Apoptosis inhibitors and therapeutic agents for neurodegenerative diseases

Abstract

To provide apoptosis inhibitors more suitable for clinical applications.SOLUTION: An apoptosis inhibitor comprising polynucleotides, as an active ingredient, encoding any one of the polypeptides (a-1) to (a-3) is prepared, where (a-1) is a polypeptide of a specific amino acid sequence; (a-2) is a polypeptide having 85% or more sequence identity to the polypeptide (a-1) and exhibits inhibitory action to apoptosis in the cell; (a-3) is a polypeptide of an amino acid sequence having one or several amino acid addition, deletion or substitution in the polypeptide (a-1) and exhibits apoptosis inhibitory action in the cell.SELECTED DRAWING: Figure 3A

Description

[Technical Field]

[0001] The present invention relates to an apoptosis inhibitor and a therapeutic agent for neurodegenerative diseases. Regarding. [Background technology]

[0002] In the brain, genetic background and cellular stress are known to cause region-specific apoptosis and neurodegeneration as aging progresses. Age-related decline in the ubiquitin-proteasome system is thought to be involved in this apoptosis. This ubiquitin-proteasome system can degrade proteins that are conjugated with multiple ubiquitins. Specifically, abnormal proteins are labeled with multiple ubiquitins, and the ubiquitin chains bind to the regulatory unit RP (PA700) of the 26S proteasome. The ubiquitin chains on the target protein are then cleaved, and the target protein is then degraded by the catalytic unit CP (20S proteasome) of the 26S proteasome (see Non-Patent Document 1).

[0003] The present inventors have been conducting research on huntingtin-associated protein 1 (HAP1). HAP1 is a protein localized in stigmoid bodies (STBs), cytoplasmic inclusions specifically distributed in the hypothalamus and other parts of the brain (see Non-Patent Document 2). Focusing on the expression pattern of HAP1 in the brain, the present inventors reported that it is hardly expressed in areas generally prone to neurodegeneration, such as the cerebral cortex, basal ganglia, hippocampus, thalamus, brainstem motor nuclei, cerebellum, and anterior horn of the spinal cord, but is specifically highly expressed in areas less prone to neurodegeneration, such as the hypothalamus and amygdala (see Non-Patent Document 3). HAP1 was identified as a molecule that binds to huntingtin, the causative molecule of Huntington's disease (see Non-Patent Document 4). The present inventors have also demonstrated that HAP1 has a protective effect against cell death in model culture cells of Huntington's disease and spinal and bulbar muscular atrophy (SBMA), and that HAP1 binds to pathogenic proteins during this process (see Non-Patent Document 5). Huntington's disease is a neurodegenerative disease, a hereditary disease caused by abnormal expansion of polyglutamine chains in the huntingtin gene.

[0004] Furthermore, the present inventors have recently demonstrated that HAP1 protects against cell death induced by proteasome inhibitors in cultured cell lines. Proteasome activity is known to decline with aging and is one of the factors that trigger the onset of numerous neurodegenerative diseases. Thus, the cytoprotective effects of HAP1 are expected to be effective not only against inherited neurodegenerative diseases caused by abnormal polyglutamine tracts, such as Huntington's disease and SBMA, but also against many other neurodegenerative diseases associated with aging. However, the mechanism of action remains unclear. Furthermore, research on HAP1, particularly human HAP1, is currently limited worldwide due to the extremely limited number of researchers. Therefore, much remains unknown about the mechanism of HAP1, and the relationship between HAP1 and STB remains unclear.

[0005] In this context, the present inventors have disclosed an apoptosis inhibitor containing a polynucleotide encoding HAP1 as an active ingredient (see Patent Document 1). However, it is thought to be highly difficult to use the entire amino acid sequence of HAP1, which is approximately 600 amino acids in length, as a therapeutic molecule for neurodegenerative diseases. For clinical application, it is thought necessary to identify the amino acid sequence of HAP1 that is essential for cytoprotective action, but no detailed findings on this point existed. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2021-158940 [Non-patent literature]

[0007] [Non-Patent Document 1] Yasushi Saeki Journal of Japanese Biochemical Society 87(6): 705-722 (2015) doi:10.14952 / SEIKAGAKU.2015.870705 [Non-patent document 2] Fujinaga et al., Histochem Cell Biol, Volume 128, 2007, pages 335-348 [Non-patent document 3] Fujinaga et al., J Comp Neurol, Volume 478, Issue 14 October 2004 Pages 88-109 [Non-patent document 4] Li et al., Nature volume 378, pages 398-402 (1995) [Non-patent document 5] Takeshita et al., Human Molecular Genetics, Volume 15, Issue 15, 1 August 2006, Pages 2298-2312 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a drug which is a fragment of HAP1 and has a high apoptosis-suppressing effect. [Means for solving the problem]

[0009] The present inventors have fragmented HAP1, expressed it in cultured cells, and observed the morphology of fragmented HAP1 in the cultured cells. During this process, they fortuitously discovered that a specific HAP1 fragment maintains a structure similar to that in vivo. They then prepared the specific HAP1 fragment and full-length HAP1, and examined whether they had an apoptosis-suppressing effect when transfected with proteasome-inhibiting stress into cultured cells. Surprisingly, they found that a specific N-terminal fragment of HAP1 alone had a higher apoptosis-suppressing effect than full-length HAP1, leading to the completion of the present invention.

[0010] That is, the present invention is as follows. [1] (a-1) a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4; (a-2) a polypeptide having 85% or more sequence identity with a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 and having an inhibitory effect on apoptosis in cells; (a-3) a polypeptide having an amino acid sequence in which one or several amino acids have been added, deleted, or substituted in a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, and which has an inhibitory effect on apoptosis in cells; An apoptosis inhibitor comprising, as an active ingredient, a polynucleotide encoding the polypeptide according to any one of (a-1) to (a-3). [2] (b-1) a polypeptide consisting of 50 to 370 consecutive amino acids from the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4; (b-2) a polypeptide having 85% or more sequence identity with a polypeptide consisting of 50 to 370 consecutive amino acids from the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, and having an inhibitory effect on apoptosis in cells; (b-3) a polypeptide comprising 50 to 370 consecutive amino acids from the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, in which one or several amino acids have been added, deleted, or substituted, and which has an inhibitory effect on apoptosis in cells; An apoptosis inhibitor comprising, as an active ingredient, a polynucleotide encoding the polypeptide according to any one of (b-1) to (b-3). [3] The apoptosis inhibitor described in [1] above, wherein the polypeptide contains at least one amino acid sequence selected from the amino acid sequence described in SEQ ID NO: 5, the amino acid sequence described in SEQ ID NO: 6, the amino acid sequence described in SEQ ID NO: 7, and the amino acid sequence described in SEQ ID NO: 8. [4] The apoptosis inhibitor described in [2] above, wherein the polypeptide contains at least one amino acid sequence selected from the amino acid sequence described in SEQ ID NO: 5, the amino acid sequence described in SEQ ID NO: 6, the amino acid sequence described in SEQ ID NO: 7, and the amino acid sequence described in SEQ ID NO: 8. [5] The apoptosis inhibitor according to any one of [1] to [4] above, wherein the polynucleotide is incorporated into an expression vector. [6] The apoptosis inhibitor according to [5] above, wherein the expression vector is a viral vector. [7] The apoptosis inhibitor according to any one of [1] to [4] above, wherein the polynucleotide is carried in a nucleic acid delivery vehicle. [8] A therapeutic agent for neurodegenerative diseases, comprising the apoptosis inhibitor according to any one of [1] to [8] above. [Effects of the Invention]

[0011] The fragmented HAP1 (1-371aa) (aa stands for amino acid) created in this study has a stronger apoptosis-suppressing effect than endogenous full-length HAP1. Therefore, by creating drugs using polynucleotides encoding polypeptides based on this fragmented HAP1 as active ingredients, it is possible to produce more effective apoptosis-suppressing drugs. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a diagram showing an alignment of the amino acid sequences set forth in SEQ ID NOs: 1 to 4. Identical amino acids are shown in gray. [Figure 2] FIG. 2 shows fragmented regions of HAP1 in Example 1. [Figure 3A] 3A shows the results of Western blot analysis of the effect of apoptosis in an immortalized hypothalamic cell line, examined by the band of cleaved poly(ADP-ribose) polymerase (cleaved PARP: cPARP), when a polynucleotide encoding full-length HAP1 (HAP1(Full)) or fragmented HAP1 (1-371aa) was introduced or not introduced, in Example 1. The three lanes on the right show the results when MG132 dissolved in DMSO was added to the cells, and the two lanes on the left show the results when DMSO was added to the cells (control without MG132). [Figure 3B] Figure 3B is a graph showing the relative values ​​of the area of ​​the cleaved PARP band when a polynucleotide encoding HAP1(1-371) is introduced in the Western blot analysis of Figure 3A in Example 1, where the area of ​​the cleaved PARP band when a polynucleotide encoding full-length HAP1 (Full) is introduced in the presence of MG132 added to cells is set to 1. Note that all values ​​were normalized based on the area of ​​the α-tubulin band in the same lane. [Figure 3C]Figure 3C shows the results of Western blot analysis in Example 1, in which the effect of apoptosis in an immortalized hypothalamic cell line upon introduction of a polynucleotide encoding three types of fragmented HAP1 (1-371aa, 474-577aa, 578-599aa) was examined using cleaved PARP bands. [Figure 4] 4 shows the results of morphological observation of cultured cells expressing polynucleotides encoding full-length HAP1 (HAP1(Full)) and fragmented HAP1 (aa 1-371, aa 474-577, aa 578-599) in Example 2. The first row shows photographs taken with the addition of DMSO, and the second to fourth rows show photographs taken with the addition of MG132. DETAILED DESCRIPTION OF THE INVENTION

[0013] The contents of all patent and non-patent literature cited herein are hereby incorporated by reference in their entirety.

[0014] One aspect of the apoptosis inhibitor herein is (a-1) a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4; (a-2) a polypeptide having 85% or more sequence identity with a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 and having an inhibitory effect on apoptosis in cells; (a-3) a polypeptide having an amino acid sequence in which one or several amino acids have been added, deleted, or substituted in a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, and which has an inhibitory effect on apoptosis in cells; Any apoptosis inhibitor A may be used, which contains as an active ingredient a polynucleotide encoding a polypeptide according to any one of (a-1) to (a-3) above (hereinafter also referred to as "polypeptide A"), and which will hereinafter also be referred to as "apoptosis inhibitor A." Another aspect of the apoptosis inhibitor herein is (b-1) a polypeptide consisting of 50 to 370 consecutive amino acids from the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4; (b-2) a polypeptide having 85% or more sequence identity with a polypeptide consisting of 50 to 370 consecutive amino acids from the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, and having an inhibitory effect on apoptosis in cells; (b-3) a polypeptide comprising 50 to 370 consecutive amino acids from the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, in which one or several amino acids have been added, deleted, or substituted, and which has an inhibitory effect on apoptosis in cells; Any apoptosis inhibitor B containing as an active ingredient a polynucleotide encoding a polypeptide described in any one of (b-1) to (b-3) above (hereinafter also referred to as "polypeptide B"), and hereinafter also referred to as "the present apoptosis inhibitor B." The present apoptosis inhibitor A and the present apoptosis inhibitor B will hereinafter be collectively referred to simply as "the present apoptosis inhibitor."

[0015] The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of amino acids 1-371 in rat HAP1 protein, in other words, the amino acid sequence of rat HAP1 protein (accession number NP_077047.2) in which amino acids from position 372 to the C-terminus are deleted and amino acids at positions 5 and 308 are substituted. Note that D (aspartic acid) at position 5 and lysine (K) at position 308 in the amino acid sequence of SEQ ID NO: 1 were derived by the present inventors through cloning analysis. The amino acid sequence set forth in SEQ ID NO: 2 is the amino acid sequence of amino acids 1-370 in the mouse HAP1 protein (accession number NP_034534.1), in other words, the amino acid sequence of the mouse HAP1 protein set forth in SEQ ID NO: 2, lacking amino acids from position 371 to the C-terminus. The amino acid sequence set forth in SEQ ID NO: 3 is the amino acid sequence of amino acids 1-398 in human HAP1 protein isoforms 1, 2, or 4 (accession numbers P54257.3, NP_817084.2, and NP_001073340.1, respectively), in other words, the amino acid sequence of human HAP1 protein isoforms 1, 2, and 4 set forth in SEQ ID NO: 3, lacking amino acids from position 399 to the C-terminus. The amino acid sequence of SEQ ID NO: 4 is the amino acid sequence of amino acids 1 to 409 in human HAP1 protein isoform 3 (accession number NP_001073339.1), in other words, the amino acid sequence of human HAP1 protein isoform 3 in which amino acids from position 410 to the C-terminus are deleted from the amino acid sequence of SEQ ID NO: 4. The amino acid sequences of SEQ ID NOs: 1 to 4 are shown in FIG. In Figure 1, the three domains surrounded by squares represent coiled-coil domains (from the N-terminus, coiled-coil domain 1 (169-205 in the case of SEQ ID NO: 1), coiled-coil domain 2 (262-305 in the case of SEQ ID NO: 1), and coiled-coil domain 3 (328-352 in the case of SEQ ID NO: 1)), the underlined domain represents the KIF5B (Kinesin Family Member 5B) interacting domain (153-320 in the case of SEQ ID NO: 1), the dotted line represents the TATA-binding protein interacting domain (158-262 in the case of SEQ ID NO: 1), and the dotted line represents the huntingtin-binding domain (278-352 in the case of SEQ ID NO: 1).

[0016] ■ The apoptosis inhibitor in question In the case of polypeptide A of the present apoptosis inhibitor A, sequence identity of 85% or more with a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 preferably includes sequence identity of 88% or more, 90% or more, 92% or more, 95% or more, 98% or more, or 99% or more with a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4.

[0017] In polypeptide A of the present apoptosis inhibitor A, the above-mentioned "one or several" in the "amino acid sequence in which one or several amino acids have been added, deleted or substituted" can include 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 3, 1 to 2, or 1.

[0018] In the polypeptide A of the present apoptosis inhibitor A, as long as it contains the polypeptide described in (a-1) to (a-3) above and can maintain its apoptosis inhibitory effect in cells, any amino acid sequence having an amino acid length of 30% or less, 20% or less, 10% or less, 5% or less, or 3% or less of the amino acid length of the polypeptide described in (a-1) to (a-3) above may be added to the N-terminus and / or C-terminus of the polypeptide described in (a-1) to (a-3) above. The length of the added amino acids can be adjusted appropriately depending on the amino acid length of the polypeptide described in (a-1) to (a-3), and examples include 1 to 150, 3 to 100, 5 to 30, and 10 to 20. Examples of such any sequence include a signal sequence and a labeling sequence.

[0019] In polypeptide B of the present apoptosis inhibitor B, the polypeptide consisting of 50 to 370 consecutive amino acids in the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 4 may be a polypeptide consisting of 50 to 370 consecutive amino acids from the N-terminus, a sequence consisting of 50 to 370 consecutive amino acids from the C-terminus, or a polypeptide consisting of 50 to 370 consecutive amino acids from a predetermined position on the N-terminus or C-terminus in the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 4. Furthermore, the 50 to 370 consecutive amino acids may be 80 to 350 consecutive amino acids, 100 to 330 consecutive amino acids, 150 to 300 consecutive amino acids, or 200 to 280 consecutive amino acids.

[0020] In polypeptide B of the present apoptosis inhibitor B, the "one or several" in the "amino acid sequence in which one or several amino acids are added, deleted or substituted" depends on the amino acid length of polypeptide B, but may include 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 3, 1 to 2, or 1.

[0021] In the polypeptide B of the present apoptosis inhibitor B, as long as it contains the polypeptide described in (b-1) to (b-3) above and can maintain its apoptosis inhibitory effect in cells, any amino acid sequence having an amino acid length of 30% or less, 20% or less, 10% or less, 5% or less, or 3% or less of the amino acid length of the polypeptide described in (b-1) to (b-3) above may be added to the N-terminus and / or C-terminus of the polypeptide described in (b-1) to (b-3) above. The length of the added amino acids can be adjusted appropriately depending on the amino acid length of the polypeptide described in (b-1) to (b-3), and examples of the length include 1 to 280, 3 to 100, 5 to 30, and 10 to 20.

[0022] Polypeptide A or B in the present apoptosis inhibitor may contain at least one, specifically one, two, three, or four predetermined domain amino acid sequences selected from the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 6, the amino acid sequence set forth in SEQ ID NO: 7, and the amino acid sequence set forth in SEQ ID NO: 8. In the amino acid sequences set forth in SEQ ID NOs: 5 to 8, "X" represents any amino acid. In the amino acid sequence of SEQ ID NO: 5, it is preferable that the 15th X from the N-terminus is N (asparagine) or S (serine), the 19th X is T (threonine) or A (alanine), the 20th X is M (methionine) or L (leucine), the 25th X is R (arginine) or K (lysine), the 30th X is H or Y, and the 33rd X is K (lysine) or H (histidine). The amino acid sequence of SEQ ID NO: 6 is such that the first X from the N-terminus is L, A or Q, the third X is H or R, the ninth X is A or T, the eleventh X is K or Q (glutamine), the twelfth X is Q or E, the fifteenth X is K or R, the twenty-second X is D or H, the twenty-third X is H or Q, the thirty-third X is H or Q, the thirty-third X is N or T, and the thirty-seventh X is K or E. In the amino acid sequence of SEQ ID NO: 7, it is preferable that the third X from the N-terminus is G or N, the ninth X is K or Q, the eleventh X is I (isoleucine) or V (valine), the twelfth X is T or A, the thirteenth X is Q or R, the seventeenth X is E or Q, the eighteenth X is I or V, and the nineteenth X is T or L. In the amino acid sequence of SEQ ID NO: 8, it is preferable that the 6th X from the N-terminus is D or H, the 7th X is H or Q, the 14th X is H or Q, the 17th X is N or T, the 21st X is K or E (glutamic acid), the 31st X is K or E, the 53rd X is G or N, the 59th X is K or Q, the 61st X is I or V, the 62nd X is T or A, the 63rd X is Q or R, the 67th X is E or Q, the 68th X is I or V, and the 69th X is T or L. The amino acid sequences set forth in SEQ ID NOS: 5, 6, and 7 are predicted to be coiled-coil domains (Simone Engelender et al., Human Molecular Genetics, 1997, Vol. 6, No. 13, pp. 2205-2212), which are predicted to function as aggregation sites between HAP1 molecules or between HAP1 and other molecules. The amino acid sequence set forth in SEQ ID NOS: 8 is the sequence of a domain disclosed to have the function of binding to huntingtin (Shi-Hua Li et al., Journal of Neuroscience, 15 February 1998, 18 (4), 1261-1269). The amino acid sequences set forth in SEQ ID NOS: 5 to 8 are shown in Table 1. In addition, polypeptide A or B in the present apoptosis inhibitor may include the sequence of a domain disclosed as a KIF5B (Kinesin Family Member 5B) interacting site set forth in any one of SEQ ID NOS: 11 to 14 (Alison E Twelvetrees et al., Neuron. 2010 Jan 14;65(1):53-65. doi: 10.1016 / j.neuron.2009.12.007.), or the sequence of a domain disclosed as a TATA-binding protein interacting site set forth in any one of SEQ ID NOS: 15 to 17.

[0023] [Table 1]

[0024] Furthermore, when polypeptide A contains the amino acid sequence of the above-mentioned predetermined domain, "(a-2) 85% or more sequence identity with a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4" can be "(a-2) 85% or more sequence identity with a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 (excluding the region of the amino acid sequence of the above-mentioned predetermined domain)," and "(a-3) an amino acid sequence in which one or several amino acids have been added, deleted, or substituted in a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4" can be "(a-3) an amino acid sequence in which one or several amino acids have been added, deleted, or substituted in a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 (excluding the amino acid sequence of the above-mentioned predetermined domain)."

[0025] Furthermore, when polypeptide B contains the amino acid sequence of the above-mentioned predetermined domain, "(b-2) 85% or more sequence identity to a polypeptide consisting of 50 to 370 consecutive amino acids from the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4" can be "85% or more sequence identity to a polypeptide consisting of 50 to 370 consecutive amino acids from the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 (excluding the amino acid sequence of the above-mentioned predetermined domain)", and "(b-3) an amino acid sequence in which one or several amino acids have been added, deleted, or substituted in a polypeptide consisting of 50 to 370 consecutive amino acids from the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4" can be "(b-3) an amino acid sequence in which one or several amino acids have been added, deleted, or substituted in a polypeptide consisting of 50 to 370 consecutive amino acids from the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 (excluding the amino acid sequence of the above-mentioned predetermined domain)".

[0026] It is preferable that the N-terminus and / or C-terminus of polypeptide A or B in the present apoptosis inhibitor is not supplemented with the amino acid sequence set forth in SEQ ID NO: 9 and / or 10, an amino acid sequence in which one or several amino acids have been added, deleted, or substituted in a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 9 and / or 10, or a partial sequence of the full length or consecutive 20 or more amino acids, preferably 30 or more, more preferably 50 or more, even more preferably 70 or more, and particularly preferably 100 or more, in the amino acid sequence set forth in SEQ ID NO: 9 and / or 10. In the above-mentioned polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 9 and / or 10, "one or several" in "an amino acid sequence in which one or several amino acids have been added, deleted, or substituted" can be 1 to 5, 1 to 3, 1 to 2, or 1.

[0027] The polynucleotide in the present apoptosis inhibitor is not particularly limited as long as it is a polynucleotide B encoding the polypeptide A described in the present apoptosis inhibitor A or the polypeptide described in the present apoptosis inhibitor B. Codons selected for encoding amino acids may be optimized appropriately depending on the type of host cell in which the polypeptide is to be expressed. Specifically, for example, an example of a polynucleotide encoding a polypeptide consisting of any of the amino acid sequences set forth in SEQ ID NO: 1 is a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 18. Furthermore, in the present apoptosis inhibitor B, polypeptide B may be a polynucleotide encoding multiple identical or different polypeptides B, specifically two, three, or four identical or different polypeptides B. In this case, the polynucleotide may encode an arbitrary amino acid sequence having an amino acid length of 30% or less, 20% or less, 10% or less, 5% or less, or 3% or less of the total amino acid length of each polypeptide B, attached to the N-terminus and / or C-terminus of any of the polypeptides B, or to the N-terminus and / or C-terminus of all of the polypeptides B, or attached between the multiple polypeptides. Examples of such arbitrary sequences include signal sequences and tagging sequences.

[0028] The apoptosis inhibitory activity of the polypeptide of the present invention can be assessed by gene transfer or other methods. For example, a polynucleotide encoding the polypeptide to be tested can be expressed in cultured cells, and the accumulation of apoptosis-indicating proteins, such as cPARP and cleaved caspase 3 (cCas3), in the presence and absence of the polynucleotide can be confirmed by known genetic engineering techniques, such as Western blot analysis. In this case, the accumulation of apoptosis-indicating proteins can be confirmed in the same manner as above, under conditions in which apoptosis is induced in cultured cells in the presence of a proteasome inhibitor such as MG132. That is, if the accumulation of apoptosis-indicating proteins, such as cPARP and cCas3, in cultured cells is reduced, preferably by 10% or more, and more preferably by 20% or more, compared to a control without the polypeptide to be tested or a full-length HAP1 polypeptide, the polypeptide to be tested can be evaluated as having apoptosis inhibitory activity. If the accumulation of apoptosis-indicating proteins, such as cPARP and cCas3, is not reduced or reduced by 5% or less compared to the control, the polypeptide can be evaluated as having no apoptosis inhibitory activity or as having a low apoptosis inhibitory activity.

[0029] "Apoptosis" in the context of this apoptosis inhibitor refers to a type of cell death induced by condensation of nuclear chromatin and DNA fragmentation. This type of cell death is distinct from necrosis, which occurs when the cell membrane is physically damaged. While apoptosis can be triggered by various factors, it is preferable that it be apoptosis caused by the accumulation of ubiquitinated proteins within the cell. In other words, it can be said to be apoptosis caused by a decrease in the activity of the ubiquitin-proteasome system.

[0030] Ubiquitin is a protein consisting of 76 amino acids. It covalently binds to a target protein, and an isopeptide can be bound between a lysine residue in the ubiquitin and the C-terminal glycine in a new ubiquitin molecule. Repeated condensation reactions between ubiquitin molecules result in the formation of a polyubiquitin chain, in which multiple ubiquitin molecules are extended in a chain-like fashion. The proteasome uses this polyubiquitin chain as a signal to degrade proteins bearing the polyubiquitin chain.

[0031] The proteasome refers to an enzyme complex present in the cytoplasm that is involved in proteolysis and acts to cleave unnecessary proteins in the cell into peptides. Specific examples include the 20S proteasome and the 26S proteasome, which is formed by binding the 19S complex and the 11S complex.

[0032] Furthermore, the apoptosis is preferably apoptosis in nerve cells. Examples of such nerve cells include nerve cells that contain acetylcholine, monoamines containing only one amino group, polypeptides, nitric oxide (NO), carbon monoxide (CO), agmatine, anandamide, dimethyltryptamine, adenosine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and taurine. Examples of the types of nerve cells include nerve cells in the central nervous system, peripheral nervous system, or enteric nervous system.

[0033] Examples of the monoamines include dopamine, noradrenaline, adrenaline, serotonin, melatonin, and histamine. Examples of the polypeptides include opioids such as adrenocorticotropic hormone, beta-lipotropin, dynorphin, endorphin, enkephalin, and leumorphin, peptides such as N-acetylaspartylglutamic acid, neuropeptide Y, pancreatic peptide, peptide YY, bombesin, gastrin-releasing peptide, neurotensin, galanin, and calcitonin gene-related peptide, gastrins such as gastrin and cholecystokinin, and posterior pituitary hormones such as vasopressin, oxytocin, neurophysin I, and neurophysin II. Examples of such neuronal cells include amino acids such as aspartic acid, glutamic acid, γ-aminobutyric acid, and glycine, peptide hormones such as bombesin, gastrin-releasing peptide (GRP), neurotensin, galanin, and calcitonin gene-related peptide (CGRP), secretins such as secretin, motilin, glucagon, vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, and growth hormone-releasing factor, and tachykinins such as somatostatin, neurokinin A, neurokinin B, neuropeptide A, gamma neuropeptide, and substance P. Of the above, preferred examples of such neuronal cells include acetylcholine-containing neurons, such as motor neurons, and neurons containing dopamine, noradrenaline, adrenaline, serotonin, melatonin, or histamine.

[0034] In the present apoptosis inhibitor, polypeptide A or polypeptide B may have the ability to bind to a ubiquitinated protein in a cell. The polypeptide that has the ability to bind to a ubiquitinated protein in a cell is not particularly limited as long as it is a peptide that can co-localize with and bind to a ubiquitinated protein when expressed in a cell. A method for confirming whether or not a polypeptide co-localizes with a ubiquitinated protein can be, for example, by fluorescently labeling the polypeptide and ubiquitin in the cell and observing them under a fluorescence microscope.

[0035] The polynucleotide is preferably incorporated into an expression vector, which may be in any form, such as circular or linear. Furthermore, in addition to the polynucleotide encoding the polypeptide described in the present apoptosis inhibitor, such an expression vector may contain other desired polynucleotides as needed. Examples of other polynucleotides include those encoding enhancer sequences, promoter sequences, ribosome binding sequences, signal peptides, etc.

[0036] The expression vector can be appropriately selected depending on the host cell to be used, and may be a viral vector such as adeno-associated virus, retrovirus, adenovirus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, or Sendai virus, or a plasmid, or a commercially available expression vector may be used.

[0037] Examples of the host cells include mammalian cells from humans, monkeys, mice, rats, hamsters, and the like.

[0038] The polynucleotide may be held in a nucleic acid delivery vehicle. The nucleic acid delivery vehicle is not particularly limited as long as it can hold the polynucleotide and deliver it to cells, particularly neurons. Specific examples of the nucleic acid delivery vehicle include membrane vesicles made of lipid bilayer membranes, surfactant peptides, lipid nanoparticles (LNPs), cationic polymers, non-cationic polymers, β-glucan, atelocollagen, PLGA nanoparticles, and superapatite. Examples of the membrane vesicles made of lipid bilayer membranes include exosomes, cationic liposomes, and non-cationic liposomes. Here, the term "held in a nucleic acid delivery vehicle" refers to a state in which the polynucleotide is attached to the nucleic acid delivery vehicle, as well as a state in which the polynucleotide is encapsulated in the nucleic acid delivery vehicle.

[0039] The present apoptosis inhibitor can be administered to mammals suffering from neurodegenerative diseases for the treatment of such diseases. Examples of such neurodegenerative diseases include tauopathies such as Alzheimer's disease, synucleinopathies such as Parkinson's disease, polyglutamine diseases such as Huntington's disease, spinal and bulbar muscular atrophy (SBMA), and spinocerebellar degeneration, and TDP-43 proteinopathies such as amyotrophic lateral sclerosis. The route of administration is not particularly limited, but parenteral administration is preferred. Parenteral administration may be systemic (e.g., intravenous) or local (e.g., intramuscular, transdermal, transmucosal, intracranial, or intrathecal) administration. The formulation may also contain pharmaceutically acceptable additives commonly used in formulations, such as excipients, binders, lubricants, disintegrants, preservatives, isotonicity agents, stabilizers, dispersants, antioxidants, colorants, flavorings, and buffers. The present apoptosis inhibitor may be administered in the form of a solid formulation, such as a powder or granules.

[0040] There are no particular limitations on the appropriate dosage when administered to mammalian cells that have developed a neurodegenerative disease, and it can be adjusted as appropriate depending on the physical condition, condition, weight, age, sex, etc. of the subject or test animal. For example, when administered in the form of an injection, the dosage can be about 0.01 mg to 60 g per day, preferably about 0.1 mg to 24 g, and more preferably about 0.1 mg to 6 g. The administration interval can be set to once to several times a day, or at intervals of one day to two weeks.

[0041] The apoptosis inhibitor of the present invention can also be used in combination with other therapeutic agents for neurodegenerative diseases. In such cases, both agents may be administered to a subject simultaneously or at different times. The term "combined use" refers to overlapping administration of two or more drugs, including but not limited to simultaneous administration.

[0042] The present invention will be described in more detail below with reference to examples. The examples are not limiting.

[0043] [Example 1] Cell death suppression effect of fragmented HAP1 First, we investigated the relationship between fragmented HAP1 and apoptosis in cultured cells. Specifically, we examined the relationship between HAP1 expression and apoptosis in cells treated with the proteasome inhibitor MG132 using the following method. Hap1-cDNA was divided into three fragments: the N-terminus (1-371), the central fragment (474-577), and the C-terminus (578-599), as shown in Figure 2. Each fragment was then incorporated into a plasmid vector containing a polynucleotide encoding GFP. Fragmented HAP1 extracted from Hap1a-cDNA was transiently transfected into immortalized mouse fetal hypothalamic cells (hereafter referred to as "cultured cells"). MG132 dissolved in DMSO was added to the culture medium at a concentration of 10 μM as a proteasome inhibitor (DMSO alone was added as a control). After 6 hours, the cells were harvested and subjected to Western blot analysis. The results of Western blot analysis are shown in Figure 3A. Figure 3B shows a graph of the relative area of ​​the cleaved PARP band when a polynucleotide encoding HAP1 (1-371) was introduced, relative to the area of ​​the cleaved PARP band when a polynucleotide encoding full-length HAP1 (Full) was introduced in the Western blot analysis of Figure 3A after MG132 was added to cells. Furthermore, Figure 3C shows the results of Western blot analysis of cells transformed with plasmid vectors containing polynucleotides encoding the N-terminus (1-371), middle (474-577), and C-terminus (578-599). MG132 is a proteasome inhibitor that inhibits the active subunit of the 26S proteasome, and treatment with MG132 inhibits the degradation of substrate proteins by the 26S proteasome.

[0044] The results in Figure 3A and B show that cultured cells transfected with fragmented HAP1 (1-371aa) exhibited a stronger inhibitory effect on cell death than cells transfected with endogenous full-length HAP1 (Full) when MG132 was added. On the other hand, the results in Figure 3C show that when a similar analysis was performed on cultured cells transfected with fragmented HAP1 (474-577aa) or fragmented HAP1 (578-599aa), no such strong inhibitory effect on cell death as with fragmented HAP1 (1-371aa) was observed.

[0045] [Example 2] Morphological characteristics of fragmented HAP1 In a previous study (Non-Patent Document 5) the present inventors obtained results suggesting that in cultured cells transfected with full-length HAP1, ubiquitinated proteins that accumulate intracellularly upon proteasome inhibition are captured by full-length HAP1. This is thought to prevent abnormal proteins, in other words, ubiquitinated proteins, from diffusing into the cytoplasm and damaging other organelles, which may contribute to the suppression of cell death.

[0046] To investigate whether fragmented HAP1 retains the same property (ubiquitinated protein binding ability), we expressed each fragmented HAP1 in cultured cells and observed their morphology. For fluorescence observation, a peptide encoding GFP was attached to the N-terminus of each fragmented HAP1. The results are shown in Figure 4. The scale bar in Figure 4 is 20 μm.

[0047] As shown in Figure 4, fragmented HAP1 (1-371) formed granular structures (stigmoid bodies (STBs)) similar to those of full-length HAP1, but not other fragmented HAP1s. Furthermore, upon MG132 addition, cells transfected with full-length HAP1 and fragmented HAP1 (1-371) showed perinuclear ring-like accumulation of HAP1. Furthermore, immunohistochemistry revealed that in cells transfected with full-length HAP1 and fragmented HAP1 (1-371), intracellular HAP1 structures colocalized with ubiquitinated proteins. These findings confirm that the amino acid sequence from positions 1 to 371 of the HAP1 amino acid sequence is involved in STB formation and binding to ubiquitinated proteins. Therefore, the cell-protective (apoptosis-inhibiting) effect of fragmented HAP1 is likely to be effective against many neurodegenerative diseases characterized by the accumulation of abnormal proteins within cells and their pathological features of ubiquitination. In other words, unlike conventional treatments that focus on "each pathogenic protein," cell death inhibitors using fragmented HAP1 are expected to be highly versatile therapeutic molecules that are independent of the type of neurodegenerative disease.

Claims

1. (a-1) a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4; (a-2) a polypeptide having 85% or more sequence identity with a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 and having an inhibitory effect on apoptosis in cells; (a-3) a polypeptide having an amino acid sequence in which one or several amino acids have been added, deleted, or substituted in a polypeptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, and which has an inhibitory effect on apoptosis in cells; An apoptosis inhibitor comprising, as an active ingredient, a polynucleotide encoding the polypeptide according to any one of (a-1) to (a-3).

2. (b-1) a polypeptide consisting of 50 to 370 consecutive amino acids from the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4; (b-2) a polypeptide having 85% or more sequence identity with a polypeptide consisting of 50 to 370 consecutive amino acids among the amino acid sequences set forth in any one of SEQ ID NOs: 1 to 4, and having an inhibitory effect on apoptosis in cells; (b-3) a polypeptide comprising 50 to 370 consecutive amino acids in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4, in which one or several amino acids have been added, deleted, or substituted, and which has an inhibitory effect on apoptosis in cells; An apoptosis inhibitor comprising, as an active ingredient, a polynucleotide encoding the polypeptide according to any one of (b-1) to (b-3).

3. The apoptosis inhibitor according to claim 1, wherein the polypeptide contains at least one amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 6, the amino acid sequence set forth in SEQ ID NO: 7, and the amino acid sequence set forth in SEQ ID NO:

8.

4. The apoptosis inhibitor according to claim 2, wherein the polypeptide contains at least one amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 6, the amino acid sequence set forth in SEQ ID NO: 7, and the amino acid sequence set forth in SEQ ID NO:

8.

5. The apoptosis inhibitor according to any one of claims 1 to 4, wherein the polynucleotide is incorporated into an expression vector.

6. The apoptosis inhibitor according to claim 5, wherein the expression vector is a viral vector.

7. The apoptosis inhibitor according to any one of claims 1 to 4, wherein the polynucleotide is carried in a nucleic acid delivery vehicle.

8. A therapeutic agent for neurodegenerative diseases, comprising the apoptosis inhibitor according to any one of claims 1 to 7.

Images