JPWO2006062173A1 - Bcl-xL heterodimer-inhibiting peptide that promotes anticancer drug action and screening method thereof - Google Patents

Abstract

A peptide having an amino acid sequence of RXXX (X is a hydrophobic amino acid), consisting of 8 to 40 amino acid residues, and the number of basic amino acid residues contained in the amino acid sequence is 5 or less, or an amino acid of YQVARMLRRVADQMAS A drug that promotes anticancer drug treatment by inhibiting heterodimer formation between Bcl-xL and Bak or Bad, which comprises a peptide comprising a sequence as an active ingredient.

Description

The present invention relates to a Bcl-x _L heterodimer-inhibiting peptide that promotes anticancer drug action and a screening method thereof.

Chemotherapy and radiation therapy are used as anticancer treatments. These treatments cause cancer cells to die and die. However, in cancer cells overexpressing apoptosis-suppressing proteins such as Bcl-2 and Bcl-x _L , these proteins inhibit apoptosis and reduce the efficiency of anticancer treatment. Therefore, a drug that inhibits the function of Bcl-2 or Bcl-x _L is expected to have a function of promoting anticancer treatment (Non-patent Document 1). For example, an example in which the effect of an anticancer agent is increased by suppressing the expression of Bcl-2 with an antisense oligonucleotide targeting the Bcl-2 gene is known (Non-patent Document 2).

Bcl-2 and Bcl-x _L form heterodimers with Bak, Bax, Bad, and Bad, which are pro-apoptotic proteins, and eliminate these pro-apoptotic actions. Therefore, it can be inferred that heterodimer formation may be inhibited in order to eliminate the apoptosis-suppressing action of Bcl-2 or Bcl-x _L. These heterodimers are formed by the BH3 domains of Bak, Bax, Bad, and Bad intercalating into the hydrophobic pockets formed by the BH1, BH2, and BH3 domains of Bcl-2 and Bcl-x _L. . A peptide consisting of about 16 residues extracted from the BH3 domain of Bak, Bax, Bad, and Bad can bind to the hydrophobic pockets of Bcl-2 and Bcl-x _L. These peptides competitively inhibit the formation of heterodimers and show pro-apoptotic effects. Thus, these peptides have the potential to facilitate anticancer therapy.

Small molecule compounds have been reported that bind to the hydrophobic pockets of Bcl-2 and Bcl-x _L. They are antimycin A, BH3I-1, and BH3I-2 (Non-patent Document 3). The affinity of the above-mentioned peptides for Bcl-2 and Bcl-x _L is about 10 ^-7 M in terms of dissociation constant, whereas the binding of these small molecule compounds is about 10 ^-6 M, Affinity is weak to use as a drug.
Huang, Oncogene (2000), 19, 6627-6631 Jansen et al., Nature Medicine (1998), 4, 232-234 Zheng, Nature Cell Biology (2001) 3, E1-E3

  An object of this invention is to provide the chemical | medical agent which promotes anticancer agent treatment by inhibiting formation of a heterodimer.

Since non-patent is described in Reference 3, the structure of compounds that bind to the hydrophobic pocket of Bcl-2 and Bcl-x _L are not similar, there is a possibility that the binding site is slightly offset. In such a case, it is important to analyze the structure of a complex in which a small molecule compound and Bcl-2 or Bcl-x _L are bound to accurately grasp the binding mode. This is because it is considered that a drug that binds to Bcl-2 or Bcl-x _L with higher affinity can be developed by designing a compound that binds in a manner that combines two different binding modes.

Based on this background, we attempted to obtain peptides that bind to Bcl-x _L using the in vitro virus method. As a result, several novel peptides that bind to Bcl-x _L competitively with the Bad protein were obtained. These peptides are different in sequence from naturally occurring peptides derived from the BH3 domain, and are presumed to have different binding modes to Bcl-x _L. Based on these findings, the present invention has been completed. The present invention provides the following.

1. A peptide having an amino acid sequence of RXXX (X is a hydrophobic amino acid) (SEQ ID NO: 48), consisting of 8 to 40 amino acid residues, wherein the number of basic amino acid residues contained in the amino acid sequence is 5 or less Bcl-x _L heterodimer formation inhibitor as an active ingredient.

2. Bcl-x _L heterodimer formation inhibitor which uses as an active ingredient the peptide which consists of one of the amino acid sequences of sequence number 4, 5, 6, 7, 9, 10, 11, 12 and 16.

3. Bcl-x _L heterodimer formation inhibitor which uses the peptide which consists of an amino acid sequence of sequence number 18 as an active ingredient.

  4). A peptide having an amino acid sequence of RXXX (X is a hydrophobic amino acid) (SEQ ID NO: 48), consisting of 8 to 40 amino acid residues, wherein the number of basic amino acid residues contained in the amino acid sequence is 5 or less Anticancer agent action promoter as an active ingredient.

  5. An anticancer agent action promoter comprising, as an active ingredient, a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 4, 5, 6, 7, 9, 10, 11, 12, and 16.

  6). An anticancer agent action promoting agent comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 18 as an active ingredient.

7). (1) Prepare a library of DNA encoding the peptide, (2) Prepare a library of molecules in which the DNA and the peptide encoded by the DNA are bound, (3) Bcl-x Select a molecule containing a peptide that binds to _L , (4) amplify the DNA by PCR using the DNA of the selected molecule as a template, (5) use the amplified DNA as a library in step (2), A method for screening a peptide that binds to Bcl-x _L , comprising repeating steps (2) to (4), wherein a library of DNA is (NNS) n (N is A, T, G And an equivalent mixture of C, S is an equivalent mixture of G and C, and n is an integer of 8 to 24).

  8). The library of DNA is XXVXRXLXXXXDXIXX (where X is an amino acid encoded by NNS (N is an equal mixture of A, T, G and C, S is an equal mixture of G and C)) (SEQ ID NO: 29) 8. The method according to 7, comprising DNA encoding a peptide having an amino acid sequence.

Schematic diagram of NNS library NNS library nucleotide sequence How to build an NNS library Bak library description In-vitro selection experiment Construction of DHFR fusion peptide Binding experiment of acquired peptide Pull-down assay experiment method Pull-down assay results (electrophoresis photo) Results of competition experiments on Bcl-x _L binding between Bad and the acquired peptide (electrophoresis photo) Results of binding experiment between GST-Bcl-x _L and fluorescent Bak peptide Results of competition experiments on Bcl-x _L binding between Bak peptide and acquired peptide

  In this specification, the one-letter symbols for bases and amino acids are those according to WIPO Standard ST.25.

  The active ingredient of the inhibitor and facilitator of the present invention has an amino acid sequence of RXXX (X is a hydrophobic amino acid) (SEQ ID NO: 48), consists of 8 to 40 amino acid residues, and is a base contained in the amino acid sequence It is a peptide having 5 or less residues of sex amino acids.

The hydrophobic amino acid is an amino acid having a hydrophobic side chain and includes phenylalanine, tryptophan, tyrosine, cysteine, methionine, alanine, valine, isoleucine, leucine, proline and the like.
The basic amino acid refers to arginine, lysine and the like. The number of basic amino acid residues is 5 or less, preferably 4 or less. In addition, the number of basic amino acid residues is preferably 25% or less of all residues constituting the peptide. When many basic amino acid content, will repel a positively charged moiety of the Bcl-x _L, it may not be possible to bind the Bcl-x _L.

Examples of the peptide include peptides consisting of any one of the amino acid sequences of SEQ ID NOs: 4, 5, 6, 7, 9, 10, 11, 12, and 16.
The peptide is preferably soluble in a biological fluid such as blood.

  The active ingredient of the inhibitor and facilitator of the present invention is also a peptide having the amino acid sequence of SEQ ID NO: 18.

  The peptide of the active ingredient 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.

The present invention also provides a method for screening a peptide that binds to Bcl-x _L , that is, (1) preparing a library of DNAs encoding the peptides, and (2) encoding DNA and DNA from the library. Prepare a library of molecules bound to the peptides to be synthesized, (3) select molecules containing peptides that bind to Bcl-x _L , and (4) amplify DNA by PCR using the DNA of the selected molecules as a template And (5) using the amplified DNA as a library in step (2) and providing a method for screening peptides that bind to Bcl-x _L , including repeating steps (2) to (4) To do.

In the method of the present invention, except that Bcl-x _L is used as a target substance and a DNA library having a specific structure is used, a screening method based on the in vitro virus method (for example, WO 02/48347, Japanese Patent Laid-Open No. 2002-176987). In vitro virus and screening method will be explained.

  In vitro virus is also referred to as a mapping molecule, and a mapping molecule by the in vitro virus method is a phenotype molecule containing a protein to be subjected to functional analysis or functional modification, and a genotype containing a nucleic acid encoding the protein. Combining with molecules. 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, a part derived from a phenotype molecule, a part derived from a spacer molecule, and a part derived from a coding molecule in the mapping molecule are referred to as 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 1-15, preferably 1-2. The nucleotide may be ribonucleotide or deoxyribonucleotide.

  The sequence at the 5 ′ end of the donor region affects the 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.

  The 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, and 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). 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 (PANS-amino acid) For example, PANS-Gly whose amino acid part is 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'-Aminoacyladenosine aminonucleoside (AANS-amino acid, 3'-Aminoacyladenosine aminonucleoside, formed by dehydration condensation of the amino group of 3'-aminoadenosine and the carboxyl group of amino acid as a chemical bond For example, AANS-Gly having an amino acid part of 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 residues of deoxyribonucleotide or ribonucleotide. 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 sequence is dC-puromycin, rC-puromycin, etc., more preferably dCdC-puromycin, rCrC-puromycin, rCdC-puromycin, dCrC-puromycin, etc., and the 3 ′ end of aminoacyl-tRNA is A mimicked CCA sequence (Philipps, GR (1969) Nature 223, 374-377) is 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, as a function modifying substance, a substance into which various separation tags such as a fluorescent substance, biotin, or His-tag are introduced 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 a 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 affecting the translation efficiency of the coding molecule include a 5 ′ UTR consisting of a transcription promoter and a translation enhancer, and a combination of a 3 ′ terminal region 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 may be part of the TMV omega sequence (O29; Gallie DR, Walbot V. (1992) Nucleic Acids Res., Vol. 20, 4631 -4638 and 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 + _An (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 ′ terminal 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 adding polyethylene glycol having the same molecular weight as the polyethylene glycol in the PEG region of the spacer molecule containing the PEG region, to the reaction system It is also possible to shorten to 0.5 to 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 problem 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.

  By adding polyethylene glycol having the same molecular weight as the PEG region of the spacer part including the PEG region during the ligation reaction, 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. Further, the nucleic acid portion of the mapping molecule of this embodiment can be made into a hybrid of RNA and DNA by reverse transcription after translation.

  The screening method based on the in vitro virus method is usually a method for screening a nucleic acid encoding a protein that interacts with a target substance from a nucleic acid library, and from the nucleic acid library by the production method of the present invention. A step of producing a library of mapping molecules, a step of mixing the library of mapping molecules and the target substance, a step of separating the mapping molecules bound to the target substance, and 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 of the nucleic acid, 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.

  Cleaving the separated linker of the mapping molecule under conditions that do not change the base sequence of the nucleic acid and liberating the nucleic acid can be carried out using a cleavage linker under the conditions corresponding to it. 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.

  The recovered 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 library of DNA used in the screening method of the present invention is (NNS) n (N is an equal mixture of A, T, G and C, S is an equal mixture of G and C, and n is 8-24. (Integer) DNA having a base sequence. The DNA library is preferably XXVXRXLXXXXDXIXX (where X is an amino acid encoded by NNS (N is an equal mixture of A, T, G and C, S is an equal mixture of G and C)) (SEQ ID NO: 29) It consists of DNA encoding the peptide consisting of the amino acid sequence.

It becomes an active ingredient of the inhibitor or facilitator of the present invention by measuring whether or not the peptide that binds to Bcl-x _L screened by the above method competes with Bak or Bad protein. Peptides can be identified. 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.

-Construction of DNA libraries Two types of DNA libraries were constructed. One is the NNS library and the other is the Bak library. The characteristics and construction methods of these DNA libraries are described below.

・ NNS library ・ Features of NNS library
N is an equal mixture of four types of nucleotides A, T, G, and C. S is a mixture of two equal amounts of G and C nucleotides. There are 32 codons that can be specified by NNS, and all 20 amino acids can be encoded. Of the 32 codons, there is one stop codon. Therefore, the probability of appearance of a stop codon is 1/32. In the case of NNN, the probability of appearance of a stop codon is 3/64, so the probability of appearance of a stop codon is lower in NNS. For example, in the case of an NNS library that encodes a peptide consisting of 16 amino acid residues, the probability of obtaining a full-length DNA molecule that does not contain a stop codon is 60%. On the other hand, it is 46% for NNN library.

・ NNS library structure
The NNS library was applied to both the in vitro virus method using a wheat germ cell-free translation system and the in vitro virus method using PURESYSTEM (Post Genome Research Institute, Inc.), which is an E. coli cell-free translation system. The full length of the NNS library for wheat germ cell-free translation system consists of Sp6 promoter, Ω29 sequence, start codon, glycine / serine linker, NNS library, flag tag and Ax6 sequence. On the other hand, the full length of the NURE library for PURESYSTEM consists of T7 promoter, SD sequence, start codon, glycine / serine linker, NNS library, flag tag and Ax6 sequence. A schematic diagram of these sequences is shown in FIG. 1, and a base sequence is shown in FIG. The NNS library contains equimolar numbers of 8 amino acids (NNS) ₈ , 16 amino acids (NNS) ₁₆ , and 24 amino acids (NNS) ₂₄ .

-Construction of NNS library Fig. 3 shows the construction method of NNS library for wheat germ cell-free translation system. First, the negative strands of DNA (G4SG4S (NNS) 8FLAGA6r, G4SG4S (NNS) 16FLAGA6r, G4SG4S (NNS) 2FLAGA6r) encoding the glycine / serine linker, NNS library, flag tag and A x 6 sequence were designed and Purchased from the company. The sequence is 5′-ttt ttt ctt atc gtc gtc atc ttt gta gtc (snn) _{8, 16, 24} tga gcc tcc gcc tcc tga acc gcc gcc acc-3 ′ (SEQ ID NO: 31, 32, 33). Next, a plus-strand DNA (priSP6OGf) containing the Sp6 promoter, Ω29 sequence, initiation codon and part of the glycine / serine linker was designed and purchased from Fasmac. The sequence is 5′-att tag gtg aca cta tag aac aac aac aac aac aaa caa caa caa aat ggg tgg cgg cgg tt-3 ′ (SEQ ID NO: 34). In addition, a negative strand (priFLAGA6r) of DNA encoding a flag tag and an Ax6 sequence was designed and purchased from Date Concept. The sequence is 5′-ttt ttt ctt atc gtc gtc atc ttt gta gtc-3 ′ (SEQ ID NO: 35). Perform PCR using G4SG4S (NNS) 8 [FLAG] A6r, G4SG4S (NNS) 16 [FLAG] A6r, G4SG4S (NNS) 24 [FLAG] A6r as template DNA, priSP6OGf and priFLAGA6r as primers A DNA library was obtained. The PCR reaction solution (100 μl) consists of 2 units of KOD DNA polymerase, 200 μM dNTP, 2 mM MgSO ₄ , 3 pmol forward and reverse primers, 1 pmol template DNA, and accompanying buffer. The reaction conditions are 1st stage (1 cycle) 94 ° C / 5 minutes, 2nd stage (15 cycles) 94 ° C / 30 seconds, 58 ° C / 30 seconds, 68 ° C / 30 seconds, 3rd stage (1 cycle) 68 It consists of ℃ / 5 minutes.

  The method of building the NNS library for PURESYSTEM is the same. However, instead of priSP6OGf, priUniv2 containing a T7 promoter sequence, SD sequence, start codon, and part of the glycine / serine linker was designed and used as a forward primer. This DNA was purchased from Fasmac. The base sequence is 5′-gaa att aat acg act cac tat agg gag acc aca acg gtt tcc ctc tag aaa taa ttt tgt tta act tta aga agg aga tat acc aat ggg tg-3 ′ (SEQ ID NO: 36).

・ Bak library ・ Features of Bak library
Bak is a member of the Bcl-2 family protein and forms a heterodimer with Bcl-x _L. A peptide consisting of 16 amino acids extracted from the BH3 domain of the Bak protein binds to Bcl-x _L with a dissociation constant of 340 nM. Some amino acids within the BH3 domain are conserved among various proteins within the family. These amino acids are assumed to be essential for binding to Bcl-x _L. Therefore, a library was constructed in which these 5-residue amino acids were fixed and the remaining 11-residue amino acids were NNS, and named Bak library. It is expected that peptides that bind to Bcl-x _L will be obtained from this library at a relatively high frequency.

・ Bak library structure
FIG. 4 (1) shows a comparison of the amino acid sequence of the 16-amino acid residue peptide derived from the Bak protein and the Bak library used in this study. The fixed amino acids are 3rd valine, 5th arginine, 7th leucine, 12th aspartic acid, and 14th isoleucine.

  The Bak library was applied only to the in vitro virus method using PURESYSTEM. The schematic diagram and base sequence of the full length are shown in FIGS. 4 (2) and 4 (3).

・ Bak library construction
The Bak library was constructed in the same way as the NNS library for PURESYSTEM. However, the template DNA used is a glycine / serine linker, a Bak library, a flag tag, and a negative strand of DNA encoding an Ax6 sequence (G4SG4SBAK16MFLAGA6r). This single-stranded DNA was purchased from Fasmac. Base sequence is 5 '-ttt ttt ctt atc gtc gtc atc ttt gta gtc snn snn aat snn atc snn snn snn snn cag snn gcg snn aac snn snn tga gcc tcc gcc tcc tga acc g' gcc gcc acc-3 ' It is.

・ Preparation of in vitro virus library
A DNA library for cell-free translation system of wheat germ having SP6 promoter was transcribed using RiboMAX Large Scale RNA Production System (Promega) and converted into an RNA library. The reaction solution (50 μl) consists of 5 μg of DNA library, attached reaction buffer, 5 μl of SP6 Enzyme mix, 5 mM substrate (ATP, UTP, CTP), 400 μM GTP, and 2 mM m7G (5 ′) ppp ( 5 ') Including G cap analog (Invitrogen). The reaction was carried out at 37 ° C. for 3 hours. In addition, a DNA library for PURESYSTEM having a T7 promoter was transcribed using a T7 RiboMAX Express Large Scale RNA Production System (Promega) and converted to an RNA library. The reaction solution (25 μl) contains 2.5 μg of the DNA library, attached reaction buffer, and 2.5 μl of Enzyme mix. The reaction was carried out at 37 ° C. for 30 minutes. The template DNA was digested with DNaseI (Promega) and purified using RNeasy Mini Kit (QIAGEN). The method followed the manual attached to the kit. RNA and spacer puromycin (p (dCp) ₂ T (FI) pPEG (2000) p (dCp) ₂ Puro (the symbols are as defined in WO 02/48347)), T4 RNA ligase (Takara Shuzo) And linked according to the method described in Example 1 of WO 02/48347. The ligation reaction was performed at 25 ° C. for 1 hour. The ligation product was purified using RNeasy Mini Kit (QIAGEN). The purified ligation product was added to a cell-free translation system and translated to construct an in vitro virus library. The composition of the cell-free translation reaction solution is 10 μl of wheat germ extract (Promega), 80 μM amino acid mixture, 50 mM potassium acetate, 10 units of RNase inhibitor (when using wheat germ system) Invitrogen), and 2 pmol ligation product. The reaction was carried out at 25 ° C. for 40 minutes. When PURESYSTEM is used, the reaction solution contains attached reaction solutions A and B, and 5 to 10 pmol of ligation product. The reaction was carried out at 37 ° C. for 30 minutes.

・ Preparation of bait
Bcl-x _L was expressed as a glutathione-S-transferase (GST) fusion protein and presented on Glutathione-binding resin via GST for use in selection experiments. Bcl-x _L was obtained from a human brain RNA library (Clontech) by reverse transcription and PCR. 60 μl of the reverse transcription reaction solution was 500 μM dNTP, 0.6 pM primer (priBcl-xLr1, 5′-tac agt ctc gag cta gtt gaa gcg ttc ctg gcc ct-3 ′ (SEQ ID NO: 38), manufactured by Date Concept), 10 mM DTT, 600 units of SuperScript II reverse transcriptase and accompanying reaction buffer (Invitrogen). The reaction was carried out at 37 ° C. for 1 hour. KOD Plus was used for PCR. PCR reaction solution (100 μl) consists of 2 units of KOD DNA polymerase, 200 μM dNTP, 2 mM MgSO ₄ , 3 pmol forward primer (priBcl-xLf1: 5'-agt atc gaa ttc atg tct cag agc aac cgg-3 '(SEQ ID NO: 39), manufactured by DateConcept, and reverse primer (priBcl-xLr1), template DNA, and attached buffer. The obtained PCR product was cloned using the TOPO TA Cloning kit (manufactured by Invitrogen), and the nucleotide sequence was confirmed by a DNA sequencer (CEQ2000, manufactured by Beckman Coulter). Glutathione-S-transferase (GST) fusion protein expression vector (pGEX-4T-1, Amersham Bioscience) digested with EcoRI and XhoI (Toyobo) after digesting the resulting DNA encoding Bcl-x _L Manufactured). Rigafirst (Promega) was used for the ligation reaction. Escherichia coli JM109 strain was transformed with the obtained vector. After liquid culture, the expression vector was recovered. After confirming the base sequence of the structural gene portion of the expression vector by DNA sequencing, the host cell for expression, E. coli BL21 (DE3) pLysS strain, was transformed. Mass expression was achieved by inducing expression with 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (manufactured by Nacalai Tesque) after reaching OD ₆₀₀ = 0.6 and culturing overnight at 25 ° C. The culture scale is usually about 5 mL. The medium contains 50 μg / ml carbenicillin and 30 μg / ml chloramphenicol. The cells recovered from 1.5 ml of the liquid medium were suspended in 200 μl of lysis buffer, and the cells were disrupted by ultrasonic waves. After centrifugation, approximately 200 μl of the soluble fraction was combined with 600 μl of the glutathione binding resin. After extensive washing, 1 ml of TBSTE buffer (Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM EDTA, 0.2% Tween20) was added to equilibrate and store the Bcl-x _L binding resin.

4. Implementation of in-vitro selection experiment Figure 5 shows the outline of the experimental procedure for in-vitro selection experiment. The translation reaction product was purified using Micro Bio-Spin Column P-30 Tris, RNase-Free (manufactured by BIO-RAD). 180 μl of TBSTE buffer was added and centrifuged at 15,000 rpm for 10 minutes. 20 μl slurry of ANTI-FLAG M2-Agarose (manufactured by SIGMA) was added to the supernatant, and the mixture was stirred at 4 ° C. for 1 hour to carry out a binding reaction. After washing 4 times with 200 μl of TBSTE buffer, reverse transcription reaction of mRNA part of in vitro virus molecule bound to resin was performed. 60 μl of the reverse transcription reaction contains 500 μM dNTP, 0.6 pM priFLAGA6r, 10 mM DTT, 600 units SuperScript II reverse transcriptase and the accompanying reaction buffer. The reaction was carried out at 37 ° C. for 1 hour. The reaction solution was purified using Micro Bio-Spin Column P-30 Tris, RNase-Free. At this time, this column was previously replaced with TBSTE buffer or TBSTE2 buffer (Tris-HCl (pH 7.4), 450 mM NaCl, 10 mM EDTA, 0.2% Tween20). Thereafter, TBSTE buffer or TBSTE2 buffer was added to make the total volume of the reaction solution 300 μl. A 15 μl slurry of Bcl-x _L binding resin was mixed with this reaction solution and stirred at 4 ° C. for 1 hour to carry out a binding reaction. Non-specifically adsorbed molecules were removed by washing 4 times with 150 μl of TBSTE buffer or TBSTE2 buffer. The following two methods were performed as a method for recovering the molecules bound to Bcl-x _L. Add 10 units of thrombin (manufactured by Amersham Bioscience), cleave between Bcl-x _L and GST and recover in vitro virus molecules together with Bcl-x _L , or act on 0.1 M KOH to make the protein part Is a method for recovering in vitro virus molecules.

  Libraries synthesized using the wheat germ cell-free translation system were bound and washed with TBSTE buffer, and thrombin was used to recover in vitro virus molecules. The library synthesized with PURESYSTEM was bound and washed with TBSTE2 buffer, and in vitro virus molecules were recovered with KOH. The recovered in vitro virus molecules were concentrated by ethanol precipitation, and the cDNA portion was amplified by PCR. PCR primers include priSP6OGf and priFLAGA6r (library for wheat germ cell-free translation system) and priSDG4Sf (5'-aag gag ata tac caa tgg gtg gcg gcg gtt-3 '(SEQ ID NO: 40), purchased from Date Concept) And priFLAGA6r (PURESYSTEM library) were used. The composition of the reaction solution is the same as in Section 1.2.3. Reaction conditions are 1st stage (1 cycle) 94 ° C / 5 minutes, 2nd stage (20-25 cycles) 94 ° C / 30 seconds, 58 ° C / 30 seconds, 68 ° C / 30 seconds, 3rd stage (1 cycle) ) It consists of 68 ℃ / 5 minutes.

-Amino acid sequence of selected peptide The cDNA portion of the selected in vitro virus molecule was amplified by PCR by the method described above and cloned using TOPO TA Cloning Kit (Invitrogen). After cloning, the base sequence was analyzed.

  In the selection experiment using the NNS library and wheat germ cell-free translation system, the nucleotide sequence was analyzed after 7 rounds of selection experiment. The amino acid sequences of the peptides obtained in this selection experiment are shown in Table 1. Clones 06 and 10 appeared 4 or 5 times, respectively. In particular, the sequences of clones 10 to 13 were highly homologous sequences differing by only one amino acid. In the 13th to 16th sites of clones 3-7 and 9-13, a motif consisting of 3 consecutive hydrophobic amino acids was observed following arginine (R).

  In the selection experiment using NNS library and PURESYSTEM, the nucleotide sequence was analyzed after 5 rounds of selection experiment. Table 2 shows the amino acid sequences of the peptides obtained in this selection experiment. Analysis of 9 clones yielded 3 and 6 clones 16 and 17, respectively. The sequence of clone 16 was identical to that of clone 04 obtained using the wheat germ cell-free translation system.

  In the selection experiment using the Bak library and PURESYSTEM, the base sequence was analyzed after 5 rounds of selection experiment. The amino acid sequences of the peptides obtained in this selection experiment are shown in Table 3. Here, three types of peptides, clones 18, 19, and 20, were obtained. Clone 18 is a sequence that appeared 7 times out of the 9 clones analyzed. In clones 18 and 19, the 14th leucine, which should have been preserved, was mutated to methionine. The 3rd to 11th amino acid sequence of clone 18 was homologous to the 4th to 12th amino acid sequence of clone 17.

・ Confirmation of affinity of selected peptides for Bcl-x _L・ Measurement of recovery rate using real-time PCR method A consensus sequence is required for accurate quantification by real-time PCR method. E. coli dihydrofolate reductase (DHFR) was used as a consensus sequence. The obtained peptide was linked to the C-terminal side of DHFR via a glycine / serine linker (FIG. 6a). A gene encoding this fusion protein was constructed by the procedure shown in FIG. 6b. The gene of DHFR was amplified by PCR from the plasmid attached to the Proteios cell-free translation kit manufactured by Toyobo. The PCR reaction solution (100 μl) consists of 2 units of KOD DNA polymerase, 200 μM dNTP, 2 mM MgSO ₄ , 3 pmol forward and reverse primers, 1 μl template DNA, and accompanying buffer. The forward primer is priSP6ODf, 5'-att tag gtg aca cta tag aac aac aac aac aac aaa caa caa caa aat gat cag tct gat tgc ggc gtt a-3 '(SEQ ID NO: 41) (manufactured by Fasmac), reverse primer is priG4S1-2Dr: 5′-tga gcc tcc gcc tcc tga acc gcc gcc acc ccg ccg ctc cag aat ct-3 ′ (SEQ ID NO: 42) (manufactured by Date Concept). The forward primer encodes the SP6 promoter, Ω29 sequence, start codon, and part of DHFR. The reverse primer encodes a glycine / serine linker and a part of DHFR. The reaction conditions are 1st stage (1 cycle) 94 ° C / 5 minutes, 2nd stage (15 cycles) 94 ° C / 30 seconds, 58 ° C / 30 seconds, 68 ° C / 30 seconds, 3rd stage (1 cycle) 68 It consists of ℃ / 5 minutes.

  The gene of the peptide whose binding ability is to be analyzed is on a cloning vector. This gene was amplified by PCR with priG4S1f, 5'-ggt ggc ggc ggt tca g-3 '(SEQ ID NO: 43) (manufactured by Date Concept) and priFLAGA6r. PCR conditions are the same as above. Since the two DNA fragments amplified here both contain a glycine / serine linker, these fragments were ligated by overlap extension PCR. PsiSP6ODf was used as a forward primer and priFLAGA6r was used as a reverse primer. PCR conditions are the same as above. The genes of the DHFR fusion peptide constructed by this method are clones 03, 04, 06, 07, 08, 09 and 10.

As a positive control, a DHFR fusion peptide of a Bad peptide consisting of 28 amino acid residues known to bind strongly to Bcl-x _L was constructed. Glycine serine linker, plus single strand DNA encoding Bad peptide and flag tag, G4SG4SmBadf, 5'-ggt ggc ggc ggt tca gga ggc gga ggc tca aat ctc tgg gca gcg cag cgc tac ggc cgt gag ctc cga agg atg agc gat gag ttt gag ggt tcc ttc aag gac tac aaa gat gac gac gat aag-3 '(SEQ ID NO: 44) (manufactured by Fasmac Co., Ltd.), chemically synthesized and subjected to PCR using priG4S1f and priFLAGA6r Got. Furthermore, the DHFR part was ligated by overlap extension PCR. PCR conditions are the same as above. The negative control was obtained by amplifying the NNS library before the selection experiment by PCR using priG4S1f and priFLAGA6r, and then ligating with the DHFR part by overlap extension PCR.

Was encoding various peptides constructed DNA was converted into in vitro virus, the experimental procedure shown in FIG. 7, it was examined the ability to bind to Bcl-x _L binding resin. This experimental procedure is the same as the procedure of the selection experiment shown in FIG. 5, except that the number of molecules of in vitro virus mixed into the resin and the number of molecules of in vitro virus recovered from the resin after washing are measured by real-time PCR. Is different.

  Real-time PCR was performed using a Roche LightCycler. The reaction solution was prepared using a light cycler fast start DNA master SYBER Green I (Roche) according to the attached manual. The reaction conditions were the first stage (1 cycle) 94 degrees / 10 minutes, the second stage (40 cycles) 94 degrees / 10 seconds, 60 degrees / 10 seconds, 72 degrees / 10 seconds. A calibration curve was prepared by preparing three types of standard substances with different concentrations by 100 times. PriEDHFRf, 5'-atg atc agt ctg att gcg gcg tta gcg gta-3 '(SEQ ID NO: 45) as forward primer, proEDHFR211r, 5'-gat cgt ccg tac ccg gtt gac tgc t-3' (SEQ ID NO: 45) 46) (both manufactured by Date Concept). These primers amplify a 1 to 211 base pair region of the DHFR gene.

  The recovery rate of the positive control was 100, the recovery rate of the negative control was 1, and the recovery rates of various peptides were normalized. The results are shown in Table 4. Although it was lower than the binding ability of the Bad peptide consisting of 28 amino acids, the peptides of clones 4 and 10 showed particularly high binding ability. By analyzing the mode of binding of these peptides, it can be expected that new peptides will be developed using these peptides as lead compounds.

-Binding experiment using pull-down assay As a clone 16, 17, 18, Bad peptide, Bak peptide, and a negative control, a sequence in which only weak binding ability was observed by binding analysis by real-time PCR (VVQPVSLREWADLWWL (SEQ ID NO: 47 )) Was constructed in vitro virus molecules for PURESYSTEM, and a pull-down assay was attempted.

The experimental procedure of the pull-down assay is shown in FIG. After the translation reaction using PURESYSTEM, the translation reaction solution was purified using a gel filtration spin column in which the buffer solution was replaced with TBSTE2 buffer solution. Approximately 200 μl of TBSTE2 buffer was added to make the total volume 300 μl. A 15 μl slurry of Bcl-x _L binding resin was mixed and subjected to a binding reaction at 4 ° C. for 1 hour, and then washed 4 times with 150 μl of TBSTE2 buffer. Thereafter, 20 μl of electrophoresis buffer was added to the resin, heated at 99 ° C. for 5 minutes, and then applied to 10% / 8 M urea SDS-PAGE. After electrophoresis, in vitro virus molecules bound to the resin were detected using a fluorescence imager (Molecular Imager FX, manufactured by Bio-Rad). As a control experiment, a Bcl-x _L- binding resin was previously treated with thrombin to prepare a resin from which most of the Bcl-x _L was removed, and a similar pull-down assay was performed. The results of these experiments are shown in FIG.

Bad peptide was clearly observed to form virus molecules in vitro. Clear binding was seen in the Bcl-x _L binding resin. Binding was also observed in the resin after thrombin treatment, but the amount was smaller than that bound to the Bcl-x _L binding resin. Therefore, there is a high possibility that the Bad peptide is specifically bound to Bcl-x _L. Binding was observed in the resin after thrombin treatment. One of the reasons may be that Bcl-x _L could not be completely removed by thrombin treatment. In the negative control peptide, the formation of in vitro virus molecules was observed, but no binding was seen on either resin. In the Bak peptide, binding was observed only to the Bcl-x _L binding resin. The above results indicate that this pull-down assay is performed accurately.

Clones 16 and 18 showed clear in vitro virus molecule formation. In particular, the in vitro virus molecule formation efficiency of clone 18 was high. Clear binding was observed for the Bcl-x _L binding resin for all clones. On the other hand, only a slight bond was observed in the resin after the thrombin treatment. Clone 17 showed a band on the high molecular side of the in vitro virus molecule compared to the others. This is probably because this peptide contains a large amount of arginine and exhibits a high isoelectric point (pI = 10). For this peptide, the band was smeared and unclear, but binding was seen in the Bcl-x _L binding resin. The above results suggest that clones 16, 17, and 18 obtained in this selection experiment have Bcl-x _L binding ability. By analyzing the mode of binding of these peptides, it can be expected that new peptides will be developed using these peptides as lead compounds.

- the selected peptides and Bad of Bcl-x _L binding for competition experiments obtained peptide promotes anticancer therapy by binding to the hydrophobic pocket of Bcl-x _L, Ya Bcl-x _L and Bak It is necessary to inhibit heterodimer formation with the Bad protein. This is because Bak and Bad proteins are released by this inhibition of heterodimer formation, and it is presumed to exert a pro-apoptotic action (Zheng, Nature Cell Biology (2000) 3, E1-E3). In this experiment, a competition experiment on Bcl-x _L binding between the peptide obtained by the selection experiment and the Bad peptide was performed, and the binding site of the obtained peptide to Bcl-x _L was verified.

In vitro virus molecules of Bad peptide, clones 16, 17 and 18 were prepared by the same procedure as described above. FIG. 10 shows a comparison of the results of the pull-down assay for Bcl-x _L- binding resin using clones 16, 17 and 18 alone and the results of the pull-down assay performed by mixing with Bad and competing. From this result, it can be seen that the band intensity of the obtained peptide is decreased in the case of competition as compared to the case of alone. This indicates that these acquired peptides and Bad peptides are competitively bound to the common site of Bcl-x _L. Therefore, it is speculated that the peptides described here have the function of inhibiting the heterodimer formation between Bcl-x _L and Bak or Bad protein, restoring the pro-apoptotic action of Bak or Bad protein, and promoting anticancer drug treatment. The

Clone 16 has the same sequence as clone 4 shown in Table 1 except for one residue. In Table 1, in clone 4 to clone 13, a common motif consisting of R, X, X, X (X is a hydrophobic amino acid) (SEQ ID NO: 48) was found on the C-terminal side of the peptide. This suggests that clone 4 to clone 13 bind to Bcl-x _L in a common manner. Since clone 16 that is almost identical to clone 4 binds to Bcl-x _L competitively with Bad peptide, clones 5 to 13 shown in Table 1 also compete with Bad peptide for Bcl-x _L. Can be guessed to be bound to. Therefore, it is speculated that clones 5 to 13 also inhibit the formation of heterodimers between Bcl-x _L and Bad or Bak proteins, restore these pro-apoptotic effects, and promote anticancer treatment.

-Inhibitory activity of obtained peptide for binding of Bak peptide and Bcl-x _{L Using} the peptide obtained by the selection experiment and the Bak peptide, competition experiment for binding to Bcl-x _L was conducted. If the binding between Bak peptide and Bcl-x _L is inhibited by this competition experiment, it can be expected that the obtained peptide inhibits heterodimer formation between Bcl-x _L and Bak or Bad protein. The fluorescently labeled F-Bak peptide, the unmodified Bak peptide, and the peptides corresponding to the obtained clones 10, 16, 17, and 18 were outsourced to Invitrogen. In addition, a FLAG tag is linked to the four types of acquired peptides in order to increase the solubility in an aqueous buffer.

8. 1. Binding of Fluorescent Bak Peptide to GST-Bcl-x _L First, F-Bak binding to GST-Bcl-x _L was analyzed by measuring the degree of fluorescence polarization (BEACON 2000, Invitrogen). . A certain amount of F-Bak and various concentrations of GST-Bcl-x _L were mixed, and the degree of polarization was measured. The A in FIG. 11 shows a B vs L _F plot. The equation (1) was used for curve fitting.

F (x) = {x * (Pmax-Pmin)} / (Kd + x) + Pmin (1)
Here, the unknown coefficients are Pmax, Pmin, and Kd (dissociation constant), and the independent variable is x (GST-Bcl-x _L concentration).

From the curve fitting, the dissociation constant was determined to be 337 ± 32 nM. Since the dissociation constant of Bak peptide binding to Bcl-x _L was reported to be 340 nM, this result was in good agreement with previous literature values.

Next, a Klots plot in which the horizontal axis represents logarithm is shown in FIG. Curve fitting can be performed using equation (1). Since these plots are on a hyperbola and a sigmoid curve, it is presumed that the binding reaction between F-Bak and GST-Bcl-x _L agrees with Clark's theory, which does not involve the cooperative effect.

Next, the result of Scatchard plotting is shown in FIG. On the horizontal axis, the ratio of bound molecules, that is, (P-Pmin) / (Pmax-Pmin) is plotted, and on the vertical axis, the value obtained by dividing this value by the GST-Bcl-x _L concentration is plotted. Plot since riding on a straight line, the binding of F-Bak and GST-Bcl-x _L is 1: can be interpreted as the first reaction. The dissociation constant obtained from the Scatchard plot was 370 nM, which was in good agreement with literature values. From the above results, it can be concluded that this analysis method using F-Bak and GST-Bcl-x _L is a suitable method for verifying the inhibitory activity of the obtained peptide. Similar results were confirmed even in the presence of 2% DMSO (D to F in FIG. 11).

8. 2. Analysis of inhibitory activity of the obtained peptide Inhibition experiments were conducted by mixing a certain amount of F-Bak (186 nM) and GST-Bcl-x _L (400 nM), and then adding various concentrations of the obtained peptide to the reaction mixture. In addition to the measurement, the degree of polarization was measured. If the obtained peptide shows an inhibitory action, F-Bak is released from GST-Bcl-x _L, and as a result, the degree of polarization decreases. The experimental results are shown in FIG. From this figure, clones 10 and 18 were found to have inhibitory activity. Next, curve fitting was performed using equation (2) to obtain IC ₅₀ .

F (x) =-{x * (Pmax-Pmin)} / (Kd + x) + Pmax (2)
Here, the unknown coefficients are Pmax, Pmin, and IC ₅₀ , and the independent variable is x (peptide concentration).

In this condition IC ₅₀ of Bak peptide was 29.7μM (PBS buffer) and 11.8μM (PBS buffer containing 2% DMSO). In contrast, the IC _{50 of} clone 18 peptide was 6.4 μM (in PBS buffer), while the IC _{50 of} clone 10 peptide was 0.9 μM (in PBS buffer containing 2% DMSO). It can be inferred that both have higher affinity for GST-Bcl-x _L than Bak peptide under the same conditions. In addition, clone 17 peptide did not show such inhibitory activity. Although some inhibitory activity was observed in the clone 16 peptide, the solubility was extremely low, analysis at a high concentration side was difficult, and IC ₅₀ could not be obtained.

Summarizing the above analysis results, it can be inferred that the peptides of clones 10 and 18 have the function of inhibiting heterodimer formation of Bcl-x _L and Bak and Bad proteins. Although clone 16 has low solubility, it can be presumed that it still has inhibitory activity. Although clone 17 showed an inhibitory activity from the pull-down assay in the state of in vitro virus molecule, such activity was not observed at the peptide level. The reason is considered as follows. Peptide clones 17, many basic amino acid content, will repel a positively charged moiety of the Bcl-x _L, it can not bind to Bcl-x _L. However, in the in vitro virus molecule state, the positive charge of the peptide is shielded by the phosphate group of the nucleic acid moiety, and it can bind without repulsion with Bcl-x _L.

Mode of inhibition acquisition peptide as described above is attached to a hydrophobic pocket of Bcl-x _L, there is a possibility that directly inhibits the binding of Bak and Bad peptide, to other parts of the Bcl-x _L By binding, it is possible that the structure of Bcl-x _L is changed, thereby inhibiting the binding of Bak and Bad peptides.

Industrial applicability

According to the present invention, it is possible to inhibit the formation of a heterodimer between Bcl-x _L and Bak or Bad protein, restore the apoptosis promoting action of Bak or Bad protein, and promote anticancer drug treatment. Moreover, if the three-dimensional structure of the complex of the active ingredient peptide and Bcl-x _L is analyzed, it becomes possible to design a new drug using these peptides as lead compounds. Moreover, there is a possibility that a drug with higher affinity can be designed by combining a partial binding mode with that of an existing peptide or small molecule compound.

Claims (8)

A peptide having an amino acid sequence of RXXX (X is a hydrophobic amino acid) (SEQ ID NO: 48), consisting of 8 to 40 amino acid residues, wherein the number of basic amino acid residues contained in the amino acid sequence is 5 or less Bcl-x _L heterodimer formation inhibitor as an active ingredient. Bcl-x _L heterodimer formation inhibitor which uses as an active ingredient the peptide which consists of one of the amino acid sequences of sequence number 4, 5, 6, 7, 9, 10, 11, 12 and 16. Bcl-x _L heterodimer formation inhibitor which uses the peptide which consists of an amino acid sequence of sequence number 18 as an active ingredient. A peptide having an amino acid sequence of RXXX (X is a hydrophobic amino acid) (SEQ ID NO: 48), consisting of 8 to 40 amino acid residues, wherein the number of basic amino acid residues contained in the amino acid sequence is 5 or less Anticancer agent action promoter as an active ingredient. An anticancer agent action promoter comprising, as an active ingredient, a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 4, 5, 6, 7, 9, 10, 11, 12, and 16. An anticancer agent action promoting agent comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 18 as an active ingredient. (1) Prepare a library of DNA encoding the peptide, (2) Prepare a library of molecules in which the DNA and the peptide encoded by the DNA are bound, (3) Bcl-x Select a molecule containing a peptide that binds to _L , (4) amplify the DNA by PCR using the DNA of the selected molecule as a template, (5) use the amplified DNA as a library in step (2), A method for screening a peptide that binds to Bcl-x _L , comprising repeating steps (2) to (4), wherein a library of DNA is (NNS) n (N is A, T, G And an equivalent mixture of C, S is an equivalent mixture of G and C, and n is an integer of 8 to 24). A library of DNA of XXVXRXLXXXXDXIXX (where X is an amino acid encoded by NNS (N is an equal mixture of A, T, G and C, S is an equal mixture of G and C)) (SEQ ID NO: 29) 8. The method according to claim 7, comprising DNA encoding a peptide having an amino acid sequence.

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