WO2006062173A1

WO2006062173A1 - Bcl-xL HETERODIMER INHIBITORY PEPTIDE ENHANCING THE EFFECT OF ANTICANCER AGENT AND METHOD OF SCREENING THE SAME

Info

Publication number: WO2006062173A1
Application number: PCT/JP2005/022585
Authority: WO
Inventors: Hiroshi Yanagawa; Toru Tsuji
Original assignee: Keio University
Priority date: 2004-12-08
Filing date: 2005-12-08
Publication date: 2006-06-15
Also published as: JPWO2006062173A1

Abstract

A drug which enhances an anticancer agent therapy by inhibiting the formation of a heterodimer of Bcl-xL with Bak or Bad and contains, as the active ingredient, a peptide having an amino acid sequence RXXX (wherein X represents a hydrophobic amino acid) and consisting of from 8 to 40 amino acid residues and the number of basic amino acid residues contained in the amino acid sequence being not more than 5, or a peptide having an amino acid sequence YQVARMLRRVADQMAS.

Description

Bel—xL heterodimer-inhibiting peptide for promoting anticancer drug action and screening method thereof

Technical field

[0001] The present invention relates to a Be 卜 X heterodimer-inhibiting peptide that promotes the action of an anticancer agent, and a scoring thereof

It relates to the leaning method.

Background art

[0002] Chemotherapy and radiation therapy are used as anticancer treatments. These treatments cause cancer cells to die and die. To suppress apoptosis such as Be 卜 2 and Be 卜 X.

Overexpressing sex proteins, in cancer cells, these proteins inhibit apoptosis and reduce the efficiency of anticancer treatment. Therefore, a drug that inhibits and inhibits the functions of Be 卜 2 and Be 卜 X is expected to have a function that promotes anticancer treatment (Non-patent Document 1). For example, there is a known example in which the effect of an anticancer agent is increased by suppressing the expression of Be2 by an antisense oligonucleotide targeting the Be2 gene (Non-patent Document 2).

[0003] Be 卜 2 and Be 卜 X are proapoptotic proteins Bak, Bax, Bad, and Bad

L

Heterodimers are formed with these, and these pro-apoptotic effects are lost. Therefore, in order to eliminate the inhibitory effect of Be 卜 2 and Be 卜 X on heterodimer formation,

L

It can be inferred that this should be inhibited. These heterodimers include Be 、 2 and Be 卜 X BH

L

It is formed by the BH3 domains of Bak, Bax, Bad, and Bad being inserted into the hydrophobic pocket formed by the 1, BH2, and BH3 domains. Bak, Bax, Bad, and Bad B H3 domain strength The extracted peptide consisting of about 16 residues can bind to the hydrophobic pockets of Be 卜 2 and Be 卜 X. These peptides competitively inhibit heterodimer formation and show pro-apoptotic action. Therefore, these peptides have the potential to facilitate anticancer treatment.

[0004] Small molecule compounds that bind to the hydrophobic pockets of Be 2 and Be X have been reported.

L

They are antimycin A, BH3I-1, and BH3I-2 (Non-patent Document 3). While affinity to Be Bok 2 and Be Bok X of peptides set forth above is about 10- ⁷ M in dissociation constant, this The binding of these small molecule compounds is about 10- ⁶ M, to be used as a drug is weak affinity.

Non-Patent Document 1: Huang, Oncogene (2000), 19, 6627-6631

Non-Patent Document 2: Jansen et al., Nature Medicine (1998), 4, 232-234

Non-Patent Document 3: Zheng, Nature Cell Biology (2001) 3, El-E3

Disclosure of the invention

[0005] An object of the present invention is to provide a pharmaceutical agent that promotes anticancer drug treatment by inhibiting the formation of heterodimer.

[0006] Non-patent document 3 describes binding to the hydrophobic pockets of Be ポケット 2 and Be 卜 X.

Since the structure of the compound is not similar, the binding site may be slightly displaced. In such cases, analyze the structure of the complex of small molecule compound and Be 2 or Be X.

Therefore, it is important to accurately grasp the coupling mode. This is because by designing a compound that binds in a manner that combines two different binding modes, a drug that binds to Be 2 or Bd-x with higher affinity can be developed.

[0007] Based on such a background, an attempt was made to obtain a peptide that binds to Bcl-x using the in vitro virus method. As a result, several kinds of novel peptides that bind to Be 卜 X competitively with the Bad protein were obtained. These peptides differ in sequence from the naturally occurring peptides derived from the BH3 domain and differ in the binding mode to Bcl-x.

Guessed. Based on these findings, the present invention has been completed. The present invention provides the following.

[0008] 1. RXXX (X is a hydrophobic amino acid) (SEQ ID NO: 48) consisting of 8 to 40 amino acid residues, and the number of basic amino acid residues contained in the amino acid sequence A Bd-x heterodimer formation inhibitor comprising a peptide having an active ingredient of 5 or less.

L

[0009] 2. A Bd-x heterodimer formation inhibitor comprising as an active ingredient a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 4, 5, 6, 7, 9, 10, 11, 12, and 16.

Shi

[0010] 3. A Be 卜 X heterodama formation inhibitor comprising a peptide having the amino acid sequence ability of SEQ ID NO: 18 as an active ingredient.

[0011] 4. It has an amino acid sequence of RXXX (X is a hydrophobic amino acid) (SEQ ID NO: 48), and has 8 to 40 amino acids. An anticancer agent action promoting agent comprising a peptide consisting of a mino acid residue, wherein the number of residues of a basic amino acid contained in the amino acid sequence is 5 or less.

[0012] 5. An anticancer agent promoting agent 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.

[0013] 6. An anticancer agent promoting agent comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 18 as an active ingredient.

[0014] 7. (1) Prepare a library of DNA encoding peptides, (2) From the library, prepare a library of molecules in which the DNA and the peptide encoded by the DNA are bound, ( 3) Select a molecule containing a peptide that binds to Bcl-x, and (4) test the DNA of the selected molecule.

L

DNA is amplified by PCR as a template, and (5) uses the amplified DNA as a library in step (2) and binds to Be 卜 X, including repeating steps (2) to (4) Peptide

L

A DNA library is a (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 an integer from 8 to 24. The method described above, comprising DNA having a base sequence of

[0015] 8. A library of DNA is encoded by XXVXRXLXXXXDXIXX (where X is an NNS (N is an equal mixture of A, T, G and C, S is an equal mixture of G and C)) ( 8. The method according to 7, wherein the DNA having the amino acid sequence ability of SEQ ID NO: 29) is encoded.

Brief Description of Drawings

[0016] [Figure 1] Schematic diagram of the NNS library

[Figure 2] Nucleotide sequence of NNS library

[Figure 3] NNS library construction method

[Figure 4] Explanation of Bak library

[Figure 5] In-vitro selection experiment

[Figure 6] Construction of DHFR fusion peptide

[Figure 7] Binding experiment of obtained peptides

[Figure 8] Pull-down experiment method

[Figure 9] Pull-down assembly test results (electrophoresis photograph)

[Fig. 10] Competition experiment results on Bd-x binding between Bad and acquired peptide (electrophoresis) [Fig. 11] Results of binding experiment between GST-Bcx and fluorescent Bak peptide.

Shi

[Fig. 12] Results of competition experiment on Be 卜 X binding between Bak peptide and obtained peptide

Shi

BEST MODE FOR CARRYING OUT THE INVENTION

[0017] In this specification, the one-letter symbols for bases and amino acids are those according to WIPO Standard ST.25.

[0018] 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), and consists of 8 to 40 amino acid residues. Is a peptide having 5 or fewer residues of basic amino acids.

[0019] Hydrophobic amino acid is an amino acid having a hydrophobic side chain, and refers to phalalanin, tryptophan, tyrosine, cysteine, methionine, alanine, norin, 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. Further, the number of residues of basic amino acids is preferably 25% or less of all residues constituting the peptide. With a high basic amino acid content, Bcl-x

But it repels the positive charge part, Bcl-x

Cannot be combined with L.

[0020] Examples of the peptides 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 biological fluids such as blood.

[0021] 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.

[0022] The peptide of the active ingredient can be made into a preparation (pharmaceutical composition) using a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers include excipients or bases. 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 anti-cancer agents that are packaged and integrated separately. The dose of the active ingredient is appropriately selected depending on the intended anticancer drug treatment, the patient's condition, and the like. The inhibitors or facilitating agents of the present invention can be administered to patients who are receiving or will receive anticancer drug treatment.

[0023] The present invention also provides a method for screening a peptide that binds to Bd-x, that is: 1) Prepare a library of DNAs encoding the peptides, (2) Prepare a library of molecules in which the DNA and the peptide encoded by the DNA are bound, and (3) Bcl -X Select the molecule containing the peptide to be bound, and (4) template the DNA of the selected molecule.

(5) A peptide that binds to Be 卜 X, including repeating steps (2) to (4) using the amplified DNA as a library in step (2). Screening

Provide a way to

[0024] In the method of the present invention, Bcl-x is used as a target substance, a DNA library is used.

Other than using one having a specific configuration as one, it can be performed according to a screening method based on the in vitro virus method (see, for example, WO 02/48347, JP 2002-176987 A). In vitro virus and screening methods are described.

[0025] In vitro virus is also referred to as a mapping molecule. A mapping molecule by the in vitro virus method includes a phenotype molecule containing a protein to be subjected to functional analysis, functional modification, etc., and a nucleic acid encoding the protein. It combines with the genotype molecule it contains. 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 part of the spacer. 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 a mapping molecule are respectively a decoding part, a part of a spacer, and a part of a spacer. Called the code part. Also, in a genotype molecule, a portion derived from a single spacer molecule and a portion derived from a coding molecule are referred to as a spacer portion and a coding portion, respectively.

[0026] The spacer molecule in this embodiment includes a donor region capable of binding to the 3 'end of a nucleic acid, a PEG region mainly composed of polyethylene glycol bound to the donor region, and a peptide bound to the PEG region. And a peptide acceptor region containing a group capable of binding to a peptide by a transfer reaction. There may be no PEG area.

[0027] 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. Nucleotides may be ribonucleotides or deoxyribonucleotides.

[0028] 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 include at least one residue, For an acceptor having a poly A sequence, at least one residue of dC (deoxycytidylic acid) or two residues of dCdC (dioxycytidylic acid) is preferred. As the type of base, OU or T>G> A is preferred in this order! /.

[0029] 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.

[0030] 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 the genotype molecule containing a part of the spacer derived from one spacer molecule is subjected to correspondence translation, post processing of correspondence translation is performed. Although necessary (Liu, R., Barrick, E., Szostak, JW, Roberts, RW (2000) Methods in Enzymology, vol. 318, 268-293), molecular weight of 1000 or more, more preferably 2000 If the above PEG is used, high-efficiency association can be performed only by association translation, so that post-translation processing is not necessary. In addition, as 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 is not as stable and unstable. Sometimes.

[0031] 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 and N-aminoacylpuromycin aminonucleoside (3'-N-Aminoacylpuromycin aminonucleoside , PANS—amino acids), for example, PANS-Gly whose amino acid part is glycine, PANS-Val of norrin, PANS-Ala of alanine, and other PANS-all amino acids corresponding to all amino acids can be used. Also, as a chemical bond, 3'-N-aminoacyl adenosine aminonucleoside (3ANS-aminoaminodenosine aminonucleoside, AANS-) linked by an amide bond formed as a result of dehydration condensation of the amino group of 3'-aminoadenosine and the carboxyl group of amino acid. Amino acids), for example, AANS-Gly with the amino acid part glycine, AANS-Val with parin, AANS_Ala with alanine, and other AANS-all amino acids corresponding to all amino acids can be used. In addition, nucleoside or ester bond of nucleoside and amino acid You can also use it. In addition, use all nucleosides or substances having a chemical structural skeleton similar to nucleosides and amino acids or substances having a chemical structural skeleton similar to amino acids that have a binding mode capable of chemically binding. Can do.

[0032] 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 a peptide in a protein translation system. The puromycin derivatives are not limited to those having a complete puromycin structure, but also include those in which a part of the puromycin structure is missing. Specific examples of the puromycin derivative include PANS-amino acid and AANS-amino acid.

[0033] The peptide acceptor region may be composed solely of puromycin, but preferably has a base sequence consisting of one or more residues of DNA and / or RNA on the 5 'end side. The sequences include dC-puromycin, rC-puromycin, etc., more preferably dCdC-puromycin, rCrC-puromycin, rCdC-puromycin, dCrC-puromycin, etc., and the 3 ′ end of aminoacyl-tRNA A CCA sequence that mimics (Philipps, G.R. (1969) Nature 223, 374-377) is suitable. The type of base is preferably C> U or T> G> A.

[0034] The spacer molecule preferably includes at least one functional unit between the donor region and the PEG region. The function-imparting unit is preferably a functional modification of at least one residue of doxyribonucleotide or ribonucleotide base. For example, a function-modifying substance may be a substance into which various separation tags such as a fluorescent substance, piotin, or His-tag are introduced.

[0035] The coding molecule in this embodiment includes a 5 'untranslated region including a transcription promoter and a translation enhancer, a protein-encoding ORF region bound to the 3' end of the 5 'untranslated region, and an ORF region 3 A nucleic acid having a poly A sequence bound to the terminal side and containing an affinity tag sequence 5 ′ upstream of the poly A sequence.

[0036] The coding molecule may be DNA or RNA. In the case of RNA, the 5 'end may or may not have a Cap structure. In addition, the coding molecule should be incorporated into any vector or plasmid. A little.

[0037] The 3 'terminal region includes an affinity tag sequence and a poly A sequence downstream thereof. As a factor that affects the ligation efficiency between a spacer molecule and a coding molecule, the polyA sequence in the 3 'end region is important, and the polyA sequence has a dA and / or rA of at least 2 residues. It is a mixed or single poly A continuous chain, preferably a poly A continuous chain of 3 residues or more, more preferably 6 or more, and even more preferably 8 residues or more.

[0038] As elements that affect the translation efficiency of the coding molecule, there is a combination of a 5'UTR that also serves as a transcription promoter and a translation enhancer, and a 3 'terminal region containing a poly A sequence. The effect of the poly A sequence in the 3 'end region is usually exerted on 10 residues or less. T7 / T3 or SP6 can be used as the 5'UTR transfer profile motor, 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 sequencer is preferably part of the omega sequence, and part of the omega sequence includes part of the TMV omega sequence (029; Gallie DR, Walbot V. (1992) Nucleic Acids Res., Vol. 20, 4631-4638 and WO 02/48347 (see FIG. 3) are preferred.

[0039] The affinity tag sequence is not particularly limited as long as it is a sequence capable of detecting a protein such as an antigen-antibody reaction and using such a means. Preferably, the flag-tag sequence or His-tag sequence, which is a tag for analyzing and analyzing the affinity by the antigen-antibody reaction, is used.

[0040] The ORF region! May consist of DNA and / or RNA! /, Or any sequence. A gene sequence, exon sequence, intron sequence, random sequence, or any natural or artificial sequence is possible, and there is no sequence limitation. In addition, by setting the 5′UTR of the coding molecule to SP 6 + 029 and the 3 ′ end region, for example, Flag + Xhol + A (n = 8), each length is about 60 bp in 5′UTR. The 3 'end region is about 32 bp, and is a length that can be incorporated into the PCR primer as an adapter region. For this reason, any vector, plasmid, or cDNA library can easily generate a coding molecule with a 5 'UTR and 3' end region by PCR. In code molecules, translation may be done beyond the ORF region. That is, there may be no stop codon at the end of the ORF region.

[0041] The coding molecule in this embodiment includes a transcriptional promoter and a translation enhancer. A 5 'untranslated region, an ORF region encoding a protein bound to the 3' end of the 5 'untranslated region, and a 3' end region containing a poly A sequence bound to the 3 'end of the ORF region. Containing nucleic acids.

[0042] The genotype molecule is obtained by converting the coding molecule, if necessary, into a form in which the base sequence of the protein-coding region can be translated (for example, after transcription), and the 3 'end of the coding molecule. In addition, the donor region of a spacer molecule can be produced by binding by a usual ligase reaction. The reaction conditions usually include a condition of 4 to 48 hours at 4 to 25 ° C, and a polyethylene glycol having the same molecular weight as the polyethylene glycol in the PEG region of a spacer molecule containing the PEG region is reacted. When added to the system, it can be shortened to 0.5-4 hours at 15 ° C.

[0043] The combination of the spacer molecule and the coding molecule has an important effect on the 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, For the UTR translation sequence, the partial sequence of the omega sequence (029) is preferred, and the partial donor region of the spacer is at least one dC (doxycytidylic acid) or two residues. dCdC (dideoxycytidylic acid) is preferred! This makes it possible to avoid the problems of DNA ligase by using RNA ligase and to maintain the efficiency at 60 to 80%.

[0044] (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 A 3 ′ end of a coding molecule, which is an RNA containing a 3 ′ end region containing a poly A sequence, and (b) a donor region of the spacer molecule consisting of RNA. It is preferable to bind with RNA ligase in the presence of a free polyethylene glycol having the same molecular weight as that of polyethylene glycol constituting the PEG region in each molecule.

[0045] 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 80 to 80% regardless of the molecular weight of the polyethylene glycol of the spacer part. The separation rate after the reaction can be omitted. [0046] The mapping molecule of this embodiment is a phenotype that is a protein encoded by an ORF region in a genotype molecule in a peptide transfer reaction by translating the above genotype molecule with a cell-free translation system. Can be linked to a molecule. 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.

[0047] 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. A step of producing a library of mapping molecules by the production method, a step of mixing the library of mapping molecules and the target substance, a step of separating the mapping molecule bound to the target substance, and a separated mapping molecule The linker is cleaved under conditions to release the nucleic acid, and the released nucleic acid is recovered.

[0048] 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.

[0049] 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 the solid phase. In this case, 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, “immobilized on a solid phase” means that the conjugate of the mapping molecule and the target substance can be separated by a non-binding molecular force. For example, when the target substance is a membrane protein, Membrane proteins expressed in cell membranes and proteins embedded in artificial membranes are also included in the target substance immobilized on the solid phase.

[0050] The linker of the separated mapping molecule does not change in the base sequence of the nucleic acid, and the nucleic acid is cleaved under the conditions to release the nucleic acid under the conditions corresponding to the conditions. line I can. 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.

[0051] The liberated nucleic acid can be collected by an ordinary method. Examples thereof include a method of collecting by electrophoresis and a method of collecting a supernatant by precipitating components other than the released nucleic acid. The collected nucleic acid is amplified and sequenced for 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.

[0052] One of the DNA libraries used in the screening method of the present invention is (NNS) n (N is an equal mixture of Α, Τ, G and C, S is an equal mixture of G and C, and n is 8 It consists of DNA having a base sequence of ˜24). The DNA library is preferably XXVXRXLXXXXDXI XX (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)) The DNA encoding the peptide having the amino acid sequence ability of (SEQ ID NO: 29) is used.

[0053] For peptides that bind to Be 卜 X screened by the above method, Bak or Shi

By measuring the force competing against the Bad protein, it is possible to identify a peptide that is an active ingredient of the inhibitor or facilitator of the present invention. The ability to compete can be measured by a method as described in the examples described later.

[0054] 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 described in the following examples. It is not limited in any way.

Example 1

[0055] · Construction of DNA library

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.

[0056] · NNS library

• NNS library features 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 with 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 the NNN library.

[0057] · NNS library structure

The NNS library was applied to both the in vitro virus method using the wheat germ cell-free translation system and the in vitro virus method using PURESYSTEM (Post Genome Research Institute, Inc.), 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 Z serine linker, NNS library, flag tag and AX6 sequence. On the other hand, the entire length of the NNS library for PURESYSTEM also includes the T7 promoter, SD sequence, start codon, glycine Z serine linker, NNS library, flag tag, and A X 6 sequence. A schematic diagram of these sequences is shown in Fig. 1, and the nucleotide sequence is shown in Fig. 2. One NNS library encodes 8 amino acids (NNS)

It contains equimolar numbers of 8, 16 amino acids (NNS) and 24 amino acids (NNS).

16 24

[0058] · NNS library construction

Fig. 3 shows how to construct an 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 Z serine linker, NNS library, flag tag and AX 6 sequence were designed and Purchased from the company. The sequence is 5 '-ttt ttt ctt at c gtc gtc ate ttt gta gtc>, snn) tga gcc tec gcc tec tga acc gcc gcc acc ~ 3' (酉 C column

8, 16, 24

Numbers 31, 32, and 33). Next, a positive-strand DNA (priSP60Gl) containing the Sp6 promoter, Ω29 sequence, start codon and part of the glycine / serine linker was designed, and Fasmac Mud Force et al. ¾ 7 people. C 酉¹ J¾, 5 ― att tag gtg aca cta tag aac aac aac aac aac aaa ca a caa caa aat ggg tgg egg egg tt-3 '(SEQ ID NO: 34). Also, a flag tag and A x 6 A negative strand of DNA (priFLAGA6r) encoding the sequence was designed and purchased from Date Concept. The sequence is 5, -ttt ttt ctt ate gtc gtc ate ttt gta gtc-3, (SEQ ID NO: 35). Perform PCR using the G4SG4S (NNS) 8 [FLAG] A6r, G4SG4S (NNS) 16 [FLAG] A6r, and G4SG4S (NNS) 24 [FLAG] A6r described above as primers and priSP60Gf and priFLAGA6r as primers! / A double-stranded DNA library was obtained. PCR reaction solution (100 μ1) is 2 units of KOD DNA polymerase, 200 μ 200 dNTP, 2 mM MgSO 3 pmol forward and reverse

Four

There will also be a primer, 1 pmol of saddle DNA, and accompanying buffer power. 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) Consists of 68 ° C / 5 minutes.

[0059] The NNS library construction method for PURESYSTEM is the same. However, instead of priSP60Gf, priUniv2 containing the T7 promoter sequence, SD sequence, start codon, and part of the glycine / serine linker was designed and used as the forward primer. This DNA is also purchased by Fasmac. Its salt is a 5 -gaa att aat acg act cac tat agg gag acc aca acg gtt tec etc tag aaa taa ttt tgt tta act tta aga agg aga tat acc aat ggg tg-3 '(SEQ ID NO: 36).

[0060] · Bak library

• Bak library features

Bak is one of the members of the Be ファミリー 2 family protein, and Bcl-χ and heterodimer

L

Form. A 16-amino acid peptide extracted from the BH3 domain of Bak protein binds to BeX with a dissociation constant of 340 nM. Some amino acids in the BH3 domain

L

Are conserved among the various proteins within the family. It is speculated that these amino acids are essential for binding to Be 卜 X. Therefore, a library was constructed in which these 5 amino acids were fixed and the remaining 11 amino acids were NNS, and named Bak library. This library effort is expected when peptides that bind to Be 卜 X are obtained relatively frequently.

Wait.

[0061] · Bak library structure

A comparison of the amino acid sequences of the 16-amino acid residue peptide derived from the Bak protein and the Bak library used in this study is shown in Fig. 4 (1). The fixed amino acid is the third palin, 5th arginine, 7th leucine, 12th aspartate, and 14th isoleucine.

[0062] The Bak library was applied only to the in vitro virus method using PURESYSTEM. A schematic diagram and base sequence of the full length are shown in FIGS. 4 (2) and (3).

[0063] · Bak library construction

The Bak library was built in the same way as the NNS library for PURESYSTEM. However, the cage DNA used is a glycine Z serine linker, a Bak library, a flag tag, and a minus strand of DNA encoding the AX6 sequence (G4SG4SBAK16MFLAGA6r). This single-stranded DNA was purchased from Fasmac. The base sequence is 5 '-ttt ttt ctt ate g tc gtc ate ttt gta gtc snn snn aat snn ate snn snn snn snn cag snn gcg snn aac snn s nn tga gcc tec gcc tec tga acc-3' &) In column number 37).

[0064] · Construction of in vitro virus library

A DNA library for wheat germ cell-free translation system having an SP6 promoter was transcribed using a RiboMAX Large Scale RNA Production System (Promega) and converted into an RNA library. Reaction solution (50 1) consists of 5 g DNA library, attached reaction buffer, 5 1 SP6 Enzyme mix, 5 mM substrate (ATP ゝ UTP ゝ CTP), 400 μΜ GTP, and 2 mM m 7G (5 ') ppp (5') G cap analog (Invitrogen) is included. 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 the T7 RiboMAX Express Large Scale RNA Production System (Promega) and converted to an RNA library. The reaction mixture (25 μ1) contains 2.5 μg of DNA library, attached reaction buffer, and 2.5 μ1 of Enzyme mix. The reaction was carried out at 37 ° C for 30 minutes. The truncated DNA was digested with DNasel (Promega) and purified using RNeasy Mini Kit (QIAGEN). The method followed the manual attached to the kit. RNA and spacer puromycin (p (dCp) T (FI) pPEG (2000) p (dCp) Puro (the symbols are as defined in WO 02/48347

twenty two

)) Was ligated according to the method described in Example 1 of WO 02/48347 using T4 RNA ligase (Takara Shuzo). 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. Cell-free translation reaction If the wheat germ system is used, the composition of the solution is 10 1 wheat germ extract 1 "manufactured by Sakai _& Co., Ltd.), 80 M amino acid mixture, 50 mM potassium acetate, 10 units RNase inhibitor (Produced by Invitrogen) and 2 pmol of ligation product.The reaction was carried out for 40 minutes at 25 ° C. When using PURESYSTEM, the reaction solution was the attached reaction solutions A and B, and 5 to 10 p mol. The reaction was carried out at 37 ° C for 30 minutes.

· Bait preparation

Bcl-x is expressed as a dartathione-S-transferase (GST) fusion protein, and

It was decided to use the selection experiment by presenting it on the dartathione-bound rosin via GST. Be 卜 X is used for reverse transcription and PCR from RNA library of human brain (Clontech).

L

More acquired. Reverse transcription reaction solution 60 μ 1 is 500 μΜ dNTP, 0.6 pM primer (priBc 卜 x Lrl, 5, — tac agt etc gag cta gtt gaa gcg ttc ctg gcc ct— 3, (酉 Self U number 38) , Dateconcept), 10 mM DTT, 600 units of Superscript II reverse transcriptase and the attached reaction buffer (Invitrogen). The reaction was carried out at 37 ° C for 1 hour. KOD Plus was used for PCR. The PCR reaction solution (100 μ 1) contains 2 units of KOD DNA polymerase, 200 μΜ d NTP mM 2 mM MgSO, 3 pmol forward primer (priBc 卜 xLfl: 5, agt ate gaa ttc

Four

atg tct cag age aac egg-3 (SEQ ID NO: 39, date concept), and reverse primer (priBc 卜 xLrl), DNADNA, and attached buffer power. The obtained PCR product was cloned using the TOPO TA Cloning kit (manufactured by Invitrogen), and then the nucleotide sequence was confirmed using a DNA sequencer (CEQ2000, manufactured by Beckman Coulter). The resulting Bcl-x-encoding DNA was digested with EcoRI and Xhol (Toyobo) and then digested with the same enzyme.

L

It was ligated with a thione-S-transferase (GST) fusion protein expression vector (PGEX-4T-1, manufactured by Amersham Bioscience). 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 nucleotide sequence of the structural gene portion of the expression vector by DNA sequencing, the host cell for expression, E. coli BL21 (DE3) pLysS, was transformed. Mass expression after reaching OD = 0.6 0.4 mM isopropyl-β-D-thiogalatatopyranoside (

600

The expression was induced by IPTG) (manufactured by Nacalai Testa) and cultured overnight at 25 ° C. The culture scale is usually about 5 mL. The medium is 50 gZml carbecillin and 3 Contains 0 / z gZml of chloramhue-coal. The bacterial cells collected from 1.5 ml of the liquid medium were suspended in 200 1 lysis buffer, and the bacterial cells were disrupted by ultrasonic waves. After centrifugation, approximately 200 1 soluble fraction was combined with 6001 slurry of dartathione binding resin. After thorough washing, add 1 ml of TBSTE buffer (Tris-HCl (pH 7.4), 150 mM NaCl, 10 mM EDTA, 0.2% Tween 20), equilibrate the Bcl-x conjugated resin, saved.

Shi

[0066] 4. Performing in vitro selection experiments

Figure 5 shows an outline of the experimental procedure for in vitro selection experiments. The translation reaction product was purified using Micro Bio-S pin Column P-30 Tris, RNase-Free (BIO-RAD). 180 1 of TBSTE buffer was added and centrifuged at 15,000 rpm for 10 minutes. Into the supernatant, 201 slurry of ANTI-FLA G M2-Agarose (manufactured by SIGMA) was added and stirred at 4 ° C for 1 hour to carry out a binding reaction. After washing 4 times with 200 1 TBSTE buffer, reverse transcription reaction of mRNA part of in vitro virus molecule bound to scab was performed. Reverse Transcription Reaction 1 contains 500 / zM dNTP, 0.6 pM priFLAGA6r, 10 mM DTT, 600 units Superscript II reverse transcriptase and attached reaction buffer. The reaction was carried out at 37 ° C for 1 hour. The reaction solution was purified using Micro Bio-Spin Collumn 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 solution or TBSTE2 buffer solution was added to make the total volume of the reaction solution 3001. This reaction solution is mixed with 15 1 slurry of Be 卜 X bond resin.

The resulting mixture was stirred at 4 ° C for 1 hour to carry out a binding reaction. Washed 4 times with 150 1 TBSTE buffer or TBS TE2 buffer to remove non-specifically adsorbed molecules. The following two methods were used to recover the molecules bound to Bcl-x. Add 10 units of thrombin (manufactured by Amersham Bioscience) and cut between Be 卜 x and GST together with Be 卜 x in vitr

L L

o A method of recovering virus molecules, or a method of recovering in vitro virus molecules by acting 0.1 M KOH to denature the protein part.

[0067] The library synthesized using the wheat germ cell-free translation system was subjected to binding and washing using TBSTE buffer, and in vitro virus molecules were collected using thrombin. The library synthesized using PURESYSTEM was subjected to binding and washing using TBSTE2 buffer, and in vitro virus molecules were recovered using KOH. The recovered in vitro virus molecule is Concentration was carried out by means of knoll precipitation, and the cDNA portion was amplified by PCR. PCR primers include priSP60Gf and priFLAGA6r (library for wheat germ cell-free translation system) and priSDG4S f (5,-aag gag ata tac caa tgg gtg gcg gcg gtt-3, SEQ ID NO: 40). Purchased) and priFLAGA6r (PURESYSTEM library). 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, 3 steps (1 cycle) 68 ° C / 5 minutes.

[0068] · Amino acid sequence of the selected peptide

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

[0069] In a selection experiment using an NNS library and a wheat germ cell-free translation system, the base sequence was analyzed after 7 rounds of selection experiment. Table 1 shows the amino acid sequences of the peptides obtained in this selection experiment. Clones 06 and 10 appeared 4 or 5 times, respectively. In particular, the clones 10 to 13 were highly homologous, differing only by one amino acid. In the 13th to 16th sites of clones 3-7 and 9-13, a motif consisting of three consecutive hydrophobic amino acids was found following arginine (R).

[0070] In the selection experiment using the NNS library and PURESYSTEM, the base 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 the sequence of clone 04 obtained using the wheat germ cell-free translation system.

[0071] In selection experiments using the Bak library and PURESYSTEM, the base sequence was analyzed after 5 rounds of selection experiments. 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. Also, clones 18 and 19 were mutated to the 14th leucine catonin that should have been preserved. The 3rd to 11th amino acid sequence of clone 18 was homologous to the 4th to 12th amino acid sequence of clone 17. [] ^ 0073

Table 1 Sequences obtained by selection experiments using NNS library and wheat germ cell-free translation system

01 ARVFGLWLRSMSD 隱 L <1> 09 SQRSTMYIAAVLRWLA <9>

10 FPRWKLLAHWADRWWF (5) 10>

03 RVLRYYLVGLALRQMA 3> 11 FLRWKLLAHWADR 丽 F 11>

04 GNRWQYWIAFRMRYVA 4> 12 FPRWKLLAHWADRWWV <12>

05 I RMMVSFLRROSRWWL <5> 13 FPRWKLLAHWADRGWC <13>

06 MHYEAVRVGRVLRFLA (4) 6> 14 AAVASMLRHLAGLFVL <14>

07 MVPSWVGLARVLRWLA <7> 15 MWVHSAAMATGLASR <15>

08—One LVLLGRMLKFVA <8>

The number in 0 indicates the number of duplicate clones

The number in <> indicates the sequence number.

[] (0072 Table 2 Sequences obtained by selection experiments using NNS library and PURESYSTEM

(16 GNRWQYWIAFRLRYVAO) <16>

17 TRRVARVLRRLARLWL (6) <17>

The numbers in 0 indicate the number of duplicates obtained.

The number in o indicates the sequence number

[0074] [Table 3] Table 3 Sequences obtained by selection experiments using Bak library and PURESYSTEM

18 Y0VARMLRRVADQ AS C7) 18>

19 KVV RQLLMIADTMAR <19>

20 LTVARRLKWVADI IFA <20>

GOVGRQLAI IGDDINR [Bak] <21>

The numbers in 0 indicate the number of duplicates obtained.

For comparison, the amino acid sequence of the peptide derived from Bak is also shown. Conserved amino acids are shown in bold.

Numbers in <> indicate sequence numbers

[0075] · Confirmation of affinity of selected peptides for Be 卜 X

Shi

• Measuring recovery using real-time PCR

A consensus sequence is required for accurate quantification by real-time PCR. E. coli dihydrofolate reductase (DHFR) was used as a common sequence. The obtained peptide was linked to the C-terminal side of DHFR via a glycine Z serine linker (FIG. 6a). A gene encoding this fusion protein was constructed by the procedure shown in FIG. 6b. The DHFR gene was amplified by PCR from the plasmid attached to the Protos cell-free translation kit manufactured by Toyobo. The PCR reaction (100 μ1) consists of 2 units of KOD DNA polymerase, 200 μ d dNTP, 2 mM MgSO 3, 3 pmol forward and reverse primers, 1 μ μ cage DNA, and

Four

Consists of attached buffer. The forward primer is priSP60Df, 5-, att tag gtg aca cta ta g aac aac aac aac aac aaa caa caa caa aat gat cag tct gat tgc ggc gtt a- 3 '(Tatsumi line number 41) (manufactured by Fasmac) The reverse primer is priG4Sl-2Dr: 5, -tga gcc tec gcc tec tga acc gcc gcc acc ccg ccg etc cag aat ct-3, (Tatsumi No. 42) (manufactured by Date Concept). Forward primer includes SP6 promoter, Ω29 sequence, start codon, and D Part of the HFR is coded. The reverse primer is encoded by glycine Z serine linker and part of DHF R. 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 ° C / 5 minutes.

[0076] The peptide gene to be analyzed for binding ability is on a cloning vector. This gene was amplified by PCR with priG4Slf, 5, -ggt ggc ggc ggt tea 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. PsiSP60Df was used as the first primer and priFLAGA6r was used as the reverse primer. The conditions for PCR 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.

[0077] As a positive control, 28 amino acids known to bind strongly to Be 卜 X

We constructed a DHFR fusion peptide of Bad peptide, which also has a residue strength. Glycine serine linker, plus single-stranded DNA encoding Bad peptide and flag tag, G4SG4SmBadf, o -ggt ggc ggc ggt tea gga ggc gga ggc tea aat etc tgg gca gcg cag cgc tac ggc cgt gag etc cga agg atg age gat gag ttt gag ggt tec ttc aag gac tac aaa gat gac gac gat a ag-3 '(SEQ ID NO: 44) (manufactured by Fasmac Co., Ltd.) was chemically synthesized and PCR was performed using priG4Slf and priFLAGA6r. ! / Double-stranded DNA was obtained. Furthermore, it was ligated to the DHFR part 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 priG4Slf and priFLAGA6r, and then ligating it with the DHFR part by overlap extension PCR.

[0078] DNAs encoding various constructed peptides were converted into in vitro virus, and the binding ability to Bcl-x-binding rosin was examined by the experimental procedure shown in FIG. This experimental procedure is shown in Figure 5.

The same force as in the selection experiment The real-time PCR is different in that the number of in vitro virus molecules mixed with the fat is measured and the number of in vitro virus molecules recovered from the fat after washing. [0079] 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 1st stage (1 cycle) 94 degrees Z10 minutes, 2nd stage (40 cycles) 94 degrees Z10 seconds, 60 degrees Z10 seconds, 72 degrees ZlO seconds. Calibration curves were prepared by preparing three types of standard substances with different concentrations by 100 times. PriEDHFRf as a forward primer, 5, —atg ate agt ctg att gcg gcg tta gcg gta—3, (Umi U number 45), proEDHFR211r as a linoleose primer, 5′-gat cgt ccg tac ccg gtt gac tgc t -3, (SEQ ID NO: 46) (both manufactured by Date Concept) was used. These primers amplify the 1 to 211 base pair region of the DHFR gene.

[0080] The recovery rate of various peptides was determined by setting the recovery rate of positive control to 100, the recovery rate of negative control to 1, and various peptide recoveries. The results are shown in Table 4. The binding ability of the peptide consisting of 28 amino acids was lower than that of the bad peptide. 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.

[0081] [Table 4] Table 4 Comparison of binding of various peptides

03 RVLRYYLVGLALRQMA 3〉 [9]

04 GNRWQYWIAFRMRYVA <4> [21]

06 MHYEAVRVGRVLRFLA <6> [10]

07 MVPSWVGLARVLRWLA <7> [11]

08 —— LVLLGRMLKFVA <8> [12]

09 SQRSTMYIAAVLRWLA <9> [10]

10 FPRWKLLAHWADRWWF <10> [32]

Positive control [100] Negative control [1]

Numbers in <> indicate sequence number [0082] · Binding experiments using pull-down assembly

As a clone 16, 17, 18, Bad peptide, Bak peptide, and negative control, the binding analysis by real-time PCR described above showed a weak binding ability and an unrecognized sequence (¥¥ (3? ¥ 3 and 1¾ ¥ An in vitro virus molecule for PURESYSTEM was constructed for ^ 0 and \ ^ (SEQ ID NO: 47), and a pull-down assay was attempted.

[0083] The experimental procedure for pull-down assembly 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. TBSTE2 buffer approximately 200 1 was added to make the total volume 300 1. After mixing Be 榭 x bound resin 15 μl ^ rally and performing the binding reaction at 4 ° C for 1 hour, 150 μl

Washed 4 times with STE2 buffer. Thereafter, electrophoresis buffer 201 was applied to the resin at 99 ° C for 5 minutes, and then applied to 10% I 8 M urea SDS-PAGE. After electrophoresis, a fluorescent imager (Bio-Rad, Molecular Imager FX) was used to detect in vitro virus molecules bound to rosin. As a control experiment, Bcl-x conjugated sacchar was pretreated with thrombin and most of the B

Shi

A sesame excluding cl-x was prepared and subjected to the same pull-down assembly. These experimental results

The results are shown in Fig. 9.

[0084] Bad peptides were clearly observed to form virus molecules in vitro. Be 卜 x

An exact bond was seen. The binding force was also observed in the scab after the thrombin treatment, and the amount was small compared to that bound to the Be-X resin. Therefore, the Bad peptide is Bd-x

There is a high possibility that it is specifically bound to. No binding was observed on the resin after thrombin treatment

One of the reasons is that Bcl-x could not be completely removed by thrombin treatment.

Possible performance. In the negative control peptide, formation of in vitro virus molecules was observed. No binding was found on either scab. In the Bak peptide, Be 卜 X was bound only to the binding resin. The above results show that this pull-down assembly is performed correctly.

[0085] 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. In all clones, clear binding was observed in the Bex binding resin. On the other hand, the resin after thrombin treatment showed only slight binding. In clone 17, in vitro virus molecules are significantly more Appeared. This is probably because this peptide contains a large amount of arginine and exhibits a high isoelectric point (pl = 10). For this peptide, the band was smeared and unclear, but binding was seen in the Bcl-x binding resin. The above results were not obtained in this selection experiment.

This suggests that clones 16, 17, and 18 have Be X binding ability. These peptides

By analyzing the mode of binding, it can be expected that a novel drug will be developed using these peptides as lead compounds.

[0086] · Competition experiment on selected peptide and Bad's Be 卜 X bond

Shi

The obtained peptide binds to the hydrophobic pocket of Bd-x to facilitate anticancer therapy.

Therefore, it is necessary to inhibit heterodimer formation between Be 卜 X and Bak and Bad proteins.

The This is because Bak and Bad proteins are released by this inhibition of heterodimer formation and are expected to exert a pro-apoptotic effect (Zheng, Nature Cell Biology (2000) 3, E 1-E3). In this experiment, a competition experiment on the binding of Be 卜 X between the peptide obtained by the selection experiment and the Bad peptide was conducted, and the binding site of the obtained peptide to Be 卜 X was verified.

L

[0087] Bad peptides, clones 16, 17 and 18 in vitro virus molecules were prepared by the same procedure as described above. Clone 16, 17 and 18 alone were used to bleed against Be 卜 X-binding oil.

Figure 10 shows a comparison between the results of the unattended and the results of the pull-down assimilated by mixing with Bad. From this result, it can be seen that the band intensity of the obtained peptide is reduced in the case of competition as compared to the case of single use. This indicates that these acquired peptides and Bad peptides are competitively bound to the common site of Bcl-x.

Show. Therefore, the peptide described here is a combination of Bcl-x and Bak and Bad proteins.

It is more speculated that it inhibits heterodimer formation, restores the pro-apoptotic action of Bak and Bad proteins, and promotes anticancer drug treatment.

[0088] 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 clones 4 to 13 bind to BeX in a common manner. This and Kushi

Because clone 16, which is almost identical to loan 4, binds Be 競合 X competitively with the Bad peptide.

Clone 5 to clone 13 shown in Table 1 also bind to Be 卜 X competitively with the Bad peptide. Can be guessed. Therefore, clone 5 to clone 13 also include Bel-X and Bad and Bak proteins.

L

It is presumed that it inhibits the formation of heterodimers with these compounds, restores these pro-apoptotic effects, and promotes anticancer treatment.

[0089] · Inhibitory activity of acquired peptide against binding of Bak peptide and Be 卜 X

Shi

Using the peptide obtained from the selection experiment and the Bak peptide, binding to Bcl-x

Competition experiments were conducted. This competition experiment inhibited the binding of Bak peptide to Be 卜 X.

The resulting peptide will form a heterodimer between Bcl-x and Bak or Bad protein.

Can be expected to inhibit The fluorescently labeled F-Bak peptide, the unmodified Bak peptide, and the peptides corresponding to the obtained clones 10, 16, 17, 18 were outsourced to Invitrogen. The four acquired peptides are linked with a FLAG tag in order to increase the solubility in an aqueous buffer solution.

[0090] 8. 1. Binding of fluorescent Bak peptide to GST-Bc 卜 X

L

First, the binding of F-Bak to GST-Bc 卜 X was analyzed by measuring the degree of fluorescence polarization (B

EACON 2000, Invitrogen). Mix a certain amount of F-Bak with various concentrations of GST-Be 卜 x.

The degree of polarization was measured. Figure 11A shows the B vs L plot. Curve fee

F

The equation (1) was used for tapping.

[0091] F (x) = {x * (Pmax-Pmin)} / (Kd + x) + Pmin (1)

Here, the unknown coefficients are Pmax, Pmin, and Kd (dissociation constants), and the independent variable is x (GST_Bc 濃 x concentration).

[0092] From the curve fitting, the dissociation constant was determined to be 337 people 32 nM. The dissociation constant for the binding of Bak peptide to Be 卜 x is reported to be 340 nM.

It was in good agreement with the value.

Next, a Klots plot in which the horizontal axis is expressed in logarithm is shown in B of FIG. Curve fitting can be performed using equation (1). Since these plots are respectively hyperbolic and sigmoid, the binding reaction between F-Bak and GST-Bc 卜 X is not Clark's theory.

It is speculated that it is consistent with the theory.

Next, the result of the Scatchard plot is shown in C of FIG. The horizontal axis shows the ratio of bound molecules, that is, (P-Pmin) / (Pmax-Pmin), and the vertical axis divides this value by the GST-Be 卜 concentration. The values were plotted. F-Bak and GST-Bc 卜 X combined because the plot was on a straight line

L

Can be interpreted as a 1: 1 reaction. The dissociation constant obtained from the Scatchard plot was 370 nM, which was in good agreement with the literature value. From the above results, it can be concluded that this analysis method using F-Bak and GST-Bc 卜 X 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).

[0095] 8. 2. Analysis of inhibitory activity of obtained peptides

Inhibition experiments consisted of mixing a certain amount of F-Bak (186 nM) and GST-Be 卜 x (400 nM)

L

This was carried out by placing the obtained peptides at various concentrations in the reaction solution and measuring the degree of polarization. If the obtained peptide shows an inhibitory action, it is released from the F-Bak force GST-Bc 卜 X and bound.

As a result, the degree of polarization decreases. Figure 12 shows the experimental results. From this figure, it was found that clones 10 and 18 have inhibitory activity. Next, curve fitting is performed using equation (2), and IC

Asked for 50.

[0096] F (x) =-{x * (Pmax-Pmin)} I (Kd + x) + Pmax (2)

Where the unknown coefficients are Pmax, Pmin, and IC, and the independent variable is x (peptide concentration).

50

[0097] Under these conditions, the IC of the Bak peptide was 29.7 μM (in PBS buffer) and 11.8 μΜ (2% D

50

PBS buffer containing MSO). In contrast, the IC of clone 18 peptide is 6.4 μΜ (

50

While the clone 10 peptide has an IC of 0.9 μΜ (containing 2% DMSO)

50

S buffer). It can be inferred that both have higher affinity for GST-Bc 卜 X than the Bak peptide under the same conditions. In addition, clone 17 peptide had no such inhibitory activity. Although clone 16 peptide showed some inhibitory activity, it was difficult to analyze on the high concentration side where solubility was extremely low, and IC could be obtained.

50

There wasn't.

[0098] Summarizing the above analysis results, the peptides of clones 10 and 18 are Be 卜 X, Bak and Bad.

More speculation of the function of inhibiting protein heterodimer formation. Although clone 16 has low solubility, it can be presumed that it still has inhibitory activity. Clone 17 was an inhibitory activity in the state of the in vitro virus molecule. The reason is considered as follows. The peptide of clone 17 repels the positively charged part of Bcl-x, which has a high basic amino acid content. It cannot be combined with Bcl-x. In the in vitro virus molecule state,

L

The acid group blocks the positive charge of the peptide and can bind without repelling Bcl-x.

Shi

[0099] As described above, the mode of inhibition is that the obtained peptide binds to the hydrophobic pocket of Be 卜 X and is baked.

Forces that may directly inhibit the binding of Bad peptides and bind to other parts of Bcl-x

In combination, the structure of Be 卜 X changes, thereby inhibiting the binding of Bak and Bad peptides.

Therefore, there is a possibility of this.

Industrial applicability

[0100] According to the present invention, formation of a heterodimer between Bcl-x and Bak or Bad protein is inhibited.

L

It can restore the pro-apoptotic effect of Bak and Bad proteins and promote anticancer drug treatment. In addition, if the three-dimensional structure of the complex of the peptide of the active ingredient and Be 卜 X is analyzed, then

It becomes possible to design a new drug using the above peptide as a lead compound. In addition, it is possible to design drugs with higher affinity by combining partial binding modes with those of existing peptides and small molecule compounds.

Claims

The scope of the claims

[1] It has an amino acid sequence of RXXX (X is a hydrophobic amino acid) (SEQ ID NO: 48). Be 卜 X heterodimer formation inhibitor containing a peptide as an active ingredient.

L

[2] A Bd-x heterodimer formation inhibitor 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.

Shi

[3] Be 卜 X heterodimer comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 18 as an active ingredient

Formation inhibitor.

[4] It has an amino acid sequence of RXXX (X is a hydrophobic amino acid) (SEQ ID NO: 48), has a force 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. Anticancer agent action promoter containing a certain peptide as an active ingredient.

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

[6] An anticancer agent promoting agent comprising a peptide having the amino acid sequence ability 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 that bind the DNA and the peptide encoded by the DNA from the library, (3) Select a molecule containing a peptide that binds to Bcl -X, and (4) template the DNA of the selected molecule.

A DNA library is composed of (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 an integer from 8 to 24. The above method comprising DNA having a base sequence of

[8] DNA library is XXVXRXLXXXXDXIXX (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: The method according to claim 7, comprising DNA encoding the peptide comprising the amino acid sequence of 29).