Modulators of G3BP2-Tau interaction for the treatment of Tau-related diseases

Abstract

The present invention relates to a method for the identification of a compound that modulates protein-protein interaction between a protein comprising the NTF2-like domain of G3BP2 having the amino acid sequence set forth in SEQ ID NO: 3 and Tau protein for the treatment of Tau-related diseases.

Description

[Technical field]

[0001] The present invention relates to methods of treating Tau-associated diseases. [Background technology]

[0002] Toxic accumulation of the microtubule-associated protein Tau is a hallmark of multiple neurodegenerative diseases, including Alzheimer's disease (AD), frontotemporal dementia with Parkinsonism-17 (FTDP-17), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and Pick's disease (PiD); these diseases are collectively known as tauopathies. In healthy brains, Tau is involved in the assembly and stabilization of microtubules, but under disease conditions, hyperphosphorylated Tau detaches from microtubules and further aggregates to form paired helical filaments (PHFs) and neurofibrillary tangles (NFTs). The current limited understanding of the mechanisms underlying tauopathies hinders the development of effective treatments for these diseases. Summary of the Invention [Problem to be solved by the invention]

[0003] Thus, there is a need for the identification of new target proteins and their use for the treatment of tauopathies. [Means for solving the problem]

[0004] In a first object, the present invention provides a method for treating a Tau-related disease, comprising administering to a subject an effective amount of a compound that modulates protein-protein interaction between a protein comprising an NTF2-like domain having the amino acid sequence set forth in SEQ ID NO:3 or a variant thereof and a Tau protein.

[0005] In one embodiment, the compound promotes / enhances protein-protein interaction between a protein comprising an NTF2-like domain and a Tau protein.

[0006] In one embodiment, the compound is a mimetic of the protein-protein interaction between a protein containing an NTF2-like domain and a Tau protein.

[0007] In one embodiment, the Tau-related disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Drunk Punch syndrome, Down's syndrome, traumatic brain injury, Guam amyotrophic lateral sclerosis / Parkinsonism dementia complex, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangle disease with calcifications, frontotemporal dementia, frontotemporal dementia linked to chromosome 17 with parkinsonism, Hallervorden-Spatz disease, Niemann-Pick disease type C, pallidal-pontocephalic-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, neurofibrillary tangle dementia, postencephalitic parkinsonism, and myotonic dystrophy.

[0008] In one embodiment, the protein comprising an NTF2-like domain is a G3PB2 protein.

[0009] In one embodiment, the protein comprising the NTF2-like domain and the Tau protein are human proteins.

[0010] In a second object, the present invention provides a compound that modulates protein-protein interaction between a protein comprising an NTF2-like domain having the amino acid sequence set forth in SEQ ID NO:3 or a variant thereof and a Tau protein, for use in the treatment of a Tau-related disease.

[0011] In one embodiment of the second object, the Tau-related disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Drunk Punch syndrome, Down's syndrome, traumatic brain injury, Guam amyotrophic lateral sclerosis / Parkinsonism dementia complex, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangle disease with calcifications, frontotemporal dementia, frontotemporal dementia linked to chromosome 17 with parkinsonism, Hallervorden-Spatz disease, Niemann-Pick disease type C, pallidal-pontocephalic-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, neurofibrillary tangle dementia, postencephalitic parkinsonism, and myotonic dystrophy.

[0012] In a third object, the present invention provides a protein comprising an NTF2-like domain having the amino acid sequence set forth in SEQ ID NO:3 or a variant thereof for use in identifying a compound that modulates protein-protein interaction between a protein comprising an NTF2-like domain and a Tau protein.

[0013] In a fourth object, the present invention provides a protein comprising an NTF2-like domain having the amino acid sequence set forth in SEQ ID NO: 3 or a variant thereof, for use as a target protein for the treatment of a Tau-related disease.

[0014] In one embodiment of the third and fourth objects, the protein is a human G3BP2 protein. [Brief description of the drawings]

[0015] [Figure 1a] G3BP2 directly interacts with Tau and inhibits Tau aggregation in vitro. 1a) SPR sensorgrams and binding affinity (Kd) of full-length Tau interacting with full-length G3BP2. Tau was immobilized on a sensor chip and G3BP2 was injected at the corresponding concentrations. [Figure 1b]1b) Kinetics and bar plots showing the effect of G3BP2 on Tau aggregation (n=6). Error bars: mean ± SD. p values ​​compared to control were obtained using one-way ANOVA followed by Dunnett's multiple comparison test (post-hoc test). [Figure 2a] Aggregated Tau species are increased upon G3BP2 knockdown via Tau seeding assay in hiPSC-derived neurons. 2a) Schematic of Tau seeding assay in hiPSC-derived neurons. OE: overexpression. Tau PFF: preformed fibrils of Tau. [Figure 2b] 2b) Quantification of aggregated Tau species in the Tau seeding assay. p-values ​​were obtained using a two-tailed unpaired Student's t-test. [Figure 3a] Tau pathology is elevated in the absence of G3BP2. 3a) Immunohistochemistry for phospho-Tau (green) and total Tau (magenta) in 4-month-old fAD(PSEN1 M139V) or fAD G3BP2 knockout human brain organoids. Nuclei were stained blue with DAPI. Scale bar: 50 μm. [Figure 3b] 3b) Whole slide image showing IHC for Tau pS214 (green), total Tau (yellow) and MAP2 (red). Scale bar: 2 mm. [Figure 3c] 3c) Quantification of Tau pT181 and pT231 by ELISA. pTau levels were normalized to total Tau levels. Each data point represents 4-5 pooled hCOs. Error bars: mean ± SD. p values ​​were obtained using a two-tailed unpaired Student's t-test. [Figure 4a] Progressively increasing G3BP2-Tau interaction in human AD brain. 4a) Representative images of PLA signals (green) showing G3BP2 interacting with Tau in Braak I, IV and VI AD cases. Total Tau was stained with Tau polyclonal antibody (magenta). Nuclei were stained with DAPI (blue). Scale bar: 10 μm. [Figure 4b]4b) PLA puncta of G3BP2-Tau per cell were quantified in patients with Braak I (n=9), II&III (n=10), IV&V (n=10) and VI (n=9) AD. For box plots, median, 25th and 75th percentiles are shown. p-values ​​were obtained using a two-tailed unpaired Student's t-test. PLA: Proximity Ligation Assay. AD: Alzheimer's Disease. [Figure 5a] The NTF2-like domain of G3BP2 can inhibit Tau aggregation. 5a) Diagram of the amino acid sequence of full-length G3BP2. [Figure 5b] 5b) Amino acid sequence of the NTF2-like domain of G3BP2 (SEQ ID NO:3). [Figure 5c] 5c) Kinetics and bar plots showing the effect of G3BP2 NTF2-like domain on Tau aggregation (n=5). Data are shown as mean ± SD. p values ​​compared to control group were obtained using one-way ANOVA followed by Dunnett's multiple comparison test (post-hoc test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] definition An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, an individual or subject is a human.

[0017] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredients contained in the preparation is effective, and that does not contain additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition will be administered.

[0018] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0019] Tau proteins normally associate with microtubules and play an important function in their assembly, stabilizing them against dynamic instability and promoting microtubule attachment to other cytoskeletal filaments. (Mactui RB and Change V. (1995) Role of microtubule-associated proteins in the control of microtubule assembly. Physiol Rev. 75(4):835-864; Mactui, RB, Barbeita L, and Munoz JP (20001). The molecular bases of Alzheimer's disease and other neurodegenerative disorders. Arch. Medical Research. 32-367-381). Tau proteins belong to the family of MAPs, i.e. microtubule-associated proteins. In humans, it is found almost exclusively in neurons (Mactui RB and Arechaga J. (1987). ''The Cytoskeleton in Cell Differentiation and Development''. Oxford University Press, UK 367 pp; Mactui RB and Change V. (1995.) Role of microtubule-associated proteins in the control of microtubule assembly. Physiol Rev. 75(4): 835-864) and occurs in six isoforms resulting from the expression of a single gene. The gene is found at position 21 of the long arm of chromosome 17 (17q21) and contains 13 exons which, by an alternative splicing process, generate six molecular isoforms with 352 to 441 amino acids (Goedert M. (2004). Tau protein and neurodegeneration. Seminars in Cell & Developmental Biology. 15: 45-49).

[0020] Exemplary Tau protein-associated diseases or disorders include, but are not limited to, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Drunk Punch syndrome, Down's syndrome, traumatic brain injury, Guam amyotrophic lateral sclerosis / Parkinson-dementia complex, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangle disease with calcifications, frontotemporal dementia, frontotemporal dementia linked to chromosome 17 with parkinsonism, Hallervorden-Spatz disease, Niemann-Pick disease type C, pallidal-pontocephalic-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, neurofibrillary tangle dementia, postencephalitic parkinsonism, and myotonic dystrophy.

[0021] As used herein, "treatment" (and grammatical variants thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed for prophylaxis or during the course of clinical pathology. Desired effects of treatment include preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, remission or alleviation of disease symptoms, and improved or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or to slow the progression of the disease.

[0022] Here, "G3BP2" is the abbreviation used for Ras GTPase-activating protein-binding protein 2. Isoform A of human G3BP2 has Uniprot ID: Q9UN86-1 (SEQ ID NO: 1), and isoform B of human G3BP2 has Uniprot ID: Q9UN86-2 (SEQ ID NO: 2). Furthermore, variant G3BP2 proteins herein also include functional fragments or derivatives thereof.

[0023] The term "NTF2-like domain" as used herein refers to a protein domain that comprises the amino acid sequence set forth in SEQ ID NO: 3, and includes NTF2-like domain variants that have at least 80%, 85%, 90%, 95% and 100% amino acid sequence identity with the NTF2-like domain amino acid sequence set forth in SEQ ID NO: 3. NTF2-like domain variants can have a shorter, the same or longer amino acid sequence length than the amino acid sequence set forth in SEQ ID NO: 3, provided that these NTF2-like domain variants retain the ability to interact with Tau protein (functional NTF2-like domain variants). NTF2 refers to nuclear transport factor 2 protein.

[0024] The terms "modulator" and "inhibitor" as used herein refer to compounds that reversibly or irreversibly promote / enhance and / or inhibit protein-protein interaction between a protein containing an NTF2-like domain and a Tau protein.

[0025] The term "mimetic" as used herein refers to a compound that structurally mimics the natural protein-protein interaction between a protein containing an NTF2-like domain and a Tau protein.

[0026] "Percentage (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to achieve maximum percent sequence identity, without considering any conservative substitutions as part of sequence identity for alignment. Alignment for determining percent amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or FASTA program package. Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences to be compared. Alternatively, percent identity values ​​can be generated using sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc. and the source code is on file in the user documentation at the US Copyright Office, Washington DC, 20559, registered under US Copyright Registration No. TXU510087, and described in WO 2001 / 007611.

[0027] Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c, or the subsequent BLOSUM50 comparison matrix. The FASTA program package is described by WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; WR Pearson (1996) "Effective protein sequence comparison," Meth. Enzymol. 266:227-258; and Pearson et.al. (1997) Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch(global protein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2), ensuring a global rather than local alignment. Percent amino acid identity is indicated in the output alignment header. EXAMPLES

[0028] material and method Figure 1a: Surface plasmon resonance (SPR) experiments were performed at 25°C at Agro-Bio (La Ferte-Saint-Aubin, France) on a BiacoreT200 (Cytiva) with Biacore™ T200 Control Software (V2.0.2, Cytiva) using HBS-EP+ (Cytiva, Cat. BR100669) as running buffer. The following recombinant proteins were used in SPR: active human recombinant Tau441 (2N4R) wild type protein monomer (StressMarq, Cat. SPR-479) and customized His-tagged human full-length G3BP2 (1-449) were produced at GenScript Biotech (Piscataway, USA).

[0029] Figure 1b and Figure 5c: To mimic Tau aggregation in vitro, recombinant Tau441(2N4R)P301L (Analytik Jena, Cat.T-1014-1) at a final concentration of 1 μM was incubated with 30 μM sodium octadecyl sulfate (ODS) and 1 μM heparin in reagent buffer (20 μM thioflavin T, 5 mM 1,4-dithioerythreitol (DTT), 100 mM sodium chloride, 10 mM HEPES pH 7.4) for 15 h at 37 °C in a black non-binding 96-well plate. Three concentrations (0.2, 1 and 5 μM) of G3BP2 were incubated with the Tau-heparin-ODS-buffer solution. Immediately after preparation, baseline measurements were performed and kinetics were monitored every 15 min over 15 h while incubating at 37 °C by using 450 nm excitation and 485 nm emission fluorescence mode on a MultimodeReader Cytation 5 (BioTek). Assays were performed at QPS Austria GmbH (Grambach, Austria). Customized His-tagged human full-length G3BP2 (1-449) and G3BP2 NTF2-like fragment (amino acids 1-139) were produced at GenScript Biotech.

[0030] Figure 2: 2×10 iCell GlutaNeuron (FUJIFILM Cellular Dynamics, Cat.R1034) 5 cells / cm 2 The cells were seeded in 96-well plates at a density of 1000 x 1000 (Citroenza S.) / mL. On day 2, the cells were treated with the corresponding siRNA and subsequently transduced with lentiviral particles expressing P301S tau under the EF1a promoter at a multiplicity of infection (MOI) of 2 (Flash Therapeutics). On day 7, sonicated Tau preformed fibrils (PFFs) (StressMarq, Cat. SPR-471) were added to the medium at a concentration of 1 μg / ml along with 0.2 μl of Lipofectamine 2000 (Invitrogen, Cat. 11668019) per well. On day 14, Tau aggregates were measured using a Tau Aggregation Assay Kit (Cisbio, Cat. 6FTAUPEG) according to the manufacturer's instructions. Total Tau content was determined by Tau AlphaLISA Kit (Perkin Elmer, Cat. AL271).

[0031] Figure 3: The fAD iPSC (SFC805-03-01) line was obtained from the StemBANCC consortium. To generate G3BP2 KO fAD iPSC, two sgRNAs, G3BP2_1 5'-UUGUAGGGCGGGAGUUUGUG-3' (SEQ ID NO: 4) and G3BP2_2 5'-GUCGUUGUUCACGCACACG-3' (SEQ ID NO: 5) (150 pmol / reaction) (Synthego, USA), and TrueCut Cas9 protein v2 (50 pmol / reaction) were used. hiPSC-derived hCOs were generated using the STEMdiff Cerebral Organoid Kit (STEMCELL Technologies, Cat. 08570) according to the manufacturer's instructions.

[0032] For immunohistochemistry, hCOs were fixed using 4% paraformaldehyde (PFA) for 3 h at RT, washed with PBS, and immersed in 30% sucrose solution overnight at 4 °C. PFA-fixed organoids were embedded in OCT compound (Sakura Finetek, Cat. 4583) and stored at -80 °C. Organoid blocks were sectioned at 10 μm on a cryostat. For IHC, sections were permeabilized with 0.1% Triton X-100 and blocked with animal-free blocker (Vector Laboratories, Cat. SP-5030) for 30 min. Tissue sections were incubated with primary antibodies diluted in blocking solution for 1 h at RT, washed three times with PBS-T, and further incubated with secondary antibodies for 1 h at RT. After washing with PBS-T, sections were incubated with PureBlu DAPI (Bio-Rad, Cat. 1351303) for 3 min and mounted with ProLong Gold antifade mounting medium (Invitrogen, Cat. P36934). The following primary antibodies were used in IHC: G3BP2 (Novus, Cat. NBP1-82976), NeuN (Boster Bio, Cat. M11954-3), Tau pT231 (abcam, Cat. ab151559), Tau pT181 (abcam, Cat. ab254409) and Tau pS214 (abcam, Cat. ab170892). Species-specific Alexa Flour-conjugated secondary antibodies were used in this study (Invitrogen). Fluorescent images were acquired using a SP8 confocal microscope (Leica, Germany) and an Olympus SLIDEVIEW VS200 (Olympus, Japan) slide scanner.

[0033] hCO lysates were prepared using CytoBuster protein extraction reagent (Millipore, Cat. 71009) supplemented with cOmplete EDTA-free protease inhibitor tablets (Roche, Cat. 11836170001) and PhosSTOP tablets (Roche, Cat. 4906845001). Total Tau levels were measured by Human Tau AlphaLISA detection kit, and Tau pT231 and Tau pT181 levels were quantified using corresponding enzyme-linked immunosorbent assay (ELISA) kits (Thermo Fisher Scientific, Cat. KHB8051 and KHO0631) according to the manufacturer's instructions.

[0034] Figure 4: Duolink Proximity Ligation Assay (PLA) assays were performed according to the manufacturer's instructions. Briefly, tissue sections were processed and incubated with antibodies as described for immunohistochemistry. PLA was performed using anti-rabbit PLUS (Sigma, Cat.DUO92002), anti-mouse MINUS (Sigma, Cat.DUO92004) probes and In Situ Detection Reagents-Green (Sigma, Cat.DUO92014). Following PLA, slides were incubated with PureBlu DAPI for 3 min and mounted with ProLong Gold antifade mounting medium. Fluorescent images were acquired with a SP8 confocal microscope. Images were scanned for quantification using an Olympus SLIDEVIEW VS200 slide scanner. The following primary antibodies were used in PLA: Tau HT7 (Thermo Fisher Scientific, Cat. MN1000), G3BP2 (Novus, Cat. NBP1-82976) and Tau polyclonal antibody (Novus, Cat. NBP2-25163).

[0035] For PLA quantification of brain sections, sections were scanned using an Olympus Slideview VS200 slide scanner, and on average, over 9000 cells were counted for each patient. Nuclei and PLA spots were quantified using Track Mate's LoG detector (ImageJ) with the same estimated blob diameter and threshold in each experiment. The number of PLA spots was then normalized to the total number of cells for each patient.

Claims

1. A method for treating a Tau-related disease, comprising administering to a subject an effective amount of a compound that modulates protein-protein interaction between a protein comprising an NTF2-like domain having the amino acid sequence set forth in SEQ ID NO:3 or a variant thereof and a Tau protein.

2. The method of claim 1, wherein said compound promotes / enhances protein-protein interaction between said protein comprising said NTF2-like domain and Tau protein.

3. The method of claim 1, wherein the compound is a mimetic of the protein-protein interaction between the protein containing an NTF2-like domain and a Tau protein.

4. 4. The method of claim 1, wherein the Tau-related disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, punch-drunk syndrome, Down's syndrome, traumatic brain injury, Guam amyotrophic lateral sclerosis / Parkinsonism dementia complex, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangle disease with calcifications, frontotemporal dementia, frontotemporal dementia linked to chromosome 17 with Parkinsonism, Hallervorden-Spatz disease, Niemann-Pick disease type C, pallidal-pontocephalic-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, neurofibrillary tangle dementia, postencephalitic parkinsonism, and myotonic dystrophy.

5. The method according to claims 1 to 4, wherein the protein containing the NTF2-like domain is a G3PB2 protein.

6. The method of claims 1 to 5, wherein the protein containing the NTF2-like domain and the Tau protein are human proteins.

7. A compound which modulates protein-protein interaction between a protein comprising an NTF2-like domain having the amino acid sequence set forth in SEQ ID NO:3 or a variant thereof and Tau protein for use in the treatment of a Tau-related disease.

8. 8. The compound for use according to claim 7, wherein said Tau-related disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Drunk Punch Disease, Down's syndrome, traumatic brain injury, Guam amyotrophic lateral sclerosis / Parkinsonism dementia complex, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangle disease with calcifications, frontotemporal dementia, frontotemporal dementia linked to chromosome 17 with Parkinsonism, Hallervorden-Spatz disease, Niemann-Pick disease type C, pallidal-pontocephalic-nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, neurofibrillary tangle dementia, postencephalitic parkinsonism, and myotonic dystrophy.

9. A protein comprising an NTF2-like domain having the amino acid sequence set forth in SEQ ID NO:3 or a variant thereof, for use in identifying a compound that modulates protein-protein interaction between a protein comprising said NTF2-like domain and a Tau protein.

10. A protein comprising an NTF2-like domain having the amino acid sequence set forth in SEQ ID NO:3 or a variant thereof for use as a target protein for the treatment of a Tau-related disease.

11. The protein for use according to claim 9 or 10, wherein the protein is a human G3BP2 protein.

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