KR101289461B1 - Novel compound and Antibiotic Composition comprising the same - Google Patents

Abstract

The present invention relates to novel antibiotic compounds and antibiotic compositions comprising the compounds, and more particularly, to compounds that inhibit microbial lipid synthesis and the use of the compounds.

Description

Novel compound and Antibiotic Composition comprising the same

The present invention relates to novel antibiotic compounds and antibiotic compositions comprising the compounds, and more particularly, to compounds that inhibit microbial lipid synthesis 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.

FabD, Xoo0880, is one of Xoo's most important drug target enzymes. The FabD gene expresses a Malonyl-CoA-acyl carrier protein transacylase (MCAT) enzyme (EC 2.3.1.39). This protein delivers the malonyl site to the holo-acyl transporter protein that forms the malonyl-ACP intermediate in bacterial type II fatty acid synthesis (Ruch, FE, and Vagelos, PR (1973) The isolation and general properties of Escherichia coli malonyl) coenzyme A-acyl carrier protein transacylase.J Biol Chem 248 , 8086-94.). This intermediate malonyl-ACP provides two carbons in the extension phase of fatty acid synthesis. Fatty acid synthesis (FAS) is essential to the survival of the organism (Magnuson, K., Jackowski, S. , Rock, CO, and Cronan, JE, Jr. (1993) Regulation of fatty acid biosynthesis in Escherichia coli. Micro biol Rev 57 , 522-42; White, SW, Zheng, J., Zhang, YM, and Rock (2005) The structural biology of type II fatty acid biosynthesis. Annu Rev Biochem 74 , 791-831.), MCAT helps to extend the length of the acyl chain by two carbons. MCAT has also been reported to be involved in the polyketide biosynthesis that produces acyl-ACP thioesters, producing one of the largest segments of secondary products such as tetracyclines and erythromycins (Keatinge-Clay, AT, Shelat). , AA, Savage, DF, Tsai, SC, Miercke, LJ, O'Connell, JD, 3rd, Khosla, C., and Stroud, RM (2003) Catalysis, specificity, and ACP docking site of Streptomyces coelicolor malonyl-CoA: ACP transacylase. Structure 11 , 147-54; Summers, RG, Ali, A., Shen, B., Wessel, WA, and Hutchinson, CR (1995) Malonyl-coenzyme A: acyl carrier protein acyltransferase of Streptomyces glaucescens : a possible link between fatty acid and polyketide biosynthesis. Biochemistry 34 , 9389-402.). Thus, MCAT is considered an essential enzyme in bacterial metabolic activity among the enzymes involved in the FAS process and is used as a potential drug target for the development of antibacterial drugs (Campbell, JW and Cronan, JE Jr. (2001) Escherichia coli FadR Positively Regulates Transcription of the fab B Fatty Acid Biosynthetic Gene. J Bacteriol . 183 , 5982-90; Miesel, L., J. Greene, and TA Black. 2003. Genetic strategies for antibacterial drug discovery. Nature . 4 , 442-456 .; White, SW, Zheng, J., Zhang, YM, and Rock (2005): The structural biology of type II fatty acid biosynthesis. Annu Rev Biochem 74 , 791-831.).

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 any one of formulas (1) to (3) or a pharmaceutically acceptable salt thereof.

[Formula 1]

[Formula 2]

(3)

In one embodiment of the invention, the compound preferably inhibits malonyl-CoA: acyl carrier protein transacylase (MCAT) 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 any one of Formulas 1 to 3 of the present invention and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a feed composition for preventing or treating infection by a pathogen comprising the compound of any one of Formulas 1 to 3 of the present invention and a physiologically acceptable carrier.

In another aspect, the present invention provides a pesticide composition for infection prevention or treatment comprising the compound of any one of Formulas 1 to 3 of the present invention and a pharmaceutically acceptable carrier.

In one embodiment of the present invention, the composition preferably has microbial lipid 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 may be formulated in a unit dosage form suitable for intrabody administration of a patient according to a conventional method in the pharmaceutical field, preferably in the form of a formulation useful for the administration of a compound pharmaceutical product, and is commonly used in the art. Oral or skin, intravenous, intramuscular, intraarterial, intramedullary, intramedullary, intraventricular, lung, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal tract, topical, sublingual, intravaginal or rectal Administration may be by parenteral administration routes including, but not limited to, routes.

Compound doses effective for bacterial treatment are from about 1 to 50 mg or 1 to 20 mg per kg body weight of receptor per day, more generally from 0.1 to about 100 mg per kg body weight of 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 antimicrobials such as Enbuvirtide (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 from 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 in 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.

Examples of raw animal feeds include fish meal (bean mill, imported wheat, hall mill, coastal wheat), fish shavings, meat meal, fish meal, blood meal, Casein, dry whey, and the like.

Examples of other feed materials include plant foliage (alfalfa, heukube, alfalfa leaf, and similar acacia powder), corn processing industry by-products (conglutin, wheat, conglutinfeed, constay prima etc.), starch products, (Cassava, bean curd, bean curd, bean curd, malt bean curd, alcohol bean curd, jangyu bean curd), agricultural production byproducts (citrus processing, tofu, coffee, cocoa, Krill, spirulina, chlorella, minerals, etc.).

Examples of the purified product include proteins (casein, albumin, etc.), amino acids, saccharides (starch, cellulose, sucrose, glucose, etc.), minerals and vitamins.

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

Figure 1 shows the type II bacterial fatty acid synthesis pathway.
Figure 2a is a SDS-PAGE picture of purified fabD (Xoo0880), 2b is a crystal picture grown in 0.1 M CHES pH9.0, 1.5 M NH ₄ SO ₄ , and 0.2 M NaCl.
3A is the overall structure of XoMCAT and 3B is a detail view of its active site.
4 is a diagram depicting a VLS experiment to search for antagonist drugs for specific target proteins.
5 is a diagram showing the process of bacterial cell growth inhibition assay with selected compounds derived from VLS.
FIG. 6 shows the 1D ¹ H-NMR spectra for Formulas 1 (FIG. 6A), 2 (FIG. 6B), and 3 (FIG. 6C) of the present invention as XoMCAT lead compounds. This spectrum shows the degree of affinity for the compound for XoMCAT.

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.

Manufacturing example Preparation of Compounds of the Invention

The compounds used in the present invention were purchased from ChemBridge Corporation (16981 Via Tazon, Suite G, San Diego, Calif., 92127, USA), USA, and used by the company's web site: http://www.chembridge.com/ . .

Example  One: Xanthomona soryzae pv . oryzae ( Xoo Derived from XoMCAT of Cloning , Expression, purification, crystallization, crystal structure determination

Cloning

The FAD coding sequence of Xoo0880 was amplified by polymerase chain reaction using a bacterial cell (Xoo KACC10331 strain) genome as a template. Oligonucleotide primer sequences were designed based on previously reported gene sequences (Lee, BM, et al. (2005) Nucleic Acids Res 33 , 577-86.). Forward and reverse primers respectively: 5'GGG GGG CAT ATG ACC GAA TCC ACT CTC GCC TTC A3 '(SEQ ID NO: 1) and 5'-GGG GGG GGA TCC TCA GTG GCC CCA CGC GTC GAG A-3' (SEQ ID NO: 2).

NdeI and BamHI Restriction sites are indicated 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

His-TEV-pET11a expression vector comprising the coding sequence of Xoo0880 was expressed in E. coli. It was put in BL21 (DE3). Cells were grown at 37 ° C. until OD ₆₀₀ reached 0.5 in Luria-Bertani medium containing 50 μg / ml ampicillin. Protein expression was induced by adding 0.5 mM Isopropyl-beta-D-thiogalactopyranoside to the culture. After induction, the cells were incubated for an additional 16 hours at the same temperature. Cultured E. coli was collected by centrifugation (Vision VS24-SMTi V5006A rotor) for 20 minutes at 6,000 rpm at 277K. The collected E. coli was then resuspended in a cold cell disruption buffer A [25 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM Imidazole] and sonicated on ice (Sonomasher, S & T Science, Korea). By crushing. The lysate was then centrifuged at 277K, 12,000 rpm for 30 minutes to obtain cell extracts (Vision VS24-SMTi V508A rotor). Only 10% of the total expressed Xoo0880 was water soluble (not shown). Supernatants containing water-soluble fabD were loaded into Ni-NTA resin (Novagen) to use affinity purification. Affinity purification was performed at 4 ° C. according to the manufacturer's protocol. The fabD protein with 6 × His-tag was then eluted with crushing buffer A containing 200 mM imidazole and treated overnight with TEV protease at 4 ° C. to remove 6 × His-tag. The protein solution thus treated was dialyzed at 4 ° C. using Buffer B [25 mM Tris-HCl pH 7.5, 15 mM NaCl] for 6 hours, and further purified using a UNO6 Q column (Bio-Rad). The purity of the purified protein was analyzed using SDS-PAGE (FIG. 2). The purified protein was concentrated to 7 mg ml ^-1 and used in the crystallization process.

Crystallization and X- Lay diffraction  Data collection

Crystallization of XoMCAT 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), high throughput (HT)] crystal screening has begun. Initially very thin needle shaped crystals were obtained under Wizard G-5 conditions (0.1 M CHES pH9.5, 1.25 M NH ₄ SO ₄ , and 0.2 M NaCl) and modified conditions (0.1 M CHES pH9.0, 1.5 M NH). ₄ SO ₄ , and 0.2 M NaCl). It was regenerated using the hanging drops method, and crystals were made for a week. The grown crystals were frozen at 100K in liquid nitrogen after adding antifreeze 20% ( v / v ) glycerol to the crystallization conditions. Used to collect data. Crystalline data were collected at a beamline 17A of the Japanese Photon Factory using an ADSC Quantum 270 CCD detector up to 1.9 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 infers the decision space group of P2 ₁ 2 ₁ 2 ₁ and the unit cell parameters a = 41.4, b = 74.6, c = 98.5.

The crystal structure of XoMCAT and its active site residues are shown in FIG. 3. The overall structure of XoMCAT consists of two domains, a larger domain and a smaller domain, and folds stably. Its active site is located in the canyon between these domains. Larger domains program DSSP (Kabsch, W. and Sander, C. (1983) Biopolymers 22, 2577-637..) 14 of the alpha with a surface area of the exciter three block 12585Å ² calculated from a-helical structure, and eight beta -It is composed of linear structure. The nucleophilic activating residue Ser-94 is located in the Sharpton located between the alpha-helix and beta-linear structures of the larger domain and the other active site residues Gln 13, Arg 119 and His 203 are located near the nucleophilic residue. Located.

Example  2: target  enzyme XoMCAT For Virtual  Library screening ( VLS ) process

Since Xoo0880 (XoMCAT) is one of the potential targeted genes for rice blight based on GlaxoSmithKline (GSK), it is antagonistic to substrate binding sites of the XoMCAT enzyme through high efficiency (HT) virtual library screening (VLS, Figure 4). Drug lead compounds were searched.

For drug screening, VLS, a computer simulation, was performed using an 18 million compound database in the ICM Molsoft modeling suit. Substrates of each flexible structure present in the compound database found via Lipinski's formula in VLS were docked into the substrate binding pocket grid of XoMCAT using ICM and assigned scores reflecting the quality of the complex. Three checks were performed in parallel at the same time to check for regeneration of each substrate binding result. 1991 compounds with top scores were selected and each of its complexes examined by visualizing (numerical) 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: Xoo , E. coli , And pseudomonas Analysis of bacterial cell growth inhibition of synthesized chemical compounds for Screened  Compound MIC  Minimum Inhibitory Concentration

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 ° C. 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%.

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

ID rescue MIC ₅₀
 (/ / mL )
7014034

C ₂₁ H ₁₇ SN ₂ O ₃

C ₂₁ H ₁₇ SN ₂ O ₃ 70
6506845

C ₁₈ H ₁₃ SN ₂ O ₄ 66
5469065

C ₁₈ H ₁₅ N ₃ O ₃ 41

Example  4: NMR Using XoMCAT target  Binding Analysis of Lead Compounds to Proteins

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

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 results

1D ¹ H-NMR spectra for titration of specific concentrations of potential lead compounds were measured (FIG. 5). 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. (Fig. 5).

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Claims (8)

delete delete A pharmaceutical composition for preventing or treating infection by a pathogen comprising a compound of Formula 1 and a pharmaceutically acceptable carrier.

[Formula 1] According to claim 3, wherein the composition is a pharmaceutical composition for preventing or treating infection by a pathogen having a microbial lipid synthesis inhibitory activity. Feed composition for preventing or treating infection by a pathogen comprising a compound of formula 1 and a feedstock acceptable carrier.

[Formula 1] According to claim 5, The composition is a feed composition for preventing or treating infection by pathogens having a microbial lipid synthesis inhibitory activity. A pesticide composition for preventing or treating infection, comprising a compound of Formula 1 and an agriculturally acceptable carrier.

[Formula 1] According to claim 7, wherein the composition is a pesticide composition for preventing or treating infection by a pathogen having a microbial lipid synthesis inhibitory activity.

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