WO2023068328A1 - Agent for inducing decomposition of target peptide - Google Patents

Abstract

The purpose of the present invention is to discover and provide a novel ubiquitin drug discovery technique whereby it becomes possible to induce a ubiquitin-proteasome system from a target peptide that is difficult to be ubiquitinated and decompose the target peptide. Provided is an agent for inducing the decomposition of a target peptide, the agent having such a structure that one or more binding factors capable of binding to the target peptide are linked to the C-terminal of ubiquitin, in which two or more binding factors are linked to each other.

Description

Targeted peptide degradation inducer

The present invention relates to a target peptide degradation inducer composed of ubiquitin and a binding factor linked to its C-terminus that binds to the target peptide, and a disease suppressor using the same.

Transcription factors are DNA-binding proteins that regulate gene expression, and dysregulation of their activity causes various diseases including cancer (Non-Patent Document 1). Therefore, transcription factors can be important drug targets in the field of drug discovery. However, most transcription factors do not have active domains like enzymes or ligand binding pockets like receptors. Therefore, it was a target molecule that was difficult to develop (undruggable) with conventional low-molecular-weight compounds and antibody drugs that inhibit the activity of the target molecule or function as an antagonist (Non-Patent Document 2). Therefore, the establishment of a new drug discovery approach targeting transcription factors has been strongly desired from the viewpoint of cell function control and disease treatment.

In recent years, as a new drug discovery strategy for such undruggable molecules, ubiquitin drug discovery technology that uses target protein degradation inducers to ubiquitinate and degrade target proteins has attracted attention (Non-Patent Document 3). This technique utilizes the ubiquitin-proteasome system (Ub-PSM system) of the protein degradation mechanism present in all cells as shown in FIG. 11 to induce degradation of target proteins. A target protein degradation inducer is a compound consisting of a binder site that binds to a target protein and an E3 ligase, respectively, and a linker site that connects them. Facilitate. The target protein whose ubiquitination is thereby promoted is degraded after being induced to the proteasome. Commercially available PROTAC (registered trademark) is one of representative target protein degradation inducers.

However, the above ubiquitin drug discovery technology also faces major challenges. As described above, target protein degradation inducers promote direct ubiquitination of target proteins by E3 ligases. However, target proteins such as transcription factors are difficult to ubiquitinate by E3 ligases. Therefore, such target proteins remained undruggable target molecules.

Lee, T. I. et. al., 2013, Cell, 152: 1237-1251. Lambert, S. A, et al., 2018, Cell, 172: 650-665. Dale, B. et al., 2021, Nat. Rev. Cancer, 21: 638-654.

The task of the present invention is to develop and provide a new ubiquitin drug discovery technology that can induce the Ub-PSM system to degrade even target proteins that are difficult to ubiquitinate, such as transcription factors.

The object of the present invention is to develop and provide a target peptide degradation inducer capable of inducing the degradation of any target protein based on the above new ubiquitin drug discovery technology.

An object of the present invention is to provide a disease-suppressing agent capable of treating, improving, preventing, etc., a disease by inducing the degradation of even a target protein that causes a disease and has been difficult to develop a drug with the prior art. is.

In order to solve the above problems, the present inventors have conducted intensive research and succeeded in developing a completely new target protein degradation technology using an indirect ubiquitination method. In this method, the Ub-PSM system is used to degrade the target protein as in the conventional ubiquitin drug discovery technology. However, it does not require a direct ubiquitination process to the target protein via ubiquitin ligase E3, and instead uses a target peptide degradation inducer consisting of a binding factor pre-linked with ubiquitin as shown in FIG. A complex of the target protein and the target peptide degradation inducer is formed by binding the binding factor of the target peptide degradation inducer to the target protein. The target protein becomes indirectly ubiquitinated in this complex, resulting in recognition by the proteasome as if the target protein had been directly ubiquitinated, followed by degradation.

With this indirect ubiquitination method, by using a molecule that binds to the target protein as a binding factor, even if the target protein is difficult to ubiquitinate directly, it can be used as a drug discovery target using the Ub-PSM system. can do.

The present invention is based on the newly developed technology for degrading target peptides by the indirect ubiquitination method, and provides the following.

(1) A target peptide degradation inducer linked to the C-terminus of one or more ubiquitins linked to a binding factor that binds to a target peptide.
(2) The target peptide degradation inducer according to (1), wherein the ubiquitin consists of any one of the amino acid sequences described in (a) to (c) below.
(a) the amino acid sequence shown in SEQ ID NO: 1,
(b) an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1, or (c) having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 1 Amino acid sequence (3) The target peptide according to (2), wherein the ubiquitin comprises an amino acid sequence containing any one of the amino acid substitutions described in (I) to (III) below in the amino acid sequence shown in SEQ ID NO: 1. Decomposition inducer.
(I) an amino acid sequence in which arginine residues at positions 72 and 74 are substituted with alanine residues and threonine residues, respectively;
(II) an amino acid sequence in which the arginine residues at positions 72 and 74 are substituted with proline and threonine residues, respectively, or (III) an amino acid sequence in which the leucine residue at position 73 is substituted with a proline residue (4 ) The target peptide degradation inducer according to any one of (1) to (3), wherein the binding factor is a nucleic acid.
(5) The target peptide degradation inducer according to (4), wherein the nucleic acid is a nucleic acid aptamer or nucleic acid-binding domain-recognizing nucleic acid.
(6) The target peptide degradation inducer according to (1) to (3), wherein the binding factor is a peptide.
(7) The target peptide degradation inducer according to (6), wherein the peptide is selected from the group consisting of ligands, receptors, ligand-binding sites thereof, antibodies, and active fragments thereof.
(8) The target peptide degradation inducer according to (1) to (3), wherein the binding factor is a low-molecular-weight compound.
(9) the ubiquitin and the binding agent are linked via a thiol group introduced by substitution or addition to the C-terminus of ubiquitin, via an azide-alkyne cycloaddition reaction, or via an oxime/hydrazone bond; The target peptide degradation inducer according to any one of (1) to (8).
(10) The target peptide degradation inducer according to (9), wherein the bond via the thiol group is a bond by Michael addition reaction, thiolene reaction or thiolyne reaction.
(11) The target peptide degradation inducer according to any one of (1) to (10), wherein the binding factor and the ubiquitin are linked via a linker factor.
(12) the target peptide is a transcription factor, amyloid, tau protein, α-synuclein, caspase, modified huntingtin, pseudokinase, K-RAS mutant, Atg factor, mTOR complex 1, GAPR-1 and orphan receptor The target peptide degradation inducer according to any one of (1) to (11), which is selected from the group consisting of:
(13) The target peptide degradation inducer according to (12), wherein the transcription factor is NF-κB.
(14) A polynucleotide encoding the target peptide degradation inducer of (6) or (7).
(15) An expression vector comprising the polynucleotide of (14).
(16) A nucleic acid type target peptide degradation inducer comprising the polynucleotide of (14) or the expression vector of (15).
(17) Any two or more selected from the group consisting of the target peptide degradation inducer according to any one of (1) to (13) and/or the nucleic acid-type target peptide degradation inducer according to (16) A target peptide degradation-inducing composition comprising:
(18) A Huntington's disease inhibitor containing the target peptide degradation inducer according to (12), wherein the target peptide is denatured huntingtin.
(19) An autophagy inhibitor containing the target peptide degradation inducer according to (12), wherein the target peptide is an Atg factor.
(20) An autophagy promoter comprising the target peptide degradation inducer according to (12), wherein the target peptide is mTOR complex 1 or GAPR-1.
(21) An anticancer agent comprising the target peptide degradation inducer of (13).
This specification includes the disclosure content of Japanese Patent Application No. 2021-172029, which is the basis of priority of this application.

According to the target peptide degradation inducer of the present invention, it is possible to induce the Ub-PSM system to degrade any undruggable target protein that was difficult to ubiquitinate directly in the prior art.

According to the disease inhibitor of the present invention, any disease-causing protein can be degraded via the Ub-PSM system to treat, ameliorate, prevent, etc. the disease.

FIG. 4 shows a gel shift assay by the binding of Ub-C77-Decoy, which is a target peptide degradation inducer of the present invention, and NF-κB p50, which is a target peptide. FIG. 10 shows the results of Ub-PSM system-mediated degradation of NF-κB p50 indirectly ubiquitinated by Ub-C77-Decoy. In the figure, MG132 is a proteasome inhibitor, and Decoy DNA and Ub-C77 show only Ub-C77-Decoy binding factor and only ubiquitin, respectively. The lower band diagrams are western blotting diagrams showing the degradation results of each sample. FIG. 10 is a diagram showing the survival rate of the target peptide NF-κB p50 at each concentration of Ub-C77-Decoy. Here, the values are shown relative to the amount of NF-κB p50 immediately after introduction of Ub-C77-Decoy into cells as 100% remaining amount. Targeted peptide degradation inducer UbR-C77-decoy of the present invention using a ubiquitin mutant (UbR) in which two amino acid residues on the C-terminal side of ubiquitin are mutated (R72A/R74T) to confer deubiquitinating enzyme resistance FIG. 3 shows the degradation results of the peptide NF-κB p50 via the Ub-PSM system. The lower band diagrams are Western blotting diagrams showing the residual NF-κB p50 levels in each sample. FIG. 10 is a diagram showing the survival rate of target peptide NF-kB p65 in cell extracts after addition of UbR-C77-decoy. The figure shows relative values when the amount of NF-κB p65 remaining when UbR-C77-decoy was not added (Control) was taken as 100%. Residual amounts were calculated as the mean of three experimental results, and error bars indicate standard deviation. FIG. 2 is a western blotting diagram showing the results of residual NF-κB p65 (upper) in Hela cells transfected with UbR-C77-decoy, which is a target peptide degradation inducer of the present invention, and in MCF-7 cells. The residual amount of NF-κB p65 was calculated as a ratio to the band intensity in the western blotting image of the housekeeping protein β-actin (bottom row). MCF-7 cells in which Ub-C77-Decoy, which is the target peptide degradation inducer of the present invention, was added directly to the medium (a to e: all in the same field), and MCF-7 into which Ub-C77-Decoy was introduced by lipofection Images of cells (f to j: all in the same field of view). a and f are fluorescence images of FAM (fluorescein) showing Ub-C77-Decoy, b and g are fluorescence images of MCF-7 cells stained with Hoechst 33258, c and h are images of a and b and f and g, respectively. d and i show the phase-contrast microscope images, and e and j show the superimposed images of a and d and f and i, respectively. Bars in each image represent 50 μm. FIG. 4 is a diagram showing the cell viability, which suggests apoptosis induction by inhibition of NF-κB activity using Ub-C77-Decoy, which is the target peptide degradation inducer of the present invention, as a relative value when blank is 1.0. FIG. 2 shows the results of degradation of target peptide streptavidin by Ub-cys-biotin, a low-molecular compound-type target peptide degradation inducer of the present invention, via the Ub-PSM system. The figure shows the relative value of the amount of streptavidin remaining when biotin-cys-Ub was added, relative to the amount of streptavidin when biotin-cys-Ub was not added as 100%. Residual amounts are shown as the mean of three experiments, and error bars indicate standard errors. FIG. 2 is a Western blotting diagram showing the results of degradation of target peptide Mcl-1 by UbR ₃ -B6 and UbR ₄ -B6, which are peptide-type target peptide degradation inducers of the present invention, via the Ub-PSM system. FIG. 2 is a conceptual diagram showing the ubiquitin-proteasome system (Ub-PSM system) of the protein degradation mechanism. In the figure, Ub represents ubiquitin, E1 represents ubiquitin activating enzyme, E2 represents ubiquitin conjugating enzyme, and E3 represents ubiquitin ligase. In this figure, ubiquitinated target peptides that are monoubiquitinated and tetraubiquitinated are exemplified as ubiquitinated substrates, but the number of ubiquitins in the ubiquitinated target peptide does not matter. BRIEF DESCRIPTION OF THE DRAWINGS It is a conceptual diagram which shows the structure (A) of the target peptide degradation inducer of this invention, and the degradation mechanism (B) of the indirect ubiquitination method of this invention via a Ub-PSM system. In the figure, the binding agent is the binding agent that binds to the target peptide. This figure exemplifies a target peptide degradation inducer in which one ubiquitin is linked to the binding factor. may be

1. Target Peptide Degradation Inducing Agent 1-1. Overview A first aspect of the present invention is a target peptide degradation inducer. The target peptide degradation inducer of the present invention has a structure in which a binding factor that binds to a target peptide is linked to the C-terminus of ubiquitin. According to the target peptide degradation inducer of the present invention, the target peptide can be guided to the proteasome by administration in vivo without requiring direct ubiquitination of the target peptide.

1-2. Definitions The following terms frequently used in this specification are defined.
As used herein, the term "peptide" refers to an amino acid polymer in which multiple amino acids are linked by peptide bonds. Any number of amino acids can be linked. Therefore, when the term "peptide" is used herein, it means a generic term including oligopeptides, polypeptides and proteins.

As used herein, the term "nucleic acid" basically refers to a biopolymer composed of nucleotides linked by phosphodiester bonds. Usually, DNA linked with deoxyribonucleotides having any of adenine (A), guanine (G), cytosine (C) and thymine (T) bases, and/or adenine, guanine, cytosine and uracil (U) A natural nucleic acid formed by connecting natural nucleotides existing in nature such as RNA to which ribonucleotides having any base are linked corresponds.

"Ubiquitin" (herein often abbreviated as "Ub") is a highly conserved protein in eukaryotes, consisting of 76 amino acids. It is expressed in all cells and functions in various biological phenomena such as protein degradation, signal transduction, DNA repair, and selective autophagy through ubiquitination of target proteins. Proteolytic degradation by the ubiquitin-proteasome system is the primary function of ubiquitin, and in this specification also, without limitation, functions relating to proteolytic degradation are mentioned unless otherwise stated.

"Ubiquitination" is also called "ubiquitin modification", and refers to protein post-translational modification by addition of ubiquitin to a target protein. A state in which multiple ubiquitins are linked and added in a chain is particularly referred to as “polyubiquitination”. In polyubiquitinated target proteins, the attached polyubiquitin chains function as degradation signals in the ubiquitin-proteasome system.

"Target protein" refers to a protein that is ubiquitinated in the ubiquitin-proteasome system and targeted for degradation. Usually, proteins that are no longer needed in vivo and should be removed correspond.

“Ubiquitin-Proteasome system” (herein often referred to as “Ub-PSM system”) is one of the ATP-dependent proteolytic systems that regulate various biological phenomena in vivo. is one. In the Ub-PSM system, a series of reactions takes place, including activation of ubiquitin by three enzymes, ubiquitin modification to target proteins, and degradation of ubiquitinated target proteins by the proteasome. Specifically, ubiquitin-activating enzyme (E1) uses ATP to thioester-bond with ubiquitin at its own cysteine (Cys) residue to form an E1-Ub complex. Next, ubiquitin in E1-Ub is transferred to the Cys residue of ubiquitin conjugating enzyme E2 via a thioester bond, forming an E2-Ub complex and ubiquitin is activated. Subsequently, the activated ubiquitin in the E2-Ub complex is transferred to the target protein by the activity of the heterocomplex ubiquitin ligase E3, and the target protein is ubiquitin-modified. At this time, an isopeptide bond is formed between the C-terminus of ubiquitin and the lysine (Lys) residue of the target protein. After that, E3-mediated condensation is repeated between the C-terminus of the new ubiquitin and the ubiquitin bound to the target protein, mainly at the 48th Lys residue, to form a polyubiquitin chain in which multiple ubiquitins are linked. be done. This polyubiquitin chain serves as a label, and the polyubiquitinated protein is recognized by the proteasome and degraded in an ATP-dependent manner. Ubiquitin used for modification is reused after being cleaved from the target protein.

As used herein, the term "target peptide" (herein often referred to as "POI" (peptide of interest)) refers to a peptide to be degraded that is induced to the proteasome by the target peptide degradation-inducing agent of the present invention. say. It is substantially synonymous with the target protein. Any type of target peptide can be used. For example, it may be a target protein that is difficult to ubiquitinate directly in a normal Ub-PSM system. Examples of target peptides include, but are not limited to, transcription factors, enzymes, ligands, receptors, constituent proteins, peptide hormones, cytokines, antibodies, antigenic proteins, and the like. More specific examples of target peptides include amyloid, tau protein, α-synuclein, caspase, modified huntingtin, pseudokinase, K-RAS mutant, Atg factor, mTOR complex 1, GAPR-1, and orphan receptor. A body etc. are mentioned.

As used herein, the term "binding agent" refers to a substance capable of binding to a target peptide. A detailed configuration of the binding factor will be described later.
As used herein, the term "living organism" refers to living cells, tissues, organs, or individuals.

1-3. Configuration 1-3-1. Constituent Factors The target peptide degradation-inducing agent of the present invention contains a binding factor and ubiquitin as essential constituent factors, and a linker factor as an optional constituent factor. Each component will be specifically described below.

(1) Binding Agent A “binding agent” refers to a molecule that can bind to a target peptide. Molecules that specifically bind the target peptide are preferred.
The binding factor may be of any type as long as it has the property of binding to the target peptide. Examples include nucleic acids, peptides, or low-molecular-weight compounds. Each of these will be explained below.

(1-1) Nucleic acid The binding factor may be composed of nucleic acid. Binding agents composed of nucleic acids are referred to herein as “nucleic acid-type binding agents”.

As mentioned above, in principle, nucleic acids correspond to natural nucleic acids, but in the present specification, non-natural nucleic acids can also be included. Accordingly, a nucleic acid-type binding agent can include naturally occurring nucleic acids and/or non-naturally occurring nucleic acids.

As used herein, the term “non-natural nucleic acid” refers to biopolymers, nucleic acid analogues, etc. containing non-natural nucleotides as structural units. As used herein, "non-natural nucleotide" refers to any nucleotide other than natural nucleotides. For example, non-naturally occurring nucleotides that are artificially constructed or artificially chemically modified and that have similar properties and/or structures to natural nucleotides are applicable. Specific examples include 2'-O- _{methyl ribose, 2'-O(CH2)2OCH3 ribose ,} 2'-F(2'-fluoro) ribose, 3'-O-methyl ribose and the like. Nucleotides. As used herein, the term "nucleic acid analogue" refers to an artificially constructed compound having a structure and/or properties similar to those of naturally occurring nucleic acids. Specifically, for example, crosslinked nucleic acid (BNA / LNA: Bridged Nucleic Acid / Locked Nucleic Acid), morpholino nucleic acid, peptide nucleic acid (PNA: Peptide Nucleic Acid), etc., methylphosphonate type DNA or RNA, phosphorothioate It can also contain type DNA or RNA, phosphoramidate type DNA or RNA.
Specific examples of nucleic acid-type binding factors include nucleic acid aptamers and nucleic acids that recognize nucleic acid-binding domains.

(nucleic acid aptamer)
"Nucleic acid aptamer" is an aptamer composed of nucleic acid, and the secondary structure and tertiary structure of a single-stranded nucleic acid molecule through hydrogen bonding, etc. A ligand molecule that has the ability to bind tightly and specifically. Therefore, in the present specification, a nucleic acid aptamer that strongly and specifically binds to a target peptide is applicable.

Nucleic acid aptamers are generally known as RNA aptamers and DNA aptamers, but the nucleic acids that constitute the nucleic acid aptamers in the present specification are not particularly limited. Any of DNA aptamers, RNA aptamers, and aptamers composed of a combination of DNA and RNA can be included.

The nucleic acid aptamer used herein can be produced by a method known in the art with a target peptide as a binding target. Examples thereof include an in vitro selection method using the SELEX (systematic evolution of ligands by exponential enrichment) method. The SELEX method is a known method, and a specific method may be performed, for example, according to Pan et al. (Proc. Natl. Acad. Sci. 1995, U.S.A.92: 11509-11513).

(Nucleic acid binding domain recognition nucleic acid)
As used herein, a "Nucleic acid-Binding Domain" (herein often abbreviated as "NBD") is included in a nucleic acid-binding protein and includes a nucleic acid-binding domain recognition sequence (herein In literature, it is often referred to as an "NBD recognition sequence") and refers to a domain that recognizes and binds to it. An NBD can be either a DNA binding domain (DNB) or an RNA binding domain (RNB). Examples of specific structural motifs that constitute NBDs include, but are not limited to, zinc fingers, leucine zippers, β-hairpins, helix-turn helices, helix-loop helices, wing-helices, HMG boxes, RRM domains, and TAL effectors. be done.

As used herein, the term "nucleic acid binding domain recognition nucleic acid" (herein often abbreviated as "NBD recognition nucleic acid") refers to a nucleic acid containing an NBD recognition sequence. An NBD-recognizing nucleic acid can bind to the NBD of a target peptide via the included NBD-recognition sequence.

The NBD-recognizing nucleic acid may consist of any nucleic acid. Examples include naturally occurring nucleic acids consisting of DNA, RNA, or a combination thereof, as well as non-natural nucleic acids in which all or part of them are replaced with non-natural nucleic acids. Furthermore, the NBD-recognizing nucleic acid may consist of a single-stranded nucleic acid, a double-stranded nucleic acid, or a combination thereof, and may have a secondary structure such as a hairpin structure, or a tertiary structure.

At least the included NBD recognition sequence should consist of a nucleic acid and a sequence that the NBD of the target peptide recognizes and binds to, and there are no particular restrictions on the specific configuration. For example, if the NBD of the target peptide is a double-stranded DNA binding domain (DNB) that recognizes a specific base sequence, the NBD-recognizing nucleic acid is composed of double-stranded DNA containing the DNB recognition sequence as the target sequence. All you have to do is

Specific examples of NBD-recognizing nucleic acids include transcription factor binding sequences contained in promoters. A more specific example is an NF-κB-binding hairpin DNA represented by SEQ ID NO: 2 or 3 and containing a nucleotide sequence to which the transcription factor NF-κB binds.

(1-2) Peptide A binding agent may be composed of a peptide. Binding agents composed of peptides are referred to herein as “peptide-type binding agents”.

Peptide-type binding agents bind to target peptides through peptide-peptide (protein-protein) interactions. This binding is preferably a stable and specific binding. The inter-peptide interaction is not limited, but may be a dissociable transient interaction. Such peptide-peptide interactions are achieved, for example, by hydrophobic bonds, van der Waals forces, or salt bridges between the amino acids that make up the peptide.

The type of the peptide-type binding factor is not limited as long as it is a peptide. For example, if the target peptide is a receptor, the peptide-type binding agent may consist of its ligand or its receptor binding site. Conversely, if the target peptide is a ligand, the peptide-type binding agent may consist of its receptor or ligand binding site. Also, if the target peptide is an antigen, the peptide-type binding agent may consist of an antibody that specifically recognizes it or an active fragment thereof. Alternatively, it may be composed of commercially available target protein degradation-inducing molecules such as PROTAC (Registered Trademark) (Proteolysis Targeting Chimeric Molecules).

As a more specific example of the peptide-type binding factor, HXR9 (Morgan R. et al., 2007, Cancer Res. 67, 5806-5813).

(1-3) Low-molecular-weight compound The binding agent may be composed of a low-molecular-weight compound. A binding agent composed of a low-molecular-weight compound is referred to herein as a “low-molecular-weight compound-type binding agent”.

"Low-molecular-weight compounds" generally refer to natural compounds or chemically synthesized compounds with a molecular weight of about several hundred to several thousand. However, the low-molecular-weight compound-type binding agent herein is a low-molecular-weight compound capable of specifically interacting and binding to the target peptide based on the three-dimensional structure of the target peptide. , and types are not particularly limited. Specific examples of the low-molecular-weight compound-type binding factor constituting the target peptide degradation inducer of the present invention include methotrexate (MTX), which is an inhibitor against dihydrofolate reductase (DHFR), biotin, etc., which has the ability to specifically recognize avidin. is mentioned.

(2) Ubiquitin “Ubiquitin” is a protein consisting of 26 amino acids highly conserved among eukaryotic species, as described above. Therefore, the biological species from which the ubiquitin constituting the target peptide degradation inducer of the present invention is derived is also not limited. It may be ubiquitin that any eukaryote has. A specific example of ubiquitin is a human-derived ubiquitin consisting of the amino acid sequence shown in SEQ ID NO: 1 (MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG), in which one or more or several amino acids are added, deleted, or substituted in the amino acid sequence shown in SEQ ID NO: 1. 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more of the amino acid sequence shown in SEQ ID NO: 1, or a mutant ubiquitin or ubiquitin ortholog consisting of an amino acid sequence Mutant ubiquitin or ubiquitin orthologues consisting of amino acid sequences having 98% or more amino acid identity or 99% or more amino acid identity are mentioned above.

Examples of mutant ubiquitin include ubiquitin mutants containing mutations that confer resistance to degradation by deubiquitinating enzymes, although this is not conclusive. As a specific example of such a ubiquitin mutant, in the human ubiquitin shown in SEQ ID NO: 1, arginine at position 72 (methionine encoded by the initiation codon is designated as position 1, hereinafter the same) and arginine at position 74 located on the C-terminal side. Ubiquitin mutants (R72A/R74T) containing amino acid substitutions in which residues are substituted with alanine and threonine residues, respectively, amino acid substitutions in which arginine residues at positions 72 and 74 are substituted with proline and threonine residues, respectively and a ubiquitin mutant containing an amino acid substitution in which the leucine residue at position 73 is replaced with a proline residue (L73P). A specific example of the ubiquitin mutant (R72A/R74T) is a ubiquitin mutant consisting of the amino acid sequence shown in SEQ ID NO: 6 (MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLALTGG). A specific example of the ubiquitin mutant (R72P/R74T) is a ubiquitin mutant consisting of the amino acid sequence shown in SEQ ID NO: 7 (MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLPLTGG). A specific example of the ubiquitin mutant (L73P) is a ubiquitin mutant consisting of the amino acid sequence shown in SEQ ID NO: 8 (MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRPRGG).

As used herein, "plurality" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. In addition, "several pieces" means 2 to 3 pieces.

As used herein, the term "substitution (of amino acids)" means that among the 20 types of amino acids that make up natural proteins, within a group of conservative amino acids with similar properties such as charge, side chain, polarity, and aromaticity It means replacement. For example, uncharged polar amino acids with low polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile , Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids ( Arg, Lys, His), substitutions within the group of aromatic amino acids (Phe, Tyr, Trp). Amino acid substitutions within these groups are preferred because they are known to be less likely to cause changes in peptide properties.

As used herein, the term "amino acid identity" refers to the amino acid sequences of two peptides to be compared, with appropriate gaps inserted in one or both of the amino acid sequences to maximize the number of matching amino acid residues. It refers to the ratio (%) of the number of matching amino acid residues to the number of all amino acid residues when aligned. Alignment of two amino acid sequences for calculating amino acid identity can be performed using known programs such as Blast, FASTA, ClustalW.

In the target peptide degradation inducer of the present invention, the number of ubiquitins linked to the binding factor is not limited. For example, it may be one, or two or more (for example, 2, 3, 4, 5, 6, 7, 8, or 9) linked polyubiquitin good. When two or more are linked, each ubiquitin may be the same or different. For example, a ubiquitin having the amino acid sequence shown in SEQ ID NO: 1 and a mutant ubiquitin having one substitution in the ubiquitin amino acid sequence having the amino acid sequence shown in SEQ ID NO: 1 may be linked.

(3) Linker Factor The term "linker factor" refers to a molecule (crosslinker) that mediates the linkage between the binding factor and ubiquitin in the target peptide degradation inducer of the present invention. In the target peptide degradation inducer containing a linker factor, the binding factor and ubiquitin are indirectly linked via the linker factor.

The type of linker factor does not matter. For example, nucleic acids, peptides, and/or low-molecular-weight compounds may be used. The basic configuration of these conforms to the configuration of nucleic acids, peptides, and/or low-molecular-weight compounds described in the aforementioned binding factor. Therefore, in the present specification, a linker factor composed of a nucleic acid is referred to as a "nucleic acid-type linker factor," a linker factor composed of a peptide is referred to as a "peptide-type linker factor," and a linker factor composed of a low-molecular-weight compound is referred to as a "low-molecular-weight linker factor." compound-type linker factor". Moreover, the linker factor may be composed of a combination of different types. Examples include a combination of a nucleic acid-type linker factor and a low-molecular-weight compound-type linker factor. In this case, each linker factor may be linked in tandem, and such linked linker factors are referred to herein as "nucleic acid/low molecular weight compound type linker factors".

The linker factor may be of the same type as either the binding factor or ubiquitin, or both, or may be of a different type. For example, when the target peptide degradation-inducing agent of the present invention is composed of a nucleic acid-type binding factor, a linker factor, and ubiquitin, the linker factor may be composed of the same nucleic acid as the nucleic acid-type binding factor, or may be composed of the same peptide as ubiquitin. may be configured. Alternatively, it may be composed of a low-molecular-weight compound different from any of them. In the previous example, when the linker factor is composed of nucleic acid, the nucleic acid-type binding factor and the linker factor may be synthesized as a single fused nucleic acid strand.

Some specific examples of linker elements are described below.
First, there is a linker factor that connects a binding factor and ubiquitin via a coordinate bond to a metal. For example, a peptide/low molecular compound type linker factor consisting of polyhistidine (His-tag) and nickel-nitrilotriacetic acid (Ni-NTA) is one example. By modifying the binding factor with Ni-NTA and attaching a His-tag to the C-terminus of ubiquitin, the binding factor and ubiquitin are linked via the binding of Ni-NTA and His-tag when both are mixed.

In addition, linker factors that connect binding factors and ubiquitin via host-guest interactions are included. For example, a low-molecular-weight compound-type linker factor consisting of adamantane, a cage-like molecule having the same carbon configuration as diamond, and β-cyclodextrin, a cyclic oligosaccharide, is one example.

The linker molecule can be bound or ligated to each within the linker molecule, for example, when direct ligation between the binding factor and ubiquitin is difficult when producing the target peptide degradation inducer of the present invention. By including the configuration, both can be easily connected.

1-3-2. Binding between Constituent Factors Direct binding between constituent factors in the target peptide degradation-inducing agent of the present invention is not particularly limited. The complex formed by the binding of the target peptide degradation inducer of the present invention administered in vivo to the target peptide (target peptide degradation inducer-target peptide complex) does not readily dissociate until it is incorporated into the proteasome. Any combination is acceptable.

"Direct binding between constituent elements" (herein, often referred to as "binding between constituent elements") means, for example, between a binding agent and ubiquitin, between a binding agent and a linker A connection between factors. Examples of such component-to-component bonds include covalent bonds or chemical bonds such as ionic bonds. Others include high affinity non-covalent binding such as the binding between biotin and avidin.

Bonding between constituent factors can be achieved using methods known in the art, depending on the type and composition of each constituent factor. Examples thereof include bonding via chemical reactions such as nucleophilic addition reactions, nucleophilic substitution reactions, or electrophilic substitution reactions between functional groups present in each factor. A functional group that contributes to such bonding is preferably, but not limited to, an active functional group having chemical activity that enables linkage with a partner molecule by covalent bonding or the like. For example, hydroxyl group (-OH), ketone group (-C(=O)-), aldehyde group (-CHO), carboxy group (-COOH), methoxy group ( _-OCH3 ), sulfo group ( _-SO3H ), amino group (-NH ₂ ), oxime (>C=N-OH), carbonyl group (-C(=O)-), thiol group (-SH), cyano group (-CN), nitro group (- NO ₂ ), or an azo group (-N=N-). A functional group that contributes to binding between constituent factors may be a functional group inherently included in each constituent factor, or may be introduced by addition or substitution as necessary. For example, cysteine (C) is added to the C-terminus of ubiquitin, or glycine (G) at position 76, which is the C-terminal amino acid of ubiquitin, is replaced with cysteine to introduce a thiol group, which is an active functional group, to the C-terminus of ubiquitin. You may

In the case of bonding between functional groups, the types of both functional groups forming a bond between constituent factors are not limited, but it is desirable to select a combination that can form a covalent bond between the functional groups. Examples include amino and aldehyde groups, thiol and maleimide groups, azido and ethynyl groups, hydrazine and ketone groups, and hydrazine and aldehyde groups.

The type of bond between functional groups that form the bond between constituent factors is not limited. For example, a bond via a thiol group, a bond by an azide-alkyne cycloaddition reaction (Huisgen cycloaddition reaction), an oxime/hydrazone bond, or the like can be mentioned. Specific examples of bonding via a thiol group include bonding by Michael addition reaction, thiolene reaction, or thiolyne reaction, or disulfide bond.

1-3-3. Labeling of Constituent Factors Constituent factors of the target peptide degradation-inducing agent of the present invention may be labeled as necessary. For labeling, a labeling substance known in the art may be used depending on the type of each factor.

For example, when labeling a nucleic acid such as a nucleic acid-type binding factor or a nucleic acid-type linker factor, the phosphate group, sugar, and/or base may be labeled. In addition, when labeling a peptide such as ubiquitin, a peptide-type binding factor, or a peptide-type linker factor, the constituent amino acid residues may be labeled. Furthermore, in the case of a low-molecular-weight compound such as a low-molecular-weight compound-type binding factor or a low-molecular-weight compound-type linker factor, the included functional groups may be labeled. The type of constituent factor to be labeled is also not limited. However, labeling of the binding agent is convenient, and is preferred because it is direct and convenient when detecting binding to the target peptide. Also, multiple constituent factors may be labeled.

In the case of any constituent factor, the labeling position may be appropriately determined according to the characteristics and purpose of use of the labeling substance, and is not particularly limited. In the case of a nucleic acid-type factor, the 5'-end and/or 3'-end is preferably used as the labeling site. In the case of a peptide-type factor, the side chain of each amino acid residue, the amino group of the N-terminal amino acid residue, or the carboxy group of the C-terminal amino acid residue is suitable as the labeling site. Furthermore, if it is a low-molecular-weight compound-type factor, the functional groups it contains can be labeled.

Any substance known in the art can be used as the labeling substance. Examples thereof include radioisotopes, fluorescent substances, quenchers, chemiluminescent substances, DIG, biotin, magnetic beads and the like.

“Radioisotope” refers to an element that emits radiation among isotopes with different mass numbers. Examples include ^32P , ^3H , ^14C .

"Fluorescent substance" refers to a substance that has the property of being excited by absorbing excitation light of a specific wavelength and emitting fluorescence when returning to the original ground state. For example, FITC, Rhodamine, Fluorescamine, Fluorescein, Texas Red®, Cy3, Cy5, Cy7, FAM, HEX, JOE, ROX, TET, Bodipy493, NBD, TAMRA, Quasar®, CAL Fluor® ) Red610, SYBR Green [registered trademark], Eva Green [registered trademark], SYTOX Green [registered trademark], and the like.

"Quencher" refers to a substance that absorbs the excitation energy of the fluorescent substance and has the property of suppressing fluorescence. Examples include AMRA, DABCYL, BHQ-1, BHQ-2, or BHQ-3.

A "chemiluminescent substance" is a substance that has the property of emitting the differential energy as light when returning to the ground state after being excited by a chemical reaction. For example, an acridinium ester etc. are mentioned.

Each constituent factor can be labeled at multiple locations with two or more of the same or different labeling substances. A component labeled with a labeling substance can be a useful tool in detecting a target peptide degradation inducer.

1-4. Effect According to the target peptide degradation inducer of the present invention, the target peptide can be indirectly ubiquitinated as a whole by binding to the target peptide to form a complex. As a result, it can be directed to the proteasome for degradation without direct ubiquitination of the target peptide. As a result, not only low-molecular-weight compounds and antibody drugs, but also target peptides that have been undraggable due to the difficulty of direct ubiquitination can be used as drug discovery targets. As a result, the field of molecular-targeted medicine will expand dramatically, and it is expected that the options for drug development and design will greatly expand.

According to the target peptide degradation inducer of the present invention, any target peptide can be degraded by the Ub-PSM system by selecting a binding factor that specifically binds to the target peptide. This enables selective degradation by the Ub-PSM system even for target peptides that have been difficult to ubiquitinate in the past. For example, if the binding factor is a molecule that binds to caspase, the target peptide degradation inducer can be an apoptosis inhibitor. When the binding factor is a molecule that binds to Atg factor, the target peptide degradation inducer can be an autophagy inhibitor. The target peptide degradation inducer can be an autophagy promoter.

2. Nucleic acid type target peptide degradation inducer 2-1. Overview A second aspect of the present invention is a nucleic acid-type target peptide degradation inducer. The nucleic acid-type target peptide degradation-inducing agent of the present invention is composed of a polynucleotide encoding the target peptide degradation-inducing agent according to the first aspect, which is composed only of peptides, or an expression vector containing the same. The nucleic acid-type target peptide degradation inducer of the present invention has relatively high stability, is easy to store, and can amplify an active target peptide degradation inducer after administration in vivo.

2-2. Structure "Nucleic acid-type target peptide degradation inducer" refers to a target peptide degradation inducer composed of a nucleic acid.
When the target peptide degradation-inducing agent according to the first aspect is composed only of a peptide, the nucleic acid-type target peptide degradation-inducing agent is composed of a polynucleotide encoding the amino acid sequence, or an expression vector containing the polynucleotide. . Polynucleotides here are, in principle, natural polynucleotides. Although composed of either DNA and/or RNA, polynucleotides composed solely of DNA are preferred.

A target peptide degradation inducer composed only of a peptide corresponds to a peptide-type binding factor and ubiquitin, or a target peptide degradation inducer composed of a peptide-type binding factor, a peptide-type linker factor, and ubiquitin.

As used herein, the term "expression vector" refers to an expression unit that contains a gene or gene fragment (in the present invention, the polynucleotide) in an expressible state and can control the expression of the gene or the like. As used herein, the term "expressible state" refers to a state in which the gene or the like is placed under the control of a promoter and the expression of the gene or the like can be induced by activation of the promoter. Any type of expression vector can be used. Any expression vector whose promoter can be activated in vivo in a subject to whom the nucleic acid-type target peptide degradation inducer of the present invention is administered can be used. For example, when the subject is human, viral vectors are included. Viral vectors include various vectors derived from retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, and the like.

The functions and effects of the nucleic acid-type target peptide degradation inducer of this aspect conform to those of the target peptide degradation inducer described in the first aspect. However, due to the structural difference that they are composed of nucleic acids, their mechanisms of action are different. That is, the target peptide degradation-inducing agent according to the first aspect does not itself have the activity of inducing degradation of the target peptide, and acts directly on the target peptide after administration to the living body, whereas the nucleic acid-type target peptide degradation-inducing agent of this aspect has no activity to induce degradation of the target peptide. After administration of the inducer, the target peptide degradation inducer encoded by the nucleic acid-type target peptide degradation inducer is expressed intracellularly, and the expressed target peptide degradation inducer acts on the target peptide. This makes it possible to amplify the target peptide degradation inducer in vivo.

3. Target Peptide Degradation Inducing Composition 3-1. Overview A third aspect of the present invention is a target peptide degradation-inducing composition. The composition of the present invention includes any two or more of the target peptide degradation inducer according to the first aspect and/or the nucleic acid-type target peptide degradation inducer according to the second aspect as essential constituents. According to the peptide degradation-inducing composition of the present invention, a target peptide degradation-inducing agent or the like can be stably provided as a pharmaceutical composition in a form that reduces the burden and invasiveness upon administration to the living body and is easier to administer. can be done.

3-2. Configuration 3-2-1. Constituent Factors The target peptide degradation-inducing composition of this embodiment (hereinbelow, often abbreviated as "this composition") comprises an active ingredient as an essential constituent factor, and a carrier and/or Contains solvent. Each component will be specifically described below.

(1) Active ingredient The present composition comprises the target peptide degradation inducer according to the first aspect and/or the nucleic acid-type target peptide degradation inducer according to the second aspect (in this specification, these are collectively referred to as "POI degradation (referred to as "inducing agent, etc.") as an essential active ingredient. The present composition can contain two or more different POI degradation inducers and the like. In addition to POI degradation inducers and the like, other known target protein degradation inducers such as PROTAC (registered trademark) may be selectively included as active ingredients.

The content of the POI decomposition inducer, etc. contained in the composition includes the type and/or effective amount of the POI decomposition inducer, etc., the dosage form of the composition, the type of carrier or additive described later, and Since it varies depending on the type of disease, it may be determined appropriately in consideration of each condition.

As used herein, the term "effective amount" refers to an amount necessary for the POI decomposition inducer, etc., as an active ingredient in the present composition, to exhibit its function, and to cause no harmful side effects to the living body to which it is applied. , refers to the amount that occurs little or not at all. This effective amount may vary depending on various conditions such as subject information, route of administration, and frequency of administration.

As used herein, the term "subject" refers to a living organism to which the POI degradation inducer or the like or the present composition is applied. For example, humans, livestock (cows, horses, sheep, goats, pigs, chickens, ostriches, etc.), racehorses, pet animals (dogs, cats, rabbits, etc.), laboratory animals (mouse, rats, guinea pigs, monkeys, etc.), etc. Applicable. Humans are preferred (in this case, they are particularly referred to as "subjects"). In addition, "subject information" refers to various individual information of the living body to be applied. Including degree and severity, age, body weight, sex, diet, drug sensitivity, presence or absence of concomitant drugs, tolerance to treatment, etc. The effective amount of the POI degradation inducer, etc. in this composition and the applicable amount calculated based thereon are ultimately determined by doctors, dentists, veterinarians, etc., depending on the information of individual subjects. determined by

(2) Solvent The composition can contain a pharmaceutically acceptable solvent. Solvents are optional constituents in the composition and may be added as needed. “Pharmaceutically acceptable” means harmless or low toxicity to living bodies, and can be used normally in the field of formulation technology, preferably in pharmaceutical compositions.

Solvents include, for example, water or aqueous solutions, or organic solvents. Pharmaceutically acceptable aqueous solutions include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, phosphate buffers, sodium acetate buffers. Examples of adjuvants here include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, and the like. Pharmaceutically acceptable organic solvents include, for example, ethanol, butanol, and the like.

(3) Carrier The composition can contain a pharmaceutically acceptable carrier. A carrier is an optional component in the composition and may be added as needed.

Carriers include, for example, suspending agents, diluents, solubilizers, dispersing agents, surfactants, emulsifying agents, soothing agents, stabilizers, preservatives, preservatives, antioxidants, buffers, and tonicity agents. agents and the like.

In addition to the above, excipients, fillers, binders, disintegrants, absorption enhancers, bulking agents, humectants, humectants, humectants, adsorbents, and disintegration inhibitors commonly used in pharmaceuticals, if necessary Agents, coating agents, coloring agents and the like may also be included as appropriate.

Such a carrier is mainly used to facilitate the formation of a dosage form, maintain the dosage form and drug effect, and make the POI degradation inducer, etc., which is an active ingredient, difficult to be decomposed in vivo. It should be used appropriately as needed.

3-2-2. Dosage form The dosage form of the present composition does not inactivate the POI degradation inducer and other known target protein degradation inducers, which are active ingredients, and can exhibit the pharmacological effect of the active ingredient in vivo after administration. There is no particular limitation as long as it is in a form. Moreover, the specific dosage form of the present composition may be appropriately selected according to the administration method and/or prescription conditions. In general, administration methods can be broadly classified into oral administration and parenteral administration, and the present composition may be formulated in dosage forms suitable for each administration method.

For oral administration, the dosage forms include solids (tablets, pills, sublinguals, capsules, drops), granules, powders, powders, liquids (internal liquids, suspensions, emulsions, syrups). including agents) and the like. Solid formulations can optionally be in the form of coatings known in the art, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multi-layer tablets. .

Parenteral administration can be divided into systemic administration and topical administration, and local administration can be further subdivided into tissue administration, transepidermal administration, transmucosal administration, and transrectal administration. The composition may also be formulated in dosage forms suitable for each administration method. For example, dosage forms suitable for systemic administration or intra-tissue administration include liquid injections. Dosage forms suitable for transepidermal or transmucosal administration include liquids (including ointments, eye drops, nasal drops, and inhalants), suspensions (including emulsions and creams), and powders (nasal drops). , inhalants), pastes, gels, ointments, plasters, and the like. Dosage forms suitable for rectal administration include suppositories and the like.

The present composition can be applied for treatment or prevention of various diseases as described in the fourth aspect. The target site for application varies depending on the target disease. For example, if the target disease is a neurodegenerative disease, it will mainly be the cerebrospinal cord, which is the central nervous system. Therefore, the method of administration is not limited, but intratissue administration or systemic administration via the circulatory system can be used. Specific examples include intratissue administration by intracerebrospinal injection, or intracirculatory administration such as intravascular injection (including intravenous injection and intraarterial injection) or intralymphatic injection. In addition, if the target disease is cancer, the application target sites are mainly primary lesions and metastatic lesions. In this case, too, the method of administration is not limited, but intra-tissue administration to primary lesions and metastatic lesions or systemic administration via the circulatory system can be used.

Injectables can be prepared by appropriately combining the aforementioned emulsifying agents, suspending agents, surfactants, stabilizers, pH adjusters, etc., and mixing them in a unit dose form generally required for pharmaceutical practice. They are often provided in unit dose ampoules or in multi-dose containers.

The specific shape and size of each dosage form described above are not particularly limited as long as they are within the range of dosage forms known in the art for each dosage form.

4. Disease Suppressant 4-1. Overview A fourth aspect of the present invention is a disease suppressing agent. The disease-suppressing agent of the present invention is one form of the target peptide degradation-inducing agent according to the first aspect, and has a specific disease-causing protein as a target peptide. The disease-suppressing agent of the present invention can treat or prevent various diseases by using proteins that cause various diseases as target peptides and inducing their degradation.

According to the disease suppressor of the present invention, even nucleic acid-binding proteins that are considered undruggable in ubiquitin drug discovery, such as transcription factors, can be targeted for degradation. As a result, the options in the development and design of molecular-targeted drugs are expanded, and a wide range of drug discovery and drug discovery technologies for various diseases can be provided.

4-2. Configuration As used herein, the term "disease suppressing agent" refers to an agent that suppresses the onset of a specific disease, or alleviates or treats the aggravation or chronicity of a specific disease.

The disease suppressor of the present invention contains a binding factor and ubiquitin as essential constituent factors, and a linker factor as an selective constituent factor. Its basic configuration conforms to the target peptide degradation inducer described in the first aspect. Therefore, the specific description of the common configuration is omitted, and the characteristic points of the disease suppressing agent of the present invention are described here.

The disease-suppressing agent of the present invention is characterized in that the binding factor binds to the disease-causing peptide. In other words, the disease-causing peptide is the target peptide in the disease-suppressing agent of this embodiment.

As used herein, the term "disease-causing peptide" refers to a peptide (including protein) that causes the onset, exacerbation, or chronicity of a disease. Such disease-causing peptides include wild-type peptides as well as mutant peptides such as gain-of-function (activation), hyperfunction, and loss-of-function.

The type of disease targeted by the disease suppressing agent of the present invention is not limited as long as the cause of the disease is a peptide. As previously mentioned, target peptides include transcription factors, amyloid, tau protein, α-synuclein, caspases, denatured huntingtin, pseudokinases, K-RAS mutants, Atg factors, mTOR complex 1, GAPR-1 and orphans. Diseases caused by receptors and the like, including receptors, can all be targets of the disease-suppressing agent of the present invention.

The target peptide degradation inducer according to the first aspect induces degradation by the Ub-PSM system by indirectly ubiquitylating the target peptide via a binding factor. Therefore, by selecting a binding factor that binds to a disease-causing peptide, it can be used as a therapeutic or preventive drug for various diseases. The relationship between the target disease of the disease-suppressing agent of the present invention and the disease-causing peptide will be described below with some examples.

(1) Anticancer agent The disease inhibitor of the present invention can be an anticancer agent. In this case, the disease-suppressing agent comprises a binding agent that binds to a cancer-causing disease-causing peptide. Examples of such disease-causing peptides include transcription factors such as NF-κB, pseudokinases such as HER3 (Xie T., et al., 2014, Nat. Chem. Biol., 10: 1006). -1011), K-RAS mutants (Bond MJ, et al., 2020, ACS Cent. Sci. 6: 1367-1375), or orphan receptors such as ERR-α, but are not limited to do not. It has been difficult to inhibit the function of these disease-causing peptides with inhibitors composed of conventional low-molecular-weight compounds. Knockdown by degradation of disease-causing peptides via the PSM system makes it possible to treat, ameliorate, or suppress progression of cancer.

Here, as an example, we will explain the composition of an anticancer drug when NF-κB is used as a disease-causing peptide. Inhibition of NF-κB activity is known to promote apoptosis induction by TNF-α (Tang F., et al., 2002, Mol. Cell. Biol., 22: 8571-8579). Therefore, by selecting a binding factor that specifically binds to NF-κB, the disease suppressor of the present invention can function as an apoptosis inducer. By applying this apoptosis inducer to cancer cells, it can function as an anticancer agent.

The specific composition of the binding factor that binds to NF-κB is not limited. p50 and p52 belonging to class I of NF-κB and p65 (RelA), c-Rel and RelB belonging to class II may be configured to bind, or a configuration that specifically binds to any of them may be used. can be Examples thereof include nucleic acid-type binding agents consisting of a nucleic acid sequence to which NF-κB binds. More specifically, for example, the NF-κB-binding hairpin DNA represented by SEQ ID NO: 2 or 3. This NF-κB-binding hairpin DNA is NF-κB Decoy DNA containing a nucleotide sequence mimicking the target nucleotide sequence of NF-κB.

(2) Alzheimer's Disease Suppressant The disease suppressor of the present invention can be an agent for preventing Alzheimer's disease (AD) or for suppressing its progress. In this case, the disease-suppressing agent comprises a binding agent that binds to a disease-causing peptide that causes Alzheimer's disease. Alzheimer's disease begins with the aggregation and accumulation of β-amyloid protein in neurons in the brain, followed by hyperphosphorylation of tau protein, a microtubule protein, resulting in fibrosis, followed by destruction of neurons and brain atrophy. . Therefore, β-amyloid, tau protein, α-synuclein, and the like can be applicable as disease-causing peptides for Alzheimer's disease. Therefore, by selecting a binding factor that specifically binds to β-amyloid, tau protein, α-synuclein, or the like, the disease inhibitor of the present invention can function as an Alzheimer's disease inhibitor.

　The specific composition of the binding factor that binds to β-amyloid and tau protein is not limited. Examples thereof include low-molecular-weight compounds capable of binding to fibrillar β-amyloid or tau protein. More specific examples include thioflavin T (ThT) as a binding factor for β-amyloid aggregates and pyridoindole as a binding factor for tau protein.

(3) Parkinson's Disease Suppressant The disease suppressor of the present invention can be a prophylactic agent for Parkinson's disease (PD) or an agent for suppressing the progression thereof. In this case, the disease-suppressing agent comprises a binding agent that binds to a disease-causing peptide that causes Parkinson's disease. The onset of Parkinson's disease is believed to be related to neuronal cell death due to multimerization and accumulation of misfolded α-synuclein, which constitutes protein aggregates called Lewy bodies. Therefore, misfolded mutant α-synuclein and the like can be applicable as disease-causing peptides for Parkinson's disease. Therefore, by selecting a binding factor that specifically binds to mutant α-synuclein, the disease suppressing agent of the present invention can function as a Parkinson's disease suppressing agent.

　The specific configuration of the binding factor that binds to mutant α-synuclein is not limited. Examples thereof include short peptides such as partial sequences of β-synuclein that selectively bind to α-synuclein. More specifically, βsyn36 consisting of the amino acid sequence (GVLYVGSKTR) shown in SEQ ID NO: 5 (Shaltiel-Karyo R., et al., 2010, PLoS One, 5, e13863.; Fun X., et al. ., 2014, Nat. Neurosci., 17, 471-480.).

(4) Agent for suppressing Huntington's disease The agent for suppressing disease of the present invention can be an agent for preventing Huntington's disease (HD) or suppressing its progression. In this case, the disease-suppressing agent comprises a binding agent that binds to the disease-causing peptide responsible for Huntington's disease. Huntington's disease is known to be involved in the onset of mutated huntingtin (mHTT), which is caused by the expression of the mutated huntingtin (mHTT) gene with an expanded number of CAG repeats and induces neuronal dysfunction and cell death. (Tomoshige S., et al., 2017, Angew. Chem. Int. Ed., 56, 11530-11533). Therefore, by selecting a binding factor that specifically binds to mutant huntingtin, the disease-suppressing agent of the present invention can function as a Huntington's disease-suppressing agent.

The specific composition of the binding factor that binds to mutant huntingtin is not limited. Examples include low-molecular-weight compounds that bind to mutant huntingtin multimers and aggregates. More specifically, for example, benzothiazole aniline (BTA) or phenyldiazenyl benzothiazole (PDB) (Tomoshige S., et al., 2017, Angew. Chem. Int. Ed., 56, 11530- 11533), etc. 

The target peptide degradation inducer of the present invention will be specifically described below with reference to examples. However, the configuration and the like described here are only one specific example of the target peptide degradation inducer of the present invention, and do not limit the scope thereof.

<Example 1: Preparation of nucleic acid type target peptide degradation inducer>
(the purpose)
A target peptide degradation inducer of the present invention is prepared, and it is verified that the target peptide is captured by the target peptide degradation inducer. In this example, NF-κB was selected as the target peptide, and two target peptide degradation inducers (Ub-cys-Decoy and Ub-C77-Decoy) is prepared.

(Method)
(1) Preparation of ubiquitin-cysteamine (Ub-cys) In order to prepare the target peptide degradation-inducing agent of the present invention, first, cysteamine used for linkage with a binding factor was added to the C-terminus of ubiquitin. 250 μM ubiquitin, 2 μM E1, 2 mM ATP, 5 mM cysteamine, and 5 mM TCEP in 1 mL (final volume) of buffer containing 100 mM Tris-HCl (pH 7.5), 250 mM sucrose, 50 mM KCl, ₃ mM MgCl at 4 °C. Incubated overnight. The reaction mixture was subsequently purified by Superdex ^™ 75 Increase 10/300GL size exclusion chromatography (Cytiva). After that, the desired Ub-cys was eluted with 1.5 column volumes of S buffer (20 mM NaPO ₄ , pH 7.2, 150 mM NaCl).

(2) Preparation of ubiquitin-decoy (Ub-cys-Decoy) conjugate using Ub-cys Subsequently, SMCC (4-(N- After addition of maleimidomethyl)cyclohexanecarboxylate (N-succinimidyl), it was linked to Ub-cysteamine prepared in (1) above to prepare Ub-Decoy, the target peptide degradation inducer of the present invention.

Specifically, first, 3 mM SMCC (Thermo Fisher Scientific) in dimethylformamide (DMF) (100 eq) was added to _NaPO4 buffer (pH 7.5) containing 30 μM NF-κB decoy DNA (1 eq). As the NF-κB decoy DNA used, two types of polynucleotides shown in SEQ ID NO: 2 or 3 below were used.
(i) NF-κB ODN1 (27mer)
5′(amino)-AGGGAAATCCCAAAATGGGATTTCCCT-3′(FAM): SEQ ID NO: 2
(ii) NF-κB ODN2 (28mer)
5′(amino)-AGGGGATTCCCAAAATGGGAATTCCCCT-3′(FAM): SEQ ID NO: 3

All of the above NF-κB Decoy DNAs have been shown to form a hairpin structure and be recognized and bound by NF-κB. In addition, both are labeled with FAM (fluorescein) at the 3'.

After incubating the reaction mixture at room temperature for 90 minutes, residual SMCC was removed by ethanol precipitation to obtain the product NF-κB Decoy DNA-SMCC.

Subsequently, 250 μM Ub-cysteamine (Ub-cys) (25 eq) prepared in (1) above, 10 μM NF-κB Decoy DNA-SMCC (1 eq), and 10 mM TCEP (Fujifilm Wako Pure Chemical Industries, Ltd.) as a reducing agent ) and incubated overnight at room temperature to obtain Ub-cys-Decoy, a target peptide degradation inducer of the present invention consisting of ubiquitin and NF-κB Decoy DNA.

(3) Preparation of ubiquitin-decoy (Ub-C77-Decoy) conjugate using Ub-C77 Here, instead of adding a functional group to the C-terminus of ubiquitin such as Ub-cys, SEQ ID NO: 1 Ub-C77 in which cysteine is added as the 77th amino acid to the C-terminus of human ubiquitin shown, and two arginine residues at positions 72 and 74 in human ubiquitin shown in SEQ ID NO: 1 are alanine residues and threonine residues, respectively. The target peptide degradation inducer of the present invention was prepared using a Ub-C77 mutant (UbR-C77) in which cysteine was added as the 77th amino acid to the C-terminus of the ubiquitin mutant shown in SEQ ID NO: 6. prepared.

Ub-C77 and UbR-C77 with a cysteine added to the C-terminus were prepared by expression in E. coli as a host using a pET-26b(+) plasmid containing genes encoding Ub-C77 and UbR-C77. .
NF-κB Decoy DNA-SMCC was prepared according to the method (2) above.

2.12 mM Ub-C77 (50 eq), 42.4 μM NF-κB Decoy DNA-SMCC (1 eq), and 10 mM TCEP were mixed and incubated overnight at room temperature. Next, Ub-C77-Decoy was purified by anion exchange chromatography HiTrap Q HP (GE Healthcare). The column was equilibrated with 5 column volumes of buffer A (50 mM ammonium acetate (pH 4.5), 0.1 mM EDTA) and the desired Ub-C77-Decoy was eluted with a stepwise gradient of NaCl (200 mM, 450 mM, 500 mM). bottom. The eluate was concentrated and dialyzed against Ub storage buffer (25 mM _NaPO4 (pH 7.2), 50 mM NaCl). Yield was 14.4%.

145 μM UbR-C77 (20 eq), 7.2 μM NF-κB Decoy DNA-SMCC (1 eq) were mixed in 20 mM sodium phosphate buffer (pH 7.0) and incubated overnight at room temperature. It was then purified by Superdex ^™ 75 Increase 10/300GL size exclusion chromatography column (Cytiva). At that time, the target UbR-C77-decoy was eluted using 1.5 column volumes of S buffer (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.2).

<Example 2: Gel shift assay by binding of Ub-C77-Decoy and NF-κB p50>
(the purpose)
It is confirmed by gel shift assay that the target peptide degradation inducer Ub-C77-Decoy of the present invention prepared in Example 1 binds to the target peptide NF-κB p50.

(Method)
500 nM NF-κB p50 (Cayman Chemical) and 25 nM Ub-C77-Decoy in binding buffer (10 mM HEPES (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 2.5 mM DTT, 10% glycerol, 0.05% NP-40) Incubate at room temperature for 30 minutes. Samples were then applied to 6% Native PAGE and run at 150V, 0.5xTBE, 4°C for 25 minutes. For control, only Ub-C77-Decoy was used. After electrophoresis, the position of each sample was confirmed based on the fluorescence of FAM modified at the 3' end of NF-κB decoy DNA.

(result)
The results are shown in FIG. Compared to lane 1 containing only Ub-C77-Decoy, lane 2 containing a mixture of Ub-C77-Decoy and NF-κB p50 clearly shows a band shift, indicating that Ub-C77-Decoy is the NF of the target peptide. It was confirmed that it binds to -κB p50.
Although not shown, similar binding results were obtained when Ub-cys-Decoy and UbR-C77-decoy were used.

<Example 3: Target peptide NF-κB p50 degradation assay (1)>
(the purpose)
It is confirmed that the target peptide is degraded by binding to the target peptide degradation inducer of the present invention prepared in Example 1 without direct ubiquitination in the presence of proteasome.

(Method)
200 nM NF-κB p50 and 1 μM Ub-C77-Decoy in binding buffer (25 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM DTT, 10% glycerol, 0.05% NP-40) for 30 minutes at 25°C. After incubation, a complex NF-κB p50/Ub-C77-Decoy in which the target peptide NF-κB p50 and the target peptide degradation inducer Ub-C77-Decoy of the present invention were bound was prepared.

Next, NF-κB p50/Ub-C77-Decoy was added with 100 nM 26S proteasome (gift from Dr. Yasushi Saeki, Tokyo Metropolitan Institute of Medical Science), 2 mM ATP, and 5 mM MgCl ₂ and incubated at 37° C. for 60 minutes. . In the meantime, a portion of the mixed solution at the start of incubation was taken.

In addition, in order to prove that the degradation is due to the Ub-PSM system, a sample with 100 μM MG132 (AdipoGen Life Sciences), a proteasome inhibitor, and a sample without 26S proteasome were prepared. Furthermore, as controls, samples were also prepared in which only NF-κB-Decoy DNA or only Ub-C77, which is a component of the target peptide degradation inducer of the present invention, was added to the 26S proteasome.

Subsequently, the mixed solution after reaction was mixed with SDS sample buffer (62.5mM Tris-HCl (pH6.8), 2% SDS, 10% Glycerol, 5% 2-mercaptoethanol, 0.002% Bromophenol blue) and After heating for 5 minutes, they were separated by SDS-PAGE. After Western blotting by a standard method, the resulting membrane was incubated with primary antibodies (Anti NFKB1, p105, p50-Specific: Proteintech) diluted 1:500 in blocking solution for 1.5 hours with agitation. After washing three times with 1×TBST (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.2% Tween-20), secondary antibodies (Anti-IgG, Mouse, Goat-Poly , HRP: R&D Systems) for 1 hour with gentle shaking, and washed 3 times with 1×TBST. The membrane was then incubated with a substrate/detector mixture (enhancer solution: chemiluminescent peroxide reagent = 1:1) for 2 minutes and analyzed with a chemiluminescent imaging system (ChemiDoc XRS Plus: BIO-RAD).

(result)
The results are shown in FIG. A marked decrease in NF-κB p50 was confirmed only in lane 2 60 minutes after NF-κB p50/Ub-C77-Decoy and 26S proteasome were mixed. On the other hand, when MG132 was added to the mixture of NF-κB p50/Ub-C77-Decoy and 26S proteasome, almost no significant decrease in NF-κB p50 was observed (lane 3). Furthermore, in the case of NF-κB p50/Ub-C77-Decoy alone (lane 4), in the case of mixing Decoy-DNA and 26S proteasome (lane 5), and in the case of mixing Ub-C77 and 26S proteasome (lane 6 ) did not significantly decrease NF-κB p50.

Based on these results, the target peptide degradation inducer of the present invention indirectly ubiquitinates the target peptide by binding to the target peptide, and degrades the target peptide using the proteasome as in the conventional Ub-PSM system. proved to be possible.
Although not shown, similar decomposition results were obtained when Ub-cys-Decoy was used.

<Example 4: Verification of NF-κB p50 decomposition rate>
(the purpose)
The target peptide degradation rate by the target peptide degradation inducer of the present invention is verified.

(Method)
The basic operation conformed to the method of Example 3. In this example, the concentration of Ub-C77-Decoy, which is a target peptide degradation inducer, was divided into 50 nM, 100 nM, 200 nM, 400 nM, 800 nM, and 1000 nM (1 μM), and based on the luminescence intensity after the reaction, at each concentration The residual amount of NF-κB p50 was measured. The residual amount was calculated as a relative value when the emission intensity when Ub-C77-Decoy was not added (0 nM) was taken as 100%, that is, the residual rate of Ub-C77-Decoy.

(result)
The results are shown in FIG. The NF-κB p50 retention rate after 60 minutes of reaction relative to the amount added before reaction decreased depending on the amount of Ub-C77-Decoy added, and was 32% at 1000 nM Ub-C77-Decoy. In other words, it suggests that the target peptide was degraded by the target peptide degradation inducer of the present invention at a degradation rate of about 70%.

On the other hand, in the case of degradation by the Ub-PSM system via direct ubiquitination to the target protein, the retention rate of the target peptide α-globulin after 1 hour of reaction was determined by Sun et al. (Sun H., et al., 2019 , Proc. Natl. Acad. Sci. USA., 116: 7805-7812), 47%. This suggests that the direct ubiquitination-mediated Ub-PSM system degraded the target peptide with a degradation rate of approximately 60%.

Therefore, although the types of target peptides are different, indirect ubiquitination of target peptides by binding of the target peptide degradation-inducing agent of the present invention can be used as a signal to mediate efficient degradation by the proteasome compared to conventional direct ubiquitination. It was clarified that the target peptide could be decomposed with a similar degree of decomposition rate.

<Example 5: NF-κB p50 degradation assay (2)>
(the purpose)
We will verify the ability of UbR-C77-Decoy with mutated ubiquitin to induce target peptide degradation.

(Method)
The basic operation conformed to Example 3. In this example, UbR-C77-decoy was mixed with NF-κB p50 at a final concentration of 1 μM, and the residual amount of NF-κB p50 was evaluated by Western blotting. The residual amount was calculated as a relative value when the band intensity when Ub-C77-Decoy was not added (0 nM) was taken as 100%. Ub-C77-decoy with a final concentration of 1 μM was used as a positive control.

(result)
The results are shown in FIG. Residual NF-κB p50 after addition of UbR-C77-decoy (lane 2) and Ub-C77-decoy (lane 3) relative to residual NF-κB p50 (lane 1) when peptide degradation inducer was not added The amounts were 34% and 25% respectively. On the other hand, when Decoy DNA (lane 4), UbR-C77 (lane 5), and Ub-C77 (lane 6) were individually mixed, no significant decrease in residual NF-κB p50 was observed. From the above, it was demonstrated that UbR-C77-decoy has a degradation-inducing ability equivalent to that of Ub-C77-decoy.

<Example 6: NF-κB p65 degradation assay in cell extract>
(the purpose)
Degradation of the cell-derived target peptide NF-κB p65 contained in the cell extract by indirect ubiquitination of UbR-C77-Decoy is verified.

(Method)
Cell extract of MDA-MB-231 cells (ATCC) adjusted to a final protein concentration of 0.7 mg/mL, UbR-C77-decoy at a final concentration of 1 μM, 26S yeast proteasome at a final concentration of 0.1 μM (Tokyo Metropolitan Institute of Medical Science) A gift from Dr. Yasushi Saeki) was mixed in a degradation assay buffer (5 mM HEPES, 10 mM NaCl, 4 mM TCEP, 10% glycerol, 0.01% NP-40) and allowed to stand at room temperature for 6 hours. The amount of residual NF-κB p65 in each sample was analyzed by Western blotting according to a standard method. The residual amount was obtained as a relative value when the band intensity of NF-κB p65 when UbR-C77-decoy was not added was taken as 100%. At that time, the band intensity of NF-κB p65 was normalized by the band intensity of β-actin.

(result)
The results are shown in FIG. In the presence of UbR-C77-decoy, a marked decrease in the residual amount of NF-κB p65 was observed, and its degradation rate reached 59%. These results demonstrate that the induction of target peptide degradation by indirect ubiquitination using the target peptide degradation inducer of the present invention can be effective even in cell extracts.

<Example 7: Target peptide NF-κB p65 degradation assay in living cells>
(the purpose)
To verify the degradation induction of NF-κB p65 by UbR-C77-Decoy in living cells.
(Method)
MCF-7 cells (RIKEN BioResource Research Center) and HeLa cells (RIKEN BioResource Research Center) were plated at 3×10 ⁴ cells/well in a 48-well plate and cultured at 37°C under 5% CO ₂ for 24 hours. and grown to 80% confluency. Next, after replacing the medium with Opti-MEM, 1 μM UbR-C77-decoy was introduced into the cells by lipofection using Lipofectamine ^™ LTX (Thermo Fisher Scientific). After that, the medium was replaced with DMEM (10% FBS, 0.5% PS), and TNF-α (final concentration 20ng/mL) and protein translation material cycloheximide (CHX)/0.5% DMSO (final concentration 50μg/mL) were added. was added to the medium and cultured at 37°C under 5% CO ₂ for 4 hours. After culturing, cells were lysed with RIPA buffer (Nacalai tesque) and analyzed for NF-κB p65 by Western blotting. At that time, the band intensity of NF-κB p65 was normalized by the band intensity of β-actin.

(result)
The results are shown in FIG. When UbR-C77-decoy was introduced, the relative amount of NF-κB p65 to β-actin was 34% and 29% in HeLa cells and MCF-7 cells, respectively, and decreased to about 30% in both cells. was found to decrease to These results demonstrate that the target peptide degradation inducer of the present invention can induce the target peptide to be degraded via the Ub-PSM system even in living cells.

<Example 8: Apoptosis induction using Ub-C77-Decoy>
(the purpose)
Inhibition of NF-κB activity promotes apoptosis induction by TNF-α as described above. Therefore, it is verified that apoptosis is induced by degradation of NF-κB by Ub-C77-Decoy, which is the target peptide degradation inducer of the present invention.

(Method)
(1) Cell culture Human breast cancer cell line MCF-7 cells were placed in DMEM containing 10% FBS and 0.5% penicillin/streptomycin (hereinafter referred to as "DMEM (FBS/PS)") in a 10 cm dish and incubated with 5% CO. It was pre-incubated at 37°C under ₂ .

(2) Confirmation of intracellular introduction of Ub-C77-Decoy by lipofection Pre-cultured MCF-7 cells were plated on a 35 mm glass bottom dish at φ = 12 mm (5 × 10 ⁴ cells/dish) (IWAKI). , incubated for 24 h at 37 °C _under 5% CO2. Then, after replacing the medium with Opti-MEM, MCF-7 cells were transiently transfected with Ub-C77-Decoy using Lipofectamine (registered trademark) LTX with Plus Reagent (Thermo Fisher Scientific). NF-κB Decoy DNA was used for control. The transfected cells were incubated at 37°C under 5% CO ₂ for 2 hours, stained with Hoechst33258, incubated again at 37°C under 5% CO ₂ for 2 hours, and then subjected to fluorescence microscopy (IX83: OLYMPUS ) to observe nuclear-stained cells.

(3) Cell Viability Assay The basic procedure followed the method described in (2) above. MCF-7 cells after preculture were adjusted to 2.0×10 ⁴ cells/200 μL with DMEM (FBS/PS). Next, the cells were seeded in 96-well plates at 200 μL/well and cultured at 37° C. under 5% CO ₂ within 24 hours to approximately 80% confluence. Subsequently, after replacing the medium with Opti-MEM, MCF-7 cells were transiently transfected with 200 nM Ub-C77-Decoy using Lipofectamine® LTX with Plus Reagent (Thermo Fisher Scientific). bottom. 200 nM NF-κB Decoy DNA was used for negative control. After transduction, cells were incubated for 1.5 hours at 37° C. under 5% CO ₂ . After that, the medium was changed to DMEM (FBS/PS) and 5 ng/mL TNF-α was added to the cell suspension and incubated at 37° C. under 5% CO ₂ for 6 hours or 24 hours. PrestoBlue ^™ Cell Viability Reagent (Thermo Fisher Scientific) was added, incubated for 30 minutes at 37°C under 5% CO ₂ for staining, and cell viability was measured using a plate reader (Cytation5: BioTek).

(result)
Microscopic images are shown in FIG. 7, and cell viability is shown in FIG.
From FIG. 7, fluorescence derived from FAM modified with Ub-C77-Decoy was observed from within the cells into which Ub-C77-Decoy was introduced by lipofection (f). In addition, the f image was superimposed on the phase-contrast microscope image (i), and showed that fluorescence was observed in most of the cell groups into which Ub-C77-Decoy was introduced by lipofection (j). . On the other hand, when the control Ub-C77-Decoy was directly added to the medium, few cells exhibited fluorescence (a, d, and e). The above results suggest that Ub-C77-Decoy can be introduced into cells with high efficiency by general lipofection.

Ub-C77-Decoy was introduced by lipofection from images (c and h) in which the fluorescence diagrams (b and g) derived from Hoechst33258 used for nuclear staining and the fluorescence (a and f) derived from FAM were superimposed. It was also suggested that some of the translocated cells translocated not only into the cell but also into the nucleus (c).

From FIG. 8, the relative survival rate when the cell survival rate in the unadded blank is 1 is 0.81 for the negative control NF-κB Decoy DNA (Decoy DNA in the figure), whereas Ub-C77 -Decoy was 0.64. These results indicate that the target peptide degradation inducer Ub-C77-Decoy of the present invention degraded the target peptide NF-κB, resulting in the induction of apoptosis in the presence of TNF-α and a decrease in cell viability. suggesting.

<Example 9: Preparation of low-molecular compound-type target peptide degradation inducer>
(the purpose)
The target peptide degradation inducer Ub-cys-biotin of the present invention is prepared using biotin, which is a low-molecular-weight compound, as a binding factor.

(Method)
(1) Preparation of carbonyl acrylic biotin A low-molecular-weight ligand, biotin, was used as a low-molecular-weight compound-type binding agent. Biotin was added to ubiquitin according to the Michael addition reaction of Bernardim et al. (Nat. Commun. 2016, 7, 13128.). First, biotin (Fujifilm Wako Pure Chemical Industries, Ltd.) and 3-benzoylacrylic acid (Alfa Aesar) were condensed via 1,2-ethylenediamine (N-Boc ethylendiamine: Sigma-Aldrich) to synthesize carbonylacrylic biotin. did 3-Benzoylacrylic acid activated by N-hydroxysuccinimide (NHS) was condensed with N-Boc ethylendiamine in the presence of diisopropylethylamine (DIPEA) in dichloromethane at room temperature. After that, the Boc group was deprotected in a dichloromethane solution containing 20(v/v)% trifluoroacetic acid. Subsequently, the obtained intermediate was reacted with NHS-activated biotin in the presence of DIPEA in dichloromethane at room temperature to obtain the desired Carbonyl acrylic biotin.

(2) Preparation of ubiquitin-biotin (Ub-cys-biotin) conjugate using Ub-Cys Addition of cysteamine to Ub followed the method described in Example 1(1). Ub-cys-biotin was prepared by Michael addition reaction between Ub-cys and carbonyl acryl biotin prepared in (1) above. Specifically, 187 μM Ub-Cys and 938 μM 3-carbonylacrylbiotin were mixed in 20 mM Tris-HCl (pH 8.0) and reacted at 25° C. for 3 hours. The desired product was then purified by Superdex ^™ 75 Increase 10/300GL size exclusion chromatography (Cytiva). The desired Ub-cys-biotin was eluted using 1.5 column volumes of Tris buffer (20 mM Tris-HCl, 150 mM NaCl, pH 8.0).

<Example 10: Target Peptide Degradation Assay Using Low-Molecular-Type Target Peptide Degradation Inducing Agent>
(the purpose)
The degradation induction of the target peptide (streptavidin) by the low-molecular compound-type target peptide degradation inducer (Ub-cys-biotin) prepared in Example 9 is verified.

(Method)
2 μM streptavidin (Fujifilm Wako Pure Chemical Industries, Ltd.), 2 μM Ub-cys-biotin in Tris-HCl buffer (50 mM Tris-HCl (pH 7.6), 100 mM NaCl, 10 mM MgCl ₂ , 2 mM ATP, 5 mM DTT, 10 mM % Glycerol) and incubated at 37° C. for 30 minutes. After that, 0.1 µM of 26S yeast proteasome was added, and the mixture was allowed to stand at 37°C for 1 hour. Each sample was subjected to size fractionation by SDS-PAGE, and the residual amount of streptavidin was analyzed by CBB staining.

(result)
The results are shown in FIG. The relative residual amount of streptavidin when Ub-cys-biotin was added (Ub-cys-biotin) was 61% when the amount of streptavidin in the control without Ub-cys-biotin was set to 100%. there were. This suggests that streptavidin was 39% degraded by Ub-cys-biotin via the Ub-PSM system.
From the above results, it was demonstrated that the target peptide degradation-inducing agent of the present invention can induce degradation of the target peptide even when a low-molecular-weight compound is used as the binding factor.

<Example 11: Preparation of peptide-type target peptide degradation inducer>
(the purpose)
A target peptide degradation inducer of the present invention is prepared using a peptide ligand as a binding agent.

(Method)
(1) Preparation of Conjugates of Linear Multiubiquitin and Peptide Ligand Targeted peptide degradation inducers Ub ₃ -B6 and Ub ₄ -B6 of the present invention were prepared using peptide sequences as binding factors.
In the linear multi-ubiquitin, mutant ubiquitin (UbR) having two mutations of R72A and R74T in the amino acid sequence shown in SEQ ID NO: 1 has a linker peptide consisting of 6 amino acid residues (GSGGGG) shown in SEQ ID NO: 9. UbR ₃ or UbR ₄ in which 3 or 4 are linked in a straight chain via is used.
The ligand peptide, which is a binding factor, is a selective binding peptide to Mcl-1, which is an anti-apoptotic protein, and B6 peptide (Journal of Biological Chemistry, 2009, 284 , 31315-31326.) was used.
UbR ₃ -B6 or UbR ₄ -B6, which is the target peptide-type target peptide degradation inducer of interest, has a structure in which UbR ₃ or UbR ₄ is linked to the peptide ligand B6 via the linker peptide. UbR ₃ -B6 or UbR ₄ -B6 was prepared by introducing an expression vector in which a gene encoding each full-length peptide was introduced into a pET15b plasmid into Escherichia coli and expressing it.

<Example 12: Target Peptide Degradation Assay Using Peptide-Type Target Peptide Degradation Inducing Agent>
(the purpose)
Degradation induction of the target peptide (Mcl-1) by the peptide-type target peptide degradation inducers (UbR ₃ -B6 and UbR ₄ -B6) prepared in Example 11 is verified.

(Method)
Development and validation of targeted peptide degradation inducers using peptide sequences as binding factors.
2 μM UbR3-B6 and UbR4-B6, 0.25 μM recombinant Mcl-1 (Novus Biologicals) and Tris-HCl buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl ₂ , 5 mM DTT, 2 mM ATP , 10% Glycerol) and incubated for 30 minutes. Then, it was mixed with 26S human proteasome (LifeSensors) at a final concentration of 0.1 mg/mL and allowed to react for 2 hours. Each sample was subjected to size fractionation by SDS-PAGE, and then analyzed by Western blotting using an anti-Mcl-1 antibody (Cell Signaling Technology) as a primary antibody.

(result)
The results are shown in FIG. When UbR ₃ -B6 and UbR ₄ -B6 were not added to the control (lane 1), the remaining amount of Mcl-1 was assumed to be 100% when UbR ₃ -B6 was added (lane 4) or when UbR ₄ -B6 was added The residual Mcl-1 levels in case (lane 2) were 56% and 9%, respectively. This indicates that UbR ₃ -B6 and UbR ₄ -B6 function as Ub-PSM system-dependent Mcl-1 degradation inducers, and that UbR ₄ in particular can function as a particularly strong degradation-inducing signal. Suggest.
From the above results, it was demonstrated that the target peptide degradation-inducing agent of the present invention can induce degradation of the target peptide even when a peptide is used as the binding factor.
All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (20)

A target peptide degradation inducer linked to the C-terminus of two or more ubiquitins linked to one or more binding factors that bind to the target peptide. The target peptide degradation inducer according to claim 1, wherein said ubiquitin consists of any one of the amino acid sequences described in (a) to (c) below.
(a) the amino acid sequence shown in SEQ ID NO: 1,
(b) an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 1, or (c) having 90% or more amino acid identity with the amino acid sequence shown in SEQ ID NO: 1 amino acid sequence The target peptide degradation-inducing agent according to claim 2, wherein the ubiquitin comprises an amino acid sequence containing any one of the following amino acid substitutions described in (1) to (3) in the amino acid sequence shown in SEQ ID NO:1.
(1) an amino acid sequence in which arginine residues at positions 72 and 74 are substituted with alanine residues and threonine residues, respectively;
(2) an amino acid sequence in which the arginine residues at positions 72 and 74 are substituted with proline residues and threonine residues, respectively; or (3) an amino acid sequence in which the leucine residue at position 73 is substituted with a proline residue The target peptide degradation-inducing agent according to any one of claims 1 to 3, wherein the binding factor is a nucleic acid. The target peptide degradation-inducing agent according to claim 4, wherein the nucleic acid is a nucleic acid aptamer or a nucleic acid-binding domain-recognizing nucleic acid. The target peptide degradation inducer according to any one of claims 1 to 3, wherein the binding factor is a peptide. The target peptide degradation-inducing agent according to claim 6, wherein the peptide is selected from the group consisting of ligands, receptors, ligand-binding sites thereof, antibodies, and active fragments thereof. The target peptide degradation-inducing agent according to any one of claims 1 to 3, wherein the binding factor is a low-molecular-weight compound. The claim, wherein the ubiquitin and the binding agent are linked via a bond via a thiol group introduced by substitution or addition to the C-terminus of ubiquitin, a bond via an azide-alkyne cycloaddition reaction, or via an oxime/hydrazone bond. The target peptide degradation inducer according to any one of 1 to 8. The target peptide degradation-inducing agent according to any one of claims 1 to 9, wherein the binding factor and the ubiquitin are linked via a linker factor. the group consisting of a transcription factor, amyloid, tau protein, α-synuclein, caspase, denatured huntingtin, pseudokinase, K-RAS mutant, Atg factor, mTOR complex 1, GAPR-1 and orphan receptor The target peptide degradation inducer according to any one of claims 1 to 10, which is selected from The target peptide degradation inducer according to claim 11, wherein the transcription factor is NF-κB. A polynucleotide encoding the target peptide degradation inducer according to claim 5 or 6. An expression vector containing the polynucleotide according to claim 13 in an expressible state. A nucleic acid-type target peptide degradation inducer comprising the polynucleotide according to claim 13 or the expression vector according to claim 14. Any two or more selected from the group consisting of the target peptide degradation inducer according to any one of claims 1 to 12 and/or the nucleic acid-type target peptide degradation inducer according to claim 15. A target peptide degradation-inducing composition. A Huntington's disease inhibitor containing the target peptide degradation inducer according to claim 11, wherein the target peptide is denatured huntingtin. The autophagy inhibitor containing the target peptide degradation inducer according to claim 11, wherein the target peptide is an Atg factor. An autophagy promoter comprising the target peptide degradation inducer according to claim 11, wherein the target peptide is mTOR complex 1 or GAPR-1. An anticancer agent comprising the target peptide degradation inducer according to claim 12.

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