Novel polypeptides and their uses

Abstract

Provided is a novel polypeptide. A polypeptide according to one embodiment of the present invention is a polypeptide comprising an amino acid sequence shown by SEQ ID NO: 1 or 3, or is a variant thereof. A polypeptide according to another embodiment of the present invention is a polypeptide comprising an amino acid sequence shown by SEQ ID NO: 9 or 11, or is a variant thereof.

Description

[Technical Field]

[0001] This invention relates to a novel polypeptide and its use, particularly to applications such as anticancer drugs. [Background technology]

[0002] In recent years, in addition to the promotion of cell proliferation and the inhibition of cell differentiation, the restructuring of the metabolic environment has attracted attention as a factor in the development of cancer. Cancer cells possess unique metabolic mechanisms that enable rapid cell proliferation even under hypoxic and nutrient-poor conditions. For example, ATP production via anaerobic glycolysis, production of biomolecular precursors (nucleic acids and proteins), production of oncometabolites through gene mutations, and acquisition of anti-apoptotic properties through oxidative stress control are known. The development of groundbreaking cancer therapies targeting such cancer-specific metabolic abnormalities is highly anticipated.

[0003] Non-Patent Document 1 reports that the ubiquitin ligase COP1 forms a complex with Tribbles (Trib1, Trib2, and Trib3), which are adapter proteins overexpressed in acute myeloid leukemia (AML). Of these, the COP1-Trib1 and COP1-Trib2 complexes promote the degradation of C / EBPα, thereby inducing the proliferation and inhibition of differentiation of myeloid cells, and contributing to the development of AML (Non-Patent Document 1).

[0004] According to Non-Patent Document 2, the COP1-Trib3 complex binds to acetyl-CoA carboxylase 1 (ACC1), a fatty acid metabolic factor, and induces COP1-mediated ubiquitination and proteolysis. This suppresses intracellular fatty acid synthesis and causes metabolic abnormalities. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Yoshida A, Kato JY, Nakamae I & Yoneda-Kato N (2013) COP1 targets C / EBP alpha for degradation and induces acute myeloid leukemia via Trib1. Blood 122(10):1750-1760 [Non-Patent Document 2] Qi L, et al. (2006) TRB3 links the E3 ubiquitin ligase COP1 to lipid metabolism. Science 312(5781):1763-1766. [Overview of the project] [Problems that the invention aims to solve]

[0006] The involvement of the COP1-Tribbles complex in cancer development still requires further investigation. In particular, the inventors believe that the relationship between the COP1-Tribbles complex and acetyl-CoA carboxylase (ACC) in cancer development holds new potential applications.

[0007] One aspect of the present invention aims to provide a novel polypeptide. Another aspect of the present invention aims to provide an inhibitor of acetyl-CoA degradation by COP1-Tribbles or an anticancer agent. [Means for solving the problem]

[0008] The present invention includes the following configuration.

[0009] [1] polypeptides listed in any of the following: (a1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 3; (a2) A polypeptide having an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted and / or added from the amino acid sequence shown in SEQ ID NO: 1 or 3, and which has binding activity to the COP1-Trib1 complex; (a3) A polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 1 or 3, and that has binding activity to the COP1-Trib1 complex; (a4) A polypeptide encoded by the nucleotide sequence shown in Sequence ID No. 2 or 4; (a5) A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the base sequence shown in SEQ ID NO: 2 or 4, and which has binding activity to the COP1-Trib1 complex; (a6) A polypeptide encoded by a polynucleotide having 90% identity with the base sequence shown in SEQ ID NO: 2 or 4, and having binding activity to the COP1-Trib1 complex; (a7) A polypeptide that encodes a nucleotide sequence in which one or more nucleotides are deleted, substituted or added in the nucleotide sequence shown in Sequence ID No. 2 or 4, and which has binding activity to the COP1-Trib1 complex.

[0010] [2] polypeptides listed in any of the following: (b1) A polypeptide comprising an amino acid sequence in which one or more amino acid residues selected from the group consisting of positions 216, 217, and 220 in the amino acid sequence shown in Sequence ID No. 5 are substituted or deleted, and which has acetyl-CoA carboxylation activity and does not have binding activity to the COP1-Trib1 complex; (b2) A polypeptide of the above (b1) in which one or more amino acid residues are substituted, deleted, inserted and / or added, which has acetyl-CoA carboxylation activity and does not have binding activity to the COP1-Trib1 complex; (b3) A polypeptide having 90% or more identity to the polypeptide of (b1), having the carboxylation activity of acetyl-CoA, and not having the binding activity to the COP1-Trib1 complex; (b4) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9.

[0011] [3] A polypeptide according to any of the following: (c1) A polypeptide consisting of an amino acid sequence in which one or more amino acid residues selected from the group consisting of the 358th, 359th, and 362nd positions in the amino acid sequence shown in SEQ ID NO: 7 are substituted or deleted, having the carboxylation activity of acetyl-CoA, and not having the binding activity to the COP1-Trib1 complex; (c2) A polypeptide in which one or several amino acid residues are substituted, deleted, inserted and / or added in the polypeptide of (c1), having the carboxylation activity of acetyl-CoA, and not having the binding activity to the COP1-Trib1 complex; (c3) A polypeptide having 90% or more identity to the polypeptide of (c1), having the carboxylation activity of acetyl-CoA, and not having the binding activity to the COP1-Trib1 complex; (c4) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11.

[0012] [4] A polynucleotide according to any of the following: (d1) A polynucleotide encoding the polypeptide according to any of [1] to [3]; (d2) A polynucleotide consisting of the base sequence shown in SEQ ID NO: 2, 4, 10 or 12; (d3) A polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 2 or 4, and encodes a polypeptide having the binding activity to the COP1-Trib1 complex; (d4) A polynucleotide having 90% identity with the base sequence shown in SEQ ID NO: 2 or 4, and encoding a polypeptide having binding activity to the COP1-Trib1 complex; (d5) A polynucleotide encoding a polypeptide having binding activity to the COP1-Trib1 complex, consisting of a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence shown in Sequence ID No. 2 or 4; (d6) A polynucleotide that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the base sequence shown in SEQ ID NO: 10 or 12, and which encodes a polypeptide having acetyl-CoA carboxylation activity and not binding activity to the COP1-Trib1 complex; (d7) A polynucleotide having 90% identity with the base sequence shown in SEQ ID NO: 10 or 12, and encoding a polypeptide that has acetyl-CoA carboxylation activity and does not have binding activity to the COP1-Trib1 complex; (d8) A polynucleotide encoding a polypeptide having binding activity to the COP1-Trib1 complex, comprising a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 10 or 12.

[0013] [5] An expression vector containing the polynucleotide described in [4].

[0014] [6] Inhibitors of the degradation of acetyl-CoA carboxylase by the COP1-Tribbles complex, comprising at least one selected from the group consisting of (i) to (iii) below as an active ingredient: (i) polypeptides as described in [1]; (ii) A polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4, or a polynucleotide shown in any of (d3) to (d5); (iii) An expression vector containing the polynucleotide described in (ii) above.

[0015] [7] An anticancer agent comprising, as an active ingredient, at least one selected from the group consisting of a polypeptide described in any of [1] to [3], a polynucleotide described in [4], and an expression vector described in [5].

[0016] [8] A method for screening inhibitors of acetyl-CoA carboxylase degradation activity by the COP1-Tribbles complex or anticancer agents, comprising either step (i) or (ii) below: (i) A step of evaluating the ability of the test substance to inhibit the binding of the polypeptide described in [1] to one or more selected from the group consisting of the COP1-Trib1 complex, COP1-Trib2, and COP1-Trib3; (ii) A step of evaluating the binding ability of the test substance to the polypeptide described in [1].

[0017] [9] A kit for carrying out the screening method of [8], comprising at least one selected from the group consisting of (i) to (iii) below: (i) polypeptides as described in [1]; (ii) A polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4, or a polynucleotide shown in any of (d3) to (d5); (iii) An expression vector containing the polynucleotide described in (ii) above. [Effects of the Invention]

[0018] According to one aspect of the present invention, a novel polypeptide is provided. According to another aspect of the present invention, an inhibitor of acetyl-CoA degradation by COP1-Tribbles or an anticancer agent is provided. [Brief explanation of the drawing]

[0019] [Figure 1](Left) This figure shows the binding activity between the polypeptide (ACC1 75aa) shown in Sequence ID No. 1 and Trib1. (Right) This figure shows the binding activity between the polypeptide (ACC1 delmut1) obtained by removing the sequence shown in Sequence ID No. 1 from ACC1 and Tribbles. [Figure 2] (Left) A diagram showing the schematic structures of two ACC1 point mutants (ACC1 Helix1mut and ACC1 Helix2mut). (Right) A diagram showing the binding activity of the two ACC1 point mutants to Tribbles. [Figure 3] This figure compares the resistance of ACC1 delmut1 and ACC1 Helix1mut to degradation by COP1-Trib1 with wild-type ACC1 and other ACC1 mutants. [Figure 4] This figure shows that cell proliferation induced by the COP1-Trib1 complex is suppressed by ACC1 Helix1mut. [Figure 5] (Left) This figure shows that the expression level of ACC1 protein increases in cells expressing ACC1 Helix1mut. (Right) This figure shows that the expression level of ACC1 RNA does not change in cells expressing ACC1 Helix1mut. [Figure 6] This figure shows that ROS levels are elevated in cells expressing ACC1 Helix1mut. [Figure 7] This figure shows that NADP+ / NADPH levels are elevated in cells expressing ACC1 Helix1mut. [Figure 8] This figure illustrates the delaying effect of ACC1 Helix1mut on the onset of AML. [Figure 9] This diagram illustrates the effect of ACC1 Helix1mut on the differentiation of AML cells. [Figure 10] This diagram illustrates that ACC1 Helix1mut promotes cell differentiation and increases neutrophils. [Figure 11]This figure compares the expression levels of Mac-1 and Gr-1, granulocyte differentiation markers, as determined by flow cytometry. [Figure 12] This diagram illustrates how ACC1 Helix1mut depletes AML stem cells. [Figure 13] This figure illustrates that cultured AML stem cells differentiate into neutrophils using ACC1 Helix1mut. [Figure 14] This figure shows that ACC1 Helix1mut suppresses the proliferation and promotes the differentiation of AML stem cells in early-phase mice. [Figure 15] This figure shows that ACC1 Helix1mut suppresses the development of leukemia caused by AML stem cells in mice that have undergone re-transplantation of AML stem cells. [Figure 16] This figure shows that ACC1 Helix1mut differentiates AML stem cells into neutrophils in mice that have undergone re-transplantation of AML stem cells. [Figure 17] (Top) A diagram comparing the amino acid sequences of the Trib1 binding region of ACC1 in various organisms. (Bottom) A diagram comparing the amino acid sequences of the Trib1 binding region of ACC2 in various organisms with the amino acid sequence of the Trib1 binding region of human ACC1. [Figure 18] This figure shows the binding activity of a polypeptide obtained by removing the sequence shown in Sequence ID No. 3 from ACC2 (ACC2 Δ346-423) or a point mutant of ACC2 (Acc2 Helix1mut) to Tribbles. [Figure 19] This figure illustrates the effects of ACC2 Δ346-423 and Acc2 Helix1mut on cell proliferation. [Figure 20] This figure shows that AML patients have a reduced expression level of ACC1 mRNA compared to healthy donors. [Figure 21] This figure shows that the expression levels of ACC1 protein and mRNA are reduced in AML cell lines. [Figure 22]This figure shows that ACC1 Helix1mut suppresses the proliferation of MLL-AF9-induced AML model cells. [Figure 23] This figure shows that ACC1 Helix1mut suppresses the proliferation of CML model cells induced by BCR-ABL. [Figure 24] This figure shows that while endogenous ACC1 expression is reduced in cells expressing Trib1-COP1, MLL-AF9, or BCR-ABL, co-expression of ACC1 Helix1mut leads to stable expression of the ACC1 Helix1mut protein. [Figure 25] This figure shows the effects of ACC1 Helix1mut on ROS levels, NADP+ / NADPH ratio, and GSH / GSSG ratio in cells expressing Trib1-COP1, MLL-AF9, or BCR-ABL. [Figure 26] This figure shows that ACC1 Helix1mut extends the survival of MLL-AF9-induced AML model mice. [Figure 27] This figure shows that ACC2 Helix1mut suppresses the proliferation of cells expressing COP1-Trib1. [Figure 28] This figure shows that ACC2 Helix1mut does not affect ROS levels in cells into which COP1-Trib1 has been introduced. [Figure 29] This figure shows that ACC2 Helix1mut does not affect ROS levels in cells introduced with COP1-Trib1, regardless of whether or not they are treated with N-acetylcysteine. [Figure 30] This figure shows that ACC2 Helix1mut increases the NADP+ / NADPH ratio in cells into which COP1-Trib1 has been introduced. [Figure 31] The diagram shows that ACC2 Helix1mut reduces mitochondrial oxygen consumption (OCR). [Figure 32]This diagram illustrates how ACC2 Helix1mut reduces the maximum mitochondrial respiration rate. [Figure 33] This figure shows the increase or decrease in ACC1 and ACC2 mRNA expression levels in various solid tumors. [Modes for carrying out the invention]

[0020] One embodiment of the present invention is described below, but the present invention is not limited thereto. The present invention is not limited to the configurations described below, and various modifications are possible within the scope of the claims, and embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention. All documents mentioned herein are incorporated herein by reference. In this specification, when a numerical range is described as "A to B", such description is intended to mean "A or greater and B or less".

[0021] This specification describes polynucleotides based on the case where the polynucleotide is DNA. If the polynucleotide is RNA, simply replace "T (thymine)" with "U (uracil)" in the following description.

[0022] [0. Discovery underlying the present invention] This invention is based on several findings newly discovered by the inventors through their research. While studying changes in lipid metabolism in cancer cells, the inventors identified that acetyl-CoA carboxylase 1 (ACC1) is a substrate of the COP1-Trib1 complex. Next, the inventors investigated which regions of ACC1 contribute to binding to and degrading the COP1-Trib1 complex.

[0023] As a result, we found that the polypeptide corresponding to positions 201 to 275 of the entire amino acid sequence of ACC1 (SEQ ID NO: 5) (SEQ ID NO: 1) is the binding region to the COP1-Trib1 complex. Furthermore, we found that among the two α-helix regions (Helix1 and Helix2) within the aforementioned binding region, three amino acid residues in the Helix1 region (proline at position 216, lysine at position 217, and glutamic acid at position 220 in the entire amino acid sequence of ACC1) particularly contribute to binding to the COP1-Trib1 complex.

[0024] Furthermore, the inventors identified the binding region of acetyl-CoA carboxylase 2 (ACC2), a member of the ACC1 family, with COP1-Trib1. This binding region is the polypeptide located at positions 343 to 417 of the entire amino acid sequence of ACC2 (SEQ ID NO: 7). Among the two helix regions (Helix1 and Helix2) present in this binding region, similar to ACC1, the inventors found that three amino acid residues in the Helix1 region (proline at position 358, lysine at position 359, and glutamic acid at position 362 of the entire amino acid sequence of ACC2) particularly contribute to binding to the COP1-Trib1 complex. In the following, when simply referred to as ACC, both ACC1 and ACC2 are intended.

[0025] One aspect of the present invention provides a polypeptide that forms a binding domain to the COP1-Tribbles complex of ACC (COP1-Trib1 complex, COP1-Trib2 complex, or COP1-Trib3 complex). This polypeptide can competitively bind to COP1-Tribbles against ACC. Therefore, this polypeptide can be an inhibitor of ACC degradation by COP1-Tribbles. Furthermore, this polypeptide can be used for screening substances that inhibit ACC degradation by COP1-Tribbles.

[0026] Another aspect of the present invention provides ACC mutants in which amino acids that particularly contribute to the binding of ACC to the COP1-Tribbles complex are substituted or deleted. These ACC mutants have the function of carboxylating acetyl-CoA while exhibiting resistance to degradation by COP1-Tribbles.

[0027] Furthermore, the inventors have also found that the above-mentioned polypeptides and ACC variants have anticancer effects (particularly against leukemias such as acute myeloid leukemia) and / or therapeutic effects. Yet another aspect of the present invention provides a novel anticancer agent.

[0028] [1. Binding domain of ACC to the COP1-Tribbles complex] A polypeptide according to one embodiment of the present invention forms a binding domain between the COP1-Trib1 complex and acetyl-CoA carboxylase (ACC1 or ACC2), and is a polypeptide having binding activity to the COP1-Trib1 complex.

[0029] More specifically, a polypeptide according to one embodiment of the present invention is a polypeptide described in any of the following: (a1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 3. (a2) A polypeptide having amino acid sequences in which one or more amino acid residues are substituted, deleted, inserted and / or added from the amino acid sequence shown in SEQ ID NO: 1 or 3, and which has binding activity to the COP1-Trib1 complex. (a3) A polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 1 or 3, and that has binding activity to the COP1-Trib1 complex. (a4) A polypeptide encoded by the nucleotide sequence shown in Sequence ID No. 2 or 4. (a5) A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the base sequence shown in SEQ ID NO: 2 or 4, and which has binding activity to the COP1-Trib1 complex. (a6) A polypeptide encoded by a polynucleotide having 90% identity with the base sequence shown in SEQ ID NO: 2 or 4, and having binding activity to the COP1-Trib1 complex. (a7) A polypeptide that encodes a nucleotide sequence in which one or more nucleotides are deleted, substituted or added in the nucleotide sequence shown in Sequence ID No. 2 or 4, and which has binding activity to the COP1-Trib1 complex.

[0030] (a1) is a polypeptide that forms the binding domain between ACC and the COP1-Trib1 complex, as identified by the inventors. (a2) and (a3) ​​represent variants of (a1). (a4) is a polypeptide that forms the binding domain between ACC and the COP1-Trib1 complex, as identified by the corresponding nucleotide sequence. (a5) to (a7) are polypeptides that form the binding domain between ACC and the COP1-Trib1 complex, as identified by the nucleotide sequences of variants of (a4).

[0031] In one embodiment, polypeptides (a2), (a3), (a5)-(a7) have one or more (one, two, or three) amino acids conserved from those corresponding to positions 16, 17, and 20 in SEQ ID NO: 1. In one embodiment, polypeptides (a2), (a3), (a5)-(a7) have one or more (one, two, or three) amino acids conserved from those corresponding to positions 16, 17, and 20 in SEQ ID NO: 3.

[0032] Regarding (a1), Sequence ID 1 corresponds to the binding domain between ACC1 and the COP1-Trib1 complex. Sequence ID 3 corresponds to the binding domain between ACC2 and the COP1-Trib1 complex.

[0033] With respect to (a2), the upper limit of the number of amino acid residues that may be substituted, deleted, inserted, and / or added is not particularly limited, as long as the polypeptide of (a2) has binding activity to the COP1-Trib1 complex. The upper limit may be, for example, 7, 6, 5, 4, 3, 2, or 1.

[0034] With respect to (a3), the amino acid sequence identity is, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0035] The identity of amino acid sequences can be determined, for example, using BLASTX. In this case, parameters such as score=50 and wordlength=3 can be used. Alternatively, the amino acid sequence can be analyzed using the Gapped BLAST program. In this case, default parameters can be used. Additions or deletions (such as gaps) may be allowed to optimally align the amino acid sequences being compared.

[0036] Regarding (a4), Sequence ID 2 is the nucleotide sequence corresponding to the binding domain between ACC1 and the COP1-Tribles complex. Sequence ID 4 is the nucleotide sequence corresponding to the binding domain between ACC2 and the COP1-Tribles complex.

[0037] Regarding (a5), "hybridizing under stringent conditions" means performing hybridization in a 6×SSC hybridization solution at 50-60°C for 16 hours, washing in a 0.1×SSC solution, and then hybridizing again. Here, the composition of 1×SSC is sodium chloride: 150 mM, sodium citrate: 15 mM.

[0038] Regarding (a6), the identity of the base sequence is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0039] Sequence identity of nucleotide sequences can be determined, for example, using BLASTN. When analyzing nucleotide sequences with BLASTN, parameters such as score=100 and wordlength=12 can be used. Specific methods for these analyses are well known. Additions or deletions may be permitted to optimally align the nucleotide sequences to be compared.

[0040] With respect to (a7), there is no particular upper limit on the number of base substitutions, deletions, insertions and / or additions. The upper limit may be 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0041] With respect to (a2), (a3), and (a5)-(a7), whether or not the polypeptide has binding activity to the COP1-Trib1 complex can be determined, for example, by the assay described in the examples below. In one embodiment, the binding activity of the polypeptides (a2), (a3), and (a5)-(a7) to the COP1-Trib1 complex is 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the binding activity to wild-type ACC1 or wild-type ACC2.

[0042] In one embodiment, the polypeptides (a1) to (a7) have binding activity to the COP1-Trib2 complex. In one embodiment, the polypeptides (a1) to (a7) have binding activity to the COP1-Trib3 complex. The binding activity of the polypeptides (a1) to (a7) to the COP1-Trib2 complex or the COP1-Trib3 complex is 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the binding activity to wild-type ACC1 or wild-type ACC2.

[0043] Polypeptides (a1) to (a7) can be produced by genetic engineering techniques using the polynucleotides shown in SEQ ID NOs. Alternatively, polypeptides (a1) to (a7) can be produced by common protein chemical synthesis methods (such as liquid-phase or solid-phase methods).

[0044] [2. ACC1 variant] A polypeptide according to one embodiment of the present invention is a variant of ACC1. This variant has acetyl-CoA carboxylation activity and does not have binding activity to the COP1-Trib1 complex.

[0045] More specifically, a polypeptide according to one embodiment of the present invention is a polypeptide described in any of the following: (b1) A polypeptide comprising an amino acid sequence in which one or more amino acid residues selected from the group consisting of positions 216, 217, and 220 in the amino acid sequence shown in Sequence ID No. 5 are substituted or deleted, and which has acetyl-CoA carboxylation activity and does not have binding activity to the COP1-Trib1 complex. (b2) A polypeptide of the above (b1) wherein one or more amino acid residues are substituted, deleted, inserted and / or added, and which has acetyl-CoA carboxylation activity and does not have binding activity to the COP1-Trib1 complex. (b3) A polypeptide that has 90% or more identity with the polypeptide of (b1) above, has carboxylation activity of acetyl-CoA, and does not have binding activity to the COP1-Trib1 complex. (b4) A polypeptide consisting of the amino acid sequence shown in Sequence ID No. 9.

[0046] (b1) is a polypeptide in which amino acid residues (proline at position 216, lysine at position 217, and glutamic acid at position 220) that significantly contribute to the binding activity with the COP1-Trib1 complex in the amino acid sequence of wild-type ACC1 are substituted or deleted. In one embodiment, the polypeptide of (b1) has two or more amino acid residues selected from the group consisting of positions 216, 217, and 220 substituted or deleted. In one embodiment, the polypeptide of (b1) has amino acid residues at positions 216, 217, and 220 substituted or deleted.

[0047] When substituting amino acid residues, the substituted amino acid residue is not particularly limited. From the viewpoint of more strongly suppressing the binding activity with the COP1-Trib1 complex, it is preferable to substitute with an uncharged amino acid residue or an amino acid residue with a different charge.

[0048] Regarding (b2), there is no particular upper limit on the number of polypeptide substitutions, deletions, insertions and / or additions. The upper limit could be, for example, 230, 210, 190, 170, 150, 130, 110, 100, 90, 80, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0049] Regarding (b3), the identity of the amino acid sequence is as explained in Section [1].

[0050] (b4) is the ACC1 Helix1mut mutant prepared in the example described later.

[0051] With respect to (b1) to (b3), whether or not the polypeptide has binding activity to the COP1-Trib1 complex can be determined, for example, by the assay described in the examples below. In one embodiment, the binding activity of the polypeptides (b1) to (b3) to the COP1-Trib1 complex is less than 40%, 30% or less, 20% or less, or 10% or less of the binding activity to wild-type ACC1 or wild-type ACC2.

[0052] With respect to (b1) to (b3), "acetyl-CoA carboxylation activity" means the activity that converts acetyl-CoA to malonyl-CoA. Acetyl-CoA carboxylation activity can be measured by known methods. For example, a commercially available Acetyl-Coenzyme A Assay kit can be used. In one embodiment, the acetyl-CoA carboxylation activity of the polypeptides (b1) to (b3) is 80% or more, 85% or more, 90% or more, or 95% or more of the activity of wild-type ACC1 or wild-type ACC2.

[0053] The acetyl-CoA carboxylation site of ACC1 is known to be located at positions 279 to 2217. Therefore, it is thought that the activity to carboxylate acetyl-CoA will not be lost even if one or more amino acid residues selected from the group consisting of positions 216, 217, and 220 are substituted or deleted.

[0054] The activity of (b1) to (b3) in carboxylating acetyl-CoA can also be confirmed by expressing (b1) to (b3) in ACC1 knockout cells. Cell proliferation is inhibited in ACC1 knockout cells. Therefore, if cell proliferation is not inhibited when (b1) to (b3) are expressed in ACC1 knockout cells, it can be concluded that (b1) to (b3) have the activity of carboxylating acetyl-CoA. In the case of the ACC1 Helix1mut mutant represented by (b4), cell proliferation is not suppressed even when this mutant is expressed in ACC1 knockout cells.

[0055] Polypeptides (b1) to (b4) can be produced by genetic engineering techniques using the polynucleotide shown in Sequence ID No. 10. Alternatively, polypeptides (b1) to (b4) can also be produced by general protein chemical synthesis methods (such as liquid-phase or solid-phase methods).

[0056] [3. ACC2 mutant] A polypeptide according to one embodiment of the present invention is a variant of ACC2. This variant has acetyl-CoA carboxylation activity and does not have binding activity to the COP1-Trib1 complex.

[0057] More specifically, a polypeptide according to one embodiment of the present invention is a polypeptide described in any of the following: (c1) A polypeptide comprising an amino acid sequence in which one or more amino acid residues selected from the group consisting of positions 358, 359, and 362 in the amino acid sequence shown in Sequence ID No. 7 are substituted or deleted, having acetyl-CoA carboxylation activity and not binding activity to the COP1-Trib1 complex. (c2) A polypeptide of the above (c1) wherein one or more amino acid residues are substituted, deleted, inserted and / or added, and which has acetyl-CoA carboxylation activity and does not have binding activity to the COP1-Trib1 complex. (c3) A polypeptide that has 90% or more identity with the polypeptide of (c1) above, has carboxylation activity of acetyl-CoA, and does not have binding activity to the COP1-Trib1 complex. (c4) A polypeptide consisting of the amino acid sequence shown in Sequence ID No. 11.

[0058] (c1) is a polypeptide obtained by substituting or deleting amino acid residues (proline at position 358, lysine at position 359, and glutamic acid at position 362) from the amino acid sequence of wild-type ACC2 that contribute significantly to the binding activity with the COP1-Trib1 complex. In one embodiment, polypeptide (b1) has two or more amino acid residues selected from the group consisting of positions 358, 359, and 362 substituted or deleted. In one embodiment, polypeptide (b1) has amino acid residues at positions 358, 359, and 362 substituted or deleted. When amino acid residues are substituted, preferred substituted amino acid residues are as described in Section [2].

[0059] With respect to (c2), there is no particular upper limit on the number of amino acid residues that may be substituted, deleted, inserted and / or added. The upper limit may be, for example, 240, 220, 200, 180, 160, 140, 120, 100, 90, 80, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0060] Regarding (c3), the amino acid sequence identity is as explained in Section [1]. Regarding (c1) to (c3), the statement that "the polypeptide does not have binding activity to the COP1-Trib1 complex" is as explained in Section [2]. Regarding (c1) to (c3), the statement that "acetyl-CoA carboxylation activity" is as explained in Section [2].

[0061] (c4) is the ACC2 Helix1mut mutant prepared in the example described later.

[0062] The acetyl-CoA carboxylation site of ACC2 is known to be located at positions 417 to 2483. Therefore, it is thought that the activity to carboxylate acetyl-CoA will not be lost even if one or more amino acid residues selected from the group consisting of positions 358, 359, and 362 are substituted or deleted.

[0063] The activity of (c1) to (c3) in carboxylating acetyl-CoA can also be confirmed by expressing (c1) to (c3) in ACC2 knockout cells. Cell proliferation is inhibited in ACC2 knockout cells. Therefore, if cell proliferation is not inhibited when (c1) to (c3) are expressed in ACC2 knockout cells, it can be concluded that (c1) to (c3) have the activity of carboxylating acetyl-CoA. In the case of the ACC2 Helix1mut mutant represented by (c4), cell proliferation is not suppressed even when this mutant is expressed in ACC2 knockout cells.

[0064] Polypeptides (c1) to (c4) can be produced by genetic engineering using the polynucleotide shown in Sequence ID No. 12. Alternatively, polypeptides (b1) to (b4) can be produced by general protein chemical synthesis methods (such as liquid-phase or solid-phase methods).

[0065] [4. Polynucleotides] A polynucleotide according to one embodiment of the present invention is a polynucleotide capable of encoding the polypeptide described in Sections [1] to [3].

[0066] More specifically, a polynucleotide according to one embodiment of the present invention is a polynucleotide described in any of the following: (d1) A polynucleotide encoding the polypeptide according to any one of claims 1 to 3. (d2) A polynucleotide consisting of the base sequence shown in SEQ ID NOs: 2, 4, 10, or 12. (d3) A polynucleotide that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the base sequence shown in SEQ ID NO: 2 or 4, and which encodes a polypeptide having binding activity to the COP1-Trib1 complex. (d4) A polynucleotide having 90% identity with the base sequence shown in SEQ ID NO: 2 or 4, and encoding a polypeptide having binding activity to the COP1-Trib1 complex. (d5) A polynucleotide encoding a polypeptide having binding activity to the COP1-Trib1 complex, consisting of a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence shown in Sequence ID No. 2 or 4. (d6) A polynucleotide that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the base sequence shown in SEQ ID NO: 10 or 12, and which encodes a polypeptide having acetyl-CoA carboxylation activity and not binding activity to the COP1-Trib1 complex. (d7) A polynucleotide having 90% identity with the base sequence shown in SEQ ID NO: 10 or 12, and encoding a polypeptide that has acetyl-CoA carboxylation activity and does not have binding activity to the COP1-Trib1 complex. (d8) A polynucleotide encoding a polypeptide consisting of a sequence in which one or more bases are deleted, substituted, or added in the sequence shown in Sequence ID No. 10 or 12, and which has carboxylation activity of acetyl-CoA and does not have binding activity to the COP1-Trib1 complex.

[0067] (d1) is a polynucleotide corresponding to the polypeptide described in sections [1] to [3]. (d2) is a polynucleotide encoding the binding domain between ACC and the COP1-Trib1 complex or an ACC variant. (d3) to (d5) are variants of the polynucleotide encoding the binding domain between ACC and the COP1-Trib1 complex. (d6) to (d8) are variants of the polynucleotide encoding an ACC variant.

[0068] Regarding (d1) to (d8), Sequence ID No. 2 is the nucleotide sequence corresponding to the polypeptide that forms the binding domain between ACC1 and the COP1-Trib1 complex. Sequence ID No. 4 is the nucleotide sequence corresponding to the polypeptide that forms the binding domain between ACC2 and the COP1-Trib1 complex. Sequence ID No. 10 is the nucleotide sequence encoding the ACC1 mutant (ACC1 Helix1mut). Sequence ID No. 12 corresponds to the nucleotide sequence of the ACC2 mutant (ACC2 Helix1mut).

[0069] Regarding (d3) and (d6), the “stringent conditions” are as described in Section [1]. Regarding (d4) and (d7), the identity of the base sequence is as described in Section [1].

[0070] With respect to (d5) and (d8), there is no particular upper limit on the number of base substitutions, deletions, insertions and / or additions. The upper limit can be, for example, 730, 700, 650, 600, 550, 500, 450, 400, 350, 300, 280, 260, 240, 220, 200, 180, 160, 140, 120, 100, 90, 80, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0071] The polynucleotides (d1), (d3) to (d8) may be codon-optimized (for example, codon-optimized to match the codon usage frequency of humans). Codon optimization means that when there are multiple codons corresponding to one amino acid, the codon most frequently used in a particular organism (such as a human) is used. Preferably, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the open reading frame of the polynucleotides (d1), (d3) to (d8) are codon-optimized.

[0072] In one embodiment, the polynucleotides (d1) to (d8) are isolated. Isolated polynucleotides are those that have been separated from components that coexist in nature (other nucleic acids, proteins, etc.). In one embodiment, isolated polynucleotides may contain promoter sequences or terminator sequences of the polynucleotides (d1) to (d8) in nature. With respect to polynucleotides (cDNA molecules, etc.) produced by genetic engineering methods, "isolated polynucleotides" may mean substantially free from cellular components and culture medium. With respect to polynucleotides produced by chemical synthesis, "isolated polynucleotides" preferably mean substantially free from precursors (dNTPs, etc.) and chemicals used in the synthesis process.

[0073] The polynucleotides (d1) to (d8) can be synthesized by genetic engineering techniques based on the base sequences of SEQ ID NOs: 2, 4, 10, or 12. Alternatively, the polynucleotides (d1) to (d8) can be synthesized by chemical polynucleotide synthesis methods (e.g., the phosphoamidite method).

[0074] [5. Expression Vectors] An expression vector according to one embodiment of the present invention is an expression vector containing polynucleotides as described in Section [4].

[0075] The specific embodiments of the expression vector according to one embodiment of the present invention are not particularly limited. Specific examples of expression vectors include non-replicating viral vectors (retroviral vectors, adenovirus vectors, lentiviral vectors, adeno-associated virus vectors, Sendai virus vectors, etc.), reproducing viral vectors (oncolytic viral vectors, etc.), non-viral vectors (plasmids, liposomes containing naked DNA, etc.), and bacterial vectors.

[0076] An expression vector according to one embodiment of the present invention may contain polynucleotides having other functions. An example of such polynucleotides is a promoter sequence. By using a promoter sequence, it is possible to express polynucleotides at high levels or to express polynucleotides only under specific conditions.

[0077] [6. Inhibitors] An inhibitor according to one embodiment of the present invention is an inhibitor of the degrading action of acetyl-CoA carboxylase by the COP1-Tribbles complex. That is, it is an agent that inhibits the action of the COP1-Tribbles complex in which it degrades acetyl-CoA carboxylase. In one embodiment, the inhibitor is an inhibitor of the degrading action of acetyl-CoA carboxylase by the COP1-Trib1 complex. In one embodiment, the inhibitor is an inhibitor of the degrading action of acetyl-CoA carboxylase by the COP1-Trib2 complex. In one embodiment, the inhibitor is an inhibitor of the degrading action of acetyl-CoA carboxylase by the COP1-Trib3 complex.

[0078] An inhibitor according to one embodiment of the present invention contains at least one selected from the group consisting of (i) to (iii) below as an active ingredient. (i) Polypeptides represented by (a1) to (a7) as described in Section [1]. (ii) A polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4, or a polynucleotide shown in any of (d3) to (d5). (iii) An expression vector containing the polynucleotides of (ii).

[0079] The content of the active ingredients described in (i) to (iii) above in the inhibitor according to one embodiment of the present invention is not particularly limited. The lower limit of the content may be 0.001% by weight or more, 0.005% by weight or more, 0.01% by weight or more, 0.05% by weight or more, 0.1% by weight or more, 0.5% by weight or more, 1% by weight or more, or 5% by weight or more, when the entire inhibitor is considered to be 100% by weight. The upper limit of the content may be 100% by weight or less, 95% by weight or less, 90% by weight or less, 80% by weight or less, 70% by weight or less, 60% by weight or less, 50% by weight or less, 40% by weight or less, 30% by weight or less, 20% by weight or less, or 10% by weight or less, when the entire inhibitor is considered to be 100% by weight.

[0080] An inhibitor according to one embodiment of the present invention may contain pharmaceutically acceptable components other than the active ingredient. Examples of such components include buffers, pH adjusters, isotonic agents, preservatives, excipients, carriers, diluents, solvents, solubilizers, stabilizers, antioxidants, high molecular weight polymers, fillers, binders, surfactants, and stabilizers.

[0081] The route of administration of the inhibitor according to one embodiment of the present invention is not particularly limited. Examples of routes of administration include parenteral administration, intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, subcutaneous administration, intranasal administration, epidural administration, oral administration, sublingual administration, intranasal administration, intracerebral administration, vaginal administration, transdermal administration, rectal administration, inhalation, and local administration.

[0082] An inhibitor according to one embodiment of the present invention can normalize intracellular fatty acid metabolism by inhibiting the degradation of ACC1 and / or ACC2 by the COP1-Tribbles complex. Therefore, an inhibitor according to one embodiment of the present invention can be used as a treatment for lifestyle-related diseases (such as obesity) associated with abnormal fatty acid metabolism, or for screening candidate substances for such treatments.

[0083] [7. Anticancer drugs] An anticancer agent according to one embodiment of the present invention contains at least one selected from the group consisting of (i) to (iii) below as an active ingredient. (i) Polypeptides represented by (a1) to (a7) as described in Section [1]. (ii) A polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4, or a polynucleotide shown in any of (d3) to (d5). (iii) An expression vector containing the polynucleotides of (ii).

[0084] With respect to the anticancer drug according to one embodiment of the present invention, the content of the active ingredient, the components other than the active ingredient, and the route of administration are as described in Section [6].

[0085] The cancers to which the anticancer drug according to one embodiment of the present invention is administered are not particularly limited. Specific examples of cancers include leukemia (acute myeloid leukemia, chronic myeloid leukemia), rhabdomyosarcoma, breast cancer, lymphoma, squamous cell carcinoma (e.g., oral squamous cell carcinoma, laryngeal squamous cell carcinoma), ovarian cancer, melanoma, neuroblastoma, lung cancer (lung adenocarcinoma), pancreatic cancer, liver cancer (hepatocellular carcinoma), thyroid cancer (medullary thyroid carcinoma), lung cancer (non-small cell lung cancer), colorectal cancer, and gastric cancer. In particular, it can be suitably administered to leukemia (acute myeloid leukemia).

[0086] [8. Screening Methods and Screening Kits] One embodiment of the present invention provides a screening method for inhibitors of acetyl-CoA carboxylase degradation by COP1-Tribbles complexes (COP1-Trib1 complex, COP1-Trib2 complex, or COP1-Trib3 complex). Another embodiment of the present invention provides a screening method for anticancer drugs.

[0087] In one embodiment, this screening method screens for compounds that inhibit the binding of the COP1-Tribbles complex to the binding domain of ACC. In this embodiment, the extent to which the binding of polypeptides (a1) to (a7) to one or more complexes selected from the group consisting of the COP1-Trib1 complex, the COP1-Trib2 complex, and the COP1-Trib3 complex is inhibited in the presence of the test substance is evaluated. In one example, the extent to which the binding of polypeptides (a1) to (a7) to the COP1-Trib1 complex is inhibited in the presence of the test substance is evaluated.

[0088] Then, if the binding of polypeptides (a1) to (a7) to one or more complexes selected from the group consisting of COP1-Trib1, COP1-Trib2, and COP1-Trib3 complexes is below a predetermined value, the test substance is determined to be a candidate inhibitor or anticancer agent. In one example, if the binding of polypeptides (a1) to (a7) to the COP1-Trib1 complex is below a predetermined value, the test substance is determined to be a candidate inhibitor or anticancer agent. The predetermined value may be 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less of the binding ability in the absence of the test substance.

[0089] In one embodiment, this screening method screens compounds that bind to the binding domain of ACC with the COP1-Tribbles complex. Such compounds may compete with the COP1-Tribbles complex for binding to ACC. Therefore, such compounds are candidates for competitive inhibitors of ACC degradation by the COP1-Tribbles complex. In this embodiment, the binding activity of the test substance to polypeptides (a1) to (a7) is evaluated. If the binding activity of the test substance to polypeptides (a1) to (a7) is above a predetermined value, the test substance is determined to be a candidate for an inhibitor or anticancer agent. The predetermined value is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the binding activity between wild-type ACC and the COP1-Tribbles complex.

[0090] A screening kit according to one embodiment of the present invention is a kit for carrying out the above-described screening method. This screening kit includes at least one selected from the group consisting of (i) to (iii) below. (i) Polypeptides represented by (a1) to (a7) as described in Section [1]. (ii) A polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4, or a polynucleotide shown in any of (d3) to (d5). (iii) An expression vector containing the polynucleotides of (ii).

[0091] According to a screening kit of one embodiment of the present invention, inhibitors of acetyl-CoA carboxylase degradation by the COP1-Tribbles complex or anticancer drugs can be efficiently screened.

[0092] The above screening method or screening kit can be performed in vitro, ex vivo, or in vivo, and is not particularly limited. Known methods can be suitably used for specific screening procedures.

[0093] [9. Other] The present invention also includes the following configurations.

[0094] [1] A method for treating (and / or preventing) cancer, comprising the step of administering a polypeptide, polynucleotide and / or expression vector as described herein to a subject. Examples of subjects include humans, mammals and non-human mammals.

[0095] [2] A method for inhibiting the degradation of acetyl-CoA carboxylase by the COP1-Tribbles complex, comprising the step of administering a polypeptide, polynucleotide and / or expression vector described herein to a subject. Examples of subjects include humans, mammals, and non-human mammals.

[0096] [3] Use of polypeptides, polynucleotides and / or expression vectors as described herein to manufacture agents for treating (curing and / or preventing) cancer. Examples of subjects to whom such agents may be administered include humans, mammals and non-human mammals.

[0097] The present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Examples]

[0098] 〔material〕 Unless otherwise specified, the materials used in the examples are as follows:

[0099] [E. coli] E. coli DH5α:F-80dlacZ Δ M15, Δ (lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk-, mk+), phoA, supE44, λ-, thi-1, rgyA96, relA1 E.coli BL21:Lac, ara, gal, mtl, recA, uvr[pREP4, lacI, kan], E.coli, F-, dcm, omp, hsdS(rm-), gal.

[0100] [cell] HEK293T cell (human Embryonic Kidney 293T cell line).

[0101] [mouse] C57BL / 6 mice (CLEA Japan Inc.).

[0102] [vector] pGEM-T Vector (Promega Inc.) pGEX-6p-2 Vector (GE Healthcare Bioscience Inc.) pFLAG-CMV-2 Vector (Sigma) pCG-N-BL Vector (Provided by Professor Fujisawa, Kansai Medical University) pMSCV-IRES-GFP (provided by Dr. Owen Witte).

[0103] [Antibodies (Western blotting)] (primary antibody) Anti-h&mACC1 Rabbit IgG Antibody (Polyclonal Antibody, CST4190, Cell Signaling Technology) anti-FLAG clone M5 mouse IgG antibody (monoclonal antibody, clone M5, Sigma) anti-mCOP1 Rabbit IgG antibody (polyclonal antibody, prepared in the inventors' laboratory) anti-HA mouse IgG antibody (monoclonal antibody, clone 12CA5, Boehringer Monnheim) anti-hMLF1 mouse IgG antibody (monoclonal antibody, produced in the inventors' laboratory) anti-γ-Tubulin mouse IgG antibody (monoclonal antibody, clone GTU-88, Sigma) (Secondary antibody) anti-mouse IgG, Horseradish Peroxidase linked whole antibody (GE Healthcare Bioscience Inc.) Protein A, Horseradish Peroxidase linked (GE Healthcare Bioscience Inc.).

[0104] [Method (1)] The general methods used in Examples 1 to 8 are described below.

[0105] [1. Cloning ACC1] (1.1.PCR) Human ACC1-cDNA was amplified by PCR. In this example, human ACC1 (7041 bp total length) was amplified by separating it into the 3' terminal region (2045 bp), the middle region (2081 bp), and the 5' terminal region (2926 bp). A cDNA library from the human chronic myeloid leukemia cell line (K562) was used as the PCR template.

[0106] The primers were designed to have a base number of 20-24b, a GC content of 45-55%, and a Tm value of 58-62°C (see Table 1 below for detailed sequences). Nonspecific binding was confirmed using the Beacon Designer Free Edition and Blood databases. The primer for the 3' terminal region has the SalI restriction enzyme recognition sequence added only to the 3' end. The primer for the 5' terminal region has the NotI restriction enzyme recognition sequence added only to the 5' end.

[0107] [Table 1]

[0108] (1.2. Purification of ACC1-cDNA) For the purification of ACC1-cDNA, the QIA quick Gel Extraction kit (QIAGEN) was used. ACC1-cDNA amplified by PCR was subjected to electrophoresis on a 1% agarose gel, and the target-sized band was excised from the gel. The excised gel was dissolved in QX1 buffer (QIAGEN), and QIAEXII beads (QIAGEN) were added. The lysate was allowed to stand at 50°C for 10 minutes, then centrifuged, and the supernatant was removed. PE buffer was added to the residue, then centrifuged, and the supernatant was removed. After air-drying the residue, TE buffer (10mM Tris-HCl (pH=8.0) and 1mM EDTA) was added. After standing at 50°C for 10 minutes, centrifugation was performed, and the supernatant was collected.

[0109] (1.3. TA cloning) Purified ACC1-cDNA was inserted into a pGEM-T vector using T4 DNA ligase. The reaction mixture containing the vector (2× Rapid Ligation Buffer, pGEM-T vector, ACC1-cDNA, and T4 DNA ligase) was allowed to stand overnight at 4°C to produce plasmid DNA.

[0110] (1.4. Transformation) The prepared plasmid was reacted with competent cells of E. coli DH5α on ice. Next, SOC (2M glucose:SOB = 1:100, SOB containing Bcto Tryptone, Bacto Yeast Extract, 2.5 mM KCl, 10 mM NaCl, and 10 mM MgCl2) was added, and the cells were cultured with shaking at 37°C. Then, the culture medium was spread onto LB agar (containing 1% Bacto Tryptone, 0.5% Bacto Yeast Extract, 0.5% NaCl, and 50 μg / μL ampicillin) and cultured at 37°C for 14 hours. In this way, plasmid-transformed colonies were obtained.

[0111] (1.5. Recovery of ACC1 plasmid) Transformed colonies were cultured on LB liquid medium (containing 50 μg / μL ampicillin) at 37°C for 14 hours. After centrifugation of the culture medium, the supernatant was removed and the residue was suspended in ice-cold Sol I (50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl (pH=8.0)). Next, Sol II (200 mM NaOH and 1% SDS) was added to the suspension and mixed by inversion. Next, ice-cold Sol III (3 M potassium acetate and 11.5% acetic acid) was added and suspended. The supernatant obtained by centrifugation of the suspension was treated with PCI (phenol:chloroform:isoamyl alcohol = 25:24:1) and CIA (chloroform:isoamyl alcohol = 24:1). After adding 70% ethanol, the mixture was stirred and centrifuged. The supernatant was removed and the residue was air-dried, then TE buffer (10 mM Tris-HCl (pH=8.0), 1 mM EDTA, and RNase A (1 μg / μL) were added. After reacting at 37°C for 20 minutes, the plasmid was recovered.

[0112] (1.6. Sequence Analysis) The recovered plasmid was added to polyethylene glycol solution and reacted on ice for 1 hour, then centrifuged and the supernatant was removed. The residue was rinsed with 70% ethanol, TE buffer was added, and the mixture was purified by PCI and CIA. 3M sodium acetate and 100% ethanol were added to the purified product and the mixture was reacted at room temperature. Next, the reaction mixture was rinsed with 70% ethanol and dissolved in TE buffer. The lysate was amplified by PCR, then 3M sodium acetate and 100% ethanol were added and the mixture was reacted at room temperature. The reaction mixture was centrifuged and the supernatant was removed. The residue was rinsed with 70% ethanol, Hi-Di formamide was added, and the mixture was reacted at 95°C for 2 minutes. The reaction mixture was sequenced using an autosequencer (ABI PRISN 310 Genetic Analyzer).

[0113] [2. Protein Analysis] (2.1.Cell culture) Human fetal kidney-derived cell line 293T cells were cultured in Dulbecco's modified Eagle medium (DMEM). The medium was supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin (GIBCO / BRL). Culture was performed at 37°C in the presence of 5% CO2.

[0114] Mouse bone marrow cells were cultured in BM medium (Dulbeccoo's modified Eagle medium, 15% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, 5% WEHI-3B conditioned medium, 6 ng / mL mouse interleukin-3, 10 ng / mL human IL-6, and 50 ng / mL mouse stem cell factor). Recombinant cytokines from R&D system Inc. were used. Mouse bone marrow cells used for analysis were cultured in 10% WEHI-3B conditioned medium (containing IL-3). Cell culture was performed at 37°C in the presence of 5% CO2.

[0115] (2.2. Transfection) Transfection was performed using the calcium phosphate method with BES buffer. Confluent cells cultured in 60 mm plates were diluted to 1 / 10 to 1 / 30 and passaged into 35 mm plates. BES buffer was prepared with pH levels adjusted in 0.05 increments within the range of 6.8 to 7.3, and plasmids with adjusted dosages (1 μg, 2 μg, 3 μg, 5 μg, 7 μg, or 10 μg) to determine the optimal conditions for plasmid transfection. The optimal conditions were pH = 7.0 and plasmid amount = 3 μg. The transformation efficiency under optimal conditions remained above 80% (percentage of GFP-positive cells). 15 to 18 hours after transfection, the cells were washed with PBS and replaced with fresh medium. Depending on the cell condition, cells were harvested 48 to 72 hours after the medium change.

[0116] (2.3. Protein extraction from cells) HEK293T cells overexpressing the expression plasmid were washed twice with PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4). Next, 100 μL / 35 mm dish of EBC buffer (50 mM Tris-HCl (pH=8.0), 120 mM NaCl, 1 mM EDTA, and 0.5% NP40) was added. The EBC buffer contained 2000 KIU / μL aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 mM sodium fluoride (NaF), 0.1 mM sodium orthovanadate (Na3VO4), and 10 mM β-glycerophosphatase. After collecting the cells with a scraper, the cells were centrifuged, and the supernatant protein extract was collected.

[0117] (2.4. Induction of GST fusion protein expression (GST-Trib1, GST-Trib2, and GST-Trib3)) For protein expression induction, a glutathione-S-transferase (GST) fusion protein expression vector (pGEX-6p-2 vector) with a Trib1-cDNA, Trib2-cDNA, or Trib3-cDNA plasmid inserted (prepared by the inventors in their prior research) was used. The pGEX-6p-2 vector with the inserted Trib1-cDNA, Trib2-cDNA, or Trib3-cDNA was transformed into E. coli strain BL-21 using the same procedure as in section (1.4). At the same time, E. coli strain DH5α was also transformed in the same manner, and after restriction enzyme treatment, the insertion of the Trib1, Trib2, or Trib3 insert into the plasmid was confirmed by checking for bands of the target size. The colonies were transferred to 2 mL of LB liquid medium (containing 50 μg / μL ampicillin) and cultured at 37°C for 14 hours. Next, the culture was transferred to 200 mL of LB liquid medium (containing 50 μg / μL ampicillin) and incubated with shaking at 37°C until the OD value reached approximately 1.0 (approximately 3 hours).

[0118] To express Trib1 or Trib2, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM, and the culture was incubated with shaking at 18°C ​​for 3 hours. To express Trib3, IPTG was added to a final concentration of 0.35 mM, and the culture was incubated with shaking at 18°C ​​for 4 hours. After centrifugation of the culture, the supernatant was removed, and the E. coli pellet was collected by adding TN buffer (100 mM NaCl and 10 mM Tris-HCl (pH=8.0)).

[0119] (2.5. GST-pulldown) GST-pulldown was performed using E. coli pellets in which GST fusion protein expression was induced. Specifically, the E. coli pellets were mixed with 1 mL of NETN buffer (100 mM NaCl, 1 mM The cells were suspended in EDTA, 20 mM Tris-HCl (pH=8.0), and 0.5% NP40. Next, E. coli cells were lysed by sonication (POWER 4, 20 seconds x 5 times) on ice. After centrifugation of the lysates, 50% glutathione beads (Glutathione Sepharose 4B, GE Healthcare Inc.) were added to the supernatant, and the mixture was rotated at 4°C for 2 hours. Furthermore, the process of adding 1 mL of ice-cold NETN buffer and centrifugation was repeated three times. After adding 1 mL of EBC buffer, the mixture was centrifuged. The extract of HEK293T cells overexpressing ACC1, recovered in section (2.3), was added, and tapping was performed at room temperature for 5 hours. After adding 1 mL of EBC buffer, the process of inversion mixing and centrifugation was repeated three times. The residue was dissolved in 2× SDS sample buffer and heat-treated at 95°C for 5 minutes.

[0120] (2.6. SDS-PAGE) For the detection of ACC1, a 6% gel was used. In experiments with ACC1 deletion mutants, gels ranging from 6% to 18% were used depending on the size of the mutant. For the detection of γ-tubulin, COP1, HA, and GST-Trib1, 2, and 3, a 12% gel was used. The gels were placed in an electrophoresis tank, and after filling the tank with 1×SDS electrophoresis buffer (25 mM Tris, 1.4% glycine, and 0.1% SDS), the samples were applied. Electrophoresis was performed at 10 mV for the concentrated gel and at 20 mV for the separated gel.

[0121] (2.7. Coomassie Brilliant Blue (CBB) staining) The gels subjected to electrophoresis were stained by immersing them in a staining solution (0.25% CBB, 40% methanol, and 10% acetic acid) while shaking for about one hour. Next, they were destained by immersing them in a destaining solution (40% methanol and 10% acetic acid) while shaking.

[0122] (2.8. Western blotting) The stained gel and a polyvinylidene fluoride (PVDF) membrane, which had been hydrophilized with 100% methanol for about 1 minute, were immersed in Transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol) for 10 minutes with shaking. Next, the gel and PVDF membrane were sandwiched between filter paper, and a 30 mA current was passed through for 30 minutes, followed by a 100 mA current overnight to perform transfer. After transfer, the PVDF membrane was immersed in 5% bovine serum albumin (BSA) and blocked by shaking at room temperature for 2 hours. Primary antibody treatment with anti-ACC1 antibody, anti-FLAG antibody, or anti-HA antibody was performed at 4°C for 12 hours. Primary antibody treatment with anti-COP1 antibody or anti-γ-tubulin antibody was performed at room temperature for 2 hours. The primary antibody-treated gel was transferred to PBS and shaken at room temperature for 5 minutes. Next, the solution was transferred to PBS-T (PBS with 0.1% Tween20 added) and shaken at room temperature for 15 minutes. Then, it was transferred back to PBS and shaken at room temperature for 5 minutes. Next, it was transferred to 15 mL of 2.5% skim milk (NACALAI TESQUE Inc.) and blocked by shaking at room temperature for 30 minutes. Then, the secondary antibody was added and shaken for 1 hour. After washing with PBS and PBS-T, the solution was photosensitive and exposed using an ECL kit (GE Healthcare Bioscience Inc.).

[0123] (2.9. RT-PCR) Cells transfected in section (2.2) or mouse bone marrow cells infected with retrovirus were washed with PBS. Total RNA was extracted from the cells using ISOGEN reagent (Nippon Gene Inc.), and reverse transcription was performed using RNase-free Superscript reverse transcriptase (Invitrogen Inc.). The primers used are shown in Table 2 below.

[0124] [Table 2]

[0125] [3. AML Mouse Model] (3.1. Bone marrow transplantation) Bone marrow cells were collected from C57BL / 6 mice (CLEA Japan Inc.) administered 5 days prior to transplantation with 5-fluorouracil (Kyowa Hakko Kogyo, dose: 150 mg / kg) and maintained in BM medium. Bone marrow cells were infected with retrovirus by spin infection in the presence of polyblen (6 μg / mL). Post-infection bone marrow cells (0.5~1 × 10⁶) 6 The solution was intravenously injected into the tail of X-ray-irradiated (10 Gy) C57BL / 6 mice (8 weeks old). Blood tests were performed periodically using an automated hematology analyzer (F-820 analyzer, Sysmex). Cytospin of peripheral blood smears or bone marrow or spleen cells was stained with May-Grunwald Giemsa solution (Merck) and observed.

[0126] (3.2. Functional analysis of primary bone marrow cells) Primary bone marrow cells were cultured for 7 days after retrovirus infection in BM medium (Dulbeccoo's modified Eagle medium, 15% thermo-inactivated fetal bovine serum, 2 mM glutamine, 100 U / mL penicillin, 100 mg / mL streptomycin, 5% WEHI-3B conditioned medium, 6 ng / mL mouse IL-3, 10 ng / mL human IL-6, and 50 ng / mL mouse stem cell factor). GFP-positive cells were sorted using FACSAria (BD Biosciences) and then maintained again in BM medium. For functional analysis, cells transferred to 10% WEHI-3B conditioned medium (containing IL-3) were used.

[0127] For colony counting, sorted GFP-positive cells were further cultured in a methylcellulose-based medium (MethoCult GF M3434, StemCell Technologies Inc.) containing various cytokines. The added cytokines were mouse IL-3 (10 ng / mL), human IL-6 (10 ng / mL), mouse SCF (50 ng / mL), and human erythropoietin (3 U / mL). After 10 days of culture, the colony count was measured, and the cells were subculturised in a methylcellulose-based medium (MethoCult GF M3534) containing mouse IL-3 (10 ng / mL), human IL-6 (10 ng / mL), and mouse SCF (50 ng / mL).

[0128] (3.3. Measurement of intracellular ROS levels and NADPH levels) GFP-positive primary myeloid cells were sorted using FACSAria, cultured in 10% WEHI-3B conditioned medium (containing IL-3), and used for analysis. When N-acetylcysteine ​​(NAC) treatment was used, cells were treated with 1 mM NAC and cultured for at least 72 hours before analysis. Intracellular ROS levels were measured using a Cellular Reactive Oxygen Species Detection Assay Kit (Deep Red Fluorescence, Abcam) after standing at 37°C for 1 hour, followed by measurement with a FACS Calibur flow cytometer (Becton Dickinson). NADP+ / NADPH levels were measured using an NADP / NADPH-Glo™ Assay (Promega) after standing at room temperature for 1 hour, and the ratios were calculated.

[0129] (3.4. Flow Cytometry) Bone marrow or splenocytes recovered from mice were hemolyzed with PharM Lyse (BD Bioscience), stained with various antibodies, and analyzed using FACSAria (BD Biosciences). The antibodies used were CD3 (145-2C11), B220 (RA3-6B2), TER-119 (TER-119), Mac-1 (CD11b and M1 / 70), Gr-1 (RB6-8C5), c-kit (ACK2), Sca-1 (D7), CD34 (RAM34), or anti-FcγR II / III (2.4G2, BD Biosciences). These antibodies were stained with phycoerythrin (PE), PE-Cy5, allophycocyanin, Pacific Blue, biotin, or streptavidin-PE-Cy7.

[0130] [Example 1] We identified the binding region between ACC1 and COP1-Tribbles by using mutants in which amino acid regions were deleted from ACC1 and point mutants of ACC1.

[0131] [Example 1a] Based on wild-type ACC1 having the amino acid sequence shown in Sequence ID No. 5, partial peptides of various lengths were created (see Table 3). Then, we investigated whether each partial peptide possessed binding activity to the COP1-Trib1 complex.

[0132] [Table 3]

[0133] (result) The results are shown in Table 3. The partial peptide consisting of amino acid residues from position 1 to 275 has binding activity to the COP1-Trib1 complex. In contrast, the partial peptide consisting of amino acid residues from position 1 to 200 does not have binding activity to the COP1-Trib1 complex. Therefore, it was found that the binding domain of AAC1 to the COP1-Trib1 complex is likely to be the polypeptide consisting of 75 amino acid residues from position 201 to 275 of AAC1 (the polypeptide represented by Sequence ID No. 1).

[0134] [Example 1b] We confirmed that the subsequence of ACC1 represented by SEQ ID NO: 1 corresponds to the binding region with Trib1. To this end, we used three types of ACC1 mutants. The first is ACC1 75aa, a 75-amino acid peptide represented by SEQ ID NO: 1. The second is ACC1 delmut1, obtained by removing the 75 amino acids represented by SEQ ID NO: 1 from wild-type ACC1. The third is ACC1 cont.mut, obtained by removing the region adjacent to the 75 amino acids represented by SEQ ID NO: 1 from wild-type ACC1. The specific experimental procedure is as follows. 1. HEK293T cells were transfected with FLAG-ACC1 expression plasmids containing ACC1 75aa, ACC1 delmut1, or ACC1 cont.mut inserted into a pFLAG tag vector. 2. The cell extract and GST-Trib1 recombinant protein were mixed and collected using glutathione beads. 3. SDS-PAGE and Western blotting were performed to analyze the binding between Trib1 and ACC1. α-FLAG was used as the antibody. 4. To confirm the protein expression level of GST-Trib1, CBB staining was performed using the same sample. A sample expressing only GST was used as a control.

[0135] (result) The results are shown in Figure 1. For ACC1 75aa, a band originating from FLAG was detected (Figure 1 left). For ACC1 delmut1, no band originating from FLAG was detected (Figure 1 right). ACC1 Regarding cont.mut, a band originating from FLAG was detected (Figure 1, right). These results, taken together, suggest that the subsequence of ACC1 represented by sequence number 1 (ACC1 75aa) is a necessary and sufficient region for the binding between ACC1 and Trib1.

[0136] Since the partial sequence of ACC1 represented by Sequence ID No. 1 (ACC1 75aa) itself has binding activity to Trib1, it is suggested that ACC1 75aa can competitively inhibit the degradation of ACC1 by the COP1-Trib1 complex. This competitive inhibition is thought to be achieved by polypeptides that have ACC1 75aa and additional amino acid sequences and are not degraded by the COP1-Trib1 complex.

[0137] Similarly, compounds exhibiting binding activity to ACC1 75aa can competitively bind to ACC1 75aa with the COP1-Trib1 complex, suggesting they are candidate compounds for inhibitors of ACC1 degradation by the COP1-Trib1 complex. In other words, ACC1 75aa can be used for screening to find candidate compounds for inhibitors of ACC1 degradation by the COP1-Trib1 complex.

[0138] [Example 1c] We investigated whether the Trib1 binding region of ACC1 identified in Example 1a corresponds to a binding region with other Tribbles (Trib2 or Trib3). Specifically, we examined the binding activity of ACC1 delmut1 or ACC1 cont.mut with Trib2 or Trib3 using the same procedure as in Example 1a.

[0139] (result) The results are shown in Figure 1. ACC1 delmut1 did not exhibit binding activity to either Trib2 or Trib3 (Figure 1, right). ACC1 cont.mut exhibited binding activity to both Trib2 and Trib3 (Figure 1, right). These results suggest that the subsequence of ACC1 represented by Sequence ID No. 1 (ACC1 75aa) is a general binding region for Tribbles.

[0140] In conjunction with the results of Example 1b, it is suggested that ACC1 75aa can competitively inhibit the degradation of ACC1 by the COP1-Tribbles complex (the same applies to polypeptides that have an additional amino acid sequence and are not degraded by the COP1-Trib1 complex). Furthermore, it is suggested that ACC1 75aa can be used for screening to find candidate compounds for inhibitors of ACC1 degradation by the COP1-Tribbles complex.

[0141] Furthermore, alignment of the amino acid sequence represented by Sequence ID No. 1 across various biological species revealed that it is a highly conserved region from humans to fruit flies (upper part of Figure 17).

[0142] Furthermore, using the overall crystal structure of ACC1, three-dimensional crystal structure analysis was performed using the molecular graphic tool PyMoL. As a result, it was found that the binding region of ACC1 contains two α-helix structures protruding outward. These structures are referred to as Helix1 (10 amino acids, positions 16-25 in SEQ ID NO: 1, and positions 216-225 in SEQ ID NO: 6) and Helix2 (21 amino acids, positions 33-53 in SEQ ID NO: 1, and positions 233-253 in SEQ ID NO: 6).

[0143] [Example 1d] Point mutants of the Trib1-ACC1 binding region were created to more precisely identify the binding site between ACC1 and Trib1. Two types of mutants were used for this purpose. The first is ACC1 Helix1mut, in which the amino acids exposed on the binding surface of Helix1—proline at position 216, lysine at position 217, and glutamic acid at position 220—were all replaced with alanine. The second is ACC1 Helix2mut, in which the amino acids exposed on the binding surface of Helix2—proline at position 233, glutamine at position 235, and tryptophan at position 238—were all replaced with alanine. The specific experimental procedure is as follows. 1. FLAG-ACC1 expression plasmids were constructed by inserting ACC1 Helix1mut or ACC1 Helix2mut into a pFLAG tag vector, and these plasmids were transfected into HEK293T cells. 2. The cell extract was mixed with GST-Trib1, GST-Trib2, and GST-Trib3 recombinant proteins and collected using glutathione beads. 3. SDS-PAGE and Western blotting were performed to analyze the binding between ACC1 Helix1mut or ACC1 Helix2mut and Tribbles. α-FLAG was used as the antibody. 4. To confirm the protein expression levels of GST-Trib1, GST-Trib2, and GST-Trib3, CBB staining was performed using the same sample. A sample expressing only GST was used as a control.

[0144] (result) The results are shown in Figure 2. ACC1 Helix2mut exhibited similar binding activity to wild-type ACC1, Trib1, and Trib3. ACC1 Helix1mut showed significantly lower binding activity to all three ACC1, Trib2, and Trib3. This suggests that amino acids exposed on the binding surface of Helix1 contribute to the binding of ACC1 to Tribbles.

[0145] [Example 2] The degree of resistance to degradation by the COP1-Trib1 complex in ACC1 delmut1 prepared in Example 1b and ACC1 Helix1mut prepared in Example 1d was investigated. For comparison, wild-type ACC1, ACC1 Helix2mut prepared in Example 1d, and a ubiquitination site point mutant (ACC1 K1759R) were used. ACC1 K1759R is a mutant in which the ubiquitination site (lysine at position 1759), which can be targeted by the COP1-Trib1 complex, is replaced with arginine. The specific procedure is as follows. 1. HEK293T cells were transfected with expression plasmids for GFP-COP1, HA-Trib1, FLAG-ACC1 WT, FLAG-K1759R, FLAG-ACC1 delmut1, and FLAG-ACC1 Helix1mut. 2. SDS-PAGE and Western blotting were performed on cell extracts to analyze ACC1 expression levels. α-FLAG, α-HA, α-COP1, and α-γTublin were used as antibodies.

[0146] (result) The results are shown in Figure 3. Cells co-expressing ACC1 delmut1 or ACC1 Helix1mut with COP1 and Trib1 showed high levels of ACC1. This level of ACC1 was comparable to or higher than that of cells co-expressing K1759R with COP1 and Trib1. On the other hand, cells co-expressing wild-type ACC1 or ACC1 Helix2mut with COP1 and Trib1 showed significantly lower levels of ACC1. These results suggest that ACC1 delmut1 and ACC1 Helix1mut exhibit resistance to degradation by the COP1-Trib1 complex, and that this resistance is comparable to or higher than that of K1759R.

[0147] [Example 3] The effects of ACC1 Helix1mut on cell proliferation were investigated using cell proliferation assays, protein expression level measurements, and RNA expression level measurements.

[0148] [Example 3a] We introduced ACC1 Helix1mut into mouse bone marrow cells and analyzed its effects on cell proliferation. The specific procedure is as follows: 1. Expression plasmids of COP1-IRES-GFP, Trib1-IRES-GFP, ACC1 WT-IRES-GFP, K1759R-IRES-GFP, and ACC1 Helix1mut-IRES-GFP were introduced into mouse bone marrow cells recovered from normal mice using retroviruses. 2. GFP-positive cells were sorted using flow cytometry, cultured in BM medium (containing SCF, IL-6, and IL-3), and their cell proliferation ability was analyzed.

[0149] (result) The results are shown in Figure 4. Compared to cells expressing COP1-Trib1 alone, cells co-expressing wild-type ACC1, K1759R, or ACC1 Helix1mut with COP1-Trib1 showed a growth inhibitory effect. Furthermore, cells co-expressing ACC1 Helix1mut with COP1-Trib1 showed a significantly higher growth inhibitory effect than cells co-expressing wild-type ACC1 with COP1-Trib1. These results suggest that ACC1 Helix1mut suppresses cell proliferation induced by the COP1-Trib1 complex, and that this suppression is significantly higher than that of wild-type ACC1 and at least as effective as or greater than that of K1759R.

[0150] [Example 3b] The inhibitory effect of ACC1 Helix1mut on cell proliferation was further investigated in relation to ACC1 protein levels. Specifically, ACC1 protein levels in mouse bone marrow cells co-expressing COP1-Trib1 with wild-type ACC1, K1759R, or ACC1 Helix1mut were analyzed by Western blotting. Bone marrow cells from normal mice were used as controls. α-ACC1 and α-γTublin antibodies were used. RNA expression levels were also compared by RT-PCR.

[0151] (result) The results are shown in Figure 5. Compared to normal bone marrow cells, ACC1 degradation was significantly accelerated in cells introduced with COP1-Trib1 (left figure). The protein levels of ACC1 differed greatly between cells co-expressing COP1-Trib1 with wild-type ACC1, K1759R, or ACC1 Helix1mut (left figure). In particular, ACC1 was present at a very high level in cells co-expressing COP1-Trib1 with ACC1 Helix1mut (left figure). On the other hand, the RNA level of ACC1 remained almost constant in cells co-expressing COP1-Trib1 with wild-type ACC1, K1759R, or ACC1 Helix1mut. These results suggest that COP1-Trib1 induces the degradation of ACC1 protein, but the ACC1 Helix1mut protein is resistant to degradation by COP1-Trib1.

[0152] [Example 4] We investigated the effects of ACC1 Helix1mut on intracellular metabolic mechanisms.

[0153] [Example 4a] The effects of ACC1 Helix1mut on metabolic mechanisms were investigated from the perspective of ROS levels. Specifically, (i) COP1-IRES-GFP and Trib1-IRES-GFP, and (ii) ACC1 WT-IRES-GFP, ACC1 K1759R-IRES-GFP, or ACC1 Helix1mut-IRES-GFP were introduced into primary mouse myeloid cells, and GFP-positive cells were sorted by flow cytometry. Subsequently, the ROS levels of the sorted cells were measured. In addition, for each sample, the ROS levels were also measured after treatment with N-acetylcysteine ​​(NAC), an antioxidant that suppresses ROS.

[0154] (result) The results are shown in Figure 6. Compared to cells expressing only COP1 and Trib1, cells co-expressing wild-type ACC1 showed elevated intracellular ROS levels. Furthermore, cells co-expressing K1759R or ACC1 Helix1mut showed a more significant increase in ROS levels than cells co-expressing wild-type ACC1. These results suggest that ACC1 Helix1mut increases ROS levels in cells expressing COP1 and Trib1, and that this effect is more pronounced than with wild-type ACC1 and equivalent to or greater than that of K1759R.

[0155] [Example 4b] The effects of ACC1 Helix1mut on metabolic mechanisms were investigated from the perspective of intracellular NADP+ / NADPH. Specifically, mouse primary myeloid cells were introduced with (i) COP1-IRES-GFP and Trib1-IRES-GFP, and (ii) ACC1 WT-IRES-GFP, ACC1 K1759R-IRES-GFP, or ACC1 Helix1mut-IRES-GFP, and GFP-positive cells were sorted by flow cytometry. Subsequently, the NADP+ / NADPH of the sorted cells was measured.

[0156] (result) The results are shown in Figure 7. Similar to ROS levels, NADP+ / NADPH levels were significantly increased in cells introduced with wild-type ACC1, K1759R, or ACC1 Helix1mut. In particular, cells introduced with ACC1 Helix1mut had significantly higher NADP+ / NADPH levels than cells introduced with wild-type ACC1. These results suggest that ACC1 Helix1mut increases NADP+ / NADPH levels in cells introduced with COP1 and Trib1, and that this increase is more pronounced than with wild-type ACC1 and comparable to or greater than that of K1759R.

[0157] [Example 5] We investigated the preventive effect of ACC1 Helix1mut on AML (the inhibitory effect of ACC1 Helix1mut on the mechanism of AML onset).

[0158] [Example 5a] The effect of ACC1 Helix1mut on delaying the onset of AML was investigated. The specific procedure is as follows: 1. Bone marrow cells recovered from normal mice were introduced using retroviruses with (i) COP1-IRES-GFP and Trib1-IRES-GFP expression plasmids, and (ii) ACC1 WT-IRES-GFP, ACC1 K1759R-IRES-GFP, or ACC1 Helix1mut-IRES-GFP expression plasmids. 2. The cells prepared in step 1 were transplanted into the bone marrow of X-ray-irradiated mice to construct an AML mouse model. 3. We regularly performed blood tests using an automated hematology analyzer and Giemsa staining of peripheral blood-derived cells to confirm the onset of AML and to analyze survival curves.

[0159] (result) The results are shown in Figure 8. Mice that received COP1-Trib1 alone developed AML within 100-200 days in all cases. In contrast, mice co-introduced with ACC1 Helix1mut showed a significant delay in the onset of AML. The effect of delaying AML onset was greater with ACC1 Helix1mut than with K1759R.

[0160] [Example 5b] Peripheral blood cells and bone marrow cells were collected from mice that developed AML and stained with Giemsa dye. Based on the staining patterns, the effect of introducing ACC1 Helix1mut on the differentiation of AML cells was investigated.

[0161] (result) The results are shown in Figures 9 and 10. In AML mice introduced with COP1-Trib1, immature blast cells accumulated in the peripheral blood and bone marrow. In AML mice introduced with ACC1 Helix1mut or K1759R, neutrophils, which are in the final stage of differentiation, were significantly increased. This suggests that ACC1 Helix1mut promotes cell differentiation, to a degree equivalent to or greater than that of K1759R.

[0162] [Example 5c] The expression levels of Mac-1 and Gr-1, granulocyte differentiation markers including neutrophils, were compared in bone marrow cells derived from AML-affected mice using flow cytometry.

[0163] (result) The results are shown in Figure 11. In AML mice co-transformed with COP1-Trib1 and ACC1 Helix1mut or K1759R, immature cell populations (Mac-1) were observed. lo , Gr-1 lo ) decreases, and the differentiated cell population (Mac-1 hi , Gr-1 hi ) was increased. These results suggest that introducing ACC1 Helix1mut into the AML mouse model delays the onset of COP1-Trib1-induced AML and promotes cell differentiation.

[0164] [Example 6] The therapeutic effect of ACC1 Helix1mut on AML was investigated.

[0165] [Example 6a] We investigated the possibility that ACC1 Helix1mut could deplete self-renewing AML stem cells by inducing cell differentiation. The specific procedure is as follows: 1. Expression plasmids of COP1-IRES-GFP, Trib1-IRES-GFP, and ACC1 Helix1mut-IRES-GFP were introduced into bone marrow cells recovered from normal mice using retroviruses. 2. GFP-positive cells were sorted by flow cytometry. 3. The sorted cells were cultured in semi-solid methylcellulose medium (containing SCF, IL-6, and IL-3), and their self-renewal ability was analyzed by colony formation assay.

[0166] (result) The results are shown in Figure 12. Compared to colonies into which only COP1-Trib1 was introduced, cells co-introduced with ACC1 Helix1mut showed a significant decrease in the number of colonies from the first to the fifth passage.

[0167] Furthermore, when the first-generation cells were harvested and Giemsa stained, and their morphology was compared, it was found that the neutrophil count was significantly increased in the colonies of cells into which ACC1 Helix1mut had been introduced (Figure 13). These results suggest that ACC1 Helix1mut inhibits the proliferation of AML stem cells and promotes their differentiation.

[0168] [Example 6b] We investigated the effects on cell differentiation in mouse bone marrow cells in the early phase, before the progression of malignancy in AML. The specific procedure is as follows: 1. Expression plasmids of COP1-IRES-GFP, Trib1-IRES-GFP, and ACC1 Helix1mut-IRES-GFP were introduced into bone marrow cells recovered from normal mice using retroviruses. 2. Bone marrow transplantation was performed on X-ray irradiated mice to construct early-phase mice (10-12 weeks old). 3. GFP-positive cells were sorted by flow cytometry, and the expression levels of Mac-1 and Gr-1 were measured. 4. In addition, sorted cells were cultured in semi-solid methylcellulose medium (containing SCF, IL-6, and IL-3), and their self-renewal ability was analyzed by colony formation assay.

[0169] (result) The results are shown in Figure 14. Analysis using flow cytometry revealed that in cells into which COP1-Trib1 or COP1-Trib1-ACC1 Helix1mut was introduced, approximately 90% of the cells accumulated as GFP-positive monoclonal cells (upper figure). In addition, in mice into which COP1-Trib1-ACC1 Helix1mut was introduced, many markers specific to the differentiated cell population were expressed (Mac-1). hi , Gr-1 hi This was shown (middle figure). From the results of the colony formation assay, cells into which COP1-Trib1-ACC1 Helix1mut was introduced showed a significant decrease in the number of colonies in both the first and second generations (bottom figure). These results suggest that ACC1 Helix1mut can promote the differentiation of AML stem cells and deplete AML stem cells even in early-phase mice.

[0170] [Example 6c] In Example 6a, we demonstrated in vitro that ACC1 Helix1mut depletes AML stem cells with self-renewal ability. We investigated whether similar depletion is possible in vivo. The specific procedure is as follows. 1. Expression plasmids of COP1-IRES-GFP, Trib1-IRES-GFP, and Helix1mut-IRES-GFP were introduced into bone marrow cells recovered from normal mice using retroviruses. 2. Bone marrow transplantation was performed on X-ray irradiated mice to construct early-phase mice (10-12 weeks old). 3. Bone marrow cells were extracted from the mice in the initial phase described above and transplanted into X-ray irradiated mice. 4. Survival curves were analyzed, and GFP-positive cells were sorted by flow cytometry, and the expression levels of Sca-1, c-Kit, and Mac-1 were measured.

[0171] (result) The results are shown in Fig. 15. All mice into which only COP1-Trib1 was introduced developed AML and died. However, in mice co-introduced with ACC1 Helix1mut, the onset of AML was significantly delayed.

[0172] Furthermore, from the results of flow cytometry (Fig. 16), in mice co-introduced with COP1-Trib1 and ACC1 Helix1mut, immature cell populations (c-Kit hi , Mac-1 lo ) decreased, and cell populations in which differentiation had progressed (c-Kit lo , Mac-1 hi ) increased. From these results, it was suggested that ACC1 Helix1mut also inhibits the proliferation of AML stem cells and promotes differentiation in vivo.

[0173] [Example 7] Acetyl-CoA carboxylase 2 (ACC2) is a family protein of ACC1. The region of ACC2 corresponding to the Trib1-binding region of ACC1 corresponds to positions 346 to 417 of SEQ ID NO: 7 (the full-length amino acid sequence of ACC2) and has the amino acid sequence represented by SEQ ID NO: 3. This region was found to have high homology with the Trib1-binding region of ACC1 and to be highly conserved across species (the lower figure in Fig. 17).

[0174] Therefore, it was also examined whether one-point mutants of ACC2 are resistant to the degradation action of ACC2 by COP1-Trib1. For this purpose, two types of ACC2 mutants were used. The first is ACC2 Δ346-423 in which the region corresponding to positions 346 to 423 of SEQ ID NO: 7 in ACC2 was deleted (the region deleted in ACC2 Δ346-423 is almost the same region as positions 343 to 417 of SEQ ID NO: 7 specified as the binding region of ACC2 in this specification). The second is ACC2 Helix1mut in which proline at position 358, lysine at position 359, and glutamate at position 362 in ACC2 were all substituted with alanine. 1. ACC2 Δ346-423 or ACC2 Helix1mut was inserted into a pFLAG tagged vector to create an expression plasmid. This expression plasmid was transfected into HEK293T cells. 2. Cell extracts were mixed with GST-Trib1, GST-Trib2, or GST-Trib3 recombinant proteins and collected using glutathione beads. 3. SDS-PAGE and Western blotting were performed to analyze the binding between ACC2 Δ346-423 and Tribbles, and between ACC2 Helix1mut and Tribbles. α-FLAG was used as the antibody. 4. To confirm the protein expression levels of GST-Trib1, GST-Trib2, and GST-Trib3, CBB staining was performed using the same sample. A sample expressing only GST was used as a control.

[0175] (result) The results are shown in Figure 18. Neither ACC2 Δ346-423 nor ACC2 Helix1mut exhibited binding activity to Trib1, Trib2, or Trib3. This suggests that the partial sequence of ACC2 shown in Sequence ID No. 3 is the binding region to the COP1-Tribbles complex.

[0176] Therefore, it is suggested that the polypeptide shown in SEQ ID NO: 3 can competitively inhibit the degradation of ACC2 by the COP1-Tribbles complex (the same applies to polypeptides that have the same polypeptide and additional amino acid sequence and are not degraded by the COP1-Trib1 complex). Furthermore, it is suggested that the polypeptide shown in SEQ ID NO: 3 can be used for screening to find candidate compounds for inhibitors of ACC1 degradation by the COP1-Tribbles complex.

[0177] Furthermore, it is suggested that ACC2 Helix1mut, like ACC1 Helix1mut, exhibits resistance to degradation by the COP1-Tribbles complex. Therefore, it is suggested that ACC2 Helix1mut also has effects that alter intracellular metabolism (see Example 4) and preventive and therapeutic effects on AML (see Examples 5 and 6).

[0178] [Example 8] We introduced ACC2 Δ346-423 or ACC2 Helix1mut into mouse bone marrow cells and analyzed their effects on cell proliferation. The specific procedure is as follows: 1. Mouse bone marrow cells recovered from normal mice were introduced using retroviruses to express COP1-IRES-GFP, Trib1-IRES-GFP, ACC2 WT-IRES-GFP, ACC2 Δ346-423-IRES-GFP, and ACC2 Helix1mut-IRES-GFP expression plasmids. 2. GFP-positive cells were sorted by flow cytometry. 3. Cells sorted in BM medium (containing SCF, IL-6, and IL-3) were cultured, and their cell proliferation ability was analyzed.

[0179] (result) The results are shown in Figure 19. Compared to cells expressing COP1-Trib1 alone, cell proliferation was significantly suppressed in cells co-expressing ACC2 Δ346-423 or ACC2 Helix1mut. This result suggests that ACC2 Helix1mut also has an effect of suppressing cell proliferation induced by the COP1-Trib1 complex.

[0180] [Method (2)] The general methods used in Examples 9 to 12 are described below.

[0181] [1. Culture of primary bone marrow cells] Primary bone marrow cells were cultured using the following procedure. 1. C57BL / 6 mice (CLEA Japan Inc.) were administered 5-fluorouracil (5-FU, Kyowa Hakko Kogyo). The dose was 150 mg / kg. 2. Five days after administration of 5-FU, bone marrow cells were collected from the mice. 3. The collected bone marrow cells were cultured in BM medium (Dulbeccoo's modified Eagle agar, containing heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U / mL penicillin, 100 mg / mL streptomycin, 5% WEHI-3B conditioned medium, 6 ng / mL mouse IL-3, 10 ng / mL human IL-6, and 50 ng / mL mouse stem cell factor). 4. Bone marrow cells were infected with retroviruses by spin infection in the presence of polyblen (6 μg / mL). 5. Post-infection bone marrow cells were cultured in BM medium for 5 days. 6. GFP-positive cells were sorted using FACSAria (BD Biosciences). 7. The selected cells were maintained in 10% WEHI-3B conditioned BM medium (containing IL-3).

[0182] To measure intracellular ROS levels and intracellular NADPH levels, bone marrow cells were transferred to a glucose (1 g / L) culture medium and treated with N-acetylcysteine ​​(NAC).

[0183] [2. Measurement of intracellular ROS levels] Intracellular ROS levels were measured using a ROS detection assay kit (Deep Red Fluorescence, Abcam, UK). The kit was used according to the manufacturer's instructions. Fluorescence emitted in response to the presence of ROS was analyzed using a flow cytometer (BD FACS Calibur, BD Biosciences, USA) and analysis software (BD CellQuest Pro (v6.0), BD Biosciences, USA), and the average fluorescence value was used as an indicator of intracellular ROS levels.

[0184] [Measurement of Intracellular NADPH Level] The intracellular NADP + level and the intracellular NADPH level were measured using NADP / NADPH-GloTM Assay (Promega) and a microplate reader (Berthold Technology). Based on the measurement results, the ratio of NADP + / NADPH was calculated.

[0185] [Measurement of Mitochondrial Respiration Level] The oxygen consumption rate (OCR) of cells was calculated using an XF HS Mini Analyzer (Seahorse Bioscience, Agilent, USA), and this was regarded as the respiration level of the cells. The specific procedure is as follows. 1. The sensor cartridge was incubated overnight in sterile water at 37°C. 2. The sterile water was replaced with Seahorse XF Calibrant and incubated at 37°C for 1 hour. 3. Cell-Tak solution was added to each well of an XFp 8-well cell culture miniplate coated with Cell-Tak and incubated at room temperature for 20 minutes. 4. The wells were washed twice with sterile water. 5. Prior to the assay, the medium for culturing primary bone marrow cells was changed to Seahorse XF basal medium supplemented with 25 mM glucose and 1 mM sodium pyruvate and cultured for 1 hour. 6. Cells were seeded into each well of the plate coated with Cell-Tak. The seeding density was 1×10 5 cells per well. Through steps 3 to 6, non-adherent primary bone marrow cells were immobilized in the wells. 7. Oligomycin A, FCCP, and antimycin A / rotenone were prepared and placed into the ports of the sensor cartridge. 8. The cartridge was placed into the analyzer and calibration was performed. 9. The cell culture microplate was placed in an analyzer, and OCR measurement was started.

[0186] The timing for measuring OCR is as follows. Measurements 1 - 3: Measured in the same state as at the start of OCR measurement. Measurements 4 - 6: After measurement 3, 1.5 μM oligomycin A was injected into the microplate and then measured. Measurements 7 - 9: After measurement 6, 1 μM FCCP was injected into the microplate and then measured. Measurements 10 - 12: After measurement 9, 2.5 μM antimycin A / rotenone was injected into the microplate and then measured.

[0187] The measured OCR values (pmol / min) were normalized for each well. The average of the measured values for measurements 1 - 3 (1 - 14 minutes) was used as the OCR baseline. The difference between the average value of the measured values for measurements 7 - 9 (40 - 53 minutes) and the average value of the measured values for measurements 10 - 12 (60 - 73 minutes) was used as the maximum OCR.

[0188] [5. Statistical analysis] Using the data of the metabolic gene batch adjustment matrix of MERAV (Metabolic gEne RApid Visualiser) 1 published by the Kyoto Encyclopedia of Genes and Genomes (KEGG), gene expression between normal tissues and primary tumors was compared. Specifically, the expression levels of ACC1 and ACC2 were calculated as relative values to the expression level of GAPDH. All data were statistically analyzed by two - tailed Student's t - test. The reference values are as follows. *: P < 0.05, **: P < 0.01, ***: P < 0.001

[0189] [Example 9] The change in the expression level of ACC1 in AML patients was examined.

[0190] [Example 9a] Microarray data analysis was performed to determine ACC1 mRNA expression levels in human AML patients. Samples from healthy donors (n=9) and AML patients (n=285) were included in the analysis. All data were obtained from the GEO dataset (published by NCBI).

[0191] (result) The results are shown in Figure 20. As can be seen from the figure, the expression level of ACC1 mRNA was significantly reduced in samples taken from human AML patients.

[0192] [Example 9b] The expression levels of ACC1 protein and mRNA were compared in human leukemia cell lines. The cell lines used for comparison were normal bone marrow cells, CML cell line (K562), and AML cell lines (HL60, OCI-AML3, THP-1, KY821, and U937). Protein expression levels were analyzed by immunoblotting using antibodies against ACC1, COP1, or γ-tubulin. mRNA expression levels were analyzed by semi-qRT-PCR using primer pairs specific to ACC1, Trib1, or GAPDH.

[0193] (result) The results are shown in Figure 21. In AML cell lines, the expression levels of both mRNA and protein were significantly reduced.

[0194] [Example 10] The effects of ACC1 Helix1mut on acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) were investigated. Cells transfected with MLL-AF9 were used as the AML model, and cells transfected with BCR-ABL were used as the CML model.

[0195] [Example 10a] Primary bone marrow cells were infected with retroviruses and expressed either (i) MLL-AF9 or (ii) BCR-ABL. For each cell type, a system was prepared with co-expression of ACC1 Helix1mut and a system without co-expression. The change in cell number was recorded daily for these four systems. The cell number at the start of culture was 1 × 10⁶ 5 It was a cell.

[0196] (result) The results are shown in Figures 22 and 23. The data are the mean of three independent experiments and are expressed as mean ± SD. As shown in these figures, cells co-expressing ACC1 Helix1mut proliferated more slowly than cells expressing MLL-AF9 or BCR-ABL alone.

[0197] [Example 10b] Primary bone marrow cells were infected with retroviruses and expressed (i) Trib1-IRES-GFP and COP1-IRES-GFP, (ii) MLL-AF9, or (iii) BCR-ABL. For each cell type, a system was prepared with co-expression of ACC1 Helix1mut and a system without co-expression. The following analyses were performed on these six systems. (a) Cell lysates of GFP-positive cells were prepared. These cell lysates were analyzed by immunoblotting using antibodies specific to ACC1 and antibodies specific to γ-tubulin. (b) Total RNA was extracted from GFP-positive cells. The obtained RNA was analyzed by semi-qRT-PCR using primer pairs specific to human and mouse ACC1 (h&m ACC1) and primer pairs specific to β-actin. (c)(b) The relative amount of expressed mRNA (h&mACC1 / β-actin) was quantified from the results. ImageJ software was used for quantification.

[0198] (result) The results are shown in Fig. 24. The results of (a) are in the upper panel, the results of (b) are in the middle panel, and the results of (c) are in the lower panel. The primary bone marrow cells expressing Trib1-COP1, MLL-AF9, and BCR-ABL had lower expression levels of endogenous ACC1 protein than normal bone marrow cells. However, co-expression of ACC1 Helix1mut enabled the expression of ACC1 protein.

[0199] [Example 10c] Primary bone marrow cells were infected with retroviruses to express (i) Trib1-IRES-GFP and COP1-IRES-GFP, (ii) MLL-AF9, or (iii) BCR-ABL. For each cell type, systems with and without co-expression of ACC1 Helix1mut were prepared. GFP-positive cells were selected from the six systems thus prepared and transferred to an IL-3-containing medium with low glucose (1 g / L). Then, ROS levels, NADP + / NADPH ratio, and GSH / GSSG ratio were measured.

[0200] (Results) The results are shown in Fig. 25. The data are the averages of three independent experiments and are presented as mean ± SD. In the system co-expressing ACC1 Helix1mut and MLL-AF9, ROS levels and NADP + / NADPH ratio increased, and the GSH / GSSG ratio decreased. In the system co-expressing ACC1 Helix1mut and BCR-ABL, ROS levels increased and the GSH / GSSG ratio decreased.

[0201] [Example 10d] The following two types of bone marrow cells were prepared and transplanted into mice, and the survival period from transplantation was measured. (i) Bone marrow cells expressing MLL-AF9-IRES-GFP (ii) Bone marrow cells co-expressing MLL-AF9-IRES-GFP and ACC1 Helix1mut <​​​The results are shown in Figure 26. Co-expression of ACC1 Helix1mut in MLL-AF9-induced AML model mice significantly extended mouse survival.

[0203] [Example 11] We investigated the various effects that ACC2 Helix1mut has on cells.

[0204] [Example 11a] We introduced ACC2 Helix1mut into mouse bone marrow cells and analyzed its effects on cell proliferation. The specific procedure is as follows: 1. Using retroviruses, the following combinations of expression plasmids were introduced into mouse bone marrow cells recovered from normal mice. (i) COP1-IRES-GFP and Trib1-IRES-GFP (ii) ACC2 WT-IRES-GFP, COP1-IRES-GFP and Trib1-IRES-GFP (iii) ACC2 Helix1mut-IRES-GFP, COP1-IRES-GFP and Trib1-IRES-GFP 2. GFP-positive cells were sorted using flow cytometry. 3. GFP-positive cells were cultured in BM medium (containing SCF, IL-6, and IL-3), and their cell proliferation ability was analyzed.

[0205] (result) The results are shown in Figure 27. Cells co-expressing ACC2 Helix1mut and COP1-Trib1 showed suppressed cell proliferation compared to cells introduced with COP1-Trib1 alone. Cells co-expressing ACC2 WT (wild-type ACC2) with COP1-Trib1 showed no difference in cell proliferation compared to cells introduced with COP1-Trib1 alone. These results suggest that ACC2 Helix1mut suppresses cell proliferation induced by the COP1-Trib1 complex.

[0206] [Example 11b] The effects of ACC2 Helix1mut on intracellular metabolic mechanisms were investigated from the perspective of ROS (Related Organisms). The specific procedure is as follows: 1. The following combinations of expression plasmids were introduced into primary mouse bone marrow cells. (i) COP1-IRES-GFP and Trib1-IRES-GFP (ii) ACC2 WT-IRES-GFP, COP1-IRES-GFP and Trib1-IRES-GFP (iii) ACC2 Helix1mut-IRES-GFP, COP1-IRES-GFP and Trib1-IRES-GFP 2. GFP-positive cells were sorted by flow cytometry. 3. ROS levels in GFP-positive cells were measured.

[0207] (result) The results are shown in Figure 28. Co-expression of wild-type ACC2 or ACC2 Helix1mut in cells introduced with COP1-Trib1 did not result in a significant increase in intracellular ROS levels.

[0208] [Example 11c] The effects of ACC2 Helix1mut on intracellular metabolic mechanisms were investigated from the perspective of ROS (Related Organisms). The specific procedure is as follows: 1. The following combinations of expression plasmids were introduced into primary mouse bone marrow cells. (i) COP1-IRES-GFP and Trib1-IRES-GFP (ii) ACC2 WT-IRES-GFP, COP1-IRES-GFP and Trib1-IRES-GFP (iii) ACC2 Helix1mut-IRES-GFP, COP1-IRES-GFP and Trib1-IRES-GFP 2. GFP-positive cells were sorted by flow cytometry. 3. GFP-positive cells were divided into an N-acetylcysteine ​​(NAC) treated group and an untreated group. NAC is an antioxidant that suppresses ROS. 4. Each group was cultured for 8 days, and the number of cells after proliferation was compared. The number of cells at the start of culture was 1 × 10⁶. 5 It was a cell.

[0209] (result) The results are shown in Figure 29. Cell proliferation was not promoted even after NAC treatment to suppress ROS. The results from Examples 11b and 11c suggest that the suppression of cell proliferation by ACC2 Helix1mut is independent of ROS levels. This is a difference from the action of ACC1 Helix1mut (see Example 4a and Figure 6).

[0210] [Example 11d] The effects of ACC2 Helix1mut on intracellular metabolic mechanisms are investigated using NADP. + The study was conducted from the perspective of the NADPH ratio. The specific procedure is as follows: 1. The following combinations of expression plasmids were introduced into primary mouse bone marrow cells. (i) COP1-IRES-GFP and Trib1-IRES-GFP (ii) ACC2 Helix1mut-IRES-GFP, COP1-IRES-GFP and Trib1-IRES-GFP 2. GFP-positive cells were sorted by flow cytometry. 3. NADP in GFP-positive cells + The NADPH ratio was measured.

[0211] (result) The results are shown in Figure 30. In cells into which ACC2 Helix 1mut was introduced, NADP + / NADPH levels were significantly increased. Combining the results of Examples 11b-11d, ACC2 Helix1mut was found to increase NADP levels in cells transfected with COP1-Trib1. + This suggests that the / NADPH ratio is increased in a manner independent of ROS activity.

[0212] [Example 11e] The effect of ACC2 Helix1mut on cellular mitochondrial respiration levels was investigated. ACC2 is a protein localized to the outer mitochondrial membrane that negatively regulates energy production by fatty acid oxidation (FAO). Example 11d showed that ACC2 Helix1mut induced intracellular NADP + Since an increase in the NADPH ratio has been shown, it is presumed that ACC2 Helix1mut affects mitochondrial respiration in cells. To test this hypothesis, mitochondrial oxygen consumption rate (OCR) was measured. OCR was measured using the XF Mitostress Test with an XF HS Mini Analyzer.

[0213] (result) The results are shown in Figures 31 and 32. Figure 31 is a graph showing the change in OCR over time. From this figure, it can be seen that cells co-expressing ACC2 Helix1mut have significantly lower OCR compared to cells with COP1-Trib1 alone. Figure 32 is a graph comparing maximal respiration. From this figure, it can be seen that cells co-expressing ACC2 Helix1mut have significantly lower maximal respiration compared to cells with COP1-Trib1 alone.

[0214] These results suggest that ACC2 Helix1mut inhibits mitochondrial respiration in cells transducing COP1-Trib1, thereby reducing cellular energy production and suppressing cell proliferation. The reduction in cellular energy production by ACC2 Helix1mut suggests that ACC2 Helix1mut inhibits NADP + This is also supported by increasing the NADPH ratio.

[0215] [Example 12] We investigated the expression levels of ACC1 and ACC2 in various human solid tumors. Specifically, we analyzed whether the amount of ACC1 and ACC2 mRNA expressed increased or decreased between normal tissue and the corresponding primary solid tumor. The data used for analysis were from the metabolic gene batch-adjusted matrix of MERAV (Metabolic gEne RApid Visualiser) 1, which is publicly available from the Kyoto Gene Genome Encyclopedia (KEGG). mRNA expression levels were expressed as relative values ​​to the expression level of GAPDH mRNA.

[0216] (result) The results are shown in Figure 33 (N: number of normal samples, T: number of primary tumor samples). ACC1 expression was significantly reduced in 7 of the 18 solid tumors analyzed (upper panel). Furthermore, ACC2 expression was significantly reduced in 15 of the 18 solid tumors analyzed (lower panel). The reduction in ACC2 expression was more pronounced than the reduction in ACC1 expression.

[0217] Example 12 suggests that cell proliferation can be suppressed even in solid tumors by applying ACC1 Helix1mut and / or ACC2 Helix1mut. In other words, it is presumed that ACC1 Helix1mut and / or ACC2 Helix1mut are also effective in treating these solid tumors. [Industrial applicability]

[0218] The present invention can be used, for example, as an inhibitor of ACC degradation by the COP1-Tribbles complex, or as an anticancer agent.

Claims

1. Polypeptide listed in any of the following: (a1) Polypeptides consisting of the amino acid sequence shown in SEQ ID NO: 1 or 3; (a2) A polypeptide having an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted and / or added from the amino acid sequence shown in SEQ ID NO: 1 or 3, and which has binding activity to the COP1-Trib1 complex; (a3) A polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 1 or 3, and that has binding activity to the COP1-Trib1 complex; (a4) A polypeptide encoded by the nucleotide sequence shown in SEQ ID NO: 2 or 4.

2. Polypeptide listed in any of the following: (b1) A polypeptide comprising an amino acid sequence in which the amino acid residues at positions 216, 217, and 220 of the amino acid sequence shown in Sequence ID No. 5 are substituted or deleted, having acetyl-CoA carboxylation activity and not having binding activity to the COP1-Trib1 complex; (b2) A polypeptide according to (b1) above, wherein 1 to 230 amino acid residues are substituted, deleted, inserted and / or added (provided that substitutions or deletions at positions 216, 217 and 220 in the amino acid sequence shown in SEQ ID NO: 5 are maintained), has acetyl-CoA carboxylation activity, and does not have binding activity to the COP1-Trib1 complex; (b3) A polypeptide having 90% or more identity with the polypeptide of (b1) above (provided that substitutions or deletions at positions 216, 217, and 220 in the amino acid sequence shown in SEQ ID NO: 5 are maintained), having acetyl-CoA carboxylation activity, and not having binding activity to the COP1-Trib1 complex; (b4) A polypeptide consisting of the amino acid sequence shown in Sequence ID No.

9.

3. Polypeptide listed in any of the following: (c1) A polypeptide comprising an amino acid sequence in which the amino acid residues at positions 358, 359, and 362 of the amino acid sequence shown in Sequence ID No. 7 are substituted or deleted, having acetyl-CoA carboxylation activity and lacking binding activity to the COP1-Trib1 complex; (c2) A polypeptide according to (c1) above, wherein 1 to 240 amino acid residues are substituted, deleted, inserted and / or added (provided that substitutions or deletions at positions 358, 359 and 362 in the amino acid sequence shown in SEQ ID NO: 7 are maintained), has acetyl-CoA carboxylation activity, and does not have binding activity to the COP1-Trib1 complex; (c3) A polypeptide having 90% or more identity with the polypeptide of (c1) above (provided that substitutions or deletions at positions 358, 359, and 362 in the amino acid sequence shown in SEQ ID NO: 7 are maintained), having acetyl-CoA carboxylation activity, and not having binding activity to the COP1-Trib1 complex; (c4) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

11.

4. Polynucleotides as listed in any of the following: (d1) A polynucleotide encoding the polypeptide according to any one of claims 1 to 3; (d2) A polynucleotide consisting of the base sequence shown in SEQ ID NOs: 2, 4, 10, or 12.

5. An expression vector comprising the polynucleotide described in claim 4.

6. A competitive inhibitor of the degradation of acetyl-CoA carboxylase by the COP1-Tribbles complex, comprising at least one selected from the group consisting of (i) to (iii) below as an active ingredient: (i) The polypeptide according to claim 1; (ii) Polynucleotides consisting of the base sequence shown in SEQ ID NO: 2 or 4; (iii) An expression vector containing the polynucleotide described in (ii) above.

7. An anticancer agent comprising, as an active ingredient, at least one selected from the group consisting of a polypeptide according to any one of claims 1 to 3, a polynucleotide according to claim 4, and an expression vector according to claim 5.

8. A method for screening inhibitors of acetyl-CoA carboxylase degradation activity by COP1-Tribbles complexes or anticancer agents, comprising either step (i) or (ii) below: (i) A step of evaluating the ability of the test substance to inhibit the binding of the polypeptide described in claim 1 to one or more selected from the group consisting of COP1-Trib1 complex, COP1-Trib2, and COP1-Trib3; (ii) A step of evaluating the binding ability of a test substance to the polypeptide described in claim 1.

9. A kit for carrying out the screening method of claim 8, comprising at least one selected from the group consisting of (i) to (iii) below: (i) The polypeptide according to claim 1; (ii) Polynucleotides consisting of the base sequence shown in SEQ ID NO: 2 or 4; (iii) An expression vector containing the polynucleotide described in (ii) above.

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