Method for screening for proteins associated with neurodegenerative diseases and therapeutic or prophylactic drugs for neurodegenerative diseases

Abstract

Provided is a method for simply and quickly screening for proteins associated with neurodegenerative diseases, or compounds that are useful for the treatment or prevention of neurodegenerative diseases. The method uses a probe formed by covalently bonding an environmentally responsive fluorophore to a dipeptide repeat sequence comprising a repeating unit selected from the group consisting of proline-arginine, glycine-arginine, proline-alanine, glycine-alanine and glycine-proline.

Description

Proteins associated with neurodegenerative diseases and screening methods for therapeutic or preventive agents for neurodegenerative diseases

[0001] The present invention relates to a protein associated with a neurodegenerative disease and a method for screening for a therapeutic or preventive agent for a neurodegenerative disease.

[0002] With the current increase in the elderly population, the number of patients with neurodegenerative diseases is on the rise, and the establishment of treatments is an urgent need. Neurodegenerative diseases include dementias such as Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD), as well as amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). A common pathological feature of neurodegenerative diseases is the accumulation or deposition of abnormal proteins called inclusion bodies. For example, in ALS and FTLD, which belongs to the same disease spectrum as ALS, abnormal aggregation of RNA-binding proteins such as FUS (fused in sarcoma) and TDP-43 (TAR DNA-binding protein 43 kDa) is observed inside and outside neurons. Furthermore, in recent years, it has become clear that the aggregation process of these proteins is related to a physical phenomenon called "liquid-liquid phase separation (LLPS)," and a relationship has also been suggested between the dysfunction of LLPS regulators such as karyopherin β2 (Kapβ2) and the pathology of neurodegenerative diseases (Non-Patent Document 1).

[0003] On the other hand, it has been reported that the abnormal expansion of a hexanucleotide (GGGGCC) repeat in intron 1 of the C9orf72 gene may contribute to the pathogenesis of ALS and FTLD. There are several hypotheses regarding the abnormal expansion of repeats and the pathogenesis of ALS / FTLD, one of which is the expression of neurotoxic peptides by start codon-independent translation (repeat-associated non-ATG (RAN) translation). Neurotoxic peptides expressed from hexanucleotide repeat sequences by RAN translation are called dipeptide repeat proteins (DPRs), and consist of repeats of proline-arginine (PR), glycine-arginine (GR), proline-alanine (PA), glycine-alanine (GA), or glycine-proline (GP). Among these, a model has been reported suggesting that arginine-rich DPRs, i.e., polyPR and polyGR, may disrupt LLPS by inhibiting the interaction between FUS and Kapβ2 (Non-Patent Document 2).

[0004] Based on these findings, it is expected that elucidating the interactions between proteins and DPRs will lead to the elucidation of the pathology of neurodegenerative diseases such as ALS and FTLD, and ultimately to the development of treatments for neurodegenerative diseases. However, conventional methods for analyzing protein-protein interactions, such as co-immunoprecipitation, NMR, X-ray crystallography, and ultracentrifugation, all require a great deal of effort, time, and money, making it unrealistic to comprehensively search for proteins that interact with DPRs using these methods.

[0005] Acta Neuropathol., 2016; 132(2):159-173Nat. Commun., 2021; 12(1):5301

[0006] An object of the present invention is to provide a method for easily and quickly screening proteins associated with neurodegenerative diseases or compounds useful for treating or preventing neurodegenerative diseases, based on the interaction between DPRs and proteins.

[0007] As a result of extensive research, the present inventors have discovered that proteins that interact with DPRs and compounds that inhibit the interaction between DPRs and proteins can be detected by using a probe comprising a polypeptide containing a dipeptide repeat sequence covalently bonded to an environmentally responsive fluorophore.

[0008] That is, according to one embodiment, the present invention provides a method for screening for proteins associated with neurodegenerative diseases, comprising: (1) contacting a sample containing a protein with a probe comprising a polypeptide having a dipeptide repeat sequence and an environmentally responsive fluorophore covalently bonded thereto, wherein the dipeptide repeat sequence is composed of a repeating unit selected from the group consisting of proline-arginine, glycine-arginine, proline-alanine, glycine-alanine, and glycine-proline; and (2) measuring the fluorescence intensity of the probe.

[0009] Furthermore, according to one embodiment, the present invention provides a method for screening for therapeutic or preventive agents for neurodegenerative diseases, comprising: (1) contacting a probe, in the presence of a candidate compound, comprising a polypeptide containing a dipeptide repeat sequence and an environmentally responsive fluorophore covalently bonded thereto, with a liquid-liquid phase separation-associated protein or a nuclear pore complex family protein, wherein the dipeptide repeat sequence is composed of a repeating unit selected from the group consisting of proline-arginine, glycine-arginine, proline-alanine, glycine-alanine, and glycine-proline; and (2) measuring the fluorescence intensity of the probe.

[0010] The sample is preferably a biological sample.

[0011] The dipeptide repeat sequence preferably contains 3 to 200 of the repeating units.

[0012] The environmentally responsive fluorophore is preferably selected from the group consisting of a fluorophore having a naphthalenesulfonic acid skeleton, a fluorophore having a benzofurazan skeleton, a fluorophore having a xanthene skeleton, a fluorophore having a pyrene skeleton, and an aggregation-induced emission fluorophore.

[0013] The liquid-liquid phase separation-associated protein is preferably FUS.

[0014] The nuclear pore complex family protein is preferably karyopherin β2.

[0015] Preferably, the neurodegenerative disease is amyotrophic lateral sclerosis or frontotemporal lobar degeneration.

[0016] According to the method of the present invention, proteins that interact with DPRs and compounds that inhibit the interaction between DPRs and proteins can be detected simply by detecting a change in the fluorescence intensity of a probe comprising a polypeptide containing a dipeptide repeat sequence and an environmentally responsive fluorophore covalently bonded to the polypeptide. Therefore, the method of the present invention allows for easy and rapid screening of proteins that interact with DPRs and compounds that inhibit the interaction between DPRs and proteins, and is useful for drug discovery for neurodegenerative diseases.

[0017] Figure 1 is a schematic diagram of a method for screening proteins that interact with DPRs. Figure 2 is a schematic diagram of a method for screening compounds that inhibit the interaction between DPRs and proteins. Figure 3 shows the structural formulas of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2). Figure 4 shows the fluorescence spectra and changes in fluorescence intensity of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2) upon contact with Kapβ2. Figure 5 shows the fluorescence spectra and changes in fluorescence intensity of Dnc-PR10 (probe 3), Dnc-GP10 (probe 4), Dnc-GA10 (probe 5), Dnc-GR10 (probe 6), and Dnc-PA10 (probe 7) upon contact with Kapβ2. Figure 6 shows the fluorescence spectra and changes in fluorescence intensity of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2) upon contact with FUS. Figure 7 shows the changes in the fluorescence spectra of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2) after contact with various proteins. Figure 8 is a graph showing the changes in fluorescence intensity of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2) after contact with various proteins. Figure 9 shows the changes in the fluorescence spectra and fluorescence intensity of a Dnc-PR20 (probe 1) / Kapβ2 mixed solution to which poly K50 or IgG was added. Figure 10 is a graph showing the changes in fluorescence intensity of a Dnc-PR20 (probe 1) / Kapβ2 mixed solution containing various additives. Figure 11 is a graph showing the changes in fluorescence intensity of a Dnc-PR20 (probe 1) / Kapβ2 mixed solution to which various protein-protein interaction (PPI) inhibitors were added. Figure 12 shows the changes in the fluorescence intensity of Compound No. 10 shows the fluorescence spectrum and changes in fluorescence intensity of a Dnc-PR20 (probe 1) / Kapβ2 mixed solution to which the inhibitor No. 8 was added.

[0018] The present invention will be described in detail below, but the present invention is not limited to the embodiments described in this specification.

[0019] According to a first embodiment, the present invention provides a method for screening for proteins associated with neurodegenerative diseases, comprising: (1) contacting a probe, which comprises a polypeptide having a dipeptide repeat sequence and an environmentally responsive fluorophore covalently bonded thereto, with a sample containing the protein, wherein the dipeptide repeat sequence is composed of a repeating unit selected from the group consisting of proline-arginine, glycine-arginine, proline-alanine, glycine-alanine, and glycine-proline; and (2) measuring the fluorescence intensity of the probe.

[0020] First, the probe used in the method of this embodiment will be described. The probe in this embodiment comprises a polypeptide containing a dipeptide repeat sequence and an environment-responsive fluorophore covalently bonded thereto.

[0021] Dipeptide repeat proteins (hereinafter, also referred to as "DPRs") are neurotoxic proteins associated with neurodegenerative diseases. DPRs have been shown to be produced due to the abnormal amplification of a hexanucleotide (GGGGCC) repeat within intron 1 of the chromosome 9 open reading frame 72 (C9orf72) gene. DPRs are translated from six reading frames in the sense or antisense orientation of the hexanucleotide repeat and therefore consist of repeating diamino acid units of proline-arginine (PR), glycine-arginine (GR), proline-alanine (PA), glycine-alanine (GA), or glycine-proline (GP).

[0022] The polypeptide used in the probe of this embodiment comprises a dipeptide repeat sequence consisting of any of the above repeating units, i.e., a dipeptide repeat sequence consisting of repeating units selected from the group consisting of PR, GR, PA, GA, and GP. The number of repeating units in the dipeptide repeat sequence is not particularly limited and may be, for example, 3 to 200, 5 to 100, or 5 to 50, preferably 5 to 30. In other words, the dipeptide repeat sequence in this embodiment is (PR) ｎ , (GR) ｎ , (PA) ｎ, (GA) ｎ and (GP) ｎ and n may be, for example, 3 to 200, 5 to 100, or 5 to 100, preferably 5 to 30.

[0023] In this embodiment, the polypeptide comprising a dipeptide repeat sequence may consist solely of the dipeptide repeat sequence, or may have an amino acid residue (e.g., a lysine residue) suitable for modification with a tag such as GST, His6, MBP, HA, or FLAG, or an environmentally responsive fluorophore, added to the N-terminus and / or C-terminus, either directly or via a linker.

[0024] The environmentally responsive fluorophore used in the probe of this embodiment is not particularly limited and may be any fluorophore whose fluorescence properties change depending on the environment around the fluorescent molecule. Examples of such fluorophores include fluorophores whose fluorescence properties change depending on the polarity around the fluorescent molecule, fluorophores whose fluorescence properties change depending on the pH around the fluorescent molecule, and fluorophores whose fluorescence properties change depending on the degree of crowding around the fluorescent molecule.

[0025] Examples of fluorophores whose fluorescent properties change depending on the polarity around the fluorescent molecule include fluorophores having a naphthalenesulfonic acid skeleton, such as 5-dimethylaminonaphthalene-1-sulfonyl (dansyl), 1-anilinonaphthalene-8-sulfonic acid (ANS), N-methyl-2-anilinonaphthalene-6-sulfonic acid (MANS), and 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS); fluorophores having a benzofurazan skeleton, such as 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD), 7-nitro-2,1,3-benzoxadiazole (NBD), 4-(aminosulfonyl)-2,1,3-benzoxadiazole (ABD), and ammonium 2,1,3-benzoxadiazole-4-sulfonate (SBD); and fluorescent derivatives thereof.

[0026] Examples of fluorophores whose fluorescence properties change depending on the pH around the fluorescent molecule include fluorophores having a xanthene skeleton, such as fluorescein, fluorescein isothiocyanate (FITC), 5(6)-carboxyfluorescein (5(6)-FAM), 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and seminaphthalodafluorescein (SNARF); fluorophores having a pyrene skeleton, such as 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HTPS); and fluorescent derivatives thereof.

[0027] Examples of fluorophores whose fluorescence properties change depending on the degree of crowding around the fluorescent molecule include aggregation-induced emission (AIE) fluorophores such as tetraphenylethylene (TPE), 10,10',11,11'-tetrahydro-5,5'-bisbenzo[a,d][7]annulenylidene (THBA), and 1,1,2,3,4,5-hexaphenylsilole (HPS), as well as fluorescent derivatives thereof.

[0028] A preferred environmentally responsive fluorophore in this embodiment may be dansyl, NBD, DBD or TPE or a fluorescent derivative thereof.

[0029] The probe of this embodiment comprises a polypeptide comprising a dipeptide repeat sequence and an environmentally responsive fluorophore covalently bonded to the polypeptide. The environmentally responsive fluorophore may be introduced at any one or more positions of the polypeptide, for example, at either or both of the N-terminus and C-terminus, and / or at the side chain of an amino acid residue in the polypeptide. In the probe of this embodiment, the environmentally responsive fluorophore is preferably introduced at the N-terminus and / or C-terminus of the polypeptide, and particularly preferably at the N-terminus.

[0030] The probe of this embodiment can be prepared by synthesizing a polypeptide and labeling it with a fluorophore by a conventionally known method. For example, the probe of this embodiment can be prepared by chemically or biosynthesizing a polypeptide containing a dipeptide repeat sequence and labeling an amino group in the polypeptide with an environment-responsive fluorophore activated by an active ester group such as an N-hydroxysuccinimide (NHS) ester group or a pentafluorophenyl (PFP) ester group, a maleimide group, an isothiocyanate group, or a halogenated alkyl group.

[0031] In the method of this embodiment, the probe is contacted with a sample containing a protein. The sample in this embodiment is not particularly limited as long as it contains a protein, and may contain one type of protein or multiple types of proteins. The type of protein is also not particularly limited and may be any protein that can be expressed in any living organism, for example, a vertebrate, preferably a mammal such as a mouse, rat, rabbit, dog, monkey, or human, particularly preferably a human, and may include not only known proteins but also unknown proteins. Furthermore, the protein targeted by the method of this embodiment may be the entire protein or a peptide fragment thereof, and may further include any post-translational modification.

[0032] The sample in this embodiment may preferably be a biological sample such as a biological fluid, an extract from a cell or tissue, or a cell or tissue culture supernatant. The biological fluid may be blood, plasma, serum, cerebrospinal fluid (CSF), saliva, urine, or the like. The cells may be neurons, glial cells, fibroblasts, stem cells, endothelial cells, pericytes, or the like, which may be primary cultured cells derived from a living organism or established cell lines. The tissue may be central nervous tissue, peripheral nervous tissue, brain organoids, or the like. The biological sample may be derived from any living organism, for example, from a vertebrate, preferably from a mammal such as a mouse, rat, rabbit, dog, monkey, or human, and particularly preferably from a human.

[0033] To contact a protein-containing sample with a probe, a reaction solution is prepared by mixing the two and incubating for a certain period of time. The ionic strength and pH of the reaction solution are not particularly limited, and the reaction solution may contain any buffer and / or salt, and may further contain an organic solvent such as ethanol or glycerin. Examples of buffers include HEPES, MOPS, MES, EPPS, Tris, phosphoric acid, acetic acid, citric acid, and glycine. Examples of salts include NaCl, KCl, and MgCl. ２ , Na ２ SO ４ , K. ２ SO ４ , MgSO ４ , NaI, NaSCN, etc.

[0034] The reaction solution in this embodiment may preferably be one that conforms to in vivo conditions. Therefore, the pH of the reaction solution is preferably 4.0 to 8.0, and the ionic strength of the reaction solution is preferably 10 to 500 mM. The reaction solution in this embodiment may be a brain tissue extract or a buffer prepared based on the composition thereof (e.g., HEPES-buffered aCSF (artificial cerebrospinal fluid)).

[0035] The final concentration of the protein in the reaction solution may be 1 nM to 1000 μM, preferably 10 nM to 100 μM. If the protein concentration in the sample to be added is unknown, the sample may be serially diluted as appropriate. The incubation time of the reaction solution may be, for example, 10 seconds to 72 hours.

[0036] Next, the fluorescence intensity of the probe is measured. In the method of this embodiment, the fluorescence intensity can be measured at an excitation wavelength of 300 to 500 nm and a fluorescence wavelength of 400 to 700 nm. In the method of this embodiment, the fluorescence intensity may be measured at one or more excitation wavelength / fluorescence wavelength sets. For example, the fluorescence intensity can be measured at one, two, three, or four excitation wavelength / fluorescence wavelength sets selected from excitation wavelength (nm) / fluorescence wavelength (nm): 340 / 520, 330 / 480, 345 / 505, 360 / 530, etc.

[0037] To determine whether a protein in a sample has bound to a probe, for example, a control that does not contain the protein (such as a solution containing only the probe) can be analyzed in parallel and compared, or the results can be compared with the analytical results of a control performed in the past. Alternatively, a reaction solution in which a sample containing only a protein that has already been shown not to interact with DPRs is mixed with the probe can be used as a control. In the method of this embodiment, if the fluorescence intensity of the probe contacted with the sample is significantly increased compared to the fluorescence intensity of the probe in the control, it can be determined that a protein that can interact with DPRs, i.e., a protein that may be associated with neurodegenerative disease, is present in the sample.

[0038] "Neurodegenerative disease" is a general term for progressive diseases in which nerve cells in the central nervous system gradually degenerate and lead to cell death.Neurodegenerative diseases in this embodiment include, but are not limited to, frontotemporal lobar degeneration (FTLD), such as frontotemporal dementia (FTD), progressive non-fluent aphasia (PNFA) and semantic dementia (SD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), Huntington's disease (HD), dementia with Lewy bodies (DLB), corticobasal syndrome (CBS), and progressive supranuclear palsy (PSP).The neurodegenerative disease in this embodiment is preferably ALS or FTLD.

[0039] An outline of the method of this embodiment is shown in FIG. 1. In FIG. 1, dansyl-introduced DPRs (Dnc-DPRs) are shown as an example of a probe. When the probe interacts with a protein that can interact with DPRs ("potential binder" in the figure), the fluorescence intensity of the probe increases (turn-on response). Therefore, according to the method of this embodiment, proteins that can interact with DPRs, i.e., proteins that may be related to neurodegenerative diseases, can be screened based on the increase in fluorescence intensity of the probe.

[0040] According to a second embodiment, the present invention provides a method for screening for a therapeutic or preventive agent for a neurodegenerative disease, comprising: (1) contacting a probe, in the presence of a candidate compound, with a liquid-liquid phase separation-associated protein or a nuclear pore complex family protein, the probe comprising a polypeptide containing a dipeptide repeat sequence and an environment-responsive fluorophore covalently bonded thereto, wherein the dipeptide repeat sequence is composed of a repeating unit selected from the group consisting of proline-arginine, glycine-arginine, proline-alanine, glycine-alanine, and glycine-proline; and (2) measuring the fluorescence intensity of the probe.

[0041] In this embodiment, the terms "dipeptide repeat sequence," "environmentally responsive fluorophore," "probe comprising a polypeptide containing a dipeptide repeat sequence and an environmentally responsive fluorophore covalently bonded together," and "neurodegenerative disease" are the same as those defined in the first embodiment.

[0042] In this embodiment, "treatment" (and grammatical variations thereof) means preventing or alleviating the progression and worsening of the pathology of a neurodegenerative disease in a subject suffering from the disease, and includes not only completely curing the disease but also alleviating the symptoms of the disease. Furthermore, in this embodiment, "prevention" (and grammatical variations thereof) means preventing a neurodegenerative disease from occurring in a subject at risk of contracting the disease. The "subject" may be any mammal, but is preferably a human. The subject may be of any age, including infants, young children, adolescents, adults, and elderly subjects.

[0043] In the method of this embodiment, a probe comprising a polypeptide containing a dipeptide repeat sequence and an environmentally responsive fluorophore covalently linked thereto is contacted with a liquid-liquid phase separation-associated protein or a nuclear pore complex family protein in the presence of a candidate compound.

[0044] "Liquid-liquid phase separation (LLPS)-associated protein" refers to a protein involved in the formation of liquid-liquid phase separation (LLPS). LLPS-associated proteins have been found in a wide range of biological species, including eukaryotes, prokaryotes, and viruses, and they commonly have intrinsically disordered regions, particularly low complexity (LC) domains. Information on LLPS-associated proteins can be obtained from databases such as PhaSepDB (http: / / db.phasep.pro / ), DrLLPS (http: / / llps.biocuckoo.cn / ), LLPSDB (http: / / bio-comp.org.cn / llpsdb / home.html), and PhaSePro (https: / / phasepro.elte.hu / ).

[0045] The LLPS-associated protein in this embodiment may preferably be an FET family protein or a heterogeneous nuclear ribonucleoprotein. An "FET family protein" is an RNA-binding protein having an LC domain at the N-terminus. FET family proteins include FUS (fused in sarcoma, also known as TLS (translocated in liposarcoma)), EWSR1 (Ewing's sarcoma breakpoint region 1, also known as RNA-binding protein EWS), and TAF15 (TATA-binding protein-associated factor 15). A "heterogeneous nuclear ribonucleoprotein (hnRNP)" is an RNA-binding protein having an LC domain at the C-terminus. Examples of hnRNPs include, but are not limited to, TDP-43 (TAR DNA-binding protein 43 kDa), hnRNP A0, hnRNP A1, hnRNP A2, hnRNP A3, hnRNP H1, hnRNP H2, hnRNP H3, AUF1, and PTBP1. The LLPS-related protein in this embodiment is particularly preferably FUS.

[0046] "Nuclear pore complex (NPC) family proteins" are proteins involved in molecular transport between the cytoplasm and the nucleus and control LLPS. Examples of NPC family proteins include, but are not limited to, karyopherin β2 (also known as importin β2 or transportin 1), karyopherin β2b (also known as importin 3 or transportin 2), karyopherin β1 (also known as importin β1), importin α1, and importin α2. The NPC family protein in this embodiment is preferably karyopherin β2 (Kapβ2).

[0047] The LLPS-related proteins and NPC family proteins in this embodiment may be derived from any vertebrate, preferably from mammals such as mice, rats, rabbits, dogs, monkeys, and humans, and particularly preferably from humans.

[0048] Information on the amino acid sequences of LLPS-related proteins and NPC family proteins and the nucleic acid sequences encoding them can be obtained from certain databases. For example, the amino acid sequence and gene sequence of human Kapβ2 are available under NCBI Gene ID: 3842, and the amino acid sequence and gene sequence of human FUS are available under NCBI Gene ID: 2521.

[0049] The LLPS-associated proteins and NPC family proteins of this embodiment may also include their variants and homologs having equivalent activity. In other words, the LLPS-associated proteins of this embodiment may include proteins consisting of amino acid sequences that share 80% or more, preferably 90% or more, and more preferably about 95% or more identity with the amino acid sequences registered in the database, as long as the LLPS-forming activity is maintained. Furthermore, the NPC family proteins of this embodiment may include proteins consisting of amino acid sequences that share 80% or more, preferably 90% or more, and more preferably about 95% or more identity with the amino acid sequences registered in the database, as long as the LLPS-regulating activity is maintained. Amino acid sequence identity can be calculated using sequence analysis software or programs commonly used in the art (e.g., FASTA, BLAST).

[0050] Furthermore, the LLPS-related proteins and NPC family proteins of this embodiment may also include proteins consisting of amino acid sequences registered in a database in which one to several amino acids have been substituted, deleted, inserted, and / or added, as long as their LLPS-forming activity or LLPS-regulating activity is maintained. Here, "one to several" may mean, for example, 1 to 30 amino acids, preferably 1 to 10 amino acids, and particularly preferably 1 to 5 amino acids.

[0051] In this embodiment, the LLPS-related protein and NPC family protein may have a tag such as GST, His6, MBP, HA, or FLAG attached to the N-terminus and / or C-terminus directly or via a linker.

[0052] The LLPS-related proteins and NPC family proteins of this embodiment can be prepared by any conventionally known genetic engineering method. Specifically, for example, expression vectors containing nucleic acids encoding the LLPS-related proteins and NPC family proteins may be prepared and then introduced into host cells such as Escherichia coli for expression.

[0053] To contact the probe with the LLPS-related protein or NPC family protein in the presence of a candidate compound, a reaction solution is prepared by mixing the probe, the LLPS-related protein or NPC family protein, and the candidate compound, and then incubating the mixture for a certain period of time. The protein concentration, ionic strength, and pH of the reaction solution in this embodiment may be the same as those defined in the first embodiment.

[0054] The "candidate compound" in this embodiment may be a low molecular weight compound, nucleic acid, protein, peptide, antibody, lipid, etc., or a mixture thereof (e.g., an extract from a cell or tissue, a culture supernatant of a cell or tissue, etc.). Furthermore, these candidate compounds may be novel or known. The final concentration of the candidate compound added to the reaction solution varies depending on the type of candidate compound, but for example, in the case of a low molecular weight compound, it can be appropriately selected from the range of 1 nM to 10 mM. The incubation time may be, for example, 10 seconds to 72 hours.

[0055] Next, the fluorescence intensity of the probe is measured. The conditions for measuring the fluorescence intensity in this embodiment may be the same as those defined in the first embodiment.

[0056] To determine whether the addition of a candidate compound has changed the fluorescence intensity of the probe, a reaction solution to which the candidate compound has not been added may be analyzed in parallel for comparison, or the results may be compared with the results of a previous analysis of a reaction solution to which the candidate compound has not been added. In the method of this embodiment, if the fluorescence intensity of the probe contacted with the sample is significantly reduced compared to the fluorescence intensity of the probe in a reaction solution to which the candidate compound has not been added, it can be determined that the candidate compound is capable of inhibiting the interaction between DPRs and LLPS-associated proteins / NPC family proteins, i.e., capable of normalizing LLPS regulation, and therefore the candidate compound can be evaluated as a promising therapeutic or preventive agent for neurodegenerative diseases.

[0057] An outline of the method of this embodiment is shown in Figure 2. When the probe interacts with an LLPS-associated protein / NPC family protein, the probe emits fluorescence; however, when the interaction is inhibited, the probe dissociates from the LLPS-associated protein / NPC family protein, and the fluorescence intensity of the probe decreases (turn-off response). Therefore, according to the method of this embodiment, it is possible to screen, based on the decrease in fluorescence intensity of the probe, for compounds that can inhibit the interaction between DPRs and LLPS-associated proteins / NPC family proteins and normalize LLPS regulation, i.e., compounds that are promising as therapeutic or preventive agents for neurodegenerative diseases.

[0058] The present invention will be further described below with reference to examples, which should not be construed as limiting the scope of the present invention.

[0059] <1. Materials and Reagents> (1-1) Probes The following probes 1 to 7 (>95% purity) were synthesized by Biologica and used without further purification. As representative examples, the structural formulae of probe 1 (SEQ ID NO: 1) and probe 2 (SEQ ID NO: 2) are shown in Figure 3. Probe 1: dansyl-NH-(Pro-Arg) ２０ -COOH (SEQ ID NO: 1) (hereinafter referred to as "Dnc-PR20") Probe 2: dansyl-NH-(Gly-Pro) ２０ —COOH (SEQ ID NO: 2) (hereinafter referred to as “Dnc-GP20”) Probe 3: dansyl-NH-(Pro-Arg) １０ -COOH (SEQ ID NO: 3) (hereinafter referred to as "Dnc-PR10") Probe 4: dansyl-NH-(Gly-Pro) １０ -COOH (SEQ ID NO: 4) (hereinafter referred to as "Dnc-GP10") Probe 5: dansyl-NH-(Gly-Ala) １０ —COOH (SEQ ID NO: 5) (hereinafter referred to as “Dnc-GA10”) Probe 6: dansyl-NH-(Gly-Arg) １０ —COOH (SEQ ID NO: 6) (hereinafter referred to as “Dnc-GR10”) Probe 7: dansyl-NH-(Pro-Ala) １０—COOH (SEQ ID NO: 7) (hereinafter referred to as “Dnc-PA10”)

[0060] (1-2) Proteins: Bovine milk-derived β-lactoglobulin, human serum-derived α1-antitrypsin, human serum-derived albumin, human serum-derived apotransferrin, and chicken egg white-derived lysozyme were purchased from Sigma-Aldrich. Human serum-derived immunoglobulin G was purchased from Equitech-Bio, Inc.

[0061] MBP-tagged human FUS (Cell, 173(3): 693-705.e22(2018); hereinafter referred to as "MBP-FUS"), human karyopherin β2 (Cell, 173(3): 693-705.e22(2018); hereinafter referred to as "Kapβ2"), C. C. elegans importin β family protein (UniProt accession number: A0A131MBF9; hereinafter referred to as "Imb-2"), human heterogeneous nuclear ribonucleoprotein A2 (Cell, 163(4): 829-839(2015); hereinafter referred to as "hnRNPA2LC"), and human peptidylprolyl cis-trans isomerase A (Cell, 163(4): 829-839(2015); hereinafter referred to as "PPIA") were kindly provided by Professor Tomohide Saio of the Institute for Advanced Enzyme Research, Tokushima University. Human importin α1 (Cell, 173(3): 693-705.e22(2018); hereinafter referred to as "Impα"), human importin β1 (Cell, 173(3): 693-705.e22(2018); hereinafter referred to as "Impβ"), GST-tagged M9M peptide (Cell, 173(3): 693-705.e22(2018); hereinafter referred to as "GST-M9M"), and human GPT-bound Ran (Q69L) (UniProt accession number: P62826; hereinafter referred to as "RanGTP") were kindly provided by Assistant Professor Takuya Yoshizawa of the College of Life Sciences, Ritsumeikan University.

[0062] (1-3) Reagents: ATP, Tris-HCl, dithiothreitol (DTT), polyamidoamine dendrimer (PAMAM), and 50-mer polylysine (poly K50) were purchased from Sigma-Aldrich. NaCl was purchased from Fujifilm Wako Pure Chemical Industries, Ltd.

[0063] <2. Changes in Probe Fluorescence Induced by Kapβ2> Probe solutions (final concentration 300 nM) of probes 1 to 7 were prepared using a buffer solution (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM DTT (final concentration)). Each probe solution was added to a 384-well microplate (Corning, 3575) at 30 μL / well using an automated pipetting device (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, the fluorescence intensity at an excitation wavelength of 340 nm (fluorescence wavelength 520 nm) and the fluorescence spectrum (fluorescence wavelength 400-700 nm) were measured using a microplate reader (Cytation5, BioTek). Thereafter, 30 μL / well of Kapβ2 solutions (Kapβ2 / pure water) at various concentrations were added using an automatic dispenser, and after incubation at 35° C. for 10 minutes, the fluorescence intensity and fluorescence spectrum were measured under the same conditions.

[0064] The results for probes 1 and 2 are shown in Figure 4. Figure 4(a) shows the fluorescence spectrum of Dnc-PR20 (probe 1), and Figure 4(b) shows the fluorescence spectrum of Dnc-GP20 (probe 2). The fluorescence intensity of Dnc-PR20 increased in a Kapβ2 concentration-dependent manner, with the fluorescence wavelength peak shifting from approximately 590 nm to 540 nm. The 540 nm fluorescence intensity of Dnc-PR20 at 1600 nM Kapβ2 increased approximately 6.1-fold compared to the fluorescence intensity at 0 nM Kapβ2. On the other hand, the fluorescence spectrum of Dnc-GP20 remained almost unchanged with the addition of Kapβ2. Figure 4(c) shows the change in 520 nm fluorescence intensity of probes 1 and 2. The X axis indicates the Kapβ2 concentration, and the Y axis indicates the mean fluorescence intensity ± standard error (n = 3). These results confirmed that Dnc-PR20 binds to Kapβ2, but Dnc-GP20 does not.

[0065] The results for probes 3 to 7 are shown in Figure 5. Figure 5(a) shows the fluorescence spectrum of Dnc-PR10 (probe 3), Figure 5(b) shows the fluorescence spectrum of Dnc-GP10 (probe 4), Figure 5(c) shows the fluorescence spectrum of Dnc-GA10 (probe 5), Figure 5(d) shows the fluorescence spectrum of Dnc-GR10 (probe 6), and Figure 5(e) shows the fluorescence spectrum of Dnc-PA10 (probe 7). Figure 5(f) shows the change in fluorescence intensity at 520 nm for probes 3 to 7. The X-axis shows the concentration of Kapβ2, and the Y-axis shows the mean value ± standard error of the fluorescence intensity (n = 3). The fluorescence intensity of Dnc-PR10 and Dnc-GR10 increased in a Kapβ2 concentration-dependent manner, whereas the fluorescence spectra of Dnc-GP10, Dnc-GA10, and Dnc-PA10 did not change. These results confirmed that Dnc-PR10 and Dnc-GR10 bind to Kapβ2, but Dnc-GP10, Dnc-GA10, and Dnc-PA10 do not. These results are consistent with previous findings (Nat. Commun., 2021;12(1):5301).

[0066] 3. Changes in Fluorescence of Probes Caused by FUS The fluorescence intensity and fluorescence spectra of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2) were measured using the same procedures as in 2 above, except that an MBP-tagged FUS (MBP-FUS) solution (MBP-FUS / pure water) was used instead of the Kapβ2 solution.

[0067] The results are shown in Figure 6. Figure 6(a) shows the fluorescence spectrum of Dnc-PR20 (probe 1), and Figure 6(b) shows the fluorescence spectrum of Dnc-GP20 (probe 2). The fluorescence intensity of Dnc-PR20 increased in an MBP-FUS concentration-dependent manner, with the fluorescence wavelength peak shifting from approximately 590 nm to 540 nm. The 540 nm fluorescence intensity of Dnc-PR20 in the presence of 1600 nM MBP-FUS increased approximately 4.6-fold compared to the fluorescence intensity in the presence of 0 nM MBP-FUS. On the other hand, the fluorescence spectrum of Dnc-GP20 showed little change with the addition of MBP-FUS. Figure 6(c) shows the change in 520 nm fluorescence intensity of probes 1 and 2. The X axis indicates the MBP-FUS concentration, and the Y axis indicates the mean ± standard error of the fluorescence intensity (n = 3). These results confirmed that Dnc-PR20 binds to MBP-FUS, but Dnc-GP20 does not.

[0068] 4. Changes in Fluorescence of Probes Induced by Various Proteins (1) The fluorescence intensity and fluorescence spectra of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2) were measured using the same procedures as in 2 above, except that an aqueous solution (1600 nM) of bovine milk-derived β-lactoglobulin, human serum-derived α1-antitrypsin, human serum-derived albumin (HSA), human serum-derived apotransferrin, chicken egg white-derived lysozyme, or human serum-derived immunoglobulin G (IgG) was used instead of the Kapβ2 solution.

[0069] The results are shown in Figure 7. Figure 7(a) shows the fluorescence spectrum of Dnc-PR20 (probe 1), and Figure 7(b) shows the fluorescence spectrum of Dnc-GP20 (probe 2). The addition of β-lactoglobulin shifted the fluorescence wavelength peak of Dnc-PR20 from approximately 590 nm to 540 nm, and the fluorescence intensity at 540 nm increased slightly. The addition of other proteins did not change the fluorescence spectrum of Dnc-PR20 very much. The fluorescence spectrum of Dnc-GP20 also did not change very much with the addition of any of the proteins. These results revealed that Dnc-PR20 interacts slightly with β-lactoglobulin, but the interaction is much weaker than with Kapβ2 or MBP-FUS.

[0070] 5. Changes in Fluorescence of Probes Induced by Various Proteins (2) In addition to the proteins tested in sections 2 to 4 above, aqueous solutions of proteins that may be involved in the formation or regulation of liquid-liquid phase separation (LLPS) were used to measure the 520 nm fluorescence intensity of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2) using the same procedure as in section 2 above. The following proteins were used as proteins that may be involved in the formation or regulation of LLPS: Imb-2, hnRNPA2LC, PPIA, Impα, Impβ, GST-M9M, and RanGTP. The concentration of each aqueous protein solution was 1600 nM (800 nM for the Imb-2 aqueous solution only).

[0071] The results are shown in Figure 8. The 520 nm fluorescence intensity of Dnc-PR20 increased to over 1,000 when Imb-2, Impα, and Impβ were added in addition to Kapβ2, MBP-FUS, and β-lactoglobulin confirmed in sections 2 to 4 above. It has previously been reported that Kapβ2, importin α / β, and FUS bind to DPRs, and these results confirmed that DPR-binding proteins can be detected by the increase in the fluorescence intensity of Dnc-PR20.

[0072] These results suggest that screening for proteins that interact with DPRs is possible using polypeptide probes containing an environmentally responsive fluorophore-DPR sequence.

[0073] <6. Screening for DPRs-Kapβ2 binding inhibitors (1)> A mixed solution of Dnc-PR20 (final concentration 300 nM) and Kapβ2 (final concentration 400 nM) was prepared using a buffer solution (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM DTT (final concentration)), and added to a 384-well microplate (Corning, 3820) at 20 μL / well using an automatic pipetting device (Andrew+, Andrew Alliance). After incubation at 35 ° C for 10 minutes, the fluorescence intensity at an excitation wavelength of 340 nm (fluorescence wavelength 520 nm) and the fluorescence spectrum (fluorescence wavelength 400-700 nm) were measured using a microplate reader (Cytation5, BioTek). Subsequently, 5 μL / well of various concentrations of additives (polyK50 or IgG / pure water) was added using an automatic pipetting device, and the plates were incubated at 35°C for 10 minutes. The fluorescence intensity and fluorescence spectrum were then measured under the same conditions. As a control, the fluorescence intensity and fluorescence spectrum of a Dnc-PR20 solution containing no Kapβ2 were also measured in the same manner.

[0074] The results are shown in Figure 9. Figure 9(a) shows the fluorescence spectrum of a Dnc-PR20 / Kapβ2 solution supplemented with polyK50 (0-300 nM), and Figure 9(b) shows the fluorescence spectrum of a Dnc-PR20 / Kapβ2 solution supplemented with IgG (0-300 nM). When polyK50 was added to the Dnc-PR20 / Kapβ2 solution, the fluorescence intensity of Dnc-PR20 attenuated in a polyK50 concentration-dependent manner, and the addition of 200 nM polyK50 restored the fluorescence intensity to the same level as the control. On the other hand, the addition of IgG hardly changed the fluorescence intensity of Dnc-PR20. Figure 9(c) shows the change in fluorescence intensity at 520 nm. The X axis indicates the concentration of the additive, and the Y axis indicates the mean value of the fluorescence intensity ± standard error (n = 3). These results demonstrate that polyK50 inhibited the interaction between Dnc-PR20 and Kapβ2. Although not wishing to be bound by a particular theory, polyK50 is cationic like PR20, but has a larger number of charges per molecule than PR20, and is therefore thought to interact more strongly with Kapβ2.

[0075] 7. Screening of DPRs-Kapβ2 Binding Inhibitors (2) The fluorescence intensity of Dnc-PR20 at 520 nm was measured using the same procedure as in 6 above, except that in addition to poly K50 and IgG, PAMAM, GST-M9M, RanGTP, ATP, and HSA were used as additives.

[0076] The results are shown in Figure 10. In the figure, "none" indicates a Dnc-PR20 solution containing no Kapβ2, and "Kapβ2" indicates a Dnc-PR20 / Kapβ2 mixed solution. The 520 nm fluorescence intensity of Dnc-PR20 was also attenuated when polyK50, PAMAM, a cationic synthetic dendrimer, or M9M peptide, a known Kapβ2 inhibitor, was added. Other additives caused almost no change in the fluorescence intensity of Dnc-PR20. These results demonstrate that compounds that inhibit the interaction between DPRs and Kapβ2 can be detected by the attenuation of Dnc-PR20 fluorescence intensity.

[0077] These results suggest that it is possible to screen for compounds that inhibit the interaction between DPRs and Kapβ2 using a polypeptide probe containing an environmentally responsive fluorophore-DPR sequence.

[0078] <8. Screening for DPRs-Kapβ2 Binding Inhibitors (3)> (8-1) Screening of PPI Inhibitor Library Using 47 protein-protein interaction (PPI) inhibitors selected from the PPI Inhibitor Library (TargetMol, L9400), a demonstration experiment was conducted to screen for compounds that inhibit the interaction between DPRs and Kapβ2. The mixed solution of Dnc-PR20 (final concentration 300 nM) and Kapβ2 (final concentration 400 nM) prepared in 6 above was added to a 384-well microplate (Corning, 3820) at 20 μL / well using an automatic pipetting device (ASSIST PLUS, Integra Biosciences). After incubation at 35°C for 10 minutes, fluorescence intensity at an excitation wavelength of 340 nm (fluorescence wavelength 520 nm) was measured using a microplate reader (Synergy H1, Agilent Technologies). Subsequently, various PPI inhibitors (compounds No. 01-47, compound / 25 vol% DMSO) were added at 5 μL / well using an automated dispenser. After incubation at 35°C for 10 minutes, fluorescence intensity was measured under the same conditions. As a control, fluorescence intensity was also measured for a Dnc-PR20 solution that did not contain Kapβ2.

[0079] The results are shown in Figure 11. The upper and lower panels show the 520 nm fluorescence intensity (mean ± standard error) of Dnc-PR20 / Kapβ2 solutions containing 10 μM and 100 μM PPI inhibitors, respectively. In the figure, "none" indicates a Dnc-PR20 solution without Kapβ2, and "Kapβ2" indicates a Dnc-PR20 / Kapβ2 mixed solution. One compound at 10 μM and 13 compounds at 100 μM significantly altered the 520 nm fluorescence intensity of the solution (vs. Dnc-PR20 / Kapβ2 mixed solution, *1 P<0.05, *2 P<0.01, *4 P<0.0001; one-way ANOVA with Tukey's post-hoc test). Furthermore, it was confirmed that 10 out of 13 compounds significantly attenuated the 520 nm fluorescence intensity of the solution (FIG. 11, black bars).

[0080] (8-2) Inhibitory Effect of Compound No. 08 on DPRs-Kapβ2 Binding Next, the fluorescence intensity of Dnc-PR20 at 520 nm was measured using Compound No. 8, which is representative of the 10 compounds that significantly attenuated the fluorescence intensity at 520 nm of the solution, in the same manner as in (8-1) above.

[0081] The results are shown in Figure 12. Figure 12(a) shows the change in 520 nm fluorescence intensity of a Dnc-PR20 / Kapβ2 solution to which Compound No. 8 (0-100 μM) was added. The X axis represents the concentration of Compound No. 8, and the Y axis represents the mean value of fluorescence intensity ± standard error (n = 3). Figure 12(b) shows the fluorescence spectrum of a Dnc-PR20 / Kapβ2 solution to which Compound No. 8 (0-100 μM) was added. The fluorescence intensity of Dnc-PR20 attenuated in a concentration-dependent manner up to 75 μM of Compound No. 8. These results demonstrate that Compound No. 8 significantly inhibited the interaction between DPRs and Kapβ2, suggesting that Compound No. 8 may be a promising inhibitor of DPRs-Kapβ2 binding.

[0082] The above results demonstrate that compounds that inhibit the interaction between DPRs and Kapβ2 can be selected by this screening method using an environmentally responsive fluorophore-DPR sequence-containing polypeptide probe.

Claims

1. A method for screening for proteins associated with neurodegenerative diseases, comprising: (1) contacting a sample containing a protein with a probe comprising a polypeptide having a dipeptide repeat sequence and an environmentally responsive fluorophore covalently bonded thereto, wherein the dipeptide repeat sequence is comprised of a repeating unit selected from the group consisting of proline-arginine, glycine-arginine, proline-alanine, glycine-alanine and glycine-proline; and (2) measuring the fluorescence intensity of the probe.

2. The method of claim 1, wherein said dipeptide repeat sequence comprises from 3 to 200 of said repeat units.

3. The method according to claim 1 or 2, wherein the environmentally responsive fluorophore is selected from the group consisting of a fluorophore having a naphthalenesulfonic acid skeleton, a fluorophore having a benzofurazan skeleton, a fluorophore having a xanthene skeleton, a fluorophore having a pyrene skeleton, and an aggregation-induced emission fluorophore.

4. The method according to any one of claims 1 to 3, wherein the sample is a biological sample.

5. The method according to any one of claims 1 to 4, wherein the neurodegenerative disease is amyotrophic lateral sclerosis or frontotemporal lobar degeneration.

6. A method for screening for a therapeutic or preventive agent for a neurodegenerative disease, comprising: (1) a step of contacting a probe, comprising a polypeptide containing a dipeptide repeat sequence and an environmentally responsive fluorophore covalently bonded thereto, with a liquid-liquid phase separation-associated protein or a nuclear pore complex family protein in the presence of a candidate compound, wherein the dipeptide repeat sequence is composed of a repeating unit selected from the group consisting of proline-arginine, glycine-arginine, proline-alanine, glycine-alanine and glycine-proline; and (2) a step of measuring the fluorescence intensity of the probe.

7. The method of claim 6, wherein said dipeptide repeat sequence comprises from 3 to 200 of said repeat units.

8. The method according to claim 6 or 7, wherein the environmentally responsive fluorophore is selected from the group consisting of a fluorophore having a naphthalenesulfonic acid skeleton, a fluorophore having a benzofurazan skeleton, a fluorophore having a xanthene skeleton, a fluorophore having a pyrene skeleton, and an aggregation-induced emission fluorophore.

9. The method according to any one of claims 6 to 8, wherein the liquid-liquid phase separation-associated protein is FUS.

10. The method according to any one of claims 6 to 8, wherein the nuclear pore complex family protein is karyopherin β2.

11. The method according to any one of claims 6 to 10, wherein the neurodegenerative disease is amyotrophic lateral sclerosis or frontotemporal lobar degeneration.

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