WO2017179897A1 - Kit pour le criblage de substance antibactérienne à l'aide de thiamine monophosphate kinase et procédé de criblage - Google Patents

Kit pour le criblage de substance antibactérienne à l'aide de thiamine monophosphate kinase et procédé de criblage Download PDF

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WO2017179897A1
WO2017179897A1 PCT/KR2017/003924 KR2017003924W WO2017179897A1 WO 2017179897 A1 WO2017179897 A1 WO 2017179897A1 KR 2017003924 W KR2017003924 W KR 2017003924W WO 2017179897 A1 WO2017179897 A1 WO 2017179897A1
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thil
atp
screening
antimicrobial
protein
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장수진
김형준
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Institut Pasteur Korea
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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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • 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
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04016—Thiamine-phosphate kinase (2.7.4.16)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/06—Diphosphotransferases (2.7.6)
    • C12Y207/06002—Thiamine diphosphokinase (2.7.6.2)

Definitions

  • the present invention relates to a screening kit and a screening method of an antimicrobial agent using thiamine monophosphate kinase (ThiL), and more particularly to screening a substance that inhibits ThiL involved in the thiamine biosynthetic pathway, an essential path of bacteria.
  • Kits and methods can be screened for substances that are effective in inhibiting and killing bacteria to provide novel antimicrobials.
  • antibiotics or antimicrobials
  • antibiotics can often be ineffective because of their inevitable resistance to antibiotics.
  • the selection and dissemination of intrinsic or acquired resistance to antibiotics between bacteria interferes with chemical treatment methods, including the various antibiotics known to date, and enhances the development of bacterial species with multiple antibiotic resistances. For this reason, the mortality rate of patients infected with bacteria has recently increased. Therefore, there is a strong need to develop new treatment and prevention strategies that can control infection by bacteria.
  • Pseudomonas aeruginosa is known to be highly adaptable to the environment that promotes ecological aptitude despite the presence of traditional antibiotic treatment methods.
  • alternatives such as phage treatment methods for treating Pseudomonas-mediated infection are emerging.
  • Korean Patent Laid-Open Publication No. 10-2015-0015589 discloses that the protein expressed by PA4834 serves as a siderophore transporter associated with the iron transport mechanism essential for the growth of Pseudomonas aeruginosa. Although methods have been disclosed for screening Pseudomonas aeruginosa antibiotics, the discovery of new targets for antibiotics of Pseudomonas aeruginosa infection is still required.
  • Biochemical pathways control the synthesis and metabolism of molecules required by all living things. Blocking metabolic pathways by inhibiting one or more enzymes in the pathway can sometimes lead to deleterious consequences for the organism or to certain afflictions or deaths, such as birth defects in metabolism. The suppression of pathways that ultimately cause the death of organisms is the priori inhibition of the pathways required for survival and is defined as an essential pathway.
  • TPP Thiamine pyrophosphate
  • TPK thiamine pyrophosphokinase
  • the transported thiamin is first converted to thiamine monophosphate (TMP n) by ThiK, after which the second phosphorylation reaction is the last enzyme in the thiamin biosynthetic pathway, the thiamine monophosphate kinase (ThiL). Takes place).
  • the present inventors have diligently sought to discover new targets of antimicrobial agents against Pseudomonas aeruginosa infections, suggesting that ThiL may be a good target for antimicrobial molecules since it is essential for TPP biosynthesis as well as thiamine salvage pathway.
  • the present invention was completed by discovering and developing a ThiL kinase assay kit.
  • the present invention has been made to solve the above problems, and provides a kit that can screen a substance that inhibits the activity of thiamine monophosphate kinase (ThiL).
  • Another object of the present invention to provide a screening method of the antimicrobial material using the screening kit.
  • thiamine monophosphate kinase thiamine monophosphate kinase, ThiL
  • Adenosine triphosphate (ATP) thiamine monophosphate (TMP)
  • TMP Thiamine monophosphate
  • the antimicrobial material may exhibit antimicrobial activity against bacteria that do not have a thiamine pyrophosphokinase (TPK) protein or a gene encoding the same.
  • TPK thiamine pyrophosphokinase
  • the bacteria may be any one selected from the group consisting of Escherichia coli , Salmonella species and Pseudomonas aeruginosa belonging to the enterobacteria.
  • the reagent capable of measuring the amount of ATP may indicate the amount of ATP through the intensity of luminescence.
  • the present invention also provides a method for screening an antimicrobial substance using the screening kit.
  • the screening method comprises the steps of: a) adding buffer, TMP and ATP in order to tube 1; b) adding distilled water, ThiL protein and candidates to tube 2; c) mixing tubes 1 and 2; d) adding the mixture of step c) to a test well comprising a reagent capable of measuring the amount of ATP; And e) measuring the light emission of the test well of step d).
  • the activity of ThiL based on the intensity of luminescence measured in step e) is inhibited by more than 70% compared to the activity of ThiL measured in the control group without addition of the candidate
  • the candidate substance may be determined as an antibacterial substance.
  • the antimicrobial agent may exhibit antimicrobial activity against thiamine pyrophosphokinase (TPK) protein or bacteria that do not have a gene encoding the same.
  • TPK thiamine pyrophosphokinase
  • the bacteria may be any one selected from the group consisting of Escherichia coli , Salmonella species and Pseudomonas aeruginosa belonging to the enterobacteria.
  • the screening kit provided in the present invention can be used to quickly and easily screen antimicrobial agents against bacteria that do not have thiamine pyrophosphokinase (TPK), including Pseudomonas aeruginosa, by measuring the level of inhibitory activity of ThiL.
  • ThiL assays based on the screening methods of the present invention are substrate specific and potent, making them very useful for detecting ThiL inhibitors. Accordingly, the screening kit and method of the present invention may provide a novel antimicrobial agent that does not have a TPK protein or a gene encoding the same, thereby effectively inhibiting or killing an essential bacterium.
  • 1 is a schematic diagram showing the thiamine biosynthetic pathway and TPP dependent enzymes of Pseudomonas aeruginosa.
  • Figure 2 is a graph showing the growth rate of wild-type and thiE mutant Pseudomonas aeruginosa according to the concentration of TPP through the OD value and a table showing the concentration of TPP in humans.
  • FIG. 3 shows a cleavage map of a recombinant vector expressing ThiL protein.
  • Figure 4 shows the enzyme activity according to ThiL protein concentration when performing the ThiL kinase assay as RLU (Relative light unit) value indicating the ATP consumption.
  • 5 is a result showing the activity of ThiL by measuring the amount of ATP over time after reacting with different substrate conditions when performing the ThiL kinase assay.
  • Figure 6 shows the activity of ThiL by measuring the amount of ATP over time after the reaction using TMP and thiamine as a substrate when performing the ThiL kinase assay.
  • FIG. 7 is a graph showing the growth rate of thiE variants at various thiamine concentrations.
  • ThiL may be a good target for antimicrobial molecules and sought to solve the above-mentioned problems by providing a kit for screening antimicrobial agents by measuring ThiL kinase activity.
  • TPK thiamine pyrophosphokinase
  • the kit of the present invention it is possible to quickly and easily screen antimicrobial agents against bacteria that do not have thiamine pyrophosphokinase (TPK) including Pseudomonas aeruginosa.
  • TPK thiamine pyrophosphokinase
  • it can provide a novel antimicrobial material that can effectively inhibit or kill bacteria that do not have a gene encoding it.
  • the present invention relates to thiamine monophosphate kinase (ThiL); Adenosine triphosphate (ATP); Thiamine monophosphate; It provides an antimicrobial screening kit comprising; and a reagent capable of measuring the amount of ATP.
  • screening kit refers to a kit used to select a substance having a particular effect from among a plurality of candidates.
  • the screening kit is not particularly limited thereto, but may be a kit used to select a substance capable of effectively inhibiting or killing bacteria by inhibiting the activity of ThiL.
  • antimicrobial material in combination, but all means a material that exhibits antimicrobial activity to bacteria.
  • Pseudomonas aeruginosa does not have a TPK homologous gene in a genome similar to Escherichia coli having only thiamine kinase (ThiK) instead of TPK.
  • ThiK thiamine kinase
  • the transported thiamin is first converted to Thiamine monophosphate by ThiK, and then the second phosphorylation reaction is via thiamine monophosphate kinase (ThiL), the last enzyme in the thiamin biosynthetic pathway.
  • ThiL is essential for TPP biosynthesis as well as thiamine salvage pathway and may be a good target for antimicrobial molecules.
  • FIG. 2 shows that thiE mutants that cannot produce TPP and must rely on extracellular TPP for growth require TPP supplementation above 100 uM for growth similar to wild type. However, ThiL inhibition will kill Pseudomonas aeruginosa in the human body because only 140 nM TPP is available for human blood.
  • the antibacterial material screened with the kit of the present invention may not have a TPK protein or a gene encoding the same, and thus may exhibit antimicrobial activity against all bacteria in which ThiL is essential.
  • Escherichia coli Salmonella belonging to the enterobacteria. ( Salmonella species) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) may exhibit antimicrobial activity against any one selected from the group consisting of, but is not limited thereto.
  • thiL gene sequence information used in the recombinant vector may be obtained from a known database, and specifically, the thiL gene sequence may be that disclosed in NCBI Reference Sequence NP_252740.1.
  • the wild-type ThiL of Pseudomonas aeruginosa isolated through the expression of the recombinant vector was purified as a recombinant protein having a His tag, and as shown in FIG. 4, the consumption of ATP increased according to the throughput of the purified protein. It can be seen that ThiL protein isolated and purified through the activity.
  • the reagent capable of measuring the amount of ATP is not particularly limited as long as it can measure the amount of ATP, but may be to measure the amount of ATP through the intensity of luminescence.
  • the amount of ATP remaining after the reaction was measured using a commercially available Kinase-glo ® reagent (Promega).
  • the present invention also provides a method for screening an antimicrobial substance using the screening kit.
  • the screening method comprises the steps of: a) adding buffer, TMP and ATP to tube 1 in order; b) adding distilled water, ThiL protein and candidates to tube 2; c) mixing tubes 1 and 2; d) adding the mixture of step c) to a test well comprising a reagent capable of measuring the amount of ATP; And e) measuring the light emission of the test well of step d).
  • step a) will be described.
  • the buffer of step a) is not particularly limited as long as it is a commonly used reaction buffer, but preferably may be made of the composition of Table 2.
  • ATP and TMP are used as substrates for ThiL, in particular ATP is used as a measure of the activity of ThiL. ATP and TMP can be used after diluting with distilled water in consideration of the amount of use.
  • step b) will be described.
  • ThiL in step b) is a kinase that produces TPP using ATP and TMP as substrates, and can be used commercially available or prepared.
  • a pET 28b thiL (BL21) vector was prepared as described above to prepare a wild type ThiL recombinant protein of Pseudomonas aeruginosa having His tag.
  • Candidate material in step b) refers to a substance that can be applied to a bacterial infection patient to improve or beneficially modify the patient's symptoms, and a substance that inhibits ThiL may be included without limitation.
  • step c) will be described.
  • Step c) is a step of mixing the tubes 1 and 2 prepared in step a) and b), for example, can be mixed using a pipette.
  • step d) is added to add the mixture to a test well containing reagents capable of measuring the amount of ATP.
  • the mixing time is set to time 0. In one embodiment of the present invention, every 5 minutes after time 0, the mixture is transferred to a test well containing a reagent capable of measuring the amount of ATP, and 10 minutes after the last test for stabilization of the luminescence signal. Step e) was performed.
  • the step e) is to measure the amount of ATP remaining after the reaction in which ThiL produces TPP using ATP and TMP as a substrate, and the intensity of luminescence represented by the reagent for measuring the amount of ATP is measured using a photometer. Can be measured.
  • the candidate When the activity of ThiL based on the intensity of luminescence measured in step e) is suppressed by 70% or more compared to the activity measured in the control group without adding the candidate, the candidate may be determined as an antimicrobial.
  • FIG. 5 shows that the antimicrobial screening method of the present invention uses a substrate-specific ThiL kinase reaction to detect only ThiL activity by ATP and TMP.
  • FIG. 6 shows that the ThiL protein (wild type) only reacts when TMP is used as a substrate and does not react when thiamin is used as a substrate.
  • the experiment was carried out as described in Example 7 to establish a criterion for how much inhibition of the activity of ThiL can be judged to be a suitable antimicrobial agent for treating bacterial infections. As a result, as shown in FIG.
  • the substance that inhibits the activity of ThiL by 70% or more inhibits the growth of Pseudomonas aeruginosa that is resistant to natural and existing antibiotics, and is an antibacterial substance suitable for combination treatment with single or other antimicrobial agents in the prevention or treatment of Pseudomonas aeruginosa infection.
  • Thiamine-phosphate synthase (thiE) transposon variant Pseudomonas aeruginosa was purchased from the University of Washington, LB supplemented with 60 ⁇ g / ml tetracycline for variant selection and 50 ⁇ M thiamine for normal growth. (Luria-Bertani) medium was maintained. As a control, PAO1 (wild type) was maintained in LB medium without supplement. Both strains were shaken at 180 rpm at 37 ° C.
  • TPP Thiamine and thiamine pyrophosphate
  • PAO1 and thiE variants were purified using M9 glucose medium (Na 2 HPO 4 6.78 g / L, KH 2 PO 4 3 g / L, NH 4 Cl 1 g / L, NaCl 0.5 g / L, MgSO 4 0.49 g / L, CaCl 2 0.014 g / L, glucose 3.6 g / L) and M9 glucose thiamine 50 ⁇ M medium, respectively.
  • M9 glucose medium Na 2 HPO 4 6.78 g / L, KH 2 PO 4 3 g / L, NH 4 Cl 1 g / L, NaCl 0.5 g / L, MgSO 4 0.49 g / L, CaCl 2 0.014 g / L, glucose 3.6 g / L
  • M9 glucose thiamine 50 ⁇ M medium M9 glucose thiamine 50 ⁇ M medium
  • bacterial cultures were diluted in M9 glucose mucin (2.5 / L) medium to obtain a final inoculation size of 0.00025 of OD 600 .
  • 45 ⁇ l of the diluted culture was transferred to a testing well containing 5 ⁇ l of TPP.
  • the test was performed in 384-well microplates (Greiner # 781091) with black transparent bottoms. Empty wells were filled with PBS to minimize edge effects by dehydration and heat distribution.
  • the plate was placed in a 37 ° C. incubator for 18 hours. Growth was measured by absorbance (600 nm) using a spectrophotometer.
  • FIG. 2 shows that thiE mutants that cannot produce TPP and must rely on extracellular TPP for growth require TPP supplementation above 100 uM for growth similar to wild type.
  • ThiL inhibition will kill Pseudomonas aeruginosa in the human body because only 140 nM TPP is available in human blood as shown in the table of FIG. 2.
  • pET 28b thiL (BL21) recombinant vector having a cleavage map of FIG. 3 was prepared by the following procedure.
  • a thiL gene of Pseudomonas aeruginosa is amplified by a polymerase chain reaction (PCR) by constructing a primer (TAGGTTCATATGGGTGAGTTCGAGCTGATCCGCC, reverse primer: TAGGTTAAGCTTTCAGTCACGTTGGGTTCCGAAATGTTGG) to include NdeI and HindIII restriction endonuclease base sequences.
  • the amplified thiL gene and the obtained pET28b vector are cut using restriction enzymes, and then ligation is performed to generate a recombinant vector.
  • the recombinant vector was transformed into E.
  • E. coli BL21 is then applied to LB agar containing 50 ⁇ g / ml of Kanamycin, a selection marker, to obtain transformed E. coli BL21. Transformation E. coli BL21 is confirmed by sequencing and PCR methods.
  • the culture was transferred to a centrifuge tube (50 ml), centrifuged at 3300 rpm for 10 minutes and the supernatant was discarded.
  • the cell pellet can then be stored at -20 ° C.
  • Cell pellets were resuspended in 5 ml of DPBS with protease inhibitor. After 10 cycles (total 10 minutes) sonication at 4 ° C., the sonicated bacteria were transferred to a 2 ml tube and centrifuged for 1 minute at 14,000 rpm. Supernatants were collected in 50 ml tubes.
  • the storage buffer in the column was removed and 10 ml of binding buffer was loaded.
  • the collected supernatants were loaded on a column and 10 ml of wash buffer. 1.5 ml elution buffer was separated and loaded three times and only the last two eluates were collected.
  • Protein concentration was measured by the Bradford method and the protein was diluted to 1 mg / ml. Once diluted, the protein was aliquoted in small doses. The protein was quickly frozen in liquid nitrogen and the frozen protein was stored in liquid nitrogen.
  • the ThiL protein assay developed in the present invention is a kinase assay system designed specifically for isolated ThiL proteins.
  • ThiL protein is a kinase that produces TPP based on both ATP and TMP. Therefore, measuring ATP remaining in the reaction quantifies kinase activity.
  • This assay utilizes a Promega kinase-glo ® luminescent kinase assay platform.
  • Kinase-glo ® is dissolved at room temperature by adding buffer (V379B) (dissolved in water for more rapid thawing). The buffer full dose was then added to the substrate bottle, gently shaken and inverted, and the frozen substrate was reconstituted. Kinase-glo ® reagents can be used immediately or aliquoted in small volumes (1 ml) and stored at -20 ° C.
  • ThiL kinase assay conditions are shown in Table 1 below.
  • the reaction buffer is prepared with the composition of Table 2 below.
  • ATP 100 mM
  • TMP 200 mM
  • Tube 1 contains ATP, TMP and reaction buffer.
  • Tube 2 contains the protein dissolved in distilled water.
  • Tube 1 is prepared by adding buffer, TMP, ATP in this order (added in the capacity shown in Table 1), and tube 2 is prepared by adding protein to distilled water (added in the capacity shown in Table 1). Mix well by pipetting tubes 1 and 2 at least five times.
  • tube 2 The entire volume of tube 2 was transferred to tube 1 and mixed well by pipetting.
  • the reaction (tubes 1 and 2) of 50 ⁇ l immediately transferred to a test well containing the Kinase-glo reagent ®. This is time 0. Every 50 minutes, 50 ⁇ l of the reaction is then transferred to a test well containing Kinase-glo ® reagent, wait 10 minutes after the last test to stabilize the luminescence signal, and then at each reaction time using the available photometer (Spectra Max M5). Luminescence was measured. The light emission signal is maintained for at least 1 hour.
  • Assay conditions 20 ⁇ M ATP; 50 ⁇ M TMP; 50 mM Tris-HCl, 5 mM MgCl 2 And a buffer comprising 0.35M KCl; 6, 17, 50 and 150 ⁇ g of ThiL protein dissolved in 300 ⁇ L of total reaction capacity, reaction at room temperature for 1 hour.
  • Assay Condition 1 20 ⁇ M ATP, 50 ⁇ M TMP and 150 ⁇ g ThiL Protein
  • Assay Condition 2 0 ⁇ M ATP, 50 ⁇ M TMP and 150 ⁇ g ThiL Protein (no ATP)
  • Assay Condition 3 20 ⁇ M ATP, 0 ⁇ M TMP and 150 ⁇ g ThiL Protein (no TMP)
  • the substrate specificity of ThiL was further verified using the ThiL protein assay of Example 3.
  • the amount of ATP remaining in the case of reacting with TMP as a substrate and the reaction with thiamin as a substrate was measured and compared.
  • the ThiL protein (wild type) reacted only when TMP was used as a substrate, but did not react when thiamin was used as a substrate. This suggests that the ThiL assay of the present invention is substrate specific and robust and useful for detecting ThiL inhibitors.
  • the thiE variants do not synthesize TPP, their growth depends on thiamine transported from the same environment as the culture medium. On the basis of this fact, in the present Example, the thiE mutant growth rate was measured in a medium containing various concentrations of thiamine to confirm the minimum concentration of thiamine at which the growth of Pseudomonas aeruginosa began to be inhibited. Was estimated.
  • M9 glucose Na 2 HPO 4 6.78 g / L, KH 2 PO 4 3 g / L supplemented with thiE variants of Pseudomonas aeruginosa described in Example 1, respectively, 50 ⁇ M, 16.7 ⁇ M, 5.6 ⁇ M and 0 ⁇ M thiamine, respectively.
  • NH 4 Cl 1 g / L NaCl 0.5 g / L
  • MgSO 4 0.49 g / L
  • CaCl 2 0.014 g / L glucose 3.6 g / L).
  • a substance that inhibits ThiL activity by more than 70% is an antimicrobial material suitable for concomitant treatment with single or other antimicrobial agents in the prevention or treatment of Pseudomonas aeruginosa infection by inhibiting the growth of Pseudomonas aeruginosa resistant to wild-type and conventional antibiotics. You can judge.

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Abstract

La présente invention se rapporte à : un kit pour le criblage d'une substance antibactérienne à l'aide de thiamine monophosphate kinase (ThiL); et un procédé de criblage et, plus précisément, selon l'invention une nouvelle substance antibactérienne peut être fournie par criblage d'une substance, qui est efficace pour l'inhibition bactérienne et la mort de bactéries, grâce à un kit et un procédé pour le criblage d'une substance inhibant ThiL impliquée dans une voie de biosynthèse de thiamine, qui est une voie bactérienne essentielle.
PCT/KR2017/003924 2016-04-12 2017-04-11 Kit pour le criblage de substance antibactérienne à l'aide de thiamine monophosphate kinase et procédé de criblage Ceased WO2017179897A1 (fr)

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