JP7198489B2 - Phosphorylated SARM1, antibody, SARM1 phosphorylation inhibitor, preventive or therapeutic drug for neurodegenerative disease, screening method, SARM1 variant and use - Google Patents

Description

The present invention relates to phosphorylated SARM1, antibodies, SARM1 phosphorylation inhibitors, preventive or therapeutic agents for neurodegenerative diseases, screening methods, SARM1 variants and uses.

SARM1 (Sterile alpha and TIR motif-containing protein 1) is one of the TIR adapter proteins with mitochondrial translocation signals and consists of 724 amino acids. SARM1 is strongly expressed mainly in cells of the nervous system (Non-Patent Document 1), and is reported to be a causative molecule for ischemic neuronal cell death and nerve axonal degeneration (Non-Patent Documents 2 and 3). Since this neuronal cell death can be significantly suppressed by knocking out SARM1, drugs that target SARM1 can suppress neuronal cell death, leading to various neurodegenerative diseases such as Parkinson's disease and amyotrophic lateral sclerosis (ALS). It is believed that it can be applied to the treatment of diseases (Patent Document 1). However, there are currently no drugs targeting SARM1.

Patent Documents 2 to 5 disclose that inhibitors of CDK8 or CDK19 are effective in treating cancer.

US20120328629 WO2015/159937 Special table 2016-501845 special table 2016-503408 special table 2015-506376

J Exp Med, 204(9), 2007 Science, 337(6093), 2012 J Neurosci, 33(33), 2013

An object of the present invention is to prevent or treat neurodegenerative diseases by suppressing the function of SARM1.

The present inventor analyzed changes and functions of SARM1 in cells and found that SARM1 inhibits ATP synthesis and induces cell death by acting on mitochondrial respiratory chain complex V. We also found that c-Jun N-terminal kinase (JNK) activates SARM1 by phosphorylating at least one serine residue selected from the group consisting of 54th, 548th and 622nd positions of SARM1. . By developing a method for detecting phosphorylation of at least one serine residue selected from the group consisting of 54th, 548th and 622nd SARM1, it can be used to confirm the active state of SARM1 and to develop inhibitors. It becomes possible to go. The present inventors further phosphorylated at least one serine residue selected from the group consisting of 54th, 548th and 622nd by using a region containing the ARM domain, SAM domain, and TIR domain in SARM1 We have developed a method for sensitive detection of Using this detection method, we also discovered a SARM1 inhibitor.

The present invention provides the following phosphorylated SARM1, antibodies, SARM1 phosphorylation inhibitors, preventive or therapeutic agents for neurodegenerative diseases, screening methods, SARM1 variants and uses.
Section 1. Phosphorylated SARM1 in which at least one Ser residue selected from the group consisting of positions 54, 548 and 622 of SARM1 (Sterile alpha and TIR motif-containing protein 1) is phosphorylated.
Section 2. Item 2. The phosphorylated SARM1 according to Item 1, wherein SARM1 is derived from human.
Item 3. An anti-phosphorylated SARM1 antibody that specifically recognizes phosphorylated Ser residues at positions 54, 548 and 622 of SARM1 and does not recognize non-phosphorylated Ser residues at positions 54, 548 and 622 of SARM1.
Section 4. A SARM1 phosphorylation inhibitor comprising, as an active ingredient, at least one inhibitor selected from the group consisting of cyclin-dependent kinase 8 (CDK8), cyclin-dependent kinase 19 (CDK19) and JNK.
Item 5. A prophylactic or therapeutic agent for neurodegenerative diseases, comprising at least one inhibitor selected from the group consisting of cyclin-dependent kinase 8 (CDK8), cyclin-dependent kinase 19 (CDK19) and JNK as an active ingredient.
Item 6. A prophylactic or therapeutic agent for neurodegenerative diseases, comprising as an active ingredient a SARM1 inhibitor selected from the group consisting of anti-SARM1 antibodies, SARM1 decoy peptides and antagonists.
Item 7. Anti-SARM1 antibody specifically recognizes the phosphorylated Ser residue at position 54, 548 or 622 of SARM1 or an antibody against non-phosphorylated SARM1 capable of suppressing phosphorylation at position 54, 548 or 622 of SARM1 7. The preventive or therapeutic drug for neurodegenerative diseases according to Item 6, which is an anti-phosphorylated SARM1 antibody that does not recognize non-phosphorylated Ser residues at positions 54, 548 and 622 of SARM1.
Item 8. Item 7. The prophylactic or therapeutic drug for neurodegenerative diseases according to Item 6, wherein the SARM1 decoy peptide is a fragment peptide containing ^Ser54 , ^Ser548 or ^Ser622 of SARM1 or a modification thereof.
Item 9. Prevention of neurodegenerative disease according to any one of items 5 to 8, wherein the neurodegenerative disease is selected from the group consisting of Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, neuropathy and Alzheimer's disease. or therapeutic agents.
Item 10. A method of screening for a preventive or therapeutic agent for neurodegenerative diseases, comprising detecting phosphorylation of at least one Ser residue selected from the group consisting of positions 54, 548 and 622 of SARM1.
Item 11. A method of screening for a SARM1 inhibitor, comprising detecting phosphorylation of at least one Ser residue selected from the group consisting of positions 54, 548 and 622 of SARM1.
Item 12. A SARM1 variant in which Ser corresponding to at least one selected from the group consisting of positions 54, 548 and 622 of human SARM1 is substituted with Glu or Asp.
Item 13. 13. Use of the SARM1 variant according to item 12 for the creation of model cells or animal models of neurodegenerative diseases.

The present invention detects the activation state of SARM1 and suppresses neuronal cell death by measuring the phosphorylation of at least one serine residue selected from the group consisting of 54th, 548th and 622nd positions of SARM1 and cell viability. It is a technology that can search for effective substances. It has been reported that SARM1 is a causative molecule of ischemic neuronal death and neuroaxonal degeneration. It can be applied to the treatment of various neurodegenerative diseases. Until now, there was no technology that could measure the intracellular activity state of SARM1, so it was not possible to develop drugs that target SARM1. The present invention enables screening of SARM1 inhibitors using SARM1 phosphorylation as an indicator. In the present invention, the ARM domain (28-405aa), SAM domain (406-550aa), TIR domain (551-724aa) contained in SARM1, or a fragment thereof containing the 54th, 548th or 622nd Ser residue was used for phosphorylation analysis, it became possible to detect phosphorylation of SARM1 serine 54th, 548th or 622nd with high sensitivity and simplicity. As a TIR domain, the core region (D594-E670 aa) containing S622, the BB-loop and DD-loop structures of TIR may be used (Fig. 10B).

In addition, we found JNK1, JNK2, and JNK3 that phosphorylate SARM1, and CDK19 and CDK8 that enhance SARM1 phosphorylation. Also, analyzing the phosphorylation state of at least one serine residue selected from the group consisting of 54th, 548th, and 622nd serines of SARM1 using neurons differentiated from iPS cells derived from neurodegenerative disease patients. Therefore, it can be applied to the evaluation of the progress of the disease.

Measurement of stress-induced cell death under SARM1 suppression conditions Cell death induction by SARM1 inhibition of mitochondrial respiration Phosphorylation of SARM1 at Serine 548 Phosphorylation and activity regulation of SARM1 by CDK19 and CDK8 Development of SARM1 inhibitors using phosphorylation as an index Phosphorylation of SARM1 by JNK Screening method for SARM1 phosphorylation inhibitors using SARM1-Ser ⁵⁴⁸ phosphorylation detection Phosphorylation of SARM1-Ser ⁵⁴ , Ser ⁵⁴⁸ , Ser ⁶²² Changes in binding proteins upon phosphorylation of SARM1 Screening method for SARM1-binding substances using SAM domain and TIR domain

As used herein, SARM1 is a 724-amino acid protein that localizes to mitochondria, and has a mitochondrial localization sequence, ARM, SAM, and TIR domains (Fig. 2A).

The amino acid sequence of human SARM1 is shown in SEQ ID NO:1. Human SARM1 has a pI of 6.1 and a molecular weight of 79,388.

Although SARM1 has a large number of phosphorylatable Thr and Ser residues, the present inventors found that Ser ⁵⁴ , Ser ⁵⁴⁸ and Ser ⁶²² , preferably Ser ⁵⁴⁸ and Ser ⁶²² , are phosphorylation sites. . At least one selected from the group consisting of Ser ⁵⁴ , Ser ⁵⁴⁸ and Ser ⁶²² , preferably Ser ⁵⁴⁸ and/or Ser ⁶²² is phosphorylated to activate SARM1, inhibit mitochondrial respiration, and induce cell death. Therefore, by inhibiting phosphorylation of at least one selected from the group consisting of Ser ⁵⁴ , Ser ⁵⁴⁸ and Ser ⁶²² , preferably Ser ⁵⁴⁸ and / or Ser ⁶²² , the inhibition of mitochondrial respiration is released and neuronal cell death can suppress the induction of Therefore, at least one selected from the group consisting of Ser ⁵⁴ , Ser ⁵⁴⁸ and Ser ⁶²² , preferably a phosphorylation inhibitor of Ser ⁵⁴⁸ and/or Ser ⁶²² is useful as a preventive or therapeutic agent for neurodegenerative diseases.

Neurodegenerative diseases include Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis and Alzheimer's disease, especially Parkinson's disease, amyotrophic lateral sclerosis (ALS).

The inventors have found that phosphorylation of SARM1 is associated with cyclin-dependent kinase 8 (CDK8) and/or cyclin-dependent kinase 19 (CDK19), as well as JNK (JNK-1, JNK-2, JNK-3, especially JNK-3). I found that it is done by By inhibiting one or both of CDK8 and CDK19 (preferably both) and JNK (JNK-1, JNK-2, JNK-3, especially JNK-3), SARM1 activation can be suppressed, resulting in neuronal It can suppress cell death and prevent or treat neurodegenerative diseases. Neurons do not regenerate after cell death even if one or both of CDK8 and CDK19 (preferably both) and JNK (JNK-1, JNK-2, JNK-3, especially JNK-3) are inhibited. and/or inhibitors of CDK19, as well as JNKs (JNK-1, JNK-2, JNK-3, especially JNK-3), are useful for preventing or halting or slowing the progression of neurodegenerative diseases. In the case of axonal degeneration, it can be regenerated with the preventive or therapeutic drug for neurodegenerative diseases of the present invention.

The phosphorylation motif of SARM1 by JNK is shown below.
Ser ⁵⁴ : REV S PGAG
Ser ⁵⁴⁸ : MLH S PLPC
Ser ⁶²² : LVL S PGAL
In addition, neurodegenerative diseases can be prevented or treated with a SARM1 inhibitor selected from the group consisting of anti-SARM1 antibodies, SARM1 decoy peptides and antagonists. SARM1 decoy peptides include fragment peptides containing ^Ser54 , ^Ser548 or ^Ser622 of SARM1 or modifications thereof. Modifications include acylated (acetylated, propionylated, etc.) and alkylated (methylated, ethylated, etc.) amino groups at the N-terminus, and esterified and amidated carboxyl groups at the C-terminus. mentioned. Such decoy peptides can be easily designed by those skilled in the art from the amino acid sequence of human SARM1 of SEQ ID NO:1. Antagonists of SARM1 include the SAM domain (molecular weight approximately 17,000), the ARM domain (molecular weight approximately 40,000), the TIR domain (molecular weight approximately 19,000), but may also be lower molecular weight antagonists. Decoy peptides are included in SARM1 antagonists.

An anti-SARM1 antibody that is effective for the prevention or treatment of neurodegenerative diseases is at least one selected from the group consisting of positions 54, 548 and 622 of SARM1, preferably Ser residues at positions 548 and/or 622. An antibody against non-phosphorylated SARM1 capable of suppressing phosphorylation or specifically recognizing at least one phosphorylated Ser residue selected from the group consisting of positions 54, 548 and 622 of SARM1, position 54 of SARM1, Examples include anti-phosphorylated SARM1 antibodies that do not recognize non-phosphorylated Ser residues at positions 548 and 622. Peptides that can be used to generate such antibodies include:
RGPREVSPGAGTEVQ (Peptides phosphorylated on the Ser(S) residue can be used to generate anti-phosphorylated SARM1 antibodies, and peptides without phosphorylation on the Ser(S) residue inhibit phosphorylation of Ser ⁵⁴ of SARM1. can be used to generate antibodies against suppressible non-phosphorylated SARM1),
AREMLHSPLPCTGGK (Peptides with phosphorylated Ser(S) residues can be used to generate anti-phosphorylated SARM1 antibodies, and peptides without phosphorylated Ser(S) residues inhibit phosphorylation of Ser ⁵⁴⁸ of SARM1.) can be used to generate antibodies against suppressible non-phosphorylated SARM1),
NFVLLVLSPGALDKCM (Peptides with phosphorylated Ser(S) residues can be used to generate anti-phosphorylated SARM1 antibodies, and peptides without phosphorylated Ser(S) residues inhibit phosphorylation of Ser ⁶²² of SARM1. can be used to generate antibodies against suppressible non-phosphorylated SARM1)
Peptides containing Ser ⁵⁴ , Ser ⁵⁴⁸ or Ser ⁶²² can be used, including but not limited to.

As used herein, "antibody against non-phosphorylated SARM1 capable of suppressing at least one phosphorylation selected from the group consisting of Ser ⁵⁴ , Ser ⁵⁴⁸ and Ser ⁶²² of SARM1" refers to an epitope containing Ser54, Ser548 or Ser622 may be an antibody that recognizes, recognizes an epitope near Ser54, Ser548 or Ser622, and can suppress phosphorylation of at least one selected from the group consisting of ^Ser54 , ^Ser548 and ^Ser622 by antibody binding It may be an antibody.

CDK8 and / or CDK19 inhibitors, for example, those described in WO2015 / 159937, US20120071477, US20120071477, WO2013 / 116786, JP-T-2015-506376, JP-T-2016-503408, which are currently known or discovered in the future All CDK8 and/or CDK19 inhibitors described herein are included in the present invention as prophylactic or therapeutic agents for neurodegenerative diseases or SARM1 phosphorylation inhibitors/activation inhibitors. In addition, the ARM domain, SAM domain, TIR domain of SARM1 or a fragment thereof containing Ser ⁵⁴ , Ser ⁵⁴⁸ or Ser ⁶²² can competitively inhibit the phosphorylation of SARM1. / useful as a prophylactic or therapeutic agent for neurodegenerative diseases.

Many inhibitors of JNKs (JNK-1, JNK-2, JNK-3, especially JNK-3) are known and currently known or discovered in the future JNKs (JNK-1, JNK-2, JNK-3). 3, especially JNK-3) inhibitors are included in the present invention as prophylactic or therapeutic agents for neurodegenerative diseases or SARM1 phosphorylation inhibitors/activation inhibitors.

By substituting Ser at position 54, 548 or 622 of SARM1 with Asp (D) or Glu (E), the effect of inducing neuronal cell death can be enhanced, and position 54, 548 or 622 of SARM1 Substitution of non-phosphorylated and non-acidic amino acids such as Ala for Ser at the position inhibits SARM1 activation, and reduces or eliminates the effect of SARM1 on inducing neuronal cell death. Asp ⁵⁴ or Glu ⁵⁴ variant, Asp ⁵⁴⁸ or Glu ⁵⁴⁸ variant, Asp ⁶²² or Glu ⁶²² variant of SARM1 can enhance the neuronal cell death-inducing action of SARM1, positions 54, 548 and 622 A SARM1 variant in which at least one Ser selected from the group consisting of Asp (D) or Glu (E) has been altered is useful for preparing model animals or model cells of neurodegenerative diseases. For example, wild-type SARM1 gene is replaced (knocked in) with Asp ⁵⁴ or Glu ⁵⁴ variant gene, Asp ⁵⁴⁸ or Glu ⁵⁴⁸ variant gene, Asp ⁶²² or Glu ⁶²² variant gene (knock-in), or Asp ⁵⁴ or Transgenic non-human mammals/cells into which the Glu ⁵⁴ variant gene, Asp ⁵⁴⁸ or Glu ⁵⁴⁸ variant gene, Asp ⁶²² or Glu ⁶²² variant gene has been introduced are model animals/model cells of neurodegenerative diseases (cells are human). including). Two or more of positions 54, 548 and 622 may be modified.

Mammals include humans, mice, rats, guinea pigs, hamsters, rabbits, goats, dogs, cats, monkeys, cows, pigs, and the like. The nucleotide and amino acid sequences of SARM1 of these mammals are known, or the gene can be easily obtained from the known nucleotide sequence of SARM1 and the amino acid sequence determined.

The anti-phosphorylated SARM1 antibody of the present invention is at least one phosphorylated SARM1 selected from the group consisting of Ser ⁵⁴ , Ser ⁵⁴⁸ and Ser ⁶²² , or a fragment thereof containing phosphorylated Ser ⁵⁴ and/or Ser ⁵⁴⁸ and/or Ser ⁶²² is a monoclonal antibody or polyclonal antibody produced from a hybridoma obtained by immunizing a mouse, preferably a monoclonal antibody. Hereinafter, "Ser ⁵⁴ and/or Ser ⁵⁴⁸ and/or Ser ⁶²² " may be abbreviated as "Ser ⁵⁴ /Ser ⁵⁴⁸ /Ser ⁶²² ".

After immunizing mammals such as mice with Ser ⁵⁴ /Ser ⁵⁴⁸ /Ser ⁶²² phosphorylated SARM1 or fragments thereof containing phosphorylated Ser ⁵⁴ /Ser ⁵⁴⁸ /Ser ⁶²² , several hybridomas are obtained. The antibodies required therein are hybridomas that produce antibodies that react with Ser ⁵⁴ /Ser ⁵⁴⁸ /Ser ⁶²² phosphorylated SARM1 but do not react with non-phosphorylated SARM1.

The anti-non-phosphorylated SARM1 antibody of the present invention is obtained from a hybridoma obtained by immunizing a mouse with ^Ser54 / ^Ser548 / ^Ser622 non-phosphorylated SARM1 or a fragment thereof containing non-phosphorylated ^Ser54 / ^Ser548 / ^Ser622 . Monoclonal or polyclonal antibodies are produced, with monoclonal antibodies being preferred. After immunizing a mammal such as a mouse with Ser ⁵⁴ /Ser ⁵⁴⁸ /Ser ⁶²² non-phosphorylated SARM1 or a fragment thereof containing non-phosphorylated Ser ⁵⁴ /Ser ⁵⁴⁸ /Ser ⁶²² , several hybridomas are obtained. The antibodies required therein are hybridomas that produce antibodies that react with Ser ⁵⁴ /Ser ⁵⁴⁸ /Ser ⁶²² non-phosphorylated SARM1 but not with Ser ⁵⁴ /Ser ⁵⁴⁸ /Ser ⁶²² phosphorylated SARM1.

Mammals used for immunization include mice, rats, rabbits, goats and the like, with mice being particularly preferred. In order to obtain the antibody of the present invention, a hybridoma is incubated with ^Ser54 / ^Ser548 / ^Ser622 phosphorylated/unphosphorylated SARM1 or a fragment thereof containing phosphorylated/unphosphorylated ^Ser54 / ^Ser548 / ^Ser622 as an immunogen. After production, hybridomas producing antibodies specifically reactive with Ser ⁵⁴ /Ser ⁵⁴⁸ /Ser ⁶²² phosphorylated/unphosphorylated SARM1 must be selected. Immunity can be induced by administering 1 ng to 10 mg of the immunogen in 1 to 5 divided doses at intervals of 10 to 14 days. After sufficient immunization, organs having antibody-producing ability (spleen and lymph nodes) are aseptically removed from the mammal and used as a parent strain for cell fusion. The spleen is the most preferable organ to be removed. Myeloma cells are used as cell fusion partners. Myeloma cells are derived from mice, rats, humans, and the like, and are preferably derived from mice. Cell fusion includes a method using polyethylene glycol, a cell electrofusion method, and the like. Hybridomas can be selected from unfused spleen cells and myeloma cells by, for example, culturing in a serum medium supplemented with HAT supplements.

Selection of hybridomas producing antibodies that specifically bind to Ser ⁵⁴ /Ser ⁵⁴⁸ /Ser ⁶²² phosphorylated/unphosphorylated SARM1 was performed by collecting the aforementioned culture supernatants and quantifying Ser ⁵⁴ /Ser ⁵⁴⁸ /Ser ⁶²² phosphorylated/ Direct ELISA on an EIA plate on which non-phosphorylated SARM1 or its fragment containing phosphorylated/non-phosphorylated Ser ⁵⁴ /Ser ⁵⁴⁸ /Ser ⁶²² and non-phosphorylated / phosphorylated SARM1 are immobilized is preferred. Direct ELISA results showed intense staining with Ser ⁵⁴ /Ser ⁵⁴⁸ /Ser ⁶²² phosphorylated/unphosphorylated SARM1 or its fragments containing phosphorylated/unphosphorylated Ser ⁵⁴ /Ser ⁵⁴⁸ /Ser ⁶²² and non-phosphorylated/phosphorylated A well that does not develop color with SARM1 is selected, and the cells in that well are subjected to cloning. Hybridomas corresponding to the culture supernatant are selected as hybridomas producing antibodies that specifically react with Ser ⁵⁴ /Ser ⁵⁴⁸ /Ser ⁶²² phosphorylated/unphosphorylated SARM1. Cloning is the work of selecting and singulating antibody-producing hybridomas, and includes the limiting dilution method, fibrin gel method, and method using a cell sorter, among which the limiting dilution method is preferred. Thus, hybridomas that produce the desired monoclonal antibody can be obtained. A monoclonal antibody can be obtained in the culture supernatant by culturing the hybridoma obtained by the above method.

Specifically, as a screening method for preventive or therapeutic drugs for neurodegenerative diseases, neurons are induced from iPS cells derived from healthy subjects and patients with neurodegenerative diseases, candidate drugs or solvent control groups are added, and then phosphorylated SARM1 It can be performed by evaluating SARM1 phosphorylation and neuronal viability by immunostaining using antibodies, Western blotting, and MTS assay. Alternatively, a candidate drug or vehicle control group was administered to neurodegenerative disease model mice, then brain cryosections were prepared, and histological staining using phosphorylated SARM1 antibody and Tyrosine hydroxylase antibody (for Parkinson's disease model) revealed SARM1 phosphorylation. Evaluation of oxidative level and neuronal viability can be performed to screen preventive or therapeutic drugs for neurodegenerative diseases.

As cells expressing SARM1, SARM1 tagged with a tag such as HA tag, His tag, or FLAG tag, and ARM domain, SAM domain, TIR domain, or Ser ⁵⁴ , Ser ⁵⁴⁸ , or Ser ⁶²² tagged with another tag. More preferred are cells co-expressing fragments thereof containing. A candidate substance is added to the culture medium of such cells to perform cell culture, immunoprecipitation of tagged SARM1 is performed using antibody-binding beads against the SARM1 tag, and whether phosphorylation is detected by the antibody of the present invention or It can be detected using an antibody such as a phosphorylated serine/threonine detection antibody. In the screening method of the present invention, a substance that suppresses SARM1 phosphorylation can be selected as a target substance.

The present invention will now be described in more detail based on examples.

Example 1
(1) Suppression of neuronal cell death by suppression of SARM1 expression
In order to confirm whether SARM1 is involved in the induction of neuronal cell death, an expression suppression experiment using siRNA was performed. Seed 3 x 10 ⁵ human neuroblastoma SH-SY5Y cells in a 12-well plate, mix 4 µl of 10 µM siRNA against control or SARM1 (QIAGEN) with 8 µl of transfection reagent Lipofectamine RNAiMAX (Thermo Fisher Scientific), Transfection was performed at a concentration of 20 nM. After 48 hours, 40 μM 6-hydroxydopamine (6-OHDA), 1 mM paraquat, and 1 μM rotenone, which are used as oxidative stress reagents to induce Parkinson's disease-like symptoms, were added. After staining, the cell death induction rate was measured (Fig. 1). Compared to the control group, suppression of SARM1 expression significantly suppressed cell death. This result indicates that stress-induced neuronal cell death may be suppressed by inhibiting SARM1 expression and function.

(2) Induction of cell death by mitochondrial respiration inhibition by SARM1 Next, we investigated the cell death induction mechanism of SARM1. Since SARM1 is a protein with a mitochondrial localization signal, we focused on mitochondrial function. As a result, we found that SARM1 inhibits mitochondrial respiration and decreases ATP synthesis. SARM1 is a 724-amino acid protein containing a mitochondrial translocation signal, an ARM domain, two SAM domains, and a TIR domain (Fig. 2A). To analyze the function of each domain, 3 × 10 ⁵ HEK293T cells were seeded in 12 well plates, and full-length SARM1 (1-724 aa), ΔN (28-724 aa) without mitochondrial translocation signal, and TIR domain without 2 μg of pDNA encoding ΔT (1-550 aa) and ΔA (406-724 aa) excluding the N-terminal side containing the ARM domain was mixed with 4 μl of transfection reagent FuGENE-HD (Promega) for transfection. . After 8 hours, the cells were detached and replated at 3×10 ⁴ cells in a 96-well plate. 48 hours after transfection, cell viability and ATP amount were measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay reagent (Promega) (Fig. 2B) and CellTiter-Glo Assay reagent (Promega) (Fig. 2C). SARM1 decreased cell viability compared to controls. This effect was somewhat attenuated when the mitochondrial translocation signal was removed (ΔN), completely abolished by ΔT, and enhanced by ΔA. Correlating with this value, the amount of cellular ATP also changed (Fig. 2C). These results suggest that the TIR domain is important for cell death induction and inhibition of ATP synthesis, and that the region containing the ARM domain is important for suppressing the activity of SARM1. Under similar transfection conditions, mitochondrial respiratory capacity (Fig. 2D) was measured using a flux analyzer XFe96 (SeahorseBioscience) and H2, _one of the reactive oxygen species, was measured using a ROS _- Glo H2O2 assay ₍ Promega). The amount of _O2 (Fig. 2E) was measured. Correlating with ATP levels, full-length SARM1 and ΔA decreased mitochondrial respiratory capacity, particularly respiratory reserve, while ΔT increased. The amount of H ₂ O ₂ was also increased by expression of full-length SARM1 and ΔA (Fig. 2E). These results indicate that SARM1 acts on the mitochondrial respiratory chain complex to inhibit mitochondrial respiration, decrease ATP synthesis, and induce cell death by inducing reactive oxygen species generation.

(3) Detection of Phosphorylation of Serine 548 of SARM1 Next, we analyzed how the activity of SARM1 was regulated. In the analysis, we found that SARM1 is phosphorylated under the condition that forced expression of SARM1 inhibits ATP synthesis. Control or SARM1-Flag (pDNA 4 μg + FuGENE-HD 8 μl) was transfected into HEK293T cells (8×10 ⁵ cells/6-well plate), and immunoprecipitation was performed 24 hours later using Flag antibody-conjugated beads. rice field. After that, Western blotting was performed using a phosphorylated serine/threonine (P-Ser/Thr) detection antibody (BD Bioscience) and a Flag antibody. A band appeared (Fig. 3A). In order to confirm which part of SARM1 is phosphorylated at serine and threonine, SARM1 was divided into three regions (ARM: 1-405aa, SAM: 406-550aa, TIR: 551-724aa) and shown in Fig. 3A. Phosphorylation was detected by the same method as described above. The SAM domain was phosphorylated in three regions as shown in Figure 3B. The SAM domain (406-550 aa) contains 12 serines and 9 threonines. To identify which of these amino acids are phosphorylated, all 21 amino acids were replaced one by one with alanine and compared with the wild-type (WT) SAM domain. Of the 21 amino acids, the band detected with the P-Ser/Thr antibody disappeared only when serine 548 was replaced with alanine (Fig. 3C). These results revealed that serine 548 is the phosphorylation site of SARM1.

(4) Increased phosphorylation of SARM1 by CDK19 and CDK8
Mass spectrometry experiments were performed to identify the kinases that regulate SARM1 phosphorylation. Control or SAM (406-550aa)-Flag (pDNA 4 μg + FuGENE-HD 8 μl) was transfected into HEK293T cells (8 × 10 ⁵ cells/6-well plate), and after 24 hours the cells were transfected using Flag antibody-conjugated beads. immunoprecipitation was performed. A band co-precipitated with SAM-Flag was detected by silver staining (Fig. 4A), and the binding protein was analyzed by mass spectrometry. As a result, we identified Cyclin dependent kinase 19 (CDK19) as a protein that binds to the SAM domain. CDK19 is one of the CDK family proteins, and it is known that CDK8 exists as a paralog with similar functions. HA-tagged CDK19 and CDK8 (HA-CDK19, HA-CDK8) expression vectors were prepared to confirm binding and phosphorylation control between the SAM domain and CDK19 and CDK8. Control, SAM-Flag, HA-CDK19 and HA-CDK8 (4 μg pDNA + 8 μl FuGENE-HD) were transfected into HEK293T cells (8×10 ⁵ cells/6-well plate), and after 24 hours, Flag antibody binding was observed. Immunoprecipitation was performed using beads. As shown in Figure 4B, CDK19 and CDK8 bound to the SAM domain and significantly enhanced the phosphorylation of the SAM domain. These results revealed that CDK19 and CDK8 enhance the phosphorylation of serine 548 of SARM1. Next, the cell survival rate when CDK19 or CDK8 was co-expressed with SARM1 was measured by MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay reagent). Forced expression of CDK19 and CDK8 reduced cell viability, and co-expression with SARM1 further reduced cell viability (Fig. 4C). These results indicate that phosphorylation of SARM1 serine 548 by CDK19 and CDK8 is important for SARM1 activation and cell death induction.

(5) Development of a SARM1 Inhibitor Using Phosphorylation as an Indicator From the results in FIG. 4, it was found that cell death may be suppressed by inhibiting the phosphorylation of SARM1 at serine 548. Therefore, we examined whether it is possible to develop a SARM1 inhibitor using the phosphorylation state of SARM1 as an index. As shown in Figure 3, the SAM domain (406-550 aa) of SARM1 is strongly phosphorylated in cells, suggesting that it functions as an antagonist that inhibits phosphorylation of full-length SARM1 in cells. Control, HA-tagged SARM1 (SARM1-HA), SARM1-HA and SAM-Flag co-expressed in HEK293T cells (8 x 10 ⁵ cells/6-well plate) (pDNA 4 μg + FuGENE-HD 8 μl ), and 24 hours later, SARM1-HA was immunoprecipitated using HA antibody-bound beads (SIGMA). As shown in FIG. 5A, phosphorylation of SARM1 was detected in the case of SARM1-HA alone, but no phosphorylation was detected in the case of co-expression of SAM-Flag. Furthermore, when cell viability was measured using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay reagent under similar transfection conditions, the rate of cell death induced by forced expression of SARM1 was reduced by co-expression of the SAM domain ( Figure 5B). Next, in order to inhibit CDK19/CDK8 phosphorylation of SARM1, the phosphorylation was examined using the CDK19/CDK8 inhibitor Senexin A (TOCRIS). HEK293T cells (8×10 ⁵ cells/6-well plate) were transfected with control or SAM-Flag (4 μg pDNA + 8 μl FuGENE-HD), and 0-10 μM Senexin A was added 8 hours later. Immunoprecipitation was performed 24 hours after transfection using Flag antibody-conjugated beads. Senexin A suppressed phosphorylation of the SAM domain in a dose-dependent manner (Fig. 5C). Next, we examined the effect of Senexin A on oxidative stress-induced cell death. 3×10 ⁴ neuroblastoma SH-SY5Y cells were plated in 96-well plates and 0-20 μM Senexin A and 1 mM paraquat were added. After 48 hours, cell viability was measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay reagent. As shown in FIG. 5D, Senexin A suppressed cell death induced by oxidative stress in a concentration-dependent manner. These results indicate that SARM1-induced cell death can be suppressed by developing a substance that inhibits SARM1 phosphorylation.

(6) Induction of SARM1 Serine 548 Phosphorylation by JNK As shown in FIG. 4, forced expression of CDK19 and CDK8 enhanced phosphorylation of the SAM domain. In vitro kinase assay was performed to confirm whether CDK19 and CDK8 directly induce phosphorylation of SAM domain. However, when the SAM domain was reacted with CDK19 or CDK8 in the presence of ATP, phosphorylation of the SAM domain could not be confirmed. Therefore, we investigated using kinase inhibitors to find other kinases that directly phosphorylate SARM1. HEK293T cells (3 × 10 ⁵ cells/12-well plate) were transfected with a SAM-Flag expression vector (2 µg of pDNA + 4 µl of FuGENE-HD), and 8 hours later, various kinase inhibitors (EGFR-I, MEK- I, p38-I, JNK-I, Akt-I) were added and cultured for 16 hours, after which the cells were collected and subjected to Western blotting. A JNK inhibitor (JNK-I: JNK inhibitor VIII (Cayman)) suppressed the phosphorylation of the SAM domain in the kinase inhibitor screening (Fig. 6A). Next, to confirm the direct phosphorylation of the SAM domain by JNK, an in vitro kinase assay was performed by reacting the GST-tagged SAM domain with kinases (p38α, JNK1, JNK2, JNK3) in the presence of ATP. As shown in FIG. 6B, it was confirmed that JNK (particularly JNK3) directly induces phosphorylation of serine 548 of the SAM domain.

An antibody was prepared to specifically detect the phosphorylation of SARM1 at serine 548. Mice were immunized with a phosphorylated peptide of AREMLHSPLPCTGGK containing serine 548 to obtain hybridomas. The reactivity of the monoclonal antibodies produced from the hybridomas was confirmed by ELISA, and clones that reacted with phosphorylated peptides but had low reactivity with non-phosphorylated peptides were selected (Fig. 6C), and the antibodies were purified. The acquired SARM1 serine 548 phosphorylation-recognizing antibody specifically recognized phosphorylation of full-length SARM1. Western blotting using this antibody detected phosphorylation of forcedly expressed SARM1, which was further enhanced by co-expression of JNK1-3 (Fig. 6D). Phosphorylation of endogenous SARM1 at serine 548 was also detectable. SH-SY5Y cells were subjected to various stress stimuli and cultured for 24 hours, and cell extracts were collected. Cell death-inducing stress stimuli (paraquat, 6-OHDA, rotenone treatment) increased phosphorylation of serine 548 of SARM1 (Fig. 6E). These results suggest that JNK is responsible for the direct phosphorylation of SARM1 serine 548, and under stress conditions that induce cell death, JNK is activated, phosphorylates SARM1, and contributes to the induction of neuronal cell death. thought to have contributed.

(7) Screening Method for SARM1 Phosphorylation Inhibitor Using SARM1-Ser548 Phosphorylation Detection As shown in FIG. 6, SARM1 was phosphorylated under a stress environment and induced cell death. Therefore, if we find a substance that inhibits SARM1 phosphorylation, it may be possible to suppress neuronal cell death induced by SARM1. Therefore, we presented a screening method for SARM1 phosphorylation inhibitors using SARM1-Ser548 phosphorylation detection. 1 μg of GST-SAM was added to a buffer of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl ₂ , 1.8 mM ATP, and 1 mM DTT, and 100 ng of JNK3, JNK inhibitors, decoy peptides, and antibodies were added. etc. were added and reacted at 37°C for 1 hour. The JNK inhibitor is JNK inhibitor VIII (Cayman), the decoy peptide is a peptide with the sequence AREMLHSPLPCTGGK containing serine 548, the mouse control IgG (mIgG) is normal mouse IgG (Wako), and the SARM1 antibody is a peptide containing serine 548. Antibodies (Nos. 1, 2, 16, 18, 30 and 33) purified from hybridomas obtained from immunizing mice were used. Then 3x SDS sample buffer was added to stop the reaction, and the level of SARM1 phosphorylation was assessed by Western blotting. The JNK inhibitor (Fig. 7A), decoy peptide (Fig. 7B), and antibody (Fig. 7C) each had an effect of suppressing SARM1-Ser548 phosphorylation. In particular, the JNK inhibitor (JNK-I) and SARM1 antibodies (No. 1, 2, 16) had a strong inhibitory effect on phosphorylation, and these substances suppressed cell death induced by SARM1. HEK293T cells were transfected with SARM1 pDNA, and 8 hours later, JNK inhibitors and antibodies were added and cultured for 16 hours. The JNK inhibitor and antibody used were the same as those used for the phosphorylation analysis. The antibody was introduced by an electroporation method (Neon transfection system, Thermo Fisher Scientific). Cell viability was then assessed by MTS assay. JNK inhibitors and SARM1 antibodies have cell death inhibitory effects (Fig. 7D, E), and by combining assays for detecting SARM1 phosphorylation and evaluating cell viability, it is possible to screen for substances that inhibit SARM1 activation. indicates that there is

(8) Induction of SARM1 Serine 54, Serine 548, and Serine 622 by JNK
JNK induced phosphorylation of SARM1 serine 548, and inhibition of JNK decreased phosphorylation of SARM1 serine 548 and suppressed cell death induction. In order to confirm the effect of phosphorylation of SARM1 serine 548 on SARM1 activity, we generated a SARM1 mutant expression vector (S548A) in which serine 548 was replaced with alanine and compared it with wild-type SARM1 by MTS assay. went. As a result, it was found that the activity of S548A was decreased compared with wild-type SARM1, but the difference was not so large (Fig. 7A). Considering the possibility that other serines and threonines of SARM1 may also be phosphorylated by JNK, we investigated the entire sequence of SARM1 and found that SARM1 has three motifs that may be phosphorylated by JNK (Ser ⁵⁴ : REV S PGAG, Ser ⁵⁴⁸ : MLH S PLPC, Ser ⁶²² : LVL S PGAL). Therefore, we prepared mutant expression vectors in which serine 54, serine 548, and serine 622 were substituted with alanine at one site or at multiple sites, and compared with wild-type SARM1 by MTS assay. As a result, S622A decreased SARM1 activity the most, followed by S548A and S54A in that order, and when all three sites were mutated, the cell death-inducing activity of SARM1 almost disappeared (Fig. 8A). Phosphorylation of serine 54 and serine 622 could not be detected by the method using the phosphorylated serine/threonine recognition antibody shown in Fig. 3, so detection by another method using Phos-tag (Wako) was performed. tried. Phos-tag is a substance that has the property of binding to a phosphate group, and by mixing it in an SDS-PAGE gel, phosphorylated proteins migrate slower than non-phosphorylated proteins, making it possible to detect phosphorylated proteins. A comparison of SARM1 mutants was performed using this method. As shown in the upper photograph of FIG. 8B, a single SARM1 band is detected by normal SDS-PAGE gel detection. However, as shown in the lower photograph of Fig. 8B, in the SDS-PAGE gel containing Phos-tag, a fast-migrating band indicated by a black arrow and a slow-migrating band indicated by a white arrow appeared, suggesting that SARM1 is phosphorylated. was confirmed (white arrow). Furthermore, the band pattern of mutants in which not only Serine 548 but also Serine 54 and Serine 622 were substituted with alanine changed compared to wild-type SARM1, suggesting that these serines are also phosphorylated. rice field. However, phosphorylation bands were also detected in the mutant in which serines 54, 548, and 622 were all replaced with alanines, suggesting that SARM1 contains amino acids that undergo phosphorylation in addition to these serines. It is suggested. To further clarify that serines 54, 548, and 622 are phosphorylated by JNK, SARM1 was expressed domain by domain, co-expressed with JNK1-3, and Phos-tag was used. detected. As shown in Fig. 8C, it was confirmed that co-expression of JNK (especially JNK3) increased phosphorylated bands in the ARM domain containing serine 54, the SAM domain containing serine 548, and the TIR domain containing serine 622. was done. These results suggest that JNK induces phosphorylation of SARM1 at Serine 54, Serine 548, and Serine 622.

(9) Changes in binding proteins by phosphorylation of SARM1
To clarify the significance of SARM1 phosphorylation, we compared binding proteins of SARM1 mutants. HEK293T cells (8×10 ⁵ /6-well plate) were transfected with control, wild-type SARM1-Flag or mutant SARM1-Flag (4 μg pDNA + 8 μl FuGENE-HD). After 24 hours, prepare a cell extract with a cell lysate containing 1% Triton-X100, perform immunoprecipitation with 20 μl of Flag antibody-conjugated beads (SIGMA) for 2 hours, and extract with a 0.1 M glycine solution (pH 2.5). Immunoprecipitates were collected. After mixing 1 M Tris solution (pH 9) and SDS solution, the mixture was developed by SDS-PAGE. A band co-precipitated with SARM1-Flag was detected with a silver staining kit (Wako) (Fig. 9A), and the binding protein was analyzed with a mass spectrometer. As a result, full-length SARM1 including wild-type and point mutations was confirmed to bind to ATP5B, ATP5C1, and ATP5O subunits of complex V of mitochondrial respiratory chain complex, and the binding was reduced by S548A mutation. On the other hand, in the ΔTIR, which has no SARM1 activity, and the S622A mutant, which has almost no SARM1 activity, a band with increased binding to SARM1 was detected at around 70 kDa, and this band was Heat shock protein 70 (HSP70). This binding was also confirmed by Western blotting after immunoprecipitation (Fig. 9B). These results suggest that SARM1 inhibits ATP synthesis through interaction with complex V (Fig. 2C), and that HSP70 suppresses its action. Furthermore, phosphorylation at serine 622 is important for dissociation with HSP70, and phosphorylation at serine 548 is considered to contribute to complex V binding.

Assuming that SARM1 binds to complex V and inhibits ATP synthesis, leading to cell death induction, we introduced an excessive amount of complex V subunits to function as decoys, thereby suppressing cell death induced by SARM1. It is speculated that it can be suppressed. HEK293T cells (3 × 10 ⁵ /12-well plate) were co-expressed with SARM1-Flag and control, ATP5B, ATP5O pDNA (pDNA 4 μg + FuGENE-HD 8 μl), and cell viability was measured by MTS assay after 24 hours. was analyzed. Co-expression of ATP5B and ATP5O with SARM1 significantly suppressed SARM1-induced cell death (Fig. 9C).These results suggest that SARM1 binds to complex V and inhibits ATP synthesis, leading to SARM1-induced cell death. Therefore, it is possible to suppress the function of SARM1 by finding a substance that inhibits the phosphorylation of SARM1, which is important for binding.

(10) Screening method for SARM1-binding substances using SAM domain and TIR domain From the results in Fig. 9, compounds that suppress the phosphorylation of serine 548, which is important for binding to complex V, and serine necessary for dissociation with HSP70. A compound that suppresses the phosphorylation of 622 is assumed to be a compound that inhibits the activation of SARM1 and is effective in suppressing neuronal cell death. This led to the development of a method for screening these substances.

The SAM domain was cloned into an expression vector pET28a with a T7 promoter (pET28a-SAM-Flag (6His-T7-SAM-Flag)) and transformed into E. coli BL21(DE3)-RP. (Kanamycin 30 μg/ml) cultured at 37° C. for 10 hours with shaking, then IPTG was added to a final concentration of 1 mM, followed by shaking culture at 25° C. for 16 hours to express the SAM domain. A lysate of E. coli was obtained by ultrasonication in a 20% by mass sucrose buffer. After centrifuging the crushed material and adding the supernatant to a cobalt column, PBS-T (Phsophate Buffered Saline with Tween 20) + 10mM imidazole was used as a washing solution, and PBS + 500mM imidazole was used as an eluent for purification, followed by dialysis against PBS. The SAM domain was purified by performing (Fig. 10A).

A partial region of the TIR domain (D594 to E670 aa) was cloned into an expression vector pGEX-6P-1 with a tac promoter (pGEX-6P-1-TIR (GST-TIR)) and transformed into E. coli BL21(DE3)-RP. The glycerol stock prepared by transformation was shake-cultured in LB-Amp (ampicillin 100 µg/ml) medium at 37°C for 10 hours, then IPTG was added to a final concentration of 1 mM and incubated at 25°C for 16 hours. Shaking culture was used to express the TIR domain. A lysate of E. coli was obtained by ultrasonication in a 20% by mass sucrose buffer. This crushed product was centrifuged, the supernatant was added to a Sephadex-4B column (GE Healthcare), PBS-T (Phsophate Buffered Saline with Tween 20) was used as a washing solution, and 50 mM Tris-HCl (pH 8) + 10 mM The TIR domain was purified by purifying reduced glutathione as an eluent and dialysis against PBS (Fig. 10B).

A Biacore sensor chip was constructed for molecular interaction analysis using purified SAM domains. 0.4 mg/ml of the SAM domain dissolved in PBS was diluted 4-fold (0.1 mg/ml) with 10 mM acetate buffer (pH 4.5) and immobilized on the CM5 sensor chip by the amine coupling method. HBS-EP buffer (GE Healthcare) was used as the running buffer to measure the interaction with the analyte, and the analyte was diluted to 20 μg/ml with the running buffer. In order to confirm the molecular interaction with the SAM domain, SARM1 antibody and SAM domain were added and analyzed (Fig. 10C). Since SAM domains undergo dimer formation, binding between SAM domains was confirmed by adding SAM domains. Antibodies that recognize the vicinity of serine 548 of some SAM domains have been confirmed to bind more strongly than the binding of the SAM domain, and these antibodies can inhibit SAM domain dimer formation and inhibit SARM1 function. It was suggested. On the other hand, control IgG and antibodies recognizing other regions of SARM1 did not bind to the SAM domain. These results indicate that molecular interaction analysis by Biacore can be used to select compounds that bind to the domain of SARM1.

Currently, there are no drugs that can prevent or treat the progression of neurodegenerative diseases (Parkinson's disease, ALS, etc.).

SARM1 is one of the molecules responsible for neuronal cell death and neuroaxonal degeneration observed in neurodegenerative diseases.

If the function of SARM1 can be inhibited, it may become possible to prevent and treat various neurodegenerative diseases.

However, since the intracellular activation mechanism of SARM1 was unclear, there were no currently available inhibitors of SARM1.

The present inventors have found that phosphorylation of Serine 54, Serine 548, and Serine 622 of SARM1 by JNK is necessary for activation of SARM1.

The present inventors developed a screening method for drugs that inhibit SARM1 activation by combining detection of SARM1 phosphorylation and measurement of cell viability.

By using the present invention, it becomes possible to develop SARM1 inhibitors that are promising for the prevention and treatment of neurodegenerative diseases.

Claims (8)

An anti-phosphorylated SARM1 antibody that specifically recognizes the phosphorylated Ser residue at position 548 of SARM1 (sterile alpha and TIR motif-containing protein 1) but does not recognize the non-phosphorylated Ser residue at position 548 of SARM1. An antibody against non-phosphorylated SARM1 that can suppress phosphorylation at position 548 of SARM1 (Sterile alpha and TIR motif-containing protein 1) or an antibody that specifically recognizes the phosphorylated Ser residue at position 548 of SARM1 A prophylactic or therapeutic drug for neurodegenerative diseases, comprising an anti-phosphorylated SARM1 antibody that does not recognize the non-phosphorylated Ser residue of SARM1 as an active ingredient. A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2, or a peptide containing deletions, substitutions, or additions of several amino acid residues of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 2, wherein SARM1 (Sterile alpha and TIR motif-containing protein 1) - A prophylactic or therapeutic agent for neurodegenerative diseases, containing a peptide having an effect of suppressing Ser548 phosphorylation as an active ingredient. Prevention of neurodegenerative diseases comprising detecting phosphorylation of at least one Ser residue selected from the group consisting of positions 54, 548 and 622 of SARM1 (Sterile alpha and TIR motif-containing protein 1) or Methods of Screening for Therapeutic Agents. A method for screening a SARM1 inhibitor, comprising detecting phosphorylation of at least one Ser residue selected from the group consisting of positions 54, 548 and 622 of SARM1 (Sterile alpha and TIR motif-containing protein 1) . A SARM1 variant in which Ser corresponding to at least one selected from the group consisting of positions 54, 548 and 622 of human SARM1 (Sterile alpha and TIR motif-containing protein 1) is replaced with Ala, Glu or Asp. Use of the SARM1 (Sterile alpha and TIR motif-containing protein 1) variant according to claim 6 for the creation of non-human model cells or non-human model animals of neurodegenerative diseases. 4. The preventive or therapeutic drug for neurodegenerative diseases according to claim 2 or 3, wherein the neurodegenerative diseases are selected from the group consisting of Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, neuropathy and Alzheimer's disease.

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