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WO2010058819A1 - Peptide capable d’inhiber l’interaction entre l’oncoprotéine mdm2 humaine et la protéine antitumorale p53 humaine, et utilisation associée - Google Patents

Peptide capable d’inhiber l’interaction entre l’oncoprotéine mdm2 humaine et la protéine antitumorale p53 humaine, et utilisation associée Download PDF

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WO2010058819A1
WO2010058819A1 PCT/JP2009/069644 JP2009069644W WO2010058819A1 WO 2010058819 A1 WO2010058819 A1 WO 2010058819A1 JP 2009069644 W JP2009069644 W JP 2009069644W WO 2010058819 A1 WO2010058819 A1 WO 2010058819A1
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mdm2
leu
peptide
protein
amino acid
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弘志 柳川
弘和 始平堂
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Keio University
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Keio University
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to peptides that inhibit the interaction between human cancer suppressor protein p53 and human cancer protein MDM2, and further to the use of such peptides in the treatment of human cancer.
  • P53 a human tumor suppressor protein
  • P53 a human tumor suppressor protein
  • the expressed p53 protein forms a tetramer, and then moves into the nucleus to activate transcription of its target.
  • p21 Waf1 / Cip1 is a protein involved in cell cycle regulation (Hideyuki Saya (1998) Cell Engineering Supplement: Molecular mechanism and clinical application of p53 cancer control; Nakayama Keiichi (2001) Understanding experimental medicine series: Understanding cell cycle, Yodosha; Gartel, AL, Radhakrishnan, SK (2005) Cancer Res., 65, 3980-3985; Chene, P., et al. (2002) FEBS Lett. , 529, 293-297).
  • the cell cycle shifts from the G1 phase to the S phase by the transcription factor E2F transcriptionally activating a gene encoding a protein important for DNA synthesis.
  • E2F The activity of E2F is suppressed by binding to RB, but when RB is phosphorylated by the cyclin / cyclin-dependent kinase 2 (CDK2) complex, it is dissociated from E2F, and the cell cycle proceeds by activated E2F (Hideyuki Saya (1998) Cell engineering separate volume: p53 Molecular mechanism and clinical application of cancer control, Shujunsha; Keiichi Nakayama (2001) Understanding experimental medicine series: Understanding cell cycle, Yodosha).
  • p21 is known as an inhibitor of CDK and mainly inhibits the cyclin / CDK2 complex, thus arresting the cell cycle in the G1 phase and repairing DNA during that time. However, depending on the degree of DNA damage, apoptosis is induced without DNA repair (Hideyuki Saya (1998) Cell Engineering Supplement: p53 Molecular Mechanism and Clinical Application of Cancer Control, Shujunsha).
  • p53 is known as a guardian of the genome because of its function of suppressing the growth of abnormal cells (Stiewe, T. (2007) Nature Rev. Cancer, 7, 165-168). That is, if this p53 loses its activity and cannot perform the cellular response as described above, it will grow cells with mutations in various genes, leading to canceration (Hideyuki Saya (1998) Cell engineering separate volume: Molecular mechanism and clinical application of p53 cancer control, Shujunsha). In fact, about 50% of human cancer cells are inactivated by mutation or deletion of p53 (Shangary, S., ⁇ (1997) Proc. Natl. Acad. Sci. USA., 105, 3933-3938 ).
  • inhibitors include those that directly act on p53 and those that inhibit apoptosis-promoting factors downstream of the p53 pathway, and it is thought that the p53 pathway is involved in almost all cancers (Shangary, S., ⁇ (1997) Proc. Natl. Acad. Sci. USA., 105, 3933-3938).
  • the oncoprotein MDM2 (mouse double minute) is one of the p53 targets that are transcriptionally activated by the expression-induced p53 protein (Shangary, S., et al. (1997) Proc. Natl. Acad. Sci. USA., 105 , 3933-3938; Hu, B., et al. (2007) Cancer Res., 67, 8810-8817; Chene, P., et al. (2000) J. Mol. Biol., 299, 245-253 (2000).
  • MDM2 then translocates into the nucleus and binds to the transcriptional activation region of p53, conversely inhibiting the activity of p53 as a transcription factor, and MDM2 also acts as a ubiquitin ligase, p53's proteasome-dependent activity By promoting degradation, they form negative feedback loops with each other (Kussie, PH, et al.
  • MDM2 interacts directly with p53, It inhibits the activity of p53 and its tumor suppressive effect, and from the crystal structure of the MDM2-p53 protein complex, the ⁇ helix and ⁇ A hydrophobic gap is formed by the peptide, and a peptide part having an ⁇ helix structure of 15 amino acid residues in the 15th to 29th regions of p53 (SQETFSDLWKLLPEN (SEQ ID NO: 1): single letter notation) binds to this region. (Kussie, PH, et al.
  • the motif FxxLW (SEQ ID NO: 2) consisting of 5 amino acid residues corresponding to the 19th to 23rd regions of p53 (where F, L, and W are one-letter codes for amino acids, meaning phenylalanine, leucine, and tryptophan, respectively).
  • Non-patent Document 1 Non-patent Document 1; Patent Document 1, Patent Document 2), p53 peptide fragment R1-XFX consisting of 10 amino acid residues corresponding to the 17th to 26th regions of p53 -R2-R3-WXX-R4 (SEQ ID NO: 3)
  • R1 is proline (P), leucine (L), glutamic acid (E), cysteine (C), or glutamine (Q)
  • X is any natural amino acid
  • R2 is arginine (R), histidine (H), glutamic acid (E), cysteine (C), serine (S), or aspartic acid (D)
  • R3 is histidine (H), phenylalanine (F), or Tyrosine (Y)
  • R4 is phenylalanine (F), glutamine (Q), or leucine (L);
  • Patent Document 3 p P53 peptide fragment pDI (LTFEHYWAQLTS
  • An object of the present invention is to provide a drug that inhibits the interaction between human cancer protein MDM2 and human cancer suppressor protein p53 and has a cancer cell growth inhibitory effect.
  • the inventors of the present invention have described the in-vitro virus (IVV) method (Nemoto, N., et al. (1997) FEBS Lett., 414, 405-408; Miyamoto-Sato, E. , ⁇ (2003) Nucleic Acids Res., 31, e78) successfully screened peptides that bind to MDM2 competitively with p53 from a random library and obtained peptides that inhibit the interaction between MDM2 and p53 The present invention has been completed.
  • IVV in-vitro virus
  • the present invention provides a human cancer protein MDM2-human cancer suppressor protein p53 interaction inhibitor or anticancer agent comprising a peptide containing the following amino acid sequence as an active ingredient.
  • Xaa1 Xaa2 Phe Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Leu Xaa11 Xaa12 (SEQ ID NO: 4)
  • Xaa1 is Lys, Pro, Arg, Ser, Leu, Ala or Met.
  • Xaa2 is Ser, Thr, Arg, Val, Gly, Tyr or Pro.
  • Xaa4 is Trp, Gln, Glu, Pro, Ala or Leu.
  • Xaa5 is Glu, Gln, Asp, Phe, Ala, Trp or Ser.
  • Xaa6 is Tyr, His, Leu or Glu.
  • Xaa7 is Trp or Leu.
  • Xaa8 is Leu, Gln, Glu, Val, Ser or Met.
  • Xaa9 is Glu, Arg, Met, Asp, Gln, Asn, or Lys.
  • Xaa11 is Met, Val, Leu, Ile or Tyr.
  • Xaa12 is Leu, Glu, Ser, Gly or Trp.
  • the peptide preferably comprises the amino acid sequence of any of SEQ ID NOs: 37 to 41, and more preferably comprises the amino acid sequence of SEQ ID NO: 37.
  • Bait DNA preparation The MDM27-300 gene was subcloned into the BamHI / XhoI site of pCMV-Fos-CBPzz. Based on this plasmid, the SP6 promoter and a part of the omega enhancer ( ⁇ 29) were added by PCR. Schematic of immobilization of bait protein to beads and protease elution.
  • the IgG binding region (ZZ) of protein A was fused to the C-terminus of MDM2 via the TEV protease cleavage site. This ZZ region allows the bait protein to be immobilized on IgG beads and eluted with TEV protease.
  • T7 tag-MDM2-ZZ protein was expressed in Wheat germ extract and incubated with IgG beads. After washing the beads 5 times, TEV protease was added to elute the bait. Each fraction was separated by 10% SDS-PAGE and then detected by Western blot using an anti-T7 tag antibody (top). I: input, F: pass through, W: fifth wash, B1: beads before elution, B2: beads after elution. (B) Each band intensity was quantified and the ratio to the input was shown.
  • A Plasmid for expressing GFP fusion peptide.
  • GFP and FLAG tags were fused to the N-terminus and C-terminus of the peptide obtained by IVV screening, respectively.
  • CMV and T7 promoters were added upstream of the ORF so that this peptide can be transcribed in cultured cells and in vitro, respectively.
  • B A GFP fusion peptide was synthesized by a cell-free transcription and translation system, and a binding assay was performed. I: Input, F: Through, B: Beads. Optimization of selection conditions (electrophoresis photograph).
  • a random IVV library consisting of 12 amino acid residues after 4 rounds of selection was translated using a cell-free translation system, and an in vitro binding assay was performed.
  • the bead fraction under three kinds of conditions was separated by 8 M urea 8% SDS-PAGE, and the fluorescence of fluorescein linked to the PEG-Puro spacer was detected.
  • the binding activity compared to the control was quantified from the band intensity.
  • Progress of selection experiment (electrophoresis photograph).
  • a random IVV library consisting of 12 amino acid residues after completion of each selection round was translated by a cell-free system, and a binding assay for MDM2 immobilized on beads was performed.
  • the binding activity compared to the initial library was quantified from the band intensity.
  • HCT116 cell ⁇ wild type p53 expressing cell
  • SW480 cell mutant p53 expressing cell
  • GFP-HL4-FLAG control
  • GFP-MIP GFP-MIP
  • the mRNA levels of p53, MDM2, and p21 were each quantified by quantitative reverse transcription PCR.
  • Cell survival inhibitory effect of Tat-MIP HCT116 cell (wild type p53-expressing cell) and Saos-2 cell (p53-deficient cell) were cultured for 24 hours in a medium containing Tat-MIP at the concentration shown in the graph. Cell viability was then assessed by WST-1 assay.
  • the active ingredient of the inhibitor and facilitator of the present invention is a peptide comprising the following amino acid sequence.
  • Xaa1 is Lys, Pro, Arg, Ser, Leu, Ala or Met, preferably Lys, Pro, Arg or Ser, and more preferably Lys or Pro.
  • Xaa2 is Ser, Thr, Arg, Val, Gly, Tyr or Pro, preferably Ser, Thr, Arg or Val, more preferably Ser, Thr or Arg.
  • Xaa4 is Trp, Gln, Glu, Ala, Pro or Leu, preferably Trp, Gln, Glu, Ala or Pro, and more preferably Trp.
  • Xaa5 is Glu, Gln, Asp, Ala, Phe, Trp or Ser, preferably Glu, Gln, Asp or Phe, and more preferably Glu.
  • Xaa6 is Tyr, His, Leu or Glu, preferably Tyr or His, and more preferably Tyr.
  • Xaa7 is Trp or Leu, preferably Trp.
  • Xaa8 is Leu, Gln, Glu, Val, Ser or Met, preferably Leu, Gln or Met, and more preferably Leu.
  • Xaa9 is Glu, Arg, Met, Asp, Gln, Asn or Lys, preferably Glu, Arg or Asp, more preferably Glu or Arg.
  • Xaa11 is Met, Val, Leu, Ile or Tyr, preferably Met, Val or Leu, more preferably Met.
  • Xaa12 is Leu, Glu, Ser, Gly or Trp, preferably Leu, Glu or Trp, more preferably Leu or Glu.
  • Xaa9-Leu-Xaa10 is preferably Arg-Leu-Met or Glu-Leu-Met.
  • Examples of the peptide include a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 37 to 41.
  • the active ingredient of the inhibitor and facilitator of the present invention is also preferably a peptide having the amino acid sequence of SEQ ID NO: 37.
  • the peptide is preferably soluble in biological fluids such as blood.
  • the active ingredient peptide can be made into a preparation (pharmaceutical composition) using a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include an excipient or a base.
  • the formulation may contain the additive used normally.
  • the dosage form is appropriately selected depending on the administration route.
  • Formulations include the active ingredient peptide and other anticancer agents that are separately packaged and integrated.
  • the dose of the active ingredient is appropriately selected depending on the intended anticancer drug treatment, the patient's condition, and the like. Inhibitors or facilitators of the invention can be administered to patients who are receiving or willing to receive anticancer drug treatment.
  • peptides Compared with low molecular weight compounds, peptides require high synthesis costs and in vivo administration by injection due to their large molecular weight, and are low in solubility and cell membrane permeability, are easily degraded in vivo and are unstable. There are many disadvantages as pharmaceuticals (Chemical & Engineering Engineering News, 83, 17-24, 2005). For this reason, natural or synthetic peptides have not been considered as realistic drugs so far (Borghouts, C., ⁇ (2005) J. Peptide Sci., 11, 713-726).
  • the peptides used in the present invention have higher specificity and affinity for the target than low molecular weight compounds, there are few bindings to proteins other than the target that cause side effects (Chemical & Engineering News, 83, 17-24 (2005 )),it is conceivable that. It is also suitable for inhibiting the interaction between large proteins, which is difficult with low molecular weight compounds (Sakurai, K., Tsuji et al. J. Am. Chem. Soc., 126, 16288-16289). . A peptide having such a more effective drug activity is expected to be useful as a pharmaceutical product.
  • the peptide used in the present invention is effective as an interaction inhibitor or anticancer agent of human cancer protein MDM2 and human cancer suppressor protein p53 for the following reasons, but the present invention is not limited thereby. Absent.
  • the cancer protein MDM2 which is overexpressed in some malignant tumors, is transcriptionally activated by the tumor suppressor protein p53.
  • the expressed MDM2 binds to the p53 protein and degrades the p53 protein through a ubiquitin / proteasome-dependent pathway, thereby promoting its canceration by inhibiting its cancer suppressive action.
  • a random peptide IVV library consisting of 16 amino acid residues was prepared, and peptides that bind to MDM2 immobilized on beads were screened.
  • three hydrophobic amino acid residues F19, W23, L26
  • these three hydrophobic amino acid residues F, W, L
  • the remaining 9 amino acid residues consisted of 12 amino acid residues that are random sequences.
  • a rally was prepared and screened for peptides that bind to MDM2.
  • a peptide having an amino acid sequence selected by this screening method is expected to highly inhibit the human cancer protein MDM2-human cancer suppressor protein p53 interaction.
  • GFP-MIP protein sequence in which GFP was fused to the N-terminus of the most frequently duplicated peptide sequence (MDM2 inhibitor sequence, MIP) was expressed in the human colon cancer cell line HCT116 cells.
  • MDM2 inhibitor sequence, MIP MDM2 inhibitor sequence
  • the active ingredient of the inhibitor and anticancer agent of the present invention is not limited by its production method.
  • a library of DNAs encoding peptides is prepared, and (2) a DNA library is prepared from the library. And a peptide library linked to the DNA is prepared, (3) a molecule containing a peptide that binds to MDM2 is selected, and (4) DNA is selected by PCR using the DNA of the selected molecule as a template. And (5) using the amplified DNA as a library in step (2) and obtaining a screening method comprising repeating steps (2) to (4).
  • a screening method based on the in vitro virus (IVV) method (for example, refer to WO 02/48347, JP 2002-176987 A) can be performed. Describe IVV and screening methods.
  • IVV is also called a mapping molecule, and a mapping molecule by the IVV method binds a phenotype molecule containing a protein to be subjected to functional analysis or functional modification and a genotype molecule containing a nucleic acid encoding the protein. Do it.
  • a genotype molecule is formed by binding a coding molecule having a region encoding a protein in such a form that the base sequence of the region can be translated, and a spacer part.
  • the part derived from the phenotype molecule, the part derived from the spacer molecule, and the part derived from the coding molecule in the mapping molecule are called a decoding part, a spacer part, and a coding part, respectively.
  • the part derived from the spacer molecule and the part derived from the coding molecule in the genotype molecule are referred to as a spacer part and a coding part, respectively.
  • the spacer molecule binds to the peptide by a peptide transfer reaction that binds to the donor region that can bind to the 3 ′ end of the nucleic acid, the PEG region mainly composed of polyethylene glycol bound to the donor region, and the PEG region. And a peptide acceptor region containing the resulting group. There may be no PEG area.
  • the donor region that can bind to the 3 ′ end of a nucleic acid usually consists of one or more nucleotides.
  • the number of nucleotides is usually 1 to 15, preferably 1 to 2.
  • the nucleotide may be ribonucleotide or deoxyribonucleotide.
  • the sequence at the 5 ′ end of the donor region affects ligation efficiency.
  • the residue dCdC (dideoxycytidylic acid) is preferred.
  • the base type is preferably C> U or T> G> A.
  • the PEG region is mainly composed of polyethylene glycol.
  • the main component means that the total number of nucleotides contained in the PEG region is 20 bases or less, or the average molecular weight of polyethylene glycol is 400 or more. Preferably, it means that the total number of nucleotides is 10 bases or less, or the average molecular weight of polyethylene glycol is 2000 or more.
  • the average molecular weight of polyethylene glycol in the PEG region is usually 400 to 30,000, preferably 1,000 to 10,000, more preferably 2,000 to 8,000.
  • a post-process of associated translation may be required ( Liu, R., Barrick, E., Szostak, JW, Roberts, RW (2000) Methods in Enzymology, vol. 318, 268-293), with a molecular weight of 1000 or more, more preferably 2000 or more. Since high-efficiency association can be performed only by translation, post-translation processing is not necessary.
  • the stability of genotype molecules tends to increase, especially when the molecular weight is 1000 or higher, and when the molecular weight is 400 or lower, the DNA spacer may not be so characteristic and unstable. .
  • the peptide acceptor region is not particularly limited as long as it can bind to the C-terminus of the peptide.
  • puromycin 3'-N-aminoacylpuromycin aminonucleoside (3'-N-aminoacylpuromycin aminonucleoside, PANS-amino acid)
  • PANS-Gly having an amino acid part of glycine, PANS-Val of valine, PANS-Ala of alanine, and other PANS-all amino acids corresponding to all amino acids can be used.
  • 3'-Aminoacyladenosineoaminonucleoside AANS-amino acid, 3'-Aminoacyladenosine aminonucleoside, formed by dehydration condensation of the amino group of 3'-aminoadenosine and the carboxyl group of the amino acid as a chemical bond.
  • AANS-Gly whose amino acid part is glycine, AANS-Val of valine, AANS-Ala of alanine, and other AANS-all amino acids corresponding to all amino acids can be used.
  • a nucleoside or a nucleoside and an amino acid ester-bonded one can be used.
  • nucleoside or a substance having a chemical structure skeleton similar to nucleoside and an amino acid or a substance having a chemical structure skeleton similar to amino acid can be used as long as they can be chemically bonded.
  • the peptide acceptor region is preferably composed of puromycin or a derivative thereof, or puromycin or a derivative thereof and one or two deoxyribonucleotides or ribonucleotides.
  • the derivative means a derivative capable of binding to the C-terminus of the peptide in the protein translation system.
  • the puromycin derivatives are not limited to those having a complete puromycin structure, but also include those lacking a part of the puromycin structure. Specific examples of the puromycin derivative include PANS-amino acid and AANS-amino acid.
  • the peptide acceptor region may be composed of puromycin alone, but preferably has a base sequence consisting of DNA and / or RNA of 1 residue or more on the 5 ′ end side.
  • the sequences are dC-puromycin, rC-puromycin, etc., more preferably dCdC-puromycin, rCrC-puromycin, rCdC-puromycin, dCrC-puromycin, etc., and the 3 ′ end of aminoacyl-tRNA is Simulated CCA sequences (Philipps, GR (1969) Nature 223, 374-377) are suitable.
  • the base type is preferably C> U or T> G> A.
  • the spacer molecule preferably includes at least one function-imparting unit between the donor region and the PEG region.
  • the function-imparting unit is preferably a functional modification of at least one residue of deoxyribonucleotide or ribonucleotide base.
  • a substance into which various separation tags such as a fluorescent substance, biotin, or His-tag are introduced as a function modifying substance can be used.
  • the coding molecule in this embodiment includes a 5 ′ untranslated region containing a transcription promoter and a translation enhancer, an ORF region encoding a protein bound to the 3 ′ end of the 5 ′ untranslated region, and the 3 ′ end of the ORF region. And a nucleic acid containing an affinity tag sequence 5 ′ upstream of the poly A sequence.
  • the coding molecule may be DNA or RNA. In the case of RNA, the 5 'end may or may not have a Cap structure. The coding molecule may be incorporated into any vector or plasmid.
  • the 3 ′ end region contains an affinity tag sequence and a poly A sequence downstream thereof.
  • the polyA sequence in the 3 ′ end region is important, and the polyA sequence may be a mixture of dA and / or rA having at least 2 residues or more.
  • the poly A continuous chain is preferably 3 or more residues, more preferably 6 or more, and even more preferably 8 residues or more.
  • Elements that affect the translation efficiency of the coding molecule include a 5 'UTR consisting of a transcription promoter and translation enhancer, and a 3' end region combination containing a poly A sequence.
  • the effect of the poly A sequence in the 3 ′ end region is usually exerted with 10 residues or less.
  • T7 / T3 or SP6 can be used as the 5 ′ UTR transcription promoter, and there is no particular limitation.
  • SP6 is preferable, and SP6 is particularly preferable when an omega sequence or a sequence containing a part of the omega sequence is used as an enhancer sequence for translation.
  • the translation enhancer is preferably part of the omega sequence, and part of the omega sequence includes part of the TMV omega sequence (O29; Gallie DR, Walbot V. (1992) Nucleic Acids Res., Vol. 20, 4631 -4638, and those including WO 02/48347 (see FIG. 3) are preferred.
  • the affinity tag sequence is not particularly limited as long as it is a sequence for using any means capable of detecting a protein such as an antigen-antibody reaction.
  • a Flag-tag sequence or a His-tag sequence which is a tag for affinity separation analysis by antigen-antibody reaction.
  • the ORF region may be any sequence consisting of DNA and / or RNA.
  • a gene sequence, exon sequence, intron sequence, random sequence, or any natural or artificial sequence is possible, and there is no sequence limitation.
  • each length is about 60 bp in 5′UTR
  • the length is about 32 bp at the 3 ′ end region and can be incorporated as an adapter region into a PCR primer.
  • a coding molecule having a 5 ′ UTR and a 3 ′ end region can be easily prepared by PCR from any vector, plasmid, or cDNA library.
  • translation may be done beyond the ORF region. That is, there may not be a stop codon at the end of the ORF region.
  • the coding molecule in this embodiment includes a 5 ′ untranslated region containing a transcription promoter and a translation enhancer, an ORF region encoding a protein bound to the 3 ′ end of the 5 ′ untranslated region, and a 3 ′ end of the ORF region.
  • a nucleic acid comprising a 3 ′ end region comprising a poly A sequence bound to
  • the genotype molecule is obtained by converting the above coding molecule into a form in which the base sequence of the protein coding region can be translated (for example, after transcription), if necessary, and the 3 ′ end of the coding molecule and the spacer molecule.
  • the donor region can be produced by binding by a normal ligase reaction.
  • the reaction conditions usually include conditions at 4 to 25 ° C. for 4 to 48 hours.
  • polyethylene glycol having the same molecular weight as the polyethylene glycol in the PEG region of the spacer molecule containing the PEG region is added to the reaction system, It can also be shortened to 0.5-4 hours at 15 ° C.
  • the combination of spacer molecule and coding molecule has an important effect on ligation efficiency.
  • the UTR translation enhancer is preferably a partial sequence (O29) of the omega sequence, and the donor region of the spacer part is at least one residue dC (deoxycytidylic acid) or two residues dCdC (dideoxycytidylic acid). ) Is preferred.
  • RNA ligase (a) a 5 ′ untranslated region containing a transcription promoter and a translation enhancer, an ORF region encoding a protein bound to the 3 ′ end of the 5 ′ untranslated region, and a 3 ′ end of the ORF region
  • the 3 'end of the coding molecule which is RNA containing the 3' end region containing the poly A sequence
  • the donor region of the spacer molecule consisting of RNA constitutes the PEG region in the spacer molecule It is preferable to bind with RNA ligase in the presence of free polyethylene glycol having the same molecular weight as polyethylene glycol.
  • the ligation efficiency is improved to 80 to 90% or more regardless of the molecular weight of the polyethylene glycol of the spacer part.
  • the separation step can also be omitted.
  • the mapping molecule in this embodiment is linked to a phenotype molecule that is a protein encoded by the ORF region in the genotype molecule by translating the genotype molecule in a cell-free translation system.
  • the cell-free translation system is preferably that of wheat germ or rabbit reticulocytes.
  • the conditions for translation may be those normally employed. For example, the conditions are 15 to 240 minutes at 25 to 37 ° C.
  • the nucleic acid portion of the mapping molecule of this embodiment can be a hybrid of RNA and DNA by reverse transcription after translation.
  • the screening method based on the IVV method is usually a method for screening a nucleic acid encoding a protein that interacts with a target substance from a nucleic acid library, which is associated with the nucleic acid library by the production method of the present invention.
  • a step of producing a library of molecules a step of mixing the library of mapping molecules and a target substance, a step of separating mapping molecules bound to the target substance, a linker of the separated mapping molecules, A step of releasing the nucleic acid by cleaving under conditions that do not change the base sequence, and a step of recovering the released nucleic acid.
  • the library of the mapping molecule and the target substance may be mixed under the condition that the target protein of the mapping molecule interacts with the target substance. This condition is appropriately selected according to the interaction to be detected and the type of target substance.
  • Separation of the mapping molecule bound to the target substance is a process of separating the mapping molecule bound to the target substance and the mapping molecule that does not bind to the target substance.
  • the target substance is immobilized on a solid phase.
  • separation can be performed by washing the solid phase on which the target molecule after mixing with the corresponding molecule is immobilized. Washing conditions are appropriately selected according to the interaction to be detected and the type of target substance.
  • immobilization on a solid phase means that the conjugate of the mapping molecule and the target substance can be separated from the non-bonded molecule.
  • the target substance is a membrane protein
  • the cell membrane of the cell Membrane proteins expressed in the above and proteins embedded in artificial membranes are also included in the target substance immobilized on the solid phase.
  • the separation of the linker of the associating molecule under the condition that the nucleotide sequence of the nucleic acid does not change to release the nucleic acid can be performed using a cleaving linker and under the conditions corresponding thereto.
  • releasing the nucleic acid is also called elution.
  • “free” is used to mean “elution”.
  • the nucleic acid to be released may be modified as long as the base sequence of the nucleic acid can be analyzed.
  • the free nucleic acid can be collected by a usual method. For example, a method for recovering by electrophoresis, a method for recovering a supernatant by precipitating components other than the released nucleic acid, and the like can be mentioned.
  • the recovered nucleic acid is subjected to amplification and sequence analysis according to purposes such as functional analysis and evolutionary engineering. Depending on the purpose, the collected DNA can be sequenced or amplified by PCR and the above steps can be repeated.
  • the DNA library used in the screening method of the present invention preferably comprises a DNA encoding an amino acid sequence in which three hydrophobic amino acids corresponding to F19, W23, and L26 of human tumor suppressor protein p53 are conserved, Examples of such an amino acid sequence include XXFXXXWXXLXX (SEQ ID NO: 5) (X is an arbitrary amino acid).
  • the peptide that becomes the active ingredient of the inhibitor or anticancer agent of the present invention is identified. can do. Whether or not there is competition can be measured by a method as described in Examples described later.
  • MDM2 inhibitory peptide MIP
  • MIP MDM2 inhibitory peptide
  • HCT116 cells which are wild-type p53-expressing cells
  • the expression level of p53 protein increased. This is presumably due to inhibition of the MDM2-p53 interaction of GFP-MIP.
  • the amount of protein and mRNA of p53 targets such as MDM2 and p21 increased, it was found that the p53 pathway was activated by the expression of GFP-MIP.
  • peptides that bind to MDM2 protein with high affinity can be screened by the IVV method. In addition, it was confirmed that the obtained peptide strongly inhibited the in vitro and in vivo interaction between MDM2 and p53, and had a cancer cell growth inhibitory activity. Details of the examples will be described below.
  • Example 1 Preparation of bait protein Using Ex Taq DNA polymerase (Takara) from cDNA library derived from A549 cells, MDM (1-294) -f and MDM (1-294) -r primers (Table 1) at 95 ° C for 60 ° C. PCR was performed with a program in which a cycle of 60 seconds at 60 ° C and 60 seconds at 72 ° C was repeated 30 times. The obtained PCR product was purified with a PCR purification kit (Qiagen), and this time was used as a template, and PCR was carried out in the same manner with 5′adaptorO29T7EcoR and Flag1A-lib primers (Table 1).
  • the PCR product was purified again with the PCR purification kit and TA-cloned into the pDrive cloning vector (Qiagen). Using this plasmid as a template, using Phusion DNA polymerase (Finnzymes), Bam-MDM-f and MDM-294-Xho-r primers were cycled at 98 ° C for 10 seconds, 62 ° C for 30 seconds, and 72 ° C for 30 seconds. PCR was performed with a program repeated 25 times. This incorporated BamHI and XhoI restriction enzyme sites on both sides of the PCR product, respectively.
  • the obtained PCR product was purified with a PCR purification kit, cleaved with BamHI and XhoI, and purified again with the PCR purification kit.
  • T4 DNA Ligase Promega
  • this fragment was transformed into a vector having a T7 tag on the N-terminal side and a TAP tag on the C-terminal side, pCMV-CBPzz (Vassilev., LY, et al. (2004) Science, 844-848). Subcloned into BamHI / XhoI site.
  • MDM2 protein used as a bait for screening was prepared.
  • MDM2 with TAP tag for immobilization / elution added to beads is expressed in a cell-free translation system.
  • This protein is immobilized on the beads with TAP tag and is eluted, and the binding activity to p53 is maintained. This was confirmed by examining the binding to the p53 full-length protein.
  • the binding assay of p53 (15-29) and full-length IVV molecules was performed on MDM2 immobilized on beads (FIG. 4).
  • the p53 (15-29) IV IVV did not bind to MDM2, whereas the full length showed binding in the protein portion.
  • the prepared bait protein MDM2 has p53-binding activity due to binding of the full-length p53 IVV.
  • the dissociation constant between p53 (15-29) 2 and MDM2 is about 600 nM (Kussie, PH, etc. (1996) Science, 274, 948-953). Therefore, it is highly possible that the peptide portion linked to the mRNA did not retain the three-dimensional structure.
  • p53 (15-29) ⁇ has an ⁇ -helical structure, and the p53 mutant (P27S) peptide fragment that easily adopts this helix structure binds to MDM2 with higher affinity than the wild type (Schon, O., et al. ( 2002) J. Mol. Biol., 323, 491-501), it is likely that the peptide will not be selected in subsequent IVV screening unless it is a peptide fragment that is easy to take a helix structure and has a high affinity for MDM2. Therefore, it is favorable conditions to obtain a peptide that competes with p53 and binds to MDM2. In addition, it is speculated that the full-length p53 may have a more retained three-dimensional structure than the peptide fragment.
  • Random library construction Single-stranded DNA, G4SG4S (NNS) 16FLAGA6r (Table 1) as a template, Ex Taq DNA polymerase, 95 ° C for 60 seconds, 60 ° C with priSP6OGf and priFLAGA6r primer (Table 1) PCR was carried out using a program in which a cycle of 60 seconds at 72 ° C. was repeated 25 times. The obtained PCR product was purified with a PCR purification kit to prepare a DNA encoding a random 16 amino acid residue.
  • Immutex-MAG (MAG2101) (JSR) 20 mg was washed 3 times with 0.01% Triton X-100, 0.25 mg / ml EDC was added, and the mixture was rotated and mixed at room temperature for 90 minutes. 0.57 mg of rabbit rabbit IgG (Jackson immunoresearch) was added thereto, and the mixture was rotated and mixed at room temperature for 16 hours. After removing the supernatant, washing buffer (PBS, 0.1% BSA, 0.01% Triton X-100) was added and incubated at room temperature for 1 hour.
  • washing buffer PBS, 0.1% BSA, 0.01% Triton X-100
  • the plate was washed 5 times with a washing buffer and suspended in a storage buffer (PBS, 0.1% BSA, 0.01% Triton X-100, 0.02% NaN 3 ) to prepare IgG-Immutex-MAG beads (2% slurry).
  • a storage buffer PBS, 0.1% BSA, 0.01% Triton X-100, 0.02% NaN 3
  • TEV protease (10 U / ⁇ l) (Invitrogen) 2 ⁇ l was mixed, and mixed by rotation at 16 ° C. for 2 hours. This supernatant was collected as an elution fraction.
  • RT-PCR kit Qiagen
  • 30 seconds at 94 ° C, 30 seconds at 60 ° C, 120 ° C at 72 ° C RT-PCR was performed with a program in which a second cycle was repeated 14-30 times. From this, a band pattern with a non-saturated amplification amount was selected, and a large amount of RT-PCR was performed under the same conditions to reconstruct the library, and a second round of screening was performed. The same operation was repeated, and screening was performed up to the fifth round.
  • the DNA library was cloned with PCR Cloning kit (Qiagen), and the sequence was analyzed with ABI3700 PRISM Genetic Analyzer (Applied Biosystems). Moreover, weblogo (http://weblogo.berkeley.edu/) was used for the analysis of the appearance frequency of amino acids in peptide sequences.
  • MDM2 inhibitory peptide The most frequently duplicated peptide sequences (PRFWEYWLRLME (SEQ ID NO: 37), hereinafter referred to as MDM2 inhibitory peptide, MIP) and p53 17-28 dissociation constants were measured (Table 4). MIP was p53 17- It was found that the affinity with MDM2 was about 100 times that of 28 . Moreover, from the appearance frequency of amino acids at each position of the peptide sequence obtained by screening (FIG. 8), Y6, E9, and M11 were well conserved except for the fixed three amino acid residues. Therefore, this time, the dissociation constant of MIP (M11A) was measured by substituting M11 in the MIP sequence with alanine.
  • the affinity with MDM2 was reduced by about 10 times compared to the original MIP.
  • This position is a proline in p53 17-28 , and it has been reported that substituting it with serine makes it easier to adopt an ⁇ -helical structure and improves the affinity for MDM2 by about 25-fold (Zondlo, SC, Et al. (2006) Biochemistry 45, 11945-11957). In this case as well, it is presumed that the helix structure is made easier by M11.
  • Y6 is well conserved in screening of random libraries using the phage display method, and is reported to be an important amino acid in terms of enhancing affinity with MDM2 (Bottger, V.,, etc. (1996) Oncogene, 13, 2141-2147).
  • GFP-MIP MIP IVV and GFP fused to the N-terminus
  • IPTG was added at a concentration of 1 mM and further cultured at 37 ° C. for 6 hours.
  • the culture was centrifuged at 6000 g for 20 minutes, and the supernatant was removed to collect the cells.
  • 20 ml of lysis buffer (TBS, pH 7.4, 1 mM ⁇ -ME), 40 ⁇ l of protease inhibitor cocktail (Sigma) and 8 ⁇ l of DNase I were added, suspended, and then sonicated twice for 15 minutes. g, The supernatant after centrifugation for 20 minutes was collected as a soluble fraction.
  • TALON Metal Affinity Resin was added to the soluble fraction and mixed by rotation at 4 ° C for 2 hours, and the entire amount was passed through the column. After washing with 80 ml of lysis buffer and 10 ml of washing buffer (TBS pH 7.4, 1 mM mM ⁇ -ME, 10 mM mMimidazole), elute with 5 ml of elution buffer (TBS, pH 7.4, 1 mM mM ⁇ -ME, 250 mM mMimidazole). did.
  • MDM2 binding peptide MIP (MDM2 inhibitory peptide, PR F WEY W LR L ME) obtained by screening by the in vitro virus (IVV) method is compared with the existing sequence pDI (LTFEHYWAQLTS (SEQ ID NO: 49)) (Hu , B., et al. (2007) Cancer Res., 67, 8810-8817), showing about 8 ⁇ 10 3 times the affinity (Table 4).
  • MIP is a sequence that differs from wild-type p53 peptide (17-ETFSDLWKLLPE-28 (SEQ ID NO: 42)) and pDI but is known to bind to the pocket of MDM2 (Phe19, Trp23, Leu26, underlined) were conserved.
  • MIP novel peptide sequence MIP that binds to human cancer protein MDM2 was identified by the IVV method.
  • MIP was found to have a much higher affinity than existing MDM2-binding peptides.
  • Arg9 and Met11 which had a high frequency of appearance in the screening, were replaced with Ala, the affinity with MDM2 was greatly reduced. Therefore, these two amino acid residues and their positions were determined as novel motifs.
  • Met11 has a very high frequency of occurrence, and since Affin substitution showed the same degree of affinity reduction as Trp7, it is highly likely that it is an amino acid important for binding to MDM2.
  • GFP fusion peptide Incubate the 5 'ends of single-stranded DNA, GFP-fus-MIPf and GFP-fus-MIPr (Table 1) using T4 polynucleotide kinase (Takara) for 30 minutes at 37 ° C, respectively. Phosphorylated with. After ethanol precipitation, the whole amount was mixed, denatured at 98 ° C. for 20 seconds, and annealed by slowly returning to room temperature.
  • Cell culture and transfection HCT116 cells are McCoy's 5A medium (Gibco) containing 10% (vol / vol) FBS (Gibco), 1% (vol / vol) penicillin / streptomycin (Gibco), and SW480 cells are 10%.
  • the cells were cultured in McCoy's 5A medium (Gibco) containing penicillin / streptomycin (Gibco).
  • SW480 cells which are p53 mutant expressing cells deficient in transcriptional activation ability, were subjected to the same operation, but activation of the p53 pathway could not be confirmed.
  • Immunoprecipitation HCT116 cells 24 hours after transfection were solubilized with 500 ⁇ l of TNE buffer (10 mM Tris-HCl, pH 7.8, 0.15 M NaCl, 1 mM EDTA, 1% NP-40), and 12,000 g Centrifugation was performed for 20 minutes, 20 ⁇ l of Agarose conjugated Anti-GFP (Medical & biological laboratories) was added to 400 ⁇ l of the supernatant, and the mixture was mixed by rotation at 4 ° C. for 2 hours. Thereafter, the cells were washed 5 times with TNE buffer, added with 1 ⁇ sample buffer, and heated at 95 ° C. for 5 minutes for elution.
  • TNE buffer 10 mM Tris-HCl, pH 7.8, 0.15 M NaCl, 1 mM EDTA, 1% NP-40
  • Centrifugation was performed for 20 minutes, 20 ⁇ l of Agarose conjugated Anti-GFP (Medical & biological laboratories) was added to 400 ⁇ l of the super
  • RNA levels of p53, MDM2, p21 and GAPDH were measured using QuantiTect SYBR Green RT-PCR kit (Qiagen).
  • the primers used were p53 F and p53 R, mdm2 F and mdm2 R, p21 F and p21 R (Table 1), and GAPDH was measured using the Light cycler primer set (Roche, sequence not disclosed). After normalization with the amount of GAPDH mRNA, the amount of mRNA of each gene was determined.
  • WST-1 assay HCT116 and Saos-2 cells (1 ⁇ 10 4 cells / well) were cultured in 96-well plates. These cells were cultured for 24 hours in a medium containing a peptide containing HIV-Tat added as a membrane permeation sequence, Tat-MIP ( YGRKKRRQRRR PRFWEYWLRLME (SEQ ID NO: 50), underlined HIV-Tat sequence) at different concentrations. Thereafter, Cell proliferation reagent WST-1 (Roche) was added at 10 ⁇ l / well and further incubated for 30 minutes. The absorbance at 440 nm (reference wavelength 600 nm) of each well was measured with a plate reader SAFIRE (Tecan).
  • Tat-MIP MIP peptide
  • IC 50 1.8 ⁇ M and 20 ⁇ M, respectively. About 10 times different (Shangary, S., et al. (2008) Proc. Natl. Acad. Sci. USA, 105, 3933-3938). In contrast, in the case of Tat-MIP, IC 50 was 13.2 ⁇ M in HCT116 cells and 19.3 ⁇ M in Saos-2 cells, which was about 1.5 times as high.
  • SJSA-1 cells have higher endogenous MDM2 expression levels than HCT116 cells, and are susceptible to apoptosis due to inhibition of MDM2-p53 interaction (Chene, P., et al. (2002) FEBS Lett ., 529, 293-297).
  • p53 15-29 2 Motif consisting of 5 amino acid residues corresponding to the 19-23th region of p53 3: Peptide consisting of 10 amino acid residues corresponding to the 17th to 26th region of p53 4: Amino acid sequence of the peptide used in the present invention 5: Amino acid sequence encoded by library DNA 6: Primer MDM (1-294) f 7: Primer MDM (1-294) r 8: Primer 5'adaptorO29T7EcoR 9: Primer Flag1A-lib 10: Primer Bam-MDM-f 11: Primer MDM294-Xho-r 12: Primer SP6-O'-T7 13: Primer 3'FosCBPzz 14: Template G4SG4S (NNS) 16FLAGA6r 15: Primer priSP6OGf 16: Primer priFLAGA6r 17: Template X12 (FWL) -r 18: Primer 5'O29-
  • the present invention it is possible to provide a drug that inhibits the interaction between human cancer protein MDM2 and human cancer suppressor protein p53 and has an effect of suppressing cancer cell growth.

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Abstract

La présente invention concerne un agent renfermant un peptide composé de 12 résidus d’acides aminés spécifiés en tant que composé actif, et démontrant une activité anticancéreuse par inhibition de l’interaction entre l’oncoprotéine MDM2 humaine et la protéine antitumorale p53 humaine.
PCT/JP2009/069644 2008-11-19 2009-11-19 Peptide capable d’inhiber l’interaction entre l’oncoprotéine mdm2 humaine et la protéine antitumorale p53 humaine, et utilisation associée Ceased WO2010058819A1 (fr)

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WO2023056069A1 (fr) 2021-09-30 2023-04-06 Angiex, Inc. Conjugués agent de dégradation-anticorps et leurs procédés d'utilisation
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HIROKAZU SHIHEIDO ET AL.: "In vitro virus-ho o Mochiita MDM2 Ketsugo Peptide no Screening", DAI 30 KAI ANNUAL MEETING OF THE MOLECULAR BIOLOGY SOCIETY OF JAPAN - DAI 80 KAI THE JAPANESE BIOCHEMICAL SOCIETY TAIKAI GODO TAIKAI KOEN YOSHISHU, 2007, pages ABSTR. - 2P-1499 *

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US9527896B2 (en) 2007-01-31 2016-12-27 Dana-Farber Cancer Institute, Inc. Stabilized p53 peptides and uses thereof
US10301351B2 (en) 2007-03-28 2019-05-28 President And Fellows Of Harvard College Stitched polypeptides
US9476041B2 (en) 2010-07-12 2016-10-25 National University Corporation Tottori University Method for producing novel hipsc by means of siRNA introduction
WO2012008301A1 (fr) * 2010-07-12 2012-01-19 国立大学法人鳥取大学 Méthode de production de nouvelles hipsc par introduction d'arnsi
JP2013046616A (ja) * 2010-07-12 2013-03-07 Tottori Univ ｓｉＲＮＡ導入による新規ｈｉＰＳＣ作製法
JP2014097059A (ja) * 2010-07-12 2014-05-29 Tottori Univ ｍｉＲＮＡ導入による新規ｈｉＰＳＣ作製法
JP5099570B2 (ja) * 2010-07-12 2012-12-19 国立大学法人鳥取大学 ｓｉＲＮＡ導入による新規ｈｉＰＳＣ作製法
JP5099571B2 (ja) * 2010-07-12 2012-12-19 国立大学法人鳥取大学 ｍｉＲＮＡ導入による新規ｈｉＰＳＣ作製法
US9790491B2 (en) 2010-07-12 2017-10-17 National University Corporation Tottori University Method for producing novel hiPSC by means of miRNA introduction
JP2013034479A (ja) * 2010-07-12 2013-02-21 Tottori Univ ｍｉＲＮＡ導入による新規ｈｉＰＳＣ作製法
WO2012008302A1 (fr) * 2010-07-12 2012-01-19 国立大学法人鳥取大学 Méthode de préparation de nouvelles hipsc par introduction d'arnmi
US8859723B2 (en) 2010-08-13 2014-10-14 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
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US9096684B2 (en) 2011-10-18 2015-08-04 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
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US9505804B2 (en) 2012-02-15 2016-11-29 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US8987414B2 (en) 2012-02-15 2015-03-24 Aileron Therapeutics, Inc. Triazole-crosslinked and thioether-crosslinked peptidomimetic macrocycles
US8927500B2 (en) 2012-02-15 2015-01-06 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10213477B2 (en) 2012-02-15 2019-02-26 Aileron Therapeutics, Inc. Peptidomimetic macrocycles
US10227380B2 (en) 2012-02-15 2019-03-12 Aileron Therapeutics, Inc. Triazole-crosslinked and thioether-crosslinked peptidomimetic macrocycles
US9604919B2 (en) 2012-11-01 2017-03-28 Aileron Therapeutics, Inc. Disubstituted amino acids and methods of preparation and use thereof
US10669230B2 (en) 2012-11-01 2020-06-02 Aileron Therapeutics, Inc. Disubstituted amino acids and methods of preparation and use thereof
US9845287B2 (en) 2012-11-01 2017-12-19 Aileron Therapeutics, Inc. Disubstituted amino acids and methods of preparation and use thereof
US10471120B2 (en) 2014-09-24 2019-11-12 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US10905739B2 (en) 2014-09-24 2021-02-02 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and formulations thereof
US10253067B2 (en) 2015-03-20 2019-04-09 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
US10023613B2 (en) 2015-09-10 2018-07-17 Aileron Therapeutics, Inc. Peptidomimetic macrocycles as modulators of MCL-1
WO2017201449A1 (fr) 2016-05-20 2017-11-23 Genentech, Inc. Conjugués anticorps-protac et procédés d'utilisation
US11091522B2 (en) 2018-07-23 2021-08-17 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and uses thereof
WO2023056069A1 (fr) 2021-09-30 2023-04-06 Angiex, Inc. Conjugués agent de dégradation-anticorps et leurs procédés d'utilisation
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