JP5803012B2 - Peptides that specifically bind to brain tumors and uses thereof - Google Patents

Description

  The present invention relates to a drug delivery system (DDS), which is a technique for transporting drugs and the like to a target site. More particularly, the present invention relates to a DDS for brain tumors using a peptide.

  In cancer treatment, a technique for transporting a drug or the like to a target cancer cell (target cancer tissue) is called a drug delivery system (DDS). DDS requires a lead material to link to a drug or the like and deliver it to a target. Conventionally, as such a lead substance, an antibody corresponding to a protein (antigen) that is specifically or abundantly expressed in the cell membrane of a target cancer cell has been used. For example, Patent Document 1 discloses an antibody / pharmaceutical complex for cancer (kidney tumor, B cell lymphoma, thymic carcinoma, nasopharyngeal carcinoma, brain tumor, etc.) using an anti-CD70 antibody as a lead substance as described above. The body is listed.

JP-T-2006-518753

  However, considerable technology and cost are required to stably supply a large amount of the antibodies (monoclonal antibodies) used in the conventional DDS as described above. If such an antibody can be artificially synthesized, it may be possible to supply a large amount at a low cost, but it is difficult to produce a long peptide such as an antibody by conventional peptide synthesis methods.

  An object of the present invention is to provide a substance that specifically binds to brain tumor cells, which can be used as a lead substance or the like in DDS targeting brain tumors and can be produced more efficiently than antibodies. And

  The present inventors have found that among peptides consisting of only 2 to 3 non-natural amino acids (non-natural peptides) that can be easily synthesized artificially, there are those that have extremely excellent selectivity for brain tumors. The headline and the present invention have been completed.

That is, the present invention includes the following inventions.
[1] A dimer non-natural peptide having the non-natural amino acid sequence of any one of (1) to (4) below.
Bph-2Np (1)
Bph-Bph (2)
Bph-Aib (3)
Bph-Thi (4)

  Where Bph is β- (4-biphenyl) -alanine, 2Np is N-β- (2-naphthyl) -alanine, Aib is α-aminoisobutyric acid, and Thi is N-β- ( 2-thienyl) -alanine.

[2] A trimeric non-natural peptide comprising the non-natural peptide according to [1] and another non-natural amino acid bonded to the C-terminal side thereof.
[3] The trimeric non-natural peptide according to [2], which has the following non-natural amino acid sequence of (5) or (6).
Bph-2Np-Sar (5)
Bph-2Np-Amb (6)
Here, Bph is β- (4-biphenyl) -alanine, 2Np is N-β- (2-naphthyl) -alanine, Sar is sarcosine, and Amb is p-aminomethylbenzoic acid.

[4] A complex comprising the non-natural peptide according to any one of [1] to [3] and a fluorescent amino acid linked thereto.
[5] The fluorescent amino acid is 3- (9-oxo-9,10-dihydro-acridin-2-yl) alanine (Acd), N-ε- (fluorescein-5,6-yl) carbonyl-lysine (Fam), N-ε- (tetramethylrhodamine-5,6-yl) carbonyl-lysine (Tmr), 3- (1-pyrenyl) alanine (Pyr), N-δ- (10-oxo-2,3 , 5,6-Tetrahydro-1H, 4H, 10H-11-oxa-3-aza-benzo [de] anthracene-9-carbonyl) -ornithine (Cm3), N-ε- (7-methoxy-coumarin-3- Yl) carbonyl-lysine (Moc), N-ε- (7-methoxy-coumarin-4-yl) acetyl-lysine (Mca), N-ε- (7-hydroxy-coumarin-3-yl) carbonyl-lysine ( Hoc), N-ε- (7-hydroxy-4-methyl-coumarin-3-yl) acetyl-lysine (Hmc), or N-ε- (7-dimethylamino-coumarin-4-yl) acetyl-lysine ( Mac), the complex according to [4].

[6] A labeling reagent for brain tumor cells, comprising the complex according to [4] or [5].
[7] For diagnosis of brain tumor, comprising a complex comprising the non-natural peptide according to any one of [1] to [3] and a drug for diagnosis or treatment of brain tumor linked thereto. Or a therapeutic drug.

  The non-natural peptide of the present invention can be synthesized from only a small number of non-natural amino acids and has excellent selectivity for brain tumor cells while being excellent in production efficiency. Such a non-natural peptide of the present invention can be easily linked to a drug, a diagnostic agent (fluorescent label), etc., and such various substances can be delivered to a brain tumor. DDS can be constructed. In addition, since non-natural amino acids are used in the production of peptides in the present invention, unlike peptides using natural amino acids, they are not degraded by proteases in vivo and are suitable for uses such as pharmaceuticals administered to living bodies. .

The chemical structure of 10 types of fluorescent amino acids (fluorescent tags) used in the fluorescence detection type peptide screening method in the examples (the N-terminus is represented by an Fmoc group). Numerical values in parentheses indicate excitation wavelength (nm) / fluorescence wavelength (nm). Abbreviated as follows. Acd: 3- (9-oxo-9,10-dihydro-acridin-2-yl) alanine, Fam: N-ε- (fluorescein-5,6-yl) carbonyl-lysine, Tmr: N-ε- ( Tetramethylrhodamine-5,6-yl) carbonyl-lysine, Pyr: 3- (1-pyrenyl) alanine, Cm3: N-δ- (10-oxo-2,3,5,6-tetrahydro-1H, 4H, 10H-11-oxa-3-aza-benzo [de] anthracene-9-carbonyl) -ornithine, Moc: N-ε- (7-methoxy-coumarin-3-yl) carbonyl-lysine, Mca: N-ε- (7-methoxy-coumarin-4-yl) acetyl-lysine, Hoc: N-ε- (7-hydroxy-coumarin-3-yl) carbonyl-lysine, Hmc: N-ε- (7-hydroxy-4-methyl -Coumarin-3-yl) acetyl-lysine, Mac: N-ε- (7-dimethylamino-coumarin-4-yl) acetyl-lysine. Chemical structures of 10 types of non-natural amino acids (units constituting a peptide library) used in the fluorescence detection type peptide screening method in the examples (the N-terminal is represented by the first amino group). Abbreviated as follows. Aib: α-aminoisobutyric acid, Amb: p-aminomethylbenzoic acid, Bph: β- (4-biphenyl) -alanine, Dap: N-α, β-diaminopropionic acid, Hyp: trans-L-hydroxyproline, 2Np: N-β- (2-naphthyl) -alanine, Pic: piperidine-4-carboxylic acid, 3Pr: N-3- (3-pyridyl) -alanine, Sar: sarcosine, Thi: N-β- (2- Thienyl) -alanine. The chemical structure of the linker of the fluorescent peptide used in the fluorescence detection type peptide screening method in the examples (the N-terminus is represented by the first amino group). In the text, it is abbreviated as Sp6. Chemical structural formula of Pyr-Bph-2Np-Sar (top). CPK model (bottom). Example An example of a MALDI-TOF Mass spectrum of a synthesized fluorescent peptide library. Screening of fluorescent peptide sub-library by fluorescence detection type peptide screening method in the examples (first round). Structure of the fluorescent peptide sub Lively (first round) is _{Ac-Fl-Sp6-X 1} -X 2 -X 3 -NH 2. The horizontal axis upper ellipsis shows the amino acid units of X _1. The ellipsis at the bottom of the horizontal axis indicates Fl. The vertical axis shows the amount of fluorescent peptide bound to all cells assayed. White bars show the results for A431 cells, gray bars show the results for MCF7 cells, and black bars show the results for U87MG cells. Screening of fluorescent peptide sub-library by fluorescence detection type peptide screening method in Example (2nd round). Structure of the fluorescent peptide sub Lively (second round) is Ac-Fl-Sp6-Bph- X 2 -X 3 -NH 2. The horizontal axis upper ellipsis shows the amino acid units of X _2. The ellipsis at the bottom of the horizontal axis indicates Fl. The vertical axis shows the amount of fluorescent peptide bound to all cells assayed. White bars show the results for A431 cells, gray bars show the results for MCF7 cells, and black bars show the results for U87MG cells. Screening of fluorescent peptide sub-library by fluorescence detection type peptide screening method in the examples (third round; final round). Structure of the fluorescent peptide sub Lively (third round) is Ac-Fl-Sp6-Bph- 2Np-X 3 -NH 2. The horizontal axis upper ellipsis shows the amino acid units of X _3. The ellipsis at the bottom of the horizontal axis indicates Fl. The vertical axis shows the amount of fluorescent peptide bound to all cells assayed. White bars show results for A431 cells and black bars show results for U87MG cells. The summary of the screening of the non-natural peptide specifically couple | bonded with a U87MG cell by the fluorescence detection type | mold peptide screening method in an Example. Structure of the fluorescent peptide is _{Ac-Fl-Sp6-X 1} -X 2 -X 3 -NH 2. The ellipsis, Bph-XX, Bph-2Np-X, and Bph-2Np-Sar on the upper horizontal axis indicate the peptides selected in each round. White bars show the results for A431 cells, gray bars show the results for MCF7 cells, and black bars show the results for U87MG cells. There is no brain tumor cell binding peptide (Pyr-Bph-2Np-Sar), non-natural peptide sequence shuffled (Pyr-Sar-Bph-2Np) and non-natural peptide cultured with U87MG cells and A431 cells in the examples The amount of peptide (Pyr: chemical structure Ac-Pyr-Sp6-NH ₂ ) bound to each cell. Dark gray bars Pyr-Bph-2Np-Sar results show a light gray bars shuffled sequence (Pyr-Sar-Bph-2Np ) results, a little dark bar Ac-Pyr-Sp6-NH ₂ results. The amount of brain tumor cell-binding peptide (Pyr-Bph-2Np-Sar) cultured together with U87MG cells, A431 cells, and HLF-1 cells in each Example. Chemical structural formula (left) of brain tumor-binding peptide (Bph-2Np-Sar) found in the present invention (Example). The N-terminus is displayed as a free amino group. The CPK model (right).

-Non-natural peptide-
In one aspect, the present invention provides a peptide composed of an unnatural amino acid that specifically binds to brain tumor cells (unnatural peptide).

  “Brain tumors” include gliomas (astrocytoma, oligodendroma, pleomorphic glioma), medulloblastoma, meningioma, adenoma (adenoma), chordoma, craniopharyngioma, epidermoid cyst and Epidermoid, ependymoma, germ cell tumor (including germinoma), hemangioblastoma, osteoma, osteosarcoma, pineal gland, pituitary adenoma, sarcoma and the like are included. The non-natural peptide according to the present invention can target these brain tumor cells, but it is particularly preferable to target glioma cells.

  Further, in the present invention, the non-natural peptide “specifically binds to a brain tumor” means that the amount of binding to brain tumor cells (typically U87MG cells) is normal cells and other tumor cells (human epithelial cancer cells). (Typically A431 cells), human breast cancer cells (typically MCF7 cells, etc.) binding amount is preferably 2 times or more, more preferably 4 times or more, particularly preferably 8 times or more. Say.

  In the present invention, the amount of non-natural peptide binding to brain tumor cells is not only relative to the amount of relative binding compared to other cells as described above, but also the absolute amount of binding to brain tumor cells. It is. For example, the amount of binding to brain tumor cells (U87MG cells) cultured in a nearly confluent state in a measurement condition of Examples described later, ie, 60 mm dish, is preferably 30 pmol or more, more preferably 100 pmol or more, and particularly preferably 500 pmol or more. is there.

The dimer non-natural peptide according to the present invention (hereinafter also referred to as “the dipeptide of the present invention”) has any of the following non-natural amino acid sequences (1) to (4).
Bph-2Np (1)
Bph-Bph (2)
Bph-Aib (3)
Bph-Thi (4)

Where Bph is β- (4-biphenyl) -alanine, 2Np is N-β- (2-naphthyl) -alanine, Aib is α-aminoisobutyric acid, and Thi is N-β- ( 2-thienyl) -alanine.
Among the dipeptides of the present invention, those having the amino acid sequence (Bph-2Np) of (1) are preferred because they have a higher ability to specifically bind to brain tumor cells than those having other amino acid sequences.

  Since the dipeptide of the present invention itself has a certain specific binding ability to brain tumor cells, it can be used as it is, or it can be used by binding several amino acids. . The amino acid to be bonded at that time is preferably an unnatural amino acid, but may be a natural amino acid. A peptide having an appropriate length including the dipeptide of the present invention (preferably an oligopeptide having 10 or less amino acids, more preferably 5 or less), which has the ability to specifically bind to brain tumor cells, It corresponds to the invention using the dipeptide of. The same can be said when the above “dipeptide” is replaced with “tripeptide” described below.

  For example, a trimeric unnatural peptide (hereinafter referred to as “tripeptide of the present invention”) comprising a dipeptide of the present invention and another unnatural amino acid, preferably another unnatural amino acid bonded to the C-terminal side. Is a preferred embodiment of the present invention that can enhance the specific binding ability to brain tumor cells as compared to the dipeptide of the present invention.

  The “non-natural amino acid” can be selected from any known non-natural amino acid. For example, α-aminoisobutyric acid (Aib), p-aminomethylbenzoic acid (Amb), β- (4 -Biphenyl) -alanine (Bph), N-α, β-diaminopropionic acid (Dap), trans-L-hydroxyproline (Hyp), N-β- (2-naphthyl) -alanine (2Np), piperidine-4 -Carboxylic acid (Pic), N-3- (3-pyridyl) -alanine (3Pr), sarcosine (Sar), N-β- (2-thienyl) -alanine (Thi) can be selected.

Preferred tripeptides according to the present invention include those having the following unnatural amino acid sequence (5) or (6).
Bph-2Np-Sar (5)
Bph-2Np-Amb (6)
Here, Bph is β- (4-biphenyl) -alanine, 2Np is N-β- (2-naphthyl) -alanine, Sar is sarcosine, and Amb is p-aminomethylbenzoic acid.

  In particular, the tripeptide having the unnatural amino acid sequence (Bph-2Np-Sar) of (5) is one of the most excellent tripeptides in the present invention with specific binding to brain tumor cells.

  Note that one or more unnatural amino acids contained in the unnatural peptide (dipeptide, tripeptide) may be replaced by unnatural amino acids having similar chemical properties. Such “conservative substitutions” are known for natural amino acids, and non-natural amino acids in the present invention are also modified accordingly, taking into account the charge of the side chain, hydrophilicity / hydrophobicity, aromaticity, etc. Substitution with other non-natural amino acids may be made within the scope of the effects of the invention.

  The non-natural peptide (dipeptide, tripeptide) of the present invention and a peptide obtained by further extending the peptide can be synthesized by a known technique. For example, the Fmoc peptide solid-phase synthesis method is known and commonly used as a typical method capable of synthesizing non-natural peptides, and is also suitable for use in the present invention.

-Complex-
In one aspect, the present invention provides a complex comprising the non-natural peptide of the present invention as described above and various substances linked to the non-natural peptide for delivery to brain tumor cells.

In one embodiment, the present invention provides such a complex comprising a non-natural peptide of the present invention as described above and a fluorescent amino acid linked thereto.
The “fluorescent amino acid” can be selected from known fluorescent amino acids, such as 3- (9-oxo-9,10-dihydro-acridin-2-yl) alanine (Acd). N-ε- (Fluorescein-5,6-yl) carbonyl-lysine (Fam), N-ε- (tetramethylrhodamine-5,6-yl) carbonyl-lysine (Tmr), 3- (1-pyrenyl ) Alanine (Pyr), N-δ- (10-oxo-2,3,5,6-tetrahydro-1H, 4H, 10H-11-oxa-3-aza-benzo [de] anthracene-9-carbonyl)- Ornithine (Cm3), N-ε- (7-methoxy-coumarin-3-yl) carbonyl-lysine (Moc), N-ε- (7-methoxy-coumarin-4-yl) acetyl-lysine (Mca), N -ε- (7-hydroxy-coumarin-3-yl) carbonyl-lysine (Hoc), N-ε- (7-hydroxy-4-methyl-coumarin-3-yl) acetyl-lysine (Hmc), N-ε -(7-Dimethylamino-coumarin-4-yl) acetyl-lysine (Mac ) Can be selected.

  In the complex, the non-natural peptide and the fluorescent amino acid may be linked via a spacer (linker). As a spacer for that purpose, a known spacer can be used, and an appropriate chemical structure and length can be selected. However, it affects the specific binding property of the non-natural peptide of the present invention to brain tumor cells ( Those which do not give at least an adverse effect) are suitable. In addition, since the complex of the present invention is delivered to brain tumor cells and performs a predetermined function, a spacer that is substantially unaffected by cleavage or the like in the extracellular environment is suitable. In the intracellular environment (for example, inside of lysosome, endosome, caveolae, etc.), it may be cleavable.

-Use-
In a further aspect, the present invention provides a diagnostic or therapeutic medicament for brain tumor, comprising a complex comprising the non-natural peptide of the present invention and a drug for diagnosing or treating brain tumor linked thereto. To do.

  In other words, the present invention provides use (method) of the non-natural peptide of the present invention or the complex of the present invention for the manufacture of a medicament for diagnosis or treatment of brain tumor. Alternatively, the present invention provides a use (method) for the diagnosis or treatment of brain tumor, comprising the step of administering the non-natural peptide of the present invention or the complex of the present invention. The pharmaceutical or use (method) as described above should be administered or applied to a human or other animal (preferably a mammal) having or possibly having a brain tumor, or a biological material collected therefrom. Can do.

  The complex of the present invention comprising the non-natural peptide of the present invention and the fluorescent amino acid linked thereto as described above can be used for the above-mentioned diagnostic medicine (labeling reagent or probe). This is a representative example of a complex that can be formed. In order to label brain tumor cells, other fluorescent substances such as fluorescent dyes and quantum dots can be used in the complex, instead of fluorescent amino acids. These phosphors can also be linked to the peptide consisting of an unnatural amino acid of the present invention in the same manner as a known technique for linking to a peptide consisting of a natural amino acid. Note that such fluorescent substances such as fluorescent amino acids are also included in the above-mentioned “drugs”.

  Moreover, it can be used as a medicament for treating brain tumors by linking drugs for treating brain tumors to the non-natural peptide of the present invention. As the “drugs” in this case, known ones for treatment of brain tumors can be used, and examples include chemotherapeutic agents, prodrug converting enzymes, radioisotopes or compounds, and cytotoxic agents such as toxins. .

  When the drug is a compound having a relatively low molecular weight, the drug can be linked to the peptide of the present invention to prepare a complex. The drug can be linked to the non-natural peptide of the present invention by a known technique depending on its chemical structure.

  Such a medicament of the present invention is prepared as a pharmaceutical composition containing a pharmaceutically acceptable carrier, excipient, wetting agent, emulsifying agent, pH buffering agent and the like, if necessary, by a known technique. And can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, deterrent formulations and the like. Moreover, what is necessary is just to adjust suitably the administration method and dosage of the pharmaceutical of this invention according to the chemical | medical agent to be used.

  Alternatively, a typical mode of use in DDS is that the peptide of the present invention is linked to the surface of a microcapsule (for example, a liposome) encapsulating or carrying a drug and used as a medicine. Such drug-encapsulated microcapsules are also included in one embodiment of the above-mentioned “drugs”.

[Example 1]
1-1: Synthesis of peptide library containing fluorescent dye In order to perform the fluorescence detection type peptide screening of the present invention, a peptide library containing a fluorescent amino acid was synthesized. These were all obtained by the conventional Fmoc peptide solid phase synthesis method. Tmr and Fam in FIG. 1 were purchased from ABD Bioquest as Fmoc derivatives. The other fluorescent amino acids (Fmoc derivatives), unnatural amino acids (Fmoc derivatives) and reagents necessary for Fmoc peptide solid phase synthesis in FIGS. 1 and 2 were purchased from Watanabe Chemical. The linker (Sp6) in FIG. 3 was purchased from Merck. All of the amino acids used in the examples which are optically active are L-forms.

Specifically, the fluorescent peptide library was synthesized as follows. The resin used in the synthesis was Fmoc-NH-SAL PEG resin (0.24 mmol / g). First, the resin was allowed to swell in dimethylformamide (DMF) for 3 hours to overnight at room temperature. A 20% piperidine DMF solution was used as a deprotection reagent, and an HBTU / NMM DMF solution was used as a coupling solution. The synthesis was carried out by hand synthesis, and the resin was washed with DMF, deprotected, and then coupled to one cycle. The peptide was extended on the resin surface by repeating this cycle. After capping (resin is exposed to 5% acetic anhydride DMF solution for 10 minutes to acetylate the N-terminus), the peptide resin extended to the target sequence is trifluoroacetic acid / water / isopropylsilane (= 95 / 2.5 / 2.5 (v / v / v)) was added to excise the peptide. The peptide solution cut out from the resin was air-dried with N ₂ gas and stored in a freezer as a DMSO solution. In this example, the following peptides were synthesized. Abbreviations and sequences of fluorescent peptides are shown below.
1. Tmr-2Np: Ac-Tmr-Sp6-2Np-X2-X3-NH2
2. Moc-Dap: Ac-Moc-Sp6-Dap-X2-X3-NH2
3. Cm3-Bph: Ac-Cm3-Sp6-Bph-X2-X3-NH2
4. Hmc-Aib: Ac-Hmc-Sp6-Aib-X2-X3-NH2
5. Pyr-Thi: Ac-Pyr-Sp6-Thi-X2-X3-NH2
6. Mca-Amb: Ac-Mca-Sp6-Amb-X2-X3-NH2
7. Fam-Sar: Ac-Fam-Sp6-Sar-X2-X3-NH2
8. Acd-Hyp: Ac-Acd-Sp6-Hyp-X2-X3-NH2
9. Mac-Pic: Ac-Mac-Sp6-Pic-X2-X3-NH2
10. Hoc-3Pr: Ac-Hoc-Sp6-3Pr-X2-X3-NH2
11. Mac-Bph-2Np: Ac-Mac-Sp6-Bph-2Np-X3-NH2
12. Mca-Bph-Dap: Ac-Mca-Sp6-Bph-Dap-X3-NH2
13. Tmr-Bph-Bph: Ac-Tmr-Sp6-Bph-Bph-X3-NH2
14. Pyr-Bph-Aib: Ac-Pyr-Sp6-Bph-Aib-X3-NH2
15. Hoc-Bph-Thi: Ac-Hoc-Sp6-Bph-Thi-X3-NH2
16. Fam-Bph-Amb: Ac-Fam-Sp6-Bph-Amb-X3-NH2
17. Acd-Bph-Sar: Ac-Acd-Sp6-Bph-Sar-X3-NH2
18. Cm3-Bph-Hyp: Ac-Cm3-Sp6-Bph-Hyp-X3-NH2
19. Hmc-Bph-Pic: Ac-Hmc-Sp6-Bph-Pic-X3-NH2
20. Moc-Bph-3Pr: Ac-Moc-Sp6-Bph-3Pr-X3-NH2
21. Mca-Bph-2Np-2Np: Ac-Mca-Sp6-Bph-2Np-2Np-NH2
22. Mac-Bph-2Np-Dap: Ac-Mac-Sp6-Bph-2Np-Dap-NH2
23. Moc-Bph-2Np-Bph: Ac-Moc-Sp6-Bph-2Np-Bph-NH2
24. Acd-Bph-2Np-Aib: Ac-Acd-Sp6-Bph-2Np-Aib-NH2
25. Cm3-Bph-2Np-Thi: Ac-Cm3-Sp6-Bph-2Np-Thi-NH2
26. Hmc-Bph-2Np-Amb: Ac-Hmc-Sp6-Bph-2Np-Amb-NH2
27. Pyr-Bph-2Np-Sar: Ac-Pyr-Sp6-Bph-2Np-Sar-NH2
28. Hoc-Bph-2Np-Hyp: Ac-Hoc-Sp6-Bph-2Np-Hyp-NH2
29. Fam-Bph-2Np-Pic: Ac-Fam-Sp6-Bph-2Np-Pic-NH2
30. Tmr-Bph-2Np-3Pr: Ac-Tmr-Sp6-Bph-2Np-3Pr-NH2
31. Pyr-Sar-Bph-2Np: Ac-Pyr-Sar-Bph-2Np
32. Ac-Pyr-Sp6-NH2

Here, Ac indicates that the N-terminus of the peptide is protected with an acetyl group. Sp6, a water-soluble linker, is introduced between the fluorescent amino acid and the trimer unnatural amino acid. X2 and X3 are equivalent mixtures of the 10 unnatural amino acids used this time. The C terminus is the primary amide. FIG. 4 shows Pyr-Bph-2Np-Sar as an example of the chemical structure of the fluorescent peptide.
Among these compounds, those with a clear structure were identified by MALDI-TOF Mass (FIG. 5).

1-2: Test Method Peptide screening was performed by the present inventors using a fluorescence detection type peptide screening method (M. Kitamatsu et al., Chem. Commun., 46, 761-763 (2010) .; M. Kitamatsu et al., Bioorg. Med. Chem. Lett., 20, 5976-5978 (2010). The experimental operation was performed according to the following procedure. First, 1 × 10 ⁵ cells (A431, MCF7, U87MG) were seeded in a 60 mm dish (BD Biocoat Collagen I dish 60 mm Falcon cat. 356401) 3 days before peptide screening. Of the cells used in this example, A431 cells are human epithelial cancer cells, MCF7 cells are human breast cancer cells, and U87MG cells are human gliomas cells (brain tumor cells). . Dulbecco's modified Eagle's medium high glucose (Sigma cat. D6429) supplemented with 10% Fetal bovine serum (Nissui cat. 171012, Lot. 10D766) and penicillin-streptomycin (gibco cat. 15140) was used as the medium. Culturing was performed at 37 ° C. and 5% CO ₂ . After culturing, peptides were screened when they became 80% confluent.

  First, 10 kinds of fluorescent peptides in each round were mixed in a PBS solution to a final concentration of 10 μM to prepare a peptide library solution. Next, a dish in which each cell was cultured on ice was prepared and washed twice with 4 mL of PBS. After washing, 1.5 mL of peptide library solution (final concentration 10 μM) was added to these dishes and incubated for 1 hour. At this time, the dish was shaken every 10 minutes. After incubation, the plate was washed 20 times with 4 mL of PBS. After thoroughly removing the washing solution, 100 μL of 100 μL of Glycine-HCl (pH 3.5) buffer was uniformly added to the whole cell, and the whole cell was exposed to the Glycine-HCl buffer using a pipette. Thereafter, the Glycine-HCl buffer was recovered in a microtube and neutralized with a small amount (5 uL) of 2M Tris buffer (pH 7.4). 100 uL of non-fluorescent methanol was added to the recovered solution. Then, centrifugation was performed using a centrifuge (eppendorf centrifuge 5415R) under the conditions of 10,000 rpm, 10 min, and 4 ° C. to precipitate cells that had detached during the assay. After centrifugation, the solution was transferred to a measurement cell with a pipetman so as not to suck the precipitated cells. Then, a two-dimensional fluorescence spectrum was measured with a two-dimensional fluorescence measuring device (manufactured by photon design). From the obtained data, the content of the fluorescent peptide in the solution was measured by the least squares analysis software.

1-3: Results FIG. 6 shows the results of the first round by the fluorescence detection type peptide screening method. Ten types of fluorescent peptides are used. Formula of the chemical structure of each of the fluorescent peptide represented by _{Ac-Fl-Sp6-X 1} -X 2 -X 3 -NH 2 ( hereinafter, referred to each peptide Fl-X _1). Here, Ac represents an acetyl group (H ₃ C—CO—). Fl represents a fluorescent amino acid unit, and is any of Tmr, Moc, Cm3, Hmc, Pyr, Mca, Fam, Acd, Dac, and Hoc. Different fluorescent amino acids are linked in 10 kinds of fluorescent peptides. Sp6 is a water-soluble linker composed of an ethylene glycol chain, and is introduced so that both the fluorescent amino acid and the peptide library portion are not affected. X ₂ and X ₃ are equivalent mixtures of 10 unnatural amino acid units. X ₁ is _one of 10 types of unnatural amino acids, and 10 types of fluorescent peptides are linked to different fluorescent amino acids. Different amounts were detected in each fluorescent peptide for A431 cells, MCF7 cells and U87MG cells. This indicates that the amount of peptide bound (adsorbed) to each cell differs depending on the cell type and the peptide sequence. With some exceptions, both fluorescent peptides appear to bind to U87MG cells more easily than A431 and MCF7 cells. Among them, Pyr-Thi is found to bind well to all of A431 cells, MCF7 cells and U87MG cells (17 pmol, 22 pmol and 21 pmol, respectively). Similarly, Cm3-Bph (10 pmol, 13 pmol and 18 pmol) and Tmr-2Np (6 pmol, 6 pmol and 9 pmol) are also likely to bind to A431 cells, MCF7 cells and U87MG cells. Here, in order to investigate the cell specificity of the peptides, the ratio of the binding amount of these peptides to U87MG cells compared to A431 cells was determined, and 1.2 times for Pyr-Thi, 1.8 times for Cm3-Bph, Tmr- It is 1.5 times with 2Np, indicating that Cm3-Bph (peptide sequence of Bph-X ₂ -X ₃ ) has the highest specificity for brain tumor cells (similar results with MCF7 cells as a reference). Hmc-Aib, Fam-Sar, and Acd-Hyp did not bind to any of A431 cells, MCF7 cells, and U87MG cells.

Based on these results, we decided to use the peptide sequence Bph-X ₂ -X ₃ which has a relatively high binding amount and the highest specificity for brain tumor cells as the second round peptide sequence.
FIG. 7 shows the results of the second round by the fluorescence detection type peptide screening method. As in the first round, 10 types of fluorescent peptides are used. Formula of the chemical structure of each of the fluorescent peptide represented by Ac-Fl-Sp6-Bph- X 2 -X 3 -NH 2 ( hereinafter, referred to each peptide Fl-Bph-X _2). Fl indicates a fluorescent amino acid unit, and different fluorescent amino acids are linked to each other by 10 kinds of fluorescent peptides as in the first round. X ₃ is an equivalent mixture of 10 unnatural amino acid units. X ₂ is any one of 10 types of unnatural amino acids, and 10 types of fluorescent peptides are linked to different fluorescent amino acids. Similar to the first round, different amounts of each fluorescent peptide were detected for A431 cells, MCF7 cells, and U87MG cells. On the other hand, the amount of peptide bound to the cells seems to increase overall. Among the fluorescent peptides, Dac-Bph-2Np bound best to U87MG cells, showing 72 pmol. Dac-Bph-2Np and Pyr-Bph-Aib bound best to A431 cells (17 pmol each). Dac-Bph-2Np also bound best to MCF7 cells (6 pmol). Furthermore, Dac-Bph-2Np was found to be 4.2 times easier to bind to U87 cells than A431 cells. In addition to Dac-Bph-2Np, Pyr-Bph-Aib, Hoc-Bph-Thi, and Tmr-Bph-Bph are easy to bind to these cells, and the ratio of binding to U87MG cells compared to A431 cells is They were 4.0 times, 4.3 times, and 4.4 times, respectively. It can be seen that these four types of peptides all have the same specificity for brain tumor cells. Mca-Bph-Dap did not bind to any of A431, MCF7 and U87MG cells. Based on the above results, we decided to use Bph-2Np-X3, one of the peptide sequences with the highest binding power and the highest specificity for brain tumor cells, as the peptide sequence in the third round.

FIG. 8 shows the results of the third round (final round) by the fluorescence detection type peptide screening method. As in the first and second rounds, 10 types of fluorescent peptides are used. Formula of the chemical structure of each of the fluorescent peptide represented by Ac-Fl-Sp6-Bph- 2Np-X 3 -NH 2 ( hereinafter, referred to each peptide _{Fl-Bph-2Np-X 3} ). Fl indicates a fluorescent amino acid unit, and different fluorescent amino acids are linked to each other in 10 kinds of fluorescent peptides similar to those in the first and second rounds. X ₃ is one of 10 types of unnatural amino acids, and 10 types of fluorescent peptides are linked to different fluorescent amino acids. Compared to the first and second rounds, peptides that bind significantly better to U87MG cells appeared. It is Pyr-Bph-2Np-Sar, and it can be seen that 580 pmol is bound to U87MG cells under the present measurement conditions. Next, the peptide that easily binds to U87MG cells is Hmc-Bph-2Np-Amb, which is 129 pmol, which is about 1/4 to 1/5 of the binding amount of Pyr-Bph-2Np-Sar. Pyr-Bph-2Np-Sar bound 38 pmol to A431 cells, but it was 15 times easier to bind to U87MG cells than A431 cells.

  From these results, we decided to find a non-natural peptide trimer sequence, Bph-2Np-Sar, which has high binding and specificity for brain tumor cells, using this fluorescent detection-type peptide screening method. Successful.

  FIG. 9 is a summary of screening of a non-natural peptide sequence of a trimer that binds to brain tumor cells by this fluorescence detection peptide screening method. Since this peptide screening finds peptides with increased binding to U87MG cells in the order of rounds 1, 2, and 3, this screening can be said to be appropriate. The amount of peptide binding increased in the order of 18 pmol, 72 pmol, and 580 pmol with respect to U87MG cells in order from rounds 1 to 3. The screening method does not know to which part of the cell the peptide found in this example is bound, but this peptide may bind to a receptor that is overexpressed on the surface of some U87MG cells. Is expensive.

Figure 10 shows that the sequence of the trimeric non-natural peptide found this time (Bph-2Np-Sar) is really important for binding to brain tumors, and how much the fluorescent amino acid unit, Pyr, is in brain tumor cells. This peptide (Pyr-Bph-2Np-Sar), the shuffle sequence of this peptide (Pyr-Sar-Bph-2Np), and the non-natural peptide part of the trimer are used to examine the effect of binding. It is the result of investigating the binding amount with respect to the U87MG cell of the peptide (Ac-Pyr-Sp6-NH ₂ ) lacking. It can be seen that both bind well to U87MG cells as compared to U87MG cells and A431 cells. For U87MG cells, the peptide of this example (Pyr-Bph-2Np-Sar; 945 pmol) binds best, and the shuffled sequence (Pyr-Sar-Bph-2Np; 28 pmol) and the missing peptide It can be seen that (Ac-Pyr-Sp6-NH ₂ ; 7 pmol) hardly binds. From these results, it was revealed that not only the selected non-natural amino acid unit but also the peptide sequence of the trimer non-natural peptide found in this example is important.

  It should be noted that the amount of peptide binding differs between Pyr-Bph-2Np-Sar (580 pmol) for U87MG cells in FIG. 9 and that in FIG. 10 (945 pmol). In FIG. 9, this was cultured in cells in a mixed state with 10 types of fluorescent peptides, whereas in FIG. 10, it was cultured in cells with a single (1 type) fluorescent peptide (Pyr-Bph-2Np-Sar). It seems that this is due to the difference in what was done. That is, in the experiment as shown in FIG. 9, it is suggested that 10 kinds of fluorescent peptides are bound to something on the surface of the U87MG cell while competing. It is guessed that there is.

  Further, the binding amount of the above complex: Pyr-Sp-Bph-2Np-Sar to human normal embryo fibroblasts (HLF-1) was measured by the same method as described above. The results are shown in FIG. 11 (in the figure, the data for A431 and U87MG are the same as those shown in FIG. 10 above). The above complex was confirmed to specifically bind to brain tumor cells not only with other tumor cells but also with normal cells.

  FIG. 12 shows the chemical structure of a trimeric unnatural peptide of a peptide that binds to brain tumor cells found in this example. The selected amino acids are relatively hydrophobic (Bph and 2Np), but also include hydrophilic Sar (which is thought to be more water soluble because it forms a second amide bond). Each of them has a structure that restricts the structure of the peptide (restricted), and seems to include the same conditions as many drugs.

Claims (7)

The dimer non-natural peptide which has a non-natural amino acid sequence in any one of following (1) , (3) and (4).
Bph-2Np (1)
B ph-Aib (3)
Bph-Thi (4)
Where Bph is β- (4-biphenyl) -alanine, 2Np is N-β- (2-naphthyl) -alanine, Aib is α-aminoisobutyric acid, and Thi is N-β- ( 2-thienyl) -alanine.   A trimeric non-natural peptide comprising the non-natural peptide according to claim 1 and another non-natural amino acid bonded to the C-terminal side thereof. The trimeric non-natural peptide according to claim 2, which has the following non-natural amino acid sequence of (5) or (6).
Bph-2Np-Sar (5)
Bph-2Np-Amb (6)
Here, Bph is β- (4-biphenyl) -alanine, 2Np is N-β- (2-naphthyl) -alanine, Sar is sarcosine, and Amb is p-aminomethylbenzoic acid.   A complex comprising the non-natural peptide according to any one of claims 1 to 3 and a fluorescent amino acid linked thereto.   The fluorescent amino acid is 3- (9-oxo-9,10-dihydro-acridin-2-yl) alanine (Acd), N-ε- (fluorescein-5,6-yl) carbonyl-lysine (Fam) N-ε- (tetramethylrhodamine-5,6-yl) carbonyl-lysine (Tmr), 3- (1-pyrenyl) alanine (Pyr), N-δ- (10-oxo-2,3,5, 6-Tetrahydro-1H, 4H, 10H-11-oxa-3-aza-benzo [de] anthracene-9-carbonyl) -ornithine (Cm3), N-ε- (7-methoxy-coumarin-3-yl) carbonyl -Lysine (Moc), N-ε- (7-methoxy-coumarin-4-yl) acetyl-lysine (Mca), N-ε- (7-hydroxy-coumarin-3-yl) carbonyl-lysine (Hoc), N-ε- (7-hydroxy-4-methyl-coumarin-3-yl) acetyl-lysine (Hmc), or N-ε- (7-dimethylamino-coumarin-4-yl) acetyl-lysine (Mac) The composite according to claim 4.   A labeling reagent for brain tumor cells, comprising the complex according to claim 4 or 5.   A medicament for diagnosis or treatment of brain tumor, comprising a complex comprising the non-natural peptide according to any one of claims 1 to 3 and a drug for diagnosis or treatment of brain tumor linked thereto. .