC ₅₀ value of the compound.

MIC ₅₀  Way( Microtiter  Plate-based Antibacterial  analysis)

3 ml of Xoo incubated overnight were inoculated into 100 ml of NB medium. The bacterial cultures were incubated to a cell density of 0.5 McFarland Standard. The culture was diluted 10-fold and 150 μl of diluted culture was aliquoted into sterile 96-well clear flat bottom plates. Selected compounds with high bacterial cell growth inhibitory activity were serially diluted and added to 96-well plates at concentrations from 600 μM to 9.5 μM. Bacteria culture with only the same concentration of DMSO was used as a negative control. The plate was incubated at 28 ° C. and an OD ₆₀₀ measurement was performed after 20 hours. Percent inhibition of bacterial growth was calculated by the equation

Percent inhibition = 100 x (OD _nc -OD _tc ) / (OD _nc -OD _pc )

[OD _nc : OD ₆₀₀ of negative control, OD _tc : OD _600] of the positive control (a known inhibitor): OD _600, OD _pc of the test compound.

MIC ₅₀ values were determined as the minimum compound concentration that inhibited bacterial cell growth of 50%.

IC ₅₀ And Ki Enzyme Analysis for

Enzymatic activity of XoDdl was performed by monitoring the D-alanine-dependent dissociation of Pi from ATP. Lanzetta et al . , 1979 colorimetric method was used to detect dissociated Pi in the culture mixture. Culture mixtures (50 μl) contain 50 mM Tris pH 7.8, 10 mM MgC12, 10 mM KCl, the desired amount of D-alanine (range of 0.1-5 mM and 10-50 mM) and ATP. The reaction was started by adding 1.5 μg enzyme amount. After the desired time incubation at 37 ^° C., the reaction was stopped with 10 mM EDTA and 250 μl of water. Then, 750 μl Ames solution (comprising 0.36% (w / v) + 1.42% ascorbic acid) prepared before the analysis was added to the mixture and incubated for 20 minutes at 45 ^° C. for color development. The concentration of Pi dissociated through color development was measured at 820 nm with a spectrometer. Pure KH ₂ PO ₄ (Sigma) was used as standard reagent. Time-lapse velocity data were collected and their velocity variables were determined by double-reciprocal plots (Neuhaus, 1962, Liu et al . , 2006). IC ₅₀ was determined under similar conditions using D-Ala (0.5 mM), ATP (1 mM) and inhibitors (0.05-0.1 mM for Compounds 1 and 2; 0.01-0.2 mM for Compound 3). K _i was determined under similar conditions with various D-Ala concentrations and was calculated from K _mapp = K _m K _i ^-1 [I] + K _m . The compounds were incubated with enzyme 10 minutes before adding the substrate to start the reaction.

Minimum inhibitory concentration ( Minimum Inhibitory Concentration )

The minimum inhibitory concentration (MIC50) of the compound required to inhibit the growth of 50% bacteria was determined by using 10 ⁴ cells / mL for E. coli or 10 ⁶ cells / mL for S. aureus. Determined by. The antibiotic was prepared on a 2-fold dilution line. Microwell plates with 96 wells consisting of the antibiotic and bacterial suspensions were incubated at 37 ^° C. for 16 hours in a stirred incubator. Absorbance (OD = 600 nm) was measured at 2 hour intervals. The plate was stirred for 30 seconds for each measurement. MIC was adopted as the minimum concentration of antibiotic that inhibited 50% growth (Table 3).

Chemical formula IC ₅₀ ([mu] M) MIC ₅₀
㎍ / mL Ki (μM) Formula 1 161.2 52 0.53 (2) 159.6 52 2.2 (3) 38.19 37 0.53

Table 1 is a table of MICs of lead compounds derived from bacterial cell growth inhibition assays.

Example 4 Xoo Using NMR , and DPI DdlA Binding Assay for Lead Compounds to Target Proteins

Screened compounds with high bacterial cell growth inhibitory activity (˜μg / ml MICs) were tested to directly bind their target proteins. Two different experimental methods, NMR and DPI, were used to confirm the direct binding between the compounds and the target protein DdlA.

NMR  Experiment

High purity purified protein stock solution (Xoo PDF) was prepared at 100 μM concentration in 25 mM Tris pH 7.5, 10 mM NaCl and 3 mM 2-ME buffer. Each inhibitor stock solution was prepared at 30 mM in 100% DMSO. For NMR experiments, 20 μM protein and 0.1 mM inhibitor were made using the same buffer solution of 10% D ₂ O and 25 mM Tris pH 7.5, 10 mM NaCl, 3 mM 2-ME. The 1D NMR spectra of the inhibitor-only solution and the two samples containing the inhibitor and the PDF were measured, respectively. The binding of the inhibitor to the target protein PDF was confirmed by a change in the 1D NMR spectrum of the inhibitor used in the experiment. NMR spectra were recorded using a 500 MHz 1D NMR Bruker DRX from the Korea Basic Science Institute (KBSI) in Daejeon.

NMR  result

1D ¹ H-NMR spectra for titration of specific concentrations of potential lead compounds were measured (FIG. 7). As the protein concentration increases, the height of the NMR signal of the substrate alone decreases by the binding between the protein and the substrate. Using this method we were able to find proteins and high affinity compounds.

DPI  Experiment

Dual polarization interferometer (DPI) is an optical sensing technology that has been used to study solution-phase interactions between immobilized protein and drug molecules. This technique is one of the powerful techniques for detecting and quantifying the binding affinity and simultaneous morphological changes of target proteins and drug molecules in real time.

After equilibrating the chip with ethanol, distilled water and running buffer (10 mM PIPES, 150 mM NaCl, pH 7.3) to fill the chip surface; 1 mM copper solution was injected at 15 / min. Unbound copper remaining on the chip was removed by washing with an additional running buffer. To bind the protein to the chip, 180 μl (100 μg / mL) of His-tag-containing protein was injected at 30 μl / min, added to the chip surface, and the running buffer was washed for 10 minutes to wash the remaining protein. Spent on the combined chip. The loop was washed after each step injected by the inventors. Each compound was dissolved in 30 mM stock in DMSO, so 3.3% absolute DMSO was used as a control. The chemical compounds were treated on DdlA fixed chip surfaces by injection of 150 μl of 50 μl / min at various concentrations (0.25 mM to 0.05 mM). The DPI profile plot (Figure 8) shows the change in the transverse magnetic (TM) and transverse electric (TE) polarization of the chip surface, which confirms the binding of DdlA to the compound. Farfield analysis using the AnaLight program shown in Table 2 shows the change in mass, thickness and density of the immobilized protein layer with the state before and after loading of the compound.

Thickness (nm) Mass (ng / mm ² ) Density (g / cm ³⁾ protein 2.5341 1.7697 0.6983 Inhibitor 0.1 mM 3.9473 2.1519 0.5452

Table 2 shows the changes in mass, thickness and density of chips with immobilized proteins before and after inhibitor loading for DPI experiments.

MIC ₅₀
/ mL
Xoo E . coli B. licheniformis Formula 1 52 > 167 > 167 (2) 52 > 181 > 181 (3) 37 66.4 103

Table 3 shows the results of Xanthomonas oryzae pv. In addition to inhibiting oryzae bacterial cell growth, oryzae (E. coli) and B.licheniformis also have the effect of inhibiting the growth of the table.

<110> Konkuk University Industrial Cooperation Corp. <120> Novel compound and Antibiotic Composition comprising the same <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ggggggcata tgcgcaagat ccgggtcggc ctca 34 <210> 2 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gggggggatc ctcagtgtag ctcgaccgcg ctgc 34

Claims (8)

A compound of formula 3 or a pharmaceutically acceptable salt thereof.

(3) The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound inhibits ATP-dependent-D-alanyl-D-alanine ligase activity. A pharmaceutical composition for preventing or treating infection by a pathogen comprising the compound of claim 1 or 2 and a pharmaceutically acceptable carrier. According to claim 3, wherein the composition is a pharmaceutical composition for preventing or treating infection by a pathogen having bacterial cell wall synthesis inhibitory activity. Feed composition for the prevention or treatment of infection by a pathogen comprising the compound of claim 1 or 2 and a physiologically acceptable carrier. According to claim 5, The composition is a feed composition for preventing or treating infection by a pathogen having a bacterial cell wall synthesis inhibitory activity. A pesticide composition for preventing or treating an infection comprising the compound of claim 1 or 2 and an agriculturally acceptable carrier. According to claim 7, wherein the composition is a pesticide composition for preventing or treating infection by a pathogen having bacterial cell wall synthesis inhibitory activity.

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