WO2010116673A1 - ミトコンドリア機能向上剤 - Google Patents
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- WO2010116673A1 WO2010116673A1 PCT/JP2010/002271 JP2010002271W WO2010116673A1 WO 2010116673 A1 WO2010116673 A1 WO 2010116673A1 JP 2010002271 W JP2010002271 W JP 2010002271W WO 2010116673 A1 WO2010116673 A1 WO 2010116673A1
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Definitions
- the present invention relates to a mitochondrial function improver and a PGC-1 ⁇ expression inducer using a lysine-specific demethylase-1 (LSD1) inhibitor.
- LSD1 lysine-specific demethylase-1
- the energy metabolism in mitochondria plays an essential role not only for the maintenance of life but also for various higher-order functions.
- tissues with high energy demand such as brain and muscle, it is known that many disorders are induced by a decrease in mitochondrial function.
- the regulation of mitochondrial function is strongly dependent on the control of the expression of metabolic enzyme genes by nuclear transcription factors. It is a factor.
- Tranylcypromine is a kind of monoamine oxidase inhibitor, has the effect of increasing substances such as dopamine in the brain by inhibiting the action of monoamine oxidase, and is effective as an antidepressant
- Patent Document 1 describes a therapeutic agent for metabolic syndrome using tranylcypromine. Furthermore, Non-Patent Document 1 describes that tranylcypromine inhibits LSD1 enzyme, and Non-Patent Document 2 describes that other monoamine oxidase inhibitors have low LSD1 inhibitory activity. Yes.
- Trans-2-Phenylcyclopropylamine is a mechanism-based inactivator of the histone demethylase LSD1. Biochemistry 46, 4408-4416. Lee, M.G., Wynder, C., Schmidt, D.M., McCafferty, D.G., and Shiekhattar, R. (2006). Histone H3 lysine
- the present invention has been made to provide a novel mitochondrial function improver and PGC-1 ⁇ expression inducer to be solved.
- histone demethylase LSD1 suppresses the expression of mitochondrial functional genes including PGC-1 ⁇ .
- PGC-1 (alpha) can be induced
- LSD1 target gene groups, such as PGC-1 (alpha) were induced
- a mitochondrial function improver comprising a lysine-specific demethylase-1 (LSD1) inhibitor.
- the lysine-specific demethylase-1 (LSD1) inhibitor is a nucleic acid or FAD synthesis inhibitor that can suppress the expression of LSD1 or BHC80 or an enzyme involved in FAD synthesis by tranylcypromine or RNAi.
- the mitochondrial function improving agent as described.
- RFK riboflavin kinase
- FADS FAD synthase
- nucleic acid capable of suppressing LSD1 expression by RNAi is an siRNA comprising the sequence set forth in SEQ ID NO: 29 and an siRNA comprising the sequence set forth in SEQ ID NO: 30
- a PGC-1 ⁇ expression inducer comprising a lysine-specific demethylase-1 (LSD1) inhibitor.
- Lysine-specific demethylase-1 (LSD1) inhibitor is capable of expressing LSD1 or BHC80 or an enzyme involved in FAD synthesis (riboflavin kinase (RFK), FAD synthase (FADS), etc.) by tranylcypromine or RNAi.
- the PGC-1 ⁇ expression inducer according to (5) which is a nucleic acid or FAD synthesis inhibitor that can be suppressed.
- RFK riboflavin kinase
- FADS FAD synthase
- LSD1 is a chromatin structure regulatory protein identified in recent years, and there are still many unclear points regarding its physiological role. In the present invention, it was found that LSD1 regulates mitochondrial function through regulation of PGC-1 ⁇ gene expression. There is no other report on a method for improving mitochondrial function by targeting a specific chromatin regulatory factor, and according to the present invention, a novel therapeutic target can be developed.
- the drug of the present invention can be expected to be used for molecular target therapy of diseases (such as cranial nerve diseases, muscle diseases, heart diseases, etc.) associated with mitochondrial function decline.
- FIG. 1 shows the results of an exhaustive search for LSD1 target genes using RNAi for LSD1, RNAi for LSD1 coupling factor BHC80, or tranylcypromine (TC) in 3T3-L1 cells.
- FIG. 2 shows the results of induction of expression of mitochondrial functional genes including PGC-1 ⁇ by LSD1 inhibition.
- FIG. 3 shows the results of analysis of 3T3-L1 intracellular histone methylation by LSD1 inhibition using chromatin immunoprecipitation (ChIP).
- FIG. 4 shows the results of quantification of LSD1 / promoter interaction in 3T3-L1 cells by chromatin immunoprecipitation (ChIP).
- FIG. 5 shows the results of evaluation of PGC-1 ⁇ promoter activity by LSD1 inhibition using a luciferase assay.
- FIG. 6a shows the result of JC-1 staining 48 hours after exposure of 3T3-L1 cells to tranylcypromine (TC).
- FIG. 6b shows the results of JC-1 staining after LSD1 inhibition.
- FIG. 7 shows the results of inducing expression of mitochondrial functional genes including PGC-1 ⁇ in mice by tranylcypromine.
- FIG. 8 shows the results of regulation of expression of mitochondrial functional genes including PGC-1 ⁇ in mice by RNAi against LSD1.
- FIG. 9 shows the results of inducing expression of the LSD1 target gene group (mitochondrial functional gene group including PGC-1 ⁇ ) by enzyme inhibition related to flavin adenosine dinucleotide (FAD) synthesis.
- FIG. 10 shows the results of comprehensive analysis of LSD1 target genes using RNAi against LSD1 and RNAi against flavin adenosine dinucleotide synthase in 3T3-L1 cells.
- FIG. 11 shows the results of a transcriptional repression function analysis of LSD1 dependent on flavin adenosine dinucleotide (FAD).
- FIG. 12 shows the results of the expression status of LSD1 and BHC80 in mouse tissues.
- FIG. 13 shows the results of an analysis of the effects of main monoamine oxidase inhibitors on the LSD1 target gene group.
- the present invention relates to an agent for improving mitochondrial function and an agent for inducing PGC-1 ⁇ expression, including a lysine-specific demethylase-1 (LSD1) inhibitor.
- the mitochondrial function improvement referred to in the present invention means an increase in mitochondrial mass and / or activation of a citrate cycle, a fatty acid ⁇ oxidation system, an electron transport system, and the like.
- the lysine-specific demethylase-1 (LSD1) inhibitor used in the present invention is not particularly limited as long as it is a substance that can inhibit the function of lysine-specific demethylase-1 (LSD1), and for example, LSD1 by tranylcypromine or RNAi. Nucleic acids that can suppress the expression of can be used. In addition, RNAi for LSD1 coupling factors (such as BHC80) and enzymes involved in FAD synthesis required for LSD1 enzyme activity can also be used. Also included are FAD synthesis inhibitors using FAD analogs and low molecular weight compounds.
- RNAi examples include siRNA or shRNA as described below.
- siRNA or shRNA examples include siRNA or shRNA as described below.
- RNAi phenomenon occurs and RNA having a homologous sequence is degraded.
- Such RNAi phenomenon is a phenomenon observed in nematodes, insects, protozoa, hydra, plants, vertebrates (including mammals).
- double-stranded RNA having a length of about 20 bases (for example, about 21 to 23 bases) or less called siRNAs.
- siRNAs double-stranded RNA having a length of about 20 bases (for example, about 21 to 23 bases) or less, called siRNAs.
- siRNA suppresses gene expression by being expressed in a cell, and can suppress the expression of a target gene (LSD1 gene in the present invention) of the siRNA.
- siRNA kit used in the present invention may be in any form as long as it can cause RNAi.
- siRNA is an abbreviation for short interfering RNA, which is artificially chemically synthesized or biochemically synthesized, synthesized in an organism, or about 40 bases.
- This is a short double-stranded RNA of 10 base pairs or more formed by decomposing the above double-stranded RNA in the body, and usually has a 5′-phosphate, 3′-OH structure, and 3 ′ The end protrudes about 2 bases.
- a specific protein binds to the siRNA cage to form an RNA-induced-silencing-complex (RISC). This complex recognizes and binds to mRNA having the same sequence as siRNAs, and cleaves mRNA at the center of siRNA by RNaseIII-like enzyme activity.
- RISC RNA-induced-silencing-complex
- the sequence of siRNA and the sequence of mRNA that is cleaved as a target match 100%.
- the cleavage activity by RNAi often remains partially, so it does not necessarily need to match 100%.
- the region having homology between the base sequence of siRNA and the base sequence of the LSD1 gene whose expression should be suppressed preferably does not include the translation start region of the LSD1 gene. This is because various transcription factors and translation factors are expected to bind to the translation initiation region, so that siRNA can not be effectively bound to mRNA and the effect is expected to be reduced. Therefore, the sequence having homology is preferably 20 bases away from the translation initiation region of the LSD1 gene, more preferably 70 bases away from the translation initiation region of the LSD1 gene. The sequence having homology may be, for example, a sequence near the 3 ′ end of the LSD1 gene.
- siRNA can be used as a factor that causes RNAi, and a factor that generates siRNA (for example, dsRNA having about 40 bases or more) can be used as such a factor.
- a factor that generates siRNA for example, at least about 70%, preferably 75% or more, more preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% with respect to a part of the nucleic acid sequence of the LSD1 gene.
- RNA containing a double-stranded portion or a variant thereof containing a sequence having 100% homology can be used most preferably.
- sequence portion having homology is usually at least about 15 nucleotides or more, preferably at least about 19 nucleotides, more preferably at least about 20 nucleotides in length, and even more preferably at least about 21 nucleotides in length.
- the base sequence of the LSD1 gene is known and is registered in, for example, NCBINCNM 015013.
- shRNA short hairpin RNA
- shRNA having a short hairpin structure having a protruding portion at the 3 ′ end
- shRNA refers to a molecule of about 20 base pairs or more that has a double-stranded structure in a molecule and a hairpin-like structure by including a partially palindromic base sequence in a single-stranded RNA.
- Such shRNA after being introduced into the cell, is degraded to a length of about 20 bases (typically, for example, 21 bases, 22 bases, 23 bases) in the cell, and causes RNAi similarly to siRNAs. be able to.
- shRNA causes RNAi similarly to siRNAs, and thus can be used effectively in the present invention.
- the shRNA preferably has a 3 ′ protruding end.
- the length of the double-stranded part is not particularly limited, but is preferably about 10 nucleotides or more, more preferably about 20 nucleotides or more.
- the 3 ′ protruding end is preferably DNA, more preferably DNA of at least 2 nucleotides, and further preferably DNA of 2 to 4 nucleotides.
- RNAi used in the present invention may be artificially chemically synthesized, or the DNA sequence of the sense strand and antisense strand.
- a DNA having a hairpin structure ligated in the reverse direction can also be prepared by synthesizing RNA in vitro with T7 RNA polymerase.
- antisense and sense RNAs can be synthesized from template DNA using T7 RNA polymerase and T7 promoter. When these are annealed in vitro and then introduced into cells, RNAi is caused and LSD1 expression is suppressed.
- RNAi can be introduced into cells using, for example, the calcium phosphate method or various transfection reagents (eg, oligofectamine, lipofectamine, lipofection, etc.).
- the administration method of the mitochondrial function enhancer and PGC-1 ⁇ expression inducer of the present invention may be oral administration or parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration) Internal administration, intravaginal administration, local administration to the affected area, skin administration, etc.).
- parenteral administration for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration
- Internal administration intravaginal administration, local administration to the affected area, skin administration, etc.
- the mitochondrial function improver and PGC-1 ⁇ expression inducer of the present invention can be combined with pharmaceutically acceptable additives as necessary when used as a pharmaceutical composition.
- pharmaceutically acceptable additives include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles. , Diluents, carriers, excipients and / or pharmaceutical adjuvants and the like.
- the preparation form of the mitochondrial function-enhancing agent and PGC-1 ⁇ expression inducer of the present invention is not particularly limited, and examples thereof include liquid agents, injections, sustained-release agents and the like.
- the solvent used for formulating the mitochondrial function-enhancing agent and PGC-1 ⁇ expression inducer of the present invention as the above-mentioned preparation may be either aqueous or non-aqueous.
- Injections can be prepared by methods well known in the art. For example, after dissolving in an appropriate solvent (physiological saline, buffer solution such as PBS, sterilized water, etc.), sterilized by filtration with a filter, and then filled into a sterile container (eg, ampoule) Can be prepared.
- This injection may contain a conventional pharmaceutical carrier, if necessary.
- Administration methods using non-invasive catheters can also be used.
- the carrier that can be used in the present invention include neutral buffered physiological saline or physiological saline mixed with serum albumin.
- the dosage of the mitochondrial function-enhancing agent and the PGC-1 ⁇ expression inducer of the present invention is determined by the purpose of use, the severity of the disease, the patient's age, weight, sex, history, or the expression of LSD1 by RNAi as an active ingredient. This can be determined by those skilled in the art in consideration of the type of nucleic acid that can be suppressed. In the case of tranylcypromine, for example, in an adult, it can be administered in the range of 1 to 100 mg per day, preferably 10 mg to 100 mg.
- the active ingredient is a substance capable of suppressing the expression of LSD1 by RNAi
- the dose is not particularly limited, and is, for example, about 0.1 ng to about 100 mg / kg, preferably about 1 ng to about 10 mg per day.
- siRNA GL3 5'-GATTTCGAGTCGTCTTAAT-3 '(SEQ ID NO: 17) lamin A / C: 5′-CTGGACTTCCAGAAGAACA-3 ′ (SEQ ID NO: 18)
- LSD1 sense 5'-CACAAGGAAAGCUAGAAGA (dT) (dT) -3 '(SEQ ID NO: 29)
- LSD1 antisense 5'-UCUUCUAGCUUUCCUUGUG (dT) (dT) -3 '(SEQ ID NO: 30)
- BHC80 sense 5'-GUUCCAGAUACAGCCAUUG (dT) (dT) -3 '(SEQ ID NO: 31)
- BHC80 antisense 5'-CAAUGGCUGUAUCUGGAAC (dT) (dT) -3 '(SEQ ID NO: 32)
- RFK sense 5'-UCUUCCAGCUGAUGUGUCU (dT) (dT) -3 '(SEQ ID NO: 33)
- RFK antisense 5'-AGACACAUCAGCUGGAAGA (dT) (dT) -3 '(SEQ ID NO: 34)
- FADS sense 5'-GAGCC
- Chromatin immunoprecipitation (ChIP) assay for detecting methylation of the 4th lysine residue (H3K4) and binding of LSD1 of histone H3 (FIGS. 3 and 4)
- the DNA-protein complex of 3T3-L1 cells was cross-linked using 1% formalin, and chromatin was fragmented with a water tank sonicator. Chromatin fragments were incubated overnight at 4 ° C. in the presence of antibodies against methyl-H3K4 or LSD1 or BHC80 and recovered using agarose beads to which proteins A and G were bound. DNA was isolated and real-time PCR was performed using the following primer set.
- PGC-1a locus region a Forward: 5′-GTCTAATTGAGACTGGCTGTG-3 ′ (SEQ ID NO: 19) Region a: Reverse: 5′-CAACATGTTGAGCAACTCAGC-3 ′ (SEQ ID NO: 20) Region b: Forward: 5'-AAGCTTGACTGGCGTCATTC-3 '(SEQ ID NO: 21) Region b: Reverse: 5′-GCTCCGGTCCTGCAATACTC-3 ′ (SEQ ID NO: 22) Region c: Forward: 5′-TCAAAGATGCCTCCTGTGAC-3 ′ (SEQ ID NO: 23) Region c: Reverse: 5′-CAAGGAGAGACCTGCTTGCT-3 ′ (SEQ ID NO: 24) PDK4: Forward: 5'-CTGGCTAGGAATGCGTGACA-3 '(SEQ ID NO: 25) PDK4: Reverse: 5'-GATCCCAGGTCGCTAGGACT-3 '(SEQ ID NO: 26) FATP
- FIG. 5 (4) Analysis of PGC-1 ⁇ promoter activity by luciferase assay (FIG. 5)
- a luciferase reporter vector containing the mPGC-1 ⁇ promoter, fragments of 3707-bps from ⁇ 3627 to +80 of the promoter were 5 ′ and 3 ′.
- Amplification was performed by PCR using primers containing MluI and XhoI sites at the ends.
- Luciferase assay was performed using Dual-Luciferase Reporter Assay System (Promega) according to the attached protocol.
- the pGL3-PGC-1 ⁇ reporter vector was cotransfected into 3T3-L1 cells together with the pRL-TK reference vector and induced for adipogenesis for 24 hours in the presence or absence of TC, and then luciferase was measured. When siRNA was applied, siRNA was introduced 24 hours prior to reporter transfection.
- the cells were contacted with 5 ⁇ g / ml JC-1 at 37 ° C. for 15 minutes in the medium, suspended in PBS and subjected to FACS analysis. Green fluorescence and red fluorescence were detected by FL1 and FL2 settings, respectively. The value indicates the average fluorescence intensity in each setting.
- FAD-dependent transcriptional repression function of LSD1 FAD-dependent transcriptional repression function of LSD1 (FIG. 11) This was performed using the Dual-Luciferase Reporter Assay System (Promega) as described above, using a luciferase reporter vector containing a GAL4 binding sequence and a promoter. This reporter vector was cotransfected into 3T3-L1 cells together with an expression vector of LSD1 (wild type or FAD binding loss type) fused with GAL4, and luciferase was measured.
- LSD1 and BHC80 Expression of LSD1 and BHC80 in mouse tissues (FIG. 12) Each tissue was isolated from C57B / 6J mice (7-week old male), and total RNA was extracted using Trizol reagent (Invitrogen). LSD1 and BHC80 expression levels were normalized to the internal control gene 36B4. Values are expressed as a multiple of white adipose tissue. WAT (white fat), BAT (brown fat), liver (liver), Sk. Muscle (skeletal muscle), brain (brain).
- LSD1 target candidates were induced more than 2-fold by LSD1 knockdown (KD), and many of them were treated with coupling factor BHC80 knockdown (KD) or tranycypromine.
- KD LSD1 knockdown
- Target genes common to the three groups of LSD1 KD, BHC80 KD, and TC are key to energy consumption and mitochondrial biogenesis such as PGC-1 ⁇ , pyruvate dehydrogenase kinase 4 (PDK4) and AMP-dependent protein kinase ⁇ 2 subunits. Many regulatory molecules were included (FIG. 1, table below). Induction of gene expression such as PGC-1 ⁇ by LSD1 or BHC80 knockdown (KD) and tranylcypromine was also confirmed by quantitative RT-PCR (FIG. 2).
- LSD1 was located near the transcription start site of the PGC-1 ⁇ promoter (indicated by site b). Similar results were obtained with promoters of other LSD1 target genes. Furthermore, the expression of the luciferase reporter gene downstream of the PGC-1 ⁇ promoter was activated (derepressed) by LSD1 knockdown or tranylcypromine treatment (FIG. 5).
- tranylcypromine is effective in activation of mitochondrial function by LSD1 inhibition
- administration of tranylcypromine affects energy homeostasis in vivo Investigate whether or not to do.
- a 7-week-old C57B / 6J mouse was given a high fat diet for 6 weeks, and 10 mg / kg body weight of tranylcypromine or PBS was administered every other day.
- the administration of tranylcypromine increased the expression of LSD1 target genes including PGC-1 ⁇ in mouse testicular fat (FIG. 7). Similar results were obtained in the liver. This data indicates that inhibition of LSD1 by tranylcypromine stimulates energy expenditure in vivo.
- FAD-dependent transcriptional repression function of LSD1 In order to examine the FAD dependency of LSD1 function, a luciferase assay was performed using a luciferase reporter vector containing a GAL4 binding sequence and a promoter (FIG. 11). When LSD1 fused with GAL4 was expressed, wild-type LSD1 repressed transcription in a dose-dependent manner, whereas FAD-binding loss-type LSD1 did not exhibit transcriptional repression ability. This data indicates that the transcriptional repression ability of LSD1 depends on FAD binding.
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Abstract
Description
(1) リジン特異的デメチラーゼ-1(LSD1)阻害剤を含む、ミトコンドリア機能向上剤。
(2) リジン特異的デメチラーゼ-1(LSD1)阻害剤が、トラニルシプロミン、又はRNAiによりLSD1又はBHC80又はFAD合成に関わる酵素の発現を抑制できる核酸、FAD合成阻害剤である、(1)に記載のミトコンドリア機能向上剤。
(3) FAD合成に関わる酵素が、リボフラビンキナーゼ(RFK)及び/又はFAD合成酵素(FADS)である、(2)に記載のミトコンドリア機能向上剤。
(4) RNAiによりLSD1の発現を抑制できる核酸が、配列番号29に記載の配列からなるsiRNAと配列番号30に記載の配列からなるsiRNAである、(1)から(3)の何れか1項に記載のミトコンドリア機能向上剤。
(5) リジン特異的デメチラーゼ-1(LSD1)阻害剤を含む、PGC-1α発現誘導剤。
(6) リジン特異的デメチラーゼ-1(LSD1)阻害剤が、トラニルシプロミン、又はRNAiによりLSD1又はBHC80又はFAD合成に関わる酵素(リボフラビンキナーゼ(RFK)、FAD合成酵素(FADS)など)の発現を抑制できる核酸、FAD合成阻害剤である、(5)に記載のPGC-1α発現誘導剤。
(7) FAD合成に関わる酵素が、リボフラビンキナーゼ(RFK)及び/又はFAD合成酵素(FADS)である、(6)に記載のPGC-1α発現誘導剤。
(8) RNAiによりLSD1の発現を抑制できる核酸が、配列番号29に記載の配列からなるsiRNAと配列番号30に記載の配列からなるsiRNAである、(5)から(7)の何れか1項に記載のPGC-1α発現誘導剤。
本発明は、リジン特異的デメチラーゼ-1(LSD1)阻害剤を含む、ミトコンドリア機能向上剤およびPGC-1α発現誘導剤に関する。本発明で言うミトコンドリア機能向上とは、ミトコンドリア量の増大および/またはクエン酸回路、脂肪酸β酸化系、電子伝達系などの活性化のことを言う。
(1)3T3-L1細胞におけるLSD1標的遺伝子のマイクロアレイによる網羅的解析(図1)
RNAimax試薬(Invitrogen)を用いたLSD1又はその共役因子BHC80に対するsiRNAの導入又は10-4 Mのトラニルシプロミン-HCl (TC, Sigma)の添加後に、3T3-L1細胞を分化誘導培地で24時間培養し、RNA分析を行った。脂質生成誘導培地は、0.5μM 3-イソブチル-1-メチルキサンチン、1μM デキサメタゾン及び5μg/mlインスリンを、10%胎児ウシ血清を含むダルベッコ改変イーグル培地に溶解したものである。全RNAは、RNeasy miniカラム(Qiagen)を用いて抽出した。GeneChip Mouse Genome Array 4302とGeneChip Hybridization, Wash and Stain Kit (Affymetrix)と組み合わせて用いて、ゲノムワイドの発現解析を行った。
全RNAをTrizol 試薬(Invitrogen)を用いて抽出した。定量的RT-PCRは、Fast SYBR Green System (Applied Biosciences)を用いて行った。PGC-1α等の発現量は、内部対照遺伝子36B4で標準化した。値は、対照siRNAを導入した試料またはビヒクルで処理した試料に対する誘導の倍数として示す。統計学的な有意差について、対照に対して、*p<0.05, **p<0.01で示す(以下、同様)。使用したプライマーは以下の通りである。
PGC-1α/フォワード:5'-AAGTGTGGAACTCTCTGGAACTG-3' (配列番号1)
PGC-1α/リバース:5'-GGGTTATCTTGGTTGGCTTTATG-3' (配列番号2)
PDK4/フォワード:5'-CAAGGAGATCTGAATCTCTA -3' (配列番号3)
PDK4/リバース:5'-GATAATGTTTGAAGGCTGAC-3' (配列番号4)
RIIα/フォワード:5'- AACTGATGAGCAGAGATGCC-3' (配列番号5)
RIIα/リバース:5'- AACATGGCATCCAGAACTTG-3' (配列番号6)
FAT1P/フォワード:5'-CGCCCAGGACTCTGCAAAG-3' (配列番号7)
FATP1/リバース:5'-CACAGAAGTCTGGACTGGGA-3' (配列番号8)
UCP1/フォワード:5'-GGCCCTTGTAAACAACAAAATAC-3' (配列番号9)
UCP1/リバース:5'-GGCAACAAGAGCTGACAGTAAAT-3' (配列番号10)
36B4/フォワード:5'- GCGTCCTGGCATTGTCTGT-3' (配列番号11)
36B4/フォワード:5'-GCAAATGCAGATGGATCAGCC-3' (配列番号12)
LSD1:5'- CACAAGGAAAGCTAGAAGA -3'(配列番号13)
BHC80:5'-GTTCCAGATACAGCCATTG-3'(配列番号14)
RFK:5'-TCTTCCAGCTGATGTGTGT-3'(配列番号15)
FADS:5'-GAGCCCTTGGAGGAATGTC-3'(配列番号16)
コントロールsiRNA
GL3:5'-GATTTCGAGTCGTCTTAAT-3'(配列番号17)
lamin A/C:5'-CTGGACTTCCAGAAGAACA-3'(配列番号18)
LSD1センス:5'-CACAAGGAAAGCUAGAAGA(dT)(dT) -3'(配列番号29)
LSD1アンチセンス:5'-UCUUCUAGCUUUCCUUGUG(dT)(dT) -3'(配列番号30)
BHC80センス:5'-GUUCCAGAUACAGCCAUUG(dT)(dT) -3'(配列番号31)
BHC80アンチセンス:5'-CAAUGGCUGUAUCUGGAAC(dT)(dT) -3'(配列番号32)
RFKセンス:5'-UCUUCCAGCUGAUGUGUCU(dT)(dT)-3'(配列番号33)
RFKアンチセンス:5'-AGACACAUCAGCUGGAAGA(dT)(dT)-3'(配列番号34)
FADSセンス:5'-GAGCCCUUGGAGGAAUGUC(dT)(dT)-3'(配列番号35)
FADSアンチセンス:5'-GACAUUCCUCCAAGGGCUC(dT)(dT)-3'(配列番号36)
GL3センス:5'- GAUUUCGAGUCGUCUUAAU(dT)(dT)-3' (配列番号37)
GL3アンチセンス:5'-AUUAAGACGACUCGAAAUC(dT)(dT)-3' (配列番号38)
lamin A/Cセンス:5'- CUGGACUUCCAGAAGAACA (dT)(dT)-3' (配列番号39)
lamin A/Cアンチセンス:5'-UGUUCUUCUGGAAGUCCAG(dT)(dT)-3' (配列番号40)
3T3-L1細胞のDNA-タンパク質複合体を、1%ホルマリンを用いて架橋し、水槽超音波処理機でクロマチンを断片化した。クロマチン断片は、メチル-H3K4 又はLSD1又はBHC80に対する抗体の存在下において4℃で一晩インキュベートし、プロテインA及びGを結合させたアガロースビーズを用いて回収した。DNAを単離し、以下のプライマーセットを用いてリアルタイムPCRを行った。
領域a: フォワード:5'-GTCTAATTGAGACTGGCTGTG-3' (配列番号19)
領域a: リバース:5'-CAACATGTTGAGCAACTCAGC-3' (配列番号20)
領域b: フォワード: 5'-AAGCTTGACTGGCGTCATTC-3' (配列番号21)
領域b: リバース: 5'- GCTCCGGTCCTGCAATACTC-3' (配列番号22)
領域c: フォワード:5'- TCAAAGATGCCTCCTGTGAC-3' (配列番号23)
領域c: リバース:5'-CAAGGAGAGACCTGCTTGCT-3' (配列番号24)
PDK4:フォワード:5'-CTGGCTAGGAATGCGTGACA -3' (配列番号25)
PDK4:リバース:5'-GATCCCAGGTCGCTAGGACT-3' (配列番号26)
FATP1:フォワード:5'-CGCCCAGGACTCTGCAAAG-3' (配列番号27)
FATP1:リバース:5'-CACAGAAGTCTGGACTGGGA-3' (配列番号28)
mPGC-1αプロモーターを含むルシフェラーゼレポーターベクターであるpGL3-PGC-1α(PGC-1α/Luc)の構築のために、プロモーターの-3627から+80までの3707-bpsの断片を、5’及び3’末端にそれぞれMluI 部位及びXhoI部位を含むプライマーを用いてPCRにより増幅した。ルシフェラーゼアッセイは、Dual-Luciferase Reporter Assay System (Promega)を添付のプロトコールに従って用いて行った。pGL3-PGC-1αレポーターベクターを、pRL-TK参照ベクターと一緒に3T3-L1細胞にコトランスフェクションし、TCの存在下又は非存在下において24時間脂肪分化を誘導した後、ルシフェラーゼを測定した。siRNAを適用する場合には、siRNAはレポーターのトランスフェクションの24時間前に導入した。
ミトコンドリア生合成の測定のために、細胞を蛍光色素JC-1 (Molecular Probes)で染色した後、フローサイトメトリーで解析した。JC-1は、ミトコンドリア内膜に結合して緑色の蛍光を発し、膜電位に依存して赤色蛍光の凝集体を形成する。従って、ミトコンドリア量は緑色蛍光を検出することによって評価でき、ミトコンドリア電子伝達の量は赤色蛍光を検出することによって評価できる。3T3-L1 細胞は、10-3 又は10-4 Mのトラニルシプロミンで処理し(図6a)、又はLSD1に対する上記のsiRNAを導入し(図6b)、24時間脂肪分化を誘導した。次いで、細胞を培地中で15分間37℃で5 μg/ml JC-1に接触させ、PBSに懸濁し、FACS解析を行った。緑色蛍光及び赤色蛍光はそれぞれFL1及びFL2セッティングにより検出した。値は、各セッティングにおける平均蛍光強度を示す。
C57B/6Jマウス(7-週齢の雄)に、高脂肪食を6週間与え、10mg/kg体重のトラニルシプロミン又はPBS (n=8)を1日おきに腹腔内に投与した。解剖直前に16時間絶食した後、組織をマウスから単離した。マウスの精巣周囲白色脂肪組織及び肝臓からの全RNAをTrizol 試薬(Invitrogen)を用いて抽出した。発現解析は、上記の通り行った。PGC-1α等の発現量は、内部対照遺伝子36B4に標準化した。値は、PBSで処理した対照マウスに対する誘導の倍数で示す。
C57B/6Jマウス(7-週齢の雄)に、高脂肪食又は通常食を6週間与え、精巣周囲白色脂肪組織をマウスから単離した。細かく切断した組織にアデノウイルスベクターを用いてLSD1 shRNAを導入し、同組織からの全RNAをTrizol 試薬(Invitrogen)を用いて抽出した。発現解析は、上記の通り行った。PGC-1α等の発現量は、内部対照遺伝子36B4に標準化した。値は、コントロールのアデノウイルスを用いた対照組織に対する誘導の倍数で示す。
リボフラビンキナーゼ(RFK)又はFAD合成酵素(FADS)に対するsiRNAを導入した3T3-L1細胞から全RNAをTrizol 試薬(Invitrogen)を用いて抽出した。定量的RT-PCRは、上記の通りに行い、PGC-1α等の発現量は、内部対照遺伝子36B4で標準化した。値は、対照siRNAを導入した試料またはビヒクルで処理した試料に対する誘導の倍数として示す。FADアッセイキット(BioVision)を用いて、FAD合成阻害下による細胞内FAD量を測定した。
RNAimax試薬(Invitrogen)を用いたRFKに対するsiRNAの導入後に、3T3-L1細胞を分化誘導培地で24時間培養し、上記の通りに、RNA分析を行った。GeneChip Mouse Genome Array 4302とGeneChip Hybridization, Wash and Stain Kit (Affymetrix)と組み合わせて用いて、ゲノムワイドの発現解析を行った。
GAL4結合配列とプロモーターを含むルシフェラーゼレポーターベクターを用いて、上記の通りに、Dual-Luciferase Reporter Assay System (Promega)を用いて行った。このレポーターベクターを、GAL4と融合したLSD1(野生型又はFAD結合喪失型)の発現ベクターと一緒に3T3-L1細胞にコトランスフェクションし、ルシフェラーゼを測定した。
C57B/6Jマウス(7-週齢の雄)から、各組織を単離し、全RNAをTrizol 試薬(Invitrogen)を用いて抽出した。LSD1とBHC80の発現量は、内部対照遺伝子36B4に標準化した。値は、白色脂肪組織に対する倍数で示す。WAT(白色脂肪)、BAT(褐色脂肪)、liver(肝臓)、Sk. muscle(骨格筋)、brain(脳)を示す。
トラニルシプロミン又はモノアミン酸化酵素(MAO)阻害剤の添加後に、3T3-L1細胞を分化誘導培地で24時間培養し、RNA分析を行った。発現解析は、上記の通り行った。PGC-1α等の発現量は、内部対照遺伝子36B4に標準化した。値は、ビークルで処理した対照に対する誘導の倍数で示す。Tranylcypromine(TC)、pargyline(parg)、phenelzine(phen)は10-4 M濃度で使用し、clorgyline(clorg)は10-5 M濃度で用いた。
(1)LSD1による脂肪細胞におけるエネルギー代謝の制御.
siRNA又は低分子化合物阻害剤であるトラニルシプロミン(TC)を用いて3T3-L1細胞からLSD1機能を除去した。トラニルシプロミンは、モノアミン酸化酵素A及びB(MAO A 及びB)の阻害剤として最初に同定され、LSD1阻害に特異性が高いことが生化学的に実証されている。これらの条件下で、マイクロアレイによる網羅的な発現解析を行い、分化中の3T3-L1細胞において標的遺伝子を同定した(図1)。LSD1の転写抑制活性と一致して、LSD1ノックダウン(KD)によって、2倍以上に誘導された標的候補は601個であり、それらの多くは共役因子BHC80ノックダウン(KD) 又はトラニルシプロミン処理によっても発現誘導された(図1、上図)。LSD1 KD, BHC80 KD, TCの3つのグループに共通した標的遺伝子には、PGC-1α, ピルビン酸デヒドロゲナーゼキナーゼ4 (PDK4)及びAMP依存性プロテインキナーゼγ2 サブユニットなどのエネルギー消費及びミトコンドリア生合成の鍵調節分子が多数含まれていた(図1、下表)。LSD1又はBHC80のノックダウン(KD)、トラニルシプロミンによるPGC-1α等の遺伝子発現の誘導は、定量的RT-PCRでも確認した(図2)。
PGC-1α遺伝子が、3T3-L1細胞においてLSD1によるH3K4脱メチル化により直接制御されているかどうかを調べた。クロマチン免疫沈降(ChIP)解析により、LSD1をノックダウンした細胞では、PGC-1α遺伝子プロモーターにおけるジメチル化H3K4の量が対照と比較して約2倍に増大することが示された(図3)。ジメチル化H3K4 の増大は、トラニルシプロミン処理下においてPGC-1αプロモーターにおいても検出された(図3)。PDK4などの他のLSD1標的遺伝子のプロモーターにおいても同様の結果であった。標的プロモーターにおけるLSD1の存在を確認するために、このタンパク質のChIP解析を行った(図4)。LSD1は、PGC-1αプロモーターの転写開始部位の近くに位置していた(部位bで示される)。他のLSD1標的遺伝子のプロモーターにおいても同様の結果であった。さらに、PGC-1αプロモーターの下流のルシフェラーゼレポーター遺伝子の発現は、LSD1のノックダウン又はトラニルシプロミン処理により、活性化(脱抑制)された(図5)。
LSD1阻害条件下での再活性化されたエネルギー消費の細胞学的影響を調べるために、PGC-1αによって活性化されることが知られているミトコンドリア機能の動態を調べた。そのために、ミトコンドリア内膜に結合して緑色蛍光を示し(FL1; ミトコンドリア量)、膜電位に依存して赤色蛍光の凝集物を形成する(FL2; ミチコンドリア活性)蛍光色素であるJC-1を用いて、トラニルシプロミンで処理した細胞を染色した(図6a)。JC-1陽性細胞のフローサイトメトリー解析により、トラニルシプロミンがミトコンドリア量と膜電位の両方に対して刺激効果を示すことが分かった。さらに、LSD1ノックダウンにおいて、同様の結果が得られた(図6b)。これらのデータは、LSD1がH3K4脱メチル化を介してエネルギー消費遺伝子を直接抑制し、ミトコンドリア機能を阻害していることを示している。同時に、LSD1阻害によって、ミトコンドリア機能とエネルギー消費を活性化できることを示している。
LSD1阻害によるミトコンドリア機能の活性化においてトラニルシプロミンが有効であることから、トラニルシプロミンの投与がインビボでエネルギー恒常性に影響するかどうかについて調べた。7週齢のC57B/6Jマウスに高脂肪食を6週間与え、10mg/kg体重のトラニルシプロミン又はPBSを1日おきに投与した。トラニルシプロミンの投与により、マウスの精巣周囲脂肪においてPGC-1αを含むLSD1標的遺伝子の発現は増大した(図7)。肝臓においても同様の結果であった。このデータは、トラニルシプロミンによるLSD1の阻害により、エネルギー消費がインビボで刺激されることを示している。
エネルギー消費向上の意義を調べるために、C57B/6Jマウス(7週齢の雄)に高脂肪食又は通常食を6週間与え、精巣周囲白色脂肪組織を細断してアデノウイルスベクター由来LSD1 shRNAでLSD1阻害を行った。PGC-1α等の発現量は、高脂肪食の条件下では増加するが、他方、通常食の条件下では低下した(図8)。このデータは、LSD1阻害は、エネルギー状態に依存して、ミトコンドリア機能を調節することを示している。すなわち、エネルギー過剰の場合には、LSD1阻害はエネルギー消費を促進し、他方、通常のエネルギー摂取の場合には、LSD1阻害はエネルギー消費を抑制することで、いずれも生体恒常性を維持することが示唆される。
LSD1は、FAD依存性の脱メチル化酵素であることから、細胞内FAD合成経路の機能について検討した。この経路においては、リボフラビンキナーゼ(RFK)とFAD合成酵素(FADS)が鍵酵素として知られている(図9)。これらに対するsiRNAを用いてFAD合成阻害すると、PGC-1α等のLSD1標的遺伝子の発現は活性化された。また、FAD合成阻害時においては、細胞内FAD量は低下していた。このデータは、FAD合成阻害によって、LSD1標的遺伝子を活性化できることを示している。
FADで調節される標的遺伝子を検討するために、LSD1又はRFKのノックダウン(KD)の条件下でマイクロアレイによる網羅的な発現解析を行い、3T3-L1細胞において標的遺伝子を同定した(図10)。LSD1の転写抑制活性と一致して、LSD1標的遺伝子の多くはRFKノックダウンによっても発現誘導された。このことから、LSD1とFAD合成に関わる酵素で発現調節される標的遺伝子は重複することが示された。
LSD1機能のFAD依存性を調べるために、GAL4結合配列とプロモーターを含むルシフェラーゼレポーターベクターを用いて、ルシフェラーゼアッセイを行った(図11)。GAL4と融合したLSD1を発現すると、野生型LSD1は量依存的に転写抑制したが、他方、FAD結合喪失型LSD1は転写抑制能を示さなかった。このデータは、LSD1の転写抑制能は、FAD結合に依存することを示している。
LSD1の生物学的な意義を調べるために、成熟マウスの各組織におけるLSD1とBHC80の発現を検討した(図12)。代謝組織の中では、白色脂肪で高く発現し、褐色脂肪、肝臓、骨格筋でも中等度の発現を認めた。一方、脳組織では極めて高く発現していることからも、各組織のエネルギー代謝においてLSD1が役割を果たすことを示唆している。
トラニルシプロミン及び既存のモノアミン酸化酵素(MAO)がLSD1標的遺伝子に対する影響について検討した(図13)。pargyline(parg)、phenelzine(phen)、clorgyline(clorg)に比較して、トラニルシプロミンがPGC-1αなどのLSD1標的遺伝子の顕著な活性化を示した。トラニルシプロミンがLSD1阻害活性をもち、他のMAO阻害剤ではその活性が低いことから、このデータは、LSD1阻害によるミトコンドリア機能遺伝子の促進を支持するとともに、LSD1阻害とMAO阻害の効果は区別できることを示している。
Claims (8)
- リジン特異的デメチラーゼ-1(LSD1)阻害剤を含む、ミトコンドリア機能向上剤。
- リジン特異的デメチラーゼ-1(LSD1)阻害剤が、トラニルシプロミン、又はRNAiによりLSD1又はBHC80又はFAD合成に関わる酵素の発現を抑制できる核酸、FAD合成阻害剤である、請求項1に記載のミトコンドリア機能向上剤。
- FAD合成に関わる酵素が、リボフラビンキナーゼ(RFK)及び/又はFAD合成酵素(FADS)である、請求項2に記載のミトコンドリア機能向上剤。
- RNAiによりLSD1の発現を抑制できる核酸が、配列番号29に記載の配列からなるsiRNAと配列番号30に記載の配列からなるsiRNAである、請求項1から3の何れか1項に記載のミトコンドリア機能向上剤。
- リジン特異的デメチラーゼ-1(LSD1)阻害剤を含む、PGC-1α発現誘導剤。
- リジン特異的デメチラーゼ-1(LSD1)阻害剤が、トラニルシプロミン、又はRNAiによりLSD1又はBHC80又はFAD合成に関わる酵素(リボフラビンキナーゼ(RFK)、FAD合成酵素(FADS)など)の発現を抑制できる核酸、FAD合成阻害剤である、請求項5に記載のPGC-1α発現誘導剤。
- FAD合成に関わる酵素が、リボフラビンキナーゼ(RFK)及び/又はFAD合成酵素(FADS)である、請求項6に記載のPGC-1α発現誘導剤。
- RNAiによりLSD1の発現を抑制できる核酸が、配列番号29に記載の配列からなるsiRNAと配列番号30に記載の配列からなるsiRNAである、請求項5から7の何れか1項に記載のPGC-1α発現誘導剤。
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EP10761379.6A EP2417985B1 (en) | 2009-04-10 | 2010-03-29 | Mitochondrial function-improving agent |
US13/263,391 US8637480B2 (en) | 2009-04-10 | 2010-03-29 | Mitochondrial function-improving agent |
JP2011508226A JP5685764B2 (ja) | 2009-04-10 | 2010-03-29 | ミトコンドリア機能向上剤 |
CA2761634A CA2761634A1 (en) | 2009-04-10 | 2010-03-29 | Mitochondrial function-improving agent |
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US (1) | US8637480B2 (ja) |
EP (1) | EP2417985B1 (ja) |
JP (1) | JP5685764B2 (ja) |
CA (1) | CA2761634A1 (ja) |
WO (1) | WO2010116673A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017130933A1 (ja) * | 2016-01-25 | 2017-08-03 | 国立大学法人熊本大学 | 神経変性疾患治療剤 |
JP2020500197A (ja) * | 2016-11-17 | 2020-01-09 | サイトーCytoo | 骨格筋肥大誘発剤としてのlsd1阻害剤 |
Families Citing this family (1)
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MA51507A (fr) | 2016-12-09 | 2020-11-11 | Constellation Pharmaceuticals Inc | Marqueurs pour un traitement personnalisé du cancer avec des inhibiteurs de lsd1 |
Citations (2)
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WO2006138475A2 (en) | 2005-06-16 | 2006-12-28 | Jenrin Discovery | Mao-b inhibitors useful for treating obesity |
JP2009509922A (ja) * | 2005-08-10 | 2009-03-12 | ジョンズ ホプキンス ユニバーシティ | 抗寄生虫治療薬および抗癌治療薬ならびにリジン特異的デメチラーゼ阻害物質として有用なポリアミン類 |
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EP1693062A3 (en) | 2005-02-18 | 2007-12-12 | Universitätsklinikum Freiburg | Androgen receptor-dependent gene expression control |
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2010
- 2010-03-29 JP JP2011508226A patent/JP5685764B2/ja not_active Expired - Fee Related
- 2010-03-29 US US13/263,391 patent/US8637480B2/en not_active Expired - Fee Related
- 2010-03-29 WO PCT/JP2010/002271 patent/WO2010116673A1/ja active Application Filing
- 2010-03-29 CA CA2761634A patent/CA2761634A1/en not_active Abandoned
- 2010-03-29 EP EP10761379.6A patent/EP2417985B1/en not_active Not-in-force
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2006138475A2 (en) | 2005-06-16 | 2006-12-28 | Jenrin Discovery | Mao-b inhibitors useful for treating obesity |
JP2009509922A (ja) * | 2005-08-10 | 2009-03-12 | ジョンズ ホプキンス ユニバーシティ | 抗寄生虫治療薬および抗癌治療薬ならびにリジン特異的デメチラーゼ阻害物質として有用なポリアミン類 |
Non-Patent Citations (6)
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BALASUBRAMANIYAN, N. ET AL.: "Regulation of peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1alpha) by lysine specific demethylasel (LSD1)-mediated lysine demethylation and its implications for FXR transactivation in liver", HEPATOLOGY, vol. 50, no. S4, October 2009 (2009-10-01), pages 624A * |
HAN, Y.S. ET AL.: "Antidepressants reveal differential effect against 1-methyl-4- phenylpyridinium toxicity in differentiated PC12 cells", EUR J PHARMACOL, vol. 604, no. 1-3, 2009, pages 36 - 44 * |
LEE, M. G., WYNDER, C., SCHMIDT, D. M., MCCAFFERTY, D. G., SHIEKHATTAR, R.: "Histone H3 lysine 4 demethylation is a target of nonselective antidepressive medications", CHEMISTRY AND BIOLOGY., vol. 13, 2006, pages 563 - 567 |
SCHMIDT, D. M., MCCAFFERTY, D. G.: "Trans-2-Phenylcyclopropylamine is a mechanism-based inactivator of the histone demethylase LSD1", BIOCHEMISTRY, vol. 46, 2007, pages 4408 - 4416 |
See also references of EP2417985A4 * |
SHINJIRO HINO ET AL.: "Histone Datsu Methyl-ka Koso LSD1 ni yoru Energy Taisha Chosetsu Kiko", DAI 82 KAI THE JAPANESE BIOCHEMICAL SOCIETY TAIKAI PROGRAM ? KOEN YOSHISHU, vol. 82, 25 September 2009 (2009-09-25), pages 3S17A-5, XP008161808 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017130933A1 (ja) * | 2016-01-25 | 2017-08-03 | 国立大学法人熊本大学 | 神経変性疾患治療剤 |
JP2020500197A (ja) * | 2016-11-17 | 2020-01-09 | サイトーCytoo | 骨格筋肥大誘発剤としてのlsd1阻害剤 |
US11382872B2 (en) | 2016-11-17 | 2022-07-12 | Cytoo | LSD1 inhibitors as skeletal muscle hypertrophy inducers |
Also Published As
Publication number | Publication date |
---|---|
US20120108648A1 (en) | 2012-05-03 |
JP5685764B2 (ja) | 2015-03-18 |
US8637480B2 (en) | 2014-01-28 |
CA2761634A1 (en) | 2010-10-14 |
EP2417985A1 (en) | 2012-02-15 |
EP2417985B1 (en) | 2016-10-05 |
JPWO2010116673A1 (ja) | 2012-10-18 |
EP2417985A4 (en) | 2013-03-20 |
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