Neurodegenerative disease-related protein and method for screening for therapeutic or prophylactic agent for neurodegenerative disease

Abstract

Provided is a method for simply and quickly screening for a neurodegenerative disease-related protein or a compound useful for the treatment or prevention of a neurodegenerative disease. In the method, a probe is used in which an environmentally responsive fluorophore is covalently linked to a dipeptide repetitive sequence comprising repetitive units selected from the group consisting of proline-arginine, glycine-arginine, proline-alanine, glycine-alanine, and glycine-proline, and a dipeptide repetitive sequence comprising repetitive units selected from the group consisting of proline-arginine, glycine-arginine, proline-alanine, glycine-alanine, and glycine-proline.

Description

TECHNICAL FIELD

[0001] The present application relates to a protein associated with a neurodegenerative disease and a screening method for a therapeutic or prophylactic drug for a neurodegenerative disease. BACKGROUND

[0002] Currently, with the increase in the number of elderly people, the number of patients with neurodegenerative diseases is increasing, and the establishment of a treatment method is a pressing matter. Neurodegenerative diseases include not only dementia such as Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD), but also amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and the like. As a common pathological feature in neurodegenerative diseases, accumulation and deposition of abnormal proteins called inclusions have been found. For example, in ALS or FTLD belonging 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 the nerve cells. In recent years, it has been clarified that a physical phenomenon called "liquid-liquid phase separation (LLPS)" is associated in the aggregation process of these proteins, and it has also been shown that the function of nuclear transport proteins β 2 (Kap β 2) and the like LLPS control factors is associated with the severity of neurodegenerative diseases (Non-Patent Literature 1).

[0003] On the other hand, there are reports that abnormal expansion of a 6-nucleotide (GGGGCC) repeat within intron 1 of the C9orf72 gene has the potential to affect the pathogenesis of ALS and FTLD. There are several hypotheses for the pathogenesis of the repeat abnormal expansion and ALS / FTLD, one of which is the expression of neurotoxic peptides caused by translation that is independent of the start codon (repeat-associated non-ATG (RAN) translation). Based on RAN translation, neurotoxic peptides expressed from a 6-nucleotide repeat sequence are called dipeptide repeat proteins (DPRs), which are composed of repeats of proline-arginine (PR), glycine-arginine (GR), proline-alanine (PA), glycine-alanine (GA), or glycine-proline (GP). Among them, there are reports that arginine-rich DPRs, namely poly-PR and poly-GR, show the possibility of disrupting LLPS by inhibiting the interaction of FUS and Kap β 2 (Non-Patent Literature 2).

[0004] Based on these insights, elucidation of the interaction of DPRs with proteins is expected to contribute to elucidation of the pathogenesis of neurodegenerative diseases such as ALS, FTLD, and further to development of therapeutic methods for neurodegenerative diseases. However, the conventional general methods for analyzing the interaction between proteins, such as co-immunoprecipitation, NMR, X-ray crystal structure analysis, and ultracentrifugation analysis, require a large amount of labor, time, and cost, and it is impractical to search for proteins interacting with DPRs using these methods.

[0005] Prior Art Documents

[0006] Non-Patent Literature

[0007] Non-Patent Literature 1: Acta Neuropathol., 2016; 132(2): 159-173

[0008] Non-Patent Literature 2: Nat. Commun., 2021; 12(1): 5301 SUMMARY

[0009] Problems to be Solved by the Invention

[0010] The object of the present application is to provide a method for screening a neurodegenerative disease-related protein or a compound useful for the treatment or prevention of a neurodegenerative disease based on the interaction of DPRs with proteins, simply and quickly.

[0011] Means for Solving the Problems

[0012] The present inventors have conducted intensive studies, and as a result, have found that a probe in which a dipeptide repeat sequence-containing polypeptide is covalently linked to an environmentally responsive fluorophore can detect proteins interacting with DPRs, and compounds inhibiting the interaction of DPRs with proteins.

[0013] That is, based on one embodiment, the present application provides a method for screening a neurodegenerative disease-related protein, comprising: (1) a step of bringing a probe in which a dipeptide repeat sequence-containing polypeptide is covalently linked to an environmentally responsive fluorophore into contact with a sample containing proteins, 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.

[0014] In addition, based on one embodiment, the present application provides a method for screening a therapeutic or prophylactic agent for a neurodegenerative disease, comprising: (1) a step of contacting a liquid-liquid phase separation-related protein or a nuclear pore complex family protein with a probe covalently bound to a dipeptide repeat sequence-containing polypeptide and an environmentally responsive fluorophore 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.

[0015] The sample is preferably a biological sample.

[0016] The dipeptide repeat sequence preferably contains 3 to 200 of the repeating units.

[0017] 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.

[0018] The liquid-liquid phase separation-related protein is preferably FUS.

[0019] The nuclear pore complex family protein is preferably nucleoporin β 2.

[0020] The neurodegenerative disease is preferably amyotrophic lateral sclerosis or frontotemporal lobar degeneration.

[0021] Effects of the Invention

[0022] According to the method of the present invention, by detecting only the change in the fluorescence intensity of a probe covalently bound to a dipeptide repeat sequence-containing polypeptide and an environmentally responsive fluorophore, it is possible to detect a protein that interacts with DPRs, or a compound that inhibits the interaction between DPRs and a protein. Therefore, the method of the present invention can be used to simply and quickly screen a protein that interacts with DPRs, or a compound that inhibits the interaction between DPRs and a protein, and can be applied to the development of a therapeutic drug for a neurodegenerative disease. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a schematic diagram of a screening method for a protein that interacts with DPRs.

[0024] Figure 2 is a schematic diagram of a screening method for a compound that inhibits the interaction between DPRs and a protein.

[0025] Figure 3 is a diagram showing the structural formula of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2).

[0026] Figure 4 is a graph showing the fluorescence spectrum and the change in fluorescence intensity of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2) in contact with Kap β 2.

[0027] Figure 5 is a graph showing the fluorescence spectrum and the change 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) in contact with Kap β 2.

[0028] Figure 6 is a graph showing the fluorescence spectrum and the change in fluorescence intensity of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2) in contact with FUS.

[0029] Figure 7 is a graph showing the change in fluorescence spectrum of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2) in contact with various proteins.

[0030] Figure 8 is a graph showing the change in fluorescence intensity of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2) in contact with various proteins.

[0031] Figure 9 is a graph showing the fluorescence spectrum and the change in fluorescence intensity of a Dnc-PR20 (probe 1) / Kap β 2 mixed solution to which polyK50 or IgG was added.

[0032] Figure 10 is a graph showing the change in fluorescence intensity of a Dnc-PR20 (probe 1) / Kap β 2 mixed solution containing various additives.

[0033] Figure 11 is a graph showing the change in fluorescence intensity of a Dnc-PR20 (probe 1) / Kap β 2 mixed solution to which various protein-protein interaction (PPI) inhibitors were added.

[0034] Figure 12 is a graph showing the fluorescence spectrum and the change in fluorescence intensity of a Dnc-PR20 (probe 1) / Kap β 2 mixed solution to which an inhibitor of compound No. 8 was added. DETAILED DESCRIPTION

[0035] Next, the present application will be described in detail, but the present application is not limited to the embodiments described in this specification.

[0036] Based on the first embodiment, the present application provides a screening method for a neurodegenerative disease-related protein, which comprises: (1) a step of contacting a sample containing a protein with a probe in which a dipeptide repeat sequence-containing polypeptide is covalently linked to an environmentally responsive fluorophore, 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.

[0037] First, the probe used in the method of the present embodiment will be described. The probe of the present embodiment is formed by covalently linking a dipeptide repeat sequence-containing polypeptide to an environmentally responsive fluorophore.

[0038] Dipeptide repeat proteins (hereinafter also referred to as "DPRs") are neurotoxic proteins associated with neurodegenerative diseases. It has been clarified that DPRs are produced due to abnormal expansion of a 6-nucleotide (GGGGCC) repeat within intron 1 of chromosome 9 open reading frame 72 (C9orf72) gene. DPRs are translated from 6 reading frames in the sense or antisense direction of the 6-nucleotide repeat, and thus composed of a dipeptide repeat unit of proline-arginine (PR), glycine-arginine (GR), proline-alanine (PA), glycine-alanine (GA), or glycine-proline (GP).

[0039] The polypeptide used in the probe of the present embodiment contains a dipeptide repeat sequence composed of any one of the above-described repeating units, that is, a dipeptide repeat sequence containing a repeating unit 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, for example, be 3 to 200, 5 to 100, or 5 to 50, and may preferably be 5 to 30. In other words, the dipeptide repeat sequence of the present embodiment is selected from the group consisting of (PR) n , (GR) n , (PA) n , (GA) n , and (GP) n , and n may, for example, be 3 to 200, 5 to 100, or 5 to 100, and may preferably be 5 to 30.

[0040] In the present embodiment, the dipeptide repeat sequence-containing polypeptide can be composed only of the dipeptide repeat sequence, or can have a tag such as GST, His6, MBP, HA, FLAG, and the like, an amino acid residue (e.g., a lysine residue) suitable for modification with an environmentally responsive fluorophore, directly or via a linker, at the N terminus and / or the C terminus.

[0041] The environmentally responsive fluorophore used in the probe of the present embodiment is not particularly limited, and can be any fluorophore as long as the fluorescent property changes depending on the environment around the fluorescent molecule. Among such fluorophores, for example, a fluorophore whose fluorescent property changes depending on the polarity around the fluorescent molecule, a fluorophore whose fluorescent property changes depending on the pH around the fluorescent molecule, a fluorophore whose fluorescent property changes depending on the crowding around the fluorescent molecule, and the like can be mentioned.

[0042] As the fluorophore whose fluorescent property changes depending on the polarity around the fluorescent molecule, for example, a fluorophore 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), 2-p-toluidinonaphthalene-6-sulfonic acid (TNS); a fluorophore 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), ammonium 2,1,3-benzoxadiazole-4-sulfonate (SBD); or a fluorescent derivative thereof, and the like can be mentioned.

[0043] As the fluorophore whose fluorescent property changes depending on the pH around the fluorescent molecule, for example, a fluorophore having a xanthene skeleton such as fluorescein, fluorescein isothiocyanate (FITC), 5(6)-carboxyfluorescein (5(6)-FAM), 2’-7’-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), seminaphthorhodafluor (SNARF); a fluorophore having a pyrene skeleton such as trisodium 8-hydroxypyrene-1,3,6-trisulfonate (HTPS); or a fluorescent derivative thereof, and the like can be mentioned.

[0044] As the fluorophore whose fluorescent property changes depending on the crowding around the fluorescent molecule, for example, an aggregation-induced emission (AIE) fluorophore such as tetraphenyl ethene (TPE), 10,10’,11,11’-tetrahydro-5,5’-bisdibenzo[a,d][7]annuleneyl (THBA), 1,1,2,3,4,5-hexaphenylsilole (HPS), or a fluorescent derivative thereof, and the like can be mentioned.

[0045] The preferred environment-responsive fluorophore of the present embodiment can be dansyl, NBD, DBD, or TPE, or a fluorescent derivative thereof.

[0046] The probe of the present embodiment is produced by introducing an environment-responsive fluorophore into a dipeptide repeat sequence-containing polypeptide using covalent bonding. The environment-responsive fluorophore can be introduced into any one or more positions of the polypeptide, such as either or both of the N-terminus and the C-terminus and / or the side chain of an amino acid residue in the polypeptide. In the probe of the present embodiment, the environment-responsive fluorophore is preferably introduced into the N-terminus and / or the C-terminus of the polypeptide, and particularly preferably into the N-terminus.

[0047] The probe of the present embodiment can be produced by synthesizing a polypeptide and labeling it with a fluorophore using a method known in the art. For example, a dipeptide repeat sequence-containing polypeptide can be chemically or biologically synthesized, and the environment-responsive fluorophore activated by an active ester group such as an N-hydroxysuccinimide (NHS) ester group, a pentafluorophenyl (PFP) ester group, a maleimide group, an isothiocyanate group, or a halogenated alkyl group, and the like, can be labeled to an amino group in the polypeptide.

[0048] In the method of the present embodiment, the above-described probe is brought into contact with a protein-containing sample. The sample of the present embodiment is not particularly limited as long as it contains a protein, and can contain one kind of protein or a plurality of kinds of proteins. The kind of protein is also not particularly limited, and can be any kind of protein that can be expressed in any organism, such as a vertebrate (preferably a mammal such as a mouse, a rat, a rabbit, a dog, a monkey, a human, and particularly preferably a human), and can include not only a known kind of protein but also an unknown kind of protein. In addition, the protein targeted by the method of the present embodiment can be the entirety of a protein, a peptide fragment of a protein, or can include any post-translational modification.

[0049] The sample of the present embodiment can be preferably a biological sample such as an extract from a biological fluid, a cell, or a tissue, a culture supernatant of a cell or a tissue, and the like. The biological fluid can be blood, plasma, serum, cerebrospinal fluid (CSF), saliva, urine, and the like. The cell can be a neural cell, a glial cell, a fibroblast, a stem cell, an endothelial cell, a pericyte, and the like, and can be a primary culture cell derived from an organism or an established cell line. The tissue can be a central nervous tissue, a peripheral nervous tissue, a brain organoid, and the like. The biological sample can be derived from any organism, such as a vertebrate, preferably a mammal such as a mouse, a rat, a rabbit, a dog, a monkey, a human, and particularly preferably a human.

[0050] To contact the protein-containing sample with the probe, it is only necessary to prepare a reaction solution in which both are mixed and incubate for a certain period. The ionic strength and pH of the reaction solution are not particularly limited, and any buffer and / or salt can be contained in the reaction solution, and an organic solvent such as ethanol, glycerol, etc. can also be contained. Examples of the buffer include HEPES, MOPS, MES, EPPS, Tris, phosphoric acid, acetic acid, citric acid, glycine, etc. Examples of the salt include NaCl, KC1, MgCl2, Na2S04, K2S04, MgS04, Nal, NaSCN, etc.

[0051] The reaction solution of the present embodiment can preferably be a reaction solution according to conditions in vivo. 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 mM to 500 mM. An extract of brain tissue or a buffer (e.g., HEPES-buffered aCSF (artificial cerebrospinal fluid)) prepared according to the composition thereof can be used for the reaction solution of the present embodiment.

[0052] The final concentration of the protein in the reaction solution can be 1 nM to 1000 μM, and can preferably be 10 nM to 100 μM. In the case where the concentration of the protein in the added sample is unknown, the sample can be appropriately diluted in stages and used. The incubation time of the reaction solution can be, for example, 10 seconds to 72 hours.

[0053] Next, the fluorescence intensity of the probe is measured. In the method of the present embodiment, the fluorescence intensity can be measured at an excitation wavelength of 300 nm to 500 nm and a fluorescence wavelength of 400 nm to 700 nm. In the method of the present embodiment, the fluorescence intensity of one or a plurality of sets of excitation wavelength / fluorescence wavelength can also be measured, and, for example, the fluorescence intensity can be measured using 1, 2, 3, or 4 sets of excitation wavelength / fluorescence wavelength selected from the excitation wavelength (nm) / fluorescence wavelength (nm): 340 / 520, 330 / 480, 345 / 505, 360 / 530, etc.

[0054] To determine whether the protein in the sample has bound to the probe, for example, a control (a solution containing only the probe, etc.) containing no protein can be analyzed and compared in parallel, and a comparison can be made with the analysis result of a control performed in the past. Alternatively, as a control, a reaction solution obtained by mixing a sample containing only a protein that has been determined not to interact with DPRs with the probe can also be used. In the method of the present embodiment, in the case where the fluorescence intensity of the probe contacted with the sample is significantly increased as compared with the fluorescence intensity of the probe of the control, it can be determined that a protein that can interact with DPRs, i.e., a protein that can be associated with a neurodegenerative disease, is present in the sample.

[0055] The so-called "neurodegenerative disease" is a general term for progressive diseases in which nerve cells of the central nervous system gradually degenerate and eventually lead to cell death. In the neurodegenerative disease of the present embodiment, for example, frontotemporal dementia (FTD), progressive nonfluent aphasia (PNFA), and semantic dementia (SD) and the like frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), Huntington's disease (HD), Lewy body dementia (DLB), corticobasal syndrome (CBS), progressive supranuclear palsy (PSP), and the like can be given, but are not limited to these. The neurodegenerative disease of the present embodiment is preferably ALS or FTLD.

[0056] Figure 1 A schematic diagram of the method of the present embodiment is shown in FIG. 1. In Figure 1 In FIG. 1, as an example of the probe, Dnc-DPRs in which a dansyl group is introduced are shown. When the probe interacts with a protein that can interact with DPRs (in the figure, "Potential binder"), the fluorescence intensity of the probe increases (Turn-on response). Therefore, according to the method of the present embodiment, it is possible to screen for a protein that can interact with DPRs, that is, a protein that can be associated with a neurodegenerative disease, based on the increase in the fluorescence intensity of the probe.

[0057] Based on the second embodiment, the present application is a screening method for a neurodegenerative disease treatment or prophylactic drug, which comprises: (1) a step of bringing a probe in which a dipeptide repeat sequence-containing polypeptide is covalently linked to an environmentally responsive fluorophore into contact with a liquid-liquid phase separation-related protein or a nuclear membrane 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.

[0058] The "dipeptide repeat sequence", "environmentally responsive fluorophore", "probe in which a dipeptide repeat sequence-containing polypeptide is covalently linked to an environmentally responsive fluorophore", and "neurodegenerative disease" of the present embodiment are the same as defined in the first embodiment.

[0059] The so-called "treatment" (and its grammatical variations) of the present embodiment means to stop or alleviate the progression and exacerbation of the disease in a subject who has developed a neurodegenerative disease, and not only includes complete cure of the disease but also alleviation of symptoms of the disease. In addition, the so-called "prevention" (and its grammatical variations) of the present embodiment means to prevent the disease from occurring in a subject who is likely to develop a neurodegenerative disease. The "subject" can be any mammal, but is preferably a human. The subject can be any age, including infants, children, adolescents, young adults, adults, and the elderly.

[0060] In the method of the present embodiment, the liquid-liquid phase separation-related protein or nuclear membrane pore complex family protein is contacted with a probe covalently linked to a dipeptide repeat sequence-containing polypeptide and an environmentally responsive fluorophore in the presence of a candidate compound.

[0061] The so-called "liquid-liquid phase separation (LLPS)-related protein" refers to a protein associated with the formation of liquid-liquid phase separation (LLPS). LLPS-related proteins are found in a wide range of biological species such as eukaryotes, prokaryotes, viruses, etc., and have an intrinsically disordered region, particularly a low complexity (LC) domain, as a commonality. Information on LLPS-related 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), PhaSePro (https: / / phasepro.elte.hu / ), etc.

[0062] The LLPS-related protein of the present embodiment can preferably be a FET family protein or a heterogeneous nuclear ribonucleoprotein. The "FET family protein" is an RNA-binding protein having an LC domain on the N-terminal side. The FET family protein includes FUS (fused in sarcoma, also known as TLS (translocated in liposarcoma)), EWSR1 (Ewing sarcoma breakpoint region 1, also known as RNA-binding protein EWS), and TAF15 (TATA box-binding protein-associated factor 15). The "heterogeneous nuclear ribonucleoprotein (hnRNP)" is an RNA-binding protein having an LC domain on the C-terminal side. Among the hnRNPs, for example, TDP-43 (TAR DNA-binding protein 43 kDa), hnRNP A0, hnRNP A1, hnRNP A2, hnRNP A3, hnRNP H1, hnRNP H2, hnRNP H3, AUF1, PTBP1, and the like can be given, but the present embodiment is not limited thereto. The LLPS-related protein of the present embodiment is particularly preferably FUS.

[0063] The "nuclear pore complex (NPC) family protein" is a protein involved in molecular transport between the cytoplasm and the nucleus, and controls LLPS. Among the NPC family proteins, for example, nucleoporin β 2 (also known as importin β 2 or transportin 1), nucleoporin β 2b (also known as importin 3 or transportin 2), nucleoporin β 1 (also known as importin β 1), importin α 1, importin α 2, and the like can be given, but the present embodiment is not limited thereto. The NPC family protein of the present embodiment is preferably nucleoporin β 2 (Kap β 2).

[0064] The LLPS-related protein and the NPC family protein of the present embodiment can be derived from any vertebrate, preferably from a mammal such as a mouse, a rat, a rabbit, a dog, a monkey, a human, and the like, and particularly preferably from a human.

[0065] The amino acid sequence and the nucleic acid sequence information of the LLPS-related protein and the NPC family protein can be obtained from a prescribed database. For example, if the amino acid sequence and the gene sequence of human Kap β 2, NCBI Gene ID: 3842 can be used, and if the amino acid sequence and the gene sequence of human FUS, NCBI Gene ID: 2521 can be used.

[0066] The LLPS-related proteins and the NPC family proteins of the present embodiment can also include variants, homologues, etc. thereof having equivalent activity. In other words, within the limit of maintaining the LLPS formation activity thereof, if it is an amino acid sequence registered in a database, the LLPS-related proteins of the present embodiment can include proteins including an amino acid sequence having 80% or more, preferably 90% or more, more preferably about 95% or more of identity. In addition, within the limit of maintaining the LLPS control activity thereof, if it is an amino acid sequence registered in a database, the NPC family proteins of the present embodiment can include proteins including an amino acid sequence having 80% or more, preferably 90% or more, more preferably about 95% or more of identity. The identity of the amino acid sequence can be calculated using a sequence analysis software or a program (FASTA, BLAST, etc.) commonly used in the field.

[0067] In addition, within the limit of maintaining the LLPS formation activity or the LLPS control activity thereof, in an amino acid sequence registered in a database, the LLPS-related proteins and the NPC family proteins of the present embodiment can include proteins including an amino acid sequence obtained by substituting, deleting, inserting, and / or adding 1 to several amino acids. Here, the "1 to several" can be, for example, 1 to 30, preferably 1 to 10, and particularly preferably 1 to 5.

[0068] The LLPS-related proteins and the NPC family proteins of the present embodiment can also be added with a tag such as GST, His6, MBP, HA, FLAG, etc. at the N terminus and / or the C terminus, directly or via a linker.

[0069] The LLPS-related proteins and the NPC family proteins of the present embodiment can be produced by any of the methods of genetic engineering conventionally known. Specifically, for example, an expression vector including a nucleic acid encoding the LLPS-related proteins and the NPC family proteins is prepared and introduced into a host cell such as Escherichia coli to express it.

[0070] To contact the probe with the LLPS-related proteins or the NPC family proteins in the presence of a candidate compound, a reaction solution obtained by mixing the probe with the LLPS-related proteins or the NPC family proteins and the candidate compound is prepared and incubated for a certain period. The protein concentration, the ionic strength, and the pH of the reaction solution of the present embodiment can be the same as defined in the first embodiment.

[0071] The "candidate compound" of the present embodiment can be a low-molecular compound, a nucleic acid, a protein, a peptide, an antibody, a lipid, or the like, and can also be a mixture thereof (for example, an extract from a cell or tissue, a culture supernatant of a cell or tissue, or the like). In addition, the candidate compound can be a novel compound, or a known compound. The final concentration of the candidate compound added to the reaction solution varies depending on the kind of the candidate compound, and for example, if it is a low-molecular compound, it can be appropriately selected within a range of 1 nM to 10 mM. The incubation time can be, for example, 10 seconds to 72 hours.

[0072] Next, the fluorescence intensity of the probe is measured. The conditions for measuring the fluorescence intensity of the present embodiment can be the same as defined in the first embodiment.

[0073] In order to determine whether the fluorescence intensity of the probe is changed by the addition of the candidate compound, the reaction solution to which the candidate compound is not added can be analyzed and compared in parallel, or the results of the analysis performed in the past on the reaction solution to which the candidate compound is not added can be compared. In the method of the present embodiment, in the case where the fluorescence intensity of the probe after the contact with the sample is significantly weakened as compared with the fluorescence intensity of the probe in the reaction solution to which the candidate compound is not added, it can be determined that the candidate compound can inhibit the interaction of DPRs with the LLPS-related protein / NPC family protein, that is, can normalize the LLPS control, and therefore, it can be evaluated that the candidate compound is promising as a therapeutic or prophylactic drug for a neurodegenerative disease.

[0074] Figure 2 A schematic diagram of the method of the present embodiment is shown in FIG. 1. The probe emits fluorescence when it interacts with the LLPS-related protein / NPC family protein, but if the interaction is inhibited, the probe is separated from the LLPS-related protein / NPC family protein, and the fluorescence intensity of the probe is weakened (Turn-off response). Therefore, according to the method of the present embodiment, it is possible to screen a compound that can inhibit the interaction of DPRs with the LLPS-related protein / NPC family protein and normalize the LLPS control, that is, a compound that is promising as a therapeutic or prophylactic drug for a neurodegenerative disease, based on the weakening of the fluorescence intensity of the probe.

[0075] Example

[0076] Next, the present application will be further described with examples. In addition, these contents do not limit the present application in any way.

[0077] <1. Materials and reagents>

[0078] (1-1) Probe

[0079] The following probes 1-7 (>95% purity) were synthesized by Biologica and used without further purification. As representative examples, Figure 3 The structural formulas of probe 1 (SEQ ID NO: 1) and probe 2 (SEQ ID NO: 2) are shown below.

[0080] Probe 1 : Dansyl-NH-(Pro-Arg) 20 -COOH (SEQ ID NO: 1) (hereinafter referred to as "Dnc-PR20")

[0081] Probe 2: Dansyl-NH-(Gly-Pro) 20 -COOH (SEQ ID NO: 2) (hereinafter referred to as "Dnc-GP20")

[0082] Probe 3: Dansyl-NH-(Pro-Arg) 10 -COOH (SEQ ID NO: 3) (hereinafter referred to as "Dnc-PR10")

[0083] Probe 4: Dansyl-NH-(Gly-Pro) 10 -COOH (SEQ ID NO: 4) (hereinafter referred to as "Dnc-GP10")

[0084] Probe 5: Dansyl-NH-(Gly-Ala) 10 -COOH (SEQ ID NO: 5) (hereinafter referred to as "Dnc-GA10")

[0085] Probe 6: Dansyl-NH-(Gly-Arg) 10 -COOH (SEQ ID NO: 6) (hereinafter referred to as "Dnc-GR10")

[0086] Probe 7: Dansyl-NH-(Pro-Ala) 10 -COOH (SEQ ID NO: 7) (hereinafter referred to as "Dnc-PA10")

[0087] (1-2) Proteins

[0088] Bovine lactalbumin, human serum β -lactoglobulin, human serum α 1 -antitrypsin, human serum albumin, human serum apotransferrin and chicken egg white lysozyme were purchased from Sigma-Aldrich. Human serum immunoglobulin G was purchased from Equitech-Bio, Inc.

[0089] Professor Tomohide Saio of the Advanced Enzymology Research Institute at Tokushima University provided MBP-tagged human FUS (see “Cell, 173(3): 693-705.e22(2018)”, hereinafter referred to as “MBP-FUS”), and human nuclear transporter protein. β 2 (See “Cell, 173(3): 693-705.e22(2018)”, hereinafter referred to as “Kap”) β 2") Caenorhabditis elegans input protein β Family proteins (see “UniProt accession number: A0A131MBF9”, hereinafter referred to as “Imb-2”), human heteronuclear ribonucleoprotein A2 (see “Cell, 163(4): 829-839(2015)”, hereinafter referred to as “hnRNPA2LC”), and human peptidylprolyl cis-trans isomerase A (see “Cell, 163(4): 829-839(2015)”, hereinafter referred to as “PPIA”). Human input proteins were provided by Assistant Professor Takuya Yoshizawa of the Faculty of Life Sciences, Ritsumeikan University, Japan. α 1 (See “Cell, 173(3): 693-705.e22(2018)”, hereinafter referred to as “Imp”) α Human input protein β 1 (See “Cell, 173(3): 693-705.e22(2018)”, hereinafter referred to as “Imp”) β “), GST-tagged M9M peptide (see “Cell, 173(3): 693-705.e22(2018)”, hereinafter referred to as “GST-M9M”) and human GPT-bound Ran(Q69L) (see “UniProt accession number: P62826”, hereinafter referred to as “RanGTP”).

[0090] (1-3) Reagents

[0091] ATP, Tris-HCl, dithiothreitol (DTT), polyamide-amine dendritic molecules (PAMAM), and 50mer polylysine (polyK50) were purchased from Sigma-Aldrich. NaCl was purchased from Fujifilm and Koujun Chemical Co., Ltd.

[0092] <2. Based on Kap β Fluorescence changes of probe 2 >

[0093] The probe solutions (final concentration 300 nM) of the above probes 1 to 7 were prepared using a buffer solution (20 mM of Tris-HCl (pH 7.5), 150 mM of NaCl, 2 mM of DTT (final concentration)). Using a full-automatic sample applicator (pipetting robot, Andrew+, Andrew Alliance), each probe solution was added to a 384-well microplate (Corning, 3575) at 30 μL / well. 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 to 700 nm) were measured using a microplate reader (Cytation 5, BioTek). Thereafter, various concentrations of Kap β 2 solution (Kap β 2 / pure water) were added at 30 μL / well using a full-automatic sample applicator, and after incubation at 35°C for 10 minutes, the fluorescence intensity and the fluorescence spectrum were measured under the same conditions.

[0094] Figure 4 The results of probes 1 and 2 are shown in FIG. 1. Figure 4 (a) of FIG. 1 shows the fluorescence spectrum of Dnc-PR20 (probe 1), Figure 4 (b) of FIG. 1 shows the fluorescence spectrum of Dnc-GP20 (probe 2). The fluorescence intensity of Dnc-PR20 increased in a Kap β 2 concentration-dependent manner, and the fluorescence wavelength peak shifted from about 590 nm to 540 nm. The 540 nm fluorescence intensity of Dnc-PR20 at 1600 nM of Kap β 2 was increased to about 6.1 times compared to the fluorescence intensity at 0 nM of Kap β 2. On the other hand, the fluorescence spectrum of Dnc-GP20 did not substantially change due to the addition of Kap β 2. Figure 4 (c) of FIG. 1 shows the change in the 520 nm fluorescence intensity of probes 1 and 2. The X axis indicates the concentration of Kap β 2, and the Y axis indicates the average value ± standard error of the fluorescence intensity (n = 3). From these results, it was confirmed that Dnc-PR20 binds to Kap β 2, whereas Dnc-GP20 does not bind to Kap β 2.

[0095] Figure 5 The results of probes 3 to 7 are shown in FIG. 2. Figure 5 (a) of FIG. 2 shows the fluorescence spectrum of Dnc-PR10 (probe 3), Figure 5 (b) of FIG. 2 shows the fluorescence spectrum of Dnc-GP10 (probe 4), Figure 5 (c) of FIG. 2 shows the fluorescence spectrum of Dnc-GA10 (probe 5), Figure 5(d) shows the fluorescence spectrum of Dnc-GR10 (probe 6). Figure 5 (e) shows the fluorescence spectrum of Dnc-PA10 (probe 7). Figure 5 (f) shows the changes in fluorescence intensity at 520 nm for probes 3–7. The X-axis represents Kap β The concentration is 2, and the Y-axis represents the mean ± standard error of fluorescence intensity (n=3). Fluorescence intensity relative to Dnc-PR10 and Dnc-GR10 is expressed in Kap... β 2. The concentration-dependent reaction increased, but the fluorescence spectra of Dnc-GP10, Dnc-GA10, and Dnc-PA10 remained unchanged. These results confirm that Dnc-PR10 and Dnc-GR10 are related to Kap β 2. Combination, but Dnc-GP10, Dnc-GA10, and Dnc-PA10 do not combine with Kap β 2. These results are also consistent with previous insights (Nat.Commun., 2021; 12(1): 5301).

[0096] <3. Fluorescence changes of FUS-based probes>

[0097] In addition to using FUS (MBP-FUS) solution (MBP-FUS / pure water) labeled with the MBP tag instead of Kap β In addition to solution 2, the fluorescence intensity and fluorescence spectrum of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2) were measured by the same steps as described in 2 above.

[0098] Figure 6 The results are shown in the figure. Figure 6 (a) shows the fluorescence spectrum of Dnc-PR20 (probe 1). Figure 6 (b) shows the fluorescence spectrum of Dnc-GP20 (probe 2). The fluorescence intensity of Dnc-PR20 increased in a concentration-dependent manner with MBP-FUS, with the peak fluorescence wavelength shifting from approximately 590 nm to 540 nm. The fluorescence intensity of Dnc-PR20 at 540 nm with 1600 nM MBP-FUS increased by approximately 4.6 times compared to the fluorescence intensity with 0 nM M MBP-FUS. On the other hand, the fluorescence spectrum of Dnc-GP20 remained essentially unchanged due to the addition of MBP-FUS. Figure 6 (c) shows the changes in fluorescence intensity at 520 nm for probes 1 and 2. The X-axis represents the concentration of MBP-FUS, and the Y-axis represents the mean ± standard error of fluorescence intensity (n=3). These results confirm that Dnc-PR20 binds to MBP-FUS, but Dnc-GP20 does not bind to MBP-FUS.

[0099] <4. Fluorescence change of probes based on various proteins (1)>

[0100] By using an aqueous solution of β - lactoglobulin, which is derived from bovine milk, α 1- antitrypsin, albumin (HSA) derived from human serum, apotransferrin derived from human serum, lysozyme derived from chicken egg white, or immunoglobulin G (IgG) derived from human serum (1600 nM) instead of Kap β 2 solution, the fluorescence intensity and the fluorescence spectrum of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2) were measured by the same procedure as in 2 above.

[0101] Figure 7 The results are shown in FIGS. 12A and 12B. Figure 7 (a) of FIG. 12A shows the fluorescence spectrum of Dnc-PR20 (probe 1), Figure 7 (b) of FIG. 12A shows the fluorescence spectrum of Dnc-GP20 (probe 2). The fluorescence spectrum of Dnc-PR20 was shifted from about 590 nm to 540 nm in the peak wavelength of fluorescence and slightly increased in the fluorescence intensity at 540 nm due to the addition of β - lactoglobulin. The fluorescence spectrum of Dnc-PR20 was not substantially changed due to the addition of proteins other than Kap. The fluorescence spectrum of Dnc-GP20 was not substantially changed due to the addition of any of the proteins. From these results, it was clarified that Dnc-PR20 also slightly interacts with β - lactoglobulin, but the interaction is considerably weaker than that with Kap β 2 or MBP-FUS.

[0102] <5. Fluorescence change of probes based on various proteins (2)>

[0103] In addition to the proteins tested in 2 to 4 above, an aqueous solution of a protein capable of being associated with the formation or control of liquid-liquid phase separation (LLPS) was used, and the 520 nm fluorescence intensity of Dnc-PR20 (probe 1) and Dnc-GP20 (probe 2) was measured by the same procedure as in 2 above. As the protein capable of being associated with the formation or control of LLPS, the following proteins were used: Imb-2, hnRNPA2LC, PPIA, Imp α , Imp β , GST-M9M, and RanGTP. The concentration of each protein aqueous solution was set to 1600 nM (only the Imb-2 aqueous solution was 800 nM).

[0104] Figure 8The results are shown in FIG. 6. In addition to Kap β 2, MBP-FUS, and β - even in the case where Imb-2, Imp α , and Imp β were added, the fluorescence intensity of Dnc-PR20 at 520 nm increased to more than 1000. It was reported in the past that Kap β 2, importin α / β , and FUS bind to DPRs, and based on these results, it was confirmed that DPRs-binding proteins can be detected based on the increase in the fluorescence intensity of Dnc-PR20.

[0105] Based on the above results, it was found that polypeptide probes containing an environmentally responsive fluorophore-DPR sequence can be used to screen proteins that interact with DPRs.

[0106] <6. Screening of DPRs-Kap β 2 binding inhibitors (1)

[0107] A mixed solution of Dnc-PR20 (final concentration 300 nM) and Kap β 2 (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 the mixed solution was added to a 384-well microplate (Corning, 3820) at 20 μL / well using a full-automatic sample dispenser (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 (Cytation 5, BioTek). Thereafter, various concentrations of additives (polyK50 or IgG / water) were added at 5 μL / well using a full-automatic sample dispenser, and after incubation at 35°C for 10 minutes, the fluorescence intensity and the fluorescence spectrum were measured under the same conditions. As a control, the fluorescence intensity and the fluorescence spectrum were also measured for a Dnc-PR20 solution that did not contain Kap β 2.

[0108] Figure 9 The results are shown in FIG. 6. Figure 9 (a) shows the fluorescence spectrum of a Dnc-PR20 / Kap β 2 solution to which polyK50 (0 nM-300 nM) was added, Figure 9 (b) shows the fluorescence spectrum of a DPRs / Kapβ 2. Fluorescence spectra of the solution. In the case where polyK50 was added to Dnc-PR20 / Kap β 2, the fluorescence intensity of Dnc-PR20 was weakened in a polyK50 concentration-dependent manner, and became the same degree of fluorescence intensity as the control due to the addition of 200 nM of polyK50. On the other hand, even if IgG was added, the fluorescence intensity of Dnc-PR20 did not substantially change. Figure 9 (c) shows the change in the 520 nm fluorescence intensity. The X axis indicates the concentration of the additive, and the Y axis indicates the mean ± standard error of the fluorescence intensity (n = 3). According to these results, it was shown that polyK50 inhibited the interaction of Dnc-PR20 with Kap β 2. Although not wishing to be bound by a particular theory, polyK50 is cationic like PR20, and has about one more molecule of charge number than PR20, and thus it is considered to interact more strongly with Kap β 2.

[0109] <7. Screening of DPRs-Kap β 2 binding inhibitors (2)

[0110] In addition to polyK50 and IgG, PAMAM, GST-M9M, RanGTP, ATP, and HSA were also used as additives, and the 520 nm fluorescence intensity of Dnc-PR20 was measured by the same procedure as in the above 6.

[0111] Figure 10 The results are shown in the graph. In the graph, "none" indicates a Dnc-PR20 solution containing no Kap β 2, "Kap β 2" indicates a Dnc-PR20 / Kap β 2 mixed solution. In the case where PAMAM, which is a synthetic dendrimer of cationic, and M9M peptide, which is a known inhibitor of Kap β 2, were added in addition to polyK50, the 520 nm fluorescence intensity of Dnc-PR20 was weakened. The fluorescence intensity of Dnc-PR20 did not substantially change due to the addition of the other additives. According to these results, it was known that a compound that inhibits the interaction of DPRs with Kap β 2 could be detected from the weakening of the fluorescence intensity of Dnc-PR20.

[0112] According to the above results, it was known that a compound that inhibits the interaction of DPRs with Kap β 2 could be screened using a polypeptide probe containing an environmentally responsive fluorophore-DPR sequence.

[0113] <8. Screening of DPRs-Kap β2 Screening of binding inhibitors (3)

[0114] (8-1) Screening of PPI inhibitor library

[0115] Using 47 kinds of protein-protein interaction (PPI) inhibitors selected from PPI inhibitor library (TargetMol, L9400), an empirical experiment of screening compounds that inhibit the interaction of DPRs and Kap β 2 was performed. Using an automatic dispenser (ASSIST PLUS, Integra Biosciences), a mixed solution of Dnc-PR20 prepared in the above 6 (final concentration 300 nM) and Kap β 2 (final concentration 400 nM) was added at 20 μL / well to a 384-well microplate (Corning, 3820). After incubation at 35°C for 10 minutes, the fluorescence intensity at an excitation wavelength of 340 nm (fluorescence wavelength 520 nm) was measured using a microplate reader (Synergy H1, Agilent Technologies). Thereafter, using an automatic dispenser, various PPI inhibitors (Compound Nos. 01 to 47, compound / 25 vol% DMSO) were added at 5 μL / well, and after incubation at 35°C for 10 minutes, the fluorescence intensity was measured under the same conditions. As a control, the fluorescence intensity was also measured for a Dnc-PR20 solution containing no Kap β 2.

[0116] Figure 11 The results are shown in the figure. The upper layer shows the 520 nm fluorescence intensity of the Dnc-PR20 / Kap β 2 solution to which 10 μM of PPI inhibitors was added (mean ± standard error), and the lower layer shows the 520 nm fluorescence intensity of the Dnc-PR20 / Kap β 2 solution to which 100 μM of PPI inhibitors was added (mean ± standard error). In the figure, "none" indicates a Dnc-PR20 solution containing no Kap β 2, and "Kap β 2" indicates a Dnc-PR20 / Kap β 2 mixed solution. One compound caused a significant change in the 520 nm fluorescence intensity of the solution at a concentration of 10 μM, and 13 compounds caused a significant change in the 520 nm fluorescence intensity of the solution at a concentration of 100 μM (relative to the Dnc-PR20 / Kap β 2 mixed solution, 1 P<0.05, 2 P<0.01, 4P<0.0001; one-way ANOVA with Tukey's post-hoc test). In addition, it was confirmed that among the 13 compounds, 10 compounds significantly attenuated the 520 nm fluorescence intensity of the solution (Fig. 8-2, black bars). Figure 11

[0117] (8-2) DPRs-Kap of Compound No. 08 β 2 Inhibitory Effect of Binding

[0118] Next, using Compound No. 8 as a representative of the 10 compounds that significantly attenuated the 520 nm fluorescence intensity of the solution, the 520 nm fluorescence intensity of Dnc-PR20 was measured by the same procedure as in (8-1) above.

[0119] Figure 12 The results are shown in Fig. 8-3. Figure 12 (a) of Fig. 8-3 shows the 520 nm fluorescence intensity of Dnc-PR20 / Kap β 2 solution to which Compound No. 8 (0 to 100 μM) was added. The X axis indicates the concentration of Compound No. 8, and the Y axis indicates the mean ± standard error of the fluorescence intensity (n = 3). Figure 12 (b) of Fig. 8-3 shows the fluorescence spectrum of Dnc-PR20 / Kap β 2 solution to which Compound No. 8 (0 to 100 μM) was added. Until 75 μM was reached, the fluorescence intensity of Dnc-PR20 was attenuated in a compound No. 8 concentration-dependent manner. From these results, it was shown that Compound No. 8 significantly inhibited the interaction of DPRs with Kap β 2, indicating that Compound No. 8 is promising as a DPRs-Kap β 2 binding inhibitor.

[0120] From the above results, it was confirmed that the present application enables screening of compounds that inhibit the interaction of DPRs with Kap β 2 by using the present screening method using a polypeptide probe containing an environmentally responsive fluorophore-DPR sequence.​

Claims

1. A method for screening a protein associated with a neurodegenerative disease, comprising: (1) a step of contacting a sample containing a protein with a probe in which a polypeptide containing a dipeptide repeat sequence composed of a repeating unit selected from the group consisting of proline-arginine, glycine-arginine, proline-alanine, glycine-alanine, and glycine-proline is covalently linked to an environmentally responsive fluorophore; and (2) a step of measuring the fluorescence intensity of the probe.

2. The method according to claim 1, wherein the dipeptide repeat sequence contains 3 to 200 of the repeating unit.

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

7. The method according to claim 6, wherein the dipeptide repeat sequence contains 3 to 200 of the repeating unit.

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 a nucleoporin β 2.

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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