JP7359366B2 - Antifungal agent screening method - Google Patents

Description

The present invention relates to a method for screening antifungal agents, and more particularly, to a method for screening antifungal agents that target fungal transcription elongation factor S-II.

More than 1 million people die each year worldwide due to deep-seated mycoses in which the fungus invades the lungs, liver, kidneys, brain, etc., but the antifungal agents used for treatment differ by action and mechanism. There are only four types of antifungal agents, and it cannot be said that there are a sufficient number of antifungal agents.
Also, superficial fungal diseases such as yellow mites, ringworm, tinea, ringworm, and cutaneous candidiasis are also serious.
Among these, opportunistic infections in which fungi infect humans with weakened resistance have become a major problem.

On the other hand, RNA polymerase II is a protein that was identified as an activity promoter of RNA API II from extracts of Ehrlich ascites tumor cells (Non-patent Document 1, etc.) and purified (Non-Patent Document 2).
Transcription elongation factor S-II (hereinafter simply abbreviated as "S-II") is a protein that promotes the elongation activity of RNA polymerase II in fungi (Non-Patent Document 1, etc.).
Proteins with homology to S-II are ubiquitously present in the nuclei of eukaryotic cells ranging from Saccharomyces cerevisiae to humans. S-II is required for maintaining transcriptional fidelity during the elongation phase of transcription.
S-II has the function of promoting the exonuclease activity of RNAPII to remove an incorrect nucleotide when it is incorporated in vitro (Non-Patent Documents 3, 4).

The reason why the promotion of RNAPII by S-II is seen to be dependent on Mn ions is thought to be because the fidelity of transcription decreases in the presence of Mn ions.
In addition, Saccharomyces cerevisiae lacking the S-II gene can survive, but under oxidative stress conditions, transcriptional fidelity decreases and growth is suppressed (Non-Patent Document 5). Therefore, S-II is considered to have the function of correcting the decrease in transcription fidelity caused by chemical modification of nucleotides due to oxidative stress from the environment.

Although S-II can proliferate even if it is deleted (Non-patent Document 6), inhibiting S-II may reduce the pathogenicity of the fungus, and therefore targeting S-II. The compounds have the potential to be excellent antifungal agents that inhibit fungal pathogenicity.

Among fungi, for example, Cryptococcus neoformans is a pathogenic fungus that causes opportunistic infections, such as respiratory diseases and meningitis. Furthermore, C. neoformans is naturally resistant to echinocandin drugs, and resistant bacteria have also been isolated, posing a clinical problem.
C. neoformans is thought to establish infection by regulating gene expression in response to environmental stimuli such as oxidative stress. C. neoformans has the DST1 gene encoding S-II in its genome (Fig. 1). However, until now, it was unclear whether S-II is involved in the pathogenicity of C. neoformans.

On the other hand, conventionally, mammals such as mice and rats have been used to evaluate the pathogenicity of fungi. However, using mammals to evaluate pathogenicity poses not only cost but also ethical issues from the viewpoint of animal welfare.
As a method to solve this problem, the present inventors used silkworms, which have been used in the sericulture industry since ancient times, and have already discovered and reported that C. neoformans kills and injures silkworms (Non-patent Document 7). ).

As mentioned above, it cannot be said that there are currently a sufficient number of antifungal agents, and some of the current antifungal agents have strong side effects.
Furthermore, if the same antifungal agent is continued to be used, drug-resistant bacteria may develop, and depending on the type of fungi, it may become ineffective, so new antifungal agents must be constantly developed.

In order to develop new antifungal agents, especially to combat infections caused by drug-resistant bacteria, it is important to consider new targets. There were also only a few screening methods for targeted antifungal agents.

Natori, S., et al. The Journal of Biochemistry 72, (1972). Sekimizu K., et al. Biochemistry; 18:1582-1588. (1979) Reinberg D, et al., J Biol Chem; 262:3331-3337. (1987) Christie KR, et.al., J Biol Chem; 269:936-943. (1994) Koyama H, et al., Genes Cells; 8:779-788. (2003) Koyama, H., et al., Genes to Cells 12, (2007) Matsumoto Y. et al. J. Appl. Microbiol. (2012)

An object of the present invention is to find a new target for an antifungal agent, and to provide a method for screening an antifungal agent targeting the discovered "new target."

As a result of intensive studies aimed at solving the above problems, the present inventors found that transcription elongation factor S-II, a protein that promotes the elongation activity of RNA polymerase II in fungi, is suitable as a new target for antifungal agents. I found that.

Fungal growth is possible even when S-II is deleted, but under oxidative stress conditions, transcriptional fidelity decreases and growth is suppressed, so antifungal agents that target S-II have no bactericidal effect. The present inventors hypothesized that, even if they do not have this ability, there is a possibility of suppressing pathogenicity caused by fungi, and the present inventors verified this hypothesis.
That is, according to the present invention, it has been found that S-II is useful as a target for antifungal infectious agents, particularly as a target for suppressing the expression of pathogenicity caused by oxidative stress, etc., and as a target for preventing and treating opportunistic infections. Ta.

The present inventors have discovered an excellent screening method for compounds that inhibit S-II and have completed the present invention.

That is, the present invention targets transcription elongation factor S-II, which is a protein that promotes the elongation activity of RNA polymerase II in fungi, and selects an antifungal agent that reduces the pathogenicity of fungi from among compounds to be evaluated. The present invention provides a method for screening antifungal agents characterized by:

The present invention also provides the above-mentioned method in which an antifungal agent is selected from among the compounds to be evaluated based on the degree to which the amount of RNA synthesis by RNA polymerase II in the presence of S-II is reduced by the addition of the compounds to be evaluated. The present invention provides a method for screening antifungal agents.

The present invention also aims to reduce the extent to which the amount of RNA synthesis by RNA polymerase II in the presence of S-II decreases to the amount of RNA synthesis by RNA polymerase II in the absence of S-II by addition of the evaluation target compound. The present invention provides a method for screening the above-mentioned antifungal agents as a measure.

Further, the present invention provides a method for screening the above-mentioned antifungal agent, wherein the disease caused by the above-mentioned pathogenicity is a superficial mycosis or a deep mycosis.

The present invention also provides a pathogenicity expression inhibitor that does not substantially inhibit the growth of fungi or does not kill fungi, but suppresses the expression of pathogenicity caused by the fungi, or a pathogenicity expression inhibitor that suppresses the growth of fungi. The present invention provides a method for screening the above-mentioned antifungal agent, which is an antifungal infectious agent that does not substantially inhibit or kill fungi, but inhibits infection by the fungi.

The present invention also provides a method of screening for the above-mentioned antifungal agent in cases where the above-mentioned disease is an opportunistic infection.

Further, the present invention provides a method for producing an antifungal agent, which is characterized in that an antifungal agent is selected using the above-described screening method for an antifungal agent.

S-II is a highly conserved protein involved in maintaining transcriptional fidelity in eukaryotes, and contributes to oxidative stress resistance in yeast. Pathogenic fungi, such as C. neoformans, establish infection by adapting to environmental stress within the host.
In the present invention, it has been found that S-II plays an important role in the stress tolerance of fungi and the ability of fungi to infect.

As shown in the Examples, the S-II gene-deficient strain of C. neoformans showed sensitivity to 6-azauracil and sensitivity to oxidative stress. Furthermore, the S-II gene-deficient strain of C. neoformans had reduced ability to kill silkworms and mice.
The above results suggest that transcription elongation factor S-II is a necessary factor for fungal infection of the host, and suggest that S-II can be a target for antifungal agents.

Until now, it was unclear whether S-II is involved in the pathogenicity of a fungus such as C. neoformans, but the present invention reveals for the first time that fungal S-II can become a new target for antifungal agents. was discovered.
In animals, fungi are attacked by oxidative stress from the host's immune system, and S-II is thought to be necessary for the normal survival of the fungi. An S-II inhibitory strain of a fungus should reduce the pathogenicity of the fungus to the host.
Therefore, "S-II, which is a novel target" discovered by the present invention is an excellent target for antifungal agents.

The present invention has revealed that the S-II gene-deficient strain of C. neoformans actually has a reduced ability to infect silkworms and mice, and therefore, S-II is a transcription factor involved in the infection mechanism of the pathogen. One thing became clear.
S-II is thought to act as a transcription elongation factor necessary for fungi to adapt to various host environments such as oxidative stress and high temperatures, so antifungal agents targeting S-II are recommended. The development of this drug is useful as an anti-infectious drug within the category of antifungal agents that does not have bactericidal or bacteriostatic effects.
According to the present invention, since S-II having the above properties is targeted, a new category of antifungal agents can be screened.

That is, a pathogenicity expression inhibitor that does not substantially inhibit the growth of fungi or does not sterilize fungi, but suppresses the expression of pathogenicity by the fungi, or does not substantially inhibit the growth of fungi or does not sterilize fungi. However, it is possible to screen for new antifungal agents called antifungal infectious agents that inhibit the fungi from infecting the fungi.

Therefore, since the antifungal agent obtained by the antifungal agent screening method of the present invention has a new target, it is effective against drug-resistant bacteria, and can also be used as a pathogenicity inhibitor for preventing opportunistic infections. It is also useful as a therapeutic agent.

According to the present invention, it is possible to screen for a new antifungal agent whose target is different from conventional antifungal agents by targeting S-II. The antifungal agents obtained using the present invention should be novel antifungal agents.

FIG. 3 shows the conservation of the amino acid sequence of S-II and shows a comparison of the amino acid sequences of eukaryotic S-II proteins. hIIS: human, mIIS: mouse, dIIS: Drosophila, cnIIS: C. neoformans, spIIS: fission yeast, scIIS: budding yeast FIG. 2 is a diagram showing the construction of a C. neoformans S-II gene-deficient strain. A shows the genome structures of the wild strain (WT), the S-II gene-deficient strain (ΔDST1) (described as ΔS-II), and the complementary strain (ΔDST1+DST1) (described as ΔS-II/S-II). B shows the results of Southern blot hybridization using the probe shown in A. It is a graph showing 6-azauracil sensitivity in C. neoformans. A serially diluted overnight culture solution of wild strain (WT), S-II gene-deficient strain (ΔDST1) (described as ΔS-II), and complementary strain (ΔDST1+DST1) (described as ΔS-II/S-II) was prepared. This is the result of spotting on each agar medium and culturing for two nights. 1 is a graph showing a decrease in silkworm killing ability of a C. neoformans S-II gene-deficient strain. Wild strain (WT), S-II gene-deficient strain (ΔDST1) (described as ΔS-II), and complementary strain (ΔDST1+DST1) (described as ΔS-II/S-II) were injected into the body fluid of silkworms and the progress was observed. This is the result (N=6/group). 1 is a graph showing a decrease in the mouse killing ability of a C. neoformans S-II gene-deficient strain. Wild strain (WT), S-II gene-deficient strain (ΔDST1) (described as ΔS-II), and complementary strain (ΔDST1+DST1) (described as ΔS-II/S-II) were injected into the tail vein of mice and the progress was observed. This is the result (N=10/group).

The present invention will be described below, but the present invention is not limited to the following specific embodiments, and can be arbitrarily modified within the scope of the technical idea.

The antifungal agent screening method of the present invention targets transcription elongation factor S-II (hereinafter simply abbreviated as "S-II"), which is a protein that promotes the elongation activity of RNA polymerase II in fungi. It is characterized by selecting an antifungal agent that reduces the pathogenicity of fungi from among compounds.

<Transcription elongation factor S-II becomes a target>
In the present invention, S-II is a factor that confers 6-azauracil resistance in the pathogenic fungus Cryptococcus neoformans (hereinafter simply referred to as "C. neoformans"), and a factor that confers resistance to 6-azauracil in silkworms and mice. (Example).
These results revealed that the S-II-mediated transcription elongation mechanism is necessary for pathogen infection.

Within the host, pathogens are exposed to many host-derived stresses, including oxidative stress.
In yeast studies, S-II maintains transcriptional fidelity under oxidative stress conditions (Non-Patent Documents 5, 6). Furthermore, in yeast, S-II is known to be involved in resistance to 6-azauracil, which inhibits GTP synthesis (Nakanishi T, et al., J Biol Chem, 1995).
In the present invention, it was found that S-II is involved in 6-azauracil resistance in C. neoformans (Example). From the above, in pathogenic fungi such as C. neoformans, S-II is thought to regulate the expression of a group of genes for adapting to various environmental factors through the maintenance of transcriptional fidelity.

In yeast, the SSM1 gene encoding pyrimidine-5'-nucleotidase has been identified as a multicopy suppressor of the 6-azauracil sensitivity phenotype in S-II gene-deficient strains, and an in vitro experimental system has been identified. reported that S-II regulates the expression of SSM1 through the release of transcriptional elongation of stalled RNAPII at two transcription stop sites within the transcription unit of the SSM1 gene (Simoaraiso M, et al., J Biol Chem, 2000).
It is thought that the regulation of SSM1 expression via S-II is involved in the 6-azauracil resistance mechanism of C. neoformans.

In the present invention, the ability of the S-II gene-deficient strain of C. neoformans to infect silkworms and mice is reduced (Example), indicating that S-II is a transcription factor involved in the infection mechanism of the pathogen. It became clear.
S-II is thought to act as a transcription elongation factor necessary for C. neoformans to adapt to various host environments such as oxidative stress and high temperature. Therefore, the development of a compound targeting S-II can be used as an anti-infectious drug that does not have bactericidal or bacteriostatic effects within the category of antifungal drugs.

<Characteristics of antibacterial agents obtained using the screening method>
As mentioned above, until now, it was unclear whether S-II is involved in the pathogenesis of pathogenic fungi such as C. neoformans.
Here, "pathogenic" is defined as a case where a fungus that has entered the body impairs a human's health condition or significantly suppresses a person's activities. Specific symptoms of pathogenicity in humans include, for example, those described below.
The antifungal agent screening method of the present invention is effective when the disease caused by the above pathogenicity is a superficial mycosis or a deep mycosis.

S-II, which was demonstrated to be a novel target in the present invention, is a protein that promotes the elongation activity of RNA polymerase II in fungi. Therefore, the antifungal agent obtained by the screening method of the present invention does not inhibit the elongation activity of RNA polymerase II in fungi, but inhibits transcription elongation factor S-II, which is a protein that promotes the elongation activity of RNA polymerase II.

As a result, the antifungal agent obtained by screening targeting S-II can be a pathogenicity expression inhibitor that suppresses the expression of fungal pathogenicity, or an antifungal infectious agent that inhibits fungal infection. Become.
Therefore, the present invention provides a pathogenicity expression inhibitor, or a pathogenicity expression inhibitor, which does not substantially inhibit the growth of fungi or does not kill fungi, but suppresses the expression of pathogenicity caused by the fungi. It is also a screening method for the above-mentioned antifungal agent which is an antifungal infectious agent that does not substantially inhibit fungi or sterilize the fungus, but inhibits infection by the fungus.
In the above, "substantially inhibiting" refers to reducing the growth rate of fungi to 50% or less.

Fungi exist in large numbers in the environment and are likely to infect people with weakened immune systems. Therefore, the present invention also provides a screening method for the above-mentioned antifungal agent, where the disease caused by the above-mentioned pathogenicity is an opportunistic infection.

<Screening method>
Screening for antifungal agents that target S-II can be carried out by methods that would naturally occur to those skilled in the art once S-II is found to be a target of an antifungal agent. Since it has been demonstrated that S-II is a target, screening methods for antifungal agents targeting S-II can be carried out according to conventional methods using previously reported methods. Examples include:

S-II is a protein that promotes elongation activity of RNA polymerase II in eukaryotic cells, and the activity of RNA polymerase II is dependent on DNA as a template; substrates such as ATP, GTP, CTP, and UTP; buffer; Mn ²⁺ (Mg ²⁺ may be present); salts such as NaCl; etc. In an RNA synthesis reaction in a container containing such substances, the amount of the substrate etc. incorporated into RNA (UTP is incorporated as UMP) is measured as an index.
S-II inhibitors do not inhibit the activity of RNA polymerase II, but reduce the amount of RNA synthesis when S-II is added.
Therefore, by measuring the amount of the above-mentioned substrate, etc. incorporated into RNA in the presence of the evaluation target compound and S-II, the degree of inhibition of S-II by the evaluation target compound can be measured. By doing so, antifungal agents can be screened from among the compounds to be evaluated.

As an example of the method for screening antifungal agents of the present invention, the extent to which the amount of RNA synthesis by RNA polymerase II in the presence of S-II is reduced by the addition of the evaluation target compound is used as a measure, and the antifungal agent is selected from among the evaluation target compounds. This includes selecting a fungicide.

Furthermore, considering that the amount of RNA synthesis by RNA polymerase II in the absence of S-II is the lowest line (destination) of the amount of RNA synthesized by S-II inhibitors, screening should be performed based on the distance to the lowest line. You can also.
Therefore, as an example of the method for screening antifungal agents of the present invention, the amount of RNA synthesized by RNA polymerase II in the presence of S-II can be increased by adding a compound to be evaluated, compared to the amount of RNA synthesized by RNA polymerase II in the absence of S-II. An example of this is to select an antifungal agent from among the compounds to be evaluated based on the degree to which the amount of RNA synthesis decreases.

An example of an inhibitor of S-II is an antibody against S-II, but the antibody has a large molecular weight and cannot enter into living fungal cells and inhibit fungal RNA synthesis. The S-II inhibitor obtained by the present invention has a smaller molecular weight than an antibody, and exhibits antifungal activity by inhibiting S-II-dependent RNA synthesis within fungal cells.
Therefore, in the present invention, it is preferable that the compound to be evaluated has a molecular weight smaller than that of the antibody molecule. Although not particularly limited, it is more preferable that the compound to be evaluated does not contain repeating units in the molecule in order to avoid increasing the molecular weight.

<Production method of antifungal agent>
The present invention also provides a method for producing an antifungal agent, which comprises selecting an antifungal agent using the antifungal agent screening method described above.
The antifungal agent is manufactured according to conventional methods according to the following dosage forms and formulations.

<Application, dosage form, and formulation of antifungal agents>
Examples of fungi to be used as the antifungal agent in the present invention include fungi belonging to the genus Cryptococcus, Aspergillus, Candida, Coccidioides, Histoplasma, and the like. Among these, it is useful for fungi of the genus Cryptococcus, especially Cryptococcus neoformans.

Antifungal agents such as pathogenicity expression inhibitors, antifungal infection agents, and antiopportunistic infection agents obtained through the screening of the present invention can prevent and/or treat fungal diseases.
Examples of diseases caused by the mycosis or the pathogenicity of the fungus include skin mycoses (superficial mycoses) such as yellowworm, ringworm, tinea, tinea, and cutaneous candidiasis; cryptococcosis, and actinomycosis. , visceral mycoses (profound mycoses) such as norcadiasis, mucormycosis, aspergillosis, candidiasis, coccidioidomycosis, and histoplasmosis.

The antifungal agent obtained using the present invention can be used for pharmaceuticals (drugs), quasi-drugs, and the like.
There are no particular restrictions on the dosage form, and it can be appropriately selected depending on, for example, the desired administration method as described below.
Specifically, examples include oral solid preparations (tablets, coated tablets, granules, powders, hard capsules, soft capsules, etc.), oral liquid preparations (oral liquid preparations, syrups, elixirs, etc.), and injections (solvents, suspensions, etc.). agents, etc.), ointments, patches, gels, creams, external powders, sprays, inhalation powders, etc.

The above pharmaceuticals (drugs), quasi-drugs, etc. are added to the antibacterial agent (fungal pathogenicity expression inhibitor), excipients, binders, disintegrants, lubricants, coloring agents, flavoring/fragrances, etc. It can be produced by adding an agent and using a conventional method.
Examples of the excipient include lactose, white sugar, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid.
Examples of the binder include water, ethanol, propanol, syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, polyvinylpyrrolidone, and the like. .
Examples of the disintegrating agent include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose.
Examples of the lubricant include purified talc, stearate, borax, polyethylene glycol, and the like.
Examples of the coloring agent include titanium oxide and iron oxide.
Examples of the flavoring/flavoring agent include white sugar, orange peel, citric acid, and tartaric acid.
Examples of the buffer include sodium citrate.
Examples of the stabilizer include tragacanth, gum arabic, and gelatin.

The above-mentioned injections may be prepared, for example, by adding a pH adjusting agent, a buffering agent, a stabilizer, an isotonizing agent, a local anesthetic, etc. to the above-mentioned active ingredients, and using the usual method for subcutaneous, intramuscular, or intravenous administration. It is possible to produce injections such as
Examples of the pH adjuster and the buffer include sodium citrate, sodium acetate, and sodium phosphate.
Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic acid.
Examples of the tonicity agent include sodium chloride, glucose, and the like.
Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.
The above-mentioned ointment can be produced by, for example, adding the above-mentioned active ingredient to a known base, stabilizer, wetting agent, preservative, etc., and mixing them in a conventional manner.
Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, and paraffin.
Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and the like.

The above-mentioned adhesive patch can be manufactured by, for example, applying the above-mentioned ointment such as cream, gel, paste, etc. to a known support by a conventional method.
Examples of the support include woven or nonwoven fabrics made of cotton, cotton, and chemical fibers; films or foam sheets made of polyvinyl chloride, polyethylene, polypropylene, polyester, polyurethane, and the like.

<Targets and usage of antibacterial agents>
The antifungal agent obtained by the present invention, pharmaceuticals containing the same, quasi-drugs, etc. can be suitably used, for example, for individuals who require prevention or treatment of diseases caused by fungi.
Examples of the individual include, but are not limited to, humans; laboratory animals such as mice and rats; monkeys; horses; livestock such as cows, pigs, goats, and chickens; pets such as cats and dogs; and the like.

There are no particular restrictions on the method of administering the antifungal agent, and it can be selected as appropriate depending on the dosage form mentioned above, including oral administration, skin administration, intraperitoneal administration, and blood injection. Examples include injection and injection into the intestine.

The dosage of the antifungal agent is not particularly limited and can be appropriately selected depending on the age, body weight, degree of desired effect, etc. of the individual to be administered.
Furthermore, the timing of administration is not particularly limited and can be appropriately selected depending on the purpose.

<Effect>
Until now, it was unclear whether S-II is involved in the expression of pathogenicity in, for example, the fungus C. neoformans.
According to the present invention, it has been found that fungal S-II is necessary for survival within an animal host when the fungus is attacked by oxidative stress or the like from the host's immune system. Therefore, fungal S-II gene-deficient strains have reduced pathogenicity to the host. Therefore, S-II can be a target for antifungal agents such as pathogenicity inhibitors or antifungal infectious agents.

Hereinafter, the present invention will be explained in more detail based on Examples and Study Examples, but the present invention is not limited to the specific scope of the Examples and the like. Hereinafter, the description "%" means "mass %" when it refers to mass.

Evaluation example 1
<Cultivation method of bacterial strains>
C. neoformans, a pathogenic fungus, was spread on a YNB agar medium and cultured at 30°C for 3 to 5 days. The grown colonies were suspended in YNB liquid medium and cultured with shaking at 30°C.

<Genetic engineering method for C. neoformans>
Genetic recombination of C. neoformans was performed according to a previous report (Shimizu K., et al., Fungal Genet Biol, 69: 13-22, 2014).
TLHM24, in which the ku70 gene of JEC21, which is a Serotype D of C. neoformans, is deleted, was used as a parent strain (FIG. 1).
The construction of the S-II gene-deficient strain and complementary strain is shown in Figure 2.

<6-Azauracil susceptibility test>
Colonies of C. neoformans were suspended in YNB liquid medium and cultured with shaking at 30°C overnight. 6-Azauracil was added to YNB liquid medium to prepare a 2-fold dilution series, and the overnight culture was diluted (final bacterial concentration, OD ₆₀₀ = 0.01) to YNB liquid medium. After culturing for 2 days, absorbance (OD ₆₀₀ ) was measured.

Evaluation example 2
<Silkworm infection experiment (method for evaluating fungal pathogenicity using silkworms)>
Eggs of silkworm cultivars (Fu, Yo x Tsukuba, Ne) purchased from Ehime Silkworm Seed Co., Ltd. were sterilized and then incubated at 25 to 27°C. Silkmate 2S, an antibiotic-containing artificial feed purchased from Nihon Nosan Kogyo Co., Ltd., was used as the feed. 4th instar sleeping silkworms were separated, and 5th instar larvae were used in experiments after being fasted overnight and molted.

The silkworm infection experiment was performed according to a previous report (Matsumoto Y et al., J Applied Microbiology, 2012).
Each 5th instar and 1 day old silkworm was fed with 1.5 g of antibiotic-free artificial feed (Nippon Nosan Kogyo Co., Ltd.) and kept overnight. Then, 100 μL of C. neoformans suspension (OD ₆₀₀ =10) was injected into the silkworm body fluid using a 1 mL tuberculin syringe (TERUMO).

Thereafter, the silkworms were raised at 37° C. on an antibiotic-free artificial feed (Nippon Nosan Kogyo Co., Ltd.), and the number of surviving silkworms was counted over time. The silkworm's head was poked with tweezers, and those that did not respond were determined to be dead.

<Mouse infection experiment (method for evaluating fungal pathogenicity using mice)>
A suspension of each strain of C. neoformans was injected into the tail vein of 8-week-old mice (ICR), and surviving mice were counted over time.

Example 1
<Comparison of pathogenicity of S-II deletion strain and that of wild type strain>
An S-II deletion strain of C. neoformans that had already been constructed was used. Fungal genomic stock center has created a library of gene-disrupted strains by introducing mutant genes synthesized in vitro from a C. neoformans gene library into wild-type strains by electroporation, etc., and sells them as a "Madhani set." ing.

The pathogenicity of this gene-disrupted strain (CNAG 0769) to silkworms was compared with that of the wild-type strain (KN99alpha) to silkworms.
As a result, it was found that the _LD50 values 48 hours and 72 hours after injection were 3 times and 4.5 times higher, respectively.

Furthermore, we decided to verify the above results by producing an S-II gene-deficient strain and a complementary strain of C. neoformans. Through the genetic modification shown in FIG. 2A, a strain capable of growing in a uracil-deficient medium was obtained. A band of the expected size could be detected by Southern blot hybridization (Figure 2B). These strains were used as S-II gene-deficient strains and complementary strains of C. neoformans.

In budding yeast (Saccharomyces cerevisiae), it is known that the S-II gene-deficient strain has reduced growth ability in the presence of 6-Azauracil (Nakanishi T, et al., J Biol Chem, 1995).
Therefore, first, we investigated whether an S-II gene-deficient strain of C. neoformans, a pathogenic fungus, also showed susceptibility to 6-azauracil (6-AU).
A serially diluted overnight culture solution of each strain was spotted on each agar medium and cultured for two nights. Control YNB medium and YNB medium supplemented with 6-azauracil (6-AU) were cultured at 30°C.

The results are shown in Figure 3. In FIG. 3, "WT" indicates the wild type strain, "ΔS-II" indicates the S-II gene-deficient strain (ΔDST1), and "ΔS-II/S-II" indicates the complementary strain (ΔDST1+DST1).

No difference was observed between the wild-type strain and the S-II gene-deficient strain in growth ability in a liquid medium at 30°C without the addition of 6-azauracil.
However, the S-II gene-deficient strain of C. neoformans suffered from stronger growth inhibition than the wild-type strain by addition of 6-azauracil at 30°C (Fig. 3). The growth inhibition of this S-II gene-deficient strain by 6-azauracil was suppressed by introduction of the wild-type S-II gene (FIG. 3).
The above results suggest that the S-II gene-deficient strain of C. neoformans is sensitive to 6-azauracil.

Example 2
<Decrease in silkworm killing ability and mouse killing ability in S-II gene-deficient strain>
C. neoformans, a pathogenic fungus, is thought to undergo oxidative stress in infected hosts. Therefore, we considered that C. neoformans lacking the S-II gene has reduced pathogenicity.
Figure 4 shows the results when using silkworms. In FIG. 4, "WT" indicates the wild type strain, "ΔS-II" indicates the S-II gene-deficient strain (ΔDST1), and "ΔS-II/S-II" indicates the complementary strain (ΔDST1+DST1).

To date, we have established a system for evaluating the pathogenicity of C. neoformans using silkworms (Matsumoto Y et al., J Applied Microbiology, 2012).
Silkworms injected with the S-II gene-deficient strain of C. neoformans had a longer survival time than those injected with the wild-type strain (Figure 4). The extension of survival time of silkworms injected with this S-II gene-deficient strain was suppressed by reintroduction of the S-II gene (Fig. 4).

Next, we examined whether the ability to infect mice was reduced in the S-II gene-deficient strain of C. neoformans.
The results are shown in Figure 5. In FIG. 5, "WT" indicates the wild type strain, "ΔS-II" indicates the S-II gene-deficient strain (ΔDST1), and "ΔS-II/S-II" indicates the complementary strain (ΔDST1+DST1).

Mice injected intravenously with the S-II gene-deficient strain of C. neoformans survived longer than mice injected with the wild-type strain, and the mice did not die even after 34 days (Figure 5).
The extension of survival time of mice injected with this S-II gene-deficient strain was suppressed by reintroduction of the S-II gene (FIG. 5).

The above results in silkworms and mice (FIGS. 4 and 5) indicate that S-II is a factor contributing to the pathogenicity of C. neoformans.
It has been found that antifungal agents that inhibit S-II, ie, target S-II, do not substantially inhibit fungal growth, but reduce fungal pathogenicity. Therefore, the transcription elongation factor S-II protein, which promotes the elongation activity of RNA polymerase II, was found to be an excellent target for antifungal agents.

Example 3
<Screening for antifungal agents after the target is determined to be S-II>
The inhibitory activity of an S-II inhibitor, that is, a substance that suppresses the activity of S-II, is detected as follows. Since the S-II inhibitor detected as described below can be an antifungal agent, the method for screening for an antifungal agent has been completed.

S-II is a protein that promotes RNA polymerase II activity in eukaryotic cells.
The activity of RNA polymerase II is determined by any of the above in an in vitro RNA synthesis reaction containing template DNA, substrates ATP, GTP, CTP, and UTP, a buffer, manganese ion Mn ²⁺ , and salts such as NaCl. It is measured using the amount of the nucleotide incorporated into RNA as an index. More preferably, it is quantified using the amount of radiolabeled UTP (Uridine triphosphate) incorporated into RNA as an index.

S-II has the function of promoting the amount of RNA synthesized by RNA polymerase II. Inhibitors of S-II do not affect the amount of RNA synthesis by RNA polymerase II, ie do not inhibit the activity of RNA polymerase II, but reduce the amount of RNA synthesis when S-II is added.
Therefore, now that S-II has been found to be a target, it is clear to those skilled in the art how to screen for antifungal agents that target S-II.

Note that an example of something that inhibits S-II is an antibody against S-II protein (Non-Patent Document 2). Antibodies that specifically bind to S-II appear in the serum of rabbits immunized with S-II. This antibody was purified from rabbit serum and inhibition of S-II activity can be seen. This antibody reduces the amount of RNA synthesis in the presence of S-II to the level of RNA polymerase II alone in the absence of S-II. Therefore, antibodies against S-II inhibit S-II.

However, antibodies have large molecular weights and cannot enter the cells of living fungi and inhibit fungal RNA synthesis.
In the present invention, if the evaluation target compound is made to have a smaller molecular weight than an antibody molecule (smaller than an antibody as a molecule), it can be suitably screened from them, and is an excellent antifungal agent (an excellent S-II inhibitor). can be obtained.
The antifungal agent thus obtained should exhibit antifungal activity by inhibiting S-II-dependent RNA synthesis within fungal cells.

In the present invention, the transcription elongation factor S-II of RNA polymerase II was found to be a new target for antifungal agents. Inhibitors of S-II do not inhibit RNA polymerase II activity, but reduce the amount of RNA synthesis in the presence of S-II.
Since the antifungal agent screening method of the present invention can screen for compounds that inhibit S-II, the antifungal agent obtained in this way can be a novel antifungal agent that has actions and efficacy different from conventional ones. It is. Therefore, the present invention can be widely utilized in fields where the fungal agent is developed, manufactured, sold, used, etc. in the industry.

Claims (6)

An antifungal agent screening method that targets transcription elongation factor S-II, a protein that promotes the elongation activity of RNA polymerase II in fungi, and selects antifungal agents that reduce fungal pathogenicity from among evaluation target compounds. There it is,
The antifungal agent is a pathogenicity expression inhibitor that does not substantially inhibit the growth of fungi or does not sterilize the fungus, but suppresses the expression of pathogenicity caused by the fungus; A method for screening an antifungal agent, characterized in that it is an antifungal infectious agent that does not kill fungi but inhibits infection by the fungi . The antifungal agent according to claim 1, wherein the antifungal agent is selected from among the compounds to be evaluated based on the degree to which the amount of RNA synthesis by RNA polymerase II in the presence of S-II is reduced by the addition of the compound to be evaluated. agent screening method. Claim 1: The measure is the degree to which the amount of RNA synthesis by RNA polymerase II in the presence of S-II is reduced to the amount of RNA synthesis by RNA polymerase II in the absence of S-II by addition of the compound to be evaluated. Or the method for screening an antifungal agent according to claim 2. The method for screening an antifungal agent according to any one of claims 1 to 3, wherein the pathogenic disease is a superficial mycosis or a deep mycosis. The method for screening an antifungal agent according to any one of claims 1 to 4, wherein the pathogenic disease is an opportunistic infection. A method for producing an antifungal agent, comprising selecting an antifungal agent using the method for screening an antifungal agent according to any one of claims 1 to 5 .

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