KR101268563B1 - 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 a novel anti-bioactive compound and an antibiotic composition comprising the compound, and more particularly, to a compound that inhibits microbial lipid synthesis and a use of the compound.

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

The widespread abuse or overuse of antibiotics leads to increased resistance to antibiotics, which contributes to serious public health problems. Antibiotic resistance occurs when the bacteria that cause the infection are not rescued by the antibiotic used to stop the infection. The bacteria survive and multiply, making them more harmful. Staphylococcus aureus, for example, is a major contributor to hospital infections that respond satisfactorily to vancomycin antibiotics, epidemiologically. However, recently many Staphylococcus aureus strains have been shown to be resistant to vancomycin. Moreover, the mortality rate of some infectious diseases, such as tuberculosis, is rising again, in part because of bacterial resistance to antibiotics.

Antibiotics are used therapeutically to treat bacterial infections. Several types of antibiotics, classified according to their mechanism of action, are currently in use. 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 synthesis steps of bacterial peptidoglycans. Penicillin is generally effective against non-resistant streptococcus, gonococcus and staphylococcus. Amoxicillin and ampicillin exhibit a broad spectrum of gram-negative bacteria. Cephalosporins are commonly used as a surgical precaution and as a penicillin replacement for gram-negative bacteria. Monobactam is generally useful for treating allergic individuals.

A number of antibiotics suitable for use in the treatment of bacterial-related diseases and disorders include, for example, 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 It is known and disclosed in.

FabD, Xoo0880, is one of Xoo's most important drug targeting enzymes. The FabD gene expresses the enzyme Malonyl-CoA-acyl carrier protein transacylase (MCAT) (EC 2.3.1.39). This protein transfers a malonyl site to a holo-acyl transport protein that forms the malonyl-ACP intermediate in bacterial type II fatty acid synthesis (Ruch, F. E., and Vagelos, P. R. (1973) The isolation and general properties ofEscherichia coli malonyl coenzyme A-acyl carrier protein transacylase.J Biol Chem 248, 8086-94.). This intermediate, malonyl-ACP, provides two carbons in the prolonged step of fatty acid synthesis. Fatty acid synthesis (FAS) is essential for the survival of living organisms (Magnuson, K., Jackowski, S., Rock, C. O., and Cronan, J. E., Jr. (1993) Regulation of fatty acid biosynthesis inEscherichia coli.Micro biol Rev 57, 522-42.; White, S. W., Zheng, J., Zhang, Y. M., and Rock (2005) The structural biology of type II fatty acid biosynthesis.Annu Rev Biochem 74, 791-831.), MCAT helps extend the length of the acyl chain by two carbons. It has also been reported that MCAT is involved in the biosynthesis of polyketides that form acyl-ACP thioesters, producing one of the largest sectors of secondary metabolites 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 ofStreptomyces coelicolor malonyl-CoA:ACP transacylase.Structure 11, 147-54.; Summers, R. G., Ali, A., Shen, B., Wessel, W. A., and Hutchinson, C. R. (1995) Malonyl-coenzyme A:acyl carrier protein acyltransferase ofStreptomyces glaucescens: a possible link between fatty acid and polyketide biosynthesis.Biochemistry 34, 9389-402.). Therefore, among the enzymes involved in the FAS process, MCAT is considered an essential enzyme in bacterial metabolism activity, and is used as a potential drug target for the development of antibacterial drugs (Campbell, J. W. and Cronan, J. E. Jr. (2001))Escherichia coli FadR Positively Regulates Transcription of the fabB Fatty Acid Biosynthetic Gene.J Bacteriol.183, 5982-90.; Miesel, L., J. Greene, and T.A. Black. 2003. Genetic strategies for antibacterial drug discovery.Nature.4, 442-456.; White, S. W., Zheng, J., Zhang, Y. M., 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 is conceived by the necessity of the above, and an object of the present invention is to provide a compound having anti-bioactivity.

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

For the above object, the present invention provides a compound of any one of Formulas 1 to 3 or a pharmaceutically acceptable salt thereof.

[Formula 1]

[Formula 2]

[Formula 3]

In one embodiment of the present invention, the compound preferably inhibits malonyl-CoA: acyl carrier protein transacylase (MCAT) activity, but is not limited thereto.

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

In addition, the present invention provides a feed composition for preventing or treating infections caused by pathogens comprising a compound of any one of Formulas 1 to 3 of the present invention and a feed rationally acceptable carrier.

In another aspect, the present invention provides an agrochemical composition for preventing or treating infection comprising a 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 an activity to inhibit microbial lipid synthesis, but is not limited thereto.

In addition, the pharmaceutical composition of the present invention may further include pharmaceutically acceptable excipients, carriers, diluents, and the like.

Carriers usable in the present invention include liposomes, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and slowly metabolized macromolecules such as 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 substances such as wetting agents, emulsifiers or pH buffering substances can be used.

A pharmaceutically acceptable carrier is described in Remingtion's Pharmaceutical Sciences, Mack Publishing Company, 1991.

In addition, the composition is formulated in a unit dosage form suitable for administration in the body of a patient according to a conventional method in the pharmaceutical field, preferably in a form useful for administration of compound drugs, and administration commonly used in the art. Oral, or skin, intravenous, intramuscular, intraarterial, intramedullary, intrameningal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, gastrointestinal, topical, sublingual, vaginal or rectal using the method. It may be administered by a parenteral route including the route, but is not limited thereto.

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

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

Tablets, troches, pills, capsules, etc. may contain one or more of the following ingredients: binders such as microcrystalline cellulose, gum tragacanth, 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 such as magnesium stearate or Sterotes; Lubricants such as colloidal silicon dioxide; Sweeteners such as sucrose, fructose, lactose, saccharin or aspartame; Flavoring agents such as peppermint, methyl salicylate, winter green oil or cherry flavor; And peptide antimicrobial agents such as Enbuvirtide (FuzeonTM).

When the unit dosage form is a capsule, it may contain, in addition to the above type of material, a liquid carrier such as vegetable oil or polyethylene glycol. Various other materials may be present as a coating agent or to alter the physical form of the solid unit dosage form.

The pharmaceutical composition of the present invention may be prepared, packaged or marketed 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 colon washing. The pharmaceutical composition of the present invention may also be prepared, packaged or marketed in a formulation suitable for vaginal administration. Such compositions may be, for example, in the form of suppositories, impregnated or coated vaginal inserts, such as tampons, washing preparations, solutions for vaginal washing.

The pharmaceutical composition of the present invention may be prepared, packaged or marketed in a formulation suitable for administration to the eye. For topical administration, the compounds of the present invention can be applied in pure form, i.e. in liquid. However, typically, the compounds can be administered to the skin as a composition or formulation combined 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 mixtures of two or more of these, wherein the compounds of the present invention can be dissolved or dispersed to an effective level, optionally with non-toxic surfactants.

The pesticide composition according to the present 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 to which the active material is bound to facilitate application, particularly to parts of plants. Therefore, this support should generally be inert and agriculturally acceptable.

Retardation can be solid or liquid. Examples of suitable supports include clays, natural or synthetic silicates, silicas, resins, waxes, solid fertilizers, water, alcohols, especially butanol, organic solvents, mineral and vegetable oils and derivatives thereof. Mixtures of the above supports may also be used.

The pesticide composition may also contain other additional ingredients. In particular, the pesticide composition may further contain a surfactant. Surfactants may be ionic or nonionic emulsifiers, dispersants or wetting agents or mixtures 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 (especially alkylphenols or arylphenols). , Sulfosuccinic acid ester salts, taurine derivatives (especially alkyl taurates), phosphoric acid esters of polyoxyethylated alcohols or phenols, fatty acid esters of polyols, and derivatives of the above compounds having sulfate, sulfonate and phosphate functional groups. When the active substance and/or inert support is water insoluble and the vector reagent for the application is water, it is generally essential that at least one surfactant is present. Preferably, the surfactant content may be 5 to 40% by weight of the composition.

Additional ingredients may also include, for example, protective colloids, tackifiers, thickeners, thixotropic agents, penetrants, stabilizers, sequestering agents. More generally, the active substance can be combined with any solid or liquid additives 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 the active substance.

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

*These compositions include heavy-duty compositions to be applied directly to plants or seeds to be treated using a suitable device such as a spray or spray device, as well as commercially available concentrated compositions that must be diluted prior to application to crops.

The pesticide composition of the present invention can be used not only for therapeutic or prophylactic control of phytopathogenic bacteria of crops, but also for treatment or prophylactic control of insects.

Accordingly, according to a further aspect of the present invention, an effective non-phytotoxic amount of the composition as defined above is applied to the seed, leaf application, stem application, or seed, plant and/or the fruit or plant of the plant is growing or growing it. Soil and/or inert substrates to be made (e.g. organic substrates (e.g. sand, rock wool, glass wool, expansive minerals (e.g. perlite, vermiculite, zeolite, expandable clay)), sockstone, granite/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. floating hydroponic systems, Nutrient Film Technique, Aeroponics), characterized by applying through drench/drip application (irrigation), not only therapeutically or prophylactically exterminating phytopathogenic bacteria of crops, but also curative or prophylactic extermination of insects Is provided.

The expression "effective non-phytotoxic amount" of the composition according to the invention is sufficient to control or eradicate pests and/or diseases present or prone to appear in the crop, but not accompanied by any significant phytotoxic symptoms to the crop. Means sheep. These amounts can vary widely depending on the pests and diseases to be combated or controlled, the type of crop, climatic conditions and the compounds to be included in the composition according to the invention. The amount can be determined by those skilled in the art, and can be determined through systematic field experiments.

The treatment method according to the invention may be useful for the treatment of propagation material, 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 aboveground parts of plants such as the body, stem or stalk, leaves, flowers and fruits of the plant concerned.

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

Further, the feed composition obtained by adding the compound of the present invention or a salt thereof is used as a feed for animals such as mammals, birds, reptiles, amphibians and fish, preferably for mammals. For example, the feed of the present invention is used as feed for pets such as dogs, cats, and mice, feed for livestock such as cattle and pigs, feed for poultry such as chickens and turkeys, and feed for farmed fish such as sea bream and yellowtail. , It is preferably used as feed for pets or feed for livestock.

The feed of the present invention can be made by appropriately blending the compound of the present invention or a salt thereof with feed raw materials. Feed raw materials include grains, crude steel, vegetable oil meal, animal feed raw materials, other feed raw materials, refined products, and the like.

Examples of grains include myro, wheat, barley, oats, rye, brown rice, buckwheat, crude, sorghum, blood, corn, soybeans, and the like.

Examples of crude bran include rice bran, skim rice bran, bran, powder, wheat, germ, barley bran, screening, pellets, corn bran, corn germ, and the like.

As vegetable oil meal, soybean meal, soybean meal, flax meal, cotton seed meal, peanut meal, safflower meal, palm leaf, palm leaf, pumpkin meal, sunflower meal, rapeseed meal, kapok meal, mustard meal, etc. are mentioned, for example.

As raw material for animal feed, for example, fish meal (northern wheat, imported wheat, whole wheat, coastal wheat), fish brush, meat meal, fish bone meal, blood meal, decomposed hair, bone meal, processed by-products for livestock, feather mill, silkworm, skim milk powder, Casein, dry whey, and the like.

Other feed ingredients include plant foliage (alfalfa, hay cube, alfalfa leaf powder, similar acacia powder, etc.), corn processing industrial by-products (corn gluten, wheat, corn gluten feed, corn staples, etc.), starch processed products, sugar, fermentation. Industrial products (yeast, beer meal, malt root, alcohol meal, Jang oil meal, etc.), agricultural production by-products (mandarin orange processed meal, tofu meal, coffee meal, cocoa meal, etc.), others (cassava, bean beans, guam wheat, seaweed, etc.) Krill, spirulina, chlorella, minerals, etc.), etc. are mentioned.

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

As can be seen through the present invention, the present inventors discovered three kinds of lead compounds that inhibit bacterial cell growth in the range of ~ ug/ml, and their direct binding to the target protein was confirmed using NMR results.

Figure 1 is a diagram showing a type II bacterial fatty acid synthesis pathway.
Fig. 2a is an SDS-PAGE picture of purified fabD (Xoo0880), and 2b is a picture of crystals grown in 0.1 M CHES pH9.0, 1.5 M NH ₄ SO ₄ , and 0.2 M NaCl.
Figure 3a is the overall structure of XoMCAT and 3b is a detailed view of its active site.
4 is a diagram schematically illustrating a VLS experiment for searching for an antagonist drug for a specific target protein.
5 is a diagram showing the process of a bacterial cell growth inhibition assay with a selected compound derived from VLS.
^{FIG. 6 shows 1D 1} H-NMR spectra for Formulas 1 (FIG. 6a ), 2 (FIG. 6b ), and 3 (FIG. 6c) of the present invention, which are XoMCAT lead compounds. This spectrum shows the degree of affinity of the compound for XoMCAT.

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention, and the scope of the present invention is not to be interpreted as being limited by the following examples.

Manufacturing example : Preparation of the compound of the present invention

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

Example 1: Xanthomona soryzae pv . oryzae XoMCAT a claw turning, expression derived from (Xoo), purification, crystallization, crystal structure determination

Cloning

The FAD coding sequence of Xoo0880 was amplified through 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 are 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 tobacco etch virus (TEV) protease cleavage site and an additional residue of 6xHis were added in front of the Nde I site in pET11a vector (Novagen) to facilitate purification of the expressed protein. It was ligated with a modified pET11a vector (His-TEV-pET11a) designed to have.

Overexpression and purification

His-TEV-pET11a expression vector containing the coding sequence of Xoo0880 was E. coli Put in BL21 (DE3). Cells were grown at 37° C. until an _{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 medium. After induction, the cells were incubated for an additional 16 hours at the same temperature. The cultured E. coli was collected by centrifugation (Vision VS24-SMTi V5006A rotor) at 6,000 rpm for 20 minutes at 277K. Then, the collected E. coli was resuspended in a lowered cell disruption buffer A [25 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM Imidazole], and ultrasonic waves (Sonomasher, S & T Science, Korea) were used on ice. And crushed. Thereafter, the lysate was centrifuged at 277K and 12,000 rpm for 30 minutes to obtain a cell extract (Vision VS24-SMTi V508A rotor). Only 10% of the total expressed Xoo0880 was water soluble (not shown). In order to use the affinity purification method, a supernatant containing water-soluble fabD was loaded into Ni-NTA resin (Novagen). Affinity purification was performed at 4° C. according to the manufacturer's protocol. Then, the fabD protein with 6xHis-tag was eluted using crushing buffer A containing 200 mM imidazole, and treated with TEV protein at 4° C. overnight to remove 6XHis-tag. The thus-treated protein solution 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 through an automated pipetting system, Hydra II e-drop (Matrix), using several screening kits such as Crystal screen HT, Index HT, and Salt Rx HT (Hampton Research). high throughput (HT)] decision screening started. 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). Regenerated using the hanging drops method, crystals were made for a week. The crystals after growth were frozen at 100K in liquid nitrogen after adding antifreeze 20% (v/v) glycerol to crystallization conditions. Used for data collection. Crystal data was collected up to a resolution of 1.9 Å using an ADSC Quantum 270 CCD detector on beamline 17A of Japan Photon Factory. 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.). Through the auto-indexing process, the decision space group of _{P2 1} 2 ₁ 2 ₁ and the unit cell parameters a= 41.4, b= 74.6, and c = 98.5 were deduced.

The crystal structure of XoMCAT and its active site residues are shown in FIG. 3. The entire structure of XoMCAT consists of two domains, a larger domain and a smaller domain, and folds stably. Its active site is located in a canyon between these domains. The larger domains are 14 alpha-helix structures and 8 betas with ^{an accessible surface area of 12585Å 2} calculated from the DSSP program (Kabsch, W. and Sander, C. (1983). Biopolymers 22, 2577-637.) -It is composed of a linear structure. Ser-94, a nucleophyllic activating residue, is located in the sharp turn located between the alpha-helix and beta-linear structure of the larger domain, while the other active site residues Gln 13, Arg 119 and His 203 are near the nucleophyllic residues. Located.

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

Xoo0880 (XoMCAT) is one of the potential target genes for rice white leaf blight based on GlaxoSmithKline (GSK), so it is antagonistic to the substrate binding site of XoMCAT enzyme through high-efficiency (HT) virtual library screening (VLS, FIG. Drug lead compounds were searched.

For drug screening, a computer simulation, VLS, was performed using a database of 18 million compounds in the ICM Molsoft modeling suite. Among the VLS, the substrates of each flexible structure existing in the compound database found through the Lipinski formula were docked to the substrate binding pocket grid of XoMCAT using ICM, and a score reflecting the quality of the complex was assigned. In order to check the reproducibility of each substrate binding result, it was performed three times in parallel at the same time. 1991 compounds with a top score were selected, and each of its complexes was examined by visualization (numerization) with factors such as shape complementarity, hydrogen bonding network, and flexibility of the ligand. 500 compounds were obtained based on the final selection step for characterization through experimental analysis.

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

3 ml of Xoo cultured overnight was inoculated into 100 ml of NB medium. The bacterial culture was incubated until an _{OD 600 reached 0.6.} 150 μl of bacterial culture was transferred to a 96 well ELISA plate containing 1 mM of the screened compound retrieved from VLS. DMSO was used to dissolve the compound into a stock compound at a concentration of 30 mM. The DMSO concentration was kept below 5% for all bacterial growth assays, and the negative control only used the same concentration of DMSO. The plate was incubated at 28° C. with continuous shaking, and the absorbance was measured at a constant interval of 0, 3, 6, 9, and 12 hours at 600 nm. In order to measure the MIC ₅₀ value of the compound, compounds that inhibit Xoo growth by 50% or more were selected.

MIC ₅₀ Way( Microtiter Plate-based Antibacterial analysis)

3 ml of Xoo cultured overnight was inoculated into 100 ml of NB medium. The bacterial culture was incubated to a cell density of 0.5 McFarland standard. The culture solution was diluted 10-fold, and 150 µl of the diluted culture solution was separated on a sterilized 96-well clear flat bottom plate. Selected compounds having high bacterial cell growth inhibitory activity were serially diluted and added to a 96-well plate at a concentration of 600 μM to 9.5 μM. A bacterial culture medium having 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. The percent inhibition of bacterial growth was calculated by the following 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.

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

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

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: XoMCAT using NMR Analysis of binding of lead compounds to target proteins

It was tested whether a screened compound with high bacterial cell growth inhibitory activity (~ μg/ml MICs) directly binds to its target protein. The NMR experimental method was used to confirm the direct binding between the compounds and the target protein XoMCAT.

NMR Experiment

A highly purified protein stock solution (Xoo PDF) was prepared in 25 mM Tris pH 7.5, 10 mM NaCl, and 3 mM 2-ME buffer at a concentration of 100 μM. 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. 1D NMR spectra were measured for the inhibitor-only solution and the two types of samples to which the inhibitor and PDF were put together. 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. The NMR spectrum was recorded using a 500 MHz 1D NMR Bruker DRX at the Korea Basic Science Institute (KBSI) in Daejeon.

NMR result

^{1D 1} H-NMR spectra were measured for titration of specific concentrations of potential lead compounds (Figure 5). As the protein concentration increases, the height of the substrate-only NMR signal decreases due to the binding between the protein and the substrate. Using this method, the inventors were able to discover proteins and high affinity compounds. (Fig. 5).

Attach electronic file of sequence list

Claims (8)

A pharmaceutical composition for preventing or treating infection by a pathogen comprising a compound of Formula 2 and a pharmaceutically acceptable carrier.

(2) delete delete According to claim 1, 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 (2) and a feedstock acceptable carrier.

(2) 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 2 and an agriculturally acceptable carrier.

(2) 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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