WO2011132938A2 - Gpcr-bpb se liant spécifiquement à gpcr - Google Patents

Gpcr-bpb se liant spécifiquement à gpcr Download PDF

Info

Publication number
WO2011132938A2
WO2011132938A2 PCT/KR2011/002838 KR2011002838W WO2011132938A2 WO 2011132938 A2 WO2011132938 A2 WO 2011132938A2 KR 2011002838 W KR2011002838 W KR 2011002838W WO 2011132938 A2 WO2011132938 A2 WO 2011132938A2
Authority
WO
WIPO (PCT)
Prior art keywords
gpcr
type
trp
thr
glu
Prior art date
Application number
PCT/KR2011/002838
Other languages
English (en)
Korean (ko)
Other versions
WO2011132938A3 (fr
Inventor
전상용
김성현
박세호
김대진
이상헌
Original Assignee
광주과학기술원
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 광주과학기술원 filed Critical 광주과학기술원
Priority to KR1020127023551A priority Critical patent/KR20130103300A/ko
Publication of WO2011132938A2 publication Critical patent/WO2011132938A2/fr
Publication of WO2011132938A3 publication Critical patent/WO2011132938A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the present invention relates to a GPCR—BPB that specifically binds to a GPCR.
  • Antibodies are immunoglobulin proteins, a type of plasma protein produced by B cells, that specifically inactivate and inactivate antigens by specifically recognizing and binding to specific sites of antigens.
  • immunoglobulin proteins a type of plasma protein produced by B cells, that specifically inactivate and inactivate antigens by specifically recognizing and binding to specific sites of antigens.
  • antibodies such as Rituximab and Hereptin have shown effectiveness in more than 50% of patients who have not responded to other treatments. Studies have shown that monoclonal antibodies have been successful in the treatment of lymphoma, colon cancer or breast cancer.
  • the total market for therapeutic antibodies is estimated to grow at an annual rate of 20%, from $ 10 billion in 2004 to $ 30 billion in 2010, and the market is expected to grow exponentially.
  • the reason why the development of new drugs using antibodies is active is that the drug development period is short, the investment cost is small, and the side effects can be easily predicted.
  • the antibody is a herbal drug, the human body is hardly affected and the half-life in the body is low molecular weight drug. Compared to the overwhelmingly long compared to the patient-friendly.
  • monoclonal antibodies in humans are recognized as foreign antigens and can cause severe allergic reactions or hypersensitivity reactions.
  • the anti-cancer monoclonal antibody is used clinically, the production cost is high, and thus the price of the therapeutic agent increases rapidly.
  • Antibody-replacement protein is a recombinant protein made to have constant and variable regions like antibodies, and a part of small and stable protein is replaced with amino acid of random sequence to make a library and screen it against the target material to make high affinity and good Substances with specificity can be found.
  • 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, can penetrate deep into cancer cells, and generally produce less immune response.
  • 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 responsiveness, relatively low patent loyalty, less exposure to unwanted immune systems, which can inhibit the production of antibodies to the peptides themselves, synthesis Deformation through is easy and accurate.
  • most peptides show low affinity and specificity for specific protein targets compared to antibodies, they 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.
  • 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.
  • GPCRs G protein-coupled receptors
  • GPCRs are known to play a pivotal role in the process of mediating various physiology or pathologies. Therefore, research on GPCR is currently used not only as an academic research subject but also as an important target of drug development.
  • many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.
  • an object of the present invention is to provide a G protein-coupled receptor (GPCR) -bipodal peptide binder (GPCR-BPB).
  • GPCR G protein-coupled receptor
  • Another object of the present invention is to provide a G protein-coupled receptor (GPCR) -bipodal peptide binder (GPCR-BPB).
  • GPCR G protein-coupled receptor
  • GPCR-BPB G protein-coupled receptor-BPB
  • the invention provides a G protein-coupled receptor (GPCR) —bipodal peptide binder (GPCR-BPB) comprising:
  • the present inventors have tried to develop a system capable of transporting various substances into or on the cell surface based on GPCR binding and specificity.
  • the structure stabilization site having a relatively rigid peptide backbone
  • BPB bipodal peptide binder
  • the basic strategy of the present invention is to connect a peptide that is bound to a target at both ends of a rigid peptide backbone.
  • the rigid peptide backbone acts to stabilize the overall structure of the bipodal peptide provider and enhances the binding of target binding site I and target binding site ⁇ 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, bi-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.
  • interstrand non-covalent bonds at the structure stabilization site include hydrogen bonds, hydrophobic interactions, van der Waals interactions, pi-pi interactions or combinations thereof.
  • disulfide bonds can be formed at the structured stabilization site to further increase the firmness of the structure stabilized site.
  • the increase in firmness by such covalent bonds is given in consideration of the specificity and affinity of the bipodal peptide binder for the target.
  • the amino acid strands of the structure stabilization site are linked by a linker.
  • linker as used to refer to the strands refers to the material that connects the strands.
  • the turn sequence on the hairpin acts as a linker
  • leucine zipper a substance connecting two C-terminus of leucine zipper (eg, Peptide linkers) serve as linkers.
  • the linker connects the 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 three strands) is connected by the linker.
  • the linker is a turn sequence or peptide linker.
  • the turn sequence is-turn, turn, ⁇ one turn, ⁇ -turn or ⁇ one loop (Venkatachalam CM (1968), Biopolymers, 6, 1425-1436; Nemethy G and Printz MP (1972), Macro / no IcuJes, 5, 755-758; Lewis PN et al., (1973), Biochi. Biophys. Acta, 303, 211-229; Toniolo C. (1980) CRC Crit. Rev. Biochem., 9, 1-44; Richardson JS. (1981), Adv.
  • the turn sequence used in the present invention is ⁇ -turn.
  • ⁇ -turn When ⁇ -turn is used as the turn sequence, it is preferably a type I, type 1 ', type ⁇ , type ⁇ ', type m or type ⁇ 'turn sequence, more preferably type I, type 1', type ⁇ , type ⁇ 'turn sequence, even more preferably type ⁇ or type ⁇ ' turn sequence, most preferably the type turn sequence (B. L ⁇ Sibanda et al ', J. Mol. Biol., 1989 , 206, 4, 759-777; BL Sibanda et al., Methods Enzymol., 1991, 202, 59-82).
  • 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.
  • What can be used as the turn sequence includes the following amino acid sequences: X-Pro-Gly-Glu-Val; Ala-X-Gly-Glu-Val (X is selected from 20 amino acids).
  • X is selected from 20 amino acids.
  • ⁇ _sheet or leucine zipper it is preferable that the peptide linker connects two strands arranged in parallel or two strands arranged in an antiparallel manner. desirable.
  • Peptide linkers can be used in any known in the art.
  • the sequence of a suitable peptide linker can be selected in consideration of the following factors: (a) the ability to be applied to a flexible extended conformat ion; (b) a secondary structure that interacts with a biological target molecule Ability to not generate; And (C) 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 described in 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.
  • the structural stabilization site is a ⁇ -sheet connected by a ⁇ -hairpin linker or a leucine zipper connected by a linker, more preferably the structural stabilization site is a -sheet connected by a ⁇ -hairpin or linker, Most preferably ⁇ -hairpin.
  • ⁇ -hairpin refers to the simplest protein motif comprising two ⁇ strands, the two ⁇ strands representing an antiparallel alignment with each other. In this ⁇ -hairpin the two ⁇ strands are generally linked by turn sequences.
  • the turn sequence applied to the ⁇ -hairpin is a type I, type ⁇ , type ⁇ , type ⁇ ', type m or type m' turn sequence, more preferably type I, type ⁇ , type ⁇ , type ⁇ 'turn sequence, even more preferably type ⁇ or type ⁇ ' turn sequence, most preferably the type turn sequence.
  • X-Pro-Gly-Glu-Val; or Ala-X-Gly-Glu Turn sequences represented by -Val can also be used for ⁇ -hairpins.
  • the type I turn sequence is Asp-Asp-Ala-Thr-Lys-Thr
  • the type ⁇ turn sequence is Glu-Asn-Gly-Lys
  • the type ⁇ turn sequence is X-Pn > -Gly-Glu-Val
  • Ala—X-Gly-Glu-Val X is selected from 20 amino acids
  • the type ⁇ 'turn sequence is Ghi "Gly-Asn-Lys or Glu-D—Pro-Asn-Lys.
  • Peptides with ⁇ -hairpin formulations are well known in the art.
  • tryptophan zipper disclosed in US Pat. No. 6,914,123 and Andrea G. Cochran et al., PNAS, 98 (10): 5578-5583, WO 2005/047503.
  • the template-fixed ⁇ -hairpin mimetic disclosed, the ⁇ -hairpin variants disclosed in US Pat. No. 5,807,979 are well known.
  • peptides with ⁇ -hairpin conformation are described in Smith & Regan (1995) Science 270: 980-982; Chou & Fassman (1978) Annu. Rev. Biochem.
  • tryptophan zipper is used when a peptide having a ⁇ _hairpin conformation is used as a structure stabilization site.
  • the tryptophan zipper used in the present invention is represented by the following general formula (I):
  • 3 ⁇ 4 is Ser or Gly-Glu
  • 3 ⁇ 4 and X ' 2 are independently of each other Thr, His, Val, lie, Phe or Tyr
  • X 3 is Trp or Tyr
  • 3 ⁇ 4 is type I, type r, type ⁇ , Type ⁇ 'or type m or type m' turn sequence
  • 3 ⁇ 4 is Trp or Phe
  • 3 ⁇ 4 is Trp or Val
  • X 7 is Lys or Thr-Glu.
  • 3 ⁇ 4 in Formula I is s e r or Gly-Glu
  • X 2 and X'2 are independently of each other Thr, His or Val, 3 ⁇ 4 is Trp or Tyr, X4 is Type I, Type ⁇ , Type ⁇ or Type ⁇ 'turn sequence, 3 ⁇ 4 is Trp or Phe, 3 ⁇ 4 Is Trp or Val and X 7 is Lys or Thr-Glu.
  • Xl is Ser or Gly-Glu
  • X 2 and X'2 are independently of each other Thr, His or Val
  • 3 ⁇ 4 is Trp
  • 3 ⁇ 4 is Trp
  • 3 ⁇ 4 is Trp
  • X 7 is Lys or Thr-Glu.
  • 3 ⁇ 4 in Formula I is Ser, 3 ⁇ 4 and
  • X'2 is Thr
  • 3 ⁇ 4 is Trp
  • 3 ⁇ 4 is Trp
  • 3 ⁇ 4 is Trp
  • X 7 is Lys.
  • Formula I is Ser, 3 ⁇ 4 and X'2 are Thr, 3 ⁇ 4 is Trp, is a type ⁇ turn sequence (ENGK) or type ⁇ 'turn sequence (EGNK), 3 ⁇ 4 is Trp 3 ⁇ 4 is Trp and X 7 is Lys.
  • ENGK type ⁇ turn sequence
  • EGNK type ⁇ 'turn sequence
  • amino acid sequences of tryptophan zippers suitable for the present invention are described in SEQ ID NOs: 1 to 3 and 5 to 10.
  • ⁇ -hairpin peptides usable as structural stabilization sites in the present invention are peptides derived from B1 domainin of protein G, ie GB1 peptides.
  • the structural stabilization site is preferably represented by the following general formula ⁇ :
  • 3 ⁇ 4 is Arg, Gly-Glu or Lys-Lys
  • X 2 is Gin or Thr
  • 3 ⁇ 4 is type I, type 1 ', type ⁇ , type ⁇ ' or type m or type ⁇ turn sequence
  • X4 is Gin, Thr-Glu or Gln-Glu.
  • the structural stabilization site of the general formula ⁇ is
  • 3 ⁇ 4 is Gly-Glu or Lys-Lys
  • 3 ⁇ 4 is type I, type 1 ', type ⁇ , type ⁇ ' or type m or type ⁇ turn sequence
  • 3 ⁇ 4 is Thr-Glu or Gln-Glu.
  • Exemplary amino acid sequences of GB1-hairpins suitable for the present invention are described in SEQ ID NO: 4 and 14 to 15 sequences.
  • ⁇ -hairpin peptides usable as structural stabilization sites in the present invention are described in SEQ ID NO: 4 and 14 to 15 sequences.
  • the structural stabilization site is preferably represented by the following general formula m:
  • 3 ⁇ 4 is Lys or Lys-Lys
  • 3 ⁇ 4 is Trp or Tyr
  • 3 ⁇ 4 is Val or Thr
  • 3 ⁇ 4 is Trp or Ala
  • 3 ⁇ 4 is Trp or Val
  • X 7 is Glu or Gln-Glu.
  • ⁇ -hairpin peptide that can be used as a structural stabilization site in the present invention is represented by the following general formula IV:
  • 3 ⁇ 4 is Lys-Thr or Gly
  • X 2 is Trp or Tyr
  • 3 ⁇ 4 is type I, type ⁇ , type II, type ⁇ 'or type m or type ⁇ turn sequence, and is Thr-Glu or Gly.
  • amino acid sequences of ⁇ -hairpins of the general formulas [pi] and IV are described in SEQ ID NO: 11 to 12, 15, and 16 to 19 sequences.
  • a ⁇ -sheet connected by a linker may be used as the structure stabilization site.
  • two or more amino acid strands, which are parallel or antiparallel, preferably antiparallel, are in an extended form, and hydrogen bonds are formed between the amino acid strands.
  • ⁇ -sheet structure two adjacent ends of two amino acid strands are connected by a linker.
  • linker various turn-sequences or peptide linkers described above may be used. If the turn-sequence is used as a linker, the ⁇ -turn sequence is most preferred.
  • leucine zippers or leucine zippers linked by linkers may be used as structural stabilization sites.
  • Leucine zippers are conserved peptide domains that cause parallel two-chain dimerization and are commonly found in proteins involved in gene expression. Dimerization domain (“Leucine scissors”. Glossary of Biochemistry and Molecular Biology (Revised). (1997). 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.
  • leucine zippers that may be used in the present invention include 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 ⁇ -chains with direct leucine contact between the ⁇ -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.
  • two adjacent ends of two amino acid strands may be linked by a linker.
  • 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 above-mentioned structural stabilization site.
  • the random amino acid sequence forms GPCR-target binding site I and GPCR-target binding site ⁇ .
  • One of the biggest features of the present invention is to prepare a peptide binder in a bipodal manner by connecting GPCR-target binding site I and GPCR-target binding site ⁇ at both ends of the structure stabilization site.
  • GPCR-target binding site I and GPCR-target binding site ⁇ cooperatively bind to the target, thereby greatly increasing the affinity for GPCR.
  • the amino acid number n of the GPCR-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 between 3 and 10.
  • the amino acid number m of the GPCR-target binding site ⁇ 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 an integer of 3-10.
  • GPCR-target binding site I and GPCR-target binding site ⁇ may each contain different or the same number of amino acid residues.
  • GPCR-target binding site I and GPCR-target binding site ⁇ may comprise different or identical amino acid sequences, and preferably include different amino acid sequences.
  • the amino acid sequence included in GPCR-target binding site I and / or GPCR-target binding site ⁇ is a linear amino acid sequence or a cyclic amino acid sequence.
  • at least one amino acid residue of the amino acid sequence included in GPCR-target binding site I and / or GPCR-target binding site ⁇ is an acetyl group, a fluorenyl methoxy carbonyl group, Formyl, palmitoyl, myristyl, stearyl or polyethylene glycol (PEG).
  • GPCR—BPB of the present invention bound to a biological target molecule can be used for the regulation of physiological reactions in vivo, detection of in vivo substances, in vivo molecular imaging, in vitro cell imaging and drug delivery targeting, escorts It can also be used as a molecule.
  • the cargo is bound to the structure stabilization site, GPCR-target binding site I or GPCR-target binding site ⁇ (more preferably, the linker of the structure stabilization site) more preferably than the structure stabilization site. It is.
  • the cargo include, but are not limited to, labels, chemicals, biopharmaceuticals or nanoparticles that generate detectable signals.
  • the label that generates the detectable signal may be a T1 contrast medium (e.g.,
  • T2 contrast agents eg, superparamagnetics (eg magnetite, Fe 3 0 4 , y-Fe 2 0 3 , manganese ferrite, cobalt ferrite and nickel ferrite)
  • radioisotopes eg, U C
  • fluorescent materials fluorescein, phycoerythrin, rhodamine, lysamine (lissamine), and Cy3 and Cy5
  • chemiluminescent groups magnetic particles, mass labels or electron-dense particles.
  • the chemicals include, for example, anti-inflammatory drugs, analgesics, anti-arthritis agents, antispasmodics, antidepressants, antipsychotics, neurostabilizers, anti-anxiety agents, drug antagonists, antiparkin's disease drugs, cholinergic agonists, anticancer agents, antiangiogenic agents, Immunosuppressants, antivirals, antibiotics, appetite suppressants, analgesics, anticholiners, antihistamines, antimigraine, hormones, coronary, cerebrovascular or peripheral vasodilators, contraceptives, antithrombotics, diuretics, antihypertensives, cardiovascular diseases , Cosmetic ingredients (eg, anti-wrinkle agents, anti-aging agents and skin lightening agents) and the like, but are not limited thereto.
  • the biopharmaceutical is insulin, IGF-K insulin—like growth factor 1), growth hormone, erythropoietin, G-CSFs (granulocyte-colony stimulating factors), GM-GSFs (gr anu 1 ocy te / macr ophage- co 1 ony stimulating factors), interferon alpha, interferon beta, interferon gamma, interleukin-1 alpha and beta, interleukin— 3, interleukin-4, interleukin-6, interleukin-2, epidermal growth factors (EGGF), calcitonin , ACTH (adrenocorticotropic hormone), TNF (tumor necrosis factor), Atobisban, buserel in, cetrorel ix, deslorelin, desmopressin (desmopressin), dynorphin A (1-13), elcatonin, eleidosin, eptifibatide, GHRH-I I (growth hormone releasing hormone
  • GPCR-target binding site I and / or GPCR-target binding site ⁇ comprises an amino acid sequence that binds to GPCR.
  • GPCRs preferably Class A (or 1) (Rhodopsin-1 ike), Class B (or 2) (Secretin receptor family), Class C (or 3) (Metabotroic glutamate / pheromone), Class D ( or 4) (Fungal mating pheromone receptors), Class E (or 5) (Cyclic AMP receptors) and Class F (or 6) (Frizzled / Smoothened) GPCRs, more preferably a luteinizing hormone receptor, a follicle stimulating hormone receptor, a thyroid stimulating hormone receptor, a calcitonin receptor, a glucagon receptor, a glucagon ⁇ 1 ike peptide 1 receptor (GLP ⁇ 1), a metabotropic glutamate receptor, a parathyroid hormone receptor, a vasoactive intestinal peptide receptor, a secret in receptor, a growth hormone releasing factor (GRF) receptor, protease—activated receptors (PARs), cholecystokin
  • GPCR-BPB of the present invention binds to the extracellular domain of GPCR, preferably GPCR, and acts as an agonist or antagonist for GPCR.
  • Agonists for GPCRs promote intracellular signaling triggered by GPCRs, and antagonists for GPCRs act to inhibit intracellular signaling triggered by GPCRs.
  • GPCR-BPB of the present invention may bind to the extracellular domain of GPCR exposed to the cell surface, but may also bind to the intracellular domain to regulate the action of GPCR.
  • GPCR-BPB targets an intracellular domain, preferably GPCR-BPB additionally comprises a cell transmembrane peptide (CPP).
  • CPP cell transmembrane peptide
  • the CPP includes various CPPs known in the art and include, for example, HIV-1 Tat protein, oligoarginine, ANTP peptide, HSV VP22 transcriptional regulator protein, MTS peptide derived from vFGF, Penetratin, Transport an, Pep -1 peptide, Pep-7 peptide, Buforin II, model amphi hatic peptide (MAP), k-FGF, Ku 70, pVEC, SynBl or HN-1.
  • MAP model amphi hatic peptide
  • 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 with a lysine residue in the loop portion at the structural stabilization site of the bipodal peptide.
  • the bipodal peptide binder of the present invention is typically referred to as "one strand of the N-GPCR-target binding site I-structure stabilization site-linker-structure stabilization site -GPCR-target binding site ⁇ — C”.
  • between the GPCR-target binding site I and one strand of the structural stabilization site and / or between the other strands of the structural stabilization site -GPCR-target binding site ⁇ in the GPCR-bipodal peptide binder of the present invention Includes a structure influence inhibiting region that blocks the cross-structural effects between the GPCR-target binding site and the structure stabilization site.
  • At the site of rotation are amino acids that are relatively free of rotation of ⁇ and 3 ⁇ 4 ⁇ in the peptide molecule.
  • the amino acids with relatively free rotation of ⁇ and ⁇ are glycine, alanine and serine. 1-10 amino acids, preferably 1-8, and more preferably 1-3 amino acid residues may be located at the structure influence inhibitory site.
  • the library of GPCR-bipodal peptide binders of the present invention having the constructs described above can be obtained by various methods known in the art.
  • the GPCR-bipodal peptide binder will have a random sequence, which has no sequence preference or designation (or immobilization) at any position of GPCR-target binding site I and / or GPCR-target binding site ⁇ . It means no amino acid residues.
  • a library of GPCR-bipodal peptide binders can be used for the split-synthesis method (Lam et al. (1991) Nature 354: 82; WO 92/00091) performed on a solid support (eg, polystyrene or polyacrylamide resin).
  • a solid support eg, polystyrene or polyacrylamide resin
  • a library of GPCR-bipodal peptide binders is constructed in a ce ii surface display manner (eg, phage display, bacterial display or yeast display).
  • the library of GPCR-bipodal peptide binders can be prepared via display methods based on plasmids, bacteriophages, phagemids, yeasts, 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, GP (1990) Science 249: 386; Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001); Clacks on and Lowman, Phage Display, Oxford University Press (2004). Random peptides are displayed by fusing the gene to be expressed in the gene ⁇ or gene uptake of the filamentous phage (eg M13).
  • Phageimide may be used for the fiji display.
  • Phageimide is a plasmid vector with one copy of the bacterial origin of replication (eg, ColEl) and the intergenic site of the bacteriophage. DNA fragments cloned in this phagemid are propagated like plasmids.
  • a preferred embodiment of the present invention comprises the following steps: (0 phage coat protein (eg, gene m or gene of filamentous phage such as M13) A fusion gene in which a gene encoding a coat coat) and a gene encoding a bipodal tempide binder are fused, and a library of expression vectors comprising a transcriptional regulatory sequence (eg, a lac promoter) operably linked to the fusion gene. ( ⁇ ) introducing the expression vector library into a suitable host cell; (iii) culturing the host cell to produce recombinant phage or phagemid virus particles. Forming so that the fusion protein is displayed on the surface; (iv) contacting the viral particles with a GPCR molecule to bind the particles to a target molecule; And (V) separating particles not bound to GPCR molecules.
  • phage coat protein eg, gene m or gene of filamentous phage such as M13
  • the method for preparing an expression vector comprising a bipodal temptide binder gene may be performed according to methods known in the art.
  • known phagemid or phage vectors e.g. pIGT2, fUSE5, fAFFl, fd-CATl, m663, fdtetDOG, HENl, pComb3, pComb8, pCANTAB 5E (Pharmacia) LamdaSurfZap, pIF4, PM48, PM52, PM54, fdH) And p8V5
  • an expression vector can be prepared.
  • phage display methods are performed using filamentous phage, lambda phage display (W0 95/34683; US Pat. No. 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) can also be used to build a library of bipodal peptide binders.
  • 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 are gram negative bacterial cells such as E. coli, and suitable E. coli hosts are JM101, E. coli 12 strain 294, E. coli strain W3110 and E. coli XL-lBlue. It is recommended that host cells be prepared with competent cells prior to transformation (Sambrook, J. et al, Molecular Cloning. A Laboratory Manual, 3rd ed. Cold). Spring Harbor Press (2001)). Selection of transformed cells generally includes antibiotics (eg, tetracycline and ampicillin).
  • antibiotics eg, tetracycline and ampicillin
  • helper phage phages include, but are not limited to, Ex helper phages, M13-K07, M13-VCS, and R408.
  • the selection of viral particles that bind to biological target molecules can be routinely performed through a biopanning process (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).
  • the present invention provides a nucleic acid molecule encoding the aforementioned GPCR-bipodal peptide binder.
  • the invention provides a vector for the expression of a GPCR-bipodal peptide binder comprising a nucleic acid molecule encoding a GPCR-bipodal peptide binder.
  • the present invention provides a transformant comprising a vector for expression of a GPCR-bipodal peptide binder.
  • nucleic acid molecule is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides that are the basic building blocks of nucleic acid molecules are naturally modified nucleotides, as well as modified sugar or base sites.
  • Analogues Schott-Propanediol, RNA-Propanediol, RNA-Propanediol, RNA-Propanediol-N-phosphatethyl-N-(2-aminotyl)
  • nucleotides that are the basic building blocks of nucleic acid molecules are naturally modified nucleotides, as well as modified sugar or base sites.
  • Analogues Schot, Nucleotide Analogs,
  • 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 GPCR-bipodal peptide binder (e.g., tac promoter, lac promoter, / adJV5 Promoter, lpp promoter, p L promoter, p / promoter, ra> promoter, shock 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.
  • the GPCR-bipodal peptide binder e.g., tac promoter, lac promoter, / adJV5 Promoter, lpp promoter, p L promoter, p / promoter, ra> promoter, shock promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.
  • the vector of the present invention signals to the 5'-direction of the nucleic acid molecule encoding the GPCR-bipodal peptide binder. Sequences (eg, pelB) may be further included.
  • the vector of the present invention further comprises a tagging sequence (eg, myc tag) for confirming that the bipodal peptidide binder is well expressed on the surface of the phage.
  • the vector of the present invention comprises a phage coat protein, preferably a gene encoding filamentous phage such as M13 or a gene VII coat protein.
  • the vector of the present invention comprises an origin of replication of bacteria (eg ColEl) and / or a bacteriophage.
  • 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 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) It includes, but is not limited to.
  • the method of carrying the vector of the present invention into a host cell includes the 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, J. Mo J. Biol., 166: 557—580 (1983)) and electroporation methods (US Pat. Nos. 5,186,800, 5,422,272, 5, 750, 373) and the like.
  • the GPCR-bipodal peptide binders of the present invention exhibit peptides with very low levels (eg, nM levels) of K D values (dissociation constants), resulting in very high affinity to GPCR molecules, as described in the Examples below.
  • the bipodal temptide binder exhibits a high affinity of about 10 2 -10 5 times (preferably about 10 3 -10 4 times) as compared to the binder produced by the monopodal method.
  • the GPCR-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.
  • the features and advantages of the present invention are as follows:
  • the present invention provides GPCR-BPB that specifically binds to GPCR.
  • the 6bibipodal peptide binders of the present invention thus exhibit very low levels (eg, nM levels) of K D values (dissociation constants), resulting in very high affinity to the target.
  • GPCR-BPB of the present invention may bind to GPCR in vivo and act as antagonist or agonist for GPCR.
  • La shows a schematic diagram of a bipodal-peptide binder and GPCR-BPB containing ⁇ -hairpin as a structural stabilization site.
  • Lb shows a schematic diagram of GPCR— ⁇ including ⁇ -sheets linked by linkers as structural stabilization sites.
  • Figure lc shows a schematic of GPCR-BPB including leucine zippers linked by linkers as structural stabilization sites.
  • Id shows a schematic of GPCR-BPB comprising a leucine-rich motif linked by linker as a structural stabilization site.
  • Figure 2 shows a strategy for cloning the GPCR-BPB library.
  • the pelB signal sequence myc tag
  • myc tag is a tagging sequence to confirm that the gene of interest is well expressed on the surface of the phage.
  • the lac promoter was used as a promoter.
  • 3 is a result of the aequorin reporter gene assay for analyzing the antagonist action on the GPCR of sex peptide receptor-BPB.
  • Figure 4 shows the structure of a bipodal peptide binder library that binds to the formyl peptide receptor like-1 protein, a GPCR protein.
  • 5 is a result of the aequorin reporter gene assay for analyzing agonist action on the formyl peptide receptor like-1, a GPCR protein.
  • Beta— F1 (5'-TTCTATGCGGCCCAGCTGGCC (NNK) 6 GGATCTTGGACATGGGAAAACGGAAAA-3 ') and Beta-Bl (5'-
  • N is A, T, G or C; K is G or T; M is C or A.
  • Beta-Fl 4 ⁇ , Beta-Bl 4 ⁇ , 2.5 mM dNTP mixture 4 ⁇ , ExTaq DNA polymerase 1 ⁇ (Takara, Seoul, Korea) and 5 ⁇ of 10XPCR buffer were mixed and a total of 50 ⁇ A total of 25 mixtures with distilled water added were made.
  • Is a common hapaek PCR reaction After creating (5 minutes at 94 ° C, 60 cycles 3C 30 sec at C, at 72 ° C for 30 seconds and 72 ° C 7 minutes) to the double-stranded PCR purification kit (GeneAll, Seoul, Korea) to obtain a bipodal peptide binder gene.
  • the restriction gene was treated to the insert gene and the pIGT2 phagemid vector.
  • E. coli 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 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 until the absorbance was 0.3-0.4 at a wavelength of 600 nm. The incubated flask was left on ice for 30 minutes, then 4 ° C.
  • Electroporation was performed by dispensing 25/100 of the phagemid vector 12 and the bipodal peptide binder into which the insert DNA 2.9 i was linked.
  • the competent cells were thawed on ice, mixed with 200 ⁇ of competent cells and mixed with solution 4 ⁇ , and placed in a prepared 0.2 cm cuvette. Placed on ice for 1 minute later.
  • An electroporator (BioRad, Hercules, CA) was programmed at 200 ⁇ at 25 uF and 2.5 kV, drained the prepared cuvettes, placed in the electroporator and pulsed (time constant is 4.5-5 msec).
  • the cells were placed in 1 LB medium containing 20 mM glucose, prepared at 37 ° C., and a total of 25 cells obtained were transferred to a 100 ra tube. After incubation at 200 rpm at 37 ° C. for one hour, 10 ⁇ was diluted and plated in ampicillin agar medium to measure the number of libraries. The remaining cells were added 20 mM glucose and 50 g / ampicillin in 1 «LB and incubated for one day at 30 ° C. Centrifugation at 4 ° C. at 4,000 ⁇ g for 20 minutes to remove all supernatants except for the precipitated cells, resuspended in 40 LB and stored at ⁇ 80 ° C. with glycerol at a final concentration of at least 20%. Recombinant Phage Production and PEG Precipitation in Libraries
  • Recombinant phage was produced in a bipodal peptide binder library stored at -80 ° C.
  • ampicillin 50 ⁇ / ⁇
  • 20 mM glucose were added to a 100 m £ LB medium, and then library 1 i stored at -80 ° C was added at 150 rpm at 37 ° C for one hour.
  • Cultures were mixed at speed.
  • Ex helper phage Ig therapy, Chuncheon, Korea
  • ixi0 u pfu was added thereto and incubated under the same conditions for one hour.
  • Biopanning was performed on the sex peptide receptor of fruit fly as a protein of GPCR. Phage peptides recovered in every biopanning order were subjected to biopanning of the BPB (Bipodal-peptide binder) library prepared in Example 1 to CH0-K1 cells transformed with each sex peptide receptor gene. Their output phage / input phage ratio was determined. Each biopanning process involves the removal of CH0-K1-specific phage by injecting phage into non-transformed CH0-K1 cells. This is called 'counter selection'. Specific bio panning method is as follows.
  • Drosophila sex peptide receptor DNA (pcDNA3.1 (+)-SPR) cloned into pcDNA3.1 (+) vector was transfected into CH0-K1 cells using 1 ipofect amine, and then cultured in a 10 Cm culture dish for 24 hours. Used for biopanning. First, 2% BSA was added to the CH0-K1 culture dish transfected with SPR gene to block the surface of the dish for 1 hr. On the other hand, 10 11 cfu or more of library phages were mixed with 2% BSA, rotated for 15 minutes, mixed and stored on ice.
  • Example 3 Fruit fly sex peptide receptor specific phage peptide detection (phage titration) After 5 times of biopanning, the recovered phages were infected with E.
  • E. coli and placed in ampicillin LB solid medium at 37 degrees for overnight incubation. Each generated E. coli colony corresponds to one phage clone. 40 colonies were randomly selected and inoculated in 1 ml LB liquid medium in a 1.5 ml tube, ampicillin and helper phage 10 10 pfu were added together, and incubated for 2 days at 37 ° C and 200 rpm to amplify phage clones. Recover supernatant by centrifugation and titrate. Add 2% BSA, rotate and mix thoroughly.
  • CH0-K1 and non-transfected CHO cells transfected with pcDNA3.1 (+)-SPR DNA one day prior to 40 wells were put into 96 well plates and cultured. At this time, the number of cells per well is adjusted to 3 ⁇ 10 3 level. 50 ul of phage solution mixed with 2% BSA was first added to non-transfected CH0 cells, stored at 37 ° C for 1 hour, and then the supernatant was carefully collected. After spin down, supernatant was added to CH0-K1 transfected with pcDNA3.1 (+)-SPR and stored at 37 ° C for 30 minutes.
  • a bipodal peptide binder peptide specific for the sex peptide receptor overlapped in DNA sequencing was synthesized (Anigen, Korea). Twenty four hours prior to assay, 3 x 10 3 CHO-Kl cells co-transfected with pcDNA3.1 (+)-SPR and pcDNA3.1 (+)-aequorin DNA were incubated. Assay method is as follows. Coelenterazine was injected into CH0-K1 cells prepared by dissolving 0.5 mM coelenterazine in 1/10 dilution of 10x assay buffer (1.25 M KC1, 50 mM MgC12, 20 mM K / PIPES, pH 6.8, 200 mM sorbitol) for 1 hour.
  • the stable beta-hairpin motif was used as the structure stabilization site of the bipodal tempide binder.
  • 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.
  • the structure stabilization site of the bipodal peptide binder may be configured in various ways as shown in FIGS.
  • a Bipodal Peptide Binder Library Bioengineers a 5th-order Biotransfer to CH0-K1 Cells Transfected with Fruit Fly Sex Peptide Receptor Panning was performed and the phage / input phage ratio of the phage peptides recovered at each panning step was determined (Table 1).
  • Fruit fly sex peptide receptor A total of 40 candidate phage clones were obtained by performing 4 specific phage peptide searches. Phage genomes were extracted from the phage clones infected with E. coli, sequenced, and identified bipodal peptide binder amino acid sequences. 40 bipodal peptide binder amino acid sequences were arranged through the ClustalW program and clones with identical sequences were detected. This yielded overlapping specific peptide sequences (Tables 2a and 2b).
  • an aequorin reporter gene assay was performed. Sex peptide receptors alter intracellular Ca ++ levels through signal transduction when they are affected by ligand.
  • the bipodal peptide sequence overlapped from genome DNA sequence of phageclone was synthesized (Anigen, Korea).
  • Beta-Bl (5'- AACAGTTTCTGCGGCCGCTCCTCCTCC (MNN) 6 TCCOTCCATGTCCATTTTCCGTT-3') and Beta-F2 (5 '-TOTATGC (: CCA (TG ( ⁇ (OT (6)) ⁇ ACATG ⁇ AAAACGGAAAA-3 ') and Bet a-B2 (5' -AACAGTTTCTGCGGCCGCTCCTCC
  • TCC (CCACCACCACCAGCGCCA) TCCOTCCATGTCCATTTTCCGn-3 ') (N is A, T, G or C; K is G or T; M is C or A).
  • Beta-Fl 4 ⁇ , Beta-Bl 4 ⁇ (or Beta-F2 4 ⁇ , Beta-R2 4 ⁇ ), 2,5 mM dNTP mixture 4 ⁇ , ExTaq DNA polymerase 1 Takara, Seoul , Korea) and 10XPCR buffer 5 / ⁇ were mixed to make a total of 25 mixtures with the addition of distilled water to a total of 50 ⁇ .
  • E. coli XL1-BLUE cells American Type Culture Collection, Manassas,
  • Electroporation was performed by dispensing 25 ⁇ into 2.9 // g. Competent cells were dissolved on ice, mixed with 200 ⁇ of competent cells and mixed with 4 ⁇ solution, placed in a 0.2 cm cuvette prepared separately and placed on ice for 1 minute.
  • the electroporator BioRad, Hercules, CA
  • the electroporator was programmed at 200 ⁇ at 25 uF 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 liquid medium containing 20 mM glucose prepared at 37 ° C. A total of 25 cells obtained were transferred to a 100 mi test tube.
  • Recombinant phage was produced in a bipodal peptide binder library stored at -80 ° C.
  • a bipodal peptide binder library stored at -80 ° C.
  • ampicillin 50 ⁇ and 20 mM glucose
  • Library 1 stored at -80 ° C for 1 hour at 150 rpm at 37 ° C.
  • lxio H pfu Ex helper phage (Ig therapy, Chuncheon, Korea) was added thereto and incubated under the same conditions for an hour again. The supernatant was removed by centrifugation for 10 minutes with l.OOOXg and Recombinant phage was produced by adding 100 ml of LB liquid medium containing ampicillin (50 nglv) and kanamycin (25) to the culture supernatant.
  • GPCR protein Another example protein of GPCR protein was biopanned against formyl peptide receptor like-1.
  • the biopodal BPB (Bipodal-peptide binder) library prepared in Example 1 was subjected to biopanning 5 times for CH0-K1 cells transformed with each formyl peptide receptor like-1 gene, and output phage / The input phage ratio was determined. Each time the output phage was injected into non-transformed CH0-K1 cells to remove the CH0-K1 specific phage. This is called 'counter selection'.
  • the specific method is as follows. Formyl peptide receptor like-1 DNA (pcDNA3.l (+)-FPRLl) cloned into pcDNA3.1 (+) vector was transferred to CH0-K1 cells using 1 ipofectamine.
  • Example 3 Formyl peptide receptor like-1 (FPRL-1) specific phage peptide detection (phage titration method)
  • the recovered phages are infected with E. coli and placed on an Ampicillin LB plate and incubated at 37 ° C overnight. Each colony of E. coli generated was one phage clone. A random selection of 40 colonies was inoculated into a 1.5 ml tube containing 1 ml LB liquid medium and ampicillin and helper phage 10 10 pfu, 37 degrees. Incubate at 200 rpm for 2 days to amplify phage clones. Recovered and titration of supernatant by centrifugation. Add 2% BSA, rotate and mix well.
  • the pcDNA3.1 (+) before the day-by and FPRL1 is turned on by each well 40 a transfection CH0-K1, non transfected CH0 cells in 96 well plate and incubated eu wherein the well 3 X 10 3 cells, the level number per Fit. 50 ul of phage solution mixed with 2% BSA first Stored at 37 ° C for 1 hour after inoculation into non-transfected CHO cells and carefully collect the supernatant. After spin down, supernatant was added to CH0-K1 transfected with pcDNA3.1 (+)-FPRLl and stored at 37 ° C for 30 minutes.
  • Example 5 Construction of Bipodal Peptide Binder Library
  • a stable beta-hairpin motif was used.
  • 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.
  • a bipodal library comprising 'WRWWWW' at the N-terminus of the skeletal tryptophan zipper and 6 random amino acids at the C-terminus, or 6 random amino acids at the N-terminus or WRW 'at the C-terminus Produced (FIG. 4).
  • the bipodal peptide binder library was subjected to biopanning 5 times on Formyl peptide receptor like-1 transfected CHO-Kl cells, and the ratio of output phage / input phage of phage peptides recovered at each panning step was determined (Table 3). .
  • Table 3
  • Example 8 aequorin reporter gene assay for Formyl peptide receptor like-1
  • Aequorin reporter gene assay was performed to investigate the effect of peptide on the activity of Formyl peptide receptor like-1, GPCR. Increased capacity of agonists over WRWWWW peptides would increase intracellular Ca ++ levels through the downstream signaling pathway of Formyl peptide receptor like-1. Through the Aequorin reporter gene assayAssay, it was confirmed that all five peptides act more strongly as agonists than the conventionally known WRWWW (FIG. 5).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Zoology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un lieur peptidique bipied de GPCR se liant spécifiquement à GPCR, comprenant : (a) une région de stabilisation de structure comprenant des brins d'acides aminés parallèles, antiparallèles, ou parallèles et antiparallèles dans lesquels une liaison non covalente inter-brins est formée ; et (b) une région de liaison cible de GPCR I et une région de liaison cible de GPCR II qui se lient respectivement aux deux extrémités de la région de stabilisation de structure et comprennent n et m acides aminés sélectionnés de façon aléatoire, respectivement. Le lieur peptidique bipied de GPCR de la présente invention présente un niveau très faible (par exemple, au niveau nM) de la valeur de KD (constante de dissociation) pour GPCR, de manière à présenter une affinité très élevée pour une cible GPCR. Le lieur peptidique bipied de GPCR de la présente invention a une utilisation médicale, peut être utilisé pour l'imagerie moléculaire in vivo, l'imagerie cellulaire in vitro et le ciblage pour administration de médicament, et peut également être utilisé très utilement en tant que molécule escorte.
PCT/KR2011/002838 2010-04-20 2011-04-20 Gpcr-bpb se liant spécifiquement à gpcr WO2011132938A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020127023551A KR20130103300A (ko) 2010-04-20 2011-04-20 Ｇｐｃｒ에 특이적으로 결합하는 ｇｐｃｒ-ｂｐｂ

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20100036599 2010-04-20
KR10-2010-0036599 2010-04-20

Publications (2)

Publication Number Publication Date
WO2011132938A2 true WO2011132938A2 (fr) 2011-10-27
WO2011132938A3 WO2011132938A3 (fr) 2012-05-10

Family

ID=44834644

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2011/002838 WO2011132938A2 (fr) 2010-04-20 2011-04-20 Gpcr-bpb se liant spécifiquement à gpcr

Country Status (2)

Country Link
KR (1) KR20130103300A (fr)
WO (1) WO2011132938A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101531944B1 (ko) * 2013-12-24 2015-06-29 광주과학기술원 Ｖｅｇｆ에 특이적으로 결합하는 ｖｅｇｆ－ｂｐｂ

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5427908A (en) * 1990-05-01 1995-06-27 Affymax Technologies N.V. Recombinant library screening methods
US6914123B2 (en) * 2001-04-17 2005-07-05 Genentech, Inc. Hairpin peptides with a novel structural motif and methods relating thereto
KR20100044128A (ko) * 2008-10-20 2010-04-29 광주과학기술원 바이포달 펩타이드 바인더

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5427908A (en) * 1990-05-01 1995-06-27 Affymax Technologies N.V. Recombinant library screening methods
US6914123B2 (en) * 2001-04-17 2005-07-05 Genentech, Inc. Hairpin peptides with a novel structural motif and methods relating thereto
KR20100044128A (ko) * 2008-10-20 2010-04-29 광주과학기술원 바이포달 펩타이드 바인더

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
2010 KOREAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY ANNUAL INTERNATIONAL CONFERENCE POSTER SESSTION A-18-85 17 May 2010, page 153 *
BAGALKOT, V. ET AL.: 'An aptamer doxorubicin physical conjugate as a novel targeted drug-delivery platform.' ANGEW. CHEM. INT. ED. vol. 45, 2006, pages 8149 - 8152 *
CLACKSON, T. ET AL.: 'Making antibody fragments using phage display libraries.' NATURE vol. 352, 15 August 1991, pages 624 - 628 *

Also Published As

Publication number Publication date
WO2011132938A3 (fr) 2012-05-10
KR20130103300A (ko) 2013-09-23

Similar Documents

Publication Publication Date Title
KR101151805B1 (ko) 바이포달 펩타이드 바인더
EP1991560B1 (fr) Peptide présentant une activité de pénétration de membrane cellulaire
JP5677454B2 (ja) 細胞内ターゲット結合用二座ペプチドバインダー
JP2016527180A (ja) ポリペプチドの修飾
WO2009149339A2 (fr) Peptides activateurs de p53
KR20200038303A (ko) 모듈형 결합 단백질
KR101323846B1 (ko) 타겟 친화도가 유지되고 안정성이 개선된 ｄ－앱타이드
WO2011132938A2 (fr) Gpcr-bpb se liant spécifiquement à gpcr
JP2023509004A (ja) ニドゲンをベースとする足場タンパク質および治療用ナノ複合体
WO2011132940A2 (fr) Rtk-bpb se liant spécifiquement à rtk
Darlot Design of modulatory peptides against chemokines
KR20130103301A (ko) 전사인자에 특이적으로 결합하는 ｔｆ-ｂｐｂ
KR101443839B1 (ko) 펩타이드의 타겟 친화도 개선방법
KR20110116930A (ko) 이온채널에 특이적으로 결합하는 이온채널―ｂｐｂ
EP1268520A2 (fr) Composes de liaison et procede d'identification de composes de liaison
US20140356285A1 (en) Compositions and methods related to tissue targeting
Richardson Design and evaluation of fluorescent and non-fluorescent bivalent ligands to explore their mode of action at neuropeptide Y receptors
WO2011132939A2 (fr) Rtk-bpb se liant spécifiquement à rtk
William et al. Peptide ligands for Methuselah, a Drosophila G protein-coupled receptor associated with extended lifespan
Trotta et al. Disulfide Bond Replacement with Non-Reducible Side Chain to Tail Macrolactamization for the Development of Potent and Selective Cxcr4 Peptide Antagonists Endowed with Flanking Binding Sites
WO2015132248A2 (fr) Séquamères - nouvelles banques de peptides non naturels aléatoirement contraints par leurs séquences primaires

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11772226

Country of ref document: EP

Kind code of ref document: A2

ENP Entry into the national phase

Ref document number: 20127023551

Country of ref document: KR

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11772226

Country of ref document: EP

Kind code of ref document: A2