WO2014178427A1 - Procede pour controler l'expression de la semaphorine 3a - Google Patents

Procede pour controler l'expression de la semaphorine 3a Download PDF

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WO2014178427A1
WO2014178427A1 PCT/JP2014/062073 JP2014062073W WO2014178427A1 WO 2014178427 A1 WO2014178427 A1 WO 2014178427A1 JP 2014062073 W JP2014062073 W JP 2014062073W WO 2014178427 A1 WO2014178427 A1 WO 2014178427A1
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sox
expression
transcription factor
semaphorin
substance
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弥生 鎌田
光俊 冨永
建二 ▲高▼森
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学校法人順天堂
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/04—Antipruritics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to an agent that enhances or suppresses the expression of semaphorin 3A using a transcription factor, and a method for screening a compound that enhances or suppresses the expression of semaphorin 3A using the inhibition or promotion of a specific transcription factor as an index. .
  • Semaphorin is identified as a guidance factor that controls cell migration such as nerve axon elongation and is involved in various biological functions and pathologies such as immune response, organogenesis, bone metabolism control, and cancer progression It is known that it is a molecular group. Sema family proteins are classified into Sema1 to Sema7 classes based on the difference in domain structure, and further subclassified (Non-patent Document 1).
  • Semaphorin 3A (Sema3A), which is one of them, is known as a typical molecule of a nerve guidance factor that determines the elongation direction of nerve axons, and the presence of Sema3A inhibits nerve axon elongation. This is thought to lead to a two-sided pharmacological action. That is, it is to promote nerve regeneration by inhibiting Sema3A and to enhance Sema3A to suppress nerve elongation. In fact, for the former, a technique as a nerve regeneration promoting agent is disclosed (Patent Document 1), and for the latter, there is a report that it may be used to reduce itchiness, particularly in skin tissue (Non-Patent Document 2).
  • Non-patent document 3 In patients with atopic dermatitis and psoriasis with disruption of skin barrier function, intraepidermal nerve fibers become dense with the decrease in Sema3A expression in normal skin, and intractable itching that does not respond to antihistamines develops (Non-patent document 3), an attempt to significantly suppress itching and inflammation by externally applying an ointment containing recombinant Sema3A protein to NC / Nga mice with atopic dermatitis (Non-patent document 3) Document 4) and a technique for using Sema3A protein itself as a therapeutic agent for pruritic skin diseases such as atopic dermatitis have been developed (Patent Document 2).
  • Sema3A is a secreted protein member of semaphorin and has three structural domains, a semaphorin (Sema) domain of 500 amino acid residues, a C-2 type immunoglobulin (Ig) domain, and a basic terminal domain. It has been shown that homodimerization is indispensable in order to exhibit physiological activity (Non-patent Document 4).
  • Sema3A When Sema3A is formulated as a pharmaceutical product, a high molecular weight protein with a molecular weight of over 100,000, which is a homodimer of a Sema domain consisting of 500 amino acid residues, is an active ingredient, but it can stably produce a high molecular weight dimeric protein. It is never easy to develop a preparation that can ensure reasonable cost and stable quality as a therapeutic agent.
  • the nerve regeneration promoting action can be induced, and if it can be enhanced, the nerve axon elongation inhibiting action can be induced.
  • a method for simply searching for or examining a substance that controls the expression of Sema3A has not been sufficiently developed.
  • skin pruritus is associated with a decrease in Sema3A in the epidermis, which has been shown to lead to the development of itch, so it is necessary to search for substances that enhance endogenous Sema3A.
  • a simple Sema3A expression regulator search method can be provided, it will be very useful for the development of refractory pruritus therapeutics.
  • the present invention provides a novel means useful for screening a substance that regulates the expression of Sema3A, evaluation of the expression regulating action, and a novel means useful for treating pruritus and treating nerve damage.
  • the purpose is to do.
  • the inventors of the present application isolated the human Sema3A gene promoter, determined its characteristics, and identified a transcription factor group in the promoter region. These transcription factors have been intensively studied and succeeded in identifying a transcription factor group that is related to Sema3A expression enhancement and a transcription factor group that is related to suppression of expression. Furthermore, the expression of the Sema3A gene is inhibited by inhibiting these transcription factors. After confirming that it can be adjusted, the present invention was completed.
  • the present invention is NF- ⁇ B, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4 ⁇ 1, XBP-1, HLF, NF-E2, ROR ⁇ , ER, AP-1, At least one transcription factor that regulates any transcription factor selected from Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABP ⁇ , JunB, JunD, Fra1, and Fra2.
  • a semaphorin 3A expression regulator comprising a substance as an active ingredient is provided.
  • the present invention also includes NF- ⁇ B, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4 ⁇ 1, XBP-1, HLF, NF-E2, ROR ⁇ , ER, AP-1, Compounds with the regulation of at least one transcription factor selected from Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABP ⁇ , JunB, JunD, Fra1, and Fra2
  • a method for screening a compound that modulates the expression of semaphorin 3A is provided.
  • the present invention provides a cell containing a nucleic acid construct containing a semaphorin 3A promoter region and a reporter gene, and a substance that regulates a transcription factor, and measuring the expression level of the reporter gene, and then measuring the expression level of the reporter gene.
  • a method for evaluating the expression-regulating action of 3A is provided.
  • the present invention relates to cultured cells, NF- ⁇ B, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4 ⁇ 1, XBP-1, HLF, NF-E2, ROR ⁇ , ER, Contact with a substance that regulates a transcription factor selected from AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABP ⁇ , JunB, JunD, Fra1, and Fra2. And a method for evaluating an action of regulating the expression of semaphorin 3A by the substance, comprising measuring the expression level of semaphorin 3A in the cell.
  • the present invention provides a Sema3A expression regulator using a transcription factor that regulates Sema3A gene expression.
  • An agent that enhances the expression of Sema3A is useful for the treatment and prevention of pruritus in pruritic skin diseases such as atopic dermatitis.
  • An agent that suppresses the expression of Sema3A is useful for promoting nerve regeneration at the site of nerve injury.
  • the present invention also provides a method for screening a compound that regulates Sema3A expression and a method for evaluating the strength of Sema3A expression enhancing action or expression inhibiting action. According to the screening method and the evaluation method of the present invention, it is possible to provide a novel pharmaceutical that exhibits an excellent therapeutic effect by regulating the expression of Sema3A.
  • FIG. 1 It is a schematic diagram which shows the 5'-flanking region of the gene encoding human Sema3A.
  • the putative transcription factor binding site was clarified by a search using a P-Match-1.0 public program (BIOBASE). It is the base sequence (SEQ ID NO: 1) of the 5'-flanking region of the gene encoding human Sema3A.
  • a putative transcription factor binding sequence is shown below the sequence.
  • siRNA small interfering RNA
  • NHEK was transfected with ROR ⁇ , Jun, Fos, Sox4, NF- ⁇ B1 siRNA (40 nM), and total RNA was isolated after 48 hours of culture.
  • A shows the expression suppression efficiency of each transcription factor.
  • B and C are Sema3A expression analysis results after each transcription factor knockdown.
  • Transcription factors GABP ⁇ , Sox9, Rel (c-Rel), CREB, Pbx1, ER, JunB, JunD, Fra1, and Fra2 were used to confirm that each transcription factor and Sema3A used siRNA to confirm Sema3A expression. It is a gene expression measurement result by real-time PCR. NHEK was transfected with siRNA (40 nM) of each transcription factor, and total RNA was isolated after 48 hours of culture. A shows the expression suppression efficiency of each transcription factor. B is the Sema3A expression analysis result after each transcription factor knockdown. * Is significantly different from control for p ⁇ 0.05 and *** for p ⁇ 0.001.
  • a “transcription factor” is a group of proteins that specifically bind to DNA, and binds to a region that regulates transcription 5 ′ upstream of the transcription region of the gene, such as a promoter or enhancer on the DNA, thereby inheriting the DNA. It is a protein that plays a role in promoting or repressing the process of transcription of information into messenger RNA.
  • a transcription factor exerts a transcriptional regulation (promotion or repression) function when one protein molecule is used alone or forms a complex with a plurality of protein molecules.
  • the term “transcription factor” in the present invention includes not only a single protein molecule but also a multimer or complex form in which a plurality of identical or different proteins are complexed. Also included are individual protein molecules that make up the body or complex. It is estimated that there are about 1,800 genes encoding transcription factors on the human genome, but there are various transcription factors for target proteins.
  • regulation of transcription factor refers to inhibition or promotion of transcription factor.
  • Transcription factor inhibition refers to any process that begins with transcription of mRNA from a gene that encodes a transcription factor and leads to regulation of Sema3A expression by binding to the promoter region of the Sema3A gene. Inhibiting this step.
  • Use of antisense RNA, microRNA, siRNA and the like for transcription factors is an example of inhibition of transcription factors.
  • Transcription factor promotion refers to any process that begins with transcription of mRNA from a gene that encodes a transcription factor and leads to regulation of Sema3A expression when the produced transcription factor binds to the promoter region of the Sema3A gene. It means to promote the step. Applying the transcription factor protein itself to the affected area, or enhancing the expression of the transcription factor gene in the affected area is an example of promoting the transcription factor.
  • the putative transcription factor binding site shown in FIG. 2 is based on the base sequence (SEQ ID NO: 1) of the promoter region upstream of the Sema3A gene newly cloned by the present inventors, and the Match-1.0-Public program and P-Match-1.0 It was identified by performing a search using the Public program (BIOBASE).
  • This program is a database that comprehensively contains information on transcription factors and experimentally proven transcription factor binding sequences, and is software that can predict new transcription factor binding sequences. By analyzing the database, it was possible to estimate an unknown transcription factor binding sequence upstream of the Sema3A gene and to predict transcription factors involved in Sema3A expression.
  • transcription factor binding sequences Based on the prediction results of transcription factor binding sequences, reporter assays of 5 'end deletion mutants in the promoter region, and the results of transcription factor knockdown experiments with siRNA, the following transcription factors are responsible for regulating Sema3A gene expression. Identified. Regulation of these transcription factors can regulate Sema3A expression.
  • NF- ⁇ B Nkx2.5, Brn-2, Elk-1, HNF-4 ⁇ 1, XBP-1, HLF, NF-E2 (also referred to as Nrf2), Fra1 (also referred to as Fosl1), ROR ⁇ , ER (also referred to as Esr1) , AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABP ⁇ , c-Rel, CREB (also called Atf2), JunB, JunD, Fra2
  • the above transcription factors can be classified into the following groups.
  • the transcription factor group (A) may preferably be NF- ⁇ B.
  • the regulation of NF- ⁇ B may be performed by the regulation of its subunit NF- ⁇ B1.
  • the transcription factor group (B) is preferably a transcription factor group consisting of ROR ⁇ , AP-1, Sox-4, Jun, Fos, STATx (especially STAT3), GABP ⁇ , Sox-9, CREB, JunB, JunD, and Fra2. More preferably a transcription factor group consisting of ROR ⁇ , AP-1, Sox-4, Jun, Fos, CREB, and JunB, more preferably a transcription factor group consisting of ROR ⁇ , AP-1, Jun, Fos, and CREB, or It may be a transcription factor group consisting of ROR ⁇ , AP-1, Jun, and Fos.
  • ROR ⁇ is a particularly preferred transcription factor as a target for regulating expression of Sema3A.
  • Jun and Fos are proteins constituting AP-1, and these are also included in the “transcription factor” in the present invention.
  • Inhibiting the transcription factor (A) enhances the expression of the Sema3A gene. Therefore, a substance that inhibits any of the transcription factors (A) can be used as a Sema3A expression enhancer.
  • Such an agent is useful as a therapeutic or prophylactic agent for a disease or condition for which enhanced expression of Sema3A is desired. Specific examples of such diseases or conditions include pruritus, allergic rhinitis and osteoporosis in skin diseases such as atopic dermatitis and xeroderma.
  • Sema3A In pruritic skin diseases such as atopic dermatitis, it is known that the expression of Sema3A inherent in normal skin is decreased, and it is known that pruritus can be suppressed by supplementing the affected area with Sema3A protein itself (Non-patent Documents 3 and 4 and Patent Document 2 etc.).
  • Sema3A In allergic rhinitis, the expression of Sema3A decreases in the nasal mucosal epithelium, peripheral nerve fibers in the epithelial layer and the lamina intestinal of the nasal turbinate increase, and symptoms of rhinitis caused by intranasal administration of Sema3A protein (Sawaki et al., J Pharmacol Sci 117, 34-44 (2011)).
  • Sema3A is a factor that increases bone mass and is known to be useful for the treatment of osteoporosis (Hayashi et al., Experimental Medicine, Vol. 31, No. 4, 2013). Therefore, it is possible to treat and prevent pruritus, allergic rhinitis, osteoporosis and the like in skin diseases by enhancing Sema3A expression by inhibiting transcription factors that suppress Sema3A gene expression. That is, the substance that inhibits any of the transcription factors in (A) is used as a therapeutic or preventive agent for pruritus, allergic rhinitis, osteoporosis, etc. in skin diseases, and in particular, treatment or prevention of pruritus or allergic rhinitis in skin diseases. It can be preferably used as an agent, particularly as an agent for treating or preventing pruritus in skin diseases.
  • the disease or condition for which enhanced expression of Sema3A is desired is not limited to the above specific examples.
  • Enhance expression of Sema3A is also possible by promoting the transcription factor (B). Therefore, a substance that promotes any of the transcription factors of (B) can be used as an Sema3A expression enhancer, as well as an inhibitor of the transcription factor of (A). It is useful as an agent for treating or preventing a condition.
  • Preferred examples of diseases or conditions for which enhanced expression of Sema3A is desired are as described above.
  • substances that promote ROR ⁇ are particularly preferred examples of substances that can be used to enhance the expression of Sema3A.
  • the expression enhancer of Sema3A may contain one or more substances that inhibit the transcription factor (A), or one or more substances that promote the transcription factor (B). Alternatively, it may contain one or more substances that inhibit the transcription factor (A) and one or more substances that promote the transcription factor (B).
  • the expression of the Sema3A gene is suppressed by inhibiting the transcription factor (B). Therefore, a substance that inhibits any of the transcription factors (B) can be used as a Sema3A expression inhibitor.
  • Such an agent is useful as a therapeutic or prophylactic agent for a disease or condition for which suppression of Sema3A expression is desired.
  • Such diseases or conditions include traumatic nerve injury, olfactory dysfunction, cerebral infarction neuropathy, facial paralysis, diabetic neuropathy, glaucoma, retinitis pigmentosa, Alzheimer's disease, Parkinson's disease, muscle Examples include spinal nerve injury or peripheral nerve damage in developmental lateral sclerosis, amyotrophic lateral sclerosis, Huntington's disease, cerebral infarction, traumatic neurodegenerative disease, and the like. It is known that nerve regeneration can be promoted by inhibiting Sema3A, and a nerve regeneration promoter using this is known (Patent Document 1).
  • Sema3A by suppressing the expression of Sema3A by inhibiting a transcription factor that promotes the expression of the Sema3A gene, nerve regeneration in the nerve damage site can be promoted. That is, a substance that inhibits any of the transcription factors (B) can be preferably used as a nerve regeneration promoter.
  • the disease or condition for which Sema3A expression suppression is desired is not limited to the above specific examples.
  • Sema3A expression suppression can also be achieved by promoting the transcription factor (A). Therefore, a substance that promotes any of the transcription factors of (A) can be used as an inhibitor of Sema3A expression, as well as an inhibitor of the transcription factor of (B), It is useful as an agent for treating or preventing a condition, such as a nerve regeneration promoter.
  • the expression inhibitor of Sema3A may contain one or more substances that promote the transcription factor (A), or one or more substances that inhibit the transcription factor (B). Alternatively, it may contain one or more substances that promote the transcription factor (A) and one or more substances that inhibit the transcription factor (B).
  • Drugs provided by the present invention (such as pruritus, allergic rhinitis or osteoporosis treatment or prevention agent for skin diseases, treatment or prevention agent for diseases or conditions in which expression enhancement of Sema3A is desired, and nerve regeneration promoting agent, etc.
  • Therapeutic or preventive agent for diseases or conditions for which suppression of Sema3A expression is desired is mainly used by local administration. For example, administration by injection or the like to the affected area where nerve regeneration is desired, administration by application to the affected area where itching should be suppressed, intranasal administration for suppressing nasal inflammation, and the like can be mentioned.
  • an agent for treating or preventing osteoporosis for example, a nucleic acid drug using a promoter that is specifically expressed in bone tissue (for example, siRNA for (A) transcription factor or (B) transcription factor itself is used as bone.
  • Systemic administration in the form of a vector or the like that is expressed under the control of a promoter specific in the tissue), and thereby the expression of Sema3A can be locally enhanced in the bone tissue.
  • it can be formulated into an appropriate dosage form such as an injection or ointment.
  • the dose is appropriately selected according to the size of the affected area, the intensity of symptoms, etc., and is not particularly limited.
  • the amount of active ingredient is about 0.01 ⁇ g to 1 g, for example, about 0.01 mg to 10 mg per administration. obtain.
  • an expression vector capable of producing such an RNA molecule for the transcription factor of interest can be prepared by a known technique, and a Sema3A expression enhancer can be formulated using the vector as an active ingredient.
  • the amino acid sequences of various transcription factors and the gene sequences encoding them are known, and vectors for producing siRNA and miRNA in living cells are known and commercially available.
  • a Sema3A expression enhancer using RNA, microRNA, siRNA or the like can be prepared.
  • Transcription factor inhibitors include IMD-0354 (Tanaka (et al, Blood, 105: 2324-31, 2005), DHMEQ (Ariga et al, J BiolChem, 277: 24625-30, 2002) Is also known to inhibit NF- ⁇ B).
  • the compound group can be screened using the inhibition of any of the transcription factors of (A) as an index and provided as an expression enhancer of Sema3A.
  • the screening of a compound using as an index the inhibition of any of the transcription factors (A) can be performed, for example, as follows.
  • Step 1 A transcription factor and a compound are contacted, and the presence or absence of binding is examined. High throughput screening is possible by examining the presence or absence of binding in a microplate using an assay robot.
  • Step 2 It is examined whether or not a compound that has been confirmed to have a binding action has an effect of increasing the expression level of the Sema3A gene.
  • This step can be performed, for example, by a reporter assay using cells transfected with a nucleic acid construct containing a Sema3A promoter region and a reporter gene.
  • the expression level of the reporter gene may be examined by bringing the cell containing the nucleic acid construct into contact with the compound confirmed to be bound in step 1 in a well of a microplate.
  • the reporter assay itself is a well-known conventional method and can be easily performed using a commercially available kit or the like.
  • the sequence of the Sema3A promoter region the sequence shown in SEQ ID NO: 1 can be preferably used.
  • the region to which the transcription factor of (A) binds is from position 1 to position 471 in SEQ ID NO: 1.
  • Such screening makes it possible to newly identify a compound that enhances Sema3A expression, which is useful as a therapeutic or prophylactic agent for a disease or condition in which Sema3A expression enhancement is desired (preferred specific examples are as described above).
  • the substance that promotes any of the transcription factors of (B) used as an expression enhancer of Sema3A may be each transcription factor protein itself as defined above, for example.
  • the transcription factor protein itself or a vector capable of expressing the transcription factor protein can be applied to the affected area where Sema3A expression is to be enhanced.
  • it may be a substance already known as a promoter for each transcription factor.
  • ROR ⁇ agonists having ROR ⁇ activation action are particularly promising as promoters of the transcription factor (B).
  • the compound group can be screened and provided as an Sema3A expression enhancer using as an index the promotion of any of the transcription factors of (B).
  • the Sema3A expression enhancer is useful as a therapeutic or prophylactic agent for pruritus, allergic rhinitis, osteoporosis, etc., particularly as a therapeutic or prophylactic agent for pruritus.
  • WO2011 / 115892 describes the use of SR1078 for the treatment of central nervous diseases, metabolic diseases, cancers, etc.
  • (B) Screening of a compound using any of the transcription factors as an index is performed by, for example, reporter gene expression by reporter assay using a cell transfected with a nucleic acid construct containing a Sema3A promoter region and a reporter gene. This can be done by screening for compounds that increase the amount.
  • the expression level of the reporter gene may be examined by bringing the compound into contact with a cell containing a nucleic acid construct containing the Sema3A promoter region and the reporter gene in a well of the microplate.
  • the Sema3A promoter region the nucleotide sequence shown in SEQ ID NO: 1 can be used, or the region to which the transcription factor (B) binds exists, -134 to -24 positions of the Sema3A gene It is also possible to use only the region (positions 1311 to 1421 in SEQ ID NO: 1, the sequence of this region is taken out and shown in SEQ ID NO: 2). Such screening makes it possible to newly identify a compound that enhances the expression of Sema3A, which is useful as a therapeutic or prophylactic agent for a disease or condition in which Sema3A expression enhancement is desired (preferred specific examples are as described above).
  • the substance that promotes any of the transcription factors (A) used as an Sema3A expression inhibitor can be the same as the substance that promotes any of the transcription factors (B) described above.
  • Each transcription factor protein itself may be a substance that is already known as a promoter of each transcription factor, or a compound that uses any of the transcription factors of (A) as an index.
  • the group can also be screened and provided as an Sema3A expression inhibitor.
  • Transcription factor promoters include isohumulones (WO 2006/043671) and sulforaphane (Thimmulappa et al, Cancer Res, 62: 5196-203,2002) (all activate NF-E2) Are known.
  • Compound screening can be carried out, for example, by screening a compound that increases the expression level of a reporter gene by a reporter assay using a cell transfected with a nucleic acid construct containing a Sema3A promoter region and a reporter gene.
  • the region to which the transcription factor of (A) binds is from position 1 to position 471 in SEQ ID NO: 1, and the base sequence shown in SEQ ID NO: 1 can be used as the Sema3A promoter region.
  • the substance that inhibits any of the transcription factors (B) used as an Sema3A expression inhibitor can be the same as the substance that inhibits any of the transcription factors (A) described above.
  • Antisense RNA, microRNA, siRNA, etc. for each transcription factor can be used, and it can be a substance already known as an inhibitor of each transcription factor, or any of the transcription factors of (B) It can also be obtained by screening a compound using as an index the inhibition.
  • (B) transcription factor inhibitors include T-5224 (Uchihashihet al, Drug Metab Dispos.39: 803-13, 2011.AP-1 inhibition), SR1001 (Crumbley et al, PLoS One. 7: e33804, 2012.
  • ROR ⁇ inverse agonist SR3335 (Solt and Burris, risTrends in Endocrinology and Metabolism. 23: 619-627, 2012. and Kumar et al., ACS Chem Biol. 18: 218-22, 2011. ROR ⁇ Inverse agonists) are known.
  • the compound screening may be performed in accordance with the above-described steps 1 and 2, similarly to the screening of the substance that inhibits the transcription factor (A).
  • the Sema3A promoter region contained in the nucleic acid construct the base sequence shown in SEQ ID NO: 1 can be used, or the region to which the transcription factor (B) binds exists, from position 1311 in SEQ ID NO: 1.
  • the base sequence shown in SEQ ID NO: 1 is the sequence of the promoter region of the Sema3A gene identified by the inventors of the present application, but is a small number of bases in SEQ ID NO: 1, particularly a cis element of Sema3A gene regulation -1444. If it is a base other than position -974 (positions 1 to 471 in SEQ ID NO: 1) and positions -134 to -24 (positions 1311 to 1421 in SEQ ID NO: 1), Even if there is some difference from the base sequence shown, it can be used as the Sema3A promoter region in the screening method.
  • SEQ ID NO: 1 the nucleotide sequence shown in SEQ ID NO: 2
  • SEQ ID NO: 2 it is highly identical to SEQ ID NO: 1 or SEQ ID NO: 2.
  • a base sequence having (identity of 90% or more, preferably 95% or more, more preferably 98% or more) can also be used as the Sema3A promoter region.
  • the “identity” of the base sequences is a percentage obtained by aligning both sequences so that the bases of the two base sequences to be compared match as much as possible, and dividing the number of matched bases by the total number of bases. It is represented by.
  • a gap is appropriately inserted in one or both of the two sequences to be compared as necessary.
  • sequence alignment can be performed using a known program such as BLAST, FASTA, CLUSTAL W, and the like.
  • the total number of bases is the number of bases obtained by counting one gap as one base.
  • the identity is calculated by dividing the total number of bases of the longer sequence and dividing the number of matched bases.
  • the sequence to be compared is in a state linked to any other sequence (for example, a state incorporated in an expression vector, a state linked to a desired gene to be expressed, etc.), it corresponds to a promoter. Only the region to be extracted is taken out, the sequences are compared, and the identity is calculated.
  • inhibitors and promoters are known for various transcription factors.
  • the present invention includes a cell comprising a nucleic acid construct comprising a semaphorin 3A promoter region and a reporter gene, NF- ⁇ B, c-Rel, CREB, Nkx2.5, Brn-2, Elk-1, HNF-4 ⁇ 1, XBP -1, HLF, NF-E2, ROR ⁇ , ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABP ⁇ , JunB, JunD, Fra1, and There is also provided a method for evaluating the action of regulating the expression of semaphorin 3A by the substance, which comprises contacting a substance that regulates any transcription factor selected from Fra2 and measuring the expression level of the reporter gene.
  • the reporter assay and the Sema3A promoter region are the same as described above for the screening method.
  • the Sema3A promoter region one containing any one of the following base sequences (1) to (3) can be used.
  • (1) Base sequence shown in SEQ ID NO: 1 (2) Base sequence shown in SEQ ID NO: 2 (3) Base sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity with (1) or (2)
  • Sema3A by the inhibitor or the promoter is used.
  • the expression enhancing action of is evaluated.
  • the promoter or the inhibitor Sema3A expression-suppressing action is evaluated.
  • a promoter region using SEQ ID NO: 2 or a nucleotide sequence having 90% or more identity to this can be used when evaluating an inhibitor or promoter of the transcription factor (B).
  • the cultured cells the transcription factor (A) (NF- ⁇ B, Nkx2.5, Brn-2, Elk-1, HNF-4 ⁇ 1, XBP-1, HLF, NF-E2, Fra1) and the above (B) transcription factors (ROR ⁇ , ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABP ⁇ , c-Rel, CREB, JunB, JunD , Fra2) is contacted with a substance that regulates (inhibits or promotes) a transcription factor selected from, and the expression level of Sema3A in the cell is measured.
  • A transcription factor
  • B transcription factor
  • B transcription factors
  • ROR ⁇ , ER, AP-1, Sox-4, Sox-5, Sox-9, Pbx1b, Jun, Fos, GABP, STATx, AREB6, GABP ⁇ , c-Rel, CREB, JunB, JunD , Fra2 is
  • the evaluation method When the evaluation method is applied to any one of the inhibitors of the transcription factor of (A) and the promoter of any of the transcription factors of (B), the expression of Sema3A by the inhibitor or the promoter is enhanced. The effect is evaluated. On the other hand, when the evaluation method is applied to any promoter of the transcription factor (A) and any inhibitor of the transcription factor (B), the promoter or the Sema3A by the inhibitor Expression inhibitory action is evaluated.
  • the evaluation of the transcription factor inhibitor in (A) and the evaluation of the transcription factor promoter in (B) can be performed in combination to identify a combination of drugs that have a very high effect of enhancing the expression of Sema3A.
  • a combination of agents can be particularly preferably used as a therapeutic or prophylactic agent for diseases or conditions in which enhanced expression of Sema3A is desired, such as a therapeutic or prophylactic agent for pruritus.
  • the evaluation of the transcription factor promoter in (A) and the evaluation of the transcription factor inhibitor in (B) can be conducted in combination to identify combinations of drugs that have a very high effect of suppressing Sema3A expression. it can.
  • a combination of drugs can be particularly preferably used as a therapeutic or prophylactic agent for a disease or condition for which suppression of Sema3A expression is desired, such as a nerve regeneration promoter.
  • the manufacturer recommended two-step PCR protocol using Ex Taqpolymelase 94 ° C for 25 seconds, 72 ° C for 4 minutes for 7 cycles, followed by 94 ° C for 25 seconds, and Primary PCR using GSP1 and adapter primer (AP) 1 was performed using 32 cycles of 4 minutes at 67 ° C. and 4 minutes at 67 ° C. for the final extension reaction.
  • the primary PCR mixture was then diluted and used as a template for secondary PCR amplification with GSP2 and AP2.
  • the secondary PCR was identical to the primary PCR except that 5 cycles were used instead of 7 cycles followed by 22 cycles instead of 32 cycles.
  • Sequential 5′-deletion mutants of the 5′-flanking region of Sema3A were generated by PCR using the primers listed in Table 1. After amplification, all PCR products were cloned into pGEM-T-Easy vector (Promega Co., Madison, Wis., USA) and sequenced using ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, Calif.) Were determined.
  • PCR was first denatured at 94 ° C for 5 minutes, 94 ° C for 1 minute, 60 ° C for 1 minute, 72 ° C for 1 minute under the following conditions: A set of specific Sema3A primers containing a restriction enzyme site for the template pGEM-T-Sema3A-1444 / -1 and KpnI or HindIII for 30 minutes in minutes and finally 5 minutes at extension 72 ° C. Sema3A-1444 KpnI and Sema3A + 1 HindIII) were used.
  • the resulting PCR product was digested with KpnI and HindIII and cloned into the pGL3-Basic vector (Promega).
  • the pGL3-Basic vector contains a firefly luciferase gene. All of the constructs were prepared using QIAGEN Plasmid Maxi Ki (QIAGEN, Duesseldolf, Germany).
  • site-directed mutagenesis was introduced into the putative transcription factor binding sequence site of Sema3A, and the ROR ⁇ binding region, Sox-5 / 9 binding region and GABP binding region were deleted. A construct was made. Table 2 shows the primers used for site-directed mutagenesis.
  • Sema3A promoter luciferase assay using NHEK Normal human epidermal keratinocytes (NHEK) were cultured according to the protocol of Lonza. NHEKs were cultured in 12-well tissue culture plates and transfected with 1 ⁇ g of each construct using X-treme GENE HD DNA transfection reagent (Roche Diagnostics, Basel, Switzerland). To correct for transfection efficiency, all cells were co-transfected with the pRK-TK vector (Promega) containing the Renilla luciferase gene under the control of the HSV-TK promoter.
  • siRNA Transcription factor ROR ⁇ , Jun, Fos, Sox4, STAT3, NF- ⁇ B1, GABP ⁇ , Sox9, Rel (c-Rel), CREB, 40 nM of siRNA (Thermo Scientific) against Pbx1, ER, JunB, JunD, Fra1, and Fra2 was transfected using LipofectamineRNAi Max (Invitrogen, Carisbad, CA), respectively.
  • the cells were cultured in antibiotic-free medium for 48 hours, after which total RNA was extracted and analyzed by real-time PCR.
  • the sequences of each siRNA used (each using a mixture of four siRNAs) are shown in Table 3 below.
  • RPS18 Ribosomal protein S18
  • Electrophoretic mobility shift analysis A double-stranded oligonucleotide probe was prepared by annealing a single-stranded biotinylated oligonucleotide and a complementary single-stranded unlabeled oligonucleotide. Table 5 shows the sequences (sense strand sequences) used for the probes. Nuclear protein extraction and EMSA were performed using NE-PER Nuclear Cytoplasmic Extraction Reagents and LightShift Chemiluminescent EMSA kit (Thermo Scientific).
  • Nuclear protein extract (5 ⁇ g) was added to 1 ⁇ binding buffer and 50 ng / ⁇ l poly (dI ⁇ dC), 50 mM KCl, 5 mM EDTA, 2.5% glycerol, 0.05% NP-40, and ⁇ 122 / of the Sema3A promoter region Incubated for 20 minutes at room temperature with biotinylated probes (50 pmol / ⁇ l) corresponding to the -93, -77 / -46, -47 / -18 regions. For competition assays, a 400-fold excess of unlabeled probe was added to the binding reaction prior to the addition of biotinylated probe.
  • ESA electrophoretic mobility shift assay
  • FIG. 6 shows the results of luciferase assay of each mutant in normal human epidermal keratinocytes (NHEK). Mutants lacking the -108 / -98 region where the ROR ⁇ binding sequence was present showed marked suppression of luciferase activity, and it was revealed that transcription factors that upregulate Sema3A expression bind to this region.
  • ROR ⁇ , Jun, Fos, Sox4, STAT3, NF- ⁇ B, GABP ⁇ , Sox9, Rel (c-Rel), CREB, Pbx1, ER, JunB, JunD, Fra1, and Fra2 are involved in Sema3A expression.
  • transcription factor expression was suppressed using small interfering RNA (siRNA), and gene expression of each transcription factor and Sema3A was analyzed using real-time PCR.
  • siRNA small interfering RNA
  • Figure 8 shows the results of knockdown of GABP ⁇ , Sox9, Rel, CREB, Pbx1, ER, JunB, JunD, Fra1, and Fra2.
  • Sema3A expression was significantly suppressed (*, p ⁇ 0.05; ***, p ⁇ 0.001) by suppressing the expression of GABP ⁇ , SSox9, CREB, JunB, JunD, and Fra2.
  • Sema3A expression was significantly suppressed when CREB and JunB expression was suppressed.
  • FIG. A is the result of the ROR ⁇ agonist SR1078, B is the result of the ROR ⁇ agonist cholesterol sulfate, and C is the result of the ROR ⁇ antagonist SR1001.
  • Sema3A expression was significantly enhanced in NHEK supplemented with ROR ⁇ agonist SR1078 and cholesterol sulfate in a concentration-dependent manner.
  • ROR ⁇ antagonist SR1001 was added, Sema3A expression was significantly suppressed in a concentration-dependent manner. Since any compound has a molecular weight of 500 Da or less that easily penetrates into the epidermal stratum corneum, it may be applicable as a topical drug.
  • mice that developed dermatitis were extracted and classified into 2 groups (4 animals each) of (i) base dimethyl sulfoxide only, (ii) 1 mM cholesterol sulfate, and then the base or cholesterol sulfate solution was applied to the affected area of the mice. It was applied for 9 consecutive days. After the external application was completed, the skin was removed, immersed in a 0.02% trypsin / EDTA solution overnight, the epidermis was peeled off, and RNA was extracted. The expression level of Sema3A in the epidermis by quantitative real-time PCR is shown in FIG. Sema3A expression was significantly (**, p ⁇ 0.01) promoted in the group externally applied with 1 mM cholesterol sulfate compared with the group externally applied with the base.

Abstract

En tant que résultats d'études intensives, les présents inventeurs ont identifié des facteurs de transcription qui contrôlent l'expression du gène de la sémaphorine 3A. En bloquant ou en favorisant ces facteurs de transcription, l'expression de la sémaphorine 3A peut être contrôlée (supprimée ou améliorée). En outre, par dépistage de composés par utilisation d'une activité consistant à bloquer ou à favoriser un facteur de transcription tel qu'un indice, un composé apte à contrôler l'expression de la sémaphorine 3A peut être obtenu. Le composé apte à contrôler l'expression de la sémaphorine 3A est utile dans le traitement du prurit dans des maladies de la peau, la rhinite allergique, l'ostéoporose et les lésions de nerf, en particulier, pour traiter le prurit.
PCT/JP2014/062073 2013-05-02 2014-05-01 Procede pour controler l'expression de la semaphorine 3a WO2014178427A1 (fr)

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CN110694051A (zh) * 2019-11-19 2020-01-17 四川大学华西医院 一种神经导向因子Sema在制备治疗骨关节炎注射剂中的应用
CN110694051B (zh) * 2019-11-19 2023-08-01 四川大学华西医院 一种神经导向因子Sema在制备治疗骨关节炎注射剂中的应用
WO2022192249A3 (fr) * 2021-03-08 2022-10-27 The Trustees Of The University Of Pennsylvania Compositions et procédés d'évaluation et de traitement d'un dysfonctionnement des lymphocytes t
WO2023238910A1 (fr) * 2022-06-09 2023-12-14 築野グループ株式会社 Agent améliorant l'état de santé de la peau
EP4357458A1 (fr) * 2022-10-19 2024-04-24 Universitätsmedizin der Johannes Gutenberg-Universität Mainz Inhibiteurs de fra-1 et fra-2 destinés à être utilisés dans le traitement de la fibrose d'organe ou de tissu
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