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WO2012087004A2 - Utilisation des gènes ire1 et hxl1 de la voie de signalisation upr pour le traitement d'une infection fongique et de la méningite - Google Patents

Utilisation des gènes ire1 et hxl1 de la voie de signalisation upr pour le traitement d'une infection fongique et de la méningite Download PDF

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WO2012087004A2
WO2012087004A2 PCT/KR2011/009862 KR2011009862W WO2012087004A2 WO 2012087004 A2 WO2012087004 A2 WO 2012087004A2 KR 2011009862 W KR2011009862 W KR 2011009862W WO 2012087004 A2 WO2012087004 A2 WO 2012087004A2
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seq
gene
hxl1
set forth
meningitis
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WO2012087004A3 (fr
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Yong Sun Bahn
Kwang Woo Jung
Hyun Ah Kang
Seon Ah Cheon
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Industry Academic Cooperation Foundation of Chung Ang University
Industry Academic Cooperation Foundation of Yonsei University
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Industry Academic Cooperation Foundation of Chung Ang University
Industry Academic Cooperation Foundation of Yonsei University
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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Definitions

  • the unfolded protein response (UPR) signaling pathway plays an important role in maintaining endoplasmic reticulum (ER) homeostasis under various environmental conditions that cause ER stress in eukaryotic cells.
  • the Ser/Thr kinase/endoribonuclease Ire1 senses ER stress and is auto-phosphorylated and activated, after which it excises an intron from HAC1 mRNA encoding the bZIP transcription factor to activate the transcription factor, so that the cells cope with ER stress. Meanwhile, it was reported that a deletion of HacA (Homologous to Hac1 transcription factor of S.
  • Antifungal agents developed to date can be broadly classified into azole-based antifungal agents and non-azole-based antifungal agents.
  • the azole-based antifungal agents include ketoconazole, fluconazole, itraconazole, voriconazole and the like, and the non-azole-based antifungal agents include terbinafine, flucytosine, amphotericin B, caspofungin and the like.
  • the azole-based drugs when administered to impaired liver function patients, can cause hepatitis, leading to death, and for this reason, a liver function test should be carried out before administration of the azole-based drugs. It was reported that flucytosine inhibits bone marrow in a dose-dependent manner, shows hepatotoxicity and can cause enterocolitis. Such side effects further increase in the case of impaired renal function patients, and thus monitoring of renal function of the patients is very important. Also, flucytosine must not be prescribed for pregnant women. The typical toxicity of Amphotericin B is glomerular nephrotoxicity resulting from renal artery vasoconstriction, which is dose-dependent.
  • Another object of the present invention is to provide a method for screening a novel antifungal agent or meningitis-treating agent which can show a synergistic effect when being co-administered with an existing antifungal agent or meningitis-treating agent.
  • sample means an unknown candidate that is used in screening to examine whether it influences the expression of a gene or the amount or activity of a protein.
  • examples of the sample include, but are not limited to, chemical substances, nucleotides, antisense-RNA, siRNA (small interference RNA) and natural extracts.
  • the measurement of a change in the expression of a gene may be carried out according to various methods known in the art, for example, using RT-PCR (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), Northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine, 102-108, CRC press), cDNA microarray hybridization (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)) or in situ hybridization (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)).
  • a change in the amount of protein can be carried out according to various immunoassay methods known in the art.
  • the immunoassay methods include, but are not limited to, radioimmunoassay, radioimmuno-precipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, and sandwich assay.
  • the immunoassay or immunostaining method is described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J. M. ed., Humana Press, NJ, 1984; and Ed Harlow and David Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.
  • a specific embodiment of the present invention includes the steps of: (i) coating an extract from sample-treated cells on the surface of a solid substrate; (ii) allowing the cellextract to react with Ire1 or a protein-specific antibody as a primary antibody; (iii) allowing the material resulting from step (ii) to react with an enzyme-conjugated secondary antibody; and (iv) measuring the activity of the enzyme.
  • the solid substrate is preferably a hydrocarbon polymer (e.g., polystyrene or polypropylene), glass, a metal or gel, and most preferably a microtiter plate.
  • the enzyme conjugated to the secondary antibody includes, but is not limited to, an enzyme that catalyzes a color-development reaction, a fluorescent reaction, a luminescent reaction or an infrared reaction. Examples of the enzymes include alkaline phosphatase, ⁇ -galactosidase, horseradish peroxidase, luciferase and cytochrome P450.
  • the substrate used may be chloronaphtol, aminoethylcarbazol, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), TMB (3,3,5,5-tetramethylbenzidine), ABTS (2,2'-azine-di[3-ethylbenzthiazoline sulfonate]), or o-phenyldiamine (OPD).
  • Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine
  • TMB 3,3,5,5-tetramethylbenzidine
  • ABTS 2,2'-azine-di[3-ethylbenzthiazoline sulfonate]
  • OPD o-phenyldiamine
  • the pharmaceutical composition of the present invention may comprise, as an active ingredient, a chemical substance, a nucleotide, antisense RNA, an siRNA oligonucleotide or a natural extract.
  • the antifungal pharmaceutical composition or antifungal composite formulation of the present invention may comprise, in addition to the active ingredient, pharmaceutically suitable and physiologically acceptable adjuvants.
  • the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, flavoring agents, solubilizers, etc.
  • the antifungal pharmaceutical composition of the present invention may also contain at least one pharmaceutically acceptable carrier, in addition to the active ingredient.
  • Examples of pharmaceutically acceptable carriers which can be used to formulate the antifungal pharmaceutical composition of the present invention in the form of liquid solutions, include saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of two or more thereof.
  • the inventive composition may also contain other conventional additives, such as antioxidants, buffers and bacteriostatic agents.
  • the inventive composition may additionally contain diluents, dispersants, surfactants, binders and lubricants in order to formulate it into injection formulations, such as aqueous solutions, suspensions and emulsions, pills, capsules, granules and tablets.
  • inventive composition may preferably be formulated depending on particular diseases and its components, using the method described in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, which is a suitable method in the relevant field of art.
  • the pharmaceutical compositions of the present invention can be formulated as granules, powders, coated tablets, tablets, capsules, suppositories, syrup, juice, suspensions, emulsions, drops, injectable liquids, and sustained-release preparations of active ingredients, etc.
  • the pharmaceutical composition of the present invention can be administered in the conventional manner via the intravenous, intraarterial, intraabdominal, intramusclar, intrasternal, percutaneous, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal route.
  • the term "effective amount" of the active ingredient of the pharmaceutical composition means the amount required for prevention or treatment of disease.
  • the effective amount can vary depending on various factors, including the kind of disease, the severity of disease, the kind and content of active ingredient and other components contained in the composition, the kind of formulation, the patient's age, weight, physical condition, sex and diet, and administration time, administration route, the secretion rate of the composition, administration period, and the kind of drug used in combination with the composition.
  • the inhibitor of the present invention when administered to adult patients once or several times per day, it may be administered at doses of 0.1 ng/kg to 10 g/kg for a compound, 0.1 ng/kg to 10 g/kg for a polypeptide, a protein or an antibody, and 0.1 ng/kg to 10 g/kg for an antisense oligonucleotide, siRNA, shRNAi, or miRNA.
  • antisense oligonucleotide refers to DNA, RNA or its derivatives, that contain nucleic acid sequences complementary to the sequences of a target mRNA, characterized in that they bind to the target mRNA and interfere with its translation to protein.
  • the antisense nucleic acid is 6-100, preferably 8-60, more preferably 10-40 nucleotides in length.
  • the antisense oligonucleotide may at least one modification in its base, sugar or backbone for its higher inhibition efficacy (De Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55(1995)).
  • the modified nucleic acid backbone may comprise phosphorothioate, phosphotriester, methyl phosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic or heterocyclic intersugar linkages.
  • the antisense oligonucleotide may also contain one or more substituted sugar moieties.
  • the antisense nucleic acid may also include modified bases.
  • modified bases examples include hypoxanthine, 6-methyladenine, 5-Me pyrimidines (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N 6 (6-aminohexyl)adenine, and 2,6-diaminopurine.
  • the antisense nucleic acids of the present invention may be chemically bonded to one or more moieties or conjugates enhancing the activity and cellular uptake of the antisense nucleic acids.
  • lipophilic moieties include, but are not limited to, a cholesterol moiety, a cholesteryl moiety, cholic acid, a thioether, a thiocholesterol, an aliphatic chain, a phospholipid, a polyamine chain, a polyethylene glycol chain, adamantane acetic acid, a palmityl moiety, an octadecylamine moiety and a hexylamino- carbonyl-oxycholesterol moiety.
  • Methods of preparing oligonucleotides comprising lipophilic moieties are well known in the art (see U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255).
  • the modified nucleic acids may have enhanced stability in the presence of nucleases and enhanced binding affinity to target mRNA.
  • the modifications described above enhance stability against nuclease degradation and increase affinity of the antisense oligonucleotide toward its target mRNA.
  • Antisense RNA may be synthesized in vitro by a conventional method and administered to the body, or may be synthesized in vivo .
  • a method for synthesizing antisense RNA in vitro employs RNA polymerase I.
  • a method for synthesizing antisense RNA in vivo involves performing transcription of antisense RNA using a vector containing a multicloning site (MCS) in the opposite direction.
  • MCS multicloning site
  • Such antisense RNA preferably contains a translation stop codon in its sequence to block translation into a peptide sequence.
  • siRNA refers to a nucleic acid molecule mediating RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99/07409 and WO 00/44914).
  • the siRNA to inhibit expression of a target gene provides effective gene knock-down method or gene therapy method. It was first used in plants, insects, Drosophila melanogaster and parasites and recently has been used for mammalian cell research (8-10).
  • the siRNA molecule of the present invention may consist of a sense RNA strand (having sequence corresponding to mRNA) and an antisense RNA strand (having sequence complementary to mRNA) and form a duplex structure.
  • the siRNA molecule of the present invention may have a single strand structure comprising self-complementary sense and antisense strands.
  • the siRNA of the present invention is not restricted to an RNA duplex of which two strands are completely paired and may comprise a non-paired portion such as a mismatched portion with non-complementary bases and bulge with no opposite bases.
  • the overall length of the siRNA is 10-100 nucleotides, preferably, 15-80 nucleotides, and more preferably, 20-70 nucleotides.
  • the siRNA may comprise either a blunt or cohesive end so long as it enables to silence the BLT2 expression due to RNAi effect.
  • the cohesive end may be prepared in 3'-end overhanging structure or 5'-end overhanging structure.
  • the siRNA may be constructed by inserting a short nucleotide sequence (e.g., about 5-15 nt) between self-complementary sense and antisense strands.
  • the siRNA expressed forms a hairpin structure by intramolecular hybridization, resulting in the formation of stem-and-loop structure.
  • the stem-and-loop structure is processed in vitro or in vivo to generate active siRNA molecule mediating RNAi.
  • shRNA small hairpin RNA
  • shRNA small hairpin RNA
  • An oligo DNA having sense sequence and its complimentary nonsense sequence of target gene siRNA linked via a linker having 3 to 10 bases is synthesized.
  • the resulting oligo DNA is cloned into a plasmid vector or lentivirus, which is a retrovirus, or adenovirus to express, thereby forming a loop sequence of the shRNA.
  • the resulting sequence is cleaved by a Dicer in the viral cell to generate siRNA, which will exhibit RNAi effects.
  • shRNA exhibits RNAi effects for a relatively long time, as compared to siRNA.
  • the present invention provides a method for screening an antifungal agent, the method comprising the steps of: (a) contacting a cell comprising a Hxl1 protein set forth in SEQ ID NO: 1 or an Ire1 protein set forth in SEQ ID NO: 3 with a sample to be analyzed; (b) measuring the amount or activity of the protein; and (c) determining the sample as an antifungal agent wherein the amount or activity of the Hxl1 protein is measured to be down-regulated.
  • the present invention provides a method for screening an antifungal agent for co-administration, the method comprising the steps of: (a) contacting a cell comprising a Hxl1 protein set forth in SEQ ID NO: 1 or an Ire1 protein set forth in SEQ ID NO: 3 with an antifungal agent, and measuring the amount or activity of the protein; (b) contacting a cell comprising a Hxl1 protein set forth in SEQ ID NO: 1 or an Ire1 protein set forth in SEQ ID NO: 3 with a sample to be analyzed and the said antifungal agent, and measuring the amount or activity of the protein; (c) comparing the value measured in step (a) with the value measured in step (b); and determining the sample as an antifungal agent for co-administration wherein the value measured in step (b) is down-regulated compared to the value measured in step (a).
  • the present invention provides a method for screening an antifungal agent for co-administration, the method comprising the steps of: SEQ ID NO: 2 or an IRE1 gene set forth in SEQ ID NO: 4 with an antifungal agent, and measuring the expression of the gene; (b) contacting a cell comprising a HXL1 gene set forth in SEQ ID NO: 2 or an IRE1 gene set forth in SEQ ID NO: 4 with a sample to be analyzed and the said antifungal agent, and measuring the expression of the gene; (c) comparing the value measured in step (a) with the value measured in step (b); and determining the sample as an antifungal agent for co-administration wherein the value measured in step (b) is down-regulated compared to the value measured in step (a).
  • the present invention provides an antifungal pharmaceutical composition
  • an antisense or siRNA (small interference RNA) oligonucleotide having a sequence complementary to a nucleotide sequence set forth in SEQ ID NO: 2 or 4, wherein the antisense or siRNA oligonucleotide has a sequence complementary to nucleotides 88 to 617 of the nucleotide sequence of SEQ ID NO: 2, and the antisense or siRNA oligonucleotide has a sequence complementary to nucleotides 1935-2421 of the nucleotide sequence of SEQ ID NO: 4.
  • an antisense or siRNA small interference RNA
  • the meningitis-treating agent is an azole-based or non-azole-based meningitis-treating agent.
  • the azole-based meningitis-treating agentfor co-administration is any one or more of fluconazole, itraconazole, voriconazole and ketoconazole.
  • the non-azole-based meningitis-treating agent for co-administration is amphotericin B or fludioxonil.
  • step (a) is carried out at a temperature ranging from 35°C to 40°C, and the cells in step (a) are Cryptococcus neoformans .
  • the present invention provides: an Hxl1 ( H AC1 and X BP1- L ike gene 1) protein set forth in SEQ ID NO: 1; an HXL1 gene encoding the protein of SEQ ID NO: 1; an HXL1 gene set forth in SEQ ID NO: 2; and a host containing a deletion of the gene of SEQ ID NO: 2.
  • meningitis as used herein is meant to include various inflammatory diseases occurring in the subarachnoid space between the arachnoid and the pia mater, for example, those caused by invasion of viruses or bacteria into the subarachnoid space, inflammation caused by a certain chemical substance, and those caused by the spread of cancer cells into the cerebrospinal fluid space.
  • FIG. 1 is a schematic diagram showing a method of preparing primers for performing PCR.
  • FIG. 3 shows a change in the specific splicing pattern of the HXL1 gene in an ire1 ⁇ mutant strain. After treatment with DTT and tunicamycin which cause UPR stress, it was confirmed by RT-PCR2 that a change in the specific splicing pattern of the HXL1 in the ire1 ⁇ mutant strain, suggesting that HXL1 is the target of Ire1 sensor kinase/ribonclease.
  • FIG. 4 shows a phylogenetic tree of target proteins of Ire1 sensor kinase.
  • An Aspergillus nidulans
  • Af Aspergillus fumigatus
  • Tr Trichoderma reesei
  • Sc Saccharomyces cerevisiae
  • Ca Candida albicans
  • Yl Yarrowia lipolytica
  • Hs Homo sapiens
  • Mm Mus musculus
  • Ce Caenorhabditis elegans
  • Cn Cryptococcus neoformans .
  • FIG. 8 shows an siRNA design for inhibiting HXL1 gene expression.
  • An ACT1 promoter and a GAL7 promoter were introduced at the ends of the sense and antisense strands of the HXL1 , and a linker was inserted into the gene such that the sense and antisense strands could form a stem-loop.
  • the linker may be the GFP protein as mentioned as the references.
  • FIG. 10 shows the effect of a deletion of the UPR signaling pathway gene HXL1 on pathogenicity in vivo . It shows that a deletion of the UPR signaling pathway gene HXL1 led to a decrease in pathogenicity in vivo .
  • the following strains were used in the experiment: wild type (WT), hxl1 ⁇ (YSB723), and hxl1 ⁇ + HXL1 (YSB762).
  • each of the strains was incubated in 5 ml of YPD medium at 30°C for 16 hours, washed, and then serially diluted in dH 2 O (degree of dilution: 1 to 10 4 ). Then, each of the incubated strains was spotted on a solid YPD medium containing an indicated concentration of an antifungal agent.
  • each of the strains was spotted on an YPD medium containing each of polyene-based drug(amphotericin B (Sigma)), azole-based drug (fluconazole (Sigma), itraconazole (Sigma), ketoconazole (Sigma)), and the phenylpyrrole-based drug Fludioxonil. After spotting, the strains were incubated at 30°Cand photographed for 2-4 days.
  • each of the strains was incubated in YPD medium at 30°C for 16 hours. Then, each strain was inoculated into 100 ml of fresh YPD medium and adjusted to an optical density of 0.15 at 600 nm (OD 600 ), after which each strain was incubated at 30°C until it reached an OD 600 of 0.5.
  • OD 600 optical density of 0.15 at 600 nm
  • 50 ml was sampled from 100 ml of the incubation medium and washed twice with DEPC-treated dH 2 O, followed by rapid freezing in liquid nitrogen. The remaining 50 ml of the incubation medium was treated with 8 ⁇ g/mlof tunicamycin or 20 mM of DTT, and after 1 or 2 hours, the strains were harvested.
  • underlined primer sequences are restriction enzyme portions for subsequence subcloning, and the sequences indicated by bold letters are complementary sequences for fusion PCR.
  • a complemented strain for each of the transformed strains containing a deletion of the UPR signaling pathway gene was produced.
  • an IRE1 complemented strain an IRE1 gene fragment comprising a 0.78-kb promoter, a 3.52-kb ORF (Open Reading Frame) and a 0.53-kb terminator was amplified by PCR using primers containing restriction enzyme sites. The PCR product was subcloned into a pTOP-V2 plasmid (purchased from Enzynomics), and the DNA base sequence thereof was analyzed.
  • the 4.8-kb IRE1 gene insert was subcloned into a pJAF12 vector (provided from James A. Fraser, Centre for Infectious Disease Research, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia) having a NEO r marker (Neomycin/G418-resistant marker) using a plasmid which base sequences are matched.
  • the subcloned plasmid was digested with Mlu1 restriction enzyme, and the IRE1 -deleted mutant strain was transformed with the digested plasmid.
  • HXL1 gene fragment comprising a 1-kb promoter, a 1.8-kb ORF (Open ReadingFrame) and a 0.6-kb terminator was amplified by PCR using primers (C35/C38) and subcloned into a pJAFS1 vector (from which the Sac1 restriction enzyme site of pJAF12 had been removed) having a NEO r marker.
  • the subcloned plasmid was digested with Sac1 restriction enzyme, the HXL1- deleted mutant strain was transformed with the resulting plasmid.
  • the spliced mRNA and unspliced mRNA of the HXL1 gene were obtained from cDNA derived from total RNA isolated from cells treated or untreated with the UPR (unfolded protein response) derivative tunicamycin.
  • DNAs were obtained from the mRNAs and subcloned into pJAFS1-HXL1PT plasmids (from which the SacI restriction enzyme site of pJAF12 had been removed) having a HXL1 promoter and a terminator, thereby obtaining a pJAFS1-HXL1u plasmid and a pJAFS1-HXL1s plasmid.
  • pJAFS1-HXL1PT plasmids from which the SacI restriction enzyme site of pJAF12 had been removed
  • Each of the plasmids was digested with Sac1 restriction enzyme and transformed into the IRE1 -deleted mutant strain.
  • mice In order to examine the effects of Ire1 sensor kinase/ribonuclease and its target gene HXL1 (which play an important role in the UPR signaling pathway) on pathogenicity in vivo , 4-6-week-old A/Jcr mice (Jackson Laboratory, 18-22 g) were used. Each of wild type, ire1 ⁇ , hxl1 ⁇ mutant strains and the restored strains was incubated in YPD medium at 24°C for 16 hours, after which each strain was washed with PBS (phosphate buffered saline) and then adjusted to a cell density of 10 6 cells/ml. 10 mice were used for each of the wild type strain, the mutant strain and the restored strain and infected by intranasal infection of 10 5 cells. The survival of the mice was observed twice a day for 6 weeks.
  • PBS phosphate buffered saline
  • candidate genes were searched through the serotype A C. neoformans genome database ( http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.html ) using the bZIP domain sequence of Hac1 transcription factor known as the target in S. cerevisiae .
  • the following five candidate genes were selected: CNAG_00871.2, CNAG_06134.2, CNAG_07560.2, CNAG_07940.2 and CNAG_03976.2.
  • Ire1 kinase/ribonuclease is characterized in that it splices the target mRNA in an unconventional manner.
  • RT-PCR primers as shown in Table 2 above were prepared. The experiment was performed by two stages, that is, RT-PCR1 and RT-PCR2 (see FIG. 1). The target gene was identified from changes in splicing patterns which appeared in RT-PCR1 and RT-PCR2 when treated with DTT and tunicamycin which cause UPR stress (see FIG. 2). The experimental results showed that CNAG_06134.2 was specifically spliced.
  • CNAG_06134.2 was treated with the UPR stress-causing DTT and tunicamycin in the same manner as above and subjected to RT-PCR2. As a result, CNAG_06134.2 was specifically spliced in the wild type strain, but was not spliced in the ire1 ⁇ mutant strain (see FIG. 3). Such results demonstrated that CNAG_06134.2 mRNA is the target gene of Ire1 sensor kinase/ribonuclease.
  • the CNAG_06134.2 gene had a significantly low sequence similarity to the known target protein, the gene was named " HXL1 ( H AC1 and X BP1 - L ike gene 1 )"based on splicing patterns similar to those of the Hac1 transcription factor in fungi and the Xbp1 transcription factor in humans.
  • Pathogenic determinants having effects on the pathogenicity of C. neoformans include capsules interfering with phagocytosis, antioxidant melanin, and the capability to grow at the body temperature of humans which are hosts. Among them, the capability to grow at the host temperature of 37°C can be said to be a very important factor in causing pathogenicity.
  • C. neoformans mutant strains having a mutation in the UPR signaling pathway are sensitive to antifungal agents was examined. As a result, it was shown that both the ire1 ⁇ mutant strain and the hxl1 ⁇ mutant strain were more sensitive to polyene-based amphotericin B than the wild type strain. Also, the amphotericin B sensitivity of the ire1 ⁇ mutant strain transformed with the HXL1 splicing gene was restored to that of the wild type strain. The amphotericin B sensitivity of the ire1 ⁇ mutant strain was shown to be dependent on Hxl1 (FIG. 6).
  • the drug sensitivities of the mutant strains to azole-based drugs were tested.
  • the mutant strains were significantly more sensitive to the drugs than the wild type strain.
  • the hxl1 ⁇ mutant strain showed more than 10 times higher sensitivity to fluconazole than the ire1 ⁇ mutant strain.
  • Hxl1 is regulated not only by Ire1, but also by other up-regulating proteins.
  • the drug sensitivity of the ire1 ⁇ mutant strain transformed with the HXL1 splicing gene was restored to the level of the wild type strain.
  • the sensitivity of the ire1 ⁇ mutant strains to the azole-based drugs also depends on Hxl1 in the UPR signaling pathway (FIG. 6).
  • siRNA can be synthesized using Silencer TM siRNA cocktail kit (RNase III; Ambion). Oligonucleotides used to synthesize dsRNA against IRE1 are as follows: 5'-GATCTCAGATACTATCATTGGTTTTGGATC-3', and 5'- CAAGTTGTTCGCCGTCGGCGCAAAGGAT-3. Also, oligonucleotides used to synthesize dsRNA against HXL1 are as follows: 5'- CCTATCAAGCGTCCTCGTCAATCTAGT -3', and 5'- CCTATCAAGCGTCCTCGTCAATCTAGT -3'.
  • siRNA had a length of 431 bp (nucleotides 1935-2421 of SEQ ID NO: 4) for IRE1 and a length of 530 bp (nucleotides 88 -617 of SEQ ID NO: 3) for HXL1 (see Ahn JH, et al: Identification of the genes differentially expressed in human dendritic cell subsets bycDNA subtraction and microarray analysis. Blood 2002, 100(5):1742-1754, and Yang YH et al: Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variations. Nucleic Acids Res 2002, 30(4): e15. 20, 21).
  • FIGS. 7 and 8 Designs of IRE1 and HXL1 siRNAs are shown in FIGS. 7 and 8 (see Hong Liu, et al: RNA interference in the pathogenic fungus C. neoformans . Genetics, 2002, 160:463-470). Each of the above-designed siRNAs was linearized and transformed into the wild type strain by electroporation. Silencing of the expression of the IRE1 or HXL1 gene by such siRNA showed effects, such as antifungal agent sensitivity, pathogenicity and temperature sensitivity, like the deletion of the genes.
  • IRE1 encodes a putative protein kinase containing a membrane-spanning domain and is required for inositol phototrophy in Saccharomyces cerevisiae . Mol Microbiol 6:1441-1446.
  • PKC1 is essential for protection against both oxidative and nitrosative stresses, cell integrity, and normal manifestation of virulence factors in the pathogenic fungus Cryptococcus neoformans . Eukaryot Cell 7:1685-1698.
  • Sequence ID No. 1 represents amino acid sequence of Hxl1 protein
  • sequence ID No. 2 represents nucleotide sequence of Hxl1 gene
  • Sequence ID No. 3 represents amino acid sequence of Ire protein
  • sequence ID No. 4 represents nucleotide sequence of IRE1 gene.

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Abstract

La présente invention concerne l'utilisation des gènes IRE1 et HXL1 de la voie de signalisation UPR pour le traitement d'une infection fongique et de la méningite. Dans l'invention, il a été nouvellement découvert que la suppression des protéines Ire1 et Hxl1 (HAC1 et gène XBP1-Like 1), nouvellement identifiées dans Cryptococcus neoformans, et des gènes codant pour les protéines, produit un effet antifongique et un effet de traitement de la méningite. Sur la base de cette découverte, un candidat qui peut présenter un effet synergique lorsqu'il est co-administré avec un agent antifongique ou un agent de traitement de la méningite existants peut être criblé et une nouvelle composition pharmaceutique ayant un effet antifongique et un effet de traitement de la méningite peut être produit.
PCT/KR2011/009862 2010-12-23 2011-12-20 Utilisation des gènes ire1 et hxl1 de la voie de signalisation upr pour le traitement d'une infection fongique et de la méningite Ceased WO2012087004A2 (fr)

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CN104164443A (zh) * 2014-07-14 2014-11-26 东华大学 一种检测活体酵母细胞upr水平的双荧光素酶监测质粒及其构建和应用
CN107106647B (zh) * 2014-10-21 2021-08-10 赫希玛有限公司 一种治疗方法
CN107106647A (zh) * 2014-10-21 2017-08-29 赫希玛有限公司 一种治疗方法
AU2021201810B2 (en) * 2014-10-21 2024-08-22 Deftbiotech Pty Ltd A method of treatment
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AU2015336933B2 (en) * 2014-10-21 2021-01-14 Deftbiotech Pty Ltd A method of treatment
EP3279330A4 (fr) * 2015-03-30 2019-02-20 Amtixbio Co., Ltd. Nouveau gène réglant la virulence decryptococcus neoformans
EP3739058A3 (fr) * 2015-03-30 2021-01-27 Amtixbio Co., Ltd. Nouveau gène réglant la virulence de cryptococcus neoformans et utilisation correspondante
WO2018015443A1 (fr) * 2016-07-22 2018-01-25 Novozymes A/S Hôte fongique filamenteux amélioré
US11046736B2 (en) 2016-07-22 2021-06-29 Novozymes A/S Filamentous fungal host
CN109476714A (zh) * 2016-07-22 2019-03-15 诺维信公司 改善的丝状真菌宿主
CN107308178B (zh) * 2017-07-31 2020-11-10 苏州大学附属儿童医院 一种用于肠外营养相关性肝病药物
CN107308178A (zh) * 2017-07-31 2017-11-03 苏州大学附属儿童医院 一种用于肠外营养相关性肝病药物

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