WO2002090548A1 - Methode exploitant l'affinite de la proteine d'adna pour l'atp pour purifier une proteine d'adna initiant la replication d'un adn chromosomique d'une bacterie pathogene, methode d'evaluation d'une activite antibacterienne, et methode utilisant l'affinite de la proteine d'adna pour l'atp pour cribler un compose ayant une act - Google Patents

Methode exploitant l'affinite de la proteine d'adna pour l'atp pour purifier une proteine d'adna initiant la replication d'un adn chromosomique d'une bacterie pathogene, methode d'evaluation d'une activite antibacterienne, et methode utilisant l'affinite de la proteine d'adna pour l'atp pour cribler un compose ayant une act Download PDF

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WO2002090548A1
WO2002090548A1 PCT/JP2002/001913 JP0201913W WO02090548A1 WO 2002090548 A1 WO2002090548 A1 WO 2002090548A1 JP 0201913 W JP0201913 W JP 0201913W WO 02090548 A1 WO02090548 A1 WO 02090548A1
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protein
dnaa
atp
activity
dnaa protein
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PCT/JP2002/001913
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Japanese (ja)
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Kazuhisa Sekimizu
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Genome Pharmaceuticals Institute Co., Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/14—Streptococcus; Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56938—Staphylococcus

Definitions

  • a method for purifying DnaA, a chromosomal DNA replication initiation protein of pathogenic bacteria using the affinity between DnaA protein and ATP, and a method for evaluating antibacterial activity and antibacterial activity using affinity between DnaA protein and ATP Method for screening compounds having
  • the present invention relates to a method for utilizing the affinity between DnaA protein and ATP in purification of DnaA protein, evaluation of antibacterial activity targeting DnaA protein, and screening of compounds having antibacterial activity.
  • DnaA protein is essential for the initiation of chromosomal DNA replication. Mutations in the gene encoding the DnaA protein (dnaA gene) are known to result in abnormal initiation of E. coli chromosomal DNA replication. In fact, many temperature-sensitive mutants of the dnaA gene have been reported in Escherichia coli (Hirota, Y., et al. (1968) Cold Spring Harbor Symp. Quant. Biol. 33: 677). . Therefore, inhibition of DnaA protein activity inhibits bacterial growth.
  • DnaA protein is widely found in bacteria including Escherichia coli, but is not present in eukaryotic cells including humans. . These properties suggest that DnaA protein is promising as a target for clinically effective antibacterial agents. In order to search for an antimicrobial agent with a specific target, a method for measuring the activity of that target must be established. In addition, the target protein must be purified in order to avoid the effect of the mixed protein on the activity measurement.
  • replication proteins are important for the function of the replication proteins, and it is impossible to replace one bacterial system such as E. coli with another. Therefore, in order to purify the DnaA protein using the replication activity as an index, it is necessary to prepare a replication protein corresponding to each of the target bacteria. Unfortunately, it is not easy.
  • an object of the present invention is to provide a method for efficiently purifying a DnaA protein while maintaining its activity. Further, the present invention provides a method for efficiently evaluating an antibacterial activity and a method for efficiently screening a compound having an antibacterial activity, using the activity of the DnaA protein thus purified as an index. Also aim. In particular, an object of the present invention is to provide a method for purifying a DnaA protein targeting a pathogenic bacterium, a method for evaluating an antibacterial activity using the protein, and a method for screening a compound having an antibacterial activity. I do.
  • the present inventors have reported that the Escherichia coli DnaA protein binds to and is activated by ATP (Sekimizu, K. et al. (1987) Cell 50: 259). Since there are many proteins having affinity for DNA, it has been considered difficult for a person skilled in the art to purify the DnaA protein using the ATP binding activity as an index. For this reason, the DnaA protein has not been purified using the ATP-binding activity of the DnaA protein as an index. However, the present inventors have noted the advantage that the ATP-binding activity of the DnaA protein can be easily measured quantitatively, and used A. aureus, which is a clinical problem for opportunistic infection, to measure the ATP-binding activity. As an indicator, we attempted to purify the DnaA protein.
  • DnaA protein and ATP are required for the replication activity of DnaA protein (Sekimizu, K. et al. (1988) J. Biol. Chem. 263: 7124; Sekimizu, K. et al. (1987) Cell 50: 259), a substance that inhibits the binding between DnaA protein and ATP is expected to suppress bacterial growth. Therefore, the binding between DnaA protein and ATP is considered to be an effective indicator for efficient evaluation of antibacterial activity.
  • the present inventors based on the high affinity between the DnaA protein and ATP, the ATP hydrolysis activity of the DnaA protein to which ATP is bound as an index, or the ATP binding activity of the DnaA protein by an activity regulator, We thought that it would be possible to efficiently evaluate antibacterial activity using the control of ATP hydrolysis activity as an index.
  • the present invention provides a method for purifying DnaA protein using affinity between DnaA protein and ATP, a method for evaluating antibacterial activity utilizing affinity between DnaA protein and ATP, and a method for screening a compound having antibacterial activity.
  • a method for purifying DnaA protein using affinity between DnaA protein and ATP a method for evaluating antibacterial activity utilizing affinity between DnaA protein and ATP, and a method for screening a compound having antibacterial activity.
  • (c) a method comprising the step of evaluating whether or not the test sample enhances the ATP hydrolysis activity of the DnaA protein, as compared to the case where the test sample is not contacted with the DnaA protein,
  • test sample has an ATP binding activity of the DnaA protein by a DnaA protein activity regulator or Evaluating whether to suppress the control of ATP hydrolysis activity, a method comprising:
  • an antibacterial agent comprising the compound according to (16) as an active ingredient
  • the present invention provides a method for purifying a DnaA protein of a pathogenic bacterium utilizing the affinity between the DnaA protein and ATP.
  • the purification method of the present invention comprises: (a) a step of introducing an expression vector having a dnaA gene of a pathogenic bacterium into a host cell; and (b) a step of purifying the expressed DnaA protein based on ATP binding activity. , And.
  • the pathogenic bacterium which is a target for purifying the DnaA protein is not particularly limited.
  • the “pathogenic bacterium” means a bacterium capable of infecting a host and causing a disease.
  • target pathogenic bacteria that infect humans include both Gram-negative and Gram-positive bacteria.
  • Gram-negative bacteria include, for example, Pseudomonas aeruginosa, cholera, and pathogenic Escherichia coli (0-157).
  • Gram-positive bacteria include, for example, staphylococcal bacteria such as Staphylococcus aureus. Means 17 types of bacteria that are already known, including Staphylococcus aureus and Staphylococcus epidermidis), but is not limited thereto.
  • the DnaA protein of the pathogenic bacterium has high affinity with ATP.
  • a high affinity for ATP generally refers to a Kd value of 0.3 M or less, more preferably 0.21 M or less (eg, 0.15 / zM).
  • Staphylococcus bacteria such as Staphylococcus aureus are suitable as targets for purification of DnaA protein because their DnaA protein has high affinity for ATP.
  • the dnaA gene of these pathogenic bacteria can be easily obtained by a method such as PCR as a gene having a common sequence with the dnaA gene of Escherichia coli. it can.
  • Pathogenic bacteria whose dnaA gene is difficult to identify by analyzing the base sequence of chromosomal DNA can be easily converted to a gene homologous to the E. coli dnaA gene by a method such as hybridization using the E. coli dnaA gene as a probe. It is possible to obtain.
  • the present inventors have used this method for the first time to isolate the dnaA gene of Staphylococcus aureus (Katayama, H. et al. (1997) Biol. Pharm. Bull. 20: 820). Using these methods, the dnaA gene of the desired pathogenic bacterium can also be cloned.
  • the dnaA gene can be linked to an expression vector and introduced into a host cell using a known genetic engineering technique.
  • the expression vector is not particularly limited as long as it can guarantee expression of the dnaA gene in the host cell. In this example, pHKO11 was used as an expression vector.
  • pHKOll is an expression plasmid for the dnaA gene of Staphylococcus aureus, which has an origin of replication derived from pBR322. Have a resistance gene.
  • the host cells are not limited as long as they do not stop growing or die due to the expression of the dnaA gene, and include Escherichia coli, Staphylococcus aureus, Bacillus subtilis, plant cells, animal cells, and the like.
  • E. coli KA450 strain was used.
  • the KA450 strain is a mutant strain of Escherichia coli lacking the chromosomal DNA origin of replication (oriC) and proliferates by a special DNA replication initiation mechanism called stable DNA replication (K ogoma, T. (1986) J. Bacteriol. 166: 361). .
  • Expression of the dnaA gene of Staphylococcus aureus suppresses the growth of wild-type Escherichia coli, but not this strain (Kata
  • E. coli KA450 is suitable as a host cell for expressing the dnaA gene of S. aureus. Expression of the dnaA gene of other pathogenic bacteria is also expected to suppress the growth of wild-type E. coli, but not this one.
  • the introduction of the dnaA gene-incorporated vector into host cells can be performed, for example, by the electroporation method (Masami Muramatsu, Labo Manual Genetic Engineering 3rd Edition (1996) Maruzen pl46-pU9) and the rubidium-manganese method (H anahan, D. (1983) J. Mol. Biol. 166: 557), but is not limited thereto.
  • Selection of the host cell into which the vector has been introduced can be performed, for example, using a drug resistance marker of the vector.
  • host cells into which the vector has been introduced can be selected on an agar medium containing 50 g / ml of ampicillin.
  • Escherichia coli dnaA gene (Fuller, R.S. and Romberg, A. (1983) Pro
  • a biochemical method for protein purification is used for purification of the DnaA protein expressed in a host cell.
  • a biochemical method for protein purification is used.
  • a protein produced in large quantities by electrophoresis is identified, and the protein is purified using the index as an index.
  • the index is an index.
  • purification is carried out using the high affinity for ATP possessed by the DnaA protein.
  • the DnaA protein and ATP-binding activity are also suitable as purification indicators because they can be easily measured quantitatively.
  • ATP in which the other site of ATP is radiolabeled with 32 P or another nuclide can be used, or another ATP derivative can be radiolabeled and used.
  • a labeling method capable of quantitative measurement such as a fluorescent label can be used. Whether a labeled compound is suitable for quantifying the activity of DnaA protein depends on whether the compound competitively inhibits ⁇ ⁇ ⁇ ⁇ binding to DnaA protein or whether the compound's binding to DnaA protein is competitively inhibited by ATP. It can be evaluated by detecting. ATP 'binding activity can be measured, for example, by the filter binding test shown in Example 4, but various other methods can be used. For example, gel filtration, Western plotting, and a method using surface plasmon resonance phenomenon SPR (BIAC0RE, Hashimoto Setsuko (1997) Bunseki 5: 362) are considered suitable methods.
  • the separation and purification of the protein may be performed by using the separation and purification methods used in ordinary protein purification, and is not limited in any way.
  • chromatographic columns, filters, ultrafiltration, salting out, solvent precipitation, immunoprecipitation, isoelectric focusing, dialysis, recrystallization, etc. can be appropriately selected and combined to separate and purify proteins. it can.
  • the chromatography include affinity mouth chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography.
  • These chromatographic methods include liquid phase chromatography, for example, liquid phase chromatography such as HPL FPLC. It can be performed using oral matography.
  • the final preparation of a protein consisting of a single polypeptide should show a single band of the expected molecular weight from the gene on SDS polyacrylamide gel electrophoresis.
  • the purity was determined based on whether a single band was shown in SDS polyacrylamide gel electrophoresis. If this condition is satisfied, it can be determined that the purification of the DnaA protein has been completed.
  • the basic property that the final preparation should exhibit is high affinity for ATP.
  • a protein having a high affinity for a substance means that the Kd value (dissociation constant) for that substance is significantly smaller than that of other proteins.
  • the method of calculating the Kd value is a basic operation of a biochemical experiment, and various methods can be considered.
  • One way to easily determine the Kd value of DnaA protein for ATP is to perform a filter-binding test in the presence of various concentrations of radiolabeled ATP, and perform a Scatchard plot, as shown in the Examples. Is good, but not limited to this.
  • the Kd value of Escherichia coli DnaA protein with respect to ATP is 0.03 ⁇ M (Sekimizu, K. et al. (1987) Cell 50: 259).
  • the high affinity for ATP specifically means that the Kd value is smaller than 0.3 M.
  • at each stage of the purification check whether the yield and specific activity have been increased.
  • the present invention also provides a DnaA protein of a pathogenic bacterium purified by the above purification method.
  • DnaA protein can be purified with high purity while retaining its activity. Therefore, the DNAA protein purified by the method of the present invention is suitable for evaluation of antibacterial activity and screening of compounds having antibacterial activity.
  • the purity of the purified DnaA protein is preferably 90% or more.
  • the affinity of the DnaA protein to ATP after purification is preferably not more than 0.3; K, more preferably not more than 0.2 (for example, 0.15 M) in Kd value.
  • a preferred embodiment of the DnaA protein of the present invention is a DnaA protein derived from a staphylococcal bacterium (for example, DnaA protein derived from D. aureus).
  • the present invention also provides a method for evaluating the antibacterial activity of a test sample against a pathogenic bacterium by utilizing the affinity between the DnaA protein of the pathogenic bacterium and ATP.
  • the first embodiment of the evaluation method of the present invention utilizing the affinity between ATP and DnaA protein of a pathogenic bacterium is a method using the ATP binding activity of DnaA protein as an index.
  • the DnaA protein binds to ATP, thereby becoming active.
  • Substances that inhibit the binding between DnaA protein and ATP are considered to inhibit the activation of DnaA protein and, consequently, the growth of bacteria. Therefore, whether or not a test sample inhibits the ATP binding activity of DnaA protein is an important indicator of antibacterial activity.
  • the first embodiment of the evaluation method of the present invention comprises: (a) a step of bringing a test sample into contact with a DnaA protein of a pathogenic bacterium; (b) a step of detecting ATP binding activity of the DnaA protein; and (c) Assessing whether or not the test sample decreases the ATP binding activity of the DnaA protein, as compared to the case where measurement is performed using a DnaA protein that has not been brought into contact with the test sample.
  • the second embodiment of the evaluation method of the present invention utilizing the affinity between DnaA protein of pathogenic bacteria and ATP is a method using the ATP hydrolysis activity of DnaA protein as an index.
  • the DnaA protein that has become active by binding ATP converts the bound ATP to ADP by its own hydrolytic activity and becomes inactive. Therefore, it is considered that a substance that enhances the ATP hydrolysis activity of DnaA protein promotes inactivation of DnaA protein, and thus inhibits bacterial growth. Therefore, whether a test sample enhances the ATP hydrolysis activity of DnaA protein is an important indicator of antibacterial activity.
  • the high affinity between DnaA protein and ATT is an important basis for detecting the ⁇ -hydrolysis activity of DnaA protein.
  • the second embodiment of the evaluation method of the present invention comprises: (a) a step of bringing a test sample into contact with a DnaA protein of a pathogenic bacterium to which ATP has been bound; (b) a step of detecting ATP hydrolysis activity of the DnaA protein; and (C) The test sample enhances the ATP-hydrolyzing activity of the DnaA protein compared to the measurement using the DnaA protein without contacting the test sample. Assessing whether or not the method is available.
  • the third embodiment of the evaluation method of the present invention utilizing the affinity between the DnaA protein of a pathogenic bacterium and ATP is a method using the control of DnaA protein activity by an activity control factor as an index.
  • DnaA protein activity in pathogenic bacterial cells is controlled by adenine nucleotides bound to the DnaA protein. That is, the ATP-bound DnaA protein has replication activity, but the ADP-bound DnaA protein is inactive (Sekimizu, K. et al. (187) Cell 50: 259)).
  • the activity of the replication initiator protein must be suppressed.
  • a third embodiment of the evaluation method of the present invention comprises: (a) a step of bringing a test sample into contact with a DnaA protein of a pathogenic bacterium; (b) an ATP binding activity or ATP of the DiiaA protein by a DnaA protein activity regulator. (C) detecting the control of hydrolysis activity; and (c) comparing the DnaA protein measured without using the DnaA protein with which the test sample is not in contact with the test sample with the DnaA of the ATP binding activity of the DnaA protein. Evaluating whether or not the control by the protein activity regulator is suppressed.
  • a DnaA protein derived from a desired pathogenic bacterium to be targeted for the development of an antibacterial agent can be used.
  • pathogenic bacteria include both gram-negative and gram-positive bacteria.
  • Gram-negative bacteria include, for example, Pseudomonas aeruginosa, cholera, and pathogenic Escherichia coli (0-157).
  • Gram-positive bacteria include, for example, bacteria belonging to the genus Budococcus such as Staphylococcus aureus. However, these are not limited.
  • the pathogenic bacterium preferably has a DnaA protein having high affinity for ⁇ .
  • High affinity generally has a Kd value of 0.3 zM or less, more preferably 0.2 M or less (eg, 0.15 M).
  • DnaA proteins derived from Staphylococcus spp. Such as Staphylococcus aureus, have a high affinity for ATP and are therefore suitable for evaluating antibacterial activity in the evaluation method of the present invention.
  • the DNA protein purified by the above-described purification method of the present invention has high purity and high affinity for ATP, and thus can be suitably used in the evaluation method of the present invention.
  • the DnaA protein used in the evaluation method of the present invention may be a partial peptide as long as it has an activity that serves as an index for evaluating the antibacterial activity described above.
  • the test sample to be brought into contact with the DNAA protein is not particularly limited, and a desired sample whose antibacterial activity is to be evaluated can be used.
  • the form of the test sample is not particularly limited and includes, for example, cell extract, cell culture supernatant, fermented microorganism product, marine organism extract, plant extract, purified or crude protein, peptide, non-peptide Examples include, but are not limited to, compounds, synthetic low molecular weight compounds, and natural compounds.
  • the detection of the ATP binding activity of the DnaA protein in the first embodiment of the evaluation method of the present invention can be carried out as described in the above-mentioned purification method of the present invention.
  • the ATP binding activity of the DnaA protein detected in the presence of the test sample was significantly (ie, within the range of the experimental error) compared to the case of detection in the absence of the test sample. If suppressed, the test sample is evaluated as having antimicrobial activity. Whether the mode of inhibition of DnaA protein ATP binding activity by a sample evaluated to have antibacterial activity is competitive inhibition or non-competitive inhibition depends on the degree of inhibition when the concentration of ATP is changed. It can be evaluated by checking whether or not to do so.
  • the ATP hydrolysis activity of the DnaA protein can be detected by any of the following methods, but is not limited to these methods.
  • the system for detecting the ATP binding activity of DnaA protein or the ATP hydrolysis activity of DnaA protein is used.
  • a biological substance that controls these activities of the DnaA protein may be added to the system for detecting the activity of DnaA, and the effect of the biological substance on the activity of the DnaA protein may be detected.
  • the biological substance include acidic phospholipids such as DNA polymerase III holoenzyme and cardiolipin, but are not particularly limited as long as they control the activity of DnaA protein.
  • DNA polymerase ⁇ I holoenzyme promotes the hydrolysis of ATP bound to Dna A protein.
  • One of the subunits of the DNA polymerase 111 holoenzyme, / 3, is immobilized on DNA, and a structure called an iS clamp is required for the hydrolysis of ATP bound to the DnaA protein. It has also been found that the activity of an unidentified protein is required for this hydrolysis (Katayama, T. et al. (1998) Cell 94:61).
  • acidic phospholipids inhibit the binding between DnaA protein and ATP (Sekimizu, K. et al. (1988) J. Biol. Chem. 263: 7131).
  • Example 5 shows the inhibition of ATP binding activity of DnaA protein using acidic phospholipids.
  • the control of DnaA protein activity by the activity control factor detected in the presence of the test sample was significantly greater than that detected in the absence of the test sample.
  • test sample is evaluated as having antimicrobial activity.
  • the present invention provides a method for screening for a compound having such antibacterial activity.
  • the compound having an antibacterial activity identified by the evaluation method or the screening method of the present invention is a strong candidate for an antibacterial agent against pathogenic bacteria.
  • mice rats, guinea pigs, egrets, chickens, cats, dogs, sheep, pigs, pigs, monkeys, baboons, and chickens.
  • the compound When used as an antibacterial agent for pansies, the compound itself can be administered in the form of a formulation by a known pharmaceutical method, in addition to being directly administered to the patient.
  • a known pharmaceutical method for example, sugar-coated tablets, capsules, elixirs, microcapsules, orally, or sterile solutions with water or other pharmaceutically acceptable liquids, if necessary. It can be used parenterally in the form of suspension injections.
  • pharmacologically acceptable carriers or vehicles specifically, sterile water or saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives It is possible to formulate a drug product by combining it with a drug, binder and the like as appropriate and mixing it in the unit dosage form generally required for pharmaceutical practice.
  • Administration to patients can be performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, etc., or intranasally, transbronchially, intramuscularly, transdermally, or orally by a method known to those skilled in the art. It can do better.
  • the dosage varies depending on the patient's condition, weight, age, administration method, and the like, but those skilled in the art will be able to appropriately select the dosage of the drug sufficient to produce the intended antibacterial effect. .
  • the disease targeted by the antibacterial agent of the present invention is not particularly limited as long as it is a disease caused by pathogenic bacteria. Specifically, opportunistic infections caused by Staphylococcus aureus and Pseudomonas aeruginosa, diarrhea caused by cholera and Shigella, sepsis caused by Pseudomonas aeruginosa and Staphylococcus aureus, skin wound infection caused by Welsh and Streptococcus pyogenes Diseases, respiratory tract infections caused by Mycobacterium tuberculosis, Influenza bacteria, Legionella bacteria, urinary tract infections caused by gonococci, enteric infections caused by enterohemorrhagic enterobacteria 0-157, and the like, but are not limited thereto. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 is a diagram (left) and a photograph (right) showing the expression of Staphylococcus aureus DnaA protein by the S. aureus DnaA protein mass production system.
  • the structure of the mass-produced plasmid PHK011 is shown on the left.
  • S. aureus DnaA protein is induced by arabinose.
  • SDS polyacrylamide gel electrophoresis of cells before and after induction 3 shows the analysis results.
  • the proteins in the gel were stained with Coomassie-Brilliant Blue-R250. It can be seen that the Staphylococcus aureus DnaA protein having a molecular weight of 52 kDa is induced.
  • FIG. 1 is a diagram (left) and a photograph (right) showing the expression of Staphylococcus aureus DnaA protein by the S. aureus DnaA protein mass production system.
  • the structure of the mass-produced plasmid PHK011 is shown on
  • FIG. 2 is a photograph showing an increase in Staphylococcus aureus DnaA protein content as the purification step proceeds.
  • the protein 5 ⁇ ig in each purification step fraction was analyzed by SDS polyacrylamide gel electrophoresis.
  • the protein in the gel was stained with Coomassie Brilliant Blue R250.
  • FIG. 3 shows the dissociation constant (Kd) of S. aureus DnaA protein with respect to ATP. ATP binding activity was measured for 2.4 pmol of DnaA protein (MonoS column fraction) purified in the presence of various concentrations of radiolabeled ATP. The Scatchard plot is shown on the right. The Kd value was calculated to be 0.15M.
  • FIG. 4 shows the inhibition of ATP binding of DnaA protein by acidic phospholipids.
  • Various amounts of cardiolipin and purified 5 pmol of DnaA protein were added to a buffer containing 1 mol of [ ⁇ - 32 P] ATP, and left on ice for 2 hours.
  • the sample was filtered through a filter (Millipore, HA, 0.45 m, diameter 25 band), and the trapped radioactivity was quantified using a liquid scintillation counter.
  • Example 1 Construction of expression vector for purification of Staphylococcus aureus DnaA protein and induction of expression of Staphylococcus aureus DnaA protein in Escherichia coli
  • pHKOll a plasmid that links the dnaA gene of Staphylococcus aureus downstream of the promoter of the arabinose operon
  • E. coli strain KA450 (Katayama, T. (1994) J. Biol. Chem. 269: 22075) was transformed.
  • the transformant was Ampici Separation was performed on LB agar medium containing 100 g / ml phosphorus and 50 ig / ml thymine.
  • Example 2 Purification of Staphylococcus aureus DnaA protein using ATP binding activity as an index All operations were performed at 4 times unless otherwise noted. The cells were collected by centrifugation (6000 rpra x 10 minutes), and the buffer (50 mM HEPES KOH pH H7.6, 1 M EDTA, 2 mM dithiothreitol, 0.25 M KC1, 20% (v / v ) Suspended in glycerol and stored frozen at -80 ° C. This was slowly thawed on ice, 0.3 mg / ml lysozyme and 20 mM spermidine were added, and left on ice for 30 minutes.
  • the buffer 50 mM HEPES KOH pH H7.6, 1 M EDTA, 2 mM dithiothreitol, 0.25 M KC1, 20% (v / v ) Suspended in glycerol and stored frozen at -80 ° C. This was slowly
  • the samples were frozen by immersion in liquid nitrogen and then slowly thawed on ice. Furthermore, the bacteria were crushed by sonication, and an extract was obtained by centrifugation (35,000 rpm x 30 minutes) (Extract fraction). Ammonium sulfate was added at 0.2 g / ml to the extract and left at 4 degrees for 3 hours. Thereafter, the precipitate was collected by centrifugation (30000 rpni x 20 minutes), and dissolved in 4 ml of buffer (50 mM HEPES K00 ⁇ 7.6, lmM EDTA, 2 dithiothreitol, 10 mM magnesium acetate, 20% (v / v) glycerol).
  • buffer 50 mM HEPES K00 ⁇ 7.6, lmM EDTA, 2 dithiothreitol, 10 mM magnesium acetate, 20% (v / v) glycerol).
  • Ammonium sulfate fraction was dialyzed against the same buffer 11 for 6 hours.
  • the sample was centrifuged (30000n) m for 20 minutes, and the supernatant (Dialysis su P fraction) was packed in a Mono S column (Amersham-Pharmacia Biotech). After washing the column with a buffer (50 mM HEPES KOH pH7.6, lmM EDTA, 2 mM dithiothreyl, 10 mM magnesium acetate, 20% (v / v) glycerol), the DnaA protein adsorbed on the column was washed with 0- It was eluted with a gradient containing 0.5 M KC1 (MonoS column fraction).
  • the ATP binding activity of the preparation at each stage of purification was measured by the method described below.
  • Buff ⁇ — 50 mM HEPES KOH pH7.6, lmM EDTA, 2 mM dithiosley 1 ⁇ , Magnesium lOmM acetate, 20% (v / v)
  • Sample 2 1 diluted in glycerol
  • binding buffer containing [ ⁇ - 32 P] ATP for 1 mol 40mM HEPES K0H ⁇ 7 ⁇ 6, 0.5m EDTA, ImM Jichiosurei , 10% (w / v) sucrose, lOOmM potassium glutamate, 0.05 Big / ml bovine serum albumin
  • the sample was filtered through a filter (MiUipore, HA, 0.5 ⁇ m, diameter 25 face), and the radioactivity trapped was quantified using a liquid scintillation counter (Sekimizu,
  • Example 4 Quantification of affinity of purified S. aureus DnaA protein for ATP ATP binding of 2.4 pmoI of DnaA protein of final preparation (MonoS column fraction) in the presence of various concentrations (0.1 M) of ATP The amount was quantified by the filter-coupled assay described above. The Kd value was calculated from the Scatteryard plot ( Figure 3), and was calculated to be 0.15 3 ⁇ 41. Although the affinity of the DnaA protein for ATP differs depending on the origin of the DnaA protein, it was found from this example that the DnaA protein of S. aureus has a high affinity for ATP.
  • Example 5 Inhibitory effect of acidic phospholipid on ATP binding activity of purified S. aureus DnaA protein
  • the ATP binding activity of the DnaA protein is required for the DNA replication initiation activity of this protein (Mizushima, T. et al. (1996) J. Biol. Chem. 271: 25178). Therefore, a substance that inhibits the ATP binding activity of the DnaA protein of a pathogenic bacterium is considered to inhibit the initiation of DNA replication of the pathogenic bacterium, and is a candidate for a compound having antibacterial activity.
  • the present invention provides an effective method for screening such an antibacterial agent.
  • cardiolipin inhibits ATP-binding activity in the Escherichia coli DnaA protein (Sekiiizu,. Et al. (1988) J. Biol. Chem. 263: 7131).
  • Cardiolipin is an acidic phospholipid present in the cell membrane of pathogenic bacterial cells. Cardiolipin derived from bovine heart was purchased from Sigma. After evaporating the solvent, it was suspended in water by sonication (Branson Ultrasonic Machine 450).
  • the sample was filtered through a filter (Millipore, HA, 0.45 mm, diameter 25 mm) and the trapped radioactivity was quantified using a liquid scintillation counter (Sekimizu, K. et al. (1987) Cell 50: 259).
  • the ATP binding activity of the DnaA protein was almost completely inhibited by 10 ⁇ g of cardiolipin (FIG. 4).
  • DNA replication is essential for the growth of pathogenic bacteria.
  • the DnaA protein is the initiation protein for DNA replication. Therefore, substances that inhibit the activity of DnaA protein may inhibit the growth of pathogenic bacteria.
  • the activity of the DnaA protein is regulated by ATP binding to this protein, and if the regulation mechanism is abnormal, pathogenic bacterial cells cannot grow (Katayama, T. et al. (1998) Cell 94:61).
  • Inhibitors of DnaA protein activity and regulatory mechanisms are expected to inhibit the growth of pathogenic bacterial cells by inhibiting DNA replication or causing abnormal replication of pathogenic bacteria. According to the present invention, it has become possible to efficiently search for such candidate compounds for inhibitors. Further, according to the present invention, it has become possible to efficiently purify high-purity DnaA protein having activity, which is useful for searching for such candidate compounds for inhibitors.

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Abstract

La protéine d'ADNa de staphylococcus aureus peut être purifiée à 99% ou plus à partir d'une fraction d'extrait brut par exploitation de l'activité de liaison à l'ATP comme indication. La protéine d'ADNa ainsi purifiée présente une haute affinité pour l'ATP. Grâce à cette affinité de la protéine d'ADNa purifiée pour l'ATP, une activité antibactérienne peut être évaluée et un composé présentant une activité antibactérienne peut être criblé efficacement.
PCT/JP2002/001913 2001-04-26 2002-03-01 Methode exploitant l'affinite de la proteine d'adna pour l'atp pour purifier une proteine d'adna initiant la replication d'un adn chromosomique d'une bacterie pathogene, methode d'evaluation d'une activite antibacterienne, et methode utilisant l'affinite de la proteine d'adna pour l'atp pour cribler un compose ayant une act WO2002090548A1 (fr)

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JP2001129934A JP2002338595A (ja) 2001-04-26 2001-04-26 ＤｎａＡ蛋白質とＡＴＰとの親和性を利用した、病原性細菌の染色体ＤＮＡ複製開始蛋白質ＤｎａＡの精製方法、並びに、ＤｎａＡ蛋白質とＡＴＰとの親和性を利用した、抗菌活性の評価方法および抗菌活性を有する化合物のスクリーニング方法

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004106539A1 (fr) * 2003-05-30 2004-12-09 Shionogi & Co., Ltd. Procede d'identification d'un inhibiteur de la croissance bacterienne
CN107022644A (zh) * 2017-06-14 2017-08-08 山东省农业科学院农业质量标准与检测技术研究所 果蔬中六种食源性致病菌多重lamp检测引物、检测试剂盒及检测方法

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
KATAYAMA H. et al., "MOLECULAR CLONING AND SEQUENCE ANALYSIS OF THE dnaA GENE OF Stapylococcus aureus". Biol. Pharm. Bull. 1997, Vol.20, No.73, pages 820 to 822 *
KUROKAWA K. et al., "A Stimulation Factor for Hydrolysis of ATP Bound to DnaA Protein, the Initiator of Chromosomal DNA Replication in Escherichia coli." Biochem. Biophys. Res. Commun. 1998, Vol. 243, No. 1, pages 90 to 95 *
SEKIMIZU K. et al., "ATP Activates dnaA Protein in Initiating Replication of Plasmids Bearing the Origin of the E.coli Chromosome". Cell 1987, Vol.50, No.2, pages 259 to 265 *
SEKIMIZU K. et al., "Cardiolipin Activation of dnaA Protein, the Initiation Protein of Replication in Escherichia coli." J. Biol. Chem. 1988, Vol.263, No.15, pages 7131 to 7135 *
SEKIMIZU K. et al., "Sequential Early Stages in the in Vitro Initiation of Replication at the Origin of the Escherichia coli Chromosome". J. Biol. Chem. 1988, Vol.263, No.15, pages 7124 to 7130 *
SEKIMIZU K. et al., "The dnaA Protein of Escherichia Coli. ABUNDANCE, IMPROVED PURIFICATION, AND MEMBRANE BINDING." J. Biol. Chem. 1988, Vol.263, No.15, pages 7136 to 7140 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004106539A1 (fr) * 2003-05-30 2004-12-09 Shionogi & Co., Ltd. Procede d'identification d'un inhibiteur de la croissance bacterienne
CN107022644A (zh) * 2017-06-14 2017-08-08 山东省农业科学院农业质量标准与检测技术研究所 果蔬中六种食源性致病菌多重lamp检测引物、检测试剂盒及检测方法

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