WO2019117273A1 - Procédé de criblage d'agents antifongiques - Google Patents

Procédé de criblage d'agents antifongiques Download PDF

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WO2019117273A1
WO2019117273A1 PCT/JP2018/046013 JP2018046013W WO2019117273A1 WO 2019117273 A1 WO2019117273 A1 WO 2019117273A1 JP 2018046013 W JP2018046013 W JP 2018046013W WO 2019117273 A1 WO2019117273 A1 WO 2019117273A1
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antifungal
erg11
growth
test substance
activity
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PCT/JP2018/046013
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Japanese (ja)
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博治 知花
美智代 佐藤
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国立大学法人千葉大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61P31/10—Antimycotics
    • 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
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
    • 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
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility

Definitions

  • the present invention relates to a method of screening antifungal drugs having a novel mechanism of action.
  • Non-Patent Document 1 Deep fungal infections develop severe opportunistic infections in the liver, kidneys, lungs, etc. of patients who become compromised due to immunosuppressants, anticancer drugs, aging, AIDS, severe diabetes etc. .
  • Non-Patent Document 2 the number of patients worldwide has reached 2 million people / year or more, of which 20-80% have died.
  • Non-patent Document 3 the number of patients worldwide has reached 2 million people / year or more, of which 20-80% have died.
  • the number of patients is about 20,000 / year, of which 20-50% is estimated to be fatal (Non-patent Document 2).
  • Non-Patent Document 1 Non-Patent Document 1
  • Antifungal agents currently used include 5-fluorocytosine which is a nucleic acid synthesis inhibitor, candin type antifungal agent which is a cell wall synthetase inhibitor, and polyene type antifungal agents such as amphotericin B which is an ergosterol binding agent.
  • the drug includes azole antifungal agents which are Ergosterol synthetase Erg11 inhibitors.
  • 5-fluorocytosine has many side effects of renal injury, and resistant bacteria have also appeared.
  • Candin-type antifungal drugs are non-water-soluble and their use is limited, and resistant bacteria are also increasing.
  • Polyene antifungal agents are non-water soluble, have limited use and have strong renal injury side effects.
  • azole antifungal agents are the most widely used antifungal agents, resistant bacteria have recently been increasing.
  • Antifungal drugs Technologies and global markets, Paul Taylor 2017 National Institute for Infectious Diseases, Miyazaki Yoshihisa, General Meeting of the Japanese Society for Medical Mycology (2016)
  • An object of the present invention is to provide a means for developing an antifungal drug with less side effects on humans by a completely new mechanism of action.
  • Fungus is a phylogenetically close organism to a metazoan including humans, and the similarity of target molecules is high between humans and fungi, making it difficult to develop drugs with high efficacy and few side effects.
  • target molecules are high between humans and fungi, making it difficult to develop drugs with high efficacy and few side effects.
  • the present inventors are required as targets for antifungal drugs (1) to be essential molecules for fungi, (2) to be common to pathogenic fungi, and (3) to have low similarity to human genes. Target molecules of antifungal drugs were investigated.
  • Ergosterol is an essential component of cell membranes of fungi, and the most common antifungal drug, the azole antifungal drug, targets the enzyme 14- ⁇ lanosterol dimethylase (Erg11) in the ergosterol synthesis pathway (Erg 11) Table 1).
  • the present inventors have found that 5 out of 12 genes of the ergosterol synthesis pathway using squalene as a substrate, ERG1 (gene encoding Erg1), ERG7 (gene encoding Erg7), ERG11 (gene encoding Erg11), ERG25 (gene) Genes encoding Erg25), ERG26 (gene encoding Erg26) were identified as growth essential genes.
  • ERG7 3 genes, ERG7, ERG25, and ERG26, are not targets for existing antifungal drugs but have potential for new targets, but ERG7 was judged to be unsuitable as a target because it is also highly homologous to human genes.
  • ERG25 and ERG26 were judged to be highly safe because they had low homology to human genes.
  • ERG25 is superior in terms of commonality with fungi but ERG26 is somewhat lower, ERG25 is the best as a new antifungal target, and ERG26 is the next best target It was considered.
  • ERG25 or ERG26 knockdown strain causes growth inhibition, but quite surprisingly, it is combined with an azole antifungal agent which is an Erg 11 inhibitor, and sterol such as serum, bile, cholesterol, ergosterol etc. It was found that the medium contained showed good growth. However, in a medium combined with an azole antifungal agent that is an Erg11 inhibitor, in a medium that does not contain a sterol, such as serum, bile, cholesterol, ergosterol, the ERG25 knockdown strain grows and the ERG26 knockdown strain does not grow I found that ( Figure 2).
  • a substance exhibiting antifungal activity is screened, and the substance is inhibited from expressing ERG11 or the activity of Erg11, for example, using the substance in combination with an azole antifungal substance, It was found that new antifungal drugs targeting Erg25 or Erg26 as a molecular target can be screened if the growth state is evaluated in a medium containing sterols or a medium containing no sterols and substances to be grown are selected. In addition, a test substance having antifungal activity is evaluated in a medium containing sterols to evaluate its action on fungi to select a substance that inhibits growth, and the selected test substance is grown in a state where expression of ERG11 is inhibited. If not, we found that new antimicrobial agents targeting ERG26 could be screened, and completed the present invention.
  • the present invention provides the following [1] to [7].
  • a fungus which is characterized in that a test substance having antimycotic activity is evaluated for an effect on growth of a fungus in a state where the expression of ERG11 is inhibited or the activity of Erg11 is inhibited, and a substance for growing the fungus is selected. Screening method of antifungal drug which targets Erg25 or Erg26 as a molecular target.
  • the test substance having antifungal activity is evaluated for its action on fungal growth in a medium containing sterols, and a test substance that inhibits fungal growth is selected, and in the medium containing sterols, the expression of ERG11 is inhibited or Erg11
  • a method of screening for an antifungal drug molecularly targeting fungal Erg25 or Erg26 which comprises selecting a substance that causes a fungus to grow with inhibition of the activity of [3]
  • Evaluate the effect on fungal growth in a state in which the expression of ERG11 is inhibited or the activity of Erg11 is inhibited in a sterol-free medium in a test substance having antifungal activity, and a substance that causes the fungus to grow is selected
  • a screening method of an antifungal drug which molecularly targets fungal Erg25 characterized in that [4]
  • a test substance having antifungal activity is evaluated for its action on fungal growth with a medium containing sterols, a test substance which inhibits
  • test substance having antifungal activity is a test substance selected by a fungal growth inhibitory action.
  • test substance having antifungal activity is a test substance selected by a fungal growth inhibitory action.
  • test substance having antifungal activity is a test substance selected by a fungal growth inhibitory action.
  • test substance having antifungal activity is a test substance selected by a fungal growth inhibitory action.
  • the screening method according to any one of [1] to [5] which is an evaluation system in which a test substance and an azole antifungal substance are used in combination in a state where the activity of Erg11 is inhibited.
  • An antifungal agent comprising a substance that inhibits the function of fungal Erg25 or Erg26 as an active ingredient.
  • antifungal agents that specifically inhibit the function of Erg25 or Erg26 as molecular targets can be selected.
  • the obtained antifungal agent is Erg25 or Erg26, which is an essential growth component of fungi, and although these targets commonly exist in fungi, they have high safety because they have very low similarity to humans. , Acting specifically on fungi.
  • Erg25 and Erg26 do not easily develop tolerance because they do not substitute for cholesterol like Erg11. Therefore, an antifungal drug which uses Erg25 or Erg26 as a molecular target as an active ingredient is highly safe and is useful as an antifungal drug which is less likely to develop resistance.
  • Figure 2 shows the TetOFF system used to determine growth essential genes in the ergosterol synthesis pathway in Candida glabrata. The effects of serum and fluconazole at knockdown of growth essential genes in the ergosterol synthesis pathway are shown.
  • the screening method for an antifungal drug molecularly targeting the fungus Erg25 or Erg26 of the present invention has an effect on the growth of the fungus with the test substance having antifungal activity being inhibited by the expression of ERG11 or the activity of Erg11. It is characterized by evaluating and selecting a substance that causes the growth of fungi.
  • the screening method of the present invention includes the case of targeting Erg25 as the molecular target and the case of targeting Erg26 as the molecular target, and specifically the following embodiments are preferable.
  • the screening method for an antifungal drug molecularly targeting the fungus Erg25 of the present invention is a method in which a test substance having antimycotic activity is inhibited in a sterol-free medium in a state in which ERG11 expression is inhibited or Erg11 activity is inhibited. To evaluate the effect on growth and to select substances that can grow a fungus. Moreover, the screening method of the antifungal which molecularly targets the fungus Erg26 of the present invention evaluates the action on the growth of the fungus by using a medium containing a sterol, and inhibits the growth of the test substance having antimycotic activity.
  • a test substance is selected, and the selected test substance is evaluated for its action on fungal growth in a cholesterol-free medium in which ERG11 expression is inhibited or Erg11 activity is inhibited, and a substance that inhibits fungal growth To select.
  • the screening method for an antifungal drug molecularly targeting the fungus Erg25 or Erg26 of the present invention evaluates the effect of a test substance having antimycotic activity on fungal growth in a medium containing sterols, and inhibits fungal growth Selecting a test substance, evaluating the action on growth of a fungus in a state where expression of ERG11 is inhibited or activity of Erg11 is inhibited in a medium containing sterols in the selected test substance, and selecting a substance that causes the fungus to grow It is characterized by
  • the test substance used in the present invention is a test substance having antifungal activity.
  • a test substance is a substance selected depending on whether or not it has a normal fungal growth inhibitory effect.
  • inhibition refers to a decrease in expression, activity, growth rate and the like.
  • the growth inhibitory action of fungi can be evaluated by a general antifungal activity evaluation method.
  • the method includes a method of culturing the fungus on a medium containing a test substance and free of sterols, and evaluating whether the test substance can inhibit the growth of the fungus.
  • sterol cholesterol, ergosterol and the like can be mentioned.
  • examples of sterol-free media include media free of serum and bile.
  • the substance which exhibits antifungal activity in combination with the drug which does not have antifungal activity per se such as a drug efflux pump inhibitor, is also included in the test substance having antifungal activity.
  • Specific fungal growth inhibition test methods include, for example, the paper disc method (Hello test) and the MIC method (method for measuring the growth inhibition minimum concentration).
  • the paper disc method the agar medium is smeared with the fungus to be tested, and a paper disc impregnated with the test substance is placed thereon. After culturing for a certain temperature and time, this method is a method of qualitatively evaluating the antibacterial activity against fungi from the size of the growth inhibition zone (halo) of the fungus around the paper disc.
  • the MIC method is a method of inoculating a test fungus in a medium to which each test substance is added and examining the minimum concentration (MIC) at which the growth of the fungus is inhibited. More specifically, the plate dilution method and the micro liquid dilution method are used. In the plate dilution method, the test substance is diluted 10 steps or more and an agar medium containing each concentration is prepared. In these media, the test fungus is inoculated and cultured, and the antifungal activity is confirmed by the difference in the size of the colony and the presence or absence thereof caused by the sensitivity of the test fungus to the test substance.
  • a test solution is diluted with liquid medium to a constant concentration to prepare a diluted solution.
  • Whether or not a test substance exhibits a growth inhibitory effect on fungi can be judged to exhibit a growth inhibitory effect, as long as the MIC ( ⁇ g / mL) is usually 10 ⁇ mg / mL or less, preferably 1 ⁇ g / mL.
  • the fungi to be evaluated are, for example, Trichophyton; Zygomycetes such as Trichoderma spp., Trichoderma spp., Ascomycota: Cryptococcus (eg Cryptococcus neoformans etc.), Malassezia sp. (Eg Malassezia furfur etc.), rust fungus etc.
  • Trichophyton rubrum eg, Trichophyton rubrum, Trichophyton mentagrophytes etc.
  • Sporolicus spp. Imperfect bacteria such as black fungus, etc .; yeast etc.
  • Ascomycota Trichophyton (eg, Trichophyton rubrum, Trichophyton mentagrophytes etc.), Sporotrix (eg, Sporothrix schenkii etc.), Aspergillus (eg, Aspergillus fumigatus), Pneumocystis (eg, Pneumocystis jirovecii etc.); Budding yeasts such as Candida (eg Candida albicans, Candida grabrata etc.), Saccharomyces (eg Saccharomyces cerevisiae etc.), yeasts such as fission yeast such as Schizosaccharomyces sp.
  • Candida eg Candida albicans, Candida grabrata etc.
  • Saccharomyces eg Saccharomyces cerevisiae etc.
  • yeasts such as fission yeast such as Schizosaccharomyces sp.
  • the test substance having antimycotic activity which can be selected by the above-mentioned normal growth inhibition test is evaluated for its effect on fungal growth in the state where the expression of ERG11 is inhibited or the activity of Erg11 is inhibited.
  • examples of a state in which expression of ERG11 is inhibited include an evaluation system using an ERG11 knockout strain, and an evaluation system using a combination of a tetracycline transcription repressor for EGR11 and tetracycline.
  • a state in which the activity of Erg11 is inhibited includes an evaluation system in which a test substance and an Erg11 inhibitor are used in combination.
  • Erg11 inhibitors include azole antifungal agents.
  • the azole antifungal agents used herein are azole antifungal agents that inhibit the activity of Erg11, and examples thereof include bifonazole, butoconazole, clomidazole, clotrimazole, croconazole, econazole, phenthiconazole, ketoconazole, isoconazole , Miconazole, neticonazole, omoconazole, oxiconazole, sulconazole, sulconazole, thioconazole, lanaconazole, fluconazole, fluconazole, hexaconazole, isacononazole, itraconazole, posaconazole, voriconazole itraconazole, voriconazole, allylamine antifungals such as terbinafine, butenafin, etc.
  • azole antifungal agent for example, phenarimol, fluotrimazole, triadimephone, triadimenol, diclobutrazol, diniconazole, trifimizole, propiconazole, flutriahol, DPX-H6573, penconazole, butiobate Prochloraz, tebuconazole, eclomesol, metconazole, hydroxyisoxazole, bitertanol, simeconazole, tetraconazole and the like.
  • This evaluation system is preferably the above-mentioned conventional fungal growth inhibition test system using a medium containing serum or a medium not containing serum.
  • a test substance that inhibits growth in a medium that contains or does not contain sterols is a state in which ERG11 expression inhibition, deficiency or Erg11 is inhibited, for example, a fungus is grown when an azole antifungal substance is used in combination with a sterol-containing medium Select the substance.
  • Growing the fungus in this state is because, for example, the MIC in the combined use is 10 times or more, preferably 100 times or more compared to the MIC in the case of using the test substance or the azole antifungal agent alone. It can confirm.
  • the substances thus selected are antimycotics which specifically inhibit the fungus Erg25 or Erg26 (FIG. 2B, C, D).
  • antifungal substances which specifically inhibit Erg25 or Erg26 selected by the above-mentioned means, a state in which the test substance is defective in ERG11 or inhibition of expression or inhibition of Erg11 activity in a sterol-free medium, for example, When used in combination with an azole antifungal agent, an inhibitor of Erg25 is grown and an inhibitor of Erg26 inhibits growth (Figure E).
  • each of the test substance and the Erg11 inhibitor (an azole antifungal substance) alone has a growth inhibitory effect on a fungus
  • the Erg11 inhibits the test substance in a sterol-free medium.
  • a substance that causes a fungus to grow when the test substance and an azole antifungal substance are used in combination in a medium that does not contain sterols is selected.
  • Growing the fungus in this state is because, for example, the MIC in the combined use is 10 times or more, preferably 100 times or more compared to the MIC in the case of using the test substance or the azole antifungal agent alone. It can confirm.
  • the substances thus selected are antimycotics which specifically inhibit the fungal Erg25.
  • a test substance having antifungal activity is evaluated for its action on growth of the fungus in a medium containing a sterol.
  • the above-mentioned general fungal growth inhibitory activity evaluation method may be carried out in a medium to which a sterol is added.
  • the medium containing sterols it is preferable to use a medium containing serum or bile, particularly a medium containing serum.
  • serum mammalian serum including human, such as human serum, bovine serum, horse serum and the like are used.
  • the amount of serum added is preferably 10% (v / v) concentration in the medium.
  • Inhibiting fungal growth in a medium containing sterols is evaluated by measuring MIC as described above.
  • a selected test substance is used in a state in which ERG11 is defective or its expression is inhibited or the activity of Erg11 is inhibited, for example, in combination with an azole antifungal agent to evaluate its action on the growth of fungi.
  • the above-mentioned general method for evaluating fungal growth inhibitory activity may be carried out except that the ERG11 expression is inhibited or the activity of Erg11 is inhibited, for example, an azole antifungal substance is used in combination.
  • inhibition of fungal growth by this condition can be confirmed, for example, by having the same MIC as in the case of using the test substance or the azole antifungal agent alone.
  • the substance thus selected is an antifungal drug which specifically inhibits fungal Erg26 as a molecular target.
  • the substance screened by the method of the present invention is a substance that specifically inhibits Erg25 or Erg26 depends on sensitivity or enzyme reaction to the ERG25 or ERG26 knockdown strain of the fungus, and knockdown strains of other growth essential genes. It can be confirmed by comparing the sensitivity to or the enzyme reaction.
  • Substances that inhibit the function of fungal Erg25 or Erg26 are highly likely to be safe for humans, effective against a wide range of fungi, and resistant to bacteria It is useful as an antifungal agent with a low possibility of appearance of That is, as shown in the test results described below, Erg25 or Erg26 is essential for fungal growth, has high fungal commonality, and low similarity to human proteins. In particular, Erg25 is superior to other ergosterol synthetases under all three conditions.
  • the antifungal agents Erg25 inhibitor and Erg26 inhibitor obtained by the present invention can not be used in combination with azole antifungal agents, but are useful as preventive and therapeutic agents for a wide range of fungal infections.
  • the target fungus is a wide range of amino acid sequences of Erg25 and Erg26, and therefore, it is a broad spectrum, and is a pathogen such as Trichophyton, Sporotrix, Aspergillus, Pneumocystis, Candida, Saccharomysis, etc. The fungi which have sex are mentioned.
  • azole antifungal drugs are known to have increased resistance in recent years, and Candida Gravulata is known to be resistant by using cholesterol instead in the presence of serum, but targeting Erg25 or Erg26
  • the antifungal drug of the present invention is effective against azole antifungal drug-resistant bacteria because the antifungal drug can not substitute for cholesterol.
  • the antifungal agent of the present invention can be administered orally or parenterally.
  • Antifungal agents can be made into pharmaceutical compositions by being combined with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers known ones such as excipients, binders, buffers, thickeners, stabilizers, emulsifiers, dispersants, suspending agents, preservatives and the like can be used. It can be formulated by the usual methods.
  • preparations for oral administration include tablets (including coated tablets, film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions and the like.
  • the preparation for oral administration can be prepared according to a known method by incorporating additives commonly used in the field of preparation.
  • additives include excipients such as lactose, mannitol and anhydrous calcium hydrogen phosphate; binders such as hydroxypropyl cellulose, methyl cellulose and polyvinyl pyrrolidone; disintegrants such as starch and carboxymethyl cellulose; magnesium stearate, Lubricants such as talc and the like can be mentioned.
  • Parenteral administration can be administered as an injection, a preparation for rectal administration, a topical administration and the like, and among them, an injection is preferred.
  • Injections include, for example, sterile solutions or suspensions. These injections are produced, for example, by dissolving or suspending the compound of the present invention or a pharmaceutically acceptable salt thereof in water for injection by Japan Post. If necessary, an isotonicity agent such as sodium chloride; a buffer such as sodium dihydrogen phosphate or sodium monohydrogen phosphate; a solubilizing agent etc. may be blended. In addition, it can be used as an injectable preparation in a soluble form (powder filling, lyophilization), and in this case, it can be manufactured by an ordinary method by adding an excipient such as mannitol or lactose.
  • an isotonicity agent such as sodium chloride
  • a buffer such as sodium dihydrogen phosphate or sodium monohydrogen phosphate
  • solubilizing agent etc. may be blended.
  • it can be used as an injectable preparation in a soluble form (powder filling, lyophilization), and in this case, it can be manufactured by
  • Suppository etc. are mentioned as a preparation for rectal administration.
  • the suppository is produced, for example, by dissolving or suspending the antifungal agent in a base such as cocoa butter or macrogol and pouring it into a mold for molding.
  • the liquid or cream may be placed in a container for injection to give a preparation for rectal administration.
  • the topical administration preparations include solutions, eye drops, creams, ointments, gel preparations, sprays, powders and the like.
  • the liquid preparation may be produced by adding the compound of the present invention or a pharmaceutically acceptable salt thereof to water, and adding a stabilizer, a solubilizer, a thickener, a dispersant, a suspending agent, etc. as necessary. it can.
  • a stabilizer e.g., a solubilizer, e.glycerin, a solubilizer, a thickener, a dispersant, a suspending agent, etc.
  • As this thickener gelatin, sodium hyaluronate, high molecular weight dextran, sodium alginate, sodium chondroitin sulfate and the like can be used.
  • Eyedrops can be produced by adding a preservative, in addition to a buffer, pH adjuster, tonicity agent.
  • Creams and ointments can be prepared using aqueous or oily bases such as water, liquid paraffin, vegetable oils (peanut oil, castor oil, etc.), macrogol and the like.
  • the gel preparation may be gelatin, pectin, carrageenan, agar, tragacanth, alginate, cellulose ether (methylcellulose, sodium carboxymethylcellulose etc.), pectin derivative, polyacrylate, polymethacrylate, polyvinyl alcohol, polyvinyl pyrrolidone etc. Can be manufactured.
  • a spray can be prepared by dissolving or suspending the compound of the present invention or a pharmaceutically acceptable salt thereof in water or the like, and then placing it in a spray container.
  • the compound of the present invention or a pharmaceutically acceptable salt thereof can be used as it is, but can be produced by mixing it with a suitable excipient.
  • the dose of the antifungal agent is appropriately determined depending on the individual case in consideration of the target disease or condition, the age, body weight, sex and the like of the administration subject.
  • the dosage of the antifungal agent per adult (body weight about 60 kg) per day is 1 to 1000 mg, preferably 1 to 800 mg, more preferably 1 to 500 mg, once or The administration is divided into 2 to 4 times.
  • the daily dose for adults is usually 0.1 to 50 mg, preferably 0.1 to 30 mg, and more preferably 0.1 to 20 mg per kg of body weight. Administration is divided into multiple doses.
  • Test Example 1 essential gene in each antifungal target gene group
  • Candida glabrata a Tet strain was prepared, and it was judged whether or not it was essential depending on the growth state at knockdown.
  • Saccharomyces cerevisiae those for which the determination of inviable was indicated in the large-scale survey information of Saccharomyce GENOME DATABASES (https://www.yeastgenome.org) were required.
  • results shown in Table 2 are those in which the gene indicated as inviable in Candida Genome Databse (http: // www. Candidagenome. Org) and Mutant phenotype information is essential.
  • Test Example 2 (Inter-species homology analysis of each antifungal target gene group) The homology between fungi species and human in each ergosterol synthetase gene of Candida glabrata was analyzed using BLASTX. The results are shown in Tables 3 and 4. From the results of Table 2, Table 3 and Table 4, the results of evaluating the suitability of each ergosterol synthetase as an antifungal target were shown in Table 3.
  • ERG25 and ERG26 are preferable as an antifungal target gene, and ERG25 is more preferable.
  • Test Example 3 Preparation of knockdown strain using Tet system ( Figure 1) Tet strain of each target gene Tet-ERG1, Tet-ERG7, Tet-ERG11 by inserting the Tet promoter upstream of the target genes ERG1, ERG7, ERG11, ERG25, ERG26 and ERG27 with the HETS 202 strain as the parent strain. , Tet-ERG25, Tet-ERG26, and Tet-ERG27 strains were often generated. This preparation method is described in the following documents.
  • TetOFF system When cultured in minimal medium (SD) in Tet strain, the downstream gene of Tet promoter is constantly expressed, and when 20 ⁇ g / mL doxycycline is added to the medium, Tet promoter (Intestinal resident yeast Candida glabrata requires Cyb2p-mediated lactate assimilation to adapt in mouse intestinal. Ueno K, Matsumoto Y, Uno J, Sasamoto K, Sekimizu K, Kinjo Y, Chibana H) .PLoS One. 2011; 6 (9): e24759.).
  • the parent strains of recombinants, HETS 202 and Tet-ERG1, Tet-ERG7, Tet-ERG11, Tet-ERG25, Tet-ERG26 and Tet-ERG27, are graded on various agar media as shown below for spotting assays Carried out.
  • SD + Dox + Serum medium contains 100 mL / L of bovine serum in addition to the components of the above SD + Dox medium.
  • FIG. A All recombinants grew well on SD medium.
  • B Addition of doxycycline resulted in transcriptional repression of the target gene in each Tet strain, resulting in inhibition of growth.
  • the parent strain HETS 202 had no growth inhibition by doxycycline because the Tet promoter was not inserted.
  • C Tet-ERG1, Tet-ERG7, Tet-ERG11, Tet-ERG27 return to growth and Tet-ERG25 by adding serum while the target gene of each Tet strain is transcriptionally repressed by the addition of doxycycline. , Growth of Tet-ERG26 did not return.
  • Fluconazole is one of azole antifungal agents, and the molecular target is Erg11.
  • Erg11 When Erg11 is inactivated or deleted, a diversion pathway occurs, and the intermediate metabolite lanosterol is modified by Erg6, Erg25, Erg26, Erg27, Erg3 to form 14 ⁇ -Methylergosta 8,24 (28) -dien-3 ⁇ , 6 ⁇ -diol. Which are cytotoxic and inhibit fungal growth.
  • transcription of the target gene of interest was suppressed in each Tet strain by the doxycycline on this medium, the growth of Tet-ERG1, Tet-ERG7, Tet-ERG11, and Tet-ERG27 was restored by the addition of serum.
  • TetERG25 and TetERG26 the addition of fluconazole, which is an inhibitor of Erg11, causes a diversion pathway to produce Eburicol and 4-Carboxyl-Eburicol, but allows cholesterol uptake and restores cell growth. it can.
  • E The target gene of each Tet strain is transcriptionally repressed by the addition of doxycycline. When fluconazole is added there, a detour route occurs, and in TetERG25, Eburicol is produced and it becomes a substitute for ergosterol and growth does not occur. On the other hand, in TetERG26, 4-Carboxyl-Eburicol is produced but growth is not restored.

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Abstract

L'invention concerne un moyen servant à développer un agent antifongique qui présente peu d'effets indésirables chez l'être humain et qui utilise un mécanisme d'action entièrement nouveau. L'invention concerne également un procédé servant à cribler un agent antifongique ciblant l'Erg25 ou l'Erg26 fongique en tant que cible moléculaire. Le procédé est caractérisé par l'évaluation d'une action d'une substance d'essai présentant une activité antifongique sur la croissance fongique dans des conditions dans lesquelles l'activité d'Erg11 est inhibée, et la sélection d'une substance qui permet la croissance fongique.
PCT/JP2018/046013 2017-12-15 2018-12-14 Procédé de criblage d'agents antifongiques WO2019117273A1 (fr)

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JP2019559216A JPWO2019117273A1 (ja) 2017-12-15 2018-12-14 抗真菌薬のスクリーニング方法

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BAUDRY, K. ET AL.: "The effect of the erg26-1 mutation on the regulation of lipid metabolism in Saccharomyces cerevisiae", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 16, 20 April 2001 (2001-04-20), pages 12702 - 12711, XP055617695 *
GACHOTTE, D. ET AL.: "A yeast sterol auxotroph (erg25) is rescued by addition of azole antifungals and reduced levels of heme", PROC. NATL. ACAD. SCI. USA, vol. 94, no. 21, 14 October 1997 (1997-10-14), pages 11173 - 11178, XP055617694 *
NOSE, H. ET AL.: "PF1163A, a novel antifungal agent, inhibit ergosterol biosynthesis at C-4 sterol methyl oxidase.", THE JOURNAL OF ANTIBIOTICS, vol. 55, no. 11, November 2002 (2002-11-01), pages 969 - 974, XP008064130 *

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