JP2018043934A - Prophylactic or therapeutic agent for neurological diseases that develop with abnormal aggregation of neurodegenerative disease protein, and screening method for the prophylactic or therapeutic agent - Google Patents

Abstract

A prophylactic or therapeutic agent for a neurological disease (for example, Alzheimer type dementia, FTLD, HD, etc.) that develops with abnormal aggregation of a neurodegenerative disease protein, and a method for screening the prophylactic or therapeutic agent. . A preventive or therapeutic agent for a neurological disease that develops with abnormal aggregation of a neurodegenerative disease protein, comprising hepta-histidine, angiotensin III, luteinizing hormone-releasing hormone peptide fragment, hepta-glutamine, kemptide acetate, A compound having an amino acid sequence of 80% or more identity with those compounds and having affinity for a protein that directly interacts with the neurodegenerative disease protein, and nordihydroguaiaretic acid ester, sphingosine-1-phosphate, And a prophylactic or therapeutic agent comprising, as an active ingredient, at least one compound selected from the group consisting of those compounds and chemically modified compounds thereof. [Selection] Figure 1

Description

  The present invention relates to a preventive or therapeutic agent for a neurological disease that develops with abnormal aggregation of neurodegenerative disease protein, and a screening method for the prophylactic or therapeutic agent.

  Accumulation of misfolded (abnormally folded) amyloid β and abnormal aggregation of tau protein are observed from the brains of patients with Alzheimer's dementia and frontotemporal lobar degeneration (FTLD). Huntington's disease (hereinafter also referred to as HD) is also known as a disease (polyglutamine disease) that develops with abnormal aggregation of misfolded polyglutamine proteins.

HD is a disease caused by mutation of a huntingtin (hereinafter also referred to as Htt) protein having 3145 amino acid residues (hereinafter also referred to as mutant Htt). The Htt gene encoding the Htt protein is located on human chromosome 4 short arm 4p16.3. HD is an autosomal dominant disorder involving CAG (glutamine-encoding) repeat extension in exon 1 of the Htt gene.
A mutant Htt protein having a prolonged amino terminal glutamine is produced from a mutant Htt gene with increased CAG repeats, and such mutant Htt protein is particularly susceptible to abnormal aggregation.
As a model for HD disease, it is generally accepted that some features of R6 / 2 mouse pathology mimic human HD. For example, R6 / 2 transgenic mice that express exon 1-Q120 ± 5 of Htt have a long history as HD disease models (for example, Non-Patent Document 1).
Ku70 is known to be a protein that directly interacts with the Htt protein (eg, Non-Patent Document 2), and transgenic overexpression of Ku70 is one of the substances that most prolong the life span of the R6 / 2 mouse HD model. It is known that there is (Non-Patent Document 2).

Mangialini, L.M. et al. Exon1 of the HDgene with an expanded CAG repeatis sufficiency to cause a progressive neurophenotype in transgenic. Cell 87, 493-506 (1996). Enokido, Y. et al. et al. Mutant huntingtin impairs Ku70-mediated DNA repair. J Cell Biol 189, 425-443 (2010).

  In view of the above prior art, the present inventor has developed mutations from the viewpoint of developing preventive or therapeutic agents for neurological diseases (eg, Alzheimer type dementia, FTLD, HD, etc.) that develop with abnormal aggregation of neurodegenerative disease proteins. As a result of screening a compound that inhibits the interaction between Ku70, a very important DNA damage repair protein in a neuronal cell whose function can be impaired by Htt, and mutant Htt, it develops with abnormal aggregation of neurodegenerative disease protein The present inventors have found candidate compounds for preventive or therapeutic agents for neurological diseases.

  That is, the present invention relates to a prophylactic or therapeutic agent for neurological diseases (eg, Alzheimer's dementia, FTLD, HD, etc.) that develop with abnormal aggregation of neurodegenerative disease proteins, and a method for screening the prophylactic or therapeutic agent. For the purpose of provision.

As a result of intensive studies on the above object, the present inventor has shown that specific compounds such as heptahistidine change the aggregation of mutant Htt peptide fragments and change the kinetics of aggregation of R3 isoform peptide fragments of tau protein. The headline and the present invention were completed.
That is, the present invention is as follows.

(1) A preventive or therapeutic agent for neurological diseases that develop with abnormal aggregation of neurodegenerative disease proteins, comprising hepta-histidine, angiotensin III, luteinizing hormone-releasing hormone peptide fragment, hepta-glutamine, kemptide acetate, and A compound comprising an amino acid sequence having an identity of 80% or more and an affinity for a protein that directly interacts with the neurodegenerative disease protein, nordihydroguaiaretic acid ester, sphingosine-1-phosphate, and A prophylactic or therapeutic agent comprising as an active ingredient at least one compound selected from the group consisting of chemically modified compounds of these compounds.
(2) The preventive or therapeutic agent according to (1) above, wherein the compound is a compound that changes the kinetics of aggregation of the neurodegenerative disease protein.
(3) The preventive or therapeutic agent according to (1) or (2), wherein the neurodegenerative disease protein is amyloid β, tau protein, or polyglutamine disease protein.
(4) The preventive or therapeutic agent according to (3), wherein the polyglutamine disease protein is an Htt protein.
(5) The preventive or therapeutic agent according to any one of (1) to (4), wherein the neurological disease that develops with abnormal aggregation of the neurodegenerative disease protein is Alzheimer's dementia, FTLD, or HD. .

(6) A method for screening a preventive or therapeutic agent for a neurological disease that develops with abnormal aggregation of the neurodegenerative disease protein, using as an index the influence of the test substance on the affinity of the protein that directly interacts with the neurodegenerative disease protein.
(7) The method for screening a prophylactic or therapeutic agent according to (6), wherein the prophylactic or therapeutic agent is a compound that changes the kinetics of aggregation of the neurodegenerative disease protein.
(8) The test substance is composed of hepta-histidine, angiotensin III, luteinizing hormone releasing hormone peptide fragment, hepta-glutamine, kemptide acetate, and an amino acid sequence having an identity of 80% or more, and the neurodegenerative disease protein A compound that has an affinity for a protein that directly interacts with the protein, and at least one compound selected from the group consisting of nordihydroguaiaretic acid ester, sphingosine-1-phosphate, and chemically modified compounds of these compounds The screening method according to (6) or (7).
(9) The screening according to any one of (6) to (8), comprising a step of measuring the affinity of the test substance for a protein that directly interacts with the neurodegenerative disease protein by single molecule fluorescence spectroscopy. how to.
(10) The screening method according to any one of (6) to (9), wherein the neurodegenerative disease protein is amyloid β, tau protein, or polyglutamine disease protein.
(11) The screening method according to (10), wherein the polyglutamine disease protein is Htt protein.
(12) The screening method according to any one of (6) to (11), wherein the neurological disease that develops with abnormal aggregation of the neurodegenerative disease protein is Alzheimer's dementia, FTLD, or HD.

  According to the present invention, a preventive or therapeutic agent for a neurological disease (for example, Alzheimer type dementia, FTLD, HD, etc.) that develops with abnormal aggregation of a neurodegenerative disease protein, and a method for screening the prophylactic or therapeutic agent are provided. Can be provided.

It is a figure which shows the outline | summary of the screening method which concerns on the 2nd aspect of this invention. (A), (b) is a figure which shows the molecular docking simulation result between polyQ of various length, and various segments of Ku70 protein, (c) is 1-10 residue with respect to the site 4 It is a figure which shows the LibDoc score of group polyamino acid, (d) is a figure which shows the LibDoc score of polyH of various length. It is a figure which shows a fly screening result. It is a figure which shows a mouse | mouth screening result. It is a figure which shows the result of the dynamic light scattering of aggregation of GST-HttExon1-110Q. It is a figure which shows the result of the dynamic light scattering of aggregation of R3 isoform peptide fragment. (A) is a diagram showing the neurite length on differentiation day 7, (b) is a diagram showing the dendrite length and the number of branch points on differentiation day 14 on differentiation day 14, (c) is a diagram showing the spine density on the 14th day of differentiation, and (d) is a diagram showing the number of Htt inclusion body positive neurons on the 14th day of differentiation. It is a figure which shows recovery | restoration of the motor dysfunction of the mutation Htt knock-in mouse | mouth by sphingosine-1-phosphate. (A) shows the results of quantifying the YAP signal intensity in 100 neurons from 4 mice in each group. As a result,% Htt inclusion body positive cells were obtained from 10 fields in the striatum from 4 mice in each group. It is a figure which shows the result of having quantified. (B) PBS (phosphate buffered saline) injected WT mouse group (control: C57BL / 6), PBS injected mutant Htt-KI mouse group and S1P injected mutant Htt-KI mouse group (N = 3). It is a figure which shows the result of having calculated the ER-CFP volume per cell and signal intensity from 420, 509, and 507 cells.

Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be implemented with appropriate modifications within the scope of the object of the present invention. .
In the present specification, amino acid residues in amino acid sequences are represented by one-letter code (for example, “G” for glycine residue) or three-letter code (for example, “Gly” for glycine residue) well known in the art. There is a case.

<Preventive or therapeutic agent for neurological diseases that develop with abnormal aggregation of neurodegenerative disease proteins>
The invention according to the first aspect of the present invention is a preventive or therapeutic agent for a neurological disease that develops with abnormal aggregation of neurodegenerative disease protein, and is a hepta-histidine (HHHHHHH (SEQ ID NO: 1)) (hereinafter referred to as 7H). ), Angiotensin III (RVYIHPF (SEQ ID NO: 2)), luteinizing hormone releasing hormone peptide fragment 4-10 (SYGLRPG (SEQ ID NO: 3) -NH ₂ ) (hereinafter also referred to as (LH-RH4-10 peptide fragment). ), Hepta-glutamine (QQQQQQQ (SEQ ID NO: 4)) (hereinafter also referred to as 7Q), kemptide acetate (LRRASLG (SEQ ID NO: 5)), and an amino acid sequence having 80% or more identity with these compounds, and A compound having affinity for a protein that directly interacts with the neurodegenerative disease protein, In addition, prevention comprising at least one compound selected from the group consisting of nordihydroguaiaretic acid ester, sphingosine-1-phosphate (hereinafter also referred to as S1P), and chemically modified compounds of these compounds as an active ingredient Or it is a therapeutic agent.

As used herein, “amino acid sequence having 80% or more identity” refers to mutations due to substitution, insertion, deletion, etc. on the amino acid sequence of 7H, angiotensin III, LH-RH4-10 peptide fragment, 7Q, kemptide acetate The peptide means that the identity of amino acids is 80% or more, and the identity is preferably 90% or more, more preferably 95% or more, and still more preferably 97% or more.
The side chains of amino acids differ in hydrophobicity, charge, size, etc., but some highly conserved in the sense that they do not substantially affect the peptide's three-dimensional structure (also called three-dimensional structure). This relationship is known from experience and physicochemical measurements. For example, substitution of amino acid residues includes glycine (Gly) and proline (Pro), Gly and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid (Glu) and glutamine (Gln). ), Aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), Thr and serine (Ser) or Ala, lysine (Lys) and arginine (Arg), and the like. In particular, substitution of at least one H in 7H with other polar side chain amino acid residues (eg, Q), substitution of at least one Q in 7Q with other polar side chain amino acid residues (eg, H), etc. Also mentioned.
As the chemical modification of the chemical modification compound of nordihydroguaiaretic acid ester, sphingosine-1-phosphate (hereinafter also referred to as S1P), carbon atoms of 1 to 3 carbon atoms at any position of the constituent carbon atoms can be used. Examples include alkylation modification with a linear or branched alkyl group (for example, methyl group, ethyl group, isopropyl group, etc.).

In the present invention, the protein that directly interacts with the neurodegenerative disease protein means a protein that directly binds to the neurodegenerative disease protein or a protein that indirectly binds to the neurodegenerative disease protein via another substance. .
Examples of the protein that directly interacts with the neurodegenerative disease protein include Ku70 protein that is a protein that directly interacts with Htt protein and is a DNA damage repair protein in nerve cells whose function can be impaired by mutant Htt.

In the prophylactic or therapeutic agent according to the first aspect, it is preferable that the compound is a compound that changes the kinetics of aggregation of the neurodegenerative disease protein, as shown in Examples described later and FIGS.
Changes in the kinetics of aggregation of the neurodegenerative disease protein can be measured by dynamic light scattering (DLS).

In the preventive or therapeutic agent according to the first aspect, the neurodegenerative disease protein is preferably amyloid β, tau protein, or polyglutamine disease protein.
Examples of the polyglutamine disease protein include Htt protein, ataxin 1, ataxin 2, ataxin 3, ataxin 6, ataxin 7, TATA binding protein, atrophin, androgen receptor, and preferably Htt protein.
It is known that the onset of Alzheimer type dementia or FTLD is accompanied by abnormal aggregation of amyloid β and tau protein, or tau protein, TDP43 protein, FUS protein and the like.
The onset of Huntington's disease is known to accompany abnormal aggregation of Htt protein.
In the preventive or therapeutic agent according to the first aspect, the neurological disease that develops with abnormal aggregation of the neurodegenerative disease protein is preferably Alzheimer's dementia, FTLD, or HD.

The prophylactic or therapeutic agent according to the first aspect can be administered systemically or locally orally or parenterally. Examples of parenteral administration methods include intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, and subcutaneous injection. The administration method can be appropriately selected depending on the age and symptoms of the patient. The dosage varies depending on the age, administration route, and number of administrations, and can be appropriately selected by those skilled in the art.
Examples of the dosage form suitable for parenteral administration include those containing additives such as stabilizers, buffers, preservatives, tonicity agents, and further containing pharmaceutically acceptable carriers and additives. But you can. Examples of such carriers and additives include water, organic solvents, polymer compounds (collagen, polyvinyl alcohol, etc.), stearic acid, human serum albumin (HSA), mannitol, thurbitol, lactose, surfactants and the like. However, it is not limited to these.

  The dose of the preventive or therapeutic agent according to the first aspect which is an active ingredient is generally in the range of about 0.1 μg to 100 mg per kg of body weight per time.

<Method for screening a preventive or therapeutic agent for neurological diseases that develop with abnormal aggregation of neurodegenerative disease proteins>
The invention according to the second aspect of the present invention relates to a neurological disease that develops with abnormal aggregation of the neurodegenerative disease protein, using as an index the influence of the test substance on the affinity of the protein that directly interacts with the neurodegenerative disease protein. This is a method of screening for preventive or therapeutic agents.
In the screening method according to the second aspect, screening means at least narrowing down the population of test substances using the influence of the test substance on the affinity of the protein that directly interacts with the neurodegenerative disease protein as an index.
In the screening method according to the second aspect, examples of the protein that directly interacts with the neurodegenerative disease protein include Ku70 protein.
In the screening method according to the second aspect, the preventive or therapeutic agent is preferably a compound that changes the kinetics of aggregation of the neurodegenerative disease protein.

Any substance can be used as the test substance used in the screening method according to the second aspect. The type of the test substance is not particularly limited, and may be a synthetic peptide (oligo or polypeptide), a nucleic acid molecule, an antibody, an individual low-molecular synthetic compound, or a compound present in a natural product extract. But you can. Alternatively, the test substance may also be a synthetic peptide (oligo or polypeptide) library, a compound library, a phage display library, or a combinatorial library, particularly when performing an in silico screening method described below. It may be a virtual library.
The construction of a synthetic peptide (oligo or polypeptide) library, compound library or virtual library is known to those skilled in the art, and a commercially available synthetic peptide (oligo or polypeptide) library, virtual library, compound library or Virtual libraries can also be used.

The test substance is preferably a synthetic peptide (synthetic peptide library), a low molecular compound (for example, a compound library), a nucleic acid molecule, or an antibody.
In Examples described later, 19468 low molecular weight compound libraries, 3010321 virtual libraries, and polyamine libraries having 10 amino acid residues or less were used.
Examples of the test substance include hepta-histidine, angiotensin III, LH-RH4-10 peptide fragment, hepta-glutamine, kemptide acetate, and an amino acid sequence having an identity of 80% or more, and the neurodegenerative disease protein. A compound having an affinity for a directly interacting protein, and at least one compound selected from the group consisting of nordihydroguaiaretic acid ester, sphingosine-1-phosphate, and chemically modified compounds of these compounds Is more preferable.

In the screening method according to the second aspect, the affinity of a test substance for a protein (for example, Ku70 protein) that directly interacts with the neurodegenerative disease protein is evaluated, and it develops with abnormal aggregation of the neurodegenerative disease protein It is preferable to select a prophylactic or therapeutic agent candidate for a neurological disease.
In the screening method according to the second aspect, the screening method includes in vivo and in vitro as long as the affinity of a test substance for a protein that directly interacts with a neurodegenerative disease protein (for example, Ku70 protein) is used as an index. Any screening method such as (in vitro) or in silico may be used.

In the screening method according to the second aspect, as a step of evaluating the affinity of a test substance for a protein that directly interacts with the neurodegenerative disease protein in vitro, the affinity of the test substance for the protein is determined by single-molecule fluorescence. It is preferable to include a step of selecting the prophylactic or therapeutic agent candidate as evaluated by spectroscopic methods.
According to single-molecule fluorescence spectroscopy, protein-protein interaction (preferably one of the proteins is fluorescently labeled) is measured in the presence of the test substance and in the absence of the test substance. , The affinity of the test substance for the protein can be measured.
In the examples described later, a Ku70 protein tagged with a His tag for protein purification (hereinafter also referred to as His tag-Ku70 protein) with an arbitrary fluorescent reagent, and GST for protein purification. The protein-protein interaction was measured using an Htt protein with a (glutathione-S-transferase) tag (hereinafter also referred to as GST-Htt protein).
MF20 (made by Olympus) etc. are mentioned as an apparatus used for single molecule fluorescence spectroscopy.

In the screening method according to the second aspect, in step of evaluating the affinity of the test substance for the protein that directly interacts with the neurodegenerative disease protein in silico, the affinity of the test substance for the protein is determined by molecular docking simulation. It is preferably a step of evaluating and selecting the prophylactic or therapeutic agent candidate.
It is preferable to perform a molecular docking simulation between Site 4 (Non-patent Document 2), which is the N-terminal region of Ku70 protein known to interact with mutant HttExon1, and the test substance.
The molecular docking simulation can be performed by, for example, Discovery Studio 2.5 (manufactured by Dassault Systems BIOVIA).
LibDock is a high throughput algorithm for docking ligands to binding sites on the receptor.
Ligand affinity can be measured by the LibDock score.

  In the molecular docking simulation, a virtual library can be used as a test substance, and examples of the virtual library include a commercially available compound database Chemical Available Purchase (CAP) 2006 (manufactured by Dassault Systems BIOVIA).

In the screening method according to the second aspect, the in vivo screening step includes administering a test substance to an Alzheimer-type dementia organism (eg, Drosophila, mouse, rat, etc.) model, FTLD biological model, or HD biological model, Examples include a screening step of evaluating the life span, body weight, motor nerve function (for example, rotarod) and the like and selecting the preventive or therapeutic agent candidate.
In the screening method according to the second aspect, the in vivo screening step includes administering a test substance to at least one model selected from the group consisting of the Drosophila HD model and the mouse HD model, and determining the life span, body weight, or motor nerve. It is preferable to be a screening step in which the function is evaluated and the prophylactic or therapeutic agent candidate is selected.

The Drosophila HD model includes “Barclay, SS et al., Systems biology analysis of Drosophila in vivo screens in the DNA. 13 H. et al. "Shiraishi, R., Tamura, T., Sone, M. & Okazawa, H. Systematic analysis of fully models with a number of different drivers. pe-specific expression. PLoS One 9, e116567 (2014). The Drosophila HD model disclosed in "can be used.
The Drosophila HD model can express Htt protein in motor neurons under the control of OK6 motor neuron specific drivers and use lifespan as a phenotypic marker in chemical or genetic screening.

  R6 / 2 transgenic mice can be used as the mouse HD model. R6 / 2 transgenic mice express exon 1 of mutant Htt and can develop the earliest and have the shortest life span. It may be the most severe symptom compared to various mouse HD models. R6 / 2 transgenic mice can be said to be the most efficient for screening multiple test substances within the shortest period (Ehrnhofer, DE, Butland, SL, Pouladi, MA & Hayden, M R. Mouse models of Huntington disease: variations on a themes.Dis Model Mech 2, 123-129 (2009).

When assessing lifespan in in vivo screening, it can be assessed by a log rank test.
For example, if the test is performed at a general 5% level, if the significance (P) at this time is less than 0.05, the null hypothesis is rejected, the test substance administration group, the test substance There may be a significant difference from the non-administration group.

The screening method according to the second aspect includes a step of evaluating the affinity of the test substance to the protein by single-molecule fluorescence spectroscopy in vitro, and selecting the prophylactic or therapeutic agent candidate. It is preferable to include the step of evaluating the affinity of the test substance for the protein by molecular docking simulation and selecting the prophylactic or therapeutic agent candidate.
In the screening method according to the second aspect, a test substance is administered to an Alzheimer-type dementia biological model, an FTLD biological model, or an HD biological model in vivo, and the life span, body weight, motor nerve function (for example, rotarod) or the like is evaluated. It is more preferable to further include a screening step for selecting the preventive or therapeutic agent candidate.

In the screening method according to the second aspect, the neurodegenerative disease protein is preferably amyloid β, tau protein, or polyglutamine disease protein.
Examples of the polyglutamine disease protein include Htt protein, ataxin 1, ataxin 2, ataxin 3, ataxin 6, ataxin 7, TATA binding protein, atrophin, androgen receptor, and preferably Htt protein.
In the screening method according to the second aspect, the neurological disease that develops with abnormal aggregation of the neurodegenerative disease protein is preferably Alzheimer's dementia, FTLD, or HD.

FIG. 1 is a diagram showing an outline of the screening method according to the second embodiment.
As shown in FIG. 1, the step of evaluating the affinity of the test substance for the protein in vitro by single-molecule fluorescence spectroscopy and selecting the prophylactic or therapeutic agent candidate, and in silico, the test substance A first screening step in combination with the step of selecting the prophylactic or therapeutic agent candidate, by evaluating the affinity for the protein by molecular docking simulation,
The second screening comprises the step of evaluating the affinity of the prophylactic or therapeutic agent selected in the first screening step by single molecule fluorescence spectroscopy and further selecting the prophylactic or therapeutic agent candidate. As a process,
In the in vivo screening step, the prophylactic or therapeutic agent candidate selected in the second screening step is administered to a Drosophila HD model, the life span is evaluated, and the prophylactic or therapeutic agent candidate is further selected. The process is the third screening process,
A screening step of administering the candidate for the prophylactic or therapeutic agent selected in the third screening step to a mouse HD model, evaluating life span, body weight or motor nerve function, and further selecting the prophylactic or therapeutic agent candidate. Can be the fourth screening step.

(Confirmation of preventive or therapeutic effects on neurological diseases that develop with abnormal aggregation of neurodegenerative disease proteins)
For example, healing of morphological abnormalities of primary neurons differentiated from iPS cells of neurological disease patients who develop with abnormal aggregation of neurodegenerative disease proteins using a microscope using any fluorescently labeled antibody that recognizes any marker This can be confirmed by observation.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated further more concretely, the scope of the present invention is not limited to these Examples.

<Preparation of GST-Htt protein and His tag-Ku70 protein>
First, GST-Htt protein and His tag-Ku70 protein used in the screening process by in vitro single molecule fluorescence spectroscopy were prepared.
(Construction of GST-Htt vector and His-Ku70 vector)
The cDNA of human Htt gene exon 1 containing 110 or 20 CAG repeats was subcloned into pGEX-3X (GE Healthcare).
The mouse Ku70 cDNA was amplified from the RIKEN full-length amplified library using the 5′-AAAGGATCCATGTCAGATGTGGAGGTCTA-3 ′ primer (SEQ ID NO: 6) and the 5′-AAACTCGAGTGTTCTTCTCCACAGTGTCTGA-3 ′ primer (SEQ ID NO: 7). (A) Subcloning into BamHI site and XhoI site (manufactured by Clontech).

(Expression and purification of GST-Htt protein and His tag-Ku70 protein)
Plasmids for GST-Htt protein and His tag-Ku70 protein were transformed into competent cells of E. coli Rosetta (DE3) (Novagen).
The transformed cells were cultured on a shaker at 37 ° C. and 200 rpm. When the OD at a wavelength of 600 nm reached 0.3, IPTG (1.0 mM final concentration) was added and further incubated at 37 ° C. for 2 hours at 200 rpm.
E. coli cells are recovered by centrifugation and contain 0.1% Tween 20, 0.1% lysozyme (Sigma), and 1/500 protease inhibitor cocktail III-EDTA free (Calbiochem) for GST-Htt protein Lysis was performed in 20 ml of PBS, and His tag-Ku70 protein was lysed in 20 ml of PBS containing 10 mM imidazole, 1% Triton X-100, and 1/500 protease inhibitor cocktail III-EDTA free (pH 8.0).

The suspension was sonicated 7 times for 15 seconds at 1 minute intervals using power level 6 (ultrasonic homogenizer: UH-50, manufactured by SMT) and centrifuged at 12,000 × g for 20 minutes at 4 ° C. did.
For GST-Htt protein, the supernatant was mixed with 4 ml of 50% glutathione sepharose 4B (GE Healthcare) equilibrated with PBS containing 0.1% Tween20, and for His tag-Ku70 protein 0 The supernatant was mixed with 4 ml of Ni-NTA agarose (Qiagen) equilibrated with PBS containing 1% Tween20. Then, it rotated slowly at 4 degreeC for 3 hours. The GST-Htt suspension was applied to a glutathione Sepharose 4B column washed with 32 ml of PBS containing 0.1% Tween 20 (pH 8.0) at 4 ° C. and 10 mM glutathione in PBS containing 0.1% Tween 20 at 4 ° C. Elute 4 times by gravity flow at 4 ml.
His tag-Ku70 suspension was applied to a Ni-NTA agarose column washed with 32 ml of 20 mM imidazole (pH 8.0) in PBS containing 1% Triton X-100 at 4 ° C. and 4 ml of 10 mM glutathione in 250 mM imidazole. And eluted 4 times by gravity flow.
Each fraction was dialyzed twice with 2 L of PBS containing 0.01% Tween 20 for 12 hours at 4 ° C. and stirred.
The purified Ku70 protein was labeled with a fluorescent dye using a protein labeling kit (488 nm and 633 nm) (manufactured by Olympus).

<In vitro screening by single molecule fluorescence spectroscopy as the first screening>
Fluorescence intensity distribution analysis polarization (FIDA-PO) analysis using MF20 (Olympus) to detect single molecule fluorescence of 40 μl / well sample of 384 well glass bottom plate using confocal laser microscope at room temperature And fluorescence correlation spectroscopy (FCS) analysis.
Each sample contained 5 nM fluorescently labeled Ku70 protein in PBS containing 0.01% Tween 20 and 1% DMSO.
The data obtained for FIDA-PO and FCS were analyzed using Bonferroni / Dan test. In total, 19,468 compounds from the Chemical Biology Screening Center (http://mechpc5.tmd.ac.jp:3000/cbdb) of Tokyo Medical and Dental University were screened as the first screening.
The top 177 of the results was subjected to the second screening, an in vitro screening by single molecule fluorescence spectroscopy again.

<In silico screening>
All docking simulations were performed using Discovery Dock 2.5 (Dassault Systems BIOVIA) and a commercially available compound database Chemical Available Purchase (CAP) 2006 (Dassault Systems BIOVIA) using LibDoc.
The poly (amino acid) structure (10 residues or less) was generated using the “Build and Edit Protein” command and used for binding simulation in Discovery Studio 3.0.
The crystal structure of Ku heterodimer bound to DNA (PDB ID: 1JEY) was downloaded from Structural Bioinformatics Research Collaboration (RCSB) Protein Data Bank (PDB), and the isolated crystal structure of Ku70 is complex (1JEY) (Http://www.rcsb.org/pdb/explore/explore.do?structureid=1JEY).

FIG. 2 (a) performed a molecular docking simulation between polyQ of various lengths and various segments of Ku70 protein (sites 1-26).
The results are shown in FIGS. 2 (a) and 2 (b).
It is known that site 4, which is the N-terminal region of Ku70 protein, interacts with mutant HttExon1 (Non-patent Document 2).
As is clear from FIG. 2 (a), it was found that polyQ binds to the depression at site 4 which is the N-terminal region.

As is clear from the results shown in FIG. 2B, it can be seen that 6Q has the highest affinity for the site 4 and the shorter the poly Q length, the lower the affinity.
PolyQ longer than 7Q could not be simulated due to possible limitations of the software algorithm.
FIG. 2 (c) is a diagram showing the LibDoc score of various polyamino acids of 1 to 10 residues for the site 4, and (d) is a diagram showing the LibDoc score of polyH of various lengths.
As is apparent from FIGS. 2C and 2D, it was simulated that 7H is a polyamino acid having the highest affinity for Site 4.

Based on the above results, the affinity of 3,010,121 compounds from the virtual library to Site 4 was screened based on the LibDock score.
The top 20 of the results were subjected to in vitro screening by single molecule fluorescence spectroscopy in the second screening.
<In vitro screening by single molecule fluorescence spectroscopy as the second screening>
The compounds selected in the first screening were further screened by the same method as in vitro screening by single molecule fluorescence spectroscopy as the first screening.
As a result, 59 compounds were selected.

<Fly screening as the third screening>
Since 3 of the 59 compounds were difficult to synthesize, 56 were subjected to in vivo screening using the Drosophila HD model as the third screening below.
(Fly preparation)
All flies are corn meal medium (9.2% corn meal, 3.85% yeast, 3.8% sucrose, 1.05% potassium tartrate, 0.09% calcium chloride, unless otherwise noted. 6% glucose, 2.416% nipagin, 1% agar) and maintained at 25 ° C. humidity 60% ± 10% in a 12 hour: 12 hour light / dark cycle.
UAS-Htt103Q transgenic flies and OK6-Gal4 transgenic flies (Tamura, T. et al. Ku70 alleviates neurogeneration in Drosophila models of Huntington's dissease. The flies were subjected to lifespan screening.
The compound or peptide for screening was dissolved in distilled water or ethanol at 5 mM, and mixed uniformly in a 9-fold amount of cornmeal medium to a final concentration of 500 μM.
Only distilled water or ethanol was added as a control medium.
Twenty virgin female flies were maintained per vial and transferred to new vials using fresh media every 2-3 days. The number of dead flies was quantified every 2-3 days.

(statistics)
The log rank test was used for the fly model lifespan assay and the mouse model lifespan assay described below. Other biological analyses, data were considered to follow a normal distribution and were expressed as mean ± standard error. Student t-test was applied for two-group comparison (chemical treatment group vs. PBS application group). Tukey's HSD test or Dunnett's comparison was applied for multiple group comparisons. The significance level was set to 1% or 5%.

The results are shown in FIG.
As shown in FIG. 3, the life extension effect was observed with the compound of 6.
That is, the life extension effect in the log rank test at p <0.01 was observed with 7H, angiotensin III, and kemptide acetate.
Moreover, the life extension effect in the log rank test at p <0.05 was observed with 7Q, LH-RH4-10 peptide fragment, and nordihydroguaiaretic acid ester.
The extension of life was not long, but the result was reproducible.
The results are summarized in Table 1 below.

<Screening using mouse HD model as fourth screening>
Among the above 6 compounds, 7H, angiotensin III, and LH-RH4-10 peptide fragments were subjected to screening using a mouse HD model as the fourth screening.
(Mouse weight test)
The above compound was intraperitoneally injected at 50 μg / g body weight, and the change in body weight was measured.
The results are shown in FIG.
As apparent from FIG. 4 (a), R6 / 2 mice began to lose weight at about 9 weeks of age.
This weight loss continues until death, but it can be seen that the weight loss is suppressed by administration of 7H and angiotensin III (p <0.01 or p <0.05 at each age of week).

(Mouse motor function test)
Mice were maintained at 22 ° C. with a 12 hour light / dark cycle (lights on at 8:00 am and lights off at 8:00 pm), water and standard feed pellets (Clear Rodent Diet CE-2, Claire Japan) Made freely). Survival curves were analyzed using a log rank test.
For the rotarod test, place the mouse on the rotarod (shaft diameter: 3.2 cm, lane width 5.7 cm, height drop 16.5 cm; Five Station Rotarod Standalone Foremouth, ENV-577M, manufactured by MED Associates). The rotational speed was increased linearly from 0 rpm to 35 rpm in 300 seconds and maintained at 35 rpm for an additional 60 seconds (for 4-12 week old mice).
Mice were tested 3 times for 3 consecutive days.

The results are shown in FIG.
As is clear from FIG. 4B, 7H showed an improvement in motor function, but Angiotensin III did not show a clear improvement in motor function.

(Life extension test by administration of test substance to R6 / 2 transgenic mice)
Male R6 / 2 mice and their background mice (CBA / J) were maintained at 22 ° C. with a 12 hour light / dark cycle (lights on at 8:00 am and lights off at 8:00 pm) for each experiment. Prior to the start of water, water and standard feed pellets (CLEA rodent diet CE-2, manufactured by CLEA Japan) were freely ingested.
Compounds dissolved in PBS were injected intraperitoneally into mice at 50 μg / g body weight once weekly from 3 weeks of age. Survival was analyzed using a log rank test.

The results are shown in FIG.
As is clear from FIG. 4 (c), both 7H and angiotensin III showed a life extension effect (p <0.05).
On the other hand, regarding the LH-RH4-10 peptide fragment, the life extension effect could not be confirmed in the three tests.
The results are summarized in Table 2.

<Confirmation of dynamic change of abnormal aggregation of neurodegenerative disease protein by dynamic light scattering (DLS)>
(DLS analysis (1))
DLS was a Zetasizer μV instrument (Malvern) and monitored the time course of huntingtin aggregation in the presence and absence of the compound. 10 μM protein was incubated with 500 μM of each of the three compounds in PBS buffer at 25 ° C. for 6 days and the DLS signal was recorded every few hours. The term “Z average size” was applied as “aggregation index (ISO-22412: 2008)”. Each measurement was repeated four times, and the average value of the four measurements was calculated.
The results are shown in FIG.
In FIG. 5, the control is 10 μM GST-HttExon1-110Q in the absence of compound.
No. No. 1 is in the presence of 500 μM 7H. 2 is in the presence of 500 μM angiotensin III. 3 is in the presence of 500 μM luteinizing hormone-releasing hormone fragment 4-10.

As is apparent from the results shown in FIG. 5, the presence of 7H, angiotensin III, and luteinizing hormone-releasing hormone fragment 4-10 significantly increases the molecular weight of the final aggregate product of GST-HttExon1-110Q. I understand. It can also be seen that during the early stages of incubation, the process of aggregate formation is slow in the presence of the compound, whereas the molecular weight increases rapidly as larger aggregation proceeds.
Surprisingly, it can be seen that 7H and angiotensin III, which showed HD preventive or therapeutic effects in fly and mouse HD models, promote rather than inhibit Htt aggregation.
From the above, it can be said that it becomes a prophylactic or therapeutic agent candidate for neurological diseases by changing at least the kinetics of aggregation of GST-HttExon1-110Q, such as 7H and angiotensin III.

(DLS analysis (2))
Using a Zetasizer μV instrument (Malvern, Inc.), as shown in FIG. 6 (a), the 306-337th R3 isoforms in the microtubule binding domain (MBD) consisting of the 244th-369th amino acid residues in the tau protein. The time course of aggregation of the foam peptide fragments was monitored in the presence and absence of compound.
The R3 isoform peptide fragments used are as follows.
Fluorescein-V ³⁰⁶ QIVYKPVDLSKVTSKCGGSLGNIHHKPGGGQ ³³⁶ -NH ₂ (SEQ ID NO: 8)

10 μM R3 isoform peptide fragment (molecular weight 3.6 kDa) was incubated with 500 μM of each of the three compounds at 25 ° C. in a buffer containing 50 mM Tris-HCl, pH 8, 1 mM heparin for 9 days. Was recorded every few hours.
The results are shown in FIG.
In FIG. 6 (b), the control is a 10 μM R3 isoform peptide fragment in the absence of the compound.
No. No. 1 is in the presence of 500 μM 7H. 2 is in the presence of 500 μM angiotensin III. 3 is in the presence of 500 μM luteinizing hormone-releasing hormone fragment 4-10.
As is clear from the results shown in FIG. No. 1 shows that the aggregation of the R3 isoform peptide fragment is suppressed to 1/10 of the control, whereas No. 1 in the presence of angiotensin III. 2 shows that the aggregation of the R3 isoform peptide fragment is increased 4 to 10 fold over the control. In addition, No. 2 in the presence of luteinizing hormone-releasing hormone fragment 4-10. 3 shows that the aggregation of the R3 isoform peptide fragment is about the same as the control.
From the above, it can be seen that 7H and angiotensin III change at least the dynamics of tau protein aggregation, and can be said to be prophylactic or therapeutic candidates for neurological diseases such as Alzheimer's dementia and FTLD.

<Verification of preventive or therapeutic effects on neurological diseases that develop with abnormal aggregation of neurodegenerative disease proteins>
7H, angiotensin III, and LH-RH4-10 peptide fragments were confirmed to have therapeutic effects on morphological abnormalities of primary neurons differentiated from iPS cells of human HD patients.
(IPS cell culture)
201B7 is derived from RIKEN BRC (https://ja.brc.riken.jp). CS92iHD-57n9 iPS cells from HD patients have been established at the Coryell Institute of Medicine (https://catalog.coriell.org).

(Karyotype analysis of iPS cells)
A standard G-banding analysis of iPS cell lines (201B7 and CS92iHD-57n9) was performed to eliminate the possibility of abnormal karyotypes that could occur during iPS cell generation.

(In vitro differentiation of iPS cells)
Chaddah, R .; , Arntfield, M .; , Runciman, S .; Clarke, L .; & Van der Kooy, D.A. Clonal neutral stem cells from human embryonic stem cell colonies. Neuronal differentiation of iPS cells was performed according to J Neurosci 32, 7771-7781 (2012).
Briefly, iPS cells were seeded in 10 cm dishes and maintained for more than 5 days with 3 μM SB431542, 3 μM CHIR99021 and 3 μM desomorphine. IPS cells are then detached from the feeder layer, dissociated into single cells, and 2 × B27 supplemented KBM with 20 ng / ml bFGF, 10 ng / mL hLIF, 10 μM Y27632, 3 μM CHIR99021 and 2 μM SB431542. Neurospheres were formed by culturing in a suspension culture condition in a 10 cm cell repellent dish with a medium (manufactured by KHOJIN BIO).
Neurospheres were passaged twice and differentiated into neurons using adhesion culture (DMEM / F12 supplemented with B27 and glutamax). Neurons were allowed to attach to poly-L-ornithine-coated and poly-L-lysine-coated coverslips for 14-21 days.

(Immune staining of iPS cells and differentiated cells)
The cells were fixed with PBS containing 4% paraformaldehyde for 15 minutes on ice and incubated with primary antibodies against the following proteins. : SSEA1 (1: 1000, Abcam, ab16285), Nanog (1: 200, RCAB0004PF, Reprocell), βIII tubulin (1: 1000, T8660 Sigma Chemical), αSMA (1: 150, M085101, Dako) , And SOX17 (1: 500, ab 84990, Abcam).
The cells were then washed with PBS and incubated with Alexafluoro488-conjugated secondary antibody, Alexafluoro555-conjugated secondary antibody, or Alexafluoro647-conjugated secondary antibody (1: 500, manufactured by Invitrogen).

As is clear from the results shown in FIG. 7 (a), it can be seen that 7H, angiotensin III, and LH-RH4-10 peptide fragments all improved neurite length on the 7th day of differentiation.
Further, as is clear from the results shown in FIG. 7B, recovery of the length of the dendrite and the number of branch points was confirmed on the 14th day of differentiation.
Further, as apparent from the results shown in FIG. 7 (c), it was revealed that the spine density was recovered by addition of 7H and angiotensin III by immunocytochemistry using PSD95 on the 14th day of differentiation.
However, as is apparent from the results shown in FIG. 7 (d), as expected from the DLS results shown in FIG. 5, the administration groups of 7H, angiotensin III, and LH-RH4-10 peptide fragments were The number of Htt inclusion body positive neurons was not reduced.
From the above, HD therapeutic effects of 7H, angiotensin III, and LH-RH4-10 peptide fragments on human neurons were confirmed regardless of Htt inclusion formation.

<Recovery of motor dysfunction of late mutant Htt knock-in mice (hereinafter also referred to as mutant Htt-KI mice) by sphingosine-1-phosphate>
Mutant Htt knock-in mice (Wheeler, VC, Auerbach, W., White, JK, Srinidhi, J., Auerbach, A., Ryan, A., Duyao, MP, Vrbanac, , Wever, M., Gusella, JF et al. (1999) Length-dependent gaming CAG repeat instability in the Huntington's disease knock-in. Intrathecal injection (200 mM, 0. 1) of sphingosine-1-phosphate (hereinafter also referred to as S1P) at week 69 (at least 2 months after the onset of symptoms at around 60 weeks). It was started 5μl / time).
The results are shown in FIGS. 8 and 9, the data are shown as mean ± standard error. P value in Tukey test: * is p <0.05, ** is p <0.01. N. s. There is no significant difference.
WT (control: C57BL / 6) mice are mice that received intrathecal injection of PBS with the same protocol as above.
As is apparent from FIG. 8, a dramatic effect was observed after one week with S1P, and the improvement continued until the observation at 73 weeks. It should be noted that no motor function decline occurred after treatment.

Hippo signaling is an evolutionarily conserved pathway that can control cell division, apoptosis and organ size in response to changes in cell density levels. At relatively low cell densities, transcriptional coactivators YAP and TAZ can bind to transcription factors and induce the expression of genes that favor cell growth and division (Yu, FX. and Guan, KL (2013) The Hippo pathway: regulators and regulations. Genes Dev., 27, 355-371.).
The Hippo pathway activates Mst and Lats, then phosphorylates YAP and prevents nuclear translocation of YAP. This means that activation of the hippo pathway inhibits cell growth and survival (Harvey, K. and Tapon, N. (2007) The Salvador-Warts-Hippo pathway-an emerging tumor-suppressor. Rev. Cancer, 7, 182-191.).

  FIG. 9 (a) shows that the signal intensity of YAP was quantified in 100 neurons from 4 mice in each group. As a result,% Htt inclusions were positive from 10 fields in the striatum from 4 mice in each group. It is a figure which shows the result of having quantified the cell.

As is clear from FIG. 9 (a), the signal intensity of nuclear YAP decreased in striatal neurons of mutant Htt-KI mice compared to WT (C57BL / 6) mice.
On the other hand, administration of S1P restored the decrease in nuclear YAP signal, but did not decrease the number of Htt inclusion body positive cells.

FIG. 9 (b) shows 420, 509 and 507 cells of PBS injected WT mouse group (control: C57BL / 6), PBS injected mutant Htt-KI mouse group and S1P injected mutant Htt-KI mouse group (N = 3). It is a figure which shows the result of having calculated the ER-CFP volume and signal intensity per cell.
As is clear from FIG. 9 (b), at 73 weeks of age, ER instability increased in RSD cortical neurons of mutant Htt-KI mice, whereas S1P restored instability.
From the results shown in FIGS. 9 (a) and 9 (b), S1P increases YAP in the nucleus by increasing the nuclear translocation of YAP regardless of the aggregate, and S1P shows the therapeutic effect by increasing YAP. Is suggested.

Claims (12)

  A preventive or therapeutic agent for a neurological disease that develops with abnormal aggregation of neurodegenerative disease protein, comprising hepta-histidine, angiotensin III, luteinizing hormone-releasing hormone peptide fragment, hepta-glutamine, kemptide acetate, and compounds thereof A compound comprising an amino acid sequence having an identity of 80% or more and having an affinity for a protein that directly interacts with the neurodegenerative disease protein, as well as nordihydroguaiaretic acid ester, sphingosine-1-phosphate, and compounds thereof A preventive or therapeutic agent comprising at least one compound selected from the group consisting of chemically modified compounds as an active ingredient.   The prophylactic or therapeutic agent according to claim 1, wherein the compound is a compound that changes the kinetics of aggregation of the neurodegenerative disease protein.   The preventive or therapeutic agent according to claim 1 or 2, wherein the neurodegenerative disease protein is amyloid β, tau protein, or polyglutamine disease protein.   The preventive or therapeutic agent according to claim 3, wherein the polyglutamine disease protein is huntingtin protein.   The prevention or treatment according to any one of claims 1 to 4, wherein the neurological disease that develops with abnormal aggregation of the neurodegenerative disease protein is Alzheimer-type dementia, frontotemporal lobar degeneration, or Huntington's disease. Agent.   A method for screening a preventive or therapeutic agent for a neurological disease that develops with abnormal aggregation of the neurodegenerative disease protein, using as an index the influence of a test substance on the affinity of the protein that directly interacts with the neurodegenerative disease protein.   The method for screening for a prophylactic or therapeutic agent according to claim 6, wherein the prophylactic or therapeutic agent is a compound that changes the kinetics of aggregation of the neurodegenerative disease protein.   The test substance consists of hepta-histidine, angiotensin III, luteinizing hormone-releasing hormone peptide fragment, hepta-glutamine, kemptide acetate, and an amino acid sequence having an identity of 80% or more and directly interacts with the neurodegenerative disease protein A compound having an affinity for an acting protein, and at least one compound selected from the group consisting of nordihydroguaiaretic acid ester, sphingosine-1-phosphate, and chemically modified compounds of these compounds 8. The screening method according to 6 or 7.   The screening method according to any one of claims 6 to 8, comprising a step of measuring the affinity of the test substance for a protein that directly interacts with the neurodegenerative disease protein by single molecule fluorescence spectroscopy.   The screening method according to any one of claims 6 to 9, wherein the neurodegenerative disease protein is amyloid β, tau protein, or polyglutamine disease protein.   The screening method according to claim 10, wherein the polyglutamine disease protein is a huntingtin protein.   The screening method according to any one of claims 6 to 11, wherein the neurological disease that develops with abnormal aggregation of the neurodegenerative disease protein is Alzheimer-type dementia, frontotemporal lobar degeneration, or Huntington's disease. .

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