WO2017126461A1 - がん治療用ペプチド及びそれを含む医薬組成物 - Google Patents
がん治療用ペプチド及びそれを含む医薬組成物 Download PDFInfo
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- C07—ORGANIC CHEMISTRY
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
- C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/03—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof
Definitions
- the present invention relates to a peptide useful for cancer treatment and a pharmaceutical composition containing the peptide.
- Estrogen receptor ⁇ (Estrogen-receptor: ER ⁇ ) plays a central role in the development and progression of breast cancer.
- Recent endocrine therapies for breast cancer use selective ER ⁇ modulators (eg, tamoxifen and raloxifene), ER ⁇ downregulators (eg, fulvestrant), and aromatase inhibitors (AI), and ER ⁇ signaling Is mainly targeted (Non-Patent Documents 1 to 3).
- tamoxifen which inhibits breast cancer cell growth through competitive binding to ER ⁇ , is a standard therapy for ER ⁇ -positive breast cancer patients.
- Non-Patent Documents 4 and 5 tamoxifen therapy is often ineffective and patients may relapse and die from endocrine resistant tumors.
- AI which blocks estrogen synthesis, also has substantial clinical effects, such as better efficacy, a significant increase in relapse-free survival, and an extended relapse period in postmenopausal women compared to tamoxifen, Some patients who have received AI therapy relapse (Non-Patent Documents 6 and 7). The exact molecular events that affect the effectiveness of these endocrine therapies have not yet been elucidated.
- a complex of brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3), which is a cancer protein, and prohibitin 2 (prohibitin 2: PHB2), which is a tumor suppressor, is a complex of ⁇ in a breast cancer. It plays a central role in estrogen signal regulation (Non-Patent Documents 8 and 9).
- BIG3 inhibits PHB2's ability to bind to PHB2 and repress estrogen-dependent transcriptional activation, resulting in constitutive ER ⁇ activity. Based on these findings, the strategy of releasing PHB2 from the complex with BIG3 by inhibiting the BIG3-PHB2 interaction and exerting the tumor suppressive activity of PHB2 can be a novel therapy for breast cancer.
- Patent Document 1 a dominant negative peptide of BIG3 that specifically inhibits the BIG3-PHB2 interaction. It has been confirmed that this peptide inhibits the ER ⁇ signal pathway that leads to breast cancer growth and represses breast cancer growth by reactivating the tumor suppressive activity of PHB2 (Patent Document 1).
- the dominant negative peptide is not highly stable, and the duration of the inhibitory effect on the BIG3-PHB2 interaction is not so long. For clinical applications, it is desirable that the inhibitory effect last for a longer period.
- an object of the present invention is to provide a peptide having a longer duration of the inhibitory effect on the BIG3-PHB2 interaction.
- the present inventors have found that the duration of the inhibitory effect on the BIG3-PHB2 interaction is improved by introducing a stapling structure into the molecule of the dominant negative peptide, thereby completing the present invention. did. That is, the present invention provides the following peptides, and uses thereof:
- a peptide comprising an amino acid sequence represented by SEQ ID NO: 9 or a partial sequence thereof, wherein n pairs (n is a natural number) of amino acid residues are replaced with n stapling structures, or a salt thereof ;
- the present invention provides a method for treating cancer comprising the step of administering the peptide according to any one of [1] to [11] above, or a salt thereof to a subject in need of treatment.
- the present invention also relates to the use of the peptide according to any one of the above [1] to [11] or a salt thereof in the production of a pharmaceutical composition for treating cancer.
- the present invention relates to the use of the peptide according to any one of [1] to [11] above or a salt thereof in the treatment of cancer.
- the present invention also relates to a method for producing a pharmaceutical composition for treating cancer, comprising a step of mixing or blending the peptide according to any one of [1] to [11] above or a salt thereof with a carrier.
- a peptide having a longer duration of the inhibitory effect on the BIG3-PHB2 interaction is provided.
- the pharmaceutical composition containing the peptide of the present invention can be applied to the treatment of cancer.
- FIG. 1 is a schematic diagram of stapled ERAP synthesis.
- FIG. 1A shows a synthetic scheme of amino acid derivatives used for stapled ERAP synthesis.
- (I) to (vi) show the reagents and amino acid synthesis conditions in each reaction: (i) 2,4-dimethoxybenzaldehyde, AcOH, MgSO 4 , CH 2 Cl 2 ; (ii) NaBH 4 , MeOH, CH 2 Cl 2 , 87% yield (2 steps); (iii) Compound 2, EDC ⁇ HCl, DIPEA, CH 2 CH 2 , 76% yield; (iv) LiOH ⁇ H 2 O, THF, MeOH, H 2 O (V) TBSOTf, 2,6-lutidine, CH 2 CH 2 ; (vi) Fmoc-OSu, Na 2 CO 3 , THF, H 2 O, 90% yield (2 steps).
- FIG. 1B shows a scheme for stapling synthesis of ERAP by ring-closing olefin metathesis.
- FIG. 1C shows a scheme for stapling synthesis of ERAP via intramolecular amidation.
- FIG. 2 shows that stapled ERAP has a long-term stable inhibitory effect on BIG3-PHB2 interaction.
- FIG. 2A shows the primary structure of ERAP (No. 9) and stapled ERAP (No. 1 to 8).
- underlined bold letters indicate amino acid residues important for PHB2 binding, and italic bold letters indicate stapled amino acid residues. All amino acid sequences are written in the C-terminal direction with the left end as the N-terminal and rightward.
- FIG. 1B shows a scheme for stapling synthesis of ERAP by ring-closing olefin metathesis.
- FIG. 1C shows a scheme for stapling synthesis of ERAP via intramolecular amidation.
- FIG. 2B shows the results of an MTT assay that evaluated the inhibitory effect of 11R-ERAP and stapled ERAP (No. 2-6) on 17 ⁇ -estradiol (E2) dependent cell proliferation of human breast cancer cell line MCF-7 cells.
- E2 addition group 10 nM of E2 was added.
- the type of peptide added is shown in the upper left of each graph.
- Each symbol in each graph represents the following: white circle ⁇ : untreated; black circle ⁇ : E2 alone; black triangle ⁇ : E2 + 0.5 ⁇ M peptide; black square ⁇ : E2 + 1 ⁇ M peptide; black diamond ⁇ : E2 + 10 ⁇ M peptide.
- FIG. 2C shows the results of an MTT assay that evaluated the inhibitory effect of 11R-ERAP and stapled ERAP (Nos. 2-6) on cell proliferation of human mammary epithelial cell line MCF-10A cells.
- the type of peptide added is shown in the upper left of each graph.
- Each symbol in each graph indicates the following; open circle: untreated; filled triangle: 0.5 ⁇ M peptide; filled square: 1 ⁇ M peptide; filled diamond: 10 ⁇ M peptide.
- Data represent the mean ⁇ SD of 3 independent experiments ( *** P ⁇ 0.001, two-sided student t-test).
- FIG. 2D shows the percent inhibition of ECF-dependent cell proliferation of MCF-7 cells and cell proliferation of MCF-10A cells inhibited by the addition of 11R-ERAP or stapled ERAP (No. 2-6).
- the numerical value of the inhibition rate was calculated from the results of the MTT assay in FIGS. 2B and 2C.
- underlined bold letters indicate amino acid residues important for PHB2 binding, and italic bold letters indicate stapled amino acid residues.
- FIG. 2E shows the results of MTT assay in which the inhibitory effect of 11R-ERAP and stapled ERAP (Nos. 2 to 6) on the cell proliferation of MCF-10A cells was evaluated. The values at 48 hours in FIG.
- 2C are shown in a bar graph.
- “( ⁇ )” indicates unprocessed.
- Data represent the mean ⁇ SD of 3 independent experiments ( *** P ⁇ 0.001, two-sided student t-test).
- 2F-1 shows stapled ERAP No. 2.
- 3 treatment and stapled ERAP No. 6 shows the results of analysis of genes that are differentially expressed in MCF-10A cells in 6 treatments.
- the above figure shows stapled ERAP No. 3 treatment or stapled ERAP No. 6 is a heat map image obtained by analyzing gene expression 24 hours and 48 hours after treatment in 6-treated MCF-10A cells.
- the figure below shows the stapled ERAP No. 48 hours after processing. Compared with the 3 treatment, the stapled ERAP No.
- FIG. 6 shows the results of DAVID-based gene annotation enrichment analysis of 284 genes that were significantly up-regulated or down-regulated 100-fold or more in MCF-10A cells by 6 treatments.
- 2F-2 shows the stapled ERAP No. 48 hours after the processing.
- the stapled ERAP No. 6 shows the results of analysis based on GeneMANIA software for 284 genes that were significantly up-regulated or down-regulated 100-fold or more in MCF-10A cells by 6 treatments.
- FIG. 2G shows the results of co-immunoprecipitation evaluating the inhibitory effect of stapled ERAP (No. 2 and No. 3) treatment on BIG3-PHB2 interaction in MCF-7 cells.
- 11R-ERAP was used as a positive control for BIG3-PHB2 interaction inhibition.
- IP represents the antibody used for immunoprecipitation
- WCL represents whole cell lysate
- ( ⁇ ) represents E2 untreated
- ⁇ represents untreated peptide.
- the antibody reacted on the membrane is shown at the right end. The number below the membrane indicates the relative value of the reaction intensity of each lane when the reaction intensity of the leftmost lane is 1.0.
- FIG. 2H shows the results of surface plasmon resonance interaction analysis evaluating the affinity of 11R-ERAP and stapled ERAP (No. 2 and No. 3) for His-tagged recombinant PHB2.
- FIG. 1 shows the results of surface plasmon resonance interaction analysis evaluating the affinity of 11R-ERAP and stapled ERAP (No. 2 and No. 3) for His-tagged recombinant PHB2.
- FIG. 2I shows the CD spectra of ERAP and stapled ERAP (No. 2 and No. 3) in 10 mM sodium phosphate buffer (pH 7.0).
- FIG. 2J shows the duration of the inhibitory effect of 11R-ERAP and stapled ERAP (No. 2 and No. 3) on ER ⁇ target gene expression measured in MCF-7 cells. The results are shown as a multiple when the expression level in untreated cells at 0 hours is 1.0.
- the upper three graphs show the expression of the TFF1 gene, and the lower three graphs show the expression of the CCND1 gene. The type of peptide added is shown in the upper left of each graph.
- FIG. 3 shows that stapled ERAP (No. 12) without olefins suppresses E2-dependent responses stably over the long term.
- 3A shows stapled ERAP No. 1 which is a stapled ERAP having no olefin. 12 and its HA tag peptide, HA tag stapled ERAP No. 12 shows the primary structure.
- FIG. 3B shows the CD spectra of ERAP and stapled ERAP (No. 3 and No. 12).
- FIG. 3C shows stapled ERAP No. 2 for ECF-dependent cell proliferation of MCF-7 cells (left panel) and cell proliferation of MCF-10A cells (right panel). The result of the MTT assay which evaluated 12 inhibitory effects is shown. In the E2 addition group, 10 nM of E2 was added.
- Each symbol in each graph indicates the following: open circle ⁇ : untreated; filled circle ⁇ : E2 alone; filled triangle ⁇ : E2 + 0.5 ⁇ M peptide (left figure) or 0.5 ⁇ M peptide (right figure); black square ⁇ : E2 + 1 ⁇ M peptide (left figure) or 1 ⁇ M peptide (right figure); black diamond ⁇ : E2 + 10 ⁇ M peptide (left figure) or 10 ⁇ M peptide (right figure).
- Data represent the mean ⁇ SD of 3 independent experiments ( *** P ⁇ 0.001, two-sided student t-test).
- FIG. 3D shows the results of co-immunoprecipitation evaluating the inhibitory effect of 11R-ERAP and stapled ERAP (No. 3 and No.
- FIG. 12 shows the inhibitory effect of 11R-ERAP and stapled ERAP (No. 3 and No. 12) on ER ⁇ target gene expression. The results are shown as a multiple when the expression level in untreated cells at each time is 1.0.
- FIG. 3F shows stapled ERAP No. 2 in the presence or absence of E2. It is the typical immunofluorescence photograph which showed the intracellular localization of 12 and PHB2. In the presence or absence of E2, MCF-7 cells were treated with HA-tagged stapled ERAP No.
- FIG. 3G shows stapled ERAP No. 1 in the presence or absence of E2. It is the typical immunofluorescence photograph which showed the intracellular localization of 12 and PHB2.
- MCF-7 cells were treated with HA-tagged stapled ERAP No.
- FIG. 3H shows HA-tagged stapled ERAP No. 1 for E2-dependent cell proliferation of MCF-7 cells. The result of the MTT assay which evaluated 12 inhibitory effects is shown.
- FIG. 3I shows the results of an MTT assay that evaluated the inhibitory effect of ERAP on E2-dependent cell proliferation of MCF-7 cells.
- Each symbol in the graph indicates the following; open circle ⁇ : untreated; filled circle ⁇ : E2 alone; filled diamond ⁇ : E2 + 10 ⁇ M peptide.
- Data represent the mean ⁇ SD of 3 independent experiments.
- FIG. 3J shows 11R-ERAP (top) and Stapled ERAP No. 5 for E2-dependent cell proliferation of Tamoxifen resistant (TAM-R) MCF-7 cells.
- TAM-R MCF-7 cells were prepared in the presence of 1 ⁇ M tamoxifen at various concentrations of 11R-ERAP or stapled ERAP No. 12 processed. In the E2 addition group, 10 nM of E2 was added.
- FIG. 3K shows stapled ERAP No. 1 for E2-dependent cell proliferation of MCF-7 cells. The result of the MTT assay which evaluated the combination inhibitory effect of 12 and tamoxifen or fulvestrant is shown. In the figure, the dark gray bar indicates the stapled ERAP No.
- FIG. 4 shows that stapled ERAP has an in vivo anti-tumor effect in a heterologous orthotopic mouse model of human ER ⁇ -positive breast cancer.
- FIG. 4A is a schematic diagram of in vivo testing.
- FIG. 4B shows 1.4 mg / kg stapled ERAP No. 1 for tumor growth in a human breast cancer cell line KPL-3C xenograft mouse model.
- 12 shows the inhibitory effect of treatment.
- the left figure shows the results in the group to which the peptide was administered daily, and the right figure shows the results in the group to which the peptide was administered every 4 days.
- Each symbol in each graph indicates the following: white circle ⁇ : untreated; black circle ⁇ : E2 alone; black triangle ⁇ : E2 + 11R-ERAP; black square ⁇ : E2 + staple ERAP No. 12.
- 4C shows 14 mg / kg stapled ERAP No. 14 for tumor growth in a human breast cancer cell line KPL-3C xenograft mouse model.
- 12 shows the inhibitory effect of treatment.
- the left figure shows the results in the group to which the peptide was administered daily, and the right figure shows the results in the group to which the peptide was administered every 4 days.
- Each symbol in each graph indicates the following: white circle ⁇ : untreated; black circle ⁇ : E2 alone; black triangle ⁇ : E2 + 11R-ERAP; black square ⁇ : E2 + staple ERAP No. 12.
- FIG. 4D shows 1.4 mg / kg of 11R-ERAP (left figure) or stapled ERAP No. 12 shows changes in body weight of KPL-3C xenograft-transplanted mice treated with 12 (right figure).
- Each symbol in each graph indicates the following; open circle ⁇ : untreated; filled circle ⁇ : E2 alone; filled triangle ⁇ : E2 + daily peptide treatment; filled square ⁇ : peptide treatment every E2 + 4 days.
- FIG. 4E shows the results of an immunoblot in which the intracellular localization of PHB2 in the tumor was examined. For immunoblotting, 1.4 mg / kg of 11R-ERAP or stapled ERAP No.
- FIG. 4F shows 14 mg / kg HA-tagged stapled ERAP No. 14 in a KPL-3C xenograft mouse model.
- FIG. 12 shows tumor growth inhibitory effects when treated daily or every 4 days.
- Each symbol in the graph indicates the following: open circle ⁇ : untreated; filled circle ⁇ : E2 alone; filled triangle ⁇ : E2 + daily peptide treatment; filled square ⁇ : peptide treatment every E2 + 4 days.
- FIG. 4G shows PHB2 and stapled ERAP No. The result of co-immunoprecipitation which investigated 12 intracellular localization is shown. For co-immunoprecipitation, 14 mg / kg HA tag stapled ERAP No.
- FIG. 4H shows PHB2 and stapled ERAP No. 12 shows representative immunohistochemical staining photographs examining 12 subcellular localizations. For immunohistochemical staining, 14 mg / kg HA-tagged stapled ERAP No.
- FIG. 4I shows stapled ERAP No. 1 versus ERa target gene expression in tumors. The box plot which evaluated 12 inhibitory effects is shown. The upper figure shows the expression of TFF1, and the lower figure shows the expression of CCND1. For the analysis, 1.4 mg / kg stapled ERAP No. Tumors removed from KPL-3C xenograft transplanted mice treated every day or every 4 days were used. The results are shown as a multiple when the expression level in the untreated tumor is 1.0.
- FIG. 4J shows the results of an immunoblot examining the phosphorylation levels of Akt and MAPK in stapled ERAP treated tumors.
- a stapled ERAP No. of 1.4 mg / kg was used.
- Tumors removed from KPL-3C xenograft transplanted mice treated daily at 12 (upper figure) or every 4 days (lower figure) were used.
- the antibody reacted on the membrane is shown at the right end. The number below the membrane indicates the relative value of the reaction intensity of each lane when the reaction intensity of the leftmost lane is 1.0.
- FIG. 4K shows a 14 mg / kg stapled ERAP No. 12 shows representative hematoxylin and eosin-stained photographs of heart, lung, liver, kidney, pancreas, and brain isolated from KPL-3C xenograft-transplanted mice treated every 12 days.
- FIG. 4L shows a 14 mg / kg HA tag stapled ERAP No. 12 shows representative hematoxylin and eosin-stained photographs of heart, lung, liver, kidney, pancreas and brain isolated from KPL-3C xenograft-transplanted mice treated daily at 12;
- FIG. 4M shows stapled ERAP No. 1 for tumor growth in KPL-3C xenograft transplanted mice.
- FIG. 5 shows the stapled ERAP No. 12 shows that the human prostate cancer cell line 22Rv1 suppresses cell proliferation and inhibits BIG3-PHB2 interaction.
- FIG. 5A shows stapled ERAP No. 22 for cell proliferation of 22Rv1 cells.
- FIG. 5B shows stapled ERAP No. 22 for endogenous BIG3-PHB2 interaction in 22Rv1 cells.
- IP indicates an antibody used for immunoprecipitation
- WCL indicates a whole cell lysate. The antibody reacted on the membrane is shown at the right end.
- FIG. 5C shows stapled ERAP No. 5 for cell proliferation of MCF-10A cells. The result of the test which evaluated the inhibitory effect of 12 is shown. Each symbol in the graph indicates the following: black circle ⁇ : untreated; black triangle ⁇ : 10 ⁇ M stapled ERAP No. 12; Black square ⁇ : 20 ⁇ M stapled ERAP No. 12; Black diamond ⁇ : 50 ⁇ M stapled ERAP No. 12.
- FIG. 6 shows stapled-D-ERAP No. 12 (D-No. 12), stapled retro inverse ERAP No. 12 (RI-No. 12), short stapled retro inverse ERAP No.
- FIG. 6A shows the primary structure of the stapled ERAP analog. In the amino acid sequence, underlined bold letters indicate amino acid residues important for PHB2 binding, and italic bold letters indicate stapled amino acid residues. Lower case letters indicate D-amino acids. All amino acid sequences are written in the C-terminal direction with the left end as the N-terminal and rightward.
- FIG. 6B shows stapled ERAP No. 2 for E2-dependent cell proliferation of MCF-7 cells (left panel) and MCF-10A cell proliferation (right panel). 12 (first stage), D-No. 12 (second stage), RI-No.
- FIG. 6C shows stapled ERAP No. 12 (upper left), D-No. 12 (upper right figure), RI-No. 12 (lower left figure) or shRI-No.
- FIG. 6 shows the inhibitory effect of the stapled ERAP on E2-dependent cell proliferation 96 hours after treatment in MCF-7 cells treated with 12 (lower right figure).
- 10 nM of E2 was added.
- the horizontal axis of the graph indicates the treatment concentration of the peptide.
- “( ⁇ )” indicates that E2 has not been processed.
- 6D shows D-No. 12 (upper figure), RI-No. 12 (below) or shRI-No.
- FIG. 12 shows the results of evaluating the inhibitory effect of the peptide on E2-dependent cell proliferation over 7 days after treatment in MCF-7 cells treated with 12 (lower figure).
- E2 addition group 10 nM of E2 was added, and 1 ⁇ M of each peptide was added.
- Each symbol in each graph indicates the following; white circle ⁇ : untreated; black circle ⁇ : E2 alone; black triangle ⁇ : E2 + No. 12; black square ⁇ : E2 + D-No. 12 (upper figure) or E2 + RI-No. 12 (below); black diamond ⁇ : E2 + shRI-No. 12.
- FIG. 6E shows D-No. For BIG3-PHB2 interaction. 12, RI-No. 12 and shRI-No.
- FIG. 6F shows RI-No. 5 for tumor growth in a KPL-3C xenograft mouse model. 12 shows the inhibitory effect of treatment every 4 days (left figure) and every 7 days (right figure). Each symbol in each graph indicates the following; white circle ⁇ : untreated; black circle ⁇ : E2 alone; black rhombus ⁇ : E2 + 0.02 mg / kg RI-No. 12; black triangle ⁇ : E2 + 0.1 mg / kg RI-No. 12: Black square ⁇ : E2 + 1 mg / kg RI-No. 12.
- FIG. 6G shows RI-No. 12 shows suppression of expression of ER ⁇ target genes TFF1 (left figure) and CCND1 (right figure) in tumors excised from KPL-3C xenograft transplanted mice treated every 12 or 4 days.
- the horizontal axis of the graph indicates the RI-No.
- the dosage in 12 single treatments is shown.
- “( ⁇ )” indicates that E2 has not been processed.
- the results are shown as a multiple when the expression level in the untreated tumor is 1.0.
- Data show the mean ⁇ SD of 5 independent tumors ( * P ⁇ 0.05, ** P ⁇ 0.01, two-sided student t-test).
- FIG. 7 shows the results of MTT assay for evaluating the inhibitory effect of ERAP-8R (No. 10; left figure) and partial ERAP-8R (No. 11; right figure) on the E2-dependent cell proliferation of MCF-7 cells. Show. In the E2 addition group, 10 nM of E2 was added. In the amino acid sequences shown at the top of each graph, underlined bold letters indicate amino acid residues important for PHB2 binding. Each symbol in each graph represents the following: white circle ⁇ : untreated; black circle ⁇ : E2 alone; black triangle ⁇ : E2 + 0.5 ⁇ M peptide; black square ⁇ : E2 + 1 ⁇ M peptide; black diamond ⁇ : E2 + 10 ⁇ M peptide.
- FIG. 8 shows the results of an immunoblot in which the phosphorylation levels of mTOR and S6K were examined in a tamoxifen resistant breast cancer cell line treated with stapled ERAP (No. 12). The antibody reacted on the membrane is shown at the right end. The number below the membrane indicates the relative value of the reaction intensity of each lane when the reaction intensity of the leftmost lane is 1.0.
- FIG. 9 shows the results of an MTT assay for evaluating the combined effect of stapled ERAP (No. 12), tamoxifen, fulvestrant, and everolimus on E2-dependent cell proliferation of MCF-7 cells.
- the dark gray bar is untreated, the light gray bar is treated with 11R-ERAP, and the white bar is stapled ERAP No. 12 shows processing.
- TAM represents tamoxifen
- Flu represents fulvestrant
- Ever represents everolimus.
- the graph shows the results 24 hours after treatment (left figure) and 96 hours (right figure). Data represent the mean ⁇ SD of three independent experiments ( ** P ⁇ 0.01, *** P ⁇ 0.001, two-sided student t test).
- FIG. 10 shows the antitumor effect of tail vein administration of stapled ERAP (No. 12).
- FIG. 11 shows the results of a co-immunoprecipitation test in which the interaction between BIG3 and PHB2 was evaluated in tumors excised from transplanted mice treated with stapled ERAP (No. 12).
- IP indicates an antibody used for immunoprecipitation
- WCL indicates a whole cell lysate. The antibody reacted on the membrane is shown at the right end.
- FIG. 12 shows the results of an immunoblot in which phosphorylated levels of Akt and MAPK were examined in a tumor isolated from a transplanted mouse treated with stapled ERAP (No. 12). The antibody reacted on the membrane is shown at the right end. The number below the membrane indicates the relative value of the reaction intensity of each lane when the reaction intensity of the leftmost lane is 1.0.
- an amino acid written in capital letters represents an L-amino acid.
- amino acids represented in lower case represent D-amino acids.
- the L-amino acid and D-amino acid represented in this specification may include those in which any of an amino group, a carboxyl group, and a side chain is modified. Examples of preferable modifications include acetylation of amino groups, amidation of carboxyl groups, and addition of tag peptides such as FLAG tags and HA tags.
- BIG3 refers to brefeldin A-inhibited guanine nucleotide-exchange protein 3 (brefeldin A-inhibited guanine nucleotide-exchange protein).
- BIG3 inhibits the function of PHB2 that suppresses E2-dependent transcriptional activation by forming a complex with PHB2.
- BIG3 is also referred to as “ARFGEF3 (ARFGEF family member 3)” or “A7322”.
- An example of a representative base sequence of the human BIG3 gene is shown in SEQ ID NO: 23 (GenBank Accession No. NM — 020340.4), and the amino acid sequence encoded thereby is shown in SEQ ID NO: 24.
- SEQ ID NO: 24 GenBank Accession No. NM — 020340.4
- PHB2 refers to prohibitin 2 (prohibitin 2).
- PHB2 binds to the estrogen receptor, inhibits the estrogen receptor signaling pathway, and suppresses estrogen-dependent cell proliferation.
- the PHB 2 is also referred to as “REA (Repressor of Estrogen Activity)”.
- Examples of representative base sequences of the human PHB2 gene are SEQ ID NO: 25 (GenBank Accession No. NM_001144831.1) and SEQ ID NO: 27 (GenBank Accession No. NM_001267700.1), and the amino acid sequence encoded by them It is shown in SEQ ID NO: 26 and SEQ ID NO: 28, respectively.
- PHB2 is not limited to those encoded by the base sequence, and includes isoforms and mutants thereof.
- estrogen receptor encompasses both estrogen receptor ⁇ (ER ⁇ ) and estrogen receptor ⁇ (ER ⁇ ).
- the estrogen receptor moves into the nucleus when estrogen binds, and binds to ERE, which is an enhancer sequence on DNA, to cause transcriptional activation of genes related to cell proliferation. This induces estrogen-dependent cell proliferation.
- ER ⁇ and ER ⁇ are encoded by the ESR1 gene and the ESR2 gene, respectively.
- the base sequence of a typical human ESR1 gene is shown in SEQ ID NO: 29 (GenBank Accession No. NM — 000015.3).
- the base sequence of a representative human ESR2 gene is shown in SEQ ID NO: 31 (GenBank Accession No. NM_001437.2).
- ER ⁇ and ER ⁇ are not limited to those encoded by the above base sequences, but also include these isoforms and mutants.
- the estrogen receptor is ER ⁇ .
- the term “ERAP” refers to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 9.
- the term “short ERAP” refers to a peptide consisting of a partial sequence of the amino acid sequence set forth in SEQ ID NO: 9.
- the amino acid sequence described in SEQ ID NO: 9 is a sequence consisting of amino acids 165 to 177 of the amino acid sequence of BIG3 (SEQ ID NO: 24), and is an amino acid residue important for binding to PHB2 (SEQ ID NO:
- the amino acid sequence described in 24 includes the 165th glutamine (Q), the 169th aspartic acid (D), and the 173rd glutamine (Q)).
- ERAP has a binding ability to PHB2, and inhibits BIG3 from forming a complex with PHB2 by competitively binding to PHB2.
- a peptide in which polyarginine is bound to the N-terminus or C-terminus of ERAP as a cell-penetrating peptide, 11R-ERAP and ERAP-8R (the number before “R” is the arginine residue). Number) etc.
- stapling structure refers to a structure in which two (one pair) amino acid residues in an amino acid sequence constituting a peptide are cross-linked.
- a peptide in which the original amino acid residue is substituted by one or more stapling structures is referred to herein as a “stapled peptide”.
- stapled ERAP is a peptide in which at least one pair of amino acid residues is replaced with a stapling structure in the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 9 (ERAP).
- the short stapled ERAP is a peptide in which at least one pair of amino acid residues is replaced with a stapling structure in a peptide (short ERAP) consisting of a partial sequence of the amino acid sequence set forth in SEQ ID NO: 9.
- short stapled ERAP is also referred to as “sh stapled ERAP”.
- treatment includes alleviation / amelioration of at least one symptom caused by a target disease, suppression of disease progression, suppression of expansion of a disease site, and the like.
- cancer treatment includes suppression of cancer cell growth, suppression of cancer progression, induction of cancer regression / remission, alleviation / improvement of symptoms associated with cancer, suppression of cancer metastasis, Includes suppression of postoperative recurrence and induction of survival.
- the peptide of the present invention is an amino acid sequence in which n pairs (n is a natural number) of amino acid residues in the amino acid sequence of SEQ ID NO: 9 or a partial sequence thereof are replaced with n stapling structures It is a peptide containing. n is preferably 3 or less, more preferably 2, and even more preferably 1. Therefore, in the present invention, n pairs of amino acid residues usually refer to 1 to 3 pairs, or 1 or 2 pairs, preferably 1 pair of amino acid residues.
- the partial sequence of the amino acid sequence shown in SEQ ID NO: 9 is preferably a sequence of 6 or more consecutive amino acid sequences shown in SEQ ID NO: 9, and a sequence of 7 or more residues. It is more preferable that
- the first glutamine (Q), the fifth aspartic acid (D) and the ninth glutamine (Q) from the N-terminus of the amino acid sequence shown in SEQ ID NO: 9 are amino acid residues important for binding to PHB2. Since it is a group, it preferably contains at least one, more preferably two or more of these amino acid residues.
- a preferred example of a partial sequence of the amino acid sequence set forth in SEQ ID NO: 9 is the amino acid sequence set forth in SEQ ID NO: 13 (QMLSDLT).
- the amino acid residue to be replaced with the stapling structure is not particularly limited, but from the viewpoint of binding affinity to PHB2, the first amino acid sequence from the N-terminus of the amino acid sequence described in SEQ ID NO: 9 (QMLSDTLQLRRQR) It is preferable to select from amino acid residues other than glutamine (Q), fifth aspartic acid (D) and ninth glutamine (Q).
- amino acid residues that are replaced with a stapling structure include, for example, the following pairs of amino acid residues; (A) the third (L) and seventh amino acid residues (T) from the N-terminus of the amino acid sequence of SEQ ID NO: 9; (B) the second (M) and sixth amino acid residues (L) from the N-terminus of the amino acid sequence of SEQ ID NO: 9; (C) 4th (S) and 8th amino acid residues (L) from the N-terminus of the amino acid sequence of SEQ ID NO: 9; and (d) 6 from the N-terminus of the amino acid sequence of SEQ ID NO: 9.
- amino acid residue pairs include the amino acid residue pairs (a) and (b).
- amino acid residues to be replaced with the stapling structure include, for example, the following amino acid residues: A pair of; (A) the third (L) and seventh amino acid residues (T) from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 13; and (b) 2 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 13.
- the stapling structure is not particularly limited. Since peptide stapling techniques are known (eg, Blakwell, H. E. et al., Angew. Chem., Int. Ed. 37, 3281-3284 (1994); Aihara, K. et al., Tetrahedron 71, 4183-4191 (2015) etc.), these known stapling techniques can be used to form a stapling structure.
- a stapling structure can be formed.
- a commercially available amino acid derivative for forming the stapling structure can be used.
- examples of a preferable stapling structure include a structure represented by the following formula (I). (In the formula, the solid and dotted double lines indicate single bonds or double bonds.)
- the formation of the stapling structure of the above formula (I) can be performed, for example, according to the scheme shown in FIG. 1B or FIG. 1C.
- the scheme shown in FIG. 1B (hereinafter, “Scheme (I)”) is an example in which a stapling structure is formed by an olefin metathesis reaction.
- the scheme shown in FIG. 1C (hereinafter, “Scheme (II)”) is an example in which a stapling structure is formed by an intramolecular amidation reaction.
- the amino acid derivative for stapling is a glutamine derivative represented by the following formula (III) (4- ⁇ allyl- [2- ( tert-butyl-dimethyl-silanyloxy) -4-methoxy-benzyl] -carbonyl ⁇ -2- (9H-fluoren-9-ylmethoxycarbonylamino) -butyric acid).
- the glutamine derivative of formula (III) can be synthesized, for example, according to the following scheme (III) (Aihara, K. et al., Tetrahedron 71, 4183-4191 (2015)).
- the methyl ester of compound 4 is then hydrolyzed to give 4- [allyl- (2-hydroxy-4-methoxy-benzyl) -carbamoyl] -2-tert-butoxycarbonylamino-butyric acid (compound 5).
- the glutamine derivative of the formula (III) can be obtained by substituting the Foc group for the Boc group of compound 5 and protecting the phenol moiety of the Hmb group with TBS. Note that commercially available reagents can be used for the reagents necessary for carrying out Scheme (III).
- the synthesis of stapled ERAP according to scheme (I) can be performed, for example, as follows using the glutamine derivative of the above formula (III).
- a pair of amino acid residues at positions where a stapling structure is to be formed is replaced with a glutamine derivative of formula (III), and standard Fmoc solid phase peptide synthesis To synthesize the peptide.
- the acetylated peptide is treated with Hoveyda-Grubbs second generation catalyst to perform an olefin metathesis reaction.
- a TFA / m-cresol / thioanisole / 1,2-ethanedithiol / H 2 O cocktail is used to deprotect the acid labile protecting group and cleave the peptide from the resin.
- the stapled ERAP or sh stapled ERAP having the stapling structure of the formula (I) (the double line of the solid line and the dotted line is a double bond) can be obtained.
- the number of amino acid residues interposed between the stapling structures is not particularly limited, but it is usually preferably 3 residues.
- amino acid derivatives for stapling include N- ⁇ - represented by the following formula (IV): (9-Fluorenylmethoxycarbonyl) -L-glutamic acid ⁇ -allyl ester and (S) -2-((((9H-fluoren-9-yl) methoxy) carbonyl) amino) represented by the following formula (V) 5-((4-(((allyloxy) carbonyl) amino) butyl) (2,4-dimethoxybenzyl) amino) -5-oxopentanoic acid can be used.
- a commercially available glutamic acid derivative of the formula (IV) can be used.
- the glutamine derivative of the formula (V) can be synthesized, for example, according to the scheme shown in FIG. 1A (hereinafter “scheme (IV)”).
- Scheme (IV) allyl (4-aminobutyl) carbamate (Compound 1) was coupled with 2,4-dimethoxybenzaldehyde to form allyl [4- ⁇ (2,4-dimethoxybenzyl) amino ⁇ butyl.
- the carbamate (compound 2) is obtained.
- compound 2 was coupled with N- ⁇ - (tert-butoxycarbonyl) -L-glutamic acid ⁇ -methyl ester (compound 3) to give (S) -methyl-5- ⁇ (4-[ ⁇ (allyloxy)) Carbonyl ⁇ amino] butyl) (2,4-dimethoxybenzyl) amino ⁇ -2- ⁇ (tert-butoxycarbonyl) amino ⁇ -5-oxopentanoate (compound 4) is obtained.
- the synthesis of the stapled ERAP according to the scheme (II) can be performed, for example, as follows using the glutamic acid derivative of the above formula (IV) and the glutamine derivative of the formula (V).
- a pair of amino acid residues at a position where a stapling structure is to be formed is replaced with a glutamic acid derivative of formula (IV) and a glutamine derivative of formula (V)
- Peptides are synthesized by standard Fmoc solid phase peptide synthesis.
- the Fmoc-protected peptide is mixed with a solution of tetrakis (triphenylphosphine) palladium (Pd (PPh 3 ) 4 ) in CHCl 3 / AcOH / N-methylmorpholine to reduce the substituent of the glutamine derivative residue.
- intramolecular amidation is performed by coupling glutamine derivative residues using N, N-diisopropylcarbodiimide (DIPCDI) and 1-hydroxy-1H-benzotriazole hydrate (HOBt ⁇ H 2 O).
- a TFA / m-cresol / thioanisole / 1,2-ethanedithiol / H 2 O cocktail is used to deprotect the acid labile protecting group and cleave the peptide from the resin.
- stapled ERAP or sh stapled ERAP having the stapling structure of formula (I) (the double line of the solid line and the dotted line is a single bond) can be obtained.
- the number of amino acid residues interposed between the stapling structures is not particularly limited, but it is usually preferably 3 residues.
- peptide of the present invention include a peptide represented by the following formula (II).
- formula (II) the solid and dotted double lines are single bonds or double bonds;
- the peptide represented by the above formula (II) is a peptide having the amino acid sequence set forth in SEQ ID NO: 9 (QMLSDTLQLRRQR), wherein a pair of amino acid residues of the following (a) or (b) is represented by the formula (I It can also be said that the peptide is replaced by the staple structure of (A) the third (L) and seventh amino acid residues (T) from the N-terminus of the amino acid sequence of SEQ ID NO: 9; and (b) 2 from the N-terminus of the amino acid sequence of SEQ ID NO: 9.
- a peptide in which a pair of amino acid residues of (c) or (d) below is replaced with the staple structure of formula (I): is there; (C) the third (L) and seventh amino acid residues (T) from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 13; and (d) 2 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 13
- peptides correspond to the following peptides;
- Th M and sixth amino acid residues (L); or (Ii) A peptide comprising the amino acid sequence set forth in SEQ ID NO: 13 (QMLSDLT), wherein the following pair of amino acid residues is replaced with the staple structure of formula (I); (C) The third (L) and seventh amino acid residues (T) from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 13.
- the peptide of the present invention includes those in which either or both of the N-terminal and C-terminal amino acid residues are modified.
- the type of modification is not particularly limited, but is preferably one that does not decrease the affinity for PHB2 or cell permeability.
- Examples of preferred modifications include acetylation of the N-terminal amino acid residue, amidation of the C-terminal amino acid residue, and addition of tag peptides such as HA tag and FLAG tag.
- tag peptides such as HA tag and FLAG tag.
- the N-terminal amino acid residue is acetylated and the C-terminal amino acid residue is amidated.
- Peptides can be mentioned. It is preferable that amino acid residues other than the N-terminal and C-terminal amino acid residues are not modified.
- the peptide of the present invention is not limited to those composed of L-amino acids, and may contain one or more D-amino acids.
- the composition ratio of L-amino acid and D-amino acid in the peptide is not particularly limited, but in order to maintain the ⁇ -helix structure, all amino acid residues are L-type (hereinafter referred to as “L-type peptide”) or all Are preferably D-type (hereinafter referred to as “D-type peptide”). Accordingly, in any of the above-described peptides of the present invention, a peptide in which all amino acid residues are replaced with D-type amino acid residues is also mentioned as a preferred embodiment of the peptide of the present invention.
- preferred peptides include, for example, peptides in which all amino acid residues are replaced with D-type amino acid residues in the peptide represented by the formula (II) Can do.
- the peptide of the present invention is a D-type peptide, among the amino acids constituting the amino acid sequence, for example, 80% or more, usually 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99 % Or more are D-type amino acid residues.
- the peptide of the present invention may be a retroinverse form of any of the peptides of the present invention described above.
- the amino acid sequence is reversed from that of the original peptide, and all amino acid residues are replaced with D-type amino acid residues. That is, the retro-inverse form is a D-type peptide having an amino acid sequence opposite to that of the original peptide. Therefore, a peptide that is a retroinverse form of any of the above-described peptides of the present invention is also mentioned as a preferred embodiment of the peptide of the present invention.
- examples of preferred peptides include peptides that are retro-inverse forms of the peptide represented by the formula (II).
- the peptide of the present invention is a retroinverse form, among the amino acids constituting the amino acid sequence, for example, 80% or more, usually 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99 % Or more are D-type amino acid residues.
- a D-type stapled ERAP or sh-stapled ERAP is synthesized by using a D-amino acid instead of an L-amino acid in the method as described above. Can do.
- amino acid derivatives for forming a stapling structure are also used in the D-type. Since some of the D-type amino acid derivatives that can be used for forming the stapling structure are commercially available, these commercially available D-type amino acid derivatives may be used.
- D-type stapled ERAP or sh stapled ERAP is synthesized according to scheme (I) shown in FIG. 1B
- a glutamine derivative represented by the formula (III) is used as an amino acid derivative for stapling.
- the D-type optical isomer hereinafter “D-glutamine derivative of the formula (II)”
- the D-glutamine derivative of the formula (II) is obtained by replacing N- ⁇ - (tert-butoxycarbonyl) -L-glutamic acid ⁇ methyl ester (compound 3) in the above scheme (III) with N- ⁇ - (tert-butoxy It can be synthesized by using (carbonyl) -D-glutamic acid ⁇ -methyl ester.
- D-type stapled ERAP or sh-stapleted ERAP can be obtained by synthesizing D-type peptide by standard Fmoc solid phase peptide synthesis and performing olefin metathesis reaction according to scheme (I).
- solid phase peptide synthesis is performed based on the amino acid sequence shown in SEQ ID NO: 9 or the amino acid sequence opposite to the partial sequence. Just do it. In this case, the amino acid residue pair at the position where the stapling structure is to be formed is replaced with the D-glutamine derivative of the formula (II), and the olefin metathesis reaction is performed after peptide synthesis as described above.
- a glutamic acid derivative represented by the formula (IIV) is used as an amino acid derivative for stapling.
- Type D optical isomer hereinafter “D-glutamic acid derivative of formula (IV)”
- D type optical isomer of glutamine derivative represented by formula (V) hereinafter “D-glutamine derivative of formula (V)”
- a commercially available D-glutamic acid derivative of the formula (IV) can be used.
- D-glutamine derivative of the formula (V) is replaced with N- ⁇ - (tert-butoxycarbonyl) -L-glutamic acid ⁇ -methyl ester (compound 3) in the scheme (IV) shown in FIG. 1A. It can be synthesized by using-(tert-butoxycarbonyl) -D-glutamic acid ⁇ -methyl ester.
- a pair of amino acid residues at positions where a stapling structure is to be formed is represented by a D-glutamic acid derivative of formula (IV) and a D-glutamine derivative of formula (V)
- D-amino acids are used to synthesize D-type peptides by standard Fmoc solid-phase peptide synthesis, followed by intramolecular amidation according to Scheme (II), thereby providing D-type stapled ERAP or sh staples.
- De ERAP can be obtained.
- solid phase peptide synthesis is performed based on the amino acid sequence shown in SEQ ID NO: 9 or the amino acid sequence opposite to the partial sequence. Just do it. At that time, the pair of amino acid residues at the position where a stapling structure is to be formed is replaced with a D-glutamic acid derivative of formula (IV) and a D-glutamine derivative of formula (V), and an intramolecular amidation reaction is performed after peptide synthesis. This is the same as above.
- the peptide of the present invention may be in the form of a salt.
- a salt is not specifically limited, It is preferable that it is a pharmaceutically acceptable salt.
- pharmaceutically acceptable salt refers to a salt that retains the pharmacological or pharmaceutical efficacy and properties of a peptide.
- Preferred examples of salts include salts with alkali metals (lithium, potassium, sodium, etc.), salts with alkaline earth metals (calcium, magnesium, etc.), and other metals (copper, iron, zinc, manganese, etc.).
- Salt salt with organic base, salt with amine, organic acid (acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, etc. ) And salts with inorganic acids (hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, nitric acid, etc.). These salts can be prepared according to known methods.
- compositions The peptide of the present invention or a salt thereof can be formulated as a pharmaceutical composition together with a pharmaceutically acceptable carrier.
- the peptide of the present invention has a binding ability to PHB2, and competitively inhibits the BIG3-PHB2 interaction. Formation of the BIG3-PHB2 complex enhances estrogen-dependent transcriptional activity and leads to cancer cell growth. Therefore, the peptide of the present invention that inhibits the BIG3-PHB2 interaction and suppresses the formation of the BIG3-PHB2 complex is particularly useful as a pharmaceutical composition for treating cancer.
- the peptide of the present invention is particularly useful as a pharmaceutical composition for treating estrogen receptor-positive cancer.
- estrogen receptor positive cancers include breast cancer, endometrial cancer, ovarian cancer, prostate cancer (Nelles JL, et al., Expert Rev Endocrinol Metab. 2011 May; 6 (3) : 437-451.), Lung cancer (especially non-small cell lung cancer) (Stabile LP, et al., Cancer Res.
- the cancer to which the pharmaceutical composition of the present invention is applied preferably expresses BIG3 and PHB2.
- estrogen receptor-positive cancer generally expresses BIG3 and PHB2. Whether or not the cancer is estrogen receptor positive can be confirmed by a known method such as ELISA or immunohistochemical staining.
- the peptide of the present invention has a growth inhibitory effect against tamoxifen-resistant estrogen receptor-positive cancer. Therefore, the pharmaceutical composition of the present invention may be applied to tamoxifen-resistant estrogen receptor-positive cancer.
- tamoxifen-resistant estrogen receptor-positive cancer to which the pharmaceutical composition of the present invention is applied include, for example, tamoxifen-resistant estrogen receptor-positive breast cancer.
- preferable administration subjects of the pharmaceutical composition of the present invention include patients having estrogen receptor positive breast cancer refractory to tamoxifen therapy.
- the peptide of the present invention also has an inhibitory effect on estrogen-independent cancer cell growth. Therefore, the peptide of the present invention is also useful as a pharmaceutical composition for treating estrogen receptor negative cancer.
- the estrogen receptor-negative cancer to which the pharmaceutical composition of the present invention is applied is not particularly limited, but it must be a cancer expressing BIG3 and PHB2. Examples of such cancers include estrogen receptor negative breast cancer and prostate cancer.
- the pharmaceutical composition of the present invention can be produced by blending the peptide of the present invention or a salt thereof and a pharmaceutically acceptable carrier using a known formulation technique.
- pharmaceutically acceptable carrier refers to an inert substance used as a diluent or solvent for a drug.
- the pharmaceutically acceptable carrier used in the pharmaceutical composition of the present invention may be appropriately selected from carriers used in general pharmaceuticals according to the dosage form of the pharmaceutical composition to be prepared.
- the dosage form of the pharmaceutical composition of the present invention is not particularly limited, and a dosage form generally used for pharmaceuticals such as liquids, tablets, elixirs, capsules, granules, powders and the like can be appropriately selected.
- additives such as excipients, stabilizers, suspensions, preservatives, surfactants, solubilizers, pH adjusters, and aggregation inhibitors may be added as appropriate. it can.
- the pharmaceutical composition of the present invention contains a pharmaceutically effective amount of the peptide of the present invention or a salt thereof.
- the pharmaceutically effective amount can be appropriately selected according to the dosage form of the pharmaceutical composition, the administration interval, the age, sex, body weight, body surface area, type of disease, etc. of the administration subject.
- examples of the content of the peptide of the present invention or a salt thereof in the pharmaceutical composition of the present invention include 0.001 mg to 1000 mg, 0.01 mg to 100 mg, 0.1 mg to 30 mg, 0.1 mg to 10 mg, and the like. However, it is not limited to these.
- the pharmaceutical composition of the present invention may optionally contain other drugs.
- other drugs include anti-inflammatory agents, analgesics, antipyretic agents, other cancer therapeutic agents and the like.
- Other cancer therapeutic agents that can be used in the pharmaceutical composition of the present invention are not particularly limited, but when used for estrogen-positive cancer, selective ER ⁇ modulators (eg, tamoxifen and raloxifene), ER ⁇ down regulators (eg, , Fulvestrant), aromatase inhibitors, LH-RH agonist preparations, progesterone preparations and other hormone therapy agents.
- selective ER ⁇ modulators eg, tamoxifen and raloxifene
- ER ⁇ down regulators eg, Fulvestrant
- aromatase inhibitors eg, LH-RH agonist preparations
- progesterone preparations e.g, progesterone preparations and other hormone therapy agents.
- These drugs may be blended in the form of a prodrug or a pharmaceutically acceptable
- the pharmaceutical composition of the present invention can be administered to a subject by appropriately selecting an appropriate administration route according to the dosage form.
- the administration route is not particularly limited, and examples thereof include oral administration and intradermal, subcutaneous, intramuscular, intraosseous, peritoneal and intravenous injection.
- any of systemic administration and local administration to the vicinity of a disease site may be used, but local administration is preferable.
- the administration interval of the pharmaceutical composition of the present invention should also be appropriately selected according to the age, sex, body weight, body surface area, type of disease, etc. of the administration subject, and the dosage form and administration route of the pharmaceutical composition. Can do. Examples of administration intervals include, but are not limited to, every day, every 4 days, every 7 days, and the like.
- the dose of the pharmaceutical composition of the present invention can also be appropriately selected according to the age, sex, body weight, body surface area, type of disease, etc. of the administration subject, and the dosage form and administration route of the pharmaceutical composition. it can.
- Examples of the dose of the peptide of the present invention or a salt thereof include, for example, 0.001 to 1000 mg / kg / day, 0.005 to 500 mg / kg / day, 0.01 to 250 mg / kg / day, and the like. However, it is not limited to these.
- the pharmaceutical composition of the present invention may be used in combination with other pharmaceuticals depending on the state of the administration target.
- the drug to be used in combination is not particularly limited, but when used for estrogen receptor positive cancer, selective ER ⁇ modulator (eg, tamoxifen and raloxifene), ER ⁇ down regulator (eg, fulvestrant), aromatase inhibitor, Mention may be made of hormone therapy agents such as LH-RH agonist preparations and progesterone preparations. Among these hormonal therapeutic agents, tamoxifen and fulvestrant are particularly preferable.
- the pharmaceutical composition of the present invention When the pharmaceutical composition of the present invention is used for cancer treatment, it may be examined whether or not the cancer to be treated expresses BIG3 and PHB2 before administration. Whether or not the cancer to be treated expresses BIG3 and PHB2 can be confirmed by detecting transcription products and translation products of these genes in a sample collected from the subject.
- a known method can be used as the detection method.
- a transcription product is detected by a probe or PCR method (eg, cDNA microarray method, Northern blot method, RT-PCR method, etc.), and a translation product is an antibody or the like. (For example, Western blotting, immunostaining, etc.) and the like can be used.
- the present invention also provides a product or kit comprising the pharmaceutical composition of the present invention.
- the product or kit of the present invention may comprise a container containing the pharmaceutical composition of the present invention.
- suitable containers include, but are not limited to, bottles, vials, and test tubes.
- the container can be formed from a variety of materials such as glass or plastic.
- a label may be attached to the container, and the label can describe a disease or a disease state in which the pharmaceutical composition of the present invention is to be used.
- the label may also indicate directions for administration and the like.
- the product or kit of the present invention may optionally further comprise a second container containing a pharmaceutically acceptable diluent in addition to the container containing the pharmaceutical composition of the present invention.
- the product or kit of the present invention further includes other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. May be included.
- the pharmaceutical composition of the present invention can also be provided in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient, if desired.
- the pack may include metal foil or plastic foil, such as a blister pack.
- the pack or dispenser device can be accompanied by instructions for administration.
- the present invention also provides the following uses and methods: (A) use of the peptide of the present invention or a salt thereof in the manufacture of a pharmaceutical composition for treating cancer; (B) the peptide of the present invention or a salt thereof for use in the treatment of cancer; (C) A method or process for producing a pharmaceutical composition for treating cancer, comprising the step of formulating the peptide of the present invention or a salt thereof and a pharmaceutically acceptable carrier.
- Example 1 Synthesis of stapled peptides Synthesis of ERAP Peptide A dominant negative peptide (11R-ERAP; 11R-GGG-QMLSDLTLQLRQR (SEQ ID NO: 9)) designed to specifically inhibit BIG3-PHB2 interaction was synthesized as previously described (T Yoshimaru, et al., Nat. Commun. 4, 2443 (2013).). All chemicals were analytical grade. “11R” constituting 11R-ERAP indicates polyarginine (polyR) composed of 11 arginine residues. “GGG” between PolyR and SEQ ID NO: 9 is a 3-residue glycine introduced as a linker for both. 11R was introduced for the purpose of imparting cell permeability to ERAP (SEQ ID NO: 9).
- amino acid derivatives for the synthesis of stapled peptides were synthesized based on the method described in Aihara et al (Tetrahedron, 71, 4183-4191 (2015)).
- the amino acid used as the raw material for the amino acid derivative was purchased from Peptide Institute, Inc. (Osaka, Japan).
- glutamic acid derivative N- ⁇ - (9-fluorenylmethoxycarbonyl) -L-glutamic acid ⁇ allyl ester
- silica gel 60N spherical, neutral, particle size 63-210 ⁇ m
- Mass spectra were recorded with Waters MICROMASSR LCT PREMIER TM (ESI-TOF). NMR spectra were measured using a JEOL GSX300 spectrometer.
- TBSOTf tert-butyldimethylsilyl trifluoromethanesulfonic acid
- the diluted solution was neutralized with 2M NaOH aqueous solution (2 mL) at 0 ° C., and 10% (w / v) aqueous Na 2 CO 3 solution (8 mL) and Fmoc-OSu (572 mg, 1.7 mmol) were added. .
- the reaction mixture was acidified with 1M aqueous HCl and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure.
- the synthesis of the olefin-supported stapled peptide was performed by ring-closing metathesis as shown in FIG. 1B.
- the N-terminal Fmoc group was cleaved and the resulting resin was treated with acetic anhydride solution and pyridine in DMF for 30 minutes at room temperature for acetylation of the N-terminal amino group.
- the N-terminal acetylated peptide on the solid support was treated with Hoveyda-Grubbs second generation catalyst 40 mM solution in degassed o-dichlorobenzene at 80 ° C. for 10 minutes. After cleavage of the peptide from the resin, the reaction was monitored by HPLC.
- Deprotection of the acid labile protecting group with peptide release from the resin was determined by TFA / m-cresol / thioanisole / 1,2-ethanedithiol / H 2 O (90: 2.5: 2.5: 2.5 : 2.5 (v / v), 50 ⁇ L / 1 mg resin) cocktail for 90 minutes at room temperature.
- the resin-bound peptide was washed with dichloromethane and dried in vacuo, after which the peptide was cleaved from the resin, purified by semi-preparative HPLC and lyophilized.
- Cleavage of the peptide from the resin was performed according to the standard Fmoc solid phase peptide synthesis protocol described above. Specifically, deprotection of the acid labile protecting group with peptide release from the resin was performed using TFA / m-cresol / thioanisole / 1,2-ethanedithiol / H 2 O (90: 2.5: 2. 5: 2.5: 2.5 (v / v), 50 ⁇ L / 1 mg resin) cocktail for 90 minutes at room temperature. The resin-bound peptide was washed with dichloromethane and dried in vacuo, after which the peptide was cleaved from the resin, purified by semi-preparative HPLC and lyophilized.
- Example 2 Effect of stapled ERAP on E2-dependent breast cancer cells
- Human breast cancer cell line MCF-7 and mammary epithelial cell line MCF-10A were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).
- the breast cancer cell line KPLC-3C J. Kurebayashi, et al., Br. J. Cancer 74, 200-207 (1996) was kindly provided by Dr. Junichi Kobayashi (Kawasaki Medical University). All cell lines were monolayer cultured in an appropriate medium supplemented with 10% FBS. Cells were maintained at 37 ° C. in an atmosphere of humidified air containing 5% CO 2 .
- each cell was seeded in a 48-well plate (2 ⁇ 10 4 cells / mL), a 6-well plate (3 ⁇ 10 5 cells / mL), or a 10 cm dish (2 ⁇ 10 6 cells / 10 mL).
- MCF-7 cells are 10% FBS (Nichirei Bioscience, Tokyo, Japan), 1% Antibiotic / Antilytic Solution (Thermo Fisher Scientific, Waltham, MA, USA), 0.1 mM NEAA (Thermo Fisher Scientific), 1 mM pyruvate. Seeds were placed in MEM (Thermo Fisher Scientific) supplemented with sodium (Thermo Fisher Scientific) and 10 ⁇ g / mL insulin (Sigma, St. Louis, MO, USA).
- KPLC-3C cells were seeded in RPMI (Thermo Fisher Scientific) supplemented with 10% FBS and 1% Antibiotic / Antilytic solution.
- MCF-10A cells were seeded in a SingleQuots kit (BPE, hydrocortisone, hEGF, insulin, gentamicin / amphotericin-B) (Lonza, Walkersville, MD, USA) and MEBM (Lonza) supplemented with 100 ng / mL cholera toxin.
- tamoxifen Sigma
- fulvestrant LKT LKlaboratories, St. Paul, MN, ⁇ 10USA
- 10 nM tamoxifen or 2 ⁇ M full is simultaneously applied with the above treatment with E2 and peptide or treatment with E2 alone.
- Cells were treated with bestland.
- Circular dichroism (CD) spectrum measurement A CD spectrum in the range of 185 to 265 nm was recorded at 25 ° C. using a quartz cuvette with an optical path length of 2 mm (Circular Dichroism Dispersometer J1500: JASCO Corporation, Tokyo, Japan). The peptide concentration was 50 ⁇ g / mL in 10 mM sodium phosphate buffer (pH 7.0). The molar ellipticity ( ⁇ ) was calculated according to the literature (T. Wieprecht, et al., Biophys. Chem. 96, 191-201 (2002)).
- PHB2 (1: 1,000) (Abcam, Cambridge, UK); Akt, phosphorylated Akt (S473) (587F11, 1: P44 / 42 MAPK, phosphorylated p44 / 42 MAPK (T202 / Y204) (1: 1,000); ⁇ / ⁇ -tubulin (1: 1,000) (Cell Signaling Technology, Danvers, MA) , USA); and LMNB1 (1: 100) (Sigma).
- HRP-labeled secondary antibody (anti-mouse IgG-HRP, 1: 5,000; anti-rat IgG-HRP, 1: 5,000; anti-rabbit IgG-HRP, 1: 1,000) (Santa Cruz Biotechnology, Dallas, TX , USA) for 1 hour, the blots were developed with an enhanced chemiluminescence (ECL) system (GE Healthcare, Buckinghamshire, UK) and scanned using Image Reader LAS-3000 mini (Fuji Film, Tokyo, Japan). . All experiments were performed at least 3 times.
- ECL enhanced chemiluminescence
- Immunoprecipitation analysis was performed as previously described (T. Yoshimaru, et al., Nat. Commun. 4, 2443 (2013)). Cell lysates were precleared for 3 hours at 4 ° C. using normal IgG and rec-Protein G Sepharose 4B (Thermo Fisher Scientific). The supernatant was then incubated for 12 hours at 4 ° C. with 5 ⁇ g of antibody against BIG3 or PHB2. Next, the antigen-antibody complex was precipitated with rec-Protein G Sepharose 4B at 4 ° C. for 1 hour. The immunoprecipitated protein complex was washed several times with lysis buffer. Subsequently, SDS-PAGE and immunoblot analysis were performed as described above.
- Nuclei / Cytoplasmic fractions Nuclei and cytoplasmic fractions of MCF-7 cells were performed as previously described using NE-PER nuclear and cytoplasmic extraction reagent (Thermo Fisher Scientific) (T. Yoshimaru, et al., Nat. Commun. 4, 2443 (2013)). ⁇ / ⁇ -tubulin and lamin B were used as loading controls for the cytoplasmic and nuclear fractions, respectively.
- the primers used for RT-PCR are as follows: TFF1 5′-GGCCTCCTTAGGCAAATGTT-3 ′ (SEQ ID NO: 17) and 5′-CCTCCTCTCTGCTCCAAAGG-3 ′ (SEQ ID NO: 18); CCND1 5′-CAGAAGTGCGAGGAGGAGGT-3 ′ (SEQ ID NO: 19) and 5′-CGGATGGAGTTGTCGGTGT-3 ′ (SEQ ID NO: 20); ⁇ 2-MG 5′-AACTTAGAGGTGGGGAGCAG-3 ′ (SEQ ID NO: 21) and 5′-CACAACCATGCCTTACTTTATC-3 ′ (SEQ ID NO: 22).
- KPL-3C cell suspension (1 ⁇ 10 7 cells / mouse) was mixed with an equal volume of Matrigel (BD) and 6 week old female BALB / c nude mice (Charles River Laboratories, Tokyo, Japan) ) Mammary fat pad (total volume 200 ⁇ L).
- the mice were housed in a sterile isolation facility with a 12-hour light-dark cycle and fed with rodent chow and water ad libitum. Tumors grew over several days and reached a size of approximately 100 mm 3 (calculated as 1/2 ⁇ (width ⁇ length 2 )).
- mice were randomized into the following 11 treatment groups (5 / group): 1) Untreated; 2) E2 (6 ⁇ g / day, daily: hereinafter the same); 3) E2 + 1.4 mg / kg / day 11R-ERAP every day; 4) E2 + 1.4 mg / kg / day 11R-ERAP every 4 days; 5) E2 + 14 mg / kg / day 11R-ERAP daily; 6) E2 + 14 mg / kg / day 11R-ERAP every 4 days; 7) E2 + 1.4 mg / kg / day Stapled ERAP No. 7 12 every day; 8) E2 + 1.4 mg / kg / day Stapled ERAP No.
- E2 was administered via application of the solution to the cervical skin, and other treatments were administered via intraperitoneal injection unless otherwise specified. Tumor volume was measured with vernier calipers for 28 days, after which the animals were killed and resected. All experiments were conducted in accordance with Tokushima University animal facility guidelines.
- ERAP a dominant negative peptide targeting BIG3-PHB2 interaction
- Chemical modifications of ERAP were performed to improve both biological and biophysical properties such as long-term stability and the ability to inhibit BIG3-PHB2 interaction.
- FIG. 2A an attempt was made to produce a series of stapled ERAPs having stapling structures at different locations. Thereafter, the prepared stapled ERAP was screened for cell growth inhibitory activity.
- Stapled ERAP No. 1, 7 and 8 could not be synthesized, but other stapled ERAP (No. 2-6) could be synthesized.
- stapled ERAP No. Treatments 2 and 3 showed no significant effect on cell proliferation of normal breast epithelial cell lines, MCF-10A cells (ER ⁇ negative, BIG3 negative) (FIGS. 2C-E).
- stapled ERAP No. It was suggested that treatment with 4, 5 and 6 may have a non-specific inhibitory effect on cell proliferation of MCF-10A cells (FIGS. 2C-E).
- stapled ERAP No. Using MCF-10A cells treated with 3 or 6, gene expression profiles at 24 hours and 48 hours after treatment were analyzed by DNA microarray. According to gene expression profile analysis using cells 48 hours after treatment, stapled ERAP No. Compared to the cells treated with No. 3, stapled ERAP No. 93 transcripts up-regulated 100 times or more and 191 transcripts down-regulated in the cells treated with 6 were identified (upper figure in FIG. 2F, Table 2).
- TAM-R Tamoxifen-resistant MCF-7 cells
- TAM-R Tamoxifen-resistant MCF-7 cells
- FIG. 3J stapled ERAP No. Treatment with 12 significantly reduced cell proliferation of tamoxifen-resistant MCF-7 cells in the presence of E2 and tamoxifen for 96 hours after treatment.
- the inhibitory effect of 11R-ERAP was maintained only for 24 hours.
- stapled ERAP No The combined effect of 12 (0.5 ⁇ M) and tamoxifen (selective ER ⁇ modulator, 10 nM) or fulvestrant (ER ⁇ down regulator, 2 ⁇ M) was examined.
- KPL-3C orthotopic breast cancer xenografts were grown in nude mice.
- stapled ERAP No. 12 (1.4 and 14 mg / kg), 11R-ERAP (1.4 and 14 mg / kg), HA-tagged stapled ERAP No. 12 (14 mg / kg) or vehicle alone was administered daily or every 4 days by intraperitoneal (ip) injection for 28 days (FIG. 4A).
- E2 treatment (6 ⁇ g / day) was also performed on animals daily. Daily E2 treatment grows KPL-3C tumors in a time-dependent manner.
- the stapled ERAP No In order to clarify the mechanism of the antitumor effect of 12 in vivo, the stapled ERAP No.
- the mice were treated with KPL-3C heterotopically transplanted mice every day or every 4 days, and the mice were planned to die at 28 days after the start of treatment, and the tumors were removed to examine the intracellular distribution of PHB2.
- Tumor cells excised from mice were fractionated into a cytoplasmic fraction and a nuclear fraction, and each fraction was subjected to co-immunoprecipitation using an anti-PHB2 antibody.
- E2 11R-ERAP or stapled ERAP No.
- stapled ERAP No. 2 for activation of nongenomic ERa signaling pathway in tumors. Twelve effects were examined. 1.4 mg / kg stapled ERAP No. Anti-phosphorylated Akt antibody and anti-phosphorylated MAPK antibody were used to detect phosphorylation levels of Akt and MAPK in tumors excised from KPL-3C xenograft transplanted mice treated daily at 12 or every 4 days. As a result, as expected, stapled ERAP No. In any of the treatments at 12, significant suppression of Akt phosphorylation and MAPK phosphorylation was observed (FIG. 4J). Unlike 11R-ERAP, stapled ERAP No. 12 clearly suppresses the E2-dependent phosphorylation level even when treated every 4 days. 12 proved to have long-term strong in vivo anti-tumor activity (FIG. 4J).
- stapled ERAP No. Twelve long-term in vivo antitumor activities were examined.
- stapled ERAP No. 12 (0.02, 0.1 and 1 mg / kg) or vehicle alone was administered by intraperitoneal (ip) injection every 4 or 7 days for 28 days (FIG. 4M).
- E2 treatment (6 ⁇ g / day) was also performed on animals daily.
- 1 mg / kg stapled ERAP No. Treatment every 7 days with 12 completely inhibited E2-dependent tumor growth until day 28 (FIG. 4M).
- Example 3 Effect of stapled ERAP on prostate cancer cells
- the human prostate cancer cell line, 22Rv1 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). 22Rv1 was monolayer cultured in an appropriate medium supplemented with 10% FBS and maintained at 37 ° C. in an atmosphere of 5% CO 2 humidified air. 22Rv1 cells are 48-well plates (3 ⁇ 10 4 cells / mL) in RPMI (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) and 1% Antibiotic / Antilytic Solution (Wako, Tokyo, Japan). Alternatively, seeded in 10 cm dishes (8 ⁇ 10 6 cells / dish). After 48 hours, the cells were treated with 10 ⁇ M (for immunoprecipitation only), 20 ⁇ M and 50 ⁇ M stapled ERAP No. 12 respectively.
- Example 4 To identify stapled ERAP type D peptides and peptides in a configuration that is more resistant to retroinverse proteolysis, stapled ERAP No. Twelve D-type peptides and retroinverse forms were synthesized (FIG. 6A; M. Chorev, et al., Trends. Biotechnol. 13, 438-445 (1995); C. Bonny, et al., Diabetes 50, 77- 82 (2001); M. Taylor, et al., Biochemistry 49, 3261-3272 (2010); T. Weeden, et al., J. Pept. Sci. 17, 47-55 (2011)).
- the produced stapled ERAP No. 12 D-type peptides (hereinafter “stapled-D-ERAP No. 12”) and stapled ERAP No. 12 Twelve retro-inverse bodies (hereinafter “RI stapled ERAP No. 12”) inhibited E2-dependent cell proliferation of MCF-7 cells in a nanomolar dose-dependent manner (FIG. 6B, left). On the other hand, cell growth of MCF-10A cells was not inhibited (FIG. 6B right).
- Stapled-D-ERAP No. 12 and RI stapled ERAP No. The IC 50 of 12 was 0.44 ⁇ M and 0.50 ⁇ M, respectively, 96 hours after treatment (FIG. 6C). On the other hand, stapled ERAP No. The IC 50 of 12 was 0.59 ⁇ M (FIG. 6C).
- the short stapled retroinverse ERAP No. 1 which is a retroinverse body of the N-terminal partial sequence of ERAP (QMLSDLT (SEQ ID NO: 13)). 12 (hereinafter “shRI stapled ERAP No. 12”) was synthesized (FIG. 6A).
- RI stapled ERAP No. in vivo. Twelve anti-tumor activities were investigated. When the tumor was completely established, RI stapled ERAP No. 12 (0.02, 0.1 and 1 mg / kg) or vehicle alone were administered by intraperitoneal (ip) injection every 4 or 7 days. E2 (6 ⁇ g / day) treatment was also performed on animals daily. 1 mg / kg or 0.1 mg / kg RI stapled ERAP No. Treatment every 4 or 7 days at 12 almost completely inhibited E2-dependent tumor growth until day 28 after treatment (FIG. 6F). Furthermore, RI stapled ERAP No. Treatment every 4 or 7 days with 12 (0.02, 0.1 and 1 mg / kg) also significantly suppressed E2-dependent expression of ER ⁇ target genes TFF1 and CCND1 in tumors (FIG. 6G).
- Example 5 ERAP with a cell-permeable polyarginine residue added to the C-terminus Peptides with a membrane-permeable polyarginine residue (8R) added to the C-terminus of ERAP and its partial sequence were designed ( Q MLS D LTL Q LRQR-8R (SEQ ID NO: 10) and Q MLS D LTL Q L-8R, respectively) (SEQ ID NO: 11); FIG. 7).
- Q MLS D LTL Q LRQR-8R SEQ ID NO: 10
- Q MLS D LTL Q L-8R Q MLS D LTL Q L-8R
- Example 6 Inhibitory effect of stapled ERAP (No. 12) on phosphorylation of mTOR and S6K in tamoxifen-resistant breast cancer cell line Tamoxifen (TAM) -resistant MCF7 cells induced significant mTOR and S6K phosphorylation in the presence of TAM
- TAM Tamoxifen
- the phosphorylation intensity of mTOR and S6K by adding E2 for 96 hours in the presence of TAM was almost the same as the 24-hour reaction, but the inhibitory effect of 11R-ERAP treatment was significantly attenuated compared to the 24-hour reaction. (FIG. 8).
- the stapled ERAP (No. 12) treatment maintained an almost complete suppressive effect even after a 96-hour reaction, and it was considered that it could be stably suppressed for a long period of time against TAM-resistant breast cancer cases.
- Example 7 Combined effect of stapled ERAP (No. 12) and tamoxifen, fulvestrant, everolimus on E2-dependent cell proliferation 11R-ERAP and stapled ERAP (No. 12) are E2-dependent in a 24-hour reaction Suppresses cell proliferation almost completely, and when used in combination with tamoxifen (antiestrogenic agent), fulvestrant (ER ⁇ modulator), everolimus (mTOR inhibitor), synergistic inhibitory effect is observed, and the number of living cells is not yet The number was reduced to the number of viable cells or less by treatment (FIG. 9, right). In the 96-hour reaction, stapled ERAP (No.
- Example 8 Anti-tumor effect by administration of stapled ERAP (No. 12) by tail vein
- stapled ERAP No. 12
- KPL-3C orthotopic breast cancer xenogeneic When the graft is grown in nude mice and the tumor is fully established, stapled ERAP (No. 12) (0.1, 1, 10 mg / kg) or vehicle alone is added every day or every 7 days. Administered by tail vein injection for 35 days. E2 (6 ⁇ g / day) was also administered daily. Daily E2 treatment allowed KPL-3C tumors to grow in a time-dependent manner (FIG. 10 left).
- administration of stapled ERAP No.
- Example 9 Interaction between BIG3 and PHB2 in a transplanted mouse isolated tumor treated with stapled ERAP (No. 12)
- the effect of stapled ERAP (No. 12) on the interaction between BIG3 and PHB2 in a transplanted mouse isolated tumor was examined.
- Co-immunoprecipitation studies were performed with anti-BIG3 antibodies on tumors removed from KPL-3C xenograft-transplanted mice treated daily or every 7 days with 10 mg / kg stapled ERAP (No. 12).
- As a result it was confirmed that BIG3 and PHB2 were firmly bound in the untreated group and the E2 daily administration group, but 35 mg daily administration of 10 mg / kg stapled ERAP (No. 12) and every 7 days were administered.
- the tumor showed almost no coprecipitation of PHB2, indicating that 10 mg / kg stapled ERAP (No. 12) almost completely inhibited the interaction between BIG3 and PHB2 and suppressed tumor growth. (FIG. 11).
- Example 10 Phosphorylation of Akt and MAPK in tumors treated with stapled ERAP (No. 12)
- tumors excised from KPL-3C xenograft-transplanted mice treated daily or every 7 days with 10 mg / kg stapled ERAP (No. 12) Akt using anti-phosphorylated Akt antibody and anti-phosphorylated MAPK antibody And the phosphorylation level of MAPK was detected.
- marked suppression of Akt phosphorylation and MAPK phosphorylation was observed in the stapled ERAP (No. 12) treatment when administered daily and every 7 days (FIG. 12).
- the blood-brain barrier permeability test of stapled ERAP (No. 12) was conducted by placing stapled ERAP (No. 12) inside the insert (blood vessel side) of the blood-brain barrier permeability kit and transferring into the brain for 30 minutes. This was carried out by measuring the concentration of stapled ERAP (No.
- the transmission coefficient (Papp) at that time was calculated (2 or less: permeability is quite low, 2-10: low, 10-20: high, 20 or more: fairly high).
- the blood-brain barrier permeability coefficient of stapled ERAP No. 12 is 2 or less, and it is considered that the possibility of transferring into the brain is low, and it is suggested that there is no concern of side effects caused by transferring into the brain. (Table 3).
- the present invention provides a peptide having a longer duration of inhibitory effect on BIG3-PHB2 interaction.
- the peptide of the present invention has cell permeability and low blood brain barrier permeability.
- the pharmaceutical composition containing the peptide of the present invention or a salt thereof can be used to treat cancer, particularly estrogen receptor positive cancer, estrogen receptor negative breast cancer and prostate cancer.
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Abstract
Description
これらの知見に基づき、BIG3-PHB2相互作用の阻害によりBIG3との複合体からPHB2を遊離させ、PHB2の腫瘍抑制活性を発揮させるという戦略は、乳がんの新規療法となりうる。この戦略に基づき、本発明者らは、以前、BIG3-PHB2相互作用を特異的に阻害するBIG3のドミナントネガティブペプチドを開発した(特許文献1)。このペプチドは、PHB2の腫瘍抑制活性を再活性化することにより、乳がん増殖をもたらすERαシグナルパスウェイを阻害し、乳がん増殖を抑制することが確認されている(特許文献1)。
[2]n対のアミノ酸残基が、以下の(a)または(b)の1対のアミノ酸残基である、[1]記載のペプチド、またはその塩:
(a)配列番号:9に記載のアミノ酸配列のN末端から3番目および7番目のアミノ酸残基;
(b)配列番号:9に記載のアミノ酸配列のN末端から2番目および6番目のアミノ酸残基;
[3]配列番号:9に記載のアミノ酸配列の部分配列が、配列番号:13に記載のアミノ酸配列である、[1]または[2]に記載のペプチド、またはその塩;
[4]n対のアミノ酸残基が、以下の(a)または(b)の1対のアミノ酸残基である、[3]記載のペプチド、またはその塩:
(a)配列番号:13に記載のアミノ酸配列のN末端から3番目および7番目のアミノ酸残基;
(b)配列番号:13に記載のアミノ酸配列のN末端から2番目および6番目のアミノ酸残基;
[5]ステープリング構造が下記式(I)で表される、[1]~[4]のいずれか一項に記載のペプチド、
(式中、実線と点線の二重線は単結合または二重結合を示す。)
またはその塩;
[6]下記式(II)で表される、[5]に記載のペプチド、
(式中、実線と点線の二重線は単結合または二重結合であり;
A1、A2およびA3の組み合わせは以下から選択される:
A1=Q、A2=LSDおよびA3=TLQLRQR(配列番号:14);
A1=QM、A2=SDLおよびA3=LQLRQR(配列番号:15);
A1=QM、A2=SDLおよびA3=-OH;ならびに
A1=Q、A2=LSDおよびA3=T。)
またはその塩;
[7]N末端およびC末端のアミノ酸残基のいずれかまたは両方が修飾されている、[1]~[6]のいずれか一項に記載のペプチド、またはその塩;
[8]N末端およびC末端のアミノ酸残基のいずれかまたは両方が、アセチル化、アミド化およびHAタグ付加のいずれかまたはこれらの組み合わせにより修飾されている、[7]に記載のペプチド、またはその塩;
[9]N末端のアミノ酸残基がアセチル化されており、かつC末端のアミノ酸残基がアミド化されている、[8]に記載のペプチド、またはその塩;
[10]全てのアミノ酸残基がD型のアミノ酸残基に置き換えられた、[1]~[9]のいずれか一項に記載のペプチド、またはその塩;
[11][1]~[9]のいずれか一項に記載のペプチドのレトロインバース体であるペプチド、またはその塩;
[12][1]~[11]のいずれか一項に記載のペプチド、またはその塩と、薬学的に許容される担体とを含む、医薬組成物;
[13]がん治療用である、[12]に記載の医薬組成物;
[14]がんが乳がんまたは前立腺がんである、[13]に記載の医薬組成物;ならびに
[15]がんがエストロゲン受容体陽性のがんである、[13]または[14]に記載の医薬組成物。
本明細書で使用する「1つの(a)」、「1つの(an)」、および「その(the)」という単語は、特に明記しない限り「少なくとも1つの」を意味する。
本発明のペプチドは、配列番号:9に記載のアミノ酸配列またはその部分配列において、n対(nは自然数)のアミノ酸残基が、n個のステープリング構造で置き換えられたアミノ酸配列を含むペプチドである。nは3以下であることが好ましく、2であることがより好ましく、1であることがさらに好ましい。したがって、本発明において、n対のアミノ酸残基とは、通常1~3対、あるいは1対または2対、好ましくは1対のアミノ酸残基をさす。
(a)配列番号:9に記載のアミノ酸配列のN末端から3番目(L)および7番目のアミノ酸残基(T);
(b)配列番号:9に記載のアミノ酸配列のN末端から2番目(M)および6番目のアミノ酸残基(L);
(c)配列番号:9に記載のアミノ酸配列のN末端から4番目(S)および8番目のアミノ酸残基(L);および
(d)配列番号:9に記載のアミノ酸配列のN末端から6番目(L)および10番目のアミノ酸残基(L)。
(a)配列番号:13に記載のアミノ酸配列のN末端から3番目(L)および7番目のアミノ酸残基(T);および
(b)配列番号:13に記載のアミノ酸配列のN末端から2番目(M)および6番目のアミノ酸残基(L)。
上記スキーム(III)において、(i)~(vi)はそれぞれ以下を示す;(i)3-アミノ-1-プロペン、AcOH、MgSO4、CH2Cl2;(ii)NaBH4、MeOH、CH2Cl2;(iii)化合物2、DCC、CH2Cl2;(iv)LiOH・H2O、THF、MeOH、H2O;(v)TBSOtf、2,6-ルチジン;(vi)Fmoc-OSu、Na2CO3、THF、H2O。
(式中、実線と点線の二重線は単結合または二重結合であり;
A1、A2およびA3の組み合わせは以下から選択される:
A1=Q、A2=LSDおよびA3=TLQLRQR(配列番号:14);
A1=QM、A2=SDLおよびA3=LQLRQR(配列番号:15);
A1=QM、A2=SDLおよびA3=-OH;ならびに
A1=Q、A2=LSDおよびA3=T。)
(a)配列番号:9に記載のアミノ酸配列のN末端から3番目(L)および7番目のアミノ酸残基(T); ならびに
(b)配列番号:9に記載のアミノ酸配列のN末端から2番目(M)および6番目のアミノ酸残基(L)。
あるいは、配列番号:13(QMLSDLT)に記載のアミノ酸配列からなるペプチドにおいて、以下の(c)または(d)の1対のアミノ酸残基が、式(I)のステープル構造で置き換えられたペプチドである;
(c)配列番号:13に記載のアミノ酸配列のN末端から3番目(L)および7番目のアミノ酸残基(T);ならびに
(d)配列番号:13に記載のアミノ酸配列のN末端から2番目(M)および6番目のアミノ酸残基(L)。
A1=Q、A2=LSDおよびA3=TLQLRQR(配列番号:14);
A1=QM、A2=SDLおよびA3=LQLRQR(配列番号:15);または
A1=QM、A2=SDLおよびA3=-OH。
これらのペプチドは、以下のペプチドに対応する;
(i)配列番号:9(QMLSDLTLQLRQR)に記載のアミノ酸配列からなるペプチドにおいて、以下の(a)または(b)の1対のアミノ酸残基が、式(I)のステープル構造で置き換えられたペプチド;
(a)配列番号:9に記載のアミノ酸配列のN末端から3番目(L)および7番目のアミノ酸残基(T);ならびに
(b)配列番号:9に記載のアミノ酸配列のN末端から2番目(M)および6番目のアミノ酸残基(L);あるいは、
(ii)配列番号:13(QMLSDLT)に記載のアミノ酸配列からなるペプチドにおいて、以下の1対のアミノ酸残基が、式(I)のステープル構造で置き換えられたペプチド;
(c)配列番号:13に記載のアミノ酸配列のN末端から3番目(L)および7番目のアミノ酸残基(T)。
本発明のペプチドまたはその塩は、薬学的に許容される担体とともに、医薬組成物として製剤化することができる。
本発明のペプチドまたはその塩の用量の例としては、例えば、0.001~1000mg/kg/日、0.005~500mg/kg/日、0.01~250mg/kg/日等が挙げられるが、これらに限定されるものではない。
(a)がんを治療するための医薬組成物の製造における、本発明のペプチドまたはその塩の使用;
(b)がんの治療において用いるための本発明のペプチドまたはその塩;
(c)がんを治療するための医薬組成物を製造するための方法または工程であって、本発明のペプチドまたはその塩と、薬学的に許容される担体とを製剤化する段階を含む方法または工程;
(d)がんを治療するための医薬組成物を製造するための方法または工程であって、本発明のペプチドまたはその塩を薬学的に許容される担体と混合する段階を含む方法または工程;および
(e)本発明のペプチドまたはその塩を対象に投与することを含む、がんを治療するための方法。
ERAPペプチドの合成
BIG3-PHB2の相互作用を特異的に阻害するように設計したドミナントネガティブペプチド(11R-ERAP;11R-GGG-QMLSDLTLQLRQR(配列番号:9))を以前記載したように合成した(T. Yoshimaru, et al., Nat. Commun. 4, 2443 (2013).)。全ての化学薬品は分析グレードのものを使用した。11R-ERAPを構成する「11R」は、11残基のアルギニン残基からなるポリアルギニン(ポリR)を示す。ポリRと配列番号:9の間にある「GGG」は、両者のリンカーとして導入された3残基のグリシンである。11Rは、ERAP(配列番号:9)に細胞透過性を付与することを目的に導入されたものである。
オレフィン担持ステープルドペプチドの合成に使用したアミノ酸誘導体は、Aihara et al(Tetrahedron, 71, 4183-4191 (2015))に記載の方法に基づき合成した。アミノ酸誘導体の原料となるアミノ酸は、株式会社ペプチド研究所(大阪、日本)から購入した。
オレフィンを有さないステープルドペプチドの合成に使用した2種のアミノ酸誘導体のうち、グルタミン酸誘導体(N-α-(9-フルオレニルメトキシカルボニル)-L-グルタミン酸γアリルエステル)は、渡辺化学工業(広島、日本)から入手した。一方、グルタミン誘導体(((S)-2-((((9H-フルオレン-9-イル)メトキシ)カルボニル)アミノ)5-((4-(((アリルオキシ)カルボニル)アミノ)ブチル)(2,4-ジメトキシベンジル)アミノ)-5-オキソペンタン酸)は、図1Aに示すスキームに従って合成した。
1H NMR(CDCl3,300MHz)δ=1.42-1.58(4H,m),2.56(2H,t,J=6.7Hz),3.15(2H,dt,J=6.0および6.0Hz),3.67(2H,s),3.77(3H,s),3.78(3H,s),4.52(2H,d,J=5.5Hz),5.17(1H,ddt J=10.5および1.5,1.5Hz),5.27(1H,ddt J=17.3,1.5および1.5Hz),5.35(1H,br s),5.89(1H,ddt,J=17.3,10.5および5.5Hz),6.40(1H,dd,J=8.1および2.4Hz),6.43(1H,d,J=2.4Hz),7.09(1H,d,J=8.1Hz);13C NMR(CDCl3,75MHz) δ=27.4,27.9,41.0,48.6,48.9,55.3,55.4,65.4,98.6,103.7,117.4,120.9,130.5,133.2,156.4,158.6,160.1;HRMS(ESI-TOF)m/z calcd for C17H27N2O4 ([M+H]+):323.1971, found:323.1963。
[α]19 D-5.33(c 1.24,MeOH);1H NMR(DMSO-d6,300MHz,80℃)δ=1.38(9H,s),1.27-1.53(2H,m),1.27-1.53(2H,m),1.46-1.92(1H,m),1.92-2.10(1H,m),2.42(2H,dt,J=4.5および6.6Hz),2.97(2H,dt,J=6.0および6.3Hz),3.19(2H,br t,J=7.0Hz),3.62(3H,s),3.76(3H,s),3.80(3H,s),3.94-4.14(1H,m),4.39(2H,br s),4.46(2H,ddd,J=5.5,1.7および1.3Hz),5.16(1H,ddt,J=10.4,1.8および1.3Hz),5.26(1H,ddt,J=17.2,1.8および1.7Hz),5.90(1H,ddt,J=17.2,10.4および5.5Hz),6.39-6.53(1H,br m),6.53-6.63(1H,br m),6.70-6.92(2H,br m),6.96(1H,br d,7.9Hz);13C NMR(DMSO-d6,75MHz,80℃)δ=24.0,25.2,26.4,26.5,27.8,28.3,41.6,44.4,45.3,46.1,51.1,53.1,54.9,55.1,63.7,77.9,78.7,98.4,104.7,116.3,117.0,128.0,128.8,133.5,154.9,155.5,157.8,159.5,159.8,171.0,172.4;HRMS(ESI-TOF)m/z calcd for C28H43N3NaO9 ([M+Na]+):588.2897,found:588.2902。
[α]18 D-0.65(c 0.950,MeOH);1H NMR(DMSO-d6,300MHz,80℃) δ=1.39(9H,s),1.27-1.53(2H,m),1.27-1.53(2H,m),1.76-1.94(1H,m),1.94-2.13(1H,m),2.44(2H,dt,J=7.5および4.2Hz),2.99(2H,dt,J=6.3および6.1Hz),3.20(2H,br t,J=7.1Hz),3.76(3H,s),3.80(3H,s),3.89-4.08(1H,m),4.41(2H,br s),4.47(2H,ddd,J=5.4,1.5および1.5Hz),5.16(1H,ddt,J=10.5,1.7および1.5Hz),5.26(1H,ddt,J=17.4,1.7および1.5Hz),5.90(1H,ddt,J=17.4,10.5および5.4Hz),6.38-6.53(1H,br m),6.56(1H,br s),6.66(1H,br s),6.78(1H,br s),6.98(1H,br d,J=8.1Hz);13C NMR(d-DMSO,75MHz,80℃)δ=24.2,25.3,26.6,26.7,27.9,28.6,39.9,41.8,44.4,45.4,46.3,53.1,55.0,55.2,63.8,77.8,98.5,104.8,116.3,117.1,117.8,128.1,128.8,133.5,155.1,155.6,157.9,159.6,159.9,171.3,173.3;HRMS(ESI-TOF)m/z calcd for C27H41N3NaO9([M+Na]+):574.2741,found:574.2740。
1H NMR(DMSO-d6,300MHz,80℃)δ=1.29-1.61(4H,m),1.87-2.02(1H,m),2.03-2.21(1H,m),2.45-2.56(2H,m),3.00(2H,dt,J=6.0および6.4Hz),3.23(2H,br t,J=6.6Hz),3.74(3H,s),3.79(3H,s),4.05-4.18(1H,m),4.22(1H,t,J=6.6Hz),4.31(2H,d,J=6.6Hz),4.43(1H,br s),4.48(2H,ddd,J=5.7,1.7および1.5Hz),5.12(1H,ddt,J=10.2,1.7および1.5Hz),5.27(1H,ddt,J=17.1,1.7および1.7Hz),5.91(1H,ddt,J=17.1,10.2および5.7Hz),6.42-6.52(1H,br m),6.56(1H,d,J=2.1Hz),6.80(1H,br s),6.91-7.10(1H,br m),7.31(2H,t,J=7.5 Hz),7.40(2H,t,J=7.2Hz),7.70(2H,br d,J=7.2Hz),7.85(2H,d,J=7.5Hz);13C NMR(DMSO-d6,75MHz,80℃)δ=24.2,25.3,26.6,28.6,39.9,41.8.44.3,45.3,46.6,53.4,54.9,55.1,63.8,65.6,98.4,104.7,108.6,116.3,117.0,117.7,119.6,120.9,124.8,126.6,126.6,126.8,127.2,128.2,128.5,128.9,133.5,139.2,140.4,140.4,143.6,143.6,155.6,155.6,157.8,159.6,159.9,171.2,173.1;HRMS(ESI-TOF)m/z calcd for C37H43N3NaO9 ([M+Na]+):696.2897,found:696.2928。
標準Fmoc固相ペプチド合成を用いて、Rink Amide AMレジン(0.62mmol アミン/g)上でペプチドを合成した。Fmoc基切断は、20%(v/v)ピペリジンDMF溶液を用いて室温で10分間行った。レジンをDMFで洗浄し、Fmoc保護アミノ酸(Fmoc-Xaa-OH)を、DMF中のN,N-ジイソプロピルカルボジイミド(DIPCDI)および1-ヒドロキシ-1H-ベンゾトリアゾール水和物(HOBt・H2O)を用いて、室温で2時間カップリングさせ、続いてDMFで洗浄した。
材料と方法
細胞株および培養条件
ヒト乳がん細胞株MCF-7、および乳腺上皮細胞株MCF-10Aは、American Type Culture Collection(ATCC, Manassas, VA, USA)から購入した。乳がん細胞株KPLC-3C株(J. Kurebayashi, et al., Br. J. Cancer 74, 200-207 (1996))は、紅林淳一博士(川崎医科大学)の厚意により提供された。全ての細胞株は、10%FBSを補充した適切な培地で単層培養した。5%CO2を含む加湿空気の雰囲気下で37℃で細胞を維持した。
MCF-7、KPL-3CおよびMCF-10Aにおける細胞増殖アッセイは、Cell Counting Kit-8(CCK-8)(同仁堂、熊本、日本)を用いて、以前記載したように行った(T. Yoshimaru, et al., Nat. Commun. 4, 2443 (2013))。データは3つの独立した実験の平均±SEで示した。
185~265nmの範囲におけるCDスペクトルを、光路長2mmの石英キュベットを用いて25℃で記録した(円二色性分散計J1500:日本分光株式会社、東京、日本)。ペプチド濃度は、10mMリン酸ナトリウムバッファー(pH7.0)中で50μg/mLとした。モル楕円率(θ)は文献に従って計算した(T. Wieprecht, et al., Biophys. Chem. 96, 191-201 (2002))。
イムノブロット解析は、以前記載したように行った(T. Yoshimaru, et al., Nat. Commun. 4, 2443 (2013))。SDS-PAGE後、タンパク質をブロットしたメンブレンを4%ブロックエース溶液(大日本製薬、大阪、日本)で3時間ブロッキングし、その後、以下のタンパク質に対する抗体とインキュベートした:BIG3(1:1,000)(T. Yoshimaru, et al., Nat. Commun. 4, 2443 (2013));PHB2(1:1,000)(Abcam, Cambridge, UK);Akt,リン酸化Akt(S473)(587F11、1:1,000);p44/42 MAPK,リン酸化p44/42 MAPK(T202/Y204)(1:1,000);α/β-チューブリン(1:1,000)(Cell Signaling Technology, Danvers, MA, USA);およびLMNB1(1:100)(Sigma)。HRP標識二次抗体(抗マウスIgG-HRP、1:5,000;抗ラットIgG-HRP、1:5,000;抗ウサギIgG-HRP、1:1,000)(Santa Cruz Biotechnology, Dallas, TX, USA)と1時間インキュベートした後、ブロットを増強化学発光(ECL)システム(GE Healthcare, Buckinghamshire, UK)で展開し、Image Reader LAS-3000 mini(富士フィルム、東京、日本)を用いてスキャンした。全ての実験は、少なくとも3回行った。
免疫沈降解析は、以前記載したように行った(T. Yoshimaru, et al., Nat. Commun. 4, 2443 (2013))。細胞溶解物を、ノーマルIgGとrec-Protein G Sepharose 4B(Thermo Fisher Scientific)を用いて、4℃で3時間プレクリアした。その後、上清を、BIG3に対する抗体またはPHB2に対する抗体5μgと4℃で12時間インキュベートした。次に、抗原抗体複合体をrec-Protein G Sepharose 4Bを用いて4℃で1時間沈降した。免疫沈降されたタンパク質複合体をライシスバッファーで数回洗浄した。続いてSDS-PAGEおよびイムノブロット解析を上記のように行った。
MCF-7細胞の核および細胞質画分をNE-PER nuclear and cytoplasmic extraction reagent(Thermo Fisher Scientific)を用いて、以前記載したように行った(T. Yoshimaru, et al., Nat. Commun. 4, 2443 (2013))。α/β-チューブリンとラミンBを、それぞれ細胞質画分と核画分のローディングコントロールとして使用した。
MCF-7細胞を5×104細胞/ウェルで8ウェルチャンバー(Laboratory-Tek II Chamber Slide System)(Nalgene, Nunc International)に播種し、48時間インキュベートした。その後、E2およびHAタグステープルドERAPまたはE2単独で24時間処理した。染色手順は、以前記載した通りに行った(T. Yoshimaru, et al., Nat. Commun. 4, 2443 (2013))。
ERα標的遺伝子(TFF1およびCCND1)の発現を、リアルタイムRT-PCRにより、以前記載したように評価した(T. Yoshimaru, et al., Nat. Commun. 4, 2443 (2013))。各サンプルをβ2-MG mRNA含量で標準化し、未処理細胞における発現量を1.0とした場合の倍数で結果を示した。データは、3つの独立した実験の平均±SDで示した。RT-PCRに用いたプライマーは以下のとおりである;
TFF1 5'-GGCCTCCTTAGGCAAATGTT-3'(配列番号:17) および
5'-CCTCCTCTCTGCTCCAAAGG-3' (配列番号:18);
CCND1 5'-CAGAAGTGCGAGGAGGAGGT-3' (配列番号:19) および
5'-CGGATGGAGTTGTCGGTGT-3' (配列番号:20);
β2-MG 5'-AACTTAGAGGTGGGGAGCAG-3' (配列番号:21) および
5'-CACAACCATGCCTTACTTTATC-3' (配列番号:22)。
KPL-3C細胞縣濁液(1×107細胞/マウス)を等量のマトリゲル(BD)と混合し、6週齢メスBALB/cヌードマウス(Charles River Laboratories、東京、日本)の乳房脂肪体に注射した(全量200μL)。該マウスを無菌隔離施設において12時間の明暗周期で収容し、げっ歯類固形飼料と水を不断給餌した。腫瘍は数日にわたって増殖し、およそ100mm3のサイズ(1/2×(幅×長さ2)として計算)に達した。マウスを以下の11処理群(5匹/群)にランダム化した:
1)未処理;
2)E2(6μg/日、毎日:以下同じ);
3)E2+1.4mg/kg/日 11R-ERAP 毎日;
4)E2+1.4mg/kg/日 11R-ERAP 4日毎;
5)E2+14mg/kg/日 11R-ERAP 毎日;
6)E2+14mg/kg/日 11R-ERAP 4日毎;
7)E2+1.4mg/kg/日 ステープルドERAP No.12 毎日;
8)E2+1.4mg/kg/日 ステープルドERAP No.12 4日毎;
9)E2+14mg/kg/日 ステープルドERAP No.12 毎日;
10)E2+14mg/kg/日 ステープルドERAP No.12 4日毎;
11)E2+14mg/kg/日 HAタグステープルドERAP No.12 毎日;
12)E2+14mg/kg/日 HAタグステープルドERAP No.12 4日毎。
E2は頸部皮膚への溶液塗布を介して投与し、他の処理は別の投与方法を明記している場合を除いて腹腔内注射を介して投与した。腫瘍体積を28日間ノギスで計測し、その後、動物を計画死させて腫瘍を切除した。全ての実験は徳島大学の動物施設ガイドラインに従って行った。
全RNAを、NucleoSpin RNA IIシステム(タカラクロンテック、日本)を用いて、製造者の説明書に従って精製した。RNA増幅とラベリングを、Agient Low-Input QuickAmpラベリングキット(Agilent Technologies, Palo Alto, CA, USA)を用いて、製造者の説明書に従って行った。簡潔に述べると、各サンプル由来の全RNAの100ngを、T7 RNAポリメラーゼを用いて、Cy3ラベルCTPを取り込ませながら増幅した。その後、Cy3ラベル化されたcRNAの600ngを断片化し、Agient Whole Human Genome Microarray 8×60Kスライド(Agilent Technologies)上でハイブリダイズさせ、65℃で18時間、回転しながらインキュベートした。その後、スライドを洗浄し、オゾン保護ドラフト内で、Agilent Microarrayスキャナーシステムによりスキャンした。該スキャンイメージファイルを、Agient Feature Extraction(version 9.5)(Agilent Technologies)を用いて抽出した。データは、GeneSpring(version 13.0)を用いて解析した。全てのチップと遺伝子のマイクロアレイデータをquantile正規化により正規化し、ベースラインを全てのサンプルの中央値に対するシグナル値に変換した。最後に、発現レベルに基づく品質コントロールとフィルタリング工程を行った。有意に発現量が変化している遺伝子を同定するために、各解析間のシグナル強度値を比較した。
統計解析
スチューデントt検定を用いて、実験群間の有意差を求めた。P値<0.05を有意とみなした。
オレフィン担持ステープルドERAPの効果
本発明者らは、以前、BIG3-PHB2相互作用を標的とするドミナントネガティブペプチドであるERAPを設計した(T. Yoshimaru, et al., Nat. Commun. 4, 2443 (2013))。長期安定性やBIG3-PHB2相互作用の阻害機能のような生物学的および生物物理学的性質の両方を改善するために、ERAPの化学修飾を行った。図2Aに示すように、異なる位置にステープリング構造を有する一連のステープルドERAPの作製を試みた。その後、作製したステープルドERAPを、細胞増殖阻害活性によりスクリーニングした。ステープルドERAP No.1、7および8は、合成できなかったが、他のステープルドERAP(No.2~6)は合成できた。
ルテニウム触媒によるオレフィンメタセシスはコストがかかるため、分子内アミド化を介して、オレフィンを有さないステープルドERAP No.3の代替物(ステープルドERAP No.12)を新規に合成した(図3A)。このステープルドERAP No.12もまたステープルドERAP No.3に匹敵する42.5%のヘリシティーを維持し(図3B)、MCF-7細胞のE2依存性細胞増殖とBIG3-PHB2相互作用を有意に長期間阻害した(図3C、3D)。一方、ステープルドERAP No.12は、MCF-10A細胞の細胞増殖は阻害しなかった(図3C)。さらに、ERα標的遺伝子であるTFF1およびCCND1の発現に対するステープルドERAP No.12の阻害的効果は、11R-ERAPと比較して、処理後96時間の時点でも高いレベルで維持されていた(図3E)。
ステープルドERAP No.12のin vivoにおける抗腫瘍効果を調べるために、KPL-3C同所性乳がん異種移植片をヌードマウス内で増殖させた。腫瘍が完全に樹立された時点で、ステープルドERAP No.12(1.4および14mg/kg)、11R-ERAP(1.4および14mg/kg)、HAタグステープルドERAP No.12(14mg/kg)または溶媒単独(vehicle alone)を、毎日または4日毎に、28日間腹腔内(i.p.)注射により投与した(図4A)。E2処理(6μg/日)もまた毎日動物に行った。毎日のE2処理は、経時依存的にKPL-3C腫瘍を増殖させる。一方、ステープルドERAP No.12または11R-ERAPでの毎日の処理は、1.4mg/kgおよび14mg/kgのいずれの用量においても、E2依存性腫瘍増殖の有意な阻害を引き起こした(図4B、図4C)。特に、ステープルドERAP No.12での処理は、11R-ERAPでの処理とは異なり、4日毎の投与であっても有意な阻害効果を持続した(図4B、4C)。このことは、ステープルドERAP No.12が、治療的観点から、優れた治療指数(therapeutic index)を有することを示唆する。なお、毒性および有意な体重減少は観察されなかった(図4D)。したがって、今回の条件の下では、有害な副作用は観察されなかったと言える。
材料と方法
細胞株および培養条件
ヒト前立腺がん細胞株、22Rv1は、American Type Culture Collection (ATCC, Manassas, VA, USA)から購入した。22Rv1は、10%FBSを補充した適切な培地で単層培養し、5%CO2加湿空気の雰囲気下で37℃で維持した。22Rv1細胞は、10%FBS(Thermo Fisher Scientific)および1%Antibiotic/Antimycotic solution(和光、東京、日本)を補充したRPMI(Thermo Fisher Scientific)中で、48ウェルプレート(3×104細胞/mL)または10cmディッシュ(8×106細胞/ディッシュ)に播種した。48時間後、細胞を10μM(免疫沈降の場合のみ)、20μMおよび50μMのステープルドERAP No.12でそれぞれ処理した。
細胞増殖アッセイは、死細胞をトリパンブルーで染色し、全細胞数をCountess II(Thermo Fisher Scientific)で評価した。細胞生存率は、Countess II自動セルカウンター(Thermo Fisher Scientific)を用いて、製造者の説明書に従って、24時間毎に測定した。
免疫沈降
免疫沈降は、実施例2と同様に行った。
前立腺がん細胞株22Rv1細胞(ERα陰性、BIG3陽性、PHB2陽性)を用いて、E2非依存性細胞増殖に対するステープルドERAP No.12の阻害効果を試験した。図5Aに示すように、ステープルドERAP No.12での処理は、経時依存的および用量依存的に、22Rv1細胞の細胞増殖を有意に抑制した。
タンパク質分解に対してより抵抗性のある立体配置のペプチドを同定するために、ステープルドERAP No.12のD型ペプチドおよびレトロインバース体を合成した(図6A; M. Chorev, et al., Trends. Biotechnol. 13, 438-445 (1995); C. Bonny, et al., Diabetes 50, 77-82 (2001); M. Taylor, et al., Biochemistry 49, 3261-3272 (2010); T. Weeden, et al., J. Pept. Sci. 17, 47-55 (2011))。レトロインバース体では、全てのL-アミノ酸をD-アミノ酸に置換することによってアミノ酸のキラリティーが逆になっているだけでなく、アミノ酸配列もまた逆になっている。ステープルドERAP No.12のD型ペプチドおよびレトロインバース体の合成は、ペプチド合成の際にL-アミノ酸に替えてD-アミノ酸を使用する以外は、L型ペプチドのステープルドERAPの合成と同様に行った。
ERAPおよびその部分配列のC末端に膜透過性ポリアルギニン残基(8R)を付加したペプチドをそれぞれ設計した(QMLSDLTLQLRQR-8R(配列番号10)およびQMLSDLTLQL-8R(配列番号:11);図7)。MCF-7細胞をこれらのペプチドで処理したところ、これらのペプチドでの処理は、MCF-7細胞のE2依存性細胞増殖に対して、11R-ERAPと類似した阻害効果を示した(それぞれIC50=7.78μMおよび7.98μM)(図7)。
タモキシフェン(TAM)耐性MCF7細胞はTAM存在下で顕著なmTORとS6Kのリン酸化を誘導し、24時間E2添加時とほぼ同じリン酸化強度であったが、ステープルドERAP(No.12)および11R-ERAPの24時間処理は各リン酸化をほぼ完全に抑制し、ネガティブ・コントロール(TAM非存在下の未処理細胞)以下であった(図8)。
また、TAM存在下で96時間のE2添加によるmTORとS6Kのリン酸化強度は24時間反応とほとんど同じであったが、11R-ERAP処理の抑制効果は24時間反応と比べて顕著に減弱していた(図8)。一方、ステープルドERAP(No.12)処理はほぼ完全な抑制効果を96時間反応でも維持しており、TAM耐性乳がん症例に対しても長期安定的に抑制できると考えられた。
24時間反応では11R-ERAPおよびステープルドERAP(No.12)はE2依存性の細胞増殖をほぼ完全に抑制し、タモキシフェン(抗エストロゲン剤)、フルベストラント(ERαモジュレーター)、エベロリムス(mTOR阻害剤)と併用することで、相乗的な抑制効果を認め、その生細胞数は未処理による生細胞数以下まで減少させた(図9右)。
96時間反応ではステープルドERAP(No.12)はほぼ完全な抑制効果を継続し、24 時間反応と同様に既存の阻害薬と相乗的な抑制効果を示した(図9左)。一方、11R-ERAPはE2依存性増殖に対して45%の抑制率に減弱していたが、既存薬との併用効果は24時間反応とほぼ同じ生細胞数になり、24時間の時点で細胞死を誘導していた可能性が考えられた。
ステープルドERAP(No.12)のin vivoにおける静脈投与による抗腫瘍効果を調べるために、KPL-3C同所性乳がん異種移植片をヌードマウス内で増殖させて、腫瘍が完全に樹立された時点で、ステープルドERAP(No.12) (0.1、1、10mg/kg)または溶媒単独を連日または7日毎に、35日間尾静脈注射により投与した。E2 (6μg/日)もまた連日投与した。毎日のE2処理は、経時依存的にKPL‐3C腫瘍を増殖させた(図10左)。一方、ステープルドERAP(No.12)の連日ならびに7日毎投与はともにE2依存性の腫瘍増大を投与量依存的に有意に抑制し、これまで得られている腹腔内投与による抗腫瘍効果と同様に、10mg/kgで、ほぼ完全な抗腫瘍効果が得られた(図10)。
移植マウス摘出腫瘍におけるBIG3とPHB2の相互作用に対するステープルドERAP(No.12)の効果を調べた。10mg/kgのステープルドERAP(No.12)で毎日または7日毎に処理したKPL‐3C異種同所性移植マウスから摘出した腫瘍において、抗BIG3抗体を用いて免疫共沈降試験を行った。その結果、未処理群およびE2連日投与群ではBIG3とPHB2が強固に結合していることを認めたが、10mg/kgのステープルドERAP(No.12)の35日連日投与および7日毎投与した腫瘍はPHB2の共沈がほとんど検出されず、10mg/kgのステープルドERAP(No.12)がBIG3とPHB2の相互作用をほぼ完全に阻害して、腫瘍の増大を抑制していることが示された(図11)。
腫瘍におけるAktとMAPKのリン酸化に対するステープルドERAP(No.12)の効果を調べた。10mg/kgのステープルドERAP(No.12)で毎日または7日毎に処理したKPL‐3C異種同所性移植マウスから摘出した腫瘍において、抗リン酸化Akt抗体および抗リン酸化MAPK抗体を用いてAktおよびMAPKのリン酸化レベルを検出した。その結果、ステープルドERAP(No.12)処理は毎日投与および7日毎投与においてAktリン酸化およびMAPKリン酸化の顕著な抑制が観察された(図12)。
BIG3は弱いながら脳にも発現している(Kim, J. W. et al., Cancer Sci. 100, 1468-1478 (2009))。このことから、ステープルドERAPが脳に移行することによる副作用の懸念があったため、ステープルドERAPの血液脳関門透過性試験を行った。ステープルドERAP(No.12)の血液脳関門透過性試験は、血液脳関門透過性キットのインサート内側(血管腔側)にステープルドERAP(No.12)を入れ、30分間に脳内移行性検定特化型フィルターを通過し、プレートのウェル内(脳実質側)に漏れでてきたステープルドERAP(No.12)の濃度を測定することで行った。また、そのときの透過係数(Papp)を算出した(2以下:透過性はかなり低い、2-10:低い、10-20:高い、20以上:かなり高い)。その結果、ステープルドERAP(No.12)の血液脳関門透過係数は2以下を示し、脳内に移行する可能性は低いと考えられ、脳内移行による副作用の懸念はないことが示唆された(表3)。
Claims (15)
- 配列番号:9に記載のアミノ酸配列またはその部分配列において、n対(nは自然数)のアミノ酸残基が、n個のステープリング構造で置き換えられたアミノ酸配列を含むペプチド、またはその塩。
- n対のアミノ酸残基が、以下の(a)または(b)の1対のアミノ酸残基である、請求項1に記載のペプチド、またはその塩:
(a)配列番号:9に記載のアミノ酸配列のN末端から3番目および7番目のアミノ酸残基;
(b)配列番号:9に記載のアミノ酸配列のN末端から2番目および6番目のアミノ酸残基。 - 配列番号:9に記載のアミノ酸配列の部分配列が、配列番号:13に記載のアミノ酸配列である、請求項1または2に記載のペプチド、またはその塩。
- n対のアミノ酸残基が、以下の(a)または(b)の1対のアミノ酸残基である、請求項3に記載のペプチド、またはその塩:
(a)配列番号:13に記載のアミノ酸配列のN末端から3番目および7番目のアミノ酸残基;
(b)配列番号:13に記載のアミノ酸配列のN末端から2番目および6番目のアミノ酸残基。 - N末端およびC末端のアミノ酸残基のいずれかまたは両方が修飾されている、請求項1~6のいずれか一項に記載のペプチド、またはその塩。
- N末端およびC末端のアミノ酸残基のいずれかまたは両方が、アセチル化、アミド化およびHAタグ付加のいずれかまたはこれらの組み合わせにより修飾されている、請求項7に記載のペプチド、またはその塩。
- N末端のアミノ酸残基がアセチル化されており、かつC末端のアミノ酸残基がアミド化されている、請求項8に記載のペプチド、またはその塩。
- 全てのアミノ酸残基がD型のアミノ酸残基に置き換えられた、請求項1~9のいずれか一項に記載のペプチド、またはその塩。
- 請求項1~9のいずれか一項に記載のペプチドのレトロインバース体であるペプチド、またはその塩。
- 請求項1~11のいずれか一項に記載のペプチド、またはその塩と、薬学的に許容される担体とを含む、医薬組成物。
- がん治療用である、請求項12に記載の医薬組成物。
- がんが乳がんまたは前立腺がんである、請求項13に記載の医薬組成物。
- がんがエストロゲン受容体陽性のがんである、請求項13または14に記載の医薬組成物。
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