WO2011132941A2 - Tf-bpb specifically binding to transcription factor - Google Patents

Abstract

The present invention relates to a TF-bipodal peptide binder specifically binding to TF, comprising: (a) a structure stabilizing region comprising parallel, antiparallel, or parallel and antiparallel amino acid strands in which an interstrand noncovalent bond is formed; and (b) a TF-target binding region I and a TF-target binding region II which bind respectively to both ends of the structure stabilizing region and comprise randomly selected n and m amino acids, respectively. The TF-bipodal peptide binder of the present invention shows a very low level (for example, nM level) of KD value (dissociation constant) to TF, thereby showing very high affinity to a TF target. The TF-bipodal peptide binder of the present invention has a medical use, can be used for in vivo molecular imaging and targeting for drug delivery, and also can be very usefully used as an escort molecule.

Description

 【Specification】

 [Name of invention]

 TF- specifically binds to transcription factors

 The present invention relates to TF 'BPB that specifically binds to transcription factor (TF).

[Background technology]

Antibodies are immunoglobulin proteins, a type of plasma protein produced by B cells, that specifically inactivate and inactivate antigens by specifically recognizing and binding specific sites of antigens from outside. The specificity and high affinity of these antigen-antibody reactions and the diversity of antibodies that can distinguish tens of millions of antigens have led to the emergence of many types of antibody products, including diagnostics and therapeutics. Currently, the FDA has approved 21 monoclonal antibodies, and antibodies such as Rituximab and Herceptin have been effective in more than 50% of patients who have not responded to other treatments. Has demonstrated successful clinical treatment of lymphoma, colon cancer or breast cancer using monoclonal antibodies. The overall market size of therapeutic antibodies is estimated to grow at an annual rate of 20%, from $ 10 billion in 2004 to $ 30 billion in 2010. The market is expected to grow exponentially. The development of new drugs using antibodies is active because the drug development period is short, investment costs are low, and side effects can be easily predicted. In addition, since the antibody is a herbal medicine, the human body is hardly affected, and the half-life in the body is overwhelmingly long compared to low molecular weight drugs, so the patient is friendly. Despite this usefulness, monoclonal antibodies in humans are recognized as foreign antigens and can cause severe allergic reactions or hypersensitivity reactions. In addition, when the anti-cancer monoclonal antibody is clinically used, the production cost is high, and thus the price of the therapeutic agent is rapidly increased. Since a wide range of technologies are protected by various intellectual property rights, expensive licensing fees are required.

 Therefore, in order to solve this problem, the development of antibody replacement proteins in the European Union, especially in the United States, is in its infancy. Antibody-replacement protein is a recombinant protein made to have constant and variable regions like an antibody. A small and stable protein is replaced with a random sequence of amino acids to make a library, which is then screened for the target material, thereby providing high affinity and good Substances with specificity can be found. For example, avimers and affibodies among antibody replacement proteins have been reported to have a picomol affinity for a target substance. It is reported that these antibody replacement proteins are small and stable and can penetrate deep into cancer cells and generally produce less immune responses. First of all, it is possible to escape from a wide range of antibody patent problems, and because it can be easily purified in large quantities from bacteria, it is economically superior to antibodies because of low production cost. There are currently 40 antibody replacement proteins that have been developed. Among them, the antibody replacement proteins that are being commercialized by venture companies and multinational pharmaceutical companies are fibronectin type m domain, lipocalin, LDLR—A domain, crystallin, protein A, and ankyrin repeat. (Ankyrin repeat), which uses a protein called BPTI and has a high affinity of several nanomolar to picomolar to the target. Among them, Adnectin, Avimer, and Kunitz domains are currently under FDA clinical trial.

The present invention focused on peptide-based antibody replacement proteins that are different from antibody replacement proteins using proteins up to now. Peptides have been widely used in place of antibody therapeutics due to their proper pharmacokinetics, mass productivity, low toxicity, antigenic inhibition and low production cost compared to antibodies. The advantages of peptides as therapeutic drugs are low production costs, high safety and reactivity, relatively low patent royalties, and less exposure to unwanted immune systems, which can inhibit the production of antibodies to the peptad itself, However, most peptides have low affinity and specificity for specific protein targets compared to antibodies, and thus cannot be used in various applications. therefore, There is a need in the art for the development of new peptide-based antibody replacement proteins that can overcome the disadvantages of peptides. Accordingly, the present inventors have tried to develop a peptide material capable of specific binding with high affinity to a biological target molecule. This is expected to be a technology that can produce new drug candidates with high affinity and specificity in a short time using peptides having low affinity reported for a large number of targets.

 On the other hand, transcript ion factor (TF) is a protein that binds to a specific DNA sequence and regulates transcription of a specific gene (Latchman DS Int. J. Biochem. Cell Biol. 29 (12): 1305-12 (1997) Karin M. New Biol. 2 (2): 126-31 (1990)). This transcription factor acts to promote or inhibit transcription. Transcription factors have one or more DNA-binding domains and bind to sequences adjacent to the gene to be controlled through these DNA-binding domains.

 Variations in transcription factors are often associated with specific diseases, and many drugs currently or developed in development target transcription factors. Many transcription factors may be tumor suppressors or oncogenes. Many of the transcription factors studied in relation to cancer in the eye are: a) NFKB and AP-1 families, (b) the STAT family and (c) steroids receptors. And patent references are cited. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly defined. It is explained.

[Detailed Description of the Invention]

The inventors have sought to develop a system capable of delivering various substances intracellularly or to the cell surface based on TP binding and specificity. As a result, the peptides were randomly bound to both ends of the structure stabilization site having a relatively rigid peptide backbone, and significantly increased when the two peptides were jointly bound to the TP molecule. By confirming that a bipodal peptide binder (BPB) having binding capacity and specificity can be obtained, the present invention was completed.

 Accordingly, an object of the present invention is to provide a transcript ion factor (TF) -bipodal peptide binder (TF-BPB).

 Another object of the present invention is to provide a TFCtranscription factor) -bipodal peptide binder (TF-BPB).

 Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings. According to one aspect of the invention, the invention comprises

TFCtranscription factor) -bipodal peptide binder (TF-BPB) is provided:

 (a) a structural stabilizing region comprising parallel lei, antiparallel or parallel and antiparallel amino acid strands having interstrand noncovalent bonds; and

 (b) TF-target binding region KTF-target binding region I) and TF-target binding region n (TF— TF—bipodal peptide binder ᅳ specifically binding to TF containing target binding region Π)

 The present inventors have tried to develop a system capable of transporting various substances into or on the cell surface based on TF binding and specificity. As a result, the structure stabilization site having a relatively rigid peptide backbone When the peptides were randomly bound at both ends and the two peptides were jointly bound to the TF molecule, it was confirmed that a bipodal peptide binder (BPB) having a greatly increased binding capacity and specificity was obtained.

The basic strategy of the present invention is to connect peptides bound to targets at both ends of a rigid peptide backbone. In this case, the rigid tempide backbone is responsible for the overall structure of the bipodal peptide provider. Stabilizes and enhances binding of target binding site I and target binding site π to the target molecule.

 Structural stabilization sites available in the present invention include parallel, antiparallel or parallel and antiparallel amino acid strands, interstrand hydrogen bonds, electrostatic interactions, hydrophobic interactions, van der Waals interactions, pi-pi Protein structure motifs in which non-covalent bonds are formed by interaction, cation-pi interaction, or a combination thereof. Non-covalent bonds formed by hydrogen bonds, electrostatic interactions, hydrophobic interactions, van der Waals interactions, pi-pi interactions, cation-pi interactions, or a combination of these strands are the rigidity of the structural stabilization site. Contribute to.

 According to a preferred embodiment of the invention, interstrand non-covalent bonds at the structure stabilization site include hydrogen bonds, hydrophobic interactions, van der Waals interactions, pi-pi interactions or combinations thereof.

 Optionally, there may be a covalent bond at the structured stabilization site. For example, disulfide bonds may be formed at the structured stabilization site to further increase the robustness of the structure stabilization site. The increase in the robustness by the covalent bond may be given in consideration of the specificity and affinity of the bipodal peptide binder to the target. do.

 According to a preferred embodiment of the invention, the amino acid strands of the structure stabilization site are linked by a linker. As used herein, the term "linker" as used to refer to the strands refers to the material that connects the strands. For example, when β-hairpin is used as a structure stabilization site, the turn sequence on the β-hairpin serves as a linker, and when leucine zipper is used, a substance connecting two C-terminus of leucine zipper (eg , Peptide linkers) serve as linkers.

Linkers link parallel, antiparallel or parallel and antiparallel amino acid strands. For example, at least two strands (preferably two strands) aligned in parallel fashion, at least two strands (preferably two strands) aligned in antiparallel fashion, and at least three strands aligned in parallel and antiparallel fashion. The linker (preferably 3 strands) is connected by the linker, According to a preferred embodiment of the invention, the linker is a turn sequence or peptide linker.

According to a preferred embodiment of the present invention, the turn sequence is β-turn, _Υ -turn, α -¾, π round or ω one loop.] (Venkatachalam CM (1968), Biopolymers, 6, 1425-1436; Nemethy G and Printz MP. (1972), Macro / no J ecu J es, 5, 755-

758; Lewis PN et al. , (1973), Biochim. Biophys. Acta, 303, 211-229;

Toniolo C. (1980) CRC Crit, Rev. Biochem. , 9, 1-44; Richardson JS.

(1981), Adv. Protein Chem. , 34, 167-339; Rose GD et al. , (1985), Adv.

Protein Chem. , 37, 1-109; Milner-White EJ and Poet R. (1987), TIBS, 12, 189-192; Wilmot CM and Thornton JM. (1988), J. Mo J. Biol. , 203, 221-

232; Milner-White EJ. (1990), J. MoL Biol. , 216, 385-397; Pavone V et al, (1996), Biopolymers, 38, 705—721; Rajashankar KR and Ramakumar S.

(1996), Protein Sci. , 5, 932-946). Most preferably, the turn sequence used in the present invention is β-turn.

 When β—turns are used as the turn sequence, preferably the type I, type Γ, type π, type π ', type m or type m' turn sequences, more preferably type I, type ι ', type π , Type π 'turn sequence, even more preferably type r or type π' turn sequence, most preferably type

Γ turn sequence (B. L. Sibanda et al., J. MoL Biol., 1989, 206, 4, 759-777; B. L. Sibanda et al., Methods Enzymol., 1991, 202, 59-82).

 According to another preferred embodiment of the present invention, what can be used as the turn sequence in the present invention is H, Jane Dyson et al., Eur. J. Biochem. 255: 462-

471 (1998), which is incorporated herein by reference. Available as turn sequences include the following amino acid sequences: Χ-Ρπ) -Gly-Glu-Val; Ala—X-Gly—Ghi-Val (X is selected from 20 amino acids). According to one embodiment of the invention, when a β-sheet or leucine zipper is used as the structure stabilization site, it is preferred that the peptide linker connects two strands arranged in a parallel manner or two strands arranged in an antiparallel manner. desirable.

Peptide linkers can be any known in the art. The sequence of a suitable peptide linker can be selected in consideration of the following factors: There are: (a) the ability to be applied to flexible extended conformat ions; (b) the ability not to create secondary structures that interact with biological target molecules; And (c) the absence of hydrophobic residues or residues with charges that interact with the biological target molecule. Preferred peptide linkers include Gly, Asn and Ser residues. Other neutral amino acids such as Thr and Ala can also be included in the linker sequence. Suitable amino acid sequences for linkers are Maratea et al., Gene 40: 39-46 (1985); Murphy et al. , Proc. Natl. Acad Sci. USA 83: 8258-8562 (1986); US Pat. Nos. 4,935,233, 4,751,180, and 5,990,275. The peptide linker sequence may consist of 1-50 amino acid residues.

 According to a preferred embodiment of the present invention, the structural stabilization site is a β-hairpin, a linker linked β_sheet or a linker leucine zipper, and more preferably the structural stabilizer site is a β-hairpin or linker linked β-sheet. And most preferably β-hairpin.

 As used herein, the term "β_hairpin" refers to the simplest protein motif comprising two β strands, the two β strands representing an antiparallel alignment with each other. In this β-hairpin the two β strands are generally linked by turn sequences.

 Preferably, the turn sequence applied to the β-hairpin is a type I, type Γ, type π, type π ', type m or type m' turn sequence, more preferably type I, type ι ', type π, Type π 'turn sequence, even more preferably type r or type π' turn sequence, most preferably type

Γ turn sequences, and turn sequences represented by X-Pro-Gly-Glu-Val; or Ala-X-Gly-Glu-Val (X is selected from 20 amino acids) can also be used for β-hairpins have.

According to an exemplary embodiment of the invention, the type I turn sequence is Asp—Asp-Ala-Thr-Lys-Thr, the type Γ turn sequence is Gki-Asn—Gly-Lys, and the type Π turn sequence is X-Pro -Gly-Gki-Val; or Ala-X-Gly-Glu— Val (X is selected from 20 amino acids), and the type Π 'turn sequence is Glu-Gly-Asn-Lys or Glu-D-Pn)- Asn-Lys, Peptides with β-hairpin formulations are well known in the art. See, for example, US Pat. No. 6,914,123 and Andrea G. Cochran et al. , PNAS, tryptophan zipper disclosed in 98 (10): 5578-5583, and the template-fixed β-hairpin mimetic disclosed in TO 2005/047503, β-hairpin variants disclosed in US Pat. No. 5,807,979. In addition, peptides with β—hairpin conformation are described by Smith & Regan (1995) Science 270: 980-982; Chou & Fassman (1978) Annu. Rev. Biochem. 47: 251-276; Kim & Berg (1993) Nature 362: 267-270; Minor & Kim (1994) Nature 367: 660-663; Minor & Kim (1993) Nature 371: 264-267; Smith et al. Biochemistry (1994) 33: 5510-5517; Searle et al. (1995) Nat. Struct. Biol. 2: 999-1006; Haque & Gel 1 man (1997) J. Am. Chem. Soc. 119: 2303-2304; Blanco et al. (1993) J. Am. Chem. Soc. 115: 5887-5888; de Alba et al. (1996) Fold. Des. 1: 133-144; de Alba et al. (1997) Protein Sci. 6: 2548-2560; Rami rez-Alvar ado et al. (1996) Nat. Struct. Biol. 3: 604-612; St anger & Gel 1 man (1998) J. Aπ Chem, Soc. 120: 4236—4237; Maynard & Searle (1997) Chem. Commun, 1297—1298; Griffiths-Jones et al. (1998) Chem. Commun. 789-790; Maynard et al. (1998) J. Am. Chem. Soc. 120: 1996- 2007; and Blanco et al. (1994) Nat. Struct. Biol. 1: 584-590, which is incorporated herein by reference.

 When the peptide having β-hairpin conformation is used as the structural stabilization site, most preferably, tryptophan zipper is used.

 According to a preferred embodiment of the present invention, the tryptophan zipper used in the present invention is represented by the following general formula (I):

 Formula I

X ₁ -Trp (X ₂ ) X3-X4-X ₅ (X'2) X6-X7

¾ is Ser or Gly-Glu, ¾ and X ' ₂ are independently of each other Thr, His, Val, lie, Phe or Tyr, ¾ is Trp or Tyr, is Type I, Type I', Type Π Type Π 'Or type m or type ΠΓ turn sequence, ¾ is Trp or Phe, ¾ is Trp or Val, and X ₇ is Lys or Thr-Glu.

 More preferably, ¾ in Formula I is Ser or Gly-Glu,

¾ and X'2 are independently of each other Thr, His or Val, and ¾ is Trp or Tyr, X4 is type I, type Γ, type Π or type Π 'turn sequence, ¾ is Trp or Phe, ¾ is Trp or Val, and X ₇ is Lys or Thr-Glu. Even more preferably, ¾ in Formula I is Ser or Gly-Glu, X ₂ and X'2 are independently of each other Thr, His or Val, ¾ is Trp, and X4 is Type I, Type Γ, Type Π or type Π 'turn sequence, ¾ is Trp, ¾ is Trp, and X ₇ is Lys or Thr-Glu.

 Even more preferably, ¾ in Formula I is Ser, ¾ and

X'2 is Thr, ¾ is Trp, X4 is type Γ or type Π 'turn sequence,

X ₅ is Trp, ¾ is Trp, and X ₇ is Lys.

Most preferably, in formula I, Xi is Ser, ¾ and X ' ₂ are

Thr is ¾ is Trp, is type Γ turn sequence (ENGK) or type Π 'turn sequence (EGNK), ¾ is Trp, ¾ is Trp and X ₇ is Lys.

 Exemplary amino acid sequences of tryptophan zippers suitable for the present invention are described in SEQ ID NOs: 1 to 3 and 5 to 10.

 Β-hairpin peptides usable as structural stabilization sites in the present invention are peptides derived from B1 domainin of protein G, ie GB1 peptides. When GB1 peptide is used in the present invention, the structural stabilization site is preferably represented by the following general formula:

 Formula Π

 X 厂 Trp— ¾-Tyr— ¾— Phe-Thr-Va 1 -¾

Xi is Arg, Gly-Glu or Lys-Lys, X ₂ is Gin or Thr, ¾ is type i, type ι ', type π, type π' or type m or type m 'turn sequence, is Gin, Thr-Glu or Gln-Glu.

 More preferably, the structural stabilization site of the general formula Π is

It is represented by ΙΓ:

 Formula Π

Xi-Trp-Thr-Tyr-X ₂ -Phe-Thr-Va 1 -¾

¾ is Gly-Glu or Lys-Lys, ¾ is type I, type Γ, type Π, type Π ′ or type ΠΠ or type ΠΓ turn sequence, ¾ is Thr-Glu or Gln-Glu. Exemplary amino acid sequences of GB1 β-hairpins suitable for the present invention are described in SEQ ID NO: 4 and 14 to 15 sequences.

 Β-hairpin peptides usable as structural stabilization sites in the present invention are HP peptides. When the HP peptide is used in the present invention, the structural stabilization site is preferably represented by the following general formula m:

 General formula m

Xi-X2-X3-Trp-X4-X ₅ -Thr-X ₆ -X7

¾ is Lys or Lys-Lys, ¾ is Trp or Tyr, ¾ is Val or Thr, is type I, type Γ, type II, type Π 'or type m or type ΠΓ turn sequence, ¾ is Trp or Ala, ¾ is Trp or Val, and X ₇ is Glu or Gln-Glu.

 Another β-hairpin peptide that can be used as a structural stabilization site in the present invention is represented by the following general formula IV:

 Formula IV

 ¾ is Lys-Thr or Gly, ¾ is Trp or Tyr, ¾ is type I, type I ', type Π, type Π' or type m or type ΠΓ turn sequence, and is Thr-Glu or Gly.

 Exemplary amino acid sequences of β-hairpins of the general formulas [pi] and IV are set forth in SEQ ID NOs: 11-12, 15-15 and 16-19.

 According to the present invention, a -sheet connected by a linker can be used as the structural stabilization site. It is an extended form of two amino acid strands, parallel or antiparallel, preferably antiparallel, in the β-sheet structure, and hydrogen bonds are formed between the amino acid strands.

 In the β-sheet structure, two adjacent ends of two amino acid strands are connected by a linker. As the linker, various turn-sequences or peptide linkers described above may be used. If the turn-sequence is used as a linker, the β-turn sequence is most preferred.

According to another variant of the invention, a leucine zipper or a leucine zipper connected by a linker may be used as the structural stabilization site. It is a conservative peptide domain that causes two parallel α-chain dimerizations, and is usually a dimerization domain found in proteins involved in gene expression ("Leucine scissors". Glossary of Biochemistry and Molecular Biology (Revised). Ed. David M. Glick.London: Portland Press; Landschulz WH, et al. (1988) Science 240: 1759—1764). Leucine zippers generally comprise a heptad repeat sequence, with the leucine residues located at the fourth or fifth. For example, leucine zippers that may be used in the present invention comprise the amino acid sequence of LEALKEK, LKALEKE, LKKLVGE, LEDKVEE, LENEVAR or LLSKNYH. Specific examples of leucine zippers used in the present invention are described in SEQ ID NO: 39 Sequence. Each half of the leucine zipper consists of short _α -hexens with direct leucine contact between the α-chains. The leucine zipper in the transcription factor generally consists of a hydrophobic leucine zipper site and a basic site (site that interacts with the main groove of the DNA molecule). When the leucine zipper is used in the present invention, the basic site is not necessarily required. In the leucine zipper structure, two adjacent ends of two amino acid strands (ie, two α-chains) may be linked by a linker. As the linker, various turn-sequences or peptide linkers described above may be used, and preferably, a peptide linker that does not affect the structure of the leucine zipper is used.

 Random amino acid sequences are joined to both ends of the structure stabilization site described above. The random amino acid sequence forms TF-target binding site I and TF—target binding site Π. One of the greatest features of the present invention is to prepare a peptide binder in a bipodal manner by connecting TF-target binding site I and TF-target binding site Π at both ends of the structure stabilization site TF-target binding site I And TF-target binding site Π cooperatively binds to the target, thereby greatly increasing the affinity for TF.

The amino acid number n of the TF-target binding site I is not particularly limited, preferably an integer of 2-100, more preferably an integer of 2-50, even more preferably an integer of 2-20, most preferably Is an integer from 3-10. The number of amino acids m of the TF-target binding site Π is not particularly limited, preferably an integer of 2-100, more preferably an integer of 2-50, even more preferably an integer of 2-20, most preferably Preferably an integer of 3-10.

 The TF-target binding site I and the TF-target binding site Π may each contain different or the same number of amino acid residues. The TF-target binding site I and the TF-target binding site Π may include different or identical amino acid sequences, and preferably include different amino acid sequences from each other.

 The amino acid sequence included in the TF-target binding site I and / or TF-target binding site Π is a linear amino acid sequence or a cyclic amino acid sequence. In order to increase the stability of the peptide sequence of the target binding site, the TF-target binding site At least one amino acid residue in the amino acid sequence included in the I and / or TF-target binding site Π may be an acetyl group, a fluorenyl methoxy carbonyl group, a formyl group, palmitoyl group, myristyl group, stearyl group or polyethylene glycol ( PEG).

 TF-BPB of the present invention bound to a biological target molecule can be used for the regulation of physiological responses in vivo, detection of substances in vivo, imaging of in vivo molecules, and targeting for drug delivery, and also as an escort molecule. Can be.

 According to a preferred embodiment of the present invention, cargo is added to the structure stabilization site, TF-target binding site I or TF-target binding site Π (more preferably, the structure stabilization site, even more preferably the linker of the structure stabilization site). Are combined. Examples of the cargo include, but are not limited to, labels, chemicals, biopharmaceuticals or nanoparticles that generate detectable signals.

Labels that generate the detectable signal include T1 contrasts (eg, Gd chelate compounds), T2 contrasts (eg, superparamagnetics such as magnetite, Fe ₃ 0 ₄ , Y-Fe ₂ 0 ₃ , manganese ferrite, cobalt). Ferrites and nickel ferrites)), radioisotopes (e.g., ^U C, ¹⁵ 0, ¹³ N, P ³² , S ³⁵ , ⁴⁴ Sc, ⁴⁵ Ti, ¹¹⁸ I, ¹³⁶ La, ¹⁹⁸ T1, ²⁰⁰ T1, ²⁰⁵ Bi and ²⁰ ¾0, fluorescent material (fluorescein, phycoerythrin, rhodamine, lysamine (lissamine), and Cy3 and Cy5), chemiluminescent groups, magnetic particles, mass labels or electron-dense particles.

 TF-target binding site I and / or TF-target binding site Π comprises an amino acid sequence that binds to TP.

 The TF to which the BPB molecule of the present invention binds includes various TFs known in the art, preferably API, AP-2, ARE, Brn-3, C / EBP, CBF, CDP, c-Myb, CREB, E2F-1, EFR, ERE, Ets, Ets-1 / PEA3, FAST-1, GAS / ISRE, GATA, GRE, HNF-4, IRF-1, MEF-1, MEF-2, Myc-Max, NF- 1, NFATc, F-E1, NF-E2, NFKB, Oct-l, p53, Pax-5, Pbxl, Pit 1, PPAR, PRE, RAR, RAR (DR-5), SIE, Smad SBE, Smad3 / 4 , SP1, SRE, Statl, StatS, Stat4, Stat4, Stat5, Stat6, TFIID, TR, TRCDR-4), USF-1, VDR (DR-3), HSE, and MRE.

 The TF-BPB of the present invention acts as an agonist or antagonist for TF. Although the TF-BPB of the present invention may bind to the extracellular domain of TF exposed to the cell surface, it also binds to the intracellular domain and acts as a GPCR. Can be adjusted.

 When TF-BPB targets an intracellular domain, preferably TF-BPB additionally comprises a cell transmembrane peptide (CPP).

 The CPP includes various CPPs known in the art, for example HIV-1 Tat protein, oligoarginine, ANTP peptide, MTS peptide derived from HSV VP22 transcriptional regulator protein vFGF, Penetratin, Transport an, Pep— 1 Peptides, Pep-7 peptides, Buforin II, model amphiphatic peptide (MAP), k-FGF, Ku 70, pVEC, SynBl or HN-1. There are various methods for binding the CPP to the bipodal peptide, for example, covalently linking the CPP and the lysine residue in the loop portion at the structural stabilization site of the bipodal peptide.

 As described above, the bipodal peptide binder of the present invention is typically referred to as "one strand of the N-TP-target binding site I-structure stabilization site, the other strand of the structural linkage-TP-target binding site Π-C". Has a construct of

According to a preferred embodiment of the invention, one strand of the TF—target binding site I and the structural stabilization site in the TF-bipodal peptide binder of the invention Between and / or between the other strands -TF-target binding site Π of the structural stabilization site, it contains a structure influence inhibiting regk »n that blocks the cross-structural effects between the TF-target binding site and the structural stabilization site. do. At the site of rotation are amino acids that are relatively free of rotation of ø and ¾ ^ in the peptide molecule. Preferably, the amino acids with relatively free rotation of 0 and ^ are glycine, alanine and serine. 1-10, preferably 1-8, more preferably 1-3 amino acid residues may be located in the structure influence inhibitory site.

 Libraries of the TF-bipodal peptide binders of the present invention having the constructs described above can be obtained by various methods known in the art. In this library, the TF-bipodal peptide binder will have a random sequence, which has no sequence preference or designation (or immobilization) at any position of TF—target binding site I and / or TF-target binding site Π. It means no amino acid residues.

 For example, a library of TF-bipodal peptide binders can be used in a split-synthesis method performed on a solid phase support (eg, polystyrene or polyacrylamide resin) (Lam et al. (1991) Nature 354: 82; WO 92/00091; Can be built according to

 According to a preferred embodiment of the invention, the library of TF-bipodal peptide binders is constructed in a cell surface display manner (eg phage display, bacterial display or yeast display). Preferably, the library of TF-bipodal peptide binder may be prepared through a display method based on plasmid, bacteriophage, phagemid, yeast, bacteria, mRNA or ribosomes.

Phage display is a technique for displaying various polypeptides in the form of proteins fused to coat proteins on the surface of the phage (Scott, JK and Smith, G, P. (1990) Science 249: 386; Sambrook, J. et al., Molecular Cloning.A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001); Clackson and Lowman, Phage Display, Oxford University Press (2004). Gene m or gene of filamentous phage (eg, M13) A random peptide is displayed by fusing the gene to be expressed in the 珊.

 Phageimide may be used for the phage display. Phageimide is a plasmid vector having one copy of the bacterial origin of replication (eg, ColEl) and the intergenic region of the bacteriophage. The DNA fragment cloned in this phagemid is propagated like a plasmid.

 When constructing a library of TF-bipodal peptide binders by phage display, a preferred embodiment of the present invention comprises the following steps: (i) a phage coat protein (e.g., gene ΙΠ of filamentous phage such as M13 or A fusion gene in which a gene encoding gene VI coat protein) and a gene encoding a bipodal piptide binder are fused; And constructing a library of expression vectors comprising transcriptional regulatory sequences (eg, lac promoters) operably linked to the fusion gene; (ii) introducing said expression vector library into a suitable host cell; (iii) culturing the host cell to form recombinant phage or phagemid virus particles so that the fusion protein is displayed on the surface; (iv) contacting the TF molecule with the viral particle to bind the particle to the target molecule; And (V) separating particles not bound to TF molecules.

 Methods for constructing and screening peptide libraries using phage display are described in US Pat. Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018, and 5,698,426. , 5,763,192 and 5,723,323,

 The method of preparing an expression vector comprising a bipodal pipide binder gene may be performed according to methods known in the art. For example, known phagemid or phage vector (eg, pIGT2, fUSE5, fAFFl, Expression vectors can be constructed by inserting bipodal peptide binder genes into fd-CAT1, m663, fdtetDOG, pHENl, pCombS, pComb8, pCANTAB 5E (Pharmacia), LamdaSurfZap, pIF4, PM48, PM52, PM54, fdH and p8V5). .

Most phage display methods are performed using filamentous phage, but lambda phage display (TO 95/34683; US Patent 5,627,024), T4 phage display (Ren et al. (1998) Gene 215: 439; Zhu (1997) CAN 33: 534) and T7 phage display (US Pat. No. 5,766,905) also construct libraries of bipodal peptide binders. It can be used to.

 The method of introducing the vector library into a suitable host cell can be carried out according to a variety of transformation methods, most preferably according to the electroporation method (see US Pat. Nos. 5,186,800, 5,422,272, 5,750,373), suitable hosts for the invention are gram negative bacterial cells such as E. coli, and suitable E. coli hosts are JM101, E. coli K12 strain 294, E. coli strain W3110 and E. coli XL-lBlue. (Stratagene), including but not limited to. Host cells are preferably prepared as competent cells prior to transformation (Sambrook, J, et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (200D). Selection of transformed cells It is generally carried out by culturing in a medium containing antibiotics such as tetracycline and ampicillin Selected transformed cells are further cultured in the presence of helper phage to generate recombinant phage or phagemid virus particles. Suitable for include, but are not limited to, Ex helper phage, M13-K07, M13-VCS, and R408. The selection of viral particles that bind to biological target molecules can typically be carried out via a biopanning process ( Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001); Clackson and Lowman, Ph age Display, Oxford University Press (2004).

 According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the aforementioned TF-bipodal peptide binder.

 According to another aspect of the invention, the invention provides a vector for the expression of a TF-bipodal peptide binder comprising a nucleic acid molecule encoding a TF-bipodal peptide binder.

According to another aspect of the present invention, the present invention provides a transformant comprising a vector for expression of a TF-bipodal peptide binder. As used herein, the term “nucleic acid molecule” is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides, which are the basic structural units in nucleic acid molecules, are modified from sugar or base sites, as well as natural nucleotides. Analogs are also included (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

According to a preferred embodiment of the present invention, the vector of the present invention is a powerful promoter capable of transferring transcription to the nucleic acid molecule in addition to the nucleic acid molecule encoding the TF-bipodal peptide binder (e.g., tac promoter, lac promoter, JadN5 promoter , // ¾σ promoter, _Ρί ^λ promoter, ρ / promoter, rad promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), ribosomal binding site and transcription / detox termination sequence for initiation of translation It includes.

 According to a preferred embodiment of the invention, the vector of the invention may further comprise a signal sequence (eg pelB) on the 5'-direction of the nucleic acid molecule encoding the TF-bipodal peptide binder. In addition, according to a preferred embodiment of the present invention, the vector of the present invention further comprises a tagging sequence (eg, myc tag) for confirming that the bipodal peptide binder is well expressed on the surface of the phage.

 According to a preferred embodiment of the invention, the vector of the invention comprises a phage coat protein, preferably a gene encoding a gene ΙΠ or gene VI coat protein of filamentous phage such as M13. According to a preferred embodiment of the present invention, the vector of the present invention comprises an origin of replication of bacteria (eg ColEl) and / or a bacteriophage. On the other hand, the vector of the present invention may include antibiotic resistance genes commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neo May include resistance genes for mycin and tetracycline,

The transformants of the present invention are preferably Gram-negative bacterial cells such as E. coli, and suitable E. coli hosts are JM101, E. coli K12 strain 294, E. coli strain W3110 and E. coli XL-lBlue (Stratagene ), But It is not limited. The method of carrying the vector of the present invention into a host cell may be performed by CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973)), one method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973); and Hanahan, D., J. Mo Biol., 166: 557-580 (1983)) and electroporation methods ( US Pat. Nos. 5,186,800, 5,422,272, 5, 750, 373), and the like.

The TF-bipodal peptide binders of the present invention exhibit very low levels (e.g., nM levels) of values (dissociation constants) to provide peptides with very high affinity to TF molecules. The bipodal temptide binder shows a high affinity of about 10 ² -10 ⁵ times (preferably about 10 ³ -10 ⁴ times) as compared to the binder made in the monopodal manner.

 The TF-bipodal peptide binder of the present invention not only has a use as a medicament, but also can be used for the detection of substances in vivo, in vivo molecular imaging, in vitro cell imaging and drug delivery, and as an escort molecule. Can be used.

 In summary, the features and advantages of the present invention are as follows:

 (i) The present invention provides TF-BPB that specifically binds to TP.

 (ii) In the TF-bipodal peptide binder of the present invention, two distant TF-target binding sites bound to both ends of the structural stability site are cooperatively and synergistically. ) To the target.

 (iii) Thus, the TF-bipodal peptide binder of the present invention exhibits very low levels (eg, nM levels) of ¾ values (dissociation constants), resulting in very high affinity to the target.

 (iv) TF-BPB of the present invention binds to TF in vivo and can act as an antagonist or agonist on TF.

[Brief Description of Drawings]

La shows a schematic diagram of a bipodal-peptide binder and TF-BPB containing β-hairpin as a structural stabilization site. FIG. Lb shows a schematic diagram of TF-BPB including β-sheets linked by linkers as structural stabilization sites.

 Figure lc shows a schematic of TF-BPB comprising leucine zippers linked by linkers as structural stabilization sites.

 Figure ID shows a schematic of TF-BPB comprising a leucine-rich motif (1 (^ 1 -1 ^ (: 11 motif)) linked by a linker as a structural stabilization site.

 2 shows a strategy for cloning the TF-BPB library. In the pIGT2 pyrimid vector map, the pelB signal sequence, myc tag, is a tagging sequence for confirming that the target gene is well expressed on the surface of the phage. The lac promoter was used as a promoter.

 Figure 3a is a result of ELISA experiments to analyze the target specificity of TF-BPB specifically binding to NF-kB.

 Figure 3b is the result of ELISA experiments analyzing the target specificity of TF-BPB specifically binding to STAT3.

 Figure 3c is the result of ELISA experiments to analyze the target specificity of TF-BPB specifically binding to AR DBD.

 4A shows certain bipodals that bind to pyronectin ED-B protein. The affinity of the peptide binder was measured.

 Figure 4b is the result of measuring the affinity for the demonstration of the synergistic effect of the bipodal peptide binder BPB.

 5 is a result of measuring the affinity of the bipodal peptide binder by replacing the structural stabilization site with a variety of β-hairpin motifs instead of tryptophan zipper in the bipodal peptide binder.

 6 is a result of measuring the affinity of the bipodal peptide binder by replacing the structural stabilization site with leucine zipper instead of tryptophan zipper in the bipodal peptide binder.

 Figure 7a is the result of a decrease in the amount of VEGF expression due to inhibition of STAT3 activity by the STAT3 bipodal peptide binder.

Figure 7b is a result of EMSA test due to the inhibition of the binding of AR DBD and DNA by AR DBD bipodal peptide binder. EXAMPLE

 Hereinafter, the present invention will be described in more detail with reference to Examples. These Examples are only for explaining the present invention in more detail, and the scope of the present invention is not limited to these Examples according to the gist of the present invention. It will be apparent to those skilled in the art to which the present invention pertains. EXAMPLE

Experimental Materials and Methods

Example 1: Construction of the Library

 Bipodal Peptide Binder Gene Production and Insertion into Phagemid Vectors

Two ligonucleotides Beta-F! (5'-TTCTATGCGGCCCAGCTGGCC (NNK) ₆ GGATCTTGGACATGGGAAAACGGAAAA-3 ') and Beta-Bl (5'-

AACAGTTTCTGCGGCCGCTCCTCC TCC (MNN) ₆ TCCCnCCATGTCCA1TrTCCGTT-3 ') (N is A, T, G or C; K is G or T; M is C or A). To make a double chain, mix beta_Fl 4 μΜ, Beta-Bl 4 μΜ, 2.5 mM dNTP mixture 4 μί, ExTaq DNA polymerase 1 / ^ (Takara, Seoul, Korea), and 10XPCR buffer 5 ^ to distilled water A total of 25 mixtures were added. It is a common hapaek PCR banung: After creating (5 minutes at 94 ^° C, 60 cycles 30 ^° 30 sec at C, 72 ^° in the C 30 sec and 72 ^° C 7 minutes) to the double-stranded PCR purification kit (GeneAll , Seoul, Korea) to obtain a bipodal peptide binder gene. In order to connect the gene to be inserted into the baapodal peptide binder to the IGT2 phagemid vector (Ig therapy, Chuncheon, Korea), the restriction gene was treated to the insert gene and the PIGT2 phagemid vector. (New England Bio labs (NEB, Ipswich) and? / (ΝΕΒ, Ipswich) were each reacted for 4 hours and purified using a PCR purification kit. Further, about 40 // g of pIGT2 phagemid vector was purified using Sfil and After each reaction with Notl for 4 hours, CIAP (Calf Intestinal Alkaline Phosphatase) (NEB, Ipswich) was added and reacted for 1 hour, and then purified using a PCR purification kit. Bioscience) to quantify 2,9 insert genes DNA ligase (Bioneer, Dae j eon, Korea) was used to connect to pIGT2 phagemid vector 12 at 18 ^° C for 15 hours, and then precipitated with ethanol to dissolve DNA with 100 ^ TE buffer. Preparation of Competent Cells

E. coll XL1—BLUE cells (American Type Culture Collection, Manassas, USA) were plated on LB agar-plates. After the inoculating colonies grown in an agar plate medium in LB medium with 5 ι heunhap at 37 ^° C at a rate of 200 rpm and incubated for one day. Cultured 10 cells were inoculated in 2 LB medium and incubated in the same manner at a wavelength of 600 nm until the absorbance was 0.3-0.4. The cultured flask was left on ice for 30 minutes and then at 4 ^° Centrifugation at 4,000 × g for 20 minutes to remove all supernatants except for the sunk cells and suspend with 1 cooled sterile distilled water. Centrifuge this again in the same way, remove the supernatant, resuspend in 1 sterile sterile distilled water, centrifuge in 10% glycerol with solution 40 in the same manner and repeat centrifugation. After suspension in 4, 200 ^ aliquots were added to the liquid nitrogen and stored at -80 ^° C. Electroporation method

Electroporation was performed by dispensing 25 phagemid vectors 12 // g and 25 100 峰 reactions in which the insert DNA 2,9 // g was connected to the bipodal peptide binder. Competent cells were dissolved on ice, mixed with 200 ^ of competent cells and mixed with solution 4 ^, and then placed in a specially prepared 02 cm cuvette and placed on ice for 1 minute. The electroporator (BioRad, Hercules, Calif.) Was programmed at 200 Ω at 25 and 2,5 kV, drained the prepared cuvettes, placed in the electroporator and pulsed (time constant is 4.5-5 msec). Thereafter immediately placed in 1 LB medium containing 20 mM glucose prepared ^at 37 ^° C. A total of 25 m cells obtained were transferred to a 100 ml test tube. Ampicillin agar by diluting 10 ^ to measure the number of libraries after incubation at 37 ^° C for 200 hours at 37 rpm. The medium was plated. The remaining cells were put in 1 LB of 20 mM glucose and 50 / ampicillin and incubated at 30 ^° C for one day. Centrifuge at 4,000Xg at 4 ^° C for 20 min to remove all supernatants except precipitated cells, resuspend in 40 m £ LB and store glycerine ^at -80 ^° C with a final concentration of at least 20% It was. Recombinant Phage Production and PEG Precipitation in Libraries

Recombinant phage was produced in a bipodal peptide binder library stored ^at -80 ^° C. Ampicillin (50 ug / mt) and 20 mM glucose was added to a 100 M LB liquid medium in a 500 M flask at -80 ^° C. heunhap for an hour by adding a stored library 1 at 37 ^° C at a rate of 150 rpm and incubated. Ex helper phage (Ig therapy, Chuncheon, Korea) of ixi0 ^u pfu was added thereto and incubated under the same conditions for one hour. Centrifuge at l, 000Xg for 10 minutes to remove supernatant, add 100 LB medium containing ampicillin (50 g / mi) and kanamycin (25 // g / m £), and incubate for one day to incubate recombinant phage. Produced. The supernatant obtained by centrifuging the culture solution at 3,000 Xg for 10 minutes was mixed with PEG / NaCl 25 and left on ice for 1 hour, and then centrifuged at 10, 000 X g for 20 minutes at 4 ^° C. Was carefully removed and the pellet resuspended in 2 n ^ PBS (pH 4). Example 2: Bio Panning

 Bio-panning was performed for NFKB, STAT3, and Androgen receptor (AR) DNA binding domains as representative receptors of TF. In addition, Fibronectin ED-B was selected as a model protein for confirming the basic operation of BPB, and also biopanning was performed.

Biopanning was performed on the BPB (Bipodal-peptide binder) library prepared in Example 1 for each protein five times and the output phage / input phage ratio of the phage peptides recovered in each panning step was determined. It was. The protein (5 / m £) was placed in 10 wells of a 96 well ELISA plate (Corning) by 50, left overnight at 4 ^° C., the next day after blocking for 2 hours at room temperature using 2% BSA The solution was discarded and washed three times with 0.1% PBST. Here, 800 ιΛ and 10% BSA 200 ≠ of bipodal peptide binder recombinant phage containing solution were mixed and transferred to 10 wells bound to the respective proteins, and allowed to stand at room temperature for 1 hour. Remove all 10 wells of the solution, wash 10 times with 0,1% PBST, elute the phage for 20 minutes with 50 ^ of 0.2 M glycine / HCKpH 2.2) 1 ^ per well and collect 1 in a tube Neutralize the solution by adding 150 ^ of Tris-base (pH 9.0). To determine the number of input and illite phages for each biopanning, mix them into XL-1 BLUE cells with 0D = 0,7 containing ampicillin. Plated in agar medium. In order to repeat the panning, 10 E ᅳ coli XL1-BLUE cells were mixed and incubated at 37 ^° C for 1 hour at a speed of 200 rpm. Ampicillin (cultured and heunhap at a rate of 50 and 20 mM and then the combined common glucose, 2X10 ¹⁰ pfu 200 rpm for one hour at 37 ^° C by the addition of Ex helper phages in. Centrifuge for 10 minutes, the culture solution in l, 000Xg The supernatant was then removed and the precipitated cells were ampicillin (50 fg /) and kanamycin (25

Resuspend with 40 LB liquid medium included and incubated for one day at 30 ^° C. at 200 rpm. Cultures were centrifuged at 4,000 × g, 20 min and 4 ^° C. 5x PEG / NaCl [20% PEG (w / v) and 15% NaCl (w / v)] of 8 was added to the supernatant and then left at 4 ^° C. for 1 hour. After centrifugation, the PEG solution was completely removed and the phage peptide pellets were dissolved in 1 PBS solution and used for the second biopanning. The same method was used for each panning step and the washing process was increased 20 times and 30 times (0,1% PBST), respectively, step by step, Example 3: NF B, STAT3, AR DBD or Fibronectin ED—B Specific Phage Peptide Search (Phase ELISA)

Phages recovered at the biopanning stage with the highest output phage / input phage ratio were infected with XL1-BLUE cells and plated at about 100-200 plaques per plate. Using a sterile tip, 60 plaques 2 LB-ampicillin (inoculated in 50 / O broth and shaken at 37 ^° C for 5 hours and added 5X10 ⁹ pfu Ex helper phage at OD = 0,8-1 for 200 hours at 37 ^° C speed heunhap and incubated in the rpm. culture for l, and then centrifuged for 10 min 000Xg supernatant was removed and the ampicillin the precipitated cells (50 ^ g / mi) and kanamycin (25 βg / i) comprises ^ύ) Resuspended in 1 LB liquid medium and incubated at 30 ^° C. at a speed of 200 rpm and incubated for one day. The supernatant was recovered by centrifuging the culture at 10,000 × g, 20 min and 4 ^° C., and then 2% skim milk was added and used for phage peptide search. In a 96 well ELISA plate, 5 g / of each protein was placed in 30 wells, 50 / ^ for each well, and 10 BSAs were 50 ^ for each well.

Put into 30 wells and left for 4 days at 4 ^° C. The following day all wells were washed three times with 0.1% PBST and blocked for 2 hours at room temperature using 2% skim milk distilled with PBS, then discarded the solution and washed three times with 0.1% PBST. 100 ^ of amplified phage peptide solutions for each clone were dispensed into all wells and left at 27 ^° C for 1 hour 30 minutes. After washing 5 times with 0.1% PBST solution, HRP-conjugate anti-M13 antibody (GE Healthcare) was diluted 1: 1,000 and reacted at 27 ^° C for 1 hour. After washing 5 times with 0.1% PBST, 100 ≠ Λ aliquots of the TMB solution were used to induce color reaction, and then the reaction was stopped by adding 100 M of 1 M HC1. Absorbances were measured at 450 nm to select clones with higher absorbance compared to BSA. Their phages were infected with XL1 cells and plated at about 100-200 plaques per plate. Plaques were inoculated into 4 LB-ampicillin (50 Mg / mO broth) using a sterile tip and shaken at 37 ^° C for 1 day to purify the plasmids using the plasmid flap kit to request simulants (Genotech, Dae j eon, Korea) The sigmoid primer used the vector sequence 5'-TGC TAA ACA ACT TTC MC-3 'Example 4 Binding Assay

A bipodal peptide binder peptide specific to ED—B, AR DBD, STAT3, and NFKB overlapped with DNA sequencing was synthesized (Anigen, Korea). Affinity measurements were performed using BIAcore X (Biacore AB, Uppsala, Sweden). Was carried out. ED-B was immobilized with as much as 2,000 RU of biotin-EDB on a straptavidin SA chip (Biacore). STAT3, AR DBD and NFKB were immobilized by adding 2,000 RU of each protein to CM5 chip (Biacore). BS PBS (pH 7, 4) was used as the running buffer, and the flow was flowed at 30 μί / min at various concentrations. After measuring the kinetics, the affinity was calculated with BIAevaluation software (Biacore AB, Uppsala, Sweden). Example 5: Inhibition of activity of bipodal peptide binder specific for intracellular NF-kB

 Since NF-KB is a protein present in the cell, 9 arginine (eel 1 penetrating peptide), 9 arginine (Anigen, Korea), was used by using the lysine residue of the loop of the bipodal peptide binder. EDC / NHS (Sigma) was used to covalently bind the bipodal peptide binder to enable cell permeation. Since the amount of M P-13 increases when NF-kB activity is activated, it is possible to determine whether NF-kB activity is inhibited by measuring the amount of MMP-13. Cartilage cells were treated with IL-lbeta (10 ng / ml) (R & D systems, Minneapolis) to activate NF-kB activity. Then, 10 μM of chondrocytes were treated with NF-kB-specific bipodal peptide binder (peptide 1 in Table 3f), mRNA was isolated after 12 hours, and RT-PCR was performed for MMP-13 and GAPDH. Intracellular protein was obtained by disrupting chondrocytes, followed by Western blotting using an Anti-MMP 13 antibody (Abeam, ab3208, Cambridge) and a semi-dry transfer machine (Amersham Bioscience, Piscataway) to measure the amount of MMP-13. Example 6 Inhibition of AR-DBD and DNA Binding by a Bipodal Peptide Binder Specific to Androgen Receptor DBD

EMSA was performed to determine whether the synthesized bipodal peptide binders inhibit the binding of AR-DBD to DNA. 5 '-CCA GAA CAT C GAA CAC-3' 5 '- GTG TTC TTG ATG TTC TGG-3 'was synthesized. Then shift buffer (4 mMTris-HCl (pH7 „5), 80 mMNaCl, 0.5 mM ZnC12, 2 ᅳ 5 mM MgS04, 1 mM EDTA, 0.5 mM DTT, 1 lg Incubate in poly (dl-dC), 4% glycerol) in AR DBD, DNA and bipodal peptide binders. Then, the retardation is confirmed by electrophoresis on the agarose gel. Example 7: Inhibition of activity of bipodal peptide binder specific for intracellular STAT3

 Since STAT3 is a protein present in cells, it is possible to fusion 9 aginine, an eel 1 penetrating peptide, to the C-term of the bipodal peptide binder (Anigen, Korea) to synthesize the cells. Made it. Since the amount of VEGF is increased when STAT3 activity is activated, the amount of VEGF can be used to determine whether STAT3 activity is inhibited. A bipodal peptide binder specific for STAT3 is activated in cells with 10 μΜ. After 12 hours of treatment, mRNA was isolated and RT-PCR was performed for VEGF and GAPDH.

Example 8: Construction of Bipodal Peptide Binder Library

 As the structure stabilization site of the bipodal peptide binder, a stable beta-hairpin motif was used. In particular, tryptophan zippers (Andrea et al., Proc. Natl. Acad. Sci. 98: 5578—5583 (2001)), which stabilize the beta-hairpin motif structure by the interaction of tryptophan-tryptophan amino acids, were used. Variable regions were created in two portions by randomly arranging six amino acids in each of the N- and C-terminal portions of the backbone tryptophan zipper (FIG. La). This is called a bipodal peptide binder and has variable regions on both sides so that it can be cooperatively attached to the antigen and thus have high affinity and specificity. In addition, the structure stabilization site of the bipodal peptide binder may be configured in various ways as shown in FIGS.

Synthesized two random sequences were synthesized into a double chain by PCR reaction and then cut into restriction enzymes S and Not \ and cloned into pIGT2 phagemid vector to construct a library of ⁸ × 10 ⁸ or more (FIG. 2). Example 9: Bio Panning Results

 The bipodal peptide binder library was subjected to biopanning three to five times for fibronectin ED-B or NF-κΒ and the ratio of output phage / input phage of phage peptides recovered at each panning step was determined (Table la, lb, lc and Id).

[Table la]

Biopanning Results for Fibronectin ED-B Protein

3 times 6 X 10 ¹¹ 4 X 10 ⁸ 15 X 10— ⁴

4 times 2 X 10 ¹² 2.1 X 10 ⁹ 100 10 ^"5

Table Id

 Bio Panning Results for AR-DBD

 Panning Step Input Phage (pfu) Illusion Phage (pfu) Output

1 time 2.8X10 ¹¹ 1.1X10 ⁷ 4X10—5

2 times 1.6X10 ¹¹ 1.0X10 ⁷ 5.1X10 ^"5

Episode 3 1.6X10 ¹¹ ι.ι ^χ ιο ⁷ 65X10 ^"5

4 times 3.2X10 ¹¹ 4.4X10 ⁸ 130X10— ⁵

5 times 3.2 × 10 ¹¹ 4.3 × 10 ⁸ 128 × 10—5 Example 10: Phage Peptide Search (Phase ELISA) and Sequencing Specific to Target

 Phages recovered at the highest output / input ratio during the panning stage of each library were obtained in the form of plaques. ELISA was performed on BSA after amplifying 60 phages from each plaque. Clones with higher absorbance than BSA were selected and requested for DNA sequencing. From this a peptide sequence specific to each overlapped protein was obtained (Tables 2a, 2b, 2c and 2d),

Table 2a

Table 2b ^" ΰ ^" Γ Specific peptide sequence for NF-κΒ Peptide 1 LGQPFL GSWTWENGKWTWKG LKPSIT

Table 2d

 Joe Ξ

^" Peptide Sequence Peptide Specific for ο ~ ττ STAT3 1 HGFQWP GSWTWENGKWTWKG AYQFLK

Table 2d

 article

^"ΟΓΤ sΓ AR-DBD-specific peptide sequence of the peptide 1-peptide 2 YGFAPFGSWTWENG WTW GYSTQKP GGHNIQGSWTWENG WTW GWQHIWG Example 11: NF-kB, STAT3, AR DBD specificity tests by podal peptide binder

 When compared with other proteins, ELISA tests were performed on whether bipodal peptide binders were specific to each protein, and specific results were shown (FIGS. 3A, 3B and 3C). Example 12: Specific to NF-kB in cells Inhibition of Bipodal Peptide Binders

The results demonstrate the effect of specific bipodal peptide binders that inhibit the activity of NF-kB present in cells. A cell penetrating peptide can be attached to a bipodal peptide binder to enter a cell. It was confirmed that DNA binding activity was similarly inhibited when treated with Etoposide and TNFa-treated K562 cells treated with 10 μΜ NF-kB-specific bipodal peptide binder and NF-kB inhibitors MG132 and Bay. This result shows that the bipodal peptide binder can be used to inhibit the activity of the intracellular target. Example 13: Inhibition of Bipodal Peptide Binder Specific to STAT3 in Cells

 This is a result showing the effect of a specific bipodal peptide binder that inhibits the activity of STAT3 present in the cell (Fig. 7a) · If a cell penetrating peptide is fused to the bipodal peptide binder, it can enter the cell. have. When the cells were treated with a 10 μM STAT3-specific bipodal peptide binder, the expression of VEGF was confirmed. This shows that the bipodal peptide binder can be used to inhibit the activity of the intracellular target. Example 14 Inhibition of AR-DBD and DNA Binding by a Bipodal Peptide Binder Specific to Androgen Receptor DBD

 It is a result of demonstrating the inhibition of interaction between AR-DBD and DNA by a bipodal peptide binder specific for AR-DBD (FIG. 7B). EMSA showed that the bipodal peptide binder binds to AR-DBD. As the BPB treatment concentration increases, AR-DBD loses its binding ability with DNA. This shows that AR can block the ability to bind DNA. Example 15 Determination of the Affinity of Fibronectin ED-B

 The peptide for fibronectin ED-B was synthesized and affinity was measured using the SPR Biacore system (Biacore AB, Uppsala, Sweden). As a result of measuring affinity for fibronectin ED-B, peptide 1 showed 620 nM, peptide 2 showed 75 nM and peptide 3 showed 2.5 μΜ (FIG. 4A Example 16: SPRCSurface Plasmon Resonance). Confirmation of synergistic effect To demonstrate the synergistic effect of the bipodal peptide binder on the antigen, affinity was synthesized by synthesizing a peptide from which only one of the N- and C-terminal portions of the peptide 2 with respect to ED-B of Table 2a, which had the best affinity, was removed. Was measured.

The N-terminus had 592 μΜ and the C-terminal portion showed 12,8 μΜ (Fig. 4b). The synergistic effect exhibited by having bipodal in the bipodal peptide binder proved to be an affinity of 43 nM (FIG. 4A). Example 17 Binding Assays for Other β-Hairpins

 In addition to the tryptophan zippers, ED β- to other β-hairpin skeletons GBlm3 and HP7

Peptides were synthesized to have N-terminal Siemens (HCSSAV) and C-terminal Siemens (IIRLEQ) of Peptide2 that specifically binds to B (Anigen, Korea). That is, a bipodal peptide binder including tryptophan zipper The sequence of HCSSAVGSWTWENGKWTWKGI IRLEQ is a bipodal peptide binder containing GBlm3 and HCSSAVGKKWTYNPATGKFTVQEGIIRLEQ and a bipodal peptide binder containing HP7 is HCSSAVGKTWNPATGKWTEGIIRLEQ. The affinity of each peptide was measured using BIAcore X (Biacore AB, Uppsala, Sweden). Fixation was performed by pouring 2,000 RU of biotin-EDB onto the straptavidin SA chip (Biacore AB, Uppsala, Sweden). PBS (pH 7.4) was used as the running buffer, and the flow was flowed at 30 ≠ per minute, the kinetics were measured at various concentrations, and affinity was calculated by BIAevaluation. As a result of measuring the affinity, it was confirmed that GBlm3 had affinity similar to tryptophan zipper (43 nM) with 70 nM and HP7 with 84 nM (FIG. 5). This is the result demonstrating that all stable β-hairpin motifs are possible as structure stabilization sites. Example 18 Binding Assay for Bipodal Peptide Binders Comprising a Leucine Zipper as Structural Stabilization Site

CSSPIQGGSMKQLEDKVEELLSKNYHLENEVARLKKLVGER and 11 RLEQGGSMKQLEDKARELLKNYHLGERNHLENEV to have a Ν-terminal sequence (HCSSAV) and C-terminal sequence (IIRLEQ) of peptide 2 that specifically binds ED-B to leucine zippers as structural stabilization sites instead of the β-hairpin backbone. Synthesized (Anigen, Korea). Both peptides were made into dimers and then affinity was measured using BIAcore X (Biacore AB, Uppsala, Sweden). The affinity measurement showed that the leucine zipper had an affinity of 5 μΜ, which was lower than the similar affinity of tryptophan zippers (43 nM), but also leucine zippers. It can be seen that it can function as the structural stabilization site of the bipodal peptide binder (FIG. 6). As described above in detail specific parts of the present invention, it is apparent to those of ordinary skill in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims

[Range of request]

 [Claim 1]

 Method for preparing a transcription factor-bipodal peptide binder (TFBPB) that specifically binds to a transcription factor (TF) comprising the following steps:

 (a) (i) a structure stabilizing region comprising parallel, antiparallel or parallel and antiparallel amino acid strands on which interstrand noncovalent bonds are formed ; (ii) TF-target binding region I and TF-target binding region n (TF), each of which binds to both ends of the structure stabilization site and comprises randomly selected n and m amino acids, respectively. providing a library of TF-bipodal peptide binders (TF-BPB) comprising -target binding region Π);

 (b) contacting said library with a transcription factor (TF) as a target; And,

 (c) selecting the TF-bipodal peptide binder (TF-BPB) bound to the TF.

[Claim 2]

 The non-covalent bond between the strands formed in the structural stabilization site is hydrogen bond, electrostatic interaction, hydrophobic interaction, van der Waals interaction, pi-pi interaction, cation-pi interaction or their Method characterized by being combination。 [claim 3]

 The method of claim 1 wherein the amino acid strands of the structural stabilization site are linked by a linker.

[Claim 4] The method of claim 1, wherein the structural stabilization site is characterized in that the β-hairpin, β-sheet connected by a linker, leucine zipper, leucine zipper connected by a linker, leucine rich motif or leucine rich motif linked by a linker Way. [Claim 5]

 The method of claim 4, wherein the structural stabilization site is β-hairpin.

[Claim 6]

 The method of claim 5, wherein the β-hairpin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 19 sequences.

[Claim 7]

 7. The method of claim 6, wherein the β-hairpin comprises an amino acid sequence selected from SEQ ID NO: 2, 14 or 18 sequence.

[Claim 8]

 The method of claim 5, wherein the -hairpin is represented by the following general formula I:

 Formula I

Xi-Trp (X ₂ ) X3-X4-X5 (X'2) X6-X7

¾ is Ser or Gly-Glu, ¾ and X ' ₂ are independently of each other Thr, His, Val, lie, Phe or Tyr, ¾ is Trp or Tyr, X4 is Type I, Type ι', Type π, Type π 'or type m or type m' turn sequence, ¾ is Trp or Phe, ¾ is Trp or Val, X ₇ is Lys or Thr-Glu, [claim 9]

The method of claim 5, wherein the β-hairpin is represented by the following general formula Π: Formula Π

Xi-Trp-X ₂ -Tyr-X3-Phe-Thr-Va 1 -¾

 Χι is Arg, Gly-Glu or Lys—Lys, ¾ is Gin or Thr, ¾ is type I, type Γ, type II, type Π 'or type m or type ΠΓ turn sequence, X4 is Gin, Thr- Glu or Gln-Glu.

[Claim 10]

 The method of claim 5, wherein the β-hairpin is represented by the following general formula ΠΙ:

 General formula m

Xi-¾-¾-Trp-X4— X ₅ -Thr-X ₆ -X ₇

¾ is Lys or Lys-Lys, ¾ is Trp or Tyr, ¾ is Val or Thr, is type I, type Γ, type Π, type Π 'or type m or type ΠΓ turn sequence, ¾ is Trp or Ala, ¾ is Trp or Val, X ₇ is Glu or Gln-Glu,

[Claim 11]

 The method of claim 5, wherein the β-hairpin is represented by the following general formula IV:

 Formula W

¾ is Lys-Thr or Gly, X ₂ is Trp or Tyr, ¾ is type I, type Γ, type Π, type Π 'or type m or type ΠΓ turn sequence and is Thr-Glu or Gly. Claim 12

According to claim 8, wherein the β-hairpin in the general formula I ¾ is Ser or Gly-Glu, ¾ and X'2 are independently of each other Thr, His or Val, ¾ is Trp or Tyr, is I, type Γ, type Π or type Π 'sequence, Χ ₅ is Trp or Phe, ¾ is Trp or Val, and X ₇ is Lys or Thr-Glu. [Claim 13]

 The method of claim 1, wherein the amino acid number n of the TF—target binding site I is 2-20.

[Claim 14]

 The method of claim 1, wherein the amino acid number m of the TF-target binding site Π is 2-20. [Claim 15]

 The method of claim 1, wherein the library is made from plasmid, bacteriophage, phagemid, yeast or bacteria. [Claim 16]

 The method of claim 1, wherein the TF-target binding site I and the TF-target binding site Π cooperatively bind to TF.

[Claim 17]

 The method according to claim 1, wherein a functional molecule is additionally bound to the structural stabilization site, TF-target binding site I or TF-target binding site Π.

[Claim 18]

 18. The method of claim 17, wherein the functional molecule is a label, chemical, biopharmaceutical, cell transmembrane peptide (CPP) or nanoparticle that generates a detectable signal.

[Claim 19]

(a) parallel, antiparallel or parallel lei with interstrand noncovalent bonds formed; Structure stabilizing region comprising antiparallel amino acid strands; and

 (b) TF-target binding site KTF-target binding region I) and TF-target binding site n (TF-target), each of which binds to both ends of the structure stabilization site and comprises randomly selected n and m amino acids, respectively. TF-bipodal peptide binder that specifically binds to TF comprising a binding region Π).

[Claim 20]

 20. The method of claim 19, wherein the non-covalent bonds between the strands formed at the structural stabilization site are hydrogen bonds, electrostatic interactions, hydrophobic interactions, van der Waals interactions, pi-pi interactions, cation-pi interactions or combinations thereof. TF-bipodal peptide binder, characterized in that the. [Claim 21]

 20. The TF-bipodal peptide binder according to claim 19, wherein the amino acid strands of the structural stabilization site are linked by a linker.

[Claim 22]

 20. The TF-bipodal peptide binder according to claim 19, wherein the structural stabilization sites are β-hairpins, β_sheets linked with linkers, leucine zippers or leucine zippers linked with linkers, and leucine rich motifs.

[Claim 23]

 20. The TF—bipodal peptide binder according to claim 19, wherein the structural stabilization site is β-hairpin.

[Claim 24]

24. The TF-bipodal peptide binder of claim 23, wherein the β-hairpin is selected from the group consisting of SEQ ID NO: 1 to 19 sequences. [Claim 25]

 The TF-bipodal peptide binder ᅳ according to claim 24, wherein the β-hairpin is selected from SEQ ID NO: 2, 14, or 18 sequences.

[Claim 26]

 The TF-bipodal peptide binder according to claim 23, wherein the β-hairpin is represented by the following general formula I:

 Formula I

X ₁ -Trp (X ₂ ) X ₃ -X4-X5 (X ^, 2) X6-X7

¾ is Ser or Gly-Glu, ¾ and X ' ₂ are independently of each other Thr, His, Val, lie, Phe or Tyr, ¾ is Trp or Tyr, is Type I, Type Γ, Type Π, Type Π 'Or type m or type ΠΓ turn sequence, ¾ is Trp or Phe, ¾ is Trp or Val, and X ₇ is Lys or Thr-Glu.

[Claim 27]

 The TF-bipodal peptide binder according to claim 23, wherein the -hairpin is represented by the following general formula:

 Formula Π

Xi-Trp-X ₂ -Tyr-X3-Phe-Thr-Va 1 _

 ¾ is Arg, Gly-Glu or Lys-Lys, ¾ is Gin or Thr, ¾ is type I, type 1 'ᅳ type Π, type Π' or type m or type ΠΓ turn sequence, is Gin, Thr- Glu or Gln-Glu.

[Claim 28]

 The TF-bipodal peptide binder according to claim 23, wherein the β-hairpin is represented by the following general formula:

 General formula m

¾-¾-¾— Trp-X4-X ₅ —Thr-X6-X ₇ ¾ is Lys or Lys-Lys, X ₂ is Trp or Tyr, ¾ is Val or Thr, X4 is type I, type 1 ', type Π, type Π' or type m or type ΠΓ turn sequence, ¾ Is Trp or Ala, ¾ is Trp or Val, and X ₇ is Glu or Gin— Glu.

[Claim 29]

 The TF-bipodal peptide binder according to claim 23, wherein the -hairpin is represented by the following general formula IV:

 Formula IV

 ¾- —¾— Trp-¾

 ¾ is Lys-Thr or Gly, ¾ is Trp or Tyr, ¾ is type I, type Γ, type Π, type Π 'or type m or type ΠΓ turn sequence and is Thr-Glu or Gly. 30】

 The method according to claim 24, wherein the β-hairpin in the general formula I ¾ is Ser or Gly-Glu, ¾ and X'2 are independently of each other Thr, His or Val, ¾ is Trp or Tyr, X4 is TF-bipodal peptide binder characterized by a type I, type Γ, type Π or type Π 'sequence, ¾ is Trp or Phe, ¾ is Trp or Val, and ¾ is Lys or Thr-Glu.

[Claim 31]

 20. The TF-bipodal peptide binder according to claim 19, wherein the amino acid number n of the TF-target binding site I is 2-20.

[Claim 32]

20. The TF-bipodal peptide binder ᅳ of claim 19, wherein the amino acid number m of the TF-target binding site Π is 2-20. 20. The TF-bipodal peptide binder according to claim 19, wherein the TF-target binding site I and the TF-target binding site Π cooperatively bind to TF.

 20. The TF-bipodal peptide binder according to claim 19, wherein a functional molecule is additionally bound to the structural stabilization site, TF-target binding site I or TF-target binding site Π. [Claim 35]

 35. The method of claim 34, wherein the functional molecule is a label, a chemical, a biopharmaceutical, a cell transmembrane peptide (CPP), or a nanoparticle that generates a detectable signal.

 A nucleic acid molecule encoding the bipodal peptide binder of any one of claims 19 to 35.

[Claim 37]

 A vector for the expression of the bipodal peptide binder comprising the nucleic acid molecule of claim 36