Use of Nav1.9 interacting protein PRMT7 and its down-regulators in the preparation of analgesic drugs

Abstract

The present invention provides a use of a Na V 1.9 interacting protein PRMT7 and its down-regulators in the preparation of analgesic drugs. It reveals a protein that specifically interacts with Na V 1.9, namely PRMT7, which has regulatory activity on Na V 1.9, can regulate the excitability of dorsal root ganglion neurons, and thus can be used as a target for pain inhibition. The present invention also provides a new use of PRMT7 inhibitors in pain treatment and the application of PRMT7 as a target in the preparation of analgesic drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and more specifically, the present invention relates to Na V 1.9 Use of the interacting protein PRMT7 and its downregulators in the preparation of analgesics. Background Art

[0002] Pain is caused by activation of the nociceptive system. Up to 10% of the world's population suffers from chronic pain, which seriously affects the quality of life of patients. At present, analgesics can be divided into antipyretic analgesics and central analgesics according to their mechanism of action. Central analgesics include opioids and other central analgesics, which mainly act on the central nervous system and are prone to dependence, addiction and tolerance with long-term use; anti-inflammatory analgesics, generally non-steroidal anti-inflammatory drugs (ibuprofen, aspirin, etc.), can temporarily relieve pain, but long-term use can damage the gastrointestinal tract, kidneys and cardiovascular system. Therefore, screening ideal analgesic drug targets is crucial.

[0003] In recent years, voltage-gated sodium channels have made important progress in the study of pain signal transduction, especially in the peripheral nervous system. V 1.7, Na V 1.8 and Na V 1.9 is expressed in large quantities in the peripheral nervous system and mainly regulates the conduction of electrical signals in peripheral neurons. V 1.7, Na V 1.8 mainly forms the ascending branch of the action potential, while Na V 1.9 acts as a threshold channel to respond to subthreshold stimulation to regulate the frequency of action potential firing. V 1.7 Gain-of-function mutations will lead to small-diameter neurofibromatosis, erythromelalgia, etc., but loss-of-function mutations will lead to congenital analgesia, so researchers believe that Na V 1.7 is an ideal analgesic target, and multiple Na V 1.7 selective inhibitors, including spider toxins, small molecule compounds, etc. However, in the study, it was found that Na V 1.7 selective inhibitors have good inhibitory effects in vitro, but their analgesic effects in vivo are limited. V 1.8 In the treatment of pain, the inhibitor A-803467 has poor solubility and low oral bioavailability, which limits its use; while other inhibitors have more side effects due to their low selectivity and inhibitory effects on other channels. V1.9. It has currently been found that it regulates the resting membrane potential by promoting sodium conductance, prolongs the depolarization response of neurons to subthreshold stimuli, and thereby reduces the single action potential threshold and increases the repetitive discharge frequency. Since 2013, mutations in this channel have been found to be associated with diseases such as episodic pain, visceral pain, and congenital painlessness. And this channel is mainly expressed in the dorsal root ganglion, and then responds to subthreshold stimuli to regulate neuronal activity, so it plays an important role in the initial stage of pain signal conduction. In the treatment research of the present inventors targeting Na V 1.9, a new spider toxin HpTx1 has been found to be able to target Na V 1.9. Unfortunately, HpTx1 is an agonist of the Na V 1.9 channel and cannot be used for analgesia.

[0004] So far, no selective inhibitor of the Na V 1.9 channel has been found. Summary of the Invention

[0005] The purpose of the present invention is to provide a new protein - arginine methyltransferase PRMT7 that specifically interacts with Na V 1.9, which can regulate the Na V 1.9 current and the excitability of dorsal root ganglion neurons.

[0006] The purpose of the present invention also lies in providing a new use of a PRMT7 down - regulator in the preparation of analgesic drugs, as well as the application of PRMT7 as a target in the preparation of analgesic drugs.

[0007] In the first aspect of the present invention, there is provided the application of arginine methyltransferase PRMT7 for use as a target for relieving or suppressing pain; or, as a target for screening drugs for relieving or suppressing pain.

[0008] In another aspect of the present invention, there is provided the application of a down - regulator of arginine methyltransferase PRMT7 for use in the preparation of a composition for relieving or suppressing pain.

[0009] In a preferred embodiment, the composition is further used for: inhibiting the interaction between arginine methyltransferase PRMT7 and the sodium channel Na V 1.9.

[0010] In another preferred embodiment, the composition is further used for: inhibiting the increase in the sodium channel Na V 1.9 current density (reducing the sodium channel Na V 1.9 current density), and inhibiting the excitability of neurons.

[0011] In another preferred embodiment, reducing the distribution of Loop1 of the sodium channel Na V 1.9 on the cell membrane.

[0012] In another preferred example, the interaction between the arginine methyltransferase PRMT7 and the sodium channel Na V 1.9 is: the interaction between the C-terminal domain of the arginine methyltransferase PRMT7 and Loop1 of the sodium channel Na V 1.9.

[0013] In another preferred example, in the interaction between the arginine methyltransferase PRMT7 and the sodium channel Na V 1.9, the C-terminal domain of the arginine methyltransferase PRMT7 acts on the 563-566th amino acids and the 519th arginine of the sodium channel Na V 1.9.

[0014] In another preferred example, the downregulator of the arginine methyltransferase PRMT7 includes (but is not limited to): a substance that downregulates the activity of the arginine methyltransferase PRMT7 or a substance that downregulates the expression, stability or reduces the effective action time of the arginine methyltransferase PRMT7.

[0015] In another preferred example, the downregulator includes (but is not limited to) selected from: a chemical small molecule antagonist or inhibitor against the arginine methyltransferase PRMT7; a reagent that interferes with or knocks out the arginine methyltransferase PRMT7; a binding molecule that specifically binds to the arginine methyltransferase PRMT7 (such as an antibody or a ligand); or, a reagent that interferes with the interaction between the arginine methyltransferase PRMT7 and an effector molecule (such as its downstream protein or interacting protein); preferably, the effector molecule is the sodium channel Na V 1.9, more preferably, the downregulator targets and interferes with the interaction between the arginine methyltransferase PRMT7 and Loop1 of the sodium channel Na V 1.9.

[0016] In another preferred example, the chemical small molecule antagonist or inhibitor against the arginine methyltransferase PRMT7 includes (but is not limited to): DS-437 (CAS No. 1674364-87-4), SGC8158 (product number Sigma-SML2340), or its salt, hydrate, isomer, analogue or derivative.

[0017] In another preferred example, the reagent for interfering with or knocking out arginine methyltransferase PRMT7 includes (but is not limited to): an interfering molecule that specifically interferes with the expression of the coding gene of arginine methyltransferase PRMT7, a CRISPR gene editing reagent targeting arginine methyltransferase PRMT7, a homologous recombination reagent or site-directed mutagenesis reagent targeting arginine methyltransferase PRMT7, and the homologous recombination reagent or site-directed mutagenesis reagent mutates arginine methyltransferase PRMT7 to cause loss of function, such as targeting its C-terminal domain for loss-of-function mutation.

[0018] In another preferred example, the interfering molecule includes shRNA, siRNA, miRNA, antisense nucleic acid, etc., or a construct capable of forming the siRNA, shRNA, miRNA, antisense nucleic acid, etc.; preferably, the interfering molecule is shRNA.

[0019] In another preferred example, the shRNA includes the forward sequence shown in SEQ ID NO:2 and the reverse sequence shown in SEQ ID NO:3.

[0020] In another preferred example, the expression construct (expression vector) for introducing downregulators such as sgRNA or interfering molecules into cells includes: viral vectors, non-viral vectors; for example, the expression vector includes: adeno-associated virus vectors, lentiviral vectors, adenoviral vectors.

[0021] In another preferred example, the targeting interference of arginine methyltransferase PRMT7 with the Loop1 of sodium channel Na V 1.9 includes: using a homologous recombination reagent or site-directed mutagenesis reagent to perform a loss-of-function mutation on the Loop1 of sodium channel Na V 1.9; preferably, mutating the 563-566th amino acids or the 519th arginine of sodium channel Na V 1.9 (including point mutation, deletion mutation or insertion mutation).

[0022] In another aspect of the present invention, there is provided a pharmaceutical composition or kit for relieving or inhibiting pain, including: a downregulator of arginine methyltransferase PRMT7; preferably, the downregulator is an interfering molecule, and the interfering molecule is shRNA; preferably, the shRNA includes the forward sequence shown in SEQ ID NO:2 and the reverse sequence shown in SEQ ID NO:3; or, the downregulator is a chemical small molecule antagonist or inhibitor targeting arginine methyltransferase PRMT7, including (but not limited to): DS-437, SGC8158, or its salt, hydrate, isomer, analogue or derivative.

[0023] In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0024] In another aspect of the present invention, there is provided a method for screening a potential substance for relieving or inhibiting pain, the method comprising:

[0025] (1) Treating an expression system expressing protein arginine methyltransferase PRMT7 with a candidate substance; and

[0026] (2) Detecting the expression or activity of protein arginine methyltransferase PRMT7 in the system; if the candidate substance downregulates (significantly downregulates, such as downregulating by more than 10%, more than 20%, more than 50%, more than 80%, etc., or renders it non-expressed or inactive) the expression or activity of protein arginine methyltransferase PRMT7 statistically, then the candidate substance is a potential substance for relieving or inhibiting pain.

[0027] In another aspect of the present invention, there is provided a method for screening a potential substance for relieving or inhibiting pain, the method comprising:

[0028] (1) Contacting a candidate with a system in which protein arginine methyltransferase PRMT7 interacts with sodium channel Na V 1.9 or its Loop1 segment;

[0029] (2) Detecting the effect of the candidate on the interaction between protein arginine methyltransferase PRMT7 and sodium channel Na V 1.9 or its Loop1 segment; wherein, if the candidate can weaken (preferably significantly weaken, such as weaken by more than 20%, preferably weaken by more than 50%; more preferably weaken by more than 80%) the interaction between the two, it indicates that the candidate is a substance for relieving or inhibiting pain.

[0030] In another preferred embodiment, the system is selected from: a cell system (such as a cell or cell culture expressing protein arginine methyltransferase PRMT7), a subcellular (culture) system, a solution system, a tissue system, an organ system or an animal system.

[0031] In another preferred embodiment, in step (1), the system is a nerve cell (culture) (such as a dorsal root ganglion cell or its culture) system; step (2) further comprises: detecting the excitability of the nerve cells in the system; if the excitability decreases (significantly decreases, such as decreases by more than 10%, more than 20%, more than 50%, more than 80%, etc.), then the candidate substance is a potential substance for relieving or inhibiting pain; or, step (2) further comprises: detecting the sodium channel Na on the cell membrane of the nerve cells VIf the distribution of Loop1 of Nav1.9 on the cell membrane decreases (significantly decreases, such as by more than 10%, more than 20%, more than 50%, more than 80%, etc.), then the candidate substance is a potential substance for relieving or inhibiting pain.

[0032] In another preferred example, detect the expression of arginine methyltransferase PRMT7, or detect arginine methyltransferase PRMT7 and sodium channel Na V Methods for interacting with Nav1.9 or its Loop1 segment include (but are not limited to): Southern blotting, Western blotting, DNA sequence analysis, polymerase chain reaction, immunoprecipitation, yeast two-hybrid method.

[0033] In another preferred example, the candidate substances include (but are not limited to): regulatory molecules or their constructs designed for PRMT7, its fragments or variants, its coding gene or its upstream and downstream molecules or signaling pathways (such as shRNA, siRNA, gene editing reagents, expression vectors, recombinant viral or non-viral constructs, etc.), chemical small molecules (such as specific inhibitors or antagonists), interacting molecules, etc.

[0034] In another preferred embodiment, the candidate substances include (but are not limited to): regulatory molecules or their constructs designed for the interaction mechanism between arginine methyltransferase PRMT7 and sodium channel Na V 1.9 or its Loop1 segment or its interaction sites (such as shRNA, siRNA, gene editing reagents, expression vectors, recombinant viral or non-viral constructs, etc.), chemical small molecules (such as specific inhibitors or antagonists), interacting molecules, etc.

[0035] In another preferred example, the method further includes: performing further cell experiments and / or animal tests on the obtained potential substances to further select and determine substances useful for inhibiting pain from the candidate substances.

[0036] In another aspect of the present invention, a complex is provided, which includes arginine methyltransferase PRMT7 and sodium channel Na V 1.9 or its Loop1 segment, and the arginine methyltransferase PRMT7 binds to the Loop1 segment of Nav1.9.

[0037] In a preferred example, the complex is used for screening substances (including potential substances) that regulate pain through the interaction of arginine methyltransferase PRMT7 and sodium channel Na V 1.9 or its Loop1 segment.

[0038] Other aspects of the present invention will be apparent to those skilled in the art from the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 、Yeast two-hybrid screening for Na V 1.9 interacting proteins. A. Schematic diagram of the Na V 1.9 channel structure and residues 402-570 of mNa V 1.9 used as bait for yeast two-hybrid screening; B. Comparison of the positions (blue, residues 594-692) of independently cloned Prmt7 repeats identified by yeast two-hybrid with the homology of full-length human PRMT7; C. Verification of the interaction between PRMT7 and mLoop1 by purifying mPRMT7 residues 594-692 from yeast clones; D. Comparison of the amino acid sequences of hLoop1 and mLoop1 using ClustalW.

[0040] Figure 2 、Verification of the interaction between PRMT7 and Na V 1.9 in vitro and in vivo. A. GST-Pull down assay to analyze the interaction between hLoop1 and hPRMT7; B. Forward and reverse co-immunoprecipitation assays to detect the interaction between hLoop1 and hPRMT7; C. Co-immunoprecipitation to verify the main intracellular domain of the interaction between hPRMT7 and hNa V 1.9; D. CO-IP to verify the interaction between mPRMT7 and mNa V 1.9 in mouse DRG tissues; E. Localization of mPRMT7 and mNa V 1.9 in mouse DRG tissues (scale bar: 100 μm).

[0041] Figure 3 、Effect of hPRMT7 on hNa V 1.9 current. A-D. mNa V 1.9 - / - Untransfected, electrotransfected hNa V 1.9 and typical whole-cell sodium currents of mock empty vector or PRMT7 in small-diameter mouse DRG neurons; E. Current-voltage relationship of hNa V 1.9; F. Average peak current density of hNa V 1.9 after co-transfection with mock or PRMT7; G. Activation curve of hNa V 1.9 after electrotransfection with mock or PRMT7; H. Steady-state fast inactivation curve of hNa V 1.9 after electrotransfection with mock or PRMT7; V 1.9; V 1.9 after electrotransfection with mock or PRMT7; V 1.9.

[0042] Figure 4, Effects of mPRMT7 on the excitability of mouse DRG neurons. A - B. mNa V 1.9 + / + Schematic diagram of action potentials of mouse DRG neurons transfected with Mock and Prmt7 by electroporation; C. mNa V 1.9 - / - and mNa V 1.9 + / + Statistical analysis of action potentials of mouse DRG neurons transfected with Mock and Prmt7 by electroporation; D. mNa V 1.9 + / + Resting membrane potential (RPM) and voltage threshold (Vthreshold) of action potential firing in mouse DRG neurons after transfection with Mock and Prmt7.

[0043] Figure 5 , hPRMT7 promotes the distribution of hLoop1 on the cell membrane. A. Schematic diagram of the construction of the HA - CD4 - hLoop1 vector; B. Detection of the expression and localization of HA - CD4 - hLoop1 by non - permeable methods (scale bar: 10 μm); C. Detection of the distribution of HA - CD4 - hLoop1 on the cell membrane (Mem: cell membrane, WCL: whole cell lysate); D. Quantitative analysis of the membrane distribution of HA - CD4 - hLoop1 by ImageJ software; E. Quantitative analysis of the total protein expression of HA - CD4 - hLoop1 in cell lysates by ImageJ software.

[0044] Figure 6 , Immunofluorescence detection shows that PRMT7 promotes the distribution of hLoop1 on the cell membrane. A. Detection of the localization of HA - CD4 - hLoop1 in HEK293T cells overexpressing HA - CD4 - hLoop1 and GFP - PRMT7 or empty GFP; B. Analysis of the relative average fluorescence intensity of co - expressed HA - CD4 - hLoop1 on the cell membrane by ImageJ software, with at least 100 cells counted.

[0045] Figure 7 , Identification of the binding site between hLoop1 and PRMT7. A. Prediction of possible binding sites of PRMT7 on the hLoop1 segment by SPPIDER software; B. Schematic diagram of hLoop1 truncations (+: yeast can grow on QDO plates, -: cannot grow); C. Schematic diagram of the overlapping alanine mutants of hLoop1 amino acids 563 - 572; D. Growth of different mutants of yeast on QDO medium; E. Statistical analysis of the proportion of the growth number of yeast clones with different truncations.

[0046] Figure 8 , PRMT7 on Na V1. Effects of methylation level. A. Detection of the methylation level of hLoop1 by co-immunoprecipitation of hPRMT7; B. Effects of the interfering plasmid shPRMT7 on the expression of endogenous PRMT7; C. Detection of the methylation level of hLoop1 after knockdown of PRMT7 expression by RNA interference; D. Identification of potential arginine residues methylated on hLoop1.

[0047] Figure 9 and the effects of hPRMT7 on hNa V 1.9 binding site (hNa V 1.9-4A) and methylated site (hNa V 1.9-R519A) mutant channels. A. mNa V 1.9 - / - Schematic diagrams of the mutant currents after electroporation of hNa V 1.9-4A and hNa V 1.9-R519A and hPRMT7 in mouse DRG neurons; B. Current-voltage curves of hNa V 1.9-4A and hNa V 1.9-R519A mutant channels.

[0048] Figure 10 and the effects of DS-437 inhibiting PRMT7 activity on mNa V 1.9 current. A-B. Schematic diagrams of mNa V 1.9 + / + currents in mouse DRG neurons treated or untreated with DS-437; C. Relationship between mNa V 1.9 current and voltage; D. Average peak current density of mNa V 1.9; E. Activation curve of mNa V 1.9; F. Steady-state fast inactivation curve of mNa V 1.9. V

[0049] Figure 11 and the effects of DS-437 inhibiting PRMT7 activity on the excitability of mNa V 1.9 + / + and mNa V 1.9 A796G / A796G in mouse DRG neurons. A-B. Effects of DS-437 on mNa V 1.9 + / + and mNa V 1.9 A796G / A796G action potentials in mouse DRG neurons; C. Effects of DS-437 on mNa V 1.9 + / + and mNa V1.9 A796G / A796G Statistical analysis of action potentials of mouse DRG neurons; D.DS-437 on mNa V 1.9 + / + and mNa V 1.9 A796G / A796G Resting membrane potential (RPM) and action potential firing voltage threshold (V threshold ) of mouse DRG neurons.

[0050] Figure 12 、Effect of PRMT7 inhibitor on mNa V 1.9 A796G / A796G Effect of PRMT7 inhibitor on mouse pain threshold. A. Effect of PRMT7 inhibitor on thermal pain threshold; B. Effect of PRMT7 inhibitor on mechanical pain threshold.

[0051] Figure 13 、Effect of PRMT7 inhibitor on pain threshold of WT mice and mNa V 1.9 A796G / A796G mouse induced by 5% formalin. A. Total time of mNa V 1.9 A796G / A796G mouse licking paws or lifting paws every 5 minutes within 45 minutes; B. mNa V 1.9 A796G / A796G Mouse formalin-induced two-phase pain analysis statistics (Phase I: 0-10 minutes; Phase II: 10-45 minutes). Detailed implementation manner

[0052] Through large-sample analysis and research, the present inventor has revealed a protein that specifically interacts with Na V 1.9 - arginine methyltransferase PRMT7, which has regulatory activity on Na V 1.9, can regulate the excitability of dorsal root ganglion neurons, and thus can be used as a target for pain inhibition. The present invention also provides a new use of PRMT7 down-regulators in pain treatment and the application of PRMT7 as a target in the preparation of analgesic drugs.

[0053] PRMT7 interacts with sodium channel Na V 1.9

[0054] Through screening for sodium channel Na V 1.9 interacting proteins, the present inventor has first discovered that PRMT7 interacts with sodium channel Na V 1.9 (especially its Loop1). After in-depth research, it was determined that PRMT7 can be used as a research target for pain.

[0055] In the specific experimental work of the present inventor, yeast two-hybrid screening was used to screen for proteins interacting with Na V1.9 interacting proteins to obtain proteins that interact with Na V The protein that interacts with 1.9-Loop1 - PRMT7, and it was found that its C-terminal domain interacts with Na V 1.9-Loop1. The inventors of the present invention confirmed the interaction between PRMT7 and Na V 1.9-Loop1 both in vitro and in vivo, and PRMT7 and Na V 1.9 are co-localized in DRG tissues. PRMT7 increases Na V 1.9 current and DRG neuron excitability, and promotes the distribution of hNa V 1.9-Loop1 on the cell membrane.

[0056] In the identification of the fine interaction sites between PRMT7 and hLoop1, the inventors of the present invention found that the key amino acids binding to hLoop1 on hPRMT7 are the amino acids Trp, Leu, Cys, and Val (WLCV) at positions 563-566 of hLoop1; moreover, hPRMT7 methylates the arginine on hLoop1, and the methylation signal of hLoop1 increases with the increase in the expression level of hPRMT7. The arginine residue at position R519 is the methylation site of hPRMT7; interference with shPRMT7 reduces the expression of hPRMT7, and the methylation signal of hLoop1 is significantly weakened. hPRMT7 increases Na V 1.91.9 current depends on the binding site and methylation site on hLoop1. Inhibiting Prmt7 activity significantly reduces the mNa V 1.9 current density, but does not affect the mNa V 1.9 channel properties.

[0057] In further research, the inventors of the present invention used a small molecule compound inhibitor of PRMT7 to inhibit it, and found that it can relieve pain hypersensitivity in animals. Therefore, PRMT7 inhibitors have therapeutic uses in pain treatment, and PRMT7 can be used as a new target for preparing analgesic drugs.

[0058] In the present invention, the amino acid sequence of human-derived PRMT7 (hPRMT7) can be substantially the same as the sequence shown in SEQ ID NO:1. The amino acid sequence of murine PRMT7 (mPRMT7) can be substantially the same as the sequence shown in GenBank accession number NP_663379.1. It should be understood that, as needed, those skilled in the art can also apply homologs of PRMT7 from other species.

[0059] In the present invention, human-derived Na V 1.9 (hNa VThe amino acid sequence of 1.9) can be substantially the same as the sequence shown in GenBank accession number NP_001336182.1. Murine Na V 1.9 (mNa V The amino acid sequence of 1.9) can be substantially the same as the sequence shown in GenBank accession number NP_036017.3. It should be understood that, as needed, those skilled in the art can also apply homologs of Na V 1.9 from other species.

[0060] When applied to the present invention, the PRMT7 or Na V 1.9 can be naturally occurring, for example, it can be isolated or purified from mammals. In addition, the PRMT7 or Na V 1.9 can also be artificially prepared, for example, recombinant PRMT7 or Na V 1.9 can be produced according to conventional genetic engineering recombination techniques for use in experiments or clinical applications. When applied, recombinant PRMT7 or Na V 1.9 can be used. The PRMT7 or Na V 1.9 includes the full-length PRMT7 or Na V 1.9 or a bioactive fragment of either of them.

[0061] The amino acid sequence of PRMT7 or Na V 1.9 formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention. PRMT7 or Na V 1.9 or its bioactive fragment includes an alternative sequence of some conserved amino acids, and the amino acid-substituted sequence does not affect its activity or retains part of its activity. Appropriate amino acid substitution is a well-known technique in the art, and the technique can be easily implemented and ensures that the biological activity of the resulting molecule is not changed. These techniques enable those skilled in the art to recognize that, generally speaking, changing a single amino acid in a non-essential region of a polypeptide basically does not change the biological activity. See Watson et al., Molecular Biology of The Gene, Fourth Edition, 1987, The Benjamin / Cummings Pub. Co. P224.

[0062] Any bioactive fragment of PRMT7 or Na V 1.9 can be applied to the present invention. Here, the meaning of the bioactive fragment of PRMT7 or Na V 1.9 refers to a polypeptide that can still maintain the full-length PRMT7 or Na VAll or part of the functions of 1.9. Usually, the said bioactive fragment retains at least 50% of the full-length PRMT7 or Na V The activity of 1.9. Under more preferred conditions, the said active fragment can retain 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length PRMT7 or Na V 1.9. Preferably, the bioactive fragment of PRMT7 includes the C-terminal domain of PRMT7. Preferably, the bioactive fragment of Na V 1.9 includes Na V The Loop1 domain of 1.9.

[0063] The present invention can also adopt modified or improved PRMT7 or Na V 1.9, for example, PRMT7 or Na V 1.9 modified or improved to promote its half-life, effectiveness, metabolism, and / or protein potency. That is to say, any variant that does not affect the biological activity of PRMT7 or Na V 1.9 can be used in the present invention.

[0064] The PRMT7 or Na V 1.9 described in the present invention can form a complex (protein complex), preferably a complex formed by the interaction (binding) of the active C-terminal domain of the former and the Loop1 region of the latter, for: serving as a target for regulating (preferably alleviating or inhibiting) pain, preparing a drug for regulating pain; serving as a target for screening drugs for regulating pain, screening drugs for regulating pain, etc.

[0065] A downregulator of PRMT7 or a downregulator of the interaction between PRMT7 and Na V 1.9 and its applications

[0066] Based on the above new findings of the present inventors, the present invention provides the use of a downregulator of PRMT7 or its coding gene for preparing a composition for alleviating or inhibiting pain.

[0067] As used herein, the terms "inhibit" or "downregulate" or "weaken" or "reduce", etc. refer to "inhibit" or "downregulate" or "weaken" or "reduce" with statistical significance. Such as compared with the control group, significantly "inhibit" or "downregulate" or "weaken" or "reduce"; more specifically, for example, more than 20%, preferably more than 50%, more preferably more than 80% of "inhibit" or "downregulate" or "weaken" or "reduce".

[0068] As used herein, the said "downregulator" includes inhibitors, antagonists, blockers, blocking agents, etc., and these terms can be used interchangeably.

[0069] The down-regulator of PRMT7 or its encoding gene refers to any substance that can reduce the activity of PRMT7, reduce the stability of PRMT7 or its encoding gene, down-regulate the expression of PRMT7, reduce the effective action time of PRMT7, or inhibit the transcription and translation of the PRMT7 gene. These substances can all be used in the present invention as substances useful for down-regulating PRMT7, and thus can be used to inhibit pain. For example, the down-regulator is: an interfering RNA molecule or antisense nucleotide that specifically interferes with the expression of the PRMT7 gene; an antibody or ligand that specifically binds to the protein encoded by the PRMT7 gene; and so on.

[0070] As a preferred embodiment of the present invention, the down-regulator is a small molecule compound targeting PRMT7. Those skilled in the art can adopt methods suitable for screening small molecule compounds to conduct the screening of such small molecule compounds. The screening can rely on various compound libraries existing in the art or to be developed, or some new compound libraries can be established by themselves.

[0071] As a preferred embodiment of the present invention, the small molecule compound is DS-437. DS-437 is a dual protein arginine methyltransferase PRMT5 / 7 inhibitor, which is selective for PRMT5 and PRMT7. DS-437 is a SAM competitive inhibitor. However, it has not been applied to pain-related treatment regimens. The present invention for the first time reveals the role of DS-437 in relieving or treating pain. The structural formula of DS-437 is as follows:

[0072]

[0073] As a preferred embodiment of the present invention, the small molecule compound is SGC8158, which has not been applied to pain-related treatment regimens. The present invention for the first time reveals the role of SGC8158 in relieving or treating pain. The structural formula of SGC8158 is as follows:

[0074]

[0075] In the present invention, the small molecule compound (such as DS-437 or SGC8158) can be a compound in a pure form, or a compound with a purity greater than 85% (preferably greater than 90%, such as greater than 95%, 98%, 99%). When the chemical structure is known, the small molecule compound can be obtained by chemical synthesis. The present invention also includes precursors of the compound. The "precursor" refers to a compound that, when taken by an appropriate method, undergoes metabolism or chemical reaction in the patient's body to be transformed into the active compound.

[0076] The present invention also includes isomers, solvates of the above DS-437 or SGC8158, or pharmaceutically acceptable salts thereof, provided that they also have the same or substantially the same function as DS-437 or SGC8158. The "pharmaceutically acceptable salts" refer to salts formed by the reaction of the compound with inorganic acids, organic acids, alkali metals or alkaline earth metals, etc. These salts include (but are not limited to): (1) salts formed with the following inorganic acids: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts formed with the following organic acids, such as acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium or magnesium) in the form of esters, carbamates, or other conventional "prodrugs". The compound has one or more asymmetric centers. Therefore, these compounds can exist as racemic mixtures, individual enantiomers, individual diastereoisomers, diastereoisomer mixtures, cis or trans isomers.

[0077] As a preferred embodiment of the present invention, the downregulator can be a PRMT7-specific interfering RNA molecule (such as siRNA, shRNA, miRNA, etc.). Those skilled in the art can understand that, based on the PRMT7 sequence information provided in the present invention, such interfering RNA molecules can be prepared. There is no particular limitation on the preparation method of the interfering RNA molecule, including but not limited to: chemical synthesis method, in vitro transcription method, etc. The interfering RNA can be delivered into cells by using an appropriate transfection reagent, or can also be delivered into cells by using a variety of techniques known in the art.

[0078] In some embodiments, RNAi is used to inhibit PRMT7. RNAi is an evolutionarily conserved cellular defense mechanism for controlling the expression of foreign genes in most eukaryotes including humans. RNAi is usually triggered by double-stranded RNA (dsRNA) and causes sequence-specific mRNA degradation of single-stranded target RNA. The mediator of mRNA degradation is small interfering RNA duplexes (siRNAs), which are usually generated by the intracellular cleavage of long dsRNA. The length of siRNAs is usually about 21 nucleotides (for example, 21-23 nucleotides). After the small RNA or RNAi is introduced into cells, it is believed that the sequence is delivered to an enzyme complex called RISC (RNA-induced silencing complex). RISC recognizes the target and cleaves it with an endonuclease. It should be noted that if a larger RNA sequence is delivered into cells, the RNAse III enzyme (Dicer) will convert the longer dsRNA into 21-23 nt ds-siRNA fragments.

[0079] In a preferred embodiment, the interference effect is achieved using shRNA technology. shRNA is an RNA sequence that can form a tight hairpin and can be used to silence gene expression through RNA interference. shRNA uses a vector introduced into cells and utilizes a promoter (such as U6) to ensure that shRNA is constantly expressed. This vector is usually passed on to daughter cells, enabling the inheritance of gene silencing. The shRNA hairpin structure is cleaved by cellular machinery into siRNA, which then binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it binds. shRNA is transcribed by RNA polymerase III.

[0080] Most preferably, the interfering molecule is shRNA; preferably, the shRNA includes the forward sequence shown in SEQ ID NO:2 and the reverse sequence shown in SEQ ID NO:3. The shRNA provides an appropriate knockdown effect, which can significantly reduce the methylation signal of hLoop1, thereby achieving significant relief or inhibition of pain.

[0081] As an alternative embodiment, an antisense compound that specifically hybridizes to one or more nucleic acids encoding PRMT7 is used to regulate PRMT7 expression. Specific hybridization of the oligomer to its target nucleic acid interferes with the normal function of the nucleic acid. This regulation of the function of the target nucleic acid by a compound that specifically hybridizes to the target nucleic acid is generally referred to as "antisense".

[0082] As an alternative aspect of the present invention, the CRISPR / Cas (such as Cas9) system can be used for targeted gene editing to knockout the PRMT7 gene in the targeted disease region. Common methods for knocking out the PRMT7 gene include: co-transfecting the sgRNA or nucleic acid capable of forming the sgRNA, Cas9 mRNA or nucleic acid capable of forming the Cas9mRNA into the targeted region or targeted cells. After determining the target site, known methods can be used to introduce the sgRNA and Cas9 into the cells. The nucleic acid capable of forming the sgRNA is a nucleic acid construct or expression vector, or the nucleic acid capable of forming the Cas9mRNA is a nucleic acid construct or expression vector, and these expression vectors are introduced into the cells to form active sgRNA and Cas9 mRNA in the cells.

[0083] As an alternative aspect of the present invention, homologous recombination can be used to specifically target PRMT7, resulting in defective expression or lack of expression. The Cre and loxp methods can also be applied to selectively knockout, reduce expression or inactivate related genes in the genomes of animals or cells.

[0084] The above are some representative ways to down-regulate PRMT7. It should be understood that the present invention provides a brand-new target. After those skilled in the art understand the general solution of the present invention, other methods known in the art or methods under development can also be adopted to regulate PRMT7, and these methods are also included in the present invention.

[0085] PRMT7 and Na V 1.9 as a drug screening target

[0086] After learning about the function and mechanism of action of the aforementioned PRMT7 and Na V 1.9, substances that inhibit the interaction between PRMT7 and Na V 1.9 can be screened based on this feature, and such substances have a positive effect on relieving or inhibiting pain.

[0087] Based on the new discovery of the inventors of the present invention, on the one hand, the present invention provides a method for screening potential substances for relieving or inhibiting pain, the method comprising: (1) treating an expression system expressing arginine methyltransferase PRMT7 with a candidate substance; and (2) detecting the expression or activity of arginine methyltransferase PRMT7 in the system; if the candidate substance down-regulates the expression or activity of arginine methyltransferase PRMT7 statistically, then the candidate substance is a potential substance for relieving or inhibiting pain.

[0088] On the other hand, the present invention provides a method for screening potential substances for relieving or inhibiting pain, the method comprising: (1) contacting a candidate with a system in which arginine methyltransferase PRMT7 interacts with sodium channel Na V 1.9 or its Loop1 segment; (2) detecting the effect of the candidate on the interaction between arginine methyltransferase PRMT7 and sodium channel Na V 1.9 or its Loop1 segment; wherein, if the candidate can weaken the interaction between the two, it indicates that the candidate is a substance for relieving or inhibiting pain.

[0089] As a preferred embodiment of the present invention, when detecting the interaction between PRMT7 and Na V 1.9, it also includes detecting the excitability of nerve cells in the system. If the excitability decreases, then the candidate substance is a potential substance for relieving or inhibiting pain.

[0090] As a preferred embodiment of the present invention, when detecting the interaction between PRMT7 and Na V 1.9, it also includes detecting the distribution of Loop1 of sodium channel Na V 1.9 on the cell membrane of nerve cells. If the distribution decreases, then the candidate substance is a potential substance for relieving or inhibiting pain.

[0091] In a preferred embodiment of the present invention, during the screening, in order to more easily observe the change in PRMT7 itself or its interaction with Na V 1.9, a control group can also be set up. The control group can be a system expressing PRMT7 Na V 1.9 without adding the candidate substance.

[0092] The system expressing PRMT7, the system expressing the interaction between PRMT7 and Na V 1.9 can be, for example, a cell (or cell culture) system. The cell can be a cell that endogenously expresses PRMT7 and / or Na V 1.9; or it can be a cell that recombinantly expresses PRMT7 and / or Na V 1.9. The system expressing PRMT7 and / or Na V 1.9 can also be (but not limited to) a subcellular system, a solution system, a tissue system, an organ system, or an animal system (such as an animal model), etc.

[0093] As a preferred embodiment of the present invention, the method further includes: performing further cell experiments and / or animal tests on the obtained potential substances to further select and determine substances that are truly useful for relieving or inhibiting pain.

[0094] The present invention places no particular limitation on the detection method for the expression, activity, abundance, and interaction of PRMT7 and Na V 1.9. Conventional gene / protein quantification or semi - quantification detection techniques, enzyme - catalyzed reactions, etc. can be used, such as (but not limited to): co - immunoprecipitation, SDS - PAGE method, Western - Blot method, ELISA, polymerase chain reaction technique (PCR), Northern blotting, etc.

[0095] On the other hand, the present invention also provides compounds, biological macromolecules, compositions, or drugs, or some potential substances obtained by the screening method. Some preliminarily screened substances can form a screening library, so that people can finally screen out substances that are truly useful for relieving or inhibiting pain, etc., and thus be used clinically.

[0096] Pharmaceutical composition

[0097] The present invention also provides a pharmaceutical composition, which contains a down - regulator of the aforementioned PRMT7 or its coding gene in an effective amount (such as 0.000001 - 50 wt%; preferably 0.00001 - 20 wt%; more preferably 0.0001 - 10 wt%), and a pharmaceutically acceptable carrier.

[0098] In a preferred embodiment of the present invention, the down-regulating agent includes, but is not limited to: an agent for knocking out or silencing PRMT7, a binding molecule (such as an antibody or ligand) that specifically binds to PRMT7, a small chemical antagonist or inhibitor against PRMT7, and the like. In a more specific embodiment, the down-regulating agent includes, but is not limited to: a CRISPR gene editing reagent against PRMT7, an interfering molecule that specifically interferes with the expression of the coding gene of PRMT7, a homologous recombination reagent or a site-directed mutagenesis reagent against PRMT7 or its effector molecule (such as sodium channel Na V 1.9 or its Loop1 segment), and the homologous recombination reagent or site-directed mutagenesis reagent causes a loss-of-function mutation in PRMT7.

[0099] As used herein, the "effective amount" refers to an amount that can produce a function or activity in a human and / or an animal and is acceptable to the human and / or the animal. The "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents. This term refers to such pharmaceutical carriers: they are not necessarily the active ingredient themselves and have no excessive toxicity after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in a composition may contain liquids, such as water, saline, buffers. Additionally, auxiliary substances may be present in these carriers, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, etc. The carrier may also contain a cell transfection reagent.

[0100] After learning the use of the down-regulating agent of PRMT7 or its coding gene, various methods well known in the art can be used to administer the down-regulating agent or its coding gene, or its pharmaceutical composition to a mammal or a human.

[0101] Preferably, gene therapy means can be adopted. For example, the down-regulating agent of PRMT7 can be directly administered to a subject by methods such as injection; or, an expression unit (such as an expression vector or a virus, etc., or siRNA) carrying the down-regulating agent of PRMT7 can be delivered to the target site through a certain route and made to express an active PRMT7 down-regulating agent. The specific situation depends on the type of the down-regulating agent, and these are all well known to those of ordinary skill in the art.

[0102] The effective amount of the down-regulating agent of PRMT7 or its coding gene according to the present invention may vary with the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art according to various factors (such as through clinical trials). Such factors include, but are not limited to: the pharmacokinetic parameters of the down-regulating agent of PRMT7 or its coding gene, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's body weight, the patient's immune status, the route of administration, etc.

[0103] In specific embodiments of the present invention, some dosing regimens for animals such as mice are given. It is easy for those skilled in the art to convert the dosing dose for animals such as mice into a dosing dose applicable to humans. For example, it can be calculated according to the Meeh-Rubner formula: Meeh-Rubner formula: A = k × (W 2 / 3 ) / 10,000. In the formula, A is the body surface area, calculated in m 2 ; W is the body weight, calculated in g; K is a constant, which varies with the animal species. Generally speaking, for mice and rats it is 9.1, for guinea pigs it is 9.8, for rabbits it is 10.1, for cats it is 9.9, for dogs it is 11.2, for monkeys it is 11.8, and for humans it is 10.6. It should be understood that according to the different drugs and clinical situations, the conversion of the dosing dose can be changed according to the evaluation of an experienced pharmacist.

[0104] The present invention also provides a medicine box containing the described pharmaceutical composition or directly containing a down-regulator of the described PRMT7 or its coding gene. In addition, the medicine box may further include an instruction manual for the usage method of the drugs in the medicine box.

[0105] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out according to conventional conditions such as those described in "Molecular Cloning: A Laboratory Manual", Third Edition, edited by J. Sambrook et al., Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0106] Materials and Methods

[0107] 1. Yeast two-hybrid screening of specifically interacting proteins between Na V 1.9 and their interaction verification

[0108] (1) Vector construction

[0109] The inventor of the present invention subcloned the first intracellular loop (amino acids 402 - 570) of mouse Na V 1.9 (GenBank accession number: NP_036017.3) into the pGBKT7 vector for yeast two-hybrid screening experiments; constructed the first intracellular loop of human Na V 1.9 (GenBank accession number: NP_001336182.1) into the pGEX-6P-1 vector for GST fusion protein expression; constructed FLAG and EGFP tag fusion proteins through the p3×FLAGCMV-7.1 and pEGFP-C1 vectors. The vector construction steps are as follows:

[0110] Primer design: By analyzing the gene sequence, restriction enzyme sites are added to design primers for amplifying the target fragment. The target fragment and the vector are digested using the restriction enzyme sites, and then ligated to a specific vector using T4 ligase.

[0111] PCR amplification of the target fragment: Using the cDNA of the cell line expressing the target gene or the DNA plasmid containing the target gene as a template to amplify the target fragment (high-fidelity DNA polymerase KOD-Plus reaction system).

[0112] Gel recovery and purification of the target fragment: The target fragment is separated by 1% agarose gel electrophoresis, and then the target band gel block is cut out and processed according to the experimental procedure of the PCR product recovery kit (Sangon).

[0113] Double digestion of the target fragment and the empty vector: The recovered target fragment and the empty vector are double-digested using the corresponding restriction enzymes, and the enzyme digestion system and conditions are referred to the instruction manual (NEB).

[0114] Purification and ligation of the double-digested products: Separated by 1% agarose gel electrophoresis, then the target band gel block is cut out, the digested target fragment and the empty vector are recovered, and the T4 ligase system and conditions are according to the instruction manual (ThermoFisherscientific).

[0115] E. coli transformation: The ligation product obtained in the previous step is added to DH5α E. coli competent cells, mixed well, left standing on ice for 30 minutes, then heat-shocked in a 42°C water bath for 90 s, immediately placed on ice for 3 minutes after taking out, and then 500 μL of LB medium without antibiotics is added, and cultured at 37°C in a shaker at 120 rpm / min for 60 minutes to resuscitate the bacteria. Then, centrifuged at 3000 rpm / min for 3 minutes, after discarding part of the supernatant, the remaining bacteria are spread on the LB solid plate with the corresponding resistance, and cultured overnight in a 37°C incubator.

[0116] PCR identification of bacterial liquid: Pick some monoclonal colonies into the LB liquid medium with the corresponding resistance, culture at 37°C in a shaker at 220 rpm / min for about 4 hours, then perform PCR screening of positive clones on the bacterial liquid, and finally send it to the company for sequencing to ensure the correct sequence.

[0117] (2) Yeast two-hybrid screening of the cDNA library of mouse DRG tissue

[0118] First, construct the pGBKT7-mLoop1 "bait" vector, transform the yeast AH109 strain, detect the expression of the bait protein, its toxic effect on the growth of host cells, and whether it can activate the expression of its own reporter gene.

[0119] Adopt SMART TMIII Library construction using the Clontech Laboratories, Inc. technology: A cDNA yeast library of mouse dorsal root ganglia (DRG) tissues was constructed. The yeast transformation method was the polyethylene glycol / lithium acetate (PEG / LiAc) method (PT3024-1, Clontech Laboratories, Inc.).

[0120] Preparation of yeast competent cells: Pick a yeast monoclonal with a diameter of 2 - 3 mm on the corresponding solid culture plate and place it in 3 - 5 mL of the corresponding liquid medium for overnight activation; take an appropriate amount of the bacterial solution and add it to the fresh enlarged medium, and place it in a shaker for cultivation when the final OD600 value of the bacterial solution is between 0.2 and 0.3; when OD600 reaches 0.4 - 0.6, collect the yeast cells under sterile conditions and wash them once with sterile water; then add an appropriate amount of TE / LiAc and gently resuspend to complete the preparation of yeast competent cells.

[0121] Transformation process: Mix the plasmid and the transformation reagent, add an appropriate amount of yeast competent cells for transformation; finally, spread the yeast containing the plasmid on the corresponding solid culture plate to observe the growth status of the yeast.

[0122] Screening method: The pGBKT7 plasmid contains the GAL4 promoter-binding domain and the tryptophan (Trp) nutritional marker. Yeast transformed with this plasmid can grow on a tryptophan-deficient culture plate. The pGADT7 plasmid contains the activation domain of GAL4 and the leucine (Leu) nutritional marker. Yeast transformed with this plasmid can grow on a tryptophan-deficient culture plate. The yeast AH109 strain has a GAL4 promoter on its genome that can express the reporter genes histidine (His), adenine (Ade), and secreted galactosidase (MEL). If the fusion proteins expressed on pGBKT7 and pGADT7 interact with each other, they will activate the expression of genes downstream of GAL4, and the yeast can grow on a medium lacking tryptophan, leucine, histidine, and adenine (SD / -Leu / -Trp / -His / -Ade). The galactosidase can react with the X-α-Gal substrate to show blue.

[0123] (3) Cell culture and transfection

[0124] HEK293T cells were used for in vitro experiments. HEK293T cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum (Gbico) at 37°C under 5% CO2. Cell passage: When the cell density reached the appropriate level, the old medium was discarded, and then the cells were washed with sterilized PBS. After discarding the PBS, an appropriate volume of trypsin was added for digestion. After digestion, an equal volume of DMEM medium containing 10% fetal bovine serum was added to terminate the digestion. The cells were gently pipetted and collected into a sterile centrifuge tube, centrifuged at 1000g for 3 min. The supernatant was discarded, and the cells were resuspended by gently pipetting with fresh medium and inoculated into a culture dish at an appropriate passage ratio for continued culture. Transfection of HEK293T cells: Before transfection, the cell density was adjusted to about 90%. An appropriate amount of serum-free DMEM medium was taken to dilute the high-purity plasmid and the corresponding amount (1 μg: 2 μL) of liposome LipoJet Transfection Reagent (SignaGen) respectively. After mixing each well, they were allowed to stand for 5 min. Then the diluted liposome was added to the diluted plasmid for mixing. After standing at room temperature for 15 min, it was added to the cells and finally gently mixed. Then the cells were continued to be cultured in the incubator for 24 - 48 h before the experiment.

[0125] (4) Co-Immunoprecipitation and Immunoblotting

[0126] Discard the culture medium from the transfected cells, add 1 mL of pre-cooled PBS, and gently wash the cells twice. Then add 1 mL of cell lysis buffer (Beyotime) to resuspend the cells, place them on ice for 30 minutes for lysis, sonicate the cells at 200 W for 2 s, 2 - 3 times, and then centrifuge at 4°C, 12,000 rpm / min for 10 minutes. Collect the supernatant into another 1.5 mL tube. Pipette 100 μL from the supernatant as a positive control (Input), divide the remaining supernatant into two equal volumes, add 2 μg of the corresponding antibody and an equal amount of IgG from the same species as the antibody source for immunoprecipitation, and incubate the antibodies overnight at 4°C on a rotating shaker. After antibody incubation, add 70 μL of Protein A agarose beads (Merck Millipore), incubate at 4°C for 4 hours, then centrifuge at 500 g / min for 30 s. After discarding the supernatant, wash the beads 3 times with 500 μL of cell lysis buffer. Finally, add 100 μL of 2× Loading buffer to each sample, pipette and mix well, then boil for 7 minutes, and store on ice or at -20°C for later use. Then prepare a 10% polyacrylamide gel and perform electrophoresis on the processed protein samples. Apply a constant voltage of 55 V until the protein molecular weight standard bands are separated, and then apply a constant voltage of 110 V to separate the proteins. After electrophoresis, transfer the proteins to a nitrocellulose membrane at a constant current of 200 mA using a wet transfer method. Then block the membrane with 5% skim milk at room temperature for 2 hours, and then add the primary antibody diluted with 3% BSA and incubate overnight at 4°C on a shaker. The next day, wash the membrane with TBST, 12 minutes per wash, 3 times; finally, incubate the membrane with the secondary antibody conjugated with HRP from the same species diluted with 5% skim milk at room temperature for 2 hours, and then wash the membrane with TBST, 12 minutes per wash, 3 times; then use the ECL immunoblotting chemiluminescent substrate (Merck Millipore) for darkroom film pressing and development detection. Antibody information is as follows: mouse anti-HA antibody (AE008, Abclonal), mouse anti-GFP antibody (AE012, Abclonal), rabbit anti-PRMT7 (A12159, Abclonal), mouse anti-FLAG antibody (M185-3 L, MBL), rabbit anti-Na V 1.9 antibody (ASC-017, Alomone Labs), rabbit anti-methyl(mono)arginine antibody (ICP0801, ImmuneChem), control mouse IgG and rabbit IgG (B900620, 30000-0-AP, ProteinTech).

[0127] (5) Fusion protein precipitation

[0128] Induced expression of GST fusion protein in prokaryotic strains: First, pGEX-6P-1-hLoop1 and the empty pGEX-6P-1 were transformed into the Rosseta Escherichia coli expression strain. Single colonies were picked and inoculated into 3 mL of LB liquid medium with ampicillin resistance, and cultured overnight at 37°C with a shaking speed of 220 rpm / min. Then, it was inoculated into 20 mL of medium for expansion culture at a ratio of 1:100. When the OD600 reached 0.6 - 0.8, induction expression was carried out using IPTG with different final concentrations and different temperatures. After collecting the bacterial cells, PBS containing the PMSF protease inhibitor was added, and then it was placed on ice for sonication. Finally, after taking the supernatant for polyacrylamide gel electrophoresis, the expression level of the fusion protein was detected by Coomassie brilliant blue staining to determine the optimal expression conditions of the fusion protein. Then, GST / GST-hLoop1 protein was expressed under the optimal conditions. First, 20 mL of bacterial cells expressing the fusion protein were collected, washed twice with pre-cooled PBS, and then resuspended in 3 mL of PBS containing the PMSF protease inhibitor. Sonication was carried out on ice at 200 W / 3 s - 3 s for 15 minutes, centrifuged at 12000 rpm / min for 10 minutes, and then an appropriate amount of glutathione agarose beads (Thermo Scientific) was added and incubated at 4°C on a shaker for 4 hours. Then, it was centrifuged at 4°C and 500 g / min for 30 seconds, the supernatant was discarded, and the beads were washed 3 times with PBS. Thus, the purified fusion protein was obtained. Finally, it was incubated overnight at 4°C on a shaker after being lysed with cells expressing PRMT7. Subsequently, it was centrifuged at 4°C and 500 g / min for 30 seconds, the beads were washed 3 times, an appropriate amount of 2×Loading buffer was added, and it was treated by boiling for 7 minutes and stored at 4°C or -20°C for later use.

[0129] (6) Immunofluorescence of tissue sections

[0130] Mouse DRGs were collected and washed in PBS, then fixed overnight at 4°C with 4% paraformaldehyde; then washed with PBS, and dehydrated with 20% - 30% sucrose until the tissue sedimented to the bottom of the tube; then frozen sections were made with a thickness of 12 μm, and the sections could be stored in a -20°C refrigerator for a long time. For immunofluorescence experiments, tissue sections were antigen - repaired by microwave using EDTA (pH = 8.0) antigen - repair solution, antigen - repaired at low heat for 10 min, and washed 3 times in PBS for 5 min each time after natural cooling. After the sections were slightly air - dried, a circle was drawn around the tissue with a histochemical pen to facilitate antibody incubation, then blocked with PBS containing 3% BSA and 0.1% Triton X - 100 at room temperature for 1 hour. The blocking solution was discarded, and the corresponding primary antibody diluted with 0.1% TritionX - 100 and 1% BSA PBS was added, and incubated overnight at 4°C in a humidified box; the primary antibody was discarded, the sections were washed 3 times in PBS for 5 min each time, then the corresponding secondary antibody was added and incubated at room temperature for 1 hour in the dark, the secondary antibody was discarded, washed 3 times in PBS for 5 min each time, stained with DAPI for nuclear staining, and incubated at room temperature for 5 min in the dark; the sections were washed 3 times in PBS for 5 min each time; finally, the sections were mounted with an anti - fluorescence quenching agent. After the samples were prepared, they were observed and imaged by an Olympus FluoView 1000 scanning confocal microscope.

[0131] 2. Identification of the Interaction Site and Methylation Site of hPRMT7 on hLoop1

[0132] (1) hPRMT7 Sequence

[0133] The amino acid sequence of hPRMT7 is as follows (SEQ ID NO:1):

[0134] MKIFCSRANPTTGSVEWLEEDEHYDYHQEIARSSYADMLHDKDRNVKYYQGIRAAVSRVKDRGQKALVLDIGTGTGLLSMMAVTAGADFCYAIEVFKPMADAAVKIVEKNGFSDKIKVINKHSTEVTVGPEGDMPCRANILVTELFDTELIGEGALPSYEHAHRHLVEENCEAVPHRATVYAQLVESGRMWSWNKLFPIHVQTSLGEQVIVPPVDVESCPGAPSVCDIQLNQVSPADFTVLSDVLPMFSIDFSKQVSSSAACHSRRFEPLTSGRAQVVLSWWDIEMDPEGKIKCTMAPFWAHSDPEEMQWRDHWMQCVYFLPQEEPVVQGSALYLVAHHDDYCVWYSLQRTSPEKNERVRQMRPVCDCQAHLLWNRPRFGEINDQDRTDRYVQALRTVLKPDSVCLCVSDGSLLSVLAHHLGVEQVFTVESSAASHKLLRKIFKANHLEDKINIIEKRPELLTNEDLQGRKVSLLLGEPFFTTSLLPWHNLYFWYVRTAVDQHLGPGAMVMPQAASLHAVVVEFRDLWRIRSPCGDCEGFDVHIMDDMIKRALDFRESREAEPHPLWEYPCRSLSEPWQILTFDFQQPVPLQPLCAEGTVELRRPGQSHAAVLWMEYHLTPECTLSTGLLEPADPEGGCCWNPHCKQAVYFFSPAPDPRALLGGPRTVSYAVEFHPDTGDIIMEFRHADTPD(692aa)

[0135] (2) Vector construction

[0136] Constructed a series of mutants pGADT7-hLoop1 with 20 and 40 amino acids deleted from both ends (N / C termini) of hLoop1 (Δ404-423 / Δ404-443 / Δ533-572 / Δ553-572); constructed a mutant pGADT7-hLoop1 (Δ563-572) with 10 amino acids deleted from the C terminus of hLoop1; constructed a yeast hybrid vector pGADT7-hLoop1 (563-566A / 565-568A / 567-570A / 569-572A), which was used to identify the interaction sites of hPRMT7 on hLoop1. Constructed an RNA interference experimental vector pSIH1-H1-CopGFP-shRNA for the PRMT7 gene, where:

[0137] Forward of PRMT7 shRNA: 5’-gatccggatgcagtgtgtgtacttccttcaagagaggaagtacacacactgcatcctttttg-3’ (SEQ ID NO:2);

[0138] Reverse of PRMT7 shRNA: 5’-aattcaaaaaggatgcagtgtgtgtacttcctctcttgaaggaagtacacacactgcatccg-3 (SEQ ID NO:3);

[0139] Constructed hLoop1-R519A, R521A and “R519A / 521A (double mutant)” mutants, which were used to identify the methylation sites of hLoop1.

[0140] (3) Identification of interaction sites by yeast two-hybrid

[0141] Transformed the above vectors into yeast and analyzed the growth of yeast on nutrient-deficient medium plates. The specific method was the same as described above.

[0142] (4) Identification of the methylation sites of hPRMT7 on hLoop1

[0143] After transfection with GFP-hPRMT7 and FLAG-hLoop1 vectors, RNA interference vectors or hLoop1-R519A, R521A and R519A / 521A mutants, the methylation level was detected by immunoprecipitation and immunoblotting.

[0144] 3. Detection of the regulation of the distribution of hLoop1 on the cell membrane by hPRMT7

[0145] (1) Cell culture and infection were the same as described above;

[0146] (2) Extraction of cell membrane and cytoplasmic proteins: After 24 hours, collect the transfected cells and separate the cell membrane and cytoplasmic proteins using a cell membrane protein and cytoplasmic protein extraction kit (P0033, Beyotime). Finally, detect by immunoblotting.

[0147] (3) Immunofluorescence assay: On the first day, inoculate an appropriate amount of cells into a glass-bottom dish, transfect the plasmid after overnight culture, and perform the experiment 24 hours after transfection. After 24 hours, discard the culture medium, wash the cells 2 times with PBS, then add 4% paraformaldehyde and fix at room temperature for 15 minutes; wash 3 times with PBS, 5 minutes each time. Perform non-permeabilized antibody incubation, directly add 5% BSA in PBS and block for 1 hour, discard the blocking solution, then add the corresponding antibody diluted with 1% BSA and incubate overnight at 4°C. Aspirate the primary antibody, wash 3 times with PBS, 5 minutes each time, then add the fluorescent secondary antibody diluted with 1% BSA and incubate at room temperature for 2 hours. After 2 hours, discard the secondary antibody, wash 3 times with PBS, 5 minutes each time, then add 5 μg / mL DAPI working solution and incubate at room temperature for 5 minutes. Finally, wash 3 times with PBS, 5 minutes each time, add 200 μL of 50% glycerol in PBS and store in the dark. After the sample is prepared, observe and image through an Olympus FluoView 1000 confocal microscope.

[0148] 4. hPRMT7 on hNa V 1.9 Current regulation

[0149] (1) Vector construction

[0150] The PRMT7 gene was cloned into the pIRES2-EGFP vector, and the SCN11A gene was constructed into the pcDNA3.1 vector and used for patch clamp recording by electroporation; the mouse Prmt7 gene (GenBank accession number NP_663379.1) was cloned into the pcDNA3.1 vector and electroporated into mouse DRG neurons to detect action potential firing. The construction method was the same as described above.

[0151] (2) Isolation and electroporation of mouse DRG cells

[0152] Coating of cell slides: Take out an 8 mm × 8 mm glass slide soaked in absolute ethanol with forceps, burn the alcohol on the flame, place it in a culture dish, and then coat the slide with poly-l-lysine (0.1%). Remove the poly-l-lysine when the DRG cells are digested, then wash the slide 2 times with ddH2O, and dry it before use.

[0153] DRG isolation: One 4-6 week-old C57BL / 6J mouse was taken and anesthetized by intraperitoneal injection of chloral hydrate. After that, the skin of the mouse was disinfected with alcohol, and the thoracic and lumbar spine of the mouse was operated on strictly according to the requirements of aseptic operation. Then, both ends were longitudinally cut open along the spine with small scissors, and the blood was washed away with pre-cooled PBS solution. Then it was placed in pre-cooled DMEM-F12 medium, the white spinal cord and its meninges in the spine were removed, the dorsal root ganglion in the intervertebral foramen was exposed, and the ganglion was picked out with forceps and placed in DMEM-F12. Under a stereomicroscope, the nerve fibers, connective tissue membrane and blood clots attached to the DRG were cut off with ophthalmic scissors. The medium was discarded, and then the trimmed DRG ganglia were minced and transferred to a 1.5 mL centrifuge tube. 1 mL of enzyme solution (trypsin I: 0.3 mg / mL and collagenase II: 1 mg / mL) was added, and it was placed in an incubator at 37 °C for digestion for 30 min. The mixture was shaken several times during digestion. After digestion, the supernatant was gently aspirated, 1 mL of DMEM-F12 + 10% FBS medium was added to terminate digestion, and then centrifuged at 1000 g / min for 3 min, and the supernatant was removed. 600 μL of DMEM-F12 + 10% FBS medium was added and pipetted until the tissue pieces disappeared and the solution became turbid.

[0154] Electroporation: The above cell suspension was centrifuged at 1000 g / min for 3 minutes, the supernatant was discarded, and then 100 μL of buffer R was added to resuspend the cells. The hNav1.9 plasmid and the empty vector or the experimental group were added to the DRG cell suspension at a ratio of 10:2, and electroporation was carried out using a Neon transfection system (Invitrogen). The electroporation program was 1200 V, 20 ms. After transfection, 500 μL of DMEM-F12 + 10% FBS medium was added, and after thorough mixing, the cells were seeded onto polylysine-coated cell slides, placed in an incubator at 37 °C for 1 hour, and then added with complete medium and cultured in a cell culture incubator. Electrophysiological experiments were performed 36 hours later.

[0155] (3) Patch clamp recording

[0156] Voltage-clamp recordings were made with an electrode resistance of 3 - 5 MΩ using an Axopatch 200b amplifier. Intracellular solution: 135 mM CsF, 10 mM NaCl, 2.5 mM MgCl2, 10 mM HEPES, 1 mM EGTA, 5 mM TEA-Cl, and 4 mM Mg-ATP (pH = 7.4, adjusted with CsOH), extracellular solution: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 10 mM HEPES, 0.1 mM CdCl2, 20 mM TEA-Cl, 0.001 mM TTX, and 10 - 30 mM glucose (pH = 7.4, adjusted with NaOH). After culturing transfected plasmids for 36 hours, small-diameter DRG neurons with green fluorescence were selected, the clamping voltage was -120 mV, and then a series of voltage pulse stimulations from -120 to -10 mV with an increment of 5 mV and a duration of 200 ms were given. The peak current at each voltage was measured. The activation curve was fitted by the Boltzmann equation G / Gmax = 1 / (1 + exp[(V 1 / 2 - Vm) / k]), where G is the voltage-dependent sodium conductance, Gmax is the maximum conductance, V 1 / 2 is the voltage at which the conductance (G) value is half of the maximum conductance value (Gmax), and k is a factor affecting the activation rate, reflecting the slope of the G-V curve; for the steady-state fast inactivation curve, first, a series of voltage pulse stimulations from -120 to 0 mV with an increment of 10 mV and a duration of 500 ms were given, and then the current was induced by a -50 mV voltage. The curve was obtained by fitting with I / Imax = 1 / (1 + exp[(Vm – V 1 / 2 ) / k]).

[0157] Current-clamp recordings were made. Intracellular solution: 140 mM KCl, 0.5 mM EGTA, 5 mM HEPES, and 2 mM Mg-ATP (pH = 7.3, adjusted with KOH), and the osmotic pressure of the solution was adjusted to 315 mOsm / L with glucose. Extracellular solution: 140 mM NaCl, 3 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES (pH = 7.3, adjusted with NaOH). A series of currents from 0 to 225 pA with an increment of 25 pA and a duration of 500 ms were injected. The action potential firing of neuron cells was recorded, and the resting membrane potential and action potential voltage threshold were statistically analyzed.

[0158] 5. Effects of the PRMT7 inhibitor DS-437 on Na V 1.9 current, DRG neuron excitability, and Na V 1.9 A796G / A796G analgesia in mice

[0159] (1) Drug treatment

[0160] DS-437 was prepared into a 100 mM stock solution with DMSO, and SGC8158 was prepared into a 20 mM stock solution with DMSO, and stored at -20 °C. Mouse dorsal root ganglion neurons were isolated, and the isolated neurons were co-incubated with 100 μM DS-437 (prepared with 10% FBS high-glucose DMEM medium) in a cell culture incubator for 1 hour; the control group was co-incubated with 10% FBS high-glucose DMEM medium containing one-thousandth DMSO in a cell culture incubator for 1 hour, and then patch clamp recording was performed.

[0161] (2) Patch clamp recording was the same as described above

[0162] (3) Formalin test

[0163] 8-week-old Na V 1.9 A796G / A796G 8-week-old Na (see Pain, 2020 Jul; 161(7):1470-1482. doi: 10.1097 / j.pain.0000000000001853) and WT mice were used for the formalin test. Mice were placed in a test cage half an hour before the experiment to adapt to the environment. Then, 1.6 mg / kg, 0.8 mg / kg DS-437, 1.4 μg / kg SGC8158 or an equal volume of solvent was injected intraperitoneally. After waiting for 1 hour, 20 μL of 5% formalin solution was injected subcutaneously into the hind paw, and then the time of paw licking and paw lifting of the mice within 45 minutes was recorded, and a data point was recorded every 5 minutes. Finally, the 0-10 minute period was defined as phase I and the 15-45 minute period as phase II for statistical analysis. The longer and more frequent the paw licking and paw lifting time indicates that the mice are sensitive to pain. This experiment was a double-blind experiment.

[0164] (4) Hot plate test and vonfrey pain measurement

[0165] 8-week-old Na V 1.9 A796G / A796GMice and WT mice were used for the formalin test. Half an hour before the experiment, the mice were placed in the test cage to adapt to the environment. First, the basic pain threshold of the test mice was measured. Then, 1.6 mg / kg, 0.8 mg / kg DS-437, 1.4 μg / kg SGC8158 or an equal amount of solvent was injected intraperitoneally. One hour and two hours after the injection, the thermal pain threshold and mechanical pain threshold of the mice were detected. Thermal pain threshold: The hot plate was adjusted to 52 °C. Timing started when the mouse was placed on the hot plate and stopped when the mouse lifted its paw, withdrew, or licked its paw. This duration reflects the sensitivity to thermal stimulation, and each mouse was tested 3 times; Mechanical pain threshold: The mouse was placed on the wire mesh, and different filament diameters were selected by the vonfrey dolorimeter in the up-down method to prick the hind paw of the mouse for 5 s. If the mouse lifted its paw, it was proved to be sensitive to the stimulation, and each mouse was measured 3 times.

[0166] Example 1, Screening of Proteins Interacting with Na V 1.9 by Yeast Two-Hybrid

[0167] The inventor constructed a cDNA library of mouse DRG neurons and used the first intracellular domain (Loop1) of murine Na V 1.9 (mNa V 1.9) as a "bait" ( Figure 1 A) to screen for proteins interacting with mNa V 1.9-Loop1. After a large amount of screening work, the inventor screened out the protein arginine methyltransferase PRMT7 that interacts with mNa V 1.9-Loop1, and its C-terminal domain interacts with mNa V 1.9-Loop1 ( Figure 1 B, C).

[0168] According to the sequence comparison results, the sequence similarity between mNa V 1.9-Loop1 and human Na V 1.9-Loop1 (hNa V 1.9-Loop1) reached 93.5% ( Figure 1 D).

[0169] Therefore, the inventor subsequently verified the interaction and function between human PRMT7 (hPRMT7) and hNa V 1.9-Loop1.

[0170] Example 2, In Vitro and In Vivo Verification of the Interaction between PRMT7 and Na V 1.9-Loop1

[0171] The inventor conducted a GST-Pull down experiment, as Figure 2A; and co-immunoprecipitation (CO-IP) experiments were performed in both forward and reverse directions, as Figure 2 B. The results confirmed that Na V 1.9-Loop1 (hLoop1) was able to interact with hPRMT7.

[0172] Further experiments demonstrated that hPRMT7 interacted only with hLoop1, rather than the N-terminus, C-terminus of Na V 1.9 and hLoop2, as Figure 2 C.

[0173] The present inventors conducted in vivo experiments, and the endogenous interaction between PRMT7 and Na V 1.9 was also demonstrated in mouse DRG tissues, as Figure 2 D.

[0174] The present inventors performed immunofluorescence experiments, and the results confirmed the co-localization of PRMT7 and Na V 1.9 in mouse DRG tissues, as Figure 2 E.

[0175] Example 3, PRMT7 increases Na V 1.9 current and DRG neuron excitability

[0176] The present inventors electrotransfected PRMT7 and SCN11A plasmids in Na V 1.9 - / - mouse DRG neurons to detect Na V 1.9 current, and found that hPRMT7 significantly increased the Na V 1.9 current density (Mock + hNa V 1.9: -51 ± 5.86 pA / pF, n = 13; hPRMT7 + hNa V 1.9: -122 ± 22.1 pA / pF, n = 15), as Figure 3 A-E; but did not affect the Na V 1.9 channel activation curve and steady-state fast inactivation, as Figure 3 F, G, H.

[0177] In addition, the present inventors electrotransfected Prmt7 plasmid in Na V 1.9 + / + mouse and Na V 1.9 - / - mouse DRG neurons to detect DRG neuron excitability. The results showed that mPRMT7 increased the firing frequency of action potentials in Na V 1.9 + / + mouse DRG neurons, as Figure 4A-C; however, it does not affect the action potential voltage threshold and the resting membrane potential of DRG neurons, such as Figure 4 D. Conversely, mPRMT7 does not affect Na V 1.9 - / - the firing frequency of action potentials in mouse DRG neurons, such as Figure 4 C.

[0178] This result proves that in mouse DRG neurons, mPRMT7 only affects neuronal excitability by regulating Na V 1.9.

[0179] Example 4. hPRMT7 promotes the distribution of hNa V 1.9-Loop1 on the cell membrane

[0180] The above experiments have demonstrated that PRMT7 increases the Na V 1.9 current, and the increase in current density generally depends on the distribution of channel proteins on the cell membrane. Therefore, the present inventors further explored the effect of hPRMT7 on the distribution of hNa V 1.9-Loop1 (hLoop1) on the cell membrane.

[0181] First, the present inventors constructed a membrane reporter molecule chimeric vector pcDNA3.1-HA-hCD4-hLoop1. Human CD4 (hCD4) is a type I single-pass transmembrane protein encoding 458 amino acids (positions 1-458 in GenBank accession number NP_000607.1). The first 25 amino acids of this protein are the signal peptide, and the amino acids at positions 397-458 are located in the cytoplasm. Using the pcDNA3.1 vector as the backbone, the cytoplasmic fragment of hCD4 (i.e., its 397-458 positions) was replaced with the hLoop1 segment, and then an HA tag was inserted between the signal peptide and the mature protein of hCD4 for easy detection of the chimeric protein, finally forming the chimeric protein HA-hCD4-Loop1, such as Figure 5 A, B.

[0182] Then, the present inventors co-transfected the pcDNA3.1-HA-hCD4-hLoop1 plasmid with the pEGFP-C1 empty vector and the pEGFP-PRMT7 plasmid into HEK293T cells, separated the cytoplasmic and cell membrane fractions, and detected the protein expression in different fractions, using Na + / K + -ATPase as the membrane protein internal reference. The results showed that overexpression of PRMT7 could significantly increase the distribution of HA-hCD4-Loop1 in the cell membrane, such as Figure 5 C, D; however, it had no effect on the expression of PRMT7 protein in the cell lysate, such as Figure 5 E.

[0183] Meanwhile, the inventor of the present invention demonstrated through immunofluorescence experiments that PRMT7 can increase the distribution of HA-hCD4-Loop1 on the cell membrane, as shown in Figure 6 A, B.

[0184] These results indicate that PRMT7 promotes the distribution of HA-hCD4-Loop1 on the cell membrane, but does not affect the expression level of HA-hCD4-Loop1 in cells.

[0185] Example 5, Identification of Fine Interaction Sites between hPRMT7 and hLoop1

[0186] The inventor of the present invention predicted the regions in the amino acid sequence of hLoop1 that are prone to binding to other proteins through bioinformatics tools, as shown in Figure 7 A.

[0187] After that, the inventor of the present invention constructed a series of mutants with 20 and 40 amino acids (10 amino acids) deleted from both the N- and C-termini of hLoop1. The results showed that the amino acids at positions 563-572 at the C-terminus of hLoop1 are crucial for its interaction with hPRMT7-C, as shown in Figure 7 B.

[0188] Furthermore, the inventor of the present invention performed overlapping alanine mutations on the amino acids at positions 563-572 of hLoop1 and found that the amino acids at positions 563-566 of hLoop1 play a key role in the interaction between the two, as shown in Figure 7 C-E.

[0189] The above experiments confirmed that the key amino acids for the binding of hPRMT7 to hLoop1 are the amino acids Trp, Leu, Cys, and Val (WLCV) at positions 563-566 of hLoop1.

[0190] Example 6, Methylation Modification of Arginine on hLoop1 by hPRMT7

[0191] The inventor of the present invention overexpressed hPRMT7 and hLoop1 in HEK293T cells and detected the methylation level of hLoop1 by hPRMT7. The results showed that the methylation signal of hLoop1 increased with the increase in the expression level of hPRMT7, as shown in Figure 8 A.

[0192] The inventor of the present invention used shPRMT7 to reduce the expression of hPRMT7 through RNA interference, as shown in Figure 8 B. The results showed that the methylation signal of hLoop1 was significantly weakened, as shown in Figure 8 C.

[0193] Since PRMT7 preferentially recognizes the RXR motif on the substrate, and there is an R in the loop1 region519 QR 521 motif. The determination results of the present inventors show that the methylation signals of the R519A and R519A / 521A mutants cannot be detected, the methylation signal of the R521A mutant can be detected, but the GFP-hPRMT7 fusion protein can be detected in both cases, indicating that the arginine residue at position R519 is the methylation site of hPRMT7, and the change of the arginine site does not affect the binding of hLoop1 to hPRMT7, as Figure 8 D.

[0194] Example 7, hPRMT7 increases Na V 1.9 current depends on the binding site and methylation site on hLoop1

[0195] Since PRMT7 regulates its current density by interacting with Na V 1.9 and methylation, the present inventors constructed the PRMT7 binding site mutant hNa V 1.9-4A(563-566A) and the methylation site mutant hNa V 1.9-R519A. Then, Na V 1.9 - / - was transfected into mouse DRG neurons. The results showed that the average peak current density of the hNa V 1.9-4A and hNa V 1.9-R519A mutants themselves was relatively low, and PRMT7 did not affect the peak current density of hNa V 1.9-4A and hNav1.9-R519A mutants, as Figure 9 A, B.

[0196] These results indicate that PRMT7 regulates the hNa V 1.9 current density depending on its binding and methylation to hNa V 1.9, and the amino acids at positions 563-566 and the arginine at position 519 are crucial for the function of the hNa V 1.9 channel.

[0197] Example 8, Inhibiting PRMT7 activity reduces Na V 1.9 current

[0198] The Na V 1.9 current was detected by inhibiting the activity of PRMT7 with the PRMT7 inhibitor DS-437. After co-incubating DRG neurons with the Prmt7 inhibitor DS-437 (10 μM, 100 μM) for 1 hour, it was found that the mNa V1.9 Peak current density (Control: -223.17 ± 35.19 pA / pF, n = 11; 10 μM: 131.48 ± 20.55 pA / pF, n = 13; 100 μM: -104.40 ± 29.40 pA / pF, n = 10, p < 0.05) was significantly decreased ( Figure 10 A - D), but did not affect mNa V 1.9 Voltage - dependent activation curve (V 1 / 2 , Control: -58.08 ± 0.84 mV; 10 μM: -57.38 ± 1.39 mV; 100 μM: -56.75 ± 0.62 mV) and steady - state fast inactivation curve (V 1 / 2 , Control: -60.59 ± 1.44 mV; 10 μM: -60.20 ± 5.59; 100 μM: -61.20 ± 1.19) ( Figure 10 E, F).

[0199] The above results indicate that inhibiting Prmt7 activity significantly reduces mNa V 1.9 current density, but does not affect mNa V 1.9 channel properties.

[0200] Example 9, Inhibiting PRMT7 Activity Reduces Na V 1.9 A796G / A796G Excitability of Mouse DRG Neurons

[0201] Since the PRMT7 inhibitor DS - 437 can significantly inhibit Na V 1.9 current, the present inventors studied whether inhibiting PRMT7 activity affects the excitability of mouse DRG neurons.

[0202] First, the present inventors co - incubated wild - type mouse DRG neurons with the PRMT7 inhibitor DS - 437 (100 μM) for 1 hour and then detected action potentials. The results showed that the PRMT7 inhibitor DS - 437 did not affect the action potential firing, voltage threshold, and resting membrane potential of wild - type mouse DRG neurons ( Figure 11 A - C), and this result was consistent with the result that Na V 1.9 knockout does not affect the action potential firing of mouse DRG neurons.

[0203] Therefore, to further verify the effect of inhibiting PRMT7 activity on the excitability of DRG neurons, the present inventors selected a pain mouse model of Na V 1.9 A796G / A796G mice for verification. The results showed that the PRMT7 inhibitor DS - 437 could significantly reduce Na V 1.9 A796G / A796GFiring frequency of action potentials in mouse DRG neurons( Figure 11 B, C), but does not affect the firing voltage threshold and resting membrane potential of action potentials( Figure 11 D).

[0204] Example 10, Inhibition of PRMT7 activity inhibits Na V 1.9 A796G / A796G Mouse pain hypersensitivity and Na V 1.9 A796G / A796G Formalin-induced pain hypersensitivity in Na

[0205] To further test the applicability of PRMT7 inhibitors in analgesia, the inventors used a pain mouse model - Na V 1.9 A796G / A796G Mice were used for testing.

[0206] Mouse behavioral experiments showed that the PRMT7 inhibitors DS-437 and SGC8158 could relieve Na V 1.9 A796G / A796G Mouse pain hypersensitivity( Figure 12 A, B), as well as Na V 1.9 A796G / A796G Mouse and WT mouse formalin-induced pain hypersensitivity( Figure 13 A, B).

[0207] The results showed that inhibiting PRMT7 activity reduced the excitability of Na V 1.9 A796G / A796G Mouse DRG neurons and relieved formalin-induced pain hypersensitivity in mice.

[0208] In summary, the present invention demonstrates a new use of PRMT7 as a target for preparing analgesic drugs, and a new therapeutic use of PRMT7 inhibitors in pain treatment.

[0209] All documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application. Sequence Listing <110> Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences <120> Use of Nav1.9 interacting protein PRMT7 and its down-regulators in the preparation of analgesic drugs <130> 212246 <160> 3 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 692 <212> PRT <213> Homo sapiens <400> 1 Met Lys Ile Phe Cys Ser Arg Ala Asn Pro Thr Thr Gly Ser Val Glu 1 5 10 15 Trp Leu Glu Glu Asp Glu His Tyr Asp Tyr His Gln Glu Ile Ala Arg 20 25 30 Ser Ser Tyr Ala Asp Met Leu His Asp Lys Asp Arg Asn Val Lys Tyr 35 40 45 Tyr Gln Gly Ile Arg Ala Ala Val Ser Arg Val Lys Asp Arg Gly Gln 50 55 60 Lys Ala Leu Val Leu Asp Ile Gly Thr Gly Thr Gly Leu Leu Ser Met 65 70 75 80 Met Ala Val Thr Ala Gly Ala Asp Phe Cys Tyr Ala Ile Glu Val Phe 85 90 95 Lys Pro Met Ala Asp Ala Ala Val Lys Ile Val Glu Lys Asn Gly Phe 100 105 110 Ser Asp Lys Ile Lys Val Ile Asn Lys His Ser Thr Glu Val Thr Val 115 120 125 Gly Pro Glu Gly Asp Met Pro Cys Arg Ala Asn Ile Leu Val Thr Glu 130 135 140 Leu Phe Asp Thr Glu Leu Ile Gly Glu Gly Ala Leu Pro Ser Tyr Glu 145 150 155 160 His Ala His Arg His Leu Val Glu Glu Asn Cys Glu Ala Val Pro His 165 170 175 Arg Ala Thr Val Tyr Ala Gln Leu Val Glu Ser Gly Arg Met Trp Ser 180 185 190 Trp Asn Lys Leu Phe Pro Ile His Val Gln Thr Ser Leu Gly Glu Gln 195 200 205 Val Ile Val Pro Pro Val Asp Val Glu Ser Cys Pro Gly Ala Pro Ser 210 215 220 Val Cys Asp Ile Gln Leu Asn Gln Val Ser Pro Ala Asp Phe Thr Val 225 230 235 240 Leu Ser Asp Val Leu Pro Met Phe Ser Ile Asp Phe Ser Lys Gln Val 245 250 255 Ser Ser Ser Ala Ala Cys His Ser Arg Arg Phe Glu Pro Leu Thr Ser 260 265 270 Gly Arg Ala Gln Val Val Leu Ser Trp Trp Asp Ile Glu Met Asp Pro 275 280 285 Glu Gly Lys Ile Lys Cys Thr Met Ala Pro Phe Trp Ala His Ser Asp 290 295 300 Pro Glu Glu Met Gln Trp Arg Asp His Trp Met Gln Cys Val Tyr Phe 305 310 315 320 Leu Pro Gln Glu Glu Pro Val Val Gln Gly Ser Ala Leu Tyr Leu Val 325 330 335 Ala His His Asp Asp Tyr Cys Val Trp Tyr Ser Leu Gln Arg Thr Ser 340 345 350 Pro Glu Lys Asn Glu Arg Val Arg Gln Met Arg Pro Val Cys Asp Cys 355 360 365 Gln Ala His Leu Leu Trp Asn Arg Pro Arg Phe Gly Glu Ile Asn Asp 370 375 380 Gln Asp Arg Thr Asp Arg Tyr Val Gln Ala Leu Arg Thr Val Leu Lys 385 390 395 400 Pro Asp Ser Val Cys Leu Cys Val Ser Asp Gly Ser Leu Leu Ser Val 405 410 415 Leu Ala His His Leu Gly Val Glu Gln Val Phe Thr Val Glu Ser Ser 420 425 430 Ala Ala Ser His Lys Leu Leu Arg Lys Ile Phe Lys Ala Asn His Leu 435 440 445 Glu Asp Lys Ile Asn Ile Ile Glu Lys Arg Pro Glu Leu Leu Thr Asn 450 455 460 Glu Asp Leu Gln Gly Arg Lys Val Ser Leu Leu Leu Gly Glu Pro Phe 465 470 475 480 Phe Thr Thr Ser Leu Leu Pro Trp His Asn Leu Tyr Phe Trp Tyr Val 485 490 495 Arg Thr Ala Val Asp Gln His Leu Gly Pro Gly Ala Met Val Met Pro 500 505 510 Gln Ala Ala Ser Leu His Ala Val Val Val Glu Phe Arg Asp Leu Trp 515 520 525 Arg Ile Arg Ser Pro Cys Gly Asp Cys Glu Gly Phe Asp Val His Ile 530 535 540 Met Asp Asp Met Ile Lys Arg Ala Leu Asp Phe Arg Glu Ser Arg Glu 545 550 555 560 Ala Glu Pro His Pro Leu Trp Glu Tyr Pro Cys Arg Ser Leu Ser Glu 565 570 575 Pro Trp Gln Ile Leu Thr Phe Asp Phe Gln Gln Pro Val Pro Leu Gln 580 585 590 Pro Leu Cys Ala Glu Gly Thr Val Glu Leu Arg Arg Pro Gly Gln Ser 595 600 605 His Ala Ala Val Leu Trp Met Glu Tyr His Leu Thr Pro Glu Cys Thr 610 615 620 Leu Ser Thr Gly Leu Leu Glu Pro Ala Asp Pro Glu Gly Gly Cys Cys 625 630 635 640 Trp Asn Pro His Cys Lys Gln Ala Val Tyr Phe Phe Ser Pro Ala Pro 645 650 655 Asp Pro Arg Ala Leu Leu Gly Gly Pro Arg Thr Val Ser Tyr Ala Val 660 665 670 Glu Phe His Pro Asp Thr Gly Asp Ile Ile Met Glu Phe Arg His Ala 675 680 685 Asp Thr Pro Asp 690 <210> 2 <211> 62 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(62) <223> shRNA <400> 2 gatccggatg cagtgtgtgt acttccttca agagaggaag tacacacact gcatcctttt 60 tg 62 <210> 3 <211> 62 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> (1)..(62) <223> shRNA <400> 3 aattcaaaaa ggatgcagtg tgtgtacttc ctctcttgaa ggaagtacac acactgcatc 60 cg 62

Claims

1. Use of a down-regulator of arginine methyltransferase PRMT7 in the preparation of a composition for relieving or inhibiting pain; the down-regulator is an inhibitor DS-437 or SGC8158 against arginine methyltransferase PRMT7.

2. The application according to claim 1, characterized in that, The described composition is also used for: inhibiting the interaction of arginine methyltransferase PRMT7 with sodium channel Na V 1.

9.

3. The application according to claim 1, characterized in that The described composition is also used for: inhibiting the sodium channel Na V to increase the current density by 1.9 and inhibit the excitability of neurons.

4. The application according to claim 1, wherein The described composition is also used for: reducing the distribution of Loop1 with a value of 1.9 on the cell membrane of sodium channels Na V 1.9 on the cell membrane.

5. Use of a pharmaceutical composition in the preparation of a kit for relieving or inhibiting pain, said pharmaceutical composition comprising: A down-regulator of arginine methyltransferase PRMT7 and a pharmaceutically acceptable carrier or excipient; The down-regulator is a small molecule chemical inhibitor against arginine methyltransferase PRMT7 selected from DS-437 or SGC8158.

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