WO2010058819A1 - Peptide capable of inhibiting interaction between human tumor protein mdm2 and human tumor suppression protein p53, and use thereof - Google Patents

Abstract

An agent which comprises a peptide composed of 12 specified amino acid residues as an active component, and which exhibits an anti-cancer activity by inhibiting the interaction between the human oncoprotein MDM2 and the human tumor suppression protein p53.

Description

Peptide for inhibiting interaction between human cancer protein MDM2 and human tumor suppressor protein p53 and use thereof

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, is usually present in cells at low concentrations, but is induced by cellular stress such as DNA damage, oncogene activation, and hypoxia (Chene, P., etc. 2003) Nature Rev. Cancer, 3, 102-109; Zondlo, SC, et al. (2006) Biochemistry, 45, 11945-11957; Schon, O., et al. (2002) J. Mol. Biol., 323, 491-501 Zhang, R., Wang, H. (2000) Curr. Pharm. Des., 6, 393-416). The expressed p53 protein forms a tetramer, and then moves into the nucleus to activate transcription of its target. One of the expressed p53 targets, 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. 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).

Thus, 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., 等 (1997) Proc. Natl. Acad. Sci. USA., 105, 3933-3938 ). In addition, in other cancer cells, despite the expression of wild-type p53, its cancer suppressive action is inhibited by intracellular inhibitors. These 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., 等 (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. (1996) Science, 274, 948-953) Thus, 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 α helix and β A hydrophobic gap is formed by the peptide, and a peptide part having an α 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. (1996) Science, 274, 948-953) Since the cancer protein MDM2 binds to the tumor suppressor protein p53 and inhibits its cancer suppressive action, anticancer drug design Targeted (Kussie, PH, et al. (1996) Science, 274, 948-953; Chene, P., et al. (2003) Nature Rev. Cancer, 3, 102-109; Zondlo, SC, et al. (2006) Biochemistry, 45, 11945-11957; Schon, O., et al. (2002) J. Mol. Biol., 323, 491-501; Zhang, R., Wang, H. (2000) Curr. Pharm. Des., 6 393-416). To date, it has been shown that p53 activation can be induced by introducing an antibody that binds to this region of MDM2 or an antisense oligonucleotide of MDM2 into cells (Chene, P., et al. (2000). ) J. Mol. Biol., 299, 245-253; Blaydes, JP, et al. (1997) Oncogene, 14, 1859-1868). From a library of low molecular weight compounds based on this structural information, Nutlin-3 was identified as a compound that binds to MDM2 and shows similar effects (Chen, L., et al. (1997) Proc. Natl. Acad. Sci. USA., 95, 195-200). In recent years, 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). , x is any amino acid) p53 peptide fragment (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; F Is phenylalanine; 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 (SEQ ID NO: 49); Non-Patent Document 2) consisting of 12 amino acid residues corresponding to the 17th to 28th region of 53 is particularly suitable for inhibiting the interaction between p53 and MDM2 However, the affinity of the various p53 peptide fragments mentioned above for MDM2 is rather weak, and even with the strongest affinity pDI, the dissociation constant (K _D ) measured by Biacore is 4 × 10 ^-4 is about M, to be used as a drug is weak affinity. Thus, strong affinity for MDM2, peptide fragments strongly inhibit MDM2-p53 interaction is required.

Special table flat 10-506525 JP2007-222170 Special table 2001-500365

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. , 等 (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.

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 (Ω29) 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. Confirmation of bait protein bead fixation and protease elution (electrophoresis photo). (A) 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. In vitro binding assay of MDM2 immobilized on beads with p53 _15-29 and full-length p53 IVV (electrophoresis photograph). IVV was produced in a cell-free system and a binding assay was performed. Each fraction was separated by 8 M urea 8% SDS-PAGE, and the fluorescence of fluorescein linked to a PEG-Puro spacer was detected. The upper and lower bands in each electropherogram correspond to mRNA-PEG-Puro spacers that are not linked to IVV and protein, respectively. I: Input, F: Through, B: Beads. In vitro binding assay of GFP fusion peptide and MDM2 immobilized on beads (electrophoresis photograph). (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. (1) Reduced binding time from 1 hour to 10 minutes. (2) Increased number of washings from 3 to 10 times. (3) Double salt concentration. 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. Amino acid appearance frequency at each position of peptides obtained from random IVV library after 5 rounds of selection. The logo for the sequence was created at WebLogo version 2.8.2 (http://weblogo.berkeley.edu/). The height of each character reflects the frequency of appearance. Binding assay of GFP-MIP and MDM2 (electrophoresis photo). (A) GFP-MIP was translated by a cell-free system, and an in vitro binding assay for MDM2 immobilized on beads was performed. (B) HCT116 cells were transfected with GFP-HL4-FLAG (control) or GFP-MIP. After 24 hours, immunoprecipitation was performed using an anti-GFP antibody, and detection was performed by Western blot using an anti-MDM2 antibody. Activation of p53 pathway by GFP-MIP (electrophoresis photograph). HCT116 cell 野生 (wild type p53 expressing cell) and SW480 cell (mutant p53 expressing cell) were transfected with GFP-FLAG (control) or GFP-MIP. 24 hours later, the cell lysate was subjected to Western blotting with antibodies against p53, MDM2, p21 and β-actin, respectively. Activation of the p53 pathway by GFP-MIP. HCT116 cell 野生 (wild type p53 expressing cell) and SW480 cell (mutant p53 expressing cell) were transfected with GFP-HL4-FLAG (control) or GFP-MIP. After 24 hours, 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.

In this specification, the one-letter symbols for base and amino acids and the three-letter symbols for amino acids are based on WIPO Standard ST.25.

The active ingredient of the inhibitor and facilitator of the present invention is a peptide comprising the following amino acid sequence.
Xaa1 Xaa2 PheXaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Leu Xaa11 Xaa12 (SEQ ID NO: 4)
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. Examples of the pharmaceutically acceptable carrier include an excipient or a base. Moreover, 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.

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., 等 (2005) J. Peptide Sci., 11, 713-726). However, in recent years, by converting to non-natural D-amino acids instead of naturally occurring L-amino acids, synthesis of peptides that have the same physiological activity as the original peptides but are not degraded by proteases in vivo (Borghouts , C., 等 (2005) J. Peptide Sci., 11, 713-726; Sakurai, K., J., etc. (2004) J. Am. Chem. Soc., 126, 16288-16289), cell membrane permeation Membrane permeability is improved by fusing sequences (Borghouts, C., 等 (2005) J. Peptide Sci., 11, 713-726; Sakurai, K., J., et al. (2004) J. Am. Chem. Soc., 126, 16288-16289; Prive, GG, Etc. (2006) Curr. Opin. Genet. Dev., 16, 71-77) improve the in vivo stability and cell membrane permeability of peptides. It has been improved, and the prospect as a peptide drug has been opened (Borghouts, C., 等 (2005) J. Peptide Sci., 11, 713-726). These techniques can be applied to the peptides used in the present invention.

Since 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.

It is considered that 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. This binding is known to involve the 15th to 29th amino acid residues of the p53 protein, and a novel peptide capable of inhibiting the MDM2-p53 interaction is expected as a candidate for an anticancer agent. Therefore, in vitro virus (IVV) method (Nemoto, N., et al. (1997) FEBS Lett., 414, 405-408; Miyamoto-Sato, E., et al. (2003) Nucleic Acids Res., 31, e78) A peptide that binds to MDM2 and suppresses the growth of cancer cells was screened. First, a random peptide IVV library consisting of 16 amino acid residues was prepared, and peptides that bind to MDM2 immobilized on beads were screened. In the obtained peptide sequence, three hydrophobic amino acid residues (F19, W23, L26) were well conserved. Next, in order to optimize this sequence, these three hydrophobic amino acid residues (F, W, L) were fixed, and 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.

Actually, a protein sequence (GFP-MIP) 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. However, binding of GFP-MIP and MDM2 in the cell, increased expression of p53 protein, and accompanying increase in mRNA and protein expression of p53 downstream genes (MDM2 and p21) were confirmed. Furthermore, the survival rate of HCT116 cells was reduced by peptide し た (Tat-MIP) し た fused with N-terminal of cell membrane permeation sequence of HIV-Tat, suggesting that MIP induces cell death.

The active ingredient of the inhibitor and anticancer agent of the present invention is not limited by its production method.For example, (1) 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).

In this method, except that MDM2 is used as a target substance, 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. For convenience of explanation, 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. Moreover, 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.

In this embodiment, 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. In order to ligate the coding part and the spacer part, it is necessary to contain at least one residue. For an acceptor having a poly A sequence, at least one residue of dC (deoxycytidylic acid) or 2 The residue dCdC (dideoxycytidylic acid) is preferred. The base type is preferably C> U or T> G> A.

PEG region is mainly composed of polyethylene glycol. Here, 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. Here, when the molecular weight of polyethylene glycol is lower than about 400, when a genotype molecule containing a spacer part derived from this spacer molecule is associated and translated, 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. In addition, when the molecular weight of polyethylene glycol increases, 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. For example, puromycin, 3'-N-aminoacylpuromycin aminonucleoside (3'-N-aminoacylpuromycin aminonucleoside, PANS-amino acid) For example, 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. In addition, 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. ) For example, 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. Further, a nucleoside or a nucleoside and an amino acid ester-bonded one can be used. In addition, 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. Here, 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. For example, 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. As a factor affecting the ligation efficiency between the spacer molecule and the coding molecule, 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. Preferably, 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. Further, by setting the 5′UTR of the coding molecule as SP6 + O29 and the 3 ′ terminal region as, for example, Flag + XhoI + A _n (n = 8), 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. For this reason, 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. In the coding molecule, 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. Is 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. When 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. In the 3 ′ terminal region of the coding part corresponding to the acceptor, there should be a poly A sequence of DNA and / or RNA of at least 2 residues, preferably 3 residues, more preferably 6-8 residues, 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. By using RNA ligase, the problems of DNA ligase can be avoided and the efficiency can be maintained at 60 to 80%.

(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, and (b) 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.

During the ligation reaction, by adding polyethylene glycol having the same molecular weight as the PEG region of the spacer part including the PEG region, 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. Can do. 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. In addition, 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. Usually, the target substance is immobilized on a solid phase. Thus, 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. Here, 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. For example, when 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. When the target substance is immobilized on a solid phase, releasing the nucleic acid is also called elution. In the present invention, “free” is used to mean “elution”. Moreover, the nucleic acid to be released may be modified as long as the base sequence of the nucleic acid can be analyzed.

回収 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).

By identifying whether or not the peptide that binds to human cancer protein MDM2 screened by the above method competes with p53 protein, 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.

Hereinafter, examples of the present invention will be specifically described. However, the following examples should be regarded as an aid for obtaining specific recognition of the present invention, and the scope of the present invention is limited by the following examples. It is not something.

From random library to MDM2 by IVV method (Nemoto, N., 等 (1997) 1997FEBS Lett., 414, 405-408; Miyamoto-Sato, E., 等 (2003) Nucleic Acids Res., 31, e78) Peptides that bind strongly were screened. First, a random peptide IVV library consisting of 16 amino residues was screened. Most of the sequences obtained in the four rounds of screening conserved three hydrophobic amino acids corresponding to F19, W23, and L26 of p53. Therefore, in order to optimize these sequences, a random peptide IVV library consisting of 12 amino acid residues with the above-mentioned 3 amino acids fixed was newly designed and screened for 5 rounds with more stringent selection pressure. As a result, the most overlapping peptide sequence PRFWEYWLRLME (SEQ ID NO: 37, hereinafter referred to as MDM2 inhibitory peptide, MIP) had a higher protein (GFP-MIP) than the wild-type p53 peptide fragment. It was confirmed that it binds to MDM2 with affinity and also binds to MDM2 in cells.

When GFP-MIP was expressed in 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. Moreover, since 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. Furthermore, the viability of HCT116 cells was reduced by Tat-MIP in which HIV-Tat, a cell membrane permeation sequence, was fused to the N-terminus (IC ₅₀ = 13.2 μM). Saos-2, which is a p53-deficient cell, had an IC ₅₀ = 19.2 μM and a wild-type p53-expressing cell specificity of about 1.5 times was observed. From this result, it was found that Tat-MIP induces p53 pathway-dependent cell death.
According to the present invention, 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]
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. Using 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. Using this as a template, using Ex Taq DNA polymerase, 20 cycles of SP6-O'-T7 and 3'FosCBPzz primer (Table 1) at 95 ° C for 60 seconds, 60 ° C for 60 seconds, 72 ° C for 120 seconds PCR was performed with a repetitive program, and the SP6 promoter and a part of the Ω sequence were added. This PCR product was purified with a PCR purification kit.

1 μg of this DNA was added to RiboMAX large-scale RNA production systems-SP6 (Promega) at 37 ° C. for 2 hours, and then DNase I (Promega) was added and further incubated at 37 ° C. for 15 minutes. The obtained mRNA was purified by RNeasy mini kit (Qiagen), and then incubated for 2 hours at 25 ° C using a wheat germ-derived cell-free translation system Wheat germ extract systems (Promega) to synthesize proteins.

First, 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 crystal structure of the complex of p53 peptide (15-29) and MDM2 (17-125) さ れ reveals that 25-109 amino acid residues of MDM2 are p53-binding domains (Kussie, PH, etc. 1996) Science, 274, 948-953). This time, MDM2 containing 7-300 amino acid residues containing this region was fused with a tag of calmodulin-binding peptide (CBP), TEV protease recognition sequence, and protein A IgG binding domain (ZZ) (Fig. 1). ) Koji protein was synthesized by cell-free translation system derived from wheat germ.

As shown in FIG. 2, it was confirmed that this protein bound to the IgG-immobilized beads via the IgG binding domain, and was cleaved and eluted by the TEV protease (FIG. 3, A). When the amount of protein in each fraction was corrected from the amount of electrophoresis, 40% of the input was immobilized on the beads (Fig. 3, B), and the remaining 60% of the protein was not immobilized on the beads or washed. Presumed to have been removed. After treatment with TEV protease, 45% of the protein immobilized on the bead is eluted, and the rest is not cleaved, or after cleaving, it binds nonspecifically to the bead outside the IgG binding domain. Therefore, it is estimated that it is not eluted.

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. It was suggested that the prepared bait protein MDM2 has p53-binding activity due to binding of the full-length p53 IVV. In addition, 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) α has an α-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.

2. 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.

In addition, using single-stranded DNA, X12 (FWL) -r (Table 1) テ ン プ レ ー ト as a template, Phusion DNA polymerase, 5'O29-T7-EcoRI and 3'Flag1A-lib primerA (Table 1) Second, 62 ° C. for 30 seconds, and 72 ° C. for 30 seconds. The obtained PCR product was purified by PCR purification kit, and DNA encoding a sequence in which 3 amino acids out of 12 amino acid residues were fixed and the remaining 9 amino acid residues were random was prepared.

1 μg of these DNAs were added using RiboMAX large-scale RNA production systems-SP6 at 37 ° C. for 2 hours, and then DNase I was added and further incubated at 37 ° C. for 15 minutes. The resulting mRNA is purified with RNeasy mini kit, and then incubated with T4 RNA Ligase (Takara) 14 for 14 hours at 16 ° C. Prepared. This was incubated at 25 ° C. for 2 hours using a wheat germ-derived cell-free translation system Wheat Germ extract systems to obtain an IVV library.

3. Preparation of IgG beads 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. Thereafter, 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).

4. IVV screening Wash 40 μl of IgG-Immutex-MAG beads 3 times with binding buffer IPP150 (1 M Tris-HCl pH 8.0, 150 mM NaCl, 0.1% NP-40), and bait MDM2 (7-300 amino acids) Residue region) -CBPzz was added, and the mixture was fixed by rotation at 4 ° C. for 1 hour. Thereafter, the plate was again washed 3 times with IPP150, and the IVV library was added and mixed by rotating at 4 ° C. for 10 minutes (5 minutes after the third round). After washing with IPP150 8 times (13 times after the 3rd round) and TEV buffer (1 M Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% NP-40, 1 mM DTT, 5 mM EDTA) twice , TEV protease (10 U / μl) (Invitrogen) 2 μl was mixed, and mixed by rotation at 16 ° C. for 2 hours. This supernatant was collected as an elution fraction.

Using 1 溶出 μl of the collected elution fraction as a template, with 5′O29-f and 3′Flag1A primers in Onestep 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. After completion of the 5th 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.

5. Identification of high-affinity MDM2-binding peptides In accordance with the above screening method, a two-step screening was performed to identify the optimal peptide sequence in terms of strong binding to MDM2. First, a library with many types of sequences was screened, and some of the amino acid residues that are highly conserved in the obtained sequences were determined as residues that are important for binding to MDM2. Subsequently, a random library in which these amino acid residues were fixed was designed, and a peptide sequence that binds to MDM2 more strongly was identified by performing a second screening.

First, a random peptide IVV library consisting of 16 amino acid residues was screened. Cloning and sequence analysis of the DNA library after 4 rounds of screening resulted in the duplication of 3 types of sequences (Table 2). IVV of these peptides bound to MDM2, and binding was observed even in the case where GFP was fused to the N-terminus (FIG. 5A) (FIG. 5, B). Since the wild-type p53 peptide fragment fused with GFP did not bind to MDM2, it can be said that a screening system capable of obtaining a peptide sequence with higher affinity for MDM2 could be constructed. In most of the 16 amino acid residue peptide sequences obtained by this screening, three hydrophobic amino acids corresponding to F19, W23, and L26 of p53 were conserved. These hydrophobic amino acids are known to be important when p53 binds to MDM2 (Kussie, PH, etc. (1996) Science, 274, 948-953; Fukuda, I., etc. (2006) Nucleic Acids Res., 34, e 127; Blaydes, JP, (1997) Oncogene, 等 14, 1859-1868), based on this result, it was suggested that the amino acid cannot be substituted for other amino acids. .

The number of possible combinations of random peptide sequences of 16 amino acid residues was 20 ¹⁶ = 6.6 × 10 ²⁰ , which was not covered by the library size (˜10 ¹³ ) of the IVV library. Therefore, in order to optimize these sequences, the above-mentioned three hydrophobic amino acids (F, W, L) were fixed, and an IVV library of 12 amino acid residues in which 9 amino acid residues were random was screened. This IVV library can sufficiently cover all the sequences of 20 ⁹ = 5.1 × 10 ¹¹ types.

After screening this library for a total of 4 rounds, the sequence was analyzed. As a result, no duplicate sequence was present. Among the sequences obtained, some IVVs bound to MDM2, indicating that the number of amino acid residues was reduced from 16 to 12, so that the binding molecules were not lost. Probably, by fixing three hydrophobic amino acids, there were many molecules with high affinity for MDM2 in the initial library, and because the selection pressure used was not appropriate, It is presumed that no concentration was observed.

Therefore, using the library after the end of the 4th round, there will be many molecules that bind to MDM2, (1) The binding time between the library and MDM2 was reduced from 1-2 hours to 10 minutes, (2) The number of washings after binding was increased from 3 to 10 times, and (3) the salt concentration in the binding buffer was doubled, and the binding assay was performed under three types of conditions. Examined (Figure 6). Conditions for shortening the binding time and increasing the number of washings when the amount of binding of the library to MDM2 was reduced were adopted as stricter selection pressures.

When a random library consisting of 12 amino acid residues was screened again at the above-mentioned selection pressure, concentration of binding molecules was observed after the completion of 3 rounds (FIG. 7). As a result of judging that the binding molecules were sufficiently concentrated at the end of the fifth round and cloning the DNA in this library and conducting sequence analysis, seven types of sequences were duplicated (Table 3).

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. As a result, 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 α-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.

The remaining 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).

It was confirmed that the MIP IVV and GFP fused to the N-terminus (hereinafter referred to as GFP-MIP) were bound to MDM2 in a test tube (FIG. 9, A). Furthermore, in order to confirm that GFP-MIP also binds to MDM2 in the cell, GFP-MIP is expressed in human colon cancer cells and HCT116 cells that are known to express wild-type p53. As a result of expression and immunoprecipitation using an α-GFP antibody, it was found that GFP-MIP binds to MDM2 (FIG. 9, B). Two bands of MDM2 were detected, but the smaller molecular weight band is assumed to be one of the splice variants of MDM2 (Bartel, F., 等 (2004) Mol. Cancer Res., 2, 29-35).

6. Mass expression and purification of biotinylated MDM2 BL21 star (DE) was transformed with plasmid DNA, MDM2-Bio-H10, plated on LB agar plate containing 50 μg / ml ampicillin and incubated at 37 ° C. for 16 hours. The formed colonies were inoculated into 5 ml of LB medium containing 50 μg / ml carbenicillin and cultured at 37 ° C. for 16 hours. The whole amount of the cultured E. coli was added to 250 ml of TB medium containing 50 μg / ml carbenicillin and cultured until OD ₆₀₀ was 0.4 to 0.5. Thereafter, 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. To this, 20 ml of lysis buffer (TBS, pH 7.4, 1 mM β-ME), 40 μl of protease inhibitor cocktail (Sigma) and 8 μl 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 β-ME, 10 mM mMimidazole), elute with 5 ml of elution buffer (TBS, pH 7.4, 1 mM mM β-ME, 250 mM mMimidazole). did.

7. By measuring the Biacore3000 (Biacore) dissociation constant measurements synthetic peptide dissociation constant of the peptide by surface plasmon resonance spectroscopy _{(K D) (K D)} , was performed at 25 ° C. with HBS-EP buffer. First, purified biotinylated MDM2 was immobilized on sensor chip SA at 4300 RU. Using this sensor chip, three different concentrations (2-200 nM) of synthesized peptides were injected to determine the dissociation constant. The injection flow rate was 20 μl / min, and the binding and dissociation times were 3 minutes each. Thereafter, the surface of the sensor chip was regenerated with 5 μl of Glycine 2.0 (Biacore). The binding data was analyzed using the 'Steady state affinty' model of BIAevaluation software ver. 4.1 (Biacore).

The 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. At the same time, the affinity of 6 kinds of peptides including these 3 hydrophobic amino acids in which 6 amino acids in the MIP sequence were substituted with Ala to MDM2 was also measured. As a result, the frequency of appearance was high among the clones obtained by screening (Fig. 8) By disposing Arg9 and Met11 to Ala, the dissociation constant value for MDM2 increased to about 50 times and 900 times that of MIP, respectively. . In addition, regarding Tyr6 (Bottger, V., et al. (1996) Oncogene, 13, 2141-2147), which is an existing motif, and Pla3, Trp7, and Leu10 corresponding to the three hydrophobic amino acids described above, Ala-substituted peptides, respectively. However, a significant increase in the dissociation constant was observed.

In the present invention, a 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. In addition, when 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. In particular, 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.

8. Preparation of 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. By cloning the resulting cassette into the HindIII / EcoRI site of pQBI25-HL4, where the peptide linker of the pQBI25f expression vector was replaced with the HL4 linker using T4 DNA Ligase, the original HindIII and EcoRI sites were removed and the insert side further In addition, EcoRI and HindIII sites were newly created. The thus obtained plasmid pQBI25-HL4-MIP was expressed with a wheat germ-derived cell-free transcription translation system TNT Coupled Wheat Germ extract systems (Promega).

9. 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%. Cultured in DMEM medium (Gibco) containing% (vol / vol) FBS and 1% penicillin / streptomycin, and Saos-2 cells were 15% (vol / vol) FBS (Gibco), 1% (vol / vol) The cells were cultured in McCoy's 5A medium (Gibco) containing penicillin / streptomycin (Gibco). Using Lipofectamine 2000 (Invitrogen) to these cells, transfection of plasmid pQBI25-HL4-MIP and plasmid DNA encoding a protein with a FLAG tag fused to the C-terminus of GFP, pQBI25-HL4-FLAG, respectively, as per the protocol. went.

According to the above method, we investigated the activation of the p53 pathway by MDM2-binding peptides. It has been shown that inhibition of MDM2-p53 interaction and suppression of MDM2 expression lead to p53 stabilization and p53 pathway activation. (Shangary, S., etc. (1997) Proc. Natl. Acad. Sci. USA., 105, 3933-3938; Hu, B., 等 (2007) Cancer Res., 67, 8810-8817; Chene, P. (2000) J. Mol. Biol., 299, 245-253; Blaydes, JP, et al. (1997) Oncogene, 14, 1859-1868; Chen, L., et al. (1997) Proc. Natl. Acad. Sci. USA., 95, 195-200; Vassilev, LT, 等 (2004) Science, 303, ence844-848). When GFP-MIP was expressed in HCT116 cells, an increase in the expression level of p53 protein, an increase in the amount of mRNA in the downstream gene M (MDM2, p21) お よ び, and induction of protein expression were observed (FIGS. 10 and 11). On the other hand, when only GFP as a control was expressed, similar results were not obtained. Therefore, these results suggest that p53 was activated and translocated to the nucleus due to inhibition of the MDM2-p53 interaction by GFP-MIP and transcriptionally activated its own targets MDM2 and p21. In addition, the amount of p53 mRNA did not change compared to the control. 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.

10. Immunoprecipitation HCT116 cells 24 hours after transfection were solubilized with 500 μl 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 μl of Agarose conjugated Anti-GFP (Medical & biological laboratories) was added to 400 μl 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.

11. Western blotting Cells were collected with 1 × sample buffer and separated by 10% SDS-PAGE, followed by Western blotting. The antibodies used were anti-p53 antibody (Cell signaling), anti-MDM2 antibody (SMP14, Santa cruz), anti-p21 antibody (SX118, BD Pharmingen), and anti-β-actin antibody (AC-15, Sigma). As the secondary antibody, an HRP-labeled antibody was used and detected by ECL chemi-luminescence reagents (Amersham). An anti-T7 tag antibody (Novagen) was used to detect the bait protein MDM2 immobilized on the beads. The protein amount quantification from the band was performed by Image J (http://rsbweb.nih.gov/ij/).

12. Real-time RT-PCR
RNA extraction from the cells was performed according to the protocol of RNeasy mini kit. Using this RNA as a template, mRNA 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.

12. 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 μl / 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).

It is known that when the p53 pathway is activated in a cell, the cell causes cell responses such as cell cycle arrest and apoptosis. MIP peptide (Tat-MIP) し た with HIV-derived Tat sequence fused to the N-terminus as a cell membrane permeation sequence, so that wild-type p53-expressing cell HCT116 survives at a lower Tat-MIP concentration than p53-deficient cell Saos-2 The wrinkles with reduced rates (Figure 12). Moreover, the survival rate of HCT116 cells did not decrease with the MIP peptide not fused with the Tat sequence. These results suggest that Tat-MIP is taken up into cells and induces p53 pathway-dependent cell death.

Like HCT116 cells, wild-type p53-expressing SJSA-1 cells and p53-deficient Saos-2 cells were treated with Nutlin-3, resulting in IC ₅₀ of 1.8 μM and 20 μM, 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 μM in HCT116 cells and 19.3 μM in Saos-2 cells, which was about 1.5 times as high. This result indicates that 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).

<Description of sequence>
1: 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-T7-EcoRI
19: Primer 5'O29-f
20: Primer 3'Flag1A
21: Primer GFP-fus-MIPf
22: Primer GFP-fus-MIPr
23: Primer p53F
24: Primer p53R
25: Primer Mdm2F
26: Primer Mdm2R
27: Primer p21F
28: Primer p21R
29: Clone X16-1
30: Clone X16-2
31: Clone X16-3
32: Clone X16-4
33: Clone X16-5
34: Clone X16-6
35: Clone X16-7
36: p53 _13-28
37: Clone X12-1, MIP
38: Clone X12-2
39: Clone X12-3
40: Clone X12-4
41: Clone X12-5
42: p53 _17-28
43: MIP variant F3A
44: MIP variant Y6A
45: MIP variant W7A
46: MIP variant R9A
47: MIP variant L10A
48: MIP variant M11A
49: PDI, a peptide consisting of 12 amino acid residues corresponding to the 17th to 28th region of p53
50: Tat-MIP

According to 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.

Claims (6)

1. A human cancer protein MDM2-human cancer suppressor protein p53 interaction inhibitor comprising a peptide comprising 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.
2. The inhibitor according to claim 1, wherein the peptide consists of the amino acid sequence of any one of SEQ ID NOs: 37 to 41.
3. The inhibitor according to claim 1, wherein the peptide consists of the amino acid sequence of SEQ ID NO: 37.
4. The anticancer agent which uses the peptide containing the following amino acid sequences 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.
5. The anticancer agent according to claim 4, wherein the peptide consists of any one of the amino acid sequences of SEQ ID NOs: 37 to 41.
6. The anticancer agent according to claim 4, wherein the peptide consists of the amino acid sequence of SEQ ID NO: 37.

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