Protein for inhibiting conserved helix of TDP-43 and use thereof

Abstract

Disclosed in the present invention are a protein for inhibiting the conserved helix of TDP-43 and the use thereof. The protein contains an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2. Further disclosed in the present invention are the use of the protein in the preparation of a drug for diagnosing, preventing and / or treating neurodegenerative diseases, and the use in the preparation of an inhibitor of a target protein containing the conserved helix of TDP-43 LCD. In the present invention, by means of using artificial intelligence-assisted protein design techniques, a high-affinity non-natural protein that precisely binds to the conserved helix of a TDP-43 protein is designed, which is used for inhibiting the participation of the conserved helix region in the formation of a β-sheet aggregation core, is mainly used in the treatment of diseases targeting the TDP-43 protein, such as amyotrophic lateral sclerosis, and is used as a research tool for TDP-43 protein phase separation experiments.

Description

A protein that inhibits the conserved TDP-43 helix and its applications

[0001] This application claims priority to Chinese Patent Application No. 2024114764391, filed on October 22, 2024. The entire contents of the aforementioned Chinese Patent Application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of protein design and macromolecular drug development, specifically relating to a protein that inhibits the conserved helix of TDP-43 and its applications. Background Technology

[0003] Protein misfolding and aggregation are the molecular basis of a variety of diseases, including neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) [1]. A key feature of these diseases is the formation of amyloid fibrils, which are formed by interchain interactions of protein fragments with a high β-fold orientation [2]. Although studies have been dedicated to understanding the link between amyloid formation and disease [3], the development of inhibitors targeting amyloid formation segments remains challenging. These segments are often disordered, making it difficult to apply traditional structure-based approaches to inhibitor design. In addition, existing inhibitors, such as antibodies and small molecule compounds, while effective in some cases, may be difficult to target specific regions and conformational states.

[0004] TDP-43 (a 43 kDa TAR DNA-binding protein) undergoes liquid-liquid phase separation under stress, forming reversible nucleosomes that protect the cell nucleus. Abnormal liquid-solid phase transitions can cause TDP-43 to condense into irreversible pathological fibers, which are the most widespread and studied pathological proteins in the ALS / FTD lineage. In disease states, TDP-43 is depleted from the cell nucleus and is found as hyperphosphorylated, aggregated intracytoplasmic inclusion bodies in approximately 97% of ALS patients and 50% of FTD patients [4]. Currently, inhibitors targeting TDP-43 mainly include small molecule compounds, antibodies, and peptides [5]. However, no drugs have yet entered the market through clinical trials. Summary of the Invention

[0005] To address the lack of an effective inhibitor targeting TDP-43 in existing technologies, this invention provides a protein that inhibits the conserved helix of TDP-43 and its applications. This invention also provides de novo design of non-natural protein drugs, primarily for the treatment of diseases targeting TDP-43, such as ALS, and as a research tool for TDP-43 protein phase separation experiments.

[0006] To solve the above-mentioned technical problems, one of the technical solutions provided by the present invention is: a non-naturally occurring protein, the protein comprising an amino acid sequence of the general formula X1-X2-X3, wherein: X1 comprises a first helical domain, the first helical domain comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:10; X2 comprises a second and a third helical domain; X3 comprises a fourth and a fifth helical domain, the fourth and fifth helical domains comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:12.

[0007] In a specific embodiment of the present invention, the second and third helical domains comprise an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:11.

[0008] In a specific embodiment of the present invention, the protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:1.

[0009] In a specific embodiment of the present invention, the protein comprises the amino acid sequence shown in SEQ ID NO:1.

[0010] In a specific embodiment of the present invention, the amino acid sequence of the protein is shown in SEQ ID NO:1.

[0011] In a specific embodiment of the present invention, the protein is capable of inhibiting the TDP-43 conserved helix; the TDP-43 conserved helix preferably contains the amino acid sequence shown in SEQ ID NO:3.

[0012] To solve the above-mentioned technical problems, the second technical solution provided by the present invention is: a non-naturally occurring protein, the protein comprising an amino acid sequence of the general formula X1-X2-X3, wherein: X1 comprises a first helical domain, the first helical domain comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:13, and changes to L15E in SEQ ID NO:13 are not permitted; X2 comprises a second, third, fourth, and fifth helical domains; X3 comprises a sixth and seventh helical domains, the sixth and seventh helical domains comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:15, and SEQ ID NO:13... Changing L28E in NO:15 is not allowed.

[0013] In a specific embodiment of the present invention, the second, third, fourth and fifth helical domains comprise an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:14.

[0014] In a specific embodiment of the present invention, the protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:2, and alterations to L15E and L112E in SEQ ID NO:2 are not permitted.

[0015] In a specific embodiment of the present invention, the protein comprises an amino acid sequence as shown in SEQ ID NO:2.

[0016] In a specific embodiment of the present invention, the amino acid sequence of the protein is shown in SEQ ID NO:2.

[0017] In a specific embodiment of the present invention, the protein is capable of inhibiting the TDP-43 conserved helix; the TDP-43 conserved helix preferably contains the amino acid sequence shown in SEQ ID NO:3.

[0018] To solve the above-mentioned technical problems, the third technical solution provided by the present invention is: an isolated nucleic acid, wherein the nucleic acid encodes a protein as described in the first or second technical solution.

[0019] To solve the above-mentioned technical problems, the fourth technical solution provided by the present invention is: a recombinant expression vector, wherein the recombinant expression vector comprises the nucleic acid as described in the third technical solution.

[0020] To solve the above-mentioned technical problems, the fifth technical solution provided by the present invention is: a transformant, wherein the transformant comprises the nucleic acid as described in the third technical solution or the recombinant expression vector as described in the fourth technical solution; the transformant is a non-animal or non-plant variety.

[0021] In a specific embodiment of the present invention, the transformant is a eukaryotic cell or a prokaryotic cell.

[0022] In a specific embodiment of the present invention, the host cell of the transformant is Escherichia coli or HEK293T cells.

[0023] To solve the above-mentioned technical problems, the sixth technical solution provided by the present invention is: a kit comprising the protein as described in technical solution one or technical solution two, the nucleic acid as described in technical solution three, the recombinant expression vector as described in technical solution four, or the transformant as described in technical solution five.

[0024] To solve the above-mentioned technical problems, the seventh technical solution provided by the present invention is: a pharmaceutical composition comprising the protein as described in one or two technical solutions, and a pharmaceutically acceptable carrier.

[0025] To solve the above-mentioned technical problems, the eighth technical solution provided by the present invention is: a method for preparing the protein as described in technical solution one or technical solution two, the method comprising culturing the transformant as described in technical solution five.

[0026] To solve the above-mentioned technical problems, the ninth technical solution provided by the present invention is: a method for studying the phase separation of target proteins, the method comprising contacting the protein as described in one or two technical solutions or the pharmaceutical composition as described in seven technical solutions with the target protein, the target protein comprising a conserved helix of TDP-43LCD; the conserved helix of TDP-43LCD preferably comprises an amino acid sequence as shown in SEQ ID NO:3.

[0027] In a specific embodiment of the present invention, the target protein comprises TDP-43LCD; the TDP-43LCD preferably comprises amino acids 274 to 414 of the sequence shown in NP_031401.1.

[0028] In a specific embodiment of the present invention, the target protein comprises TDP-43CTF; the TDP-43CTF preferably comprises amino acids 208 to 414 of the sequence shown in NP_031401.1.

[0029] In a specific embodiment of the present invention, the target protein is TDP-43, which preferably contains an amino acid sequence as shown in NP_031401.1.

[0030] In a specific embodiment of the present invention, the target protein is a TDP-43 mutant, wherein the TDP-43 mutant does not contain amino acid substitutions of M322D, A326P, and L330R relative to the sequence shown in NP_031401.1; the TDP-43 mutant preferably contains one or more amino acid substitutions selected from P112H, K181E, and K263E on the amino acid sequence shown in NP_031401.1.

[0031] In a specific embodiment of the present invention, the method is not for diagnostic, preventive and / or therapeutic purposes.

[0032] To solve the above-mentioned technical problems, the tenth technical solution provided by the present invention is: a method for inhibiting a target protein, the method comprising contacting the target protein with a protein as described in technical solution one or technical solution two or a pharmaceutical composition as described in technical solution seven, wherein the target protein comprises a conserved helix of TDP-43LCD; the conserved helix of TDP-43LCD preferably comprises an amino acid sequence as shown in SEQ ID NO:3.

[0033] In a specific embodiment of the present invention, the target protein comprises TDP-43LCD; the TDP-43LCD preferably comprises amino acids 274 to 414 of the sequence shown in NP_031401.1.

[0034] In a specific embodiment of the present invention, the target protein comprises TDP-43CTF; the TDP-43CTF preferably comprises amino acids 208 to 414 of the sequence shown in NP_031401.1.

[0035] In a specific embodiment of the present invention, the target protein is TDP-43, which preferably contains an amino acid sequence as shown in NP_031401.1.

[0036] In a specific embodiment of the present invention, the target protein is a TDP-43 mutant, wherein the TDP-43 mutant does not contain amino acid substitutions of M322D, A326P, and L330R relative to the sequence shown in NP_031401.1; the TDP-43 mutant preferably contains one or more amino acid substitutions selected from P112H, K181E, and K263E on the amino acid sequence shown in NP_031401.1.

[0037] In a specific embodiment of the present invention, the inhibited target protein is the target protein that inhibits fibrotic aggregation.

[0038] In a specific embodiment of the present invention, the method is not for diagnostic, preventive and / or therapeutic purposes.

[0039] To solve the above-mentioned technical problems, the eleventh technical solution provided by the present invention is: a set of medicine boxes, the set of medicine boxes including medicine box A, the medicine box A containing the protein as described in technical solution one or technical solution two or the drug composition as described in technical solution seven.

[0040] In a specific embodiment of the present invention, the kit further includes a box B containing other drugs for diagnosing, preventing and / or treating neurodegenerative diseases and / or inhibitors targeting TDP-43.

[0041] In a specific embodiment of the present invention, the neurodegenerative disease is ALS or frontotemporal lobe degeneration, and / or the inhibitor is selected from one or more small molecule compounds, antibodies, and peptides.

[0042] To solve the above-mentioned technical problems, the twelfth technical solution provided by the present invention is: the use of the protein as described in technical solution one or technical solution two, the nucleic acid as described in technical solution three, the recombinant expression vector as described in technical solution four, the transformant as described in technical solution five, the kit as described in technical solution six, or the pharmaceutical composition as described in technical solution seven in the preparation of a drug for diagnosing, preventing and / or treating neurodegenerative diseases.

[0043] In a specific embodiment of the present invention, the neurodegenerative disease is ALS or frontotemporal lobe degeneration.

[0044] To solve the above-mentioned technical problems, the thirteenth technical solution provided by the present invention is: the use of the protein as described in technical solution one or technical solution two, the nucleic acid as described in technical solution three, the recombinant expression vector as described in technical solution four, the transformant as described in technical solution five, the kit as described in technical solution six, or the pharmaceutical composition as described in technical solution seven in the preparation of an inhibitor of a target protein, wherein the target protein comprises a conserved helix of TDP-43LCD; the conserved helix of TDP-43LCD preferably comprises the amino acid sequence shown in SEQ ID NO:3.

[0045] In a specific embodiment of the present invention, the inhibitor is capable of inhibiting the fibrotic aggregation of the target protein.

[0046] In a specific embodiment of the present invention, the target protein comprises TDP-43LCD; the TDP-43LCD preferably comprises amino acids 274 to 414 of the sequence shown in NP_031401.1.

[0047] In a specific embodiment of the present invention, the target protein comprises TDP-43CTF; the TDP-43CTF preferably comprises amino acids 208 to 414 of the sequence shown in NP_031401.1.

[0048] In a specific embodiment of the present invention, the target protein is TDP-43, which preferably contains an amino acid sequence as shown in NP_031401.1.

[0049] In a specific embodiment of the present invention, the target protein is a TDP-43 mutant, wherein the TDP-43 mutant does not contain amino acid substitutions of M322D, A326P, and L330R relative to the sequence shown in NP_031401.1; the TDP-43 mutant preferably contains one or more amino acid substitutions selected from P112H, K181E, and K263E on the amino acid sequence shown in NP_031401.1.

[0050] To solve the above-mentioned technical problems, the fourteenth technical solution provided by the present invention is: a method for diagnosing, preventing and / or treating neurodegenerative diseases, the method comprising administering to a subject in need an effective amount of a protein as described in one or two technical solutions, a nucleic acid as described in three technical solutions, a recombinant expression vector as described in four technical solutions, a transformant as described in five technical solutions, a kit as described in six technical solutions, or a pharmaceutical composition as described in seven technical solutions.

[0051] In a specific embodiment of the present invention, the neurodegenerative disease is ALS or frontotemporal lobe degeneration.

[0052] To solve the above-mentioned technical problems, the present invention provides the following fifteenth technical solution: a protein as described in technical solution one or technical solution two, a nucleic acid as described in technical solution three, a recombinant expression vector as described in technical solution four, a transformant as described in technical solution five, a kit as described in technical solution six, or a pharmaceutical composition as described in technical solution seven, which are used for the diagnosis, prevention and / or treatment of neurodegenerative diseases.

[0053] In a specific embodiment of the present invention, the neurodegenerative disease is ALS or frontotemporal lobe degeneration.

[0054] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0055] The reagents and raw materials used in this invention are all commercially available.

[0056] The positive and progressive effects of this invention are as follows:

[0057] This invention utilizes artificial intelligence-assisted protein design technology to design a high-affinity non-natural protein that precisely binds to the conserved helix of TDP-43 protein. This protein is used to inhibit the conserved helix region from participating in the formation of the β-sheet core, thereby inhibiting the fibrosis of TDP-43 protein and achieving the therapeutic effect of treating ALS. The results have been verified at the in vitro biochemical and cellular levels. Attached Figure Description

[0058] Figure 1 is a schematic diagram of the TDP-43 protein sequence structure and target selection, in which:

[0059] The upper part of Figure 1: Schematic diagram of the TDP-43 protein sequence and secondary structure, with the conserved helix 319-335 (SEQ ID NO:3, NPAMMAAAQAALQSSWG) as the target for inhibitor design;

[0060] The lower half of Figure 1: (left) predicted alphafold structure of the full-length TDP-43 protein, (middle) top and side view of the TDP-43 aggregation core (PDB number: 7py2), and (right) predicted alphafold structure of the conserved helix.

[0061] Figures 2A-2C show the in vitro validation of the candidate proteins, in which:

[0062] Figure 2A shows the ThT aggregation kinetics curves of TDP-43LC (20 μM) with different candidate proteins (10 μM). The data are presented as mean ± standard error, n = 3.

[0063] Figure 2B shows the quantitative statistical results of fluorescence values ​​at the endpoint of the ThT experiment in Figure 2A.

[0064] Figure 2C is a TEM image of TDP-43LC amyloid fibers at the endpoint of the ThT experiment in Figure 2A, with a scale bar of 200 nm.

[0065] Figures 3A-3I show the in vitro validation of TDP-43 protein inhibitors B1 and B9, where:

[0066] Figure 3A, Figure 3B: Size exclusion chromatograms of B1 (Figure 3A) and B9 (Figure 3B);

[0067] Figure 3C, Figure 3E: ThT kinetic curves of TDP-43LC (20 μM) with different concentrations of B1 (Figure 3C) or B9 (Figure 3E). Data are presented as mean ± standard error, n = 3.

[0068] Quantitative statistical results of fluorescence values ​​at the endpoint of the ThT experiment in Figures 3D, 3F, 3C, and 3E.

[0069] Figure 3G: Transmission electron microscope images of TDP-43LC amyloid fibers at the endpoint of the ThT experiment in Figures 3C and 3E, scale bar 500 nm;

[0070] Figure 3H, Figure 3I: Quantitative analysis of the affinity of B1 and B9 in the biolayer interference experiment. The affinity of B1 (Figure 3H) is 25 μM, and the affinity of B9 (Figure 3I) is 86 nM.

[0071] Figures 4A-4H show the NMR and alphafold structures of B1 and B9, where:

[0072] Figure 4A: Titration using B9 at molar ratios of 1:0.8, 1:0.6, 1:0.4, and 1:0.2 15 The HSQC spectrum of N-labeled TDP-43LC;

[0073] Figure 4B; HSQC spectra of amino acid residues within the TDP-43 domain targeted by B9;

[0074] Figure 4C: In Figure 4A, the signal intensity changes of each amino acid in TDP-43LC after B9 titration. The domain of B9 targeting TDP-43 is shown in light color.

[0075] Figure 4D: Comparison of the design results of the TDP-43 and B9 complex with the prediction results of alphafold;

[0076] Figure 4E: HSQC spectra obtained by titrating 15N-labeled TDP-43LC with B1 at molar ratios of 1:0.8, 1:0.6, 1:0.4 and 1:0.2.

[0077] Figure 4F: HSQC spectra of amino acid residues within the TDP-43 domain targeted by B1;

[0078] Figure 4G: In Figure 4E, the signal intensity changes of each amino acid in TDP-43LC after B1 titration. The domain of B1 targeting TDP-43 is shown in light color.

[0079] Figure 4H: Comparison of the design results of the TDP-43 and B1 complex with the prediction results of alphafold.

[0080] Figures 5A-5F show the mutation verification of the TDP-43 inhibitor B9, where:

[0081] Figure 5A: Biolayer interference experiment affinity determination of leucine at position 112 and glutamate at position 15, which are important interacting amino acids of the TDP-43 inhibitor B9.

[0082] Figure 5B: Biolayer interference experiment affinity determination of the important interacting amino acids of TDP-43LC, namely, methionine at position 322 is mutated to aspartic acid, alanine at position 326 is mutated to proline, and leucine at position 330 is mutated to arginine.

[0083] Figures 5C-5E: ThT aggregation kinetics curves and transmission electron microscope images of the corresponding endpoints of the TDP-43LC mutant with and without B9, scale bar 500 nm, including A326P (Figure 5C), L330R (Figure 5D) and M322D (Figure 5E), data are presented as mean ± standard error, n=3;

[0084] Figure 5F: ThT aggregation kinetics curves and corresponding transmission electron microscopy images of TDP-43LC with and without different types of B9, scale bar 500 nm, including L15E, L112E and WT. Data are presented as mean ± standard error, n = 3.

[0085] Figures 6A-6F show the cellular validation of the inhibitory effects of B1 and B9 on TDP-43 protein aggregation, where:

[0086] Figure 6A: Co-transfection of EGFP-TDP-43 in HEK 293T cells CTF TDP-43 in the cytoplasm was observed using confocal microscopy imaging with Myc empty vector, Myc-B1 or Myc-B9;

[0087] Figure 6B: TDP-43 appears in HEK 293T cells. CTFStatistical analysis of the proportion of clustered cells, with data presented as mean ± standard error, based on three biologically independent samples, with more than 200 cells counted in each dataset.

[0088] Figure 6C: Protein expression levels were detected by Western blotting, with GAPDH as an internal control protein;

[0089] Figure 6D: Co-transfection of EGFP-TDP-43 in HEK 293T cells K181E TDP-43 aggregated in cell nuclei was observed using confocal microscopy imaging with Myc empty vector, Myc-B1, or Myc-B9.

[0090] Figure 6E: TDP-43 appears in HEK 293T cells. K181E Statistical analysis of the proportion of clustered cells, with data presented as mean ± standard error, based on three biologically independent samples, with more than 200 cells counted in each dataset.

[0091] Figure 6F: Protein expression levels were detected by Western blotting, with GAPDH used as an internal reference protein.

[0092] Figure 7A shows the circular dichroism chromatograms of B9 at different temperatures.

[0093] Figure 7B shows the circular dichroism chromatograms of B1 at different temperatures.

[0094] Figure 8 shows a transmission electron microscope image of the formation of full-length TDP-43 fibers with or without binding proteins (B1 or B9), where the scale bar is 100 nm. Detailed Implementation

[0095] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, the following definitions of key terms are provided:

[0096] In this invention, NP_031401.1 refers to the NCBI Reference Sequence number of the TDP-43 protein (TAR DNA-binding protein 43 [Homo sapiens]) in the National Center for Biotechnology Information (NCBI) database. Those skilled in the art can obtain the amino acid sequence of the TDP-43 protein by searching for this number.

[0097] In this invention, "TDP-43LC" and "TDP-43LCD" both refer to the low complexity domain (LCD) of the TDP-43 protein, for example, it can correspond to amino acids 274 to 414 of the sequence shown in NP_031401.1; "TDP-43CTF" and "TDP-43" CTF "Both refer to the truncated C-terminal fragment (CTF) of the TDP-43 protein, for example, it can correspond to amino acids 208-414 of the sequence shown in NP_031401.1. "Conserved helix of TDP-43" and "Conserved helix of TDP-43LCD" both refer to the conserved region in the LC domain (LCD) of the TDP-43 protein, for example, it can correspond to amino acids 319-335 of the sequence shown in NP_031401.1.

[0098] In this invention, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the target sequence means that compared to the target sequence, it contains one or more mutations, such as a single amino acid insertion, a single amino acid deletion, a single amino acid substitution, or a combination thereof, and retains the original protein's activity in inhibiting the TDP-43 conserved helix, or has a superior activity in inhibiting the TDP-43 conserved helix compared to the original protein.

[0099] In this invention, "nucleic acid" refers to a nucleotide chain of any length and includes DNA and RNA. A nucleotide can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or its analogues, or any substrate capable of being incorporated into the chain by DNA or RNA polymerase.

[0100] In this invention, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, permits the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors of this invention are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of this invention can contain any type of nucleotide, including but not limited to DNA and RNA that can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors can contain naturally occurring or non-naturally occurring nucleotide linkages, or both. In an exemplary aspect, modified nucleotides or non-naturally occurring nucleotide linkages do not impede transcription or replication of the vector.

[0101] The recombinant expression vector of the present invention can be any suitable recombinant expression vector capable of being used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably to express the genes or sequences in the host cell. Suitable vectors include those designed for amplification and expansion or for expression or both of the above, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0102] In this invention, the term "host cell" refers to any type of cell that may contain the nucleic acids or vectors described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, animal, fungus, or algae; or it may be a prokaryotic cell, such as a bacterium or protozoan.

[0103] In this invention, a "pharmaceutical composition" may comprise a suitable pharmaceutically acceptable carrier, such as pharmaceutical excipients, including buffers, as known in the art. A "pharmaceuticalally acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextran, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The pharmaceutical compositions of this invention can be prepared by mixing the proteins of this invention, having the desired purity, with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution.

[0104] The pharmaceutical compositions of the present invention may also comprise more than one active ingredient required for a specific indication to be treated, preferably those having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other diagnoses, preventative and / or therapeutic drugs for neurodegenerative diseases and / or inhibitors targeting TDP-43, etc. The active ingredients are suitably combined in amounts effective for the intended use. Sustained-release formulations can be prepared, suitable examples of which include a semi-permeable matrix of a solid hydrophobic polymer containing the protein of the present invention, said matrix being in the form of a shaped article, such as a film or microcapsule.

[0105] In this invention, the application scenarios for "non-diagnostic, preventive and / or therapeutic purposes" include, but are not limited to: inhibiting target proteins containing the conserved helix of TDP-43LCD for research purposes in the laboratory (e.g., the application scenarios in the embodiments of this invention); or using it as a positive control to screen other inhibitors that inhibit target proteins containing the conserved helix of TDP-43LCD; or competing with other inhibitors that inhibit target proteins containing the conserved helix of TDP-43LCD for binding, etc.

[0106] In this invention, the term "effective amount" refers to the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of a disease or condition, compared to a corresponding subject who did not receive that amount. Within its scope, the term also includes amounts that effectively enhance normal physiological function.

[0107] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0108] Preliminary example: Computer-aided and AI-assisted design and screening of non-natural TDP-43 protein aggregation inhibitors

[0109] This invention uses an AI-assisted backbone generation and sequence optimization method to design a non-natural protein that specifically binds to the conserved helical sequence of TDP-43 to inhibit the fibrotic aggregation of TDP-43 protein (Figure 1).

[0110] The inventors conducted in vitro validation of the designed candidate proteins (B1, B4-5, B7-9, B11, and B13) using the same experimental methods as in Example 1. The experimental results are shown in Figures 2A to 2C (where the control is a blank control without candidate proteins). The results show that proteins B1 and B9 can significantly inhibit the fibrotic aggregation of TDP-43 protein.

[0111] The detailed protein sequence information is as follows:

[0112] B1 (SEQ ID NO:1):

[0113] In this sequence, the N-terminus of the bold sequence represents the first helical domain X1, the bold sequence represents the second and third helical domains X2, and the C-terminus of the bold sequence represents the fourth and fifth helical domains X3.

[0114] B9 (SEQ ID NO:2):

[0115] In this sequence, the N-terminus of the bold sequence represents the first helical domain X1, the bold sequence represents the second, third, fourth, and fifth helical domains X2, and the C-terminus of the bold sequence represents the sixth and seventh helical domains X3.

[0116] B4 (SEQ ID NO:4):

[0117] B5 (SEQ ID NO:5):

[0118] B7 (SEQ ID NO:6):

[0119] B8 (SEQ ID NO:7):

[0120] B11 (SEQ ID NO:8):

[0121] B13 (SEQ ID NO:9):

[0122] Example 1: Purification and affinity experiment of B1 and B9 proteins

[0123] This invention uses an Escherichia coli expression system to obtain highly homogeneous monomeric proteins by size exclusion chromatography (SEC) (Figures 3A-3B), and determines the affinity of B1 and B9 for the TDP-43LC domain by biological layer interferometry (BLI).

[0124] 1. Experimental materials and instruments:

[0125] The proteins of this invention are B1 (molecular weight: 12.9 KD) and B9 (molecular weight: 14.63 KD).

[0126] The target protein TDP-43LC (NCBI Reference Sequence: NP_031401.1, amino acids 274-414, molecular weight: 17.2KD, exhibits aggregation tendency)

[0127] Biolayer Interferometry (BLI) Sensor (ForteBio Octet System)

[0128] 2. Experimental Methods:

[0129] 2.1 Determination of the binding affinity of B1 and B9 to target proteins using the BLI method:

[0130] 2.1.1 Affinity determination of B1

[0131] (1) The target protein TDP-43LC was immobilized on the sensor using the ForteBio Octet system.

[0132] (2) Add B1 to the sensor in a concentration gradient (30 μM, 10 μM, 3 μM, 1 μM).

[0133] Record the binding and dissociation curves, analyze the data using Octet software, and calculate the binding affinity of B1 to the target protein TDP-43LC (KD value = 25 μM) (Figure 3H).

[0134] 2.1.2 Affinity determination of B9

[0135] (1) The target protein TDP-43LC was immobilized on the sensor using the ForteBio Octet system.

[0136] (2) B9 was added to the sensor in a concentration gradient (729 nM, 243 nM, 81 nM, 27 nM, 9 nM).

[0137] Binding and dissociation curves were recorded, and the data were analyzed using Octet software to calculate the binding affinity of B9 to the target protein TDP-43LC (KD value = 86 nM) (Figure 3I).

[0138] 3. Experimental Results:

[0139] Both B1 and B9 proteins can bind to the LC domain of TDP-43 protein. The affinity of B1 was measured to be 25 μM and that of B9 was 86 nM by the BLI method.

[0140] Example 2: Testing the inhibitory effects of B1 and B9 on target protein aggregation

[0141] 1. Experimental materials and instruments:

[0142] The proteins of this invention are B1 (molecular weight: 12.9 KD) and B9 (molecular weight: 14.63 KD).

[0143] The target protein TDP-43LC (molecular weight: 17.2KD, with a tendency to aggregate)

[0144] Thioflavin T (ThT) assay kit (ThT (CAS no. 2390-54-7) Sigma-Aldrich (T3516), containing ThT dye and buffer 20 mM MES, 100 mM NaCl, pH 6.0)

[0145] Microplate reader (BMG LABTECH) and 96-well plate (Thermo Fisher Scientific)

[0146] 120kV transmission electron microscope (FEI Company); 3% w / v uranyl acetate; copper mesh (Beijing Zhongjing Keyi)

[0147] 2. Experimental Methods:

[0148] 2.1.1 ThT assay to quantitatively detect the inhibitory effects of B1 and B9 on target protein aggregation

[0149] (1) Mix TDP-43LC (20 μM) with different concentrations of B1 or B9 (0 μM, 5 μM, 10 μM, 20 μM), then mix in 50 μM ThT dye, and add 50 μl of the mixture to a 96-well plate.

[0150] (2) Place the 96-well plate in a microplate reader and monitor it continuously at 37°C with a rotation speed of 900 rpm for 8 hours. Record the fluorescence value (Ex 440nm, Em 480nm) every five minutes. Analyze the data using GraphPad Prism 8 (Figures 3C-3F, controls are without B1 or B9).

[0151] 2.1.2 Transmission electron microscopy characterization of fiber growth

[0152] A 5 μL fiber sample was dropped onto a copper grid and incubated for 45 seconds. The copper grid was then washed with 5 μL of double-distilled water and 3% uranium acetate, and then stained with 3% uranium acetate for 45 seconds. The copper grid was then dried in air and observed using a 120 kV transmission electron microscope (Figure 3G, control is without B1 or B9).

[0153] 3. Experimental Results:

[0154] Both B1 and B9 proteins can be obtained with high purity and uniformity using an *E. coli* expression system. During ThT experiments, it was found that both B1 and B9 significantly inhibited the aggregation behavior of wild-type TDP-43. Electron microscopy revealed that the introduction of B1 and B9 significantly reduced the occurrence of TDP-43 protein fibrillary aggregation compared to the control group. Biomembrane interference technology confirmed that B1 and B9 can bind to the LC domain of TDP-43 protein, with B1 having an affinity of 25 μM and B9 an affinity of 86 nM.

[0155] Example 3: Determination of the binding sites of B1 and B9 proteins with the target protein TDP-43

[0156] 1. Experimental materials and instruments:

[0157] The proteins of this invention are B1 (molecular weight: 12.9 KD) and B9 (molecular weight: 14.63 KD).

[0158] The target protein TDP-43LC (molecular weight: 17.2KD, with a tendency to aggregate)

[0159] Nuclear Magnetic Resonance System (Agilent 800MHz)

[0160] Alphafold protein structure prediction software

[0161] Thioflavin T (ThT) assay kit (includes ThT dye and buffer: 20 mM MES, 100 mM NaCl, pH 6.0)

[0162] 120kV transmission electron microscope (FEI Company); 3% w / v uranyl acetate; copper mesh (Beijing Zhongjing Keyi)

[0163] Biolayer Interferometry (BLI) Sensor (ForteBio Octet System)

[0164] 2. Experimental Methods:

[0165] 2.1 HSQC spectra of amino acid residues within the TDP-43 domain determined by nuclear magnetic resonance (NMR) method

[0166] 2.1.1 15N-labeled TDP-43LC was titrated with B1 at molar ratios of 1:0.8, 1:0.6, 1:0.4, and 1:0.2, and the HSQC spectra were measured (Figure 4E). After B1 titration, the changes in signal intensity of each amino acid in TDP-43LC were consistent with the designed target amino acids (Figure 4F). The domains of TDP-43 targeted by B1 were shown in light color (Figure 4G).

[0167] 2.1.2 15N-labeled TDP-43LC was titrated with B9 at molar ratios of 1:0.8, 1:0.6, 1:0.4, and 1:0.2, and the HSQC spectra were measured (Figure 4A). After B9 titration, the changes in signal intensity of each amino acid in TDP-43LC were consistent with the designed target amino acids (Figure 4B). The domains of TDP-43 targeted by B9 were shown in light color (Figure 4C).

[0168] 2.2 Predicting the binding modes of B1, B9, and full-length TDP-43 using Alphafold structure prediction software.

[0169] 2.2.1 The complex of B9 and full-length TDP-43 protein was predicted using the alphafold complex prediction network. Five complex networks were used, and five results were generated for each network, for a total of 25 prediction results. The binding sites of the conserved helix of TDP-43 and B9 were consistent with the design results (Figure 4D).

[0170] 2.2.2 The complex of B1 and full-length TDP-43 protein was predicted using the alphafold complex prediction network. Five complex networks were used, and five results were generated for each network, for a total of 25 prediction results. The binding sites of the conserved helix of TDP-43 and B1 were consistent with the design results (Figure 4H).

[0171] 2.3 Mutation verification of the B9-TDP-43 binding site

[0172] 2.3.1 Mutations in TDP-43

[0173] Mutations of key interacting amino acids in TDP-43 with the B9 protein (M322D, A326P, L330R) revealed that the binding ability of the TDP-43 mutant to the B9 protein was almost completely lost through BLI experiments (Figure 5B). ThT experiments showed that B9 could not inhibit the conserved helical mutant of TDP-43, and no decrease in the aggregation ratio was observed under transmission electron microscopy (Figures 5C-5E).

[0174] 2.3.2 Mutations in the B9 protein

[0175] Mutations were made in the key interacting amino acids of the B9 protein with TDP-43 (L15E, L112E). BLI assays revealed a significant decrease in the binding affinity of the TDP-43 mutant to the B9 protein (Figure 5A). ThT assays showed that the B9 mutant failed to inhibit TDP-43 aggregation, and no decrease in the aggregation ratio was observed under transmission electron microscopy (Figure 5F).

[0176] 3. Experimental Results

[0177] Experiments have shown that both B1 and B9 bind to the conserved helical region of the TDP-43 protein (319-335, SEQ ID NO:3, NPAMMAAAQAALQSSWG).

[0178] Protein B1 is composed of a non-naturally occurring polypeptide. Based on the Alphafold predicted structure (Figure 4H), the inventors described it according to the general formula X1-X2-X3. Here, X1 is the first helical domain (AAAEAEARAARLEAFRAFAEALLKAVQAAV, SEQ ID NO:10), X2 is the second and third helical domains (PDLPAEELKAWEAFVAELLKLIEAGDLAGLRALLEEELARTREDP, SEQ ID NO:11), and X3 is the fourth and fifth helical domains (AYAPRLAAAVYALRLLGEDEAAAKAVAAMEALA, SEQ ID NO:12). Based on Alphafold predictions (Fig. 4H, where B1 forms a complex with TDP-43, with the X1 and X3 regions of B1 interacting with the conserved helices of the TDP-43 protein) and NMR data (Figs. 4E-4G, where Fig. 4E shows titrations at different concentration ratios (1:0, 1:0.2, 1:0.4, 1:0.6, 1:0.8) 15 The HSQC signal changes of each amino acid in TDP-43LC labeled with N are shown in Figure 4G. It can be seen that the main region of intensity change of TDP-43LC is at the conserved helical position of TDP-43, which indicates that B1 protein binds to the conserved helical region of TDP-43 protein. X1 and X3 are identified as key regions that bind to the conserved helical region of TDP-43 protein and exert inhibitory activity.

[0179] Protein B9 is composed of a non-naturally occurring polypeptide. Based on the Alphafold predicted structure (Figure 4D), the inventors described it according to the general formula X1-X2-X3. Wherein, X1 is the first helical domain (SETKRRITELALEALTLIATD, SEQ ID NO:13), X2 is the second, third, fourth, and fifth helical domains (PSARAEVRALIDEIAEINKGKKATEVATILAEEIPEYADFFESIKDLTAEELAELTLKLIDAG, SEQ ID NO:14), and X3 is the sixth and seventh helical domains (DDRAVLALFALALLEFSREEQRAAARTLAEQLKAAA, SEQ ID NO:15). Based on the predicted structure of the Alphafold complex (Figure 4D, where B9 forms a complex with TDP-43, and the X1 and X3 regions of B9 interact with the conserved helices of the TDP-43 protein), the NMR spectra (Figures 4A-4C, where Figure 4A shows titrations at different concentration ratios (1:0, 1:0.2, 1:0.4, 1:0.6, 1:0.8) are obtained. 15 The HSQC signal changes of each amino acid in TDP-43LC labeled with N are shown in Figure 4C. It can be seen that the main region of intensity change of TDP-43LC is at the conserved helical position of TDP-43, which indicates that B9 protein binds to the conserved helical region of TDP-43 protein. The BLI of the mutant and electron microscopy results (Figures 5A-5F) confirm that X1 and X3 are the key regions that bind to the conserved helical region of TDP-43 protein and exert inhibitory activity.

[0180] Example 4: Cellular experiments demonstrating the inhibitory effect of B1 and B9 on TDP-43 protein aggregation.

[0181] 1. Experimental materials and instruments:

[0182] Reagents: DMEM cell culture medium (Gibco), Fetal bovine serum (Gibco), Penicillin-Streptomycin (Gibco), PolyJet™ transfection kit (SignaGen Laboratories), DAPI dye (Thermo Fisher Cat#P36935).

[0183] Instruments: CO2 cell culture incubator, Olympus FV3000 laser confocal microscope, eBlot L1 rapid wet transfer instrument, Molecular Image Gel Doc XR+ gel imaging system.

[0184] Antibodies: anti-GFP (AB_2619674, Cat#M20004, Abmart); anti-pS409 / 410-TDP-43 (Cat#66318-1-lg, Proteintech), GAPDH (Cat#AT0002, Engibody). HRP-coupled secondary antibodies: goat anti-mouse (Sigma, A4416), goat anti-rabbit (Sigma, A9169). Fluorescent secondary antibodies: goat anti-rabbit-Alexa Flour 488 (Life Technologies, A11034), goat anti-rabbit-Alexa Flour 568 (Life Technologies, A11011), goat anti-mouse-Alexa Flour 568 (Life Technologies, A11031), goat anti-mouse Flour 488 (Life Technologies, A10680).

[0185] Plasmid DNA: Myc empty vector (pCMV-myc-Atg7(addgene Catalog#24921)); EGFP-TDP-43 CTF (The plasmid backbone is the Myc empty vector, containing the EGFP-tagged TDP-43) CTF (The sequence of amino acids 208-414 of NCBI Reference Sequence: NP_031401.1); EGFP-TDP-43 K181E (The plasmid backbone is the Myc empty vector, containing the EGFP-tagged TDP-43) K181E (NCBI Reference Sequence: NP_031401.1, full length, with the K mutation at position 181 being replaced by E, which is an ALS / FTD-related mutation); Myc-B1 (plasmid backbone is Myc empty vector, containing B1 protein sequence); Myc-B9 (plasmid backbone is Myc empty vector, containing B9 protein sequence).

[0186] 2. Experimental Methods:

[0187] 2.1.1 Cell Culture and Plasmid Transfection

[0188] (1) In the experiment of HEK293T cell culture, DMEM medium containing 10% FBS and 1% PS is usually used, and cell culture is carried out at 37°C and 5% CO2. Cells are usually grown in culture dishes with a diameter of 10 cm, and passaged every 1 to 2 days at a ratio of 1:3 to 1:5, depending on the cell growth status. During passage, the medium is first discarded, and the cells are washed with preheated PBS to remove residual medium. Then, 1 ml of 0.25% trypsin is added and digested at 37°C for 2 minutes. Subsequently, 1 ml of medium containing FBS is added to stop the digestion and the cells are gently pipetted to resuspend them. Afterward, the cell suspension is centrifuged at 800 rpm for 3 minutes, the supernatant is discarded, the cells are resuspended in fresh medium, and reseeded into culture dishes.

[0189] (2) In cell transfection experiments, taking a 24-well plate as an example, the amount of plasmid DNA transfected into each well is usually 0.2 to 0.5 μg. Transfection is performed when the cells in the 24-well plate reach a suitable density (approximately 60%). For transfection, first disperse the required plasmid DNA in 50 μl of serum-free culture medium. Then, dilute the appropriate amount of Polyjet transfection reagent with 50 μl of serum-free culture medium at a volume (μl) to mass (μg) ratio of 3:1. Add the mixture of transfection reagent and culture medium to the culture medium containing the plasmid, incubate at room temperature for 15 minutes, and then add it to the designated wells. Subsequent experimental detection is usually performed 24 hours after transfection.

[0190] 2.1.2 Cellular Immunofluorescence

[0191] First, cells were seeded onto glass slides placed in 24-well plates using the smear method. Sample preparation began by removing the culture medium and washing with PBS to remove residual medium. Cell samples were then fixed with 4% PFA at room temperature for 10 minutes. After fixation, the PFA was discarded, and the cells were washed with 0.1% PBST buffer to remove residual PFA. Next, cells were treated with 0.5% PBST to enhance permeabilization. Cells were then blocked for 30 minutes on a shaker at room temperature using 3% GS buffer (prepared with 0.1% PBST). After blocking, 200 μl of primary antibody diluted in 3% GS solution was added to each well, and the cells were incubated overnight at 4°C. The next day, the primary antibody was recovered, and the cells were washed with 0.1% PBST buffer to remove residual primary antibody. Subsequently, 200 μl of fluorescently labeled secondary antibody diluted in 3% GS solution was added to each well, and the cells were incubated on a shaker at room temperature for 1 hour. After incubation, the secondary antibody was removed, and the cells were washed with 0.1% PBST buffer to remove residual secondary antibody. Next, the samples were mounted using mounting medium containing DAPI and the edges were sealed with colorless nail polish. Finally, the samples were observed under an Olympus FV3000 laser confocal microscope (Fig. 6A, Fig. 6B, Fig. 6D, Fig. 6E).

[0192] 2.1.3 Western blot assay

[0193] The cultured cells were resuspended in protein loading buffer and boiled for 15 minutes. Samples were subjected to SDS-PAGE gel electrophoresis. Proteins were transferred to a PVDF membrane using an eBlot L1 wet transfer instrument. The membrane was incubated with rapid blocking buffer at room temperature for 15 minutes, followed by overnight incubation with primary antibody at 4°C. The primary antibody was discarded, and the membrane was washed twice with wash buffer (PBST). The membrane was then washed once on a shaker for 15 minutes, followed by three more 10-minute washes. The wash buffer was discarded, and the membrane was incubated with secondary antibody for 1-2 hours. The secondary antibody was discarded, and the membrane was washed again following the steps described above. Imaging was performed using a Molecular Image Gel Doc XR+ gel imaging system (Figures 6C and 6F).

[0194] 3. Experimental Results:

[0195] In cell experiments, this invention tested two different types of TDP-43 mutants (truncated TDP-43). CTF Or point mutation TDP-43 K181E The aggregation phenomenon was validated using B1 and B9 for inhibition. EGFP-labeled TDP-43 mutants were used to characterize its aggregation state. CTF and TDP-43 K181EIn both types, the EGFP spot size decreased, and the phosphorylation state was also less (p-TDP-43), with the inhibition effect on B9 protein being more significant. Red fluorescence labeled B1 and B9 proteins; both proteins were normally expressed intracellularly, with B9 showing higher intracellular expression levels.

[0196] Example 5: Thermal stability experiment of B1 and B9 proteins

[0197] 1. Experimental Materials and Instruments

[0198] 1.1 Protein samples: purified binding proteins B1 and B9.

[0199] 1.2 Buffer: 20 mM MES buffer (pH 6.0) containing 5 mM NaCl.

[0200] 1.3 Instruments and Equipment: Chirascan-Plus circular dichroism chromatograph (Applied Photophysics).

[0201] 1.4 Experimental conditions: protein concentration 0.1–0.2 mg / mL, scanning wavelength range 260–190 nm, scanning step size 1 nm.

[0202] 2. Experimental Methods

[0203] 2.1 Wavelength Scanning

[0204] B1 and B9 proteins were diluted to 0.1–0.2 mg / mL and placed in 20 mM MES buffer (pH 6.0, containing 5 mM NaCl). Wavelength scanning was performed using a Chirascan-Plus circular dichroism chromatograph, with a scan range of 260–190 nm and a step size of 1 nm. Each sample was measured three times, and the resulting spectra were averaged.

[0205] 2.2 Thermal denaturation test

[0206] The thermal stability of B1 and B9 was assessed by monitoring the CD signal at 220 nm. During the experiment, the temperature was gradually increased from 20 °C to 99 °C at a rate of 2 °C per minute, with each temperature increment being 2 °C, and signal changes were recorded in real time. Subsequently, after cooling back to 20 °C, wavelength scanning was performed again to assess the reversibility of protein folding.

[0207] 3. Experimental Results

[0208] As shown in Figures 7A and 7B, neither B1 nor B9 exhibited significant changes in defolding signals when heated to 96℃, indicating that both maintain high structural stability under high-temperature conditions. After cooling to 20℃, their spectral curves remained largely consistent with their initial states, demonstrating that B1 and B9 possess good thermal reversibility and conformational stability.

[0209] Example 6: Testing the inhibitory effects of B1 and B9 on the formation of full-length TDP-43 fibers

[0210] 1. Experimental Materials and Instruments

[0211] 1.1 Protein samples: Recombinant expressed and purified full-length TDP-43 protein (concentration 10 μM), and binding proteins B1 and B9 (concentration 5 μM).

[0212] 1.2 Buffer: 20 mM MES buffer (pH 6.0).

[0213] 1.3 Experimental conditions: 37℃, static condition (no oscillation).

[0214] 1.4 Instruments and Equipment: 120kV transmission electron microscope (FEI Company).

[0215] 1.5 Reagents and consumables: 3% (w / v) uranyl acetate solution, double-distilled water, carbon film copper mesh (Beijing Zhongjing Scientific Instruments Co., Ltd.).

[0216] 2. Experimental Methods

[0217] 2.1 TDP-43 fiber induction

[0218] Full-length TDP-43 protein (10 μM) was mixed with binding proteins B1 or B9 (5 μM) and incubated in 20 mM MES buffer (pH 6.0) at 37°C for one week to induce TDP-43 filament formation. The control group consisted of TDP-43 samples without the addition of binding proteins, under the same conditions.

[0219] 2.2 Sample Preparation for Transmission Electron Microscopy

[0220] Five μL of each sample was dropped onto a carbon film copper grid treated with glow discharge and allowed to stand at room temperature for 45 s to adsorb protein fibers. The sample was then washed twice with 5 μL of double-distilled water and stained with 3% (w / v) uranium acetate solution for 45 s. After staining, excess liquid was removed, and the sample was air-dried for later use.

[0221] 2.3 Observation by transmission electron microscopy

[0222] The dried samples were imaged using a transmission electron microscope (FEI Company) at an accelerating voltage of 120 kV to record the morphology of each group of TDP-43 fibers (corresponding to Figure 8).

[0223] 3. Experimental Results

[0224] Numerous long and dense TDP-43 fibrous structures were observed in the control group samples, indicating that full-length TDP-43 can spontaneously form typical amyloid fibers under these conditions. In contrast, fiber formation was significantly reduced in samples with added binding proteins B1 or B9, with only a few short or irregular aggregates observed and no obvious mature fibrous structures. These results suggest that both B1 and B9 can effectively inhibit the fibrosis process of full-length TDP-43, hinting at their potential application value in blocking TDP-43 aggregation-related pathologies.

[0225] References:

[0226] 1. Kim, G., et al., ALS Genetics: Gains, Losses, and Implications for Future Therapies. Neuron, 2020.108(5):p.822-842.

[0227] 2. Chiti, F. and CMDobson, Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem, 2006.75: p.333-66.

[0228] 3.Knowles, TP, M. Vendruscolo, and CMDobson, The amyloid state and its association with protein misfolding diseases. Nat Rev Mol Cell Biol, 2014.15(6):p.384-96.

[0229] 4.Gu,J.,et al.,Hsp70 chaperones TDP-43in dynamic,liquid-like phase and prevents it from amyloid aggregation.Cell Research,2021.31(9):p.1024-1027.

[0230] 5.Francois-Moutal, L., DDScott, and M.Khanna, Direct targeting of TDP-43, from small molecules to biologics: the therapeutic landscape. RSC Chem Biol, 2021.2(4):p.1158-1166.

[0231] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A non-naturally occurring protein, characterized in that, The protein comprises an amino acid sequence of the general formula X1-X2-X3, wherein: X1 includes a first helical domain, the first helical domain comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:10; X2 contains second and third spiral structure domains; X3 contains a fourth and a fifth helical domain, the fourth and fifth helical domains containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:

12.

2. The protein as described in claim 1, characterized in that, The second and third helical domains contain amino acid sequences that have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:

11.

3. The protein as described in claim 1 or 2, characterized in that, The protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:

1.

4. The protein according to any one of claims 1 to 3, characterized in that, The protein contains the amino acid sequence shown in SEQ ID NO:

1.

5. The protein according to any one of claims 1 to 4, characterized in that, The protein can inhibit the TDP-43 conserved helix; the TDP-43 conserved helix preferably contains the amino acid sequence shown in SEQ ID NO:

3.

6. A non-naturally occurring protein, characterized in that, The protein comprises an amino acid sequence of the general formula X1-X2-X3, wherein: X1 includes a first helical domain containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:13, and changes to L15E in SEQ ID NO:13 are not permitted; X2 contains second, third, fourth and fifth helical structure domains; X3 contains a sixth and a seventh helical domain, which contain an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:15, and any alteration of L28E in SEQ ID NO:15 is not permitted.

7. The protein as described in claim 6, characterized in that, The second, third, fourth and fifth helical domains contain amino acid sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

14.

8. The protein as described in claim 6 or 7, characterized in that, The protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO:2, and alterations to L15E and L112E in SEQ ID NO:2 are not permitted.

9. The protein according to any one of claims 6 to 8, characterized in that, The protein contains the amino acid sequence shown in SEQ ID NO:

2.

10. The protein according to any one of claims 6 to 9, characterized in that, The protein can inhibit the TDP-43 conserved helix; the TDP-43 conserved helix preferably contains the amino acid sequence shown in SEQ ID NO:

3.

11. An isolated nucleic acid, characterized in that, The nucleic acid encodes the protein as described in any one of claims 1 to 10.

12. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 11.

13. A transformant, characterized in that, The transformant comprises the nucleic acid as described in claim 11 or the recombinant expression vector as described in claim 12; the transformant is a non-animal or non-plant variety. Preferably, the transformant is a eukaryotic cell or a prokaryotic cell; More preferably, the host cell of the transformant is Escherichia coli or HEK293T cells.

14. A reagent kit, characterized in that, The kit comprises the protein as described in any one of claims 1 to 10, the nucleic acid as described in claim 11, the recombinant expression vector as described in claim 12, or the transformant as described in claim 13.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the protein as described in any one of claims 1 to 10, and a pharmaceutically acceptable carrier.

16. A method for preparing the protein according to any one of claims 1 to 10, characterized in that, The method includes culturing the transformant as described in claim 13.

17. A method for studying phase separation of target proteins, characterized in that, The method includes contacting the protein as described in any one of claims 1 to 10 or the pharmaceutical composition as described in claim 15 with the target protein, the target protein comprising a conserved helix of TDP-43LCD; the conserved helix of TDP-43LCD preferably comprising the amino acid sequence shown in SEQ ID NO:3; Preferably, the target protein comprises TDP-43LCD; the TDP-43LCD preferably comprises amino acids 274 to 414 of the sequence shown in NP_031401.1; More preferably, the target protein comprises TDP-43CTF; the TDP-43CTF preferably comprises amino acids 208 to 414 of the sequence shown in NP_031401.1; More preferably, the target protein is TDP-43, which preferably contains the amino acid sequence shown in NP_031401.1; or, the target protein is a TDP-43 mutant, which does not contain amino acid substitutions of M322D, A326P, and L330R relative to the sequence shown in NP_031401.1; the TDP-43 mutant preferably contains one or more amino acid substitutions selected from P112H, K181E, and K263E on the amino acid sequence shown in NP_031401.

1.

18. The method as described in claim 17, characterized in that, The method is not for diagnostic, preventive and / or therapeutic purposes.

19. A method for inhibiting a target protein, characterized in that, The method includes contacting the protein as described in any one of claims 1 to 10 or the pharmaceutical composition as described in claim 15 with the target protein, the target protein comprising a conserved helix of TDP-43LCD; the conserved helix of TDP-43LCD preferably comprising the amino acid sequence shown in SEQ ID NO:3; Preferably, the target protein comprises TDP-43LCD; the TDP-43LCD preferably comprises amino acids 274 to 414 of the sequence shown in NP_031401.1; More preferably, the target protein comprises TDP-43CTF; the TDP-43CTF preferably comprises amino acids 208 to 414 of the sequence shown in NP_031401.1; More preferably, the target protein is TDP-43, which preferably contains the amino acid sequence shown in NP_031401.1; or, the target protein is a TDP-43 mutant, which does not contain amino acid substitutions of M322D, A326P, and L330R relative to the sequence shown in NP_031401.1; the TDP-43 mutant preferably contains one or more amino acid substitutions selected from P112H, K181E, and K263E on the amino acid sequence shown in NP_031401.

1.

20. The method as described in claim 19, characterized in that, The inhibition of the target protein is to inhibit the fibrotic aggregation of the target protein; and / or, the method is not for diagnostic, preventive and / or therapeutic purposes.

21. A medicine box set, characterized in that, The kit includes a medicine box A, wherein the medicine box A contains the protein as described in any one of claims 1 to 10 or the pharmaceutical composition as described in claim 15; Preferably, the kit also includes a box B containing other drugs for the diagnosis, prevention and / or treatment of neurodegenerative diseases and / or inhibitors targeting TDP-43. More preferably, the neurodegenerative disease is ALS or frontotemporal lobe degeneration, and / or the inhibitor is selected from one or more small molecule compounds, antibodies, and peptides.

22. The use of the protein of any one of claims 1 to 10, the nucleic acid of claim 11, the recombinant expression vector of claim 12, the transformant of claim 13, the kit of claim 14, or the pharmaceutical composition of claim 15 in the preparation of medicaments for the diagnosis, prevention, and / or treatment of neurodegenerative diseases; Preferably, the neurodegenerative disease is ALS or frontotemporal lobe degeneration.

23. The use of the protein of any one of claims 1 to 10, the nucleic acid of claim 11, the recombinant expression vector of claim 12, the transformant of claim 13, the kit of claim 14, or the pharmaceutical composition of claim 15 in the preparation of an inhibitor of a target protein, wherein the target protein comprises a conserved helix of TDP-43LCD; the conserved helix of TDP-43LCD preferably comprises the amino acid sequence shown in SEQ ID NO:3; and the inhibitor preferably inhibits the fibrotic aggregation of the target protein; Preferably, the target protein comprises TDP-43LCD; the TDP-43LCD preferably comprises amino acids 274 to 414 of the sequence shown in NP_031401.1; More preferably, the target protein comprises TDP-43CTF; the TDP-43CTF preferably comprises amino acids 208 to 414 of the sequence shown in NP_031401.1; More preferably, the target protein is TDP-43, which preferably contains the amino acid sequence shown in NP_031401.1; or, the target protein is a TDP-43 mutant, which does not contain amino acid substitutions of M322D, A326P, and L330R relative to the sequence shown in NP_031401.1; the TDP-43 mutant preferably contains one or more amino acid substitutions selected from P112H, K181E, and K263E on the amino acid sequence shown in NP_031401.

1.

24. A method for diagnosing, preventing, and / or treating neurodegenerative diseases, characterized in that, The method includes administering to a subject in need an effective amount of the protein as described in any one of claims 1 to 10, the nucleic acid as described in claim 11, the recombinant expression vector as described in claim 12, the transformant as described in claim 13, the kit as described in claim 14, or the pharmaceutical composition as described in claim 15; Preferably, the neurodegenerative disease is ALS or frontotemporal lobe degeneration.

25. The protein as described in any one of claims 1 to 10, the nucleic acid as described in claim 11, the recombinant expression vector as described in claim 12, the transformant as described in claim 13, the kit as described in claim 14, or the pharmaceutical composition as described in claim 15, for the diagnosis, prevention, and / or treatment of neurodegenerative diseases; Preferably, the neurodegenerative disease is ALS or frontotemporal lobe degeneration.

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