WO1996034879A1 - Identification de ligands enantiomeres - Google Patents

Identification de ligands enantiomeres Download PDF

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Publication number
WO1996034879A1
WO1996034879A1 PCT/US1996/006155 US9606155W WO9634879A1 WO 1996034879 A1 WO1996034879 A1 WO 1996034879A1 US 9606155 W US9606155 W US 9606155W WO 9634879 A1 WO9634879 A1 WO 9634879A1
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Prior art keywords
amino acid
macromolecule
domain
binds
peptide
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PCT/US1996/006155
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English (en)
Inventor
Antonius Nicolaas Maria Schumacher
Peter S. Kim
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Whitehead Institute For Biomedical Research
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Priority claimed from US08/627,497 external-priority patent/US5780221A/en
Application filed by Whitehead Institute For Biomedical Research filed Critical Whitehead Institute For Biomedical Research
Priority to EP96915457A priority Critical patent/EP0825997A1/fr
Priority to JP8533492A priority patent/JPH11505527A/ja
Publication of WO1996034879A1 publication Critical patent/WO1996034879A1/fr

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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures

Definitions

  • the enantiomers of these naturally occurring macromolecules are not good substrates for naturally occurring proteases and nucleases.
  • the enantiomers of naturally occurring molecules do not elicit an efficient immune response.
  • Availability of D-peptides and L-nucleic acids for use as drugs is desirable.
  • the present invention is a method of identifying enantiomeric macromolecules (proteins, peptides, oligonucleotides, nucleic acids, sugars and macromolecular complexes, such as RNA-protein complexes and protein-lipid complexes) , which are not of the natural handedness (not of the chirality as they occur in nature or as a wild type molecule) and which are ligands for other chiral macromolecules, which are referred to as target or desired macromolecules.
  • Target or desired macromolecules include oligonucleotides (DNA, RNA) and proteins (e.g., polypeptides and peptides) , such as hormones, enzymes, antibodies and antigens.
  • the present invention is a method of identifying D-amino acid peptide ligands which bind a target or desired L amino acid peptide.
  • this invention is a method of identifying peptides comprised of D-amino acid residues that are ligands for oligonucleotides (RNA or DNA) .
  • the present invention is a method of identifying RNA or DNA oligonucleotides "which are of the opposite chirality from that which occurs in nature. DNA occurs in nature as a D isomer.
  • the present invention relates to a method of producing a macromolecule of non-natural handedness that binds to a target macromolecule of natural handedness (e.g., peptide, oligonucleotide) , which is performed as follows : an enantiomer of the target macromolecule or of a domain characteristic of the target molecule is provided and contacted with a library of macromolecules of natural handedness, under conditions appropriate for binding of a macromolecule of natural handedness in the library with the enantiomer; as a result, the enantiomer binds a macromolecule of natural handedness present in the library.
  • a target macromolecule of natural handedness e.g., peptide, oligonucleotide
  • the enantiomer of the macromolecule of natural handedness which is bound to the enantiomer of the target macromolecule is produced;
  • the enantiomer of the macromolecule of natural handedness is a macromolecule of non-natural handedness which binds to the target macromolecule of natural handedness. That is, the enantiomer of a macromolecule which is present in the library and binds to the enantiomer of the target molecule is produced; the result is a macromolecule of non-natural handedness which binds the target molecule (of natural handedness) .
  • the target macromolecule is a protein (e.g., peptide)
  • a D amino acid peptide that binds to a target L peptide is produced.
  • the method is performed as follows: a D amino acid peptide of the target L macromolecule or of a domain characteristic thereof and a library of L amino acid peptides are provided.
  • the library and the D amino acid peptide of the target macromolecule are contacted under conditions appropriate for binding of an L amino acid peptide in the library with the D amino acid peptide; as a result, the D amino acid peptide binds an L amino acid peptide present in the library.
  • An L amino acid peptide present in the library which is bound to the D amino acid peptide is identified and sequenced.
  • a D amino acid peptide of the L amino acid peptide identified in the library or of a characteristic domain thereof is produced; the resulting D amino acid peptide binds to the target L macromolecule of natural handedness. That is, the enantiomer of the L amino acid peptide which is present in the library and binds to the D amino acid peptide of the target L macromolecule is produced; the result is a D amino acid peptide which binds the L-target macromolecule, which in this embodiment is an L amino acid peptide.
  • an L oligonucleotide that binds to a target L protein is produced.
  • the method is performed as follows: a D amino acid peptide of the target L macromolecule or of a domain characteristic thereof and a library of D oligonucleotides are provided.
  • the library is contacted with the D amino acid peptide under conditions appropriate for binding of a D oligonucleotides in the library with the D amino acid peptide, whereby the peptide binds a D oligonucleotides present in the library.
  • a D oligonucleotide which is bound to the D amino acid peptide is identified and sequenced.
  • An L oligonucleotide of the D oligonucleotide identified in the library or of a characteristic domain thereof, is produced; the L oligonucleotide binds to the target L macromolecule of non- natural handedness. That is, the enantiomer of the D oligonucleotide which is present in the library and binds to the D amino acid peptide of the target L macromolecule is produced; the result is an L oligonucleotide which binds the L-target macromolecule, which in this embodiment is an L amino acid peptide.
  • the present invention relates to a method of producing a macromolecule of non-natural handedness that binds to a target macromolecule of natural handedness, which is performed as follows: an enantiomer of the target macromolecule or of a domain characteristic of the target molecule is provided and contacted with a library of macromolecules of natural handedness, under conditions appropriate for binding of a macromolecule of natural handedness in the library with the enantiomer; as a result, the enantiomer binds a macromolecule of natural handedness present in the library.
  • a macromolecule of natural handedness which is bound to the enantiomer is identified and sequenced.
  • the enantiomer of the macromolecule of non-natural handedness which is bound to the enantiomer of the macromolecule of natural handedness or of a characteristic domain thereof, is produced; the resulting enantiomer of the macromolecule of natural handedness is a macromolecule of non-natural handedness which binds to the target macromolecule of natural handedness.
  • an L amino acid peptide which binds a D amino acid peptide of interest is identified as follows: a phage display library which comprises L amino acid peptides displayed on phage surfaces is provided and contacted with the D amino acid peptide of interest, under conditions appropriate for binding of L amino acid peptides displayed on phage surfaces with the D amino acid peptide of interest. Phage which have on their surfaces the D amino acid peptide of interest, bound to an L amino acid peptide displayed on the surface (i.e., which have on their surfaces a D amino acid peptide-displayed L amino acid peptide complex) are identified. The displayed L amino acid peptide in the complex is an L amino acid peptide which binds the D amino acid of interest.
  • the amino acid sequence of the L amino acid peptide displayed on the surface of the phage' can be determined and the D amino acid peptide which corresponds to the amino acid sequence of the L amino acid peptide can be synthesized, resulting in production of a D amino acid peptide which corresponds to the L amino acid peptide displayed on the phage surface.
  • L amino acid peptides displayed on phage surfaces bind D amino acid peptides of a class of proteins, specifically the SRC homology 3 domain (SH3 domain) .
  • a further embodiment is a method of making a D amino acid protein which corresponds to a target L amino acid protein, which can be any protein (including polypeptides, peptides) for which a binding peptide is desired.
  • a phage display library which comprises a mixture of proteins displayed on phage surfaces is contacted with a D amino acid peptide corresponding to the target L amino acid protein or corresponding to a domain characteristic of the target L amino acid protein, under conditions appropriate for binding of L amino acid peptides displayed on phage surfaces with D amino acid proteins.
  • the mixture comprises the target L amino acid protein or a characteristic L amino acid peptide domain thereof.
  • Phage which have on their surfaces the D amino acid peptide bound to an L amino acid peptide displayed on the surface are identified.
  • the amino acid sequences of the L amino acid peptides displayed on the surfaces of phages identified are determined and a D amino acid protein which corresponds to an amino acid sequence of an L amino acid peptide is synthesized, resulting in production of a D amino acid peptide which corresponds to the target L amino acid protein.
  • the present invention also relates to a method of obtaining an L oligonucleotide nucleic acid sequence which binds an L amino acid peptide of interest.
  • a collection of D nucleic acid sequences (e.g., a DNA library) is provided and contacted with a D amino acid peptide of interest, under conditions appropriate for binding of the D nucleic acid with the D amino acid peptide of interest.
  • a D nucleic acid which binds to the D amino acid peptide is isolated and the nucleotide sequence of the D nucleic acid is determined.
  • the D nucleic acid sequence which binds to the D amino acid peptide of interest is prepared using L nucleotides, resulting in the production of an L nucleic acid sequence which binds an L amino acid peptide.
  • a further embodiment of the invention relates to a method of obtaining an L nucleic acid sequence which binds a D nucleic acid which comprises providing a collection of D nucleic acid sequences and contacting the D nucleic acid sequences with an L nucleic acid sequence.
  • a D nucleic acid sequence which binds to the L nucleic acid sequence, thereby producing a D nucleic acid sequence - L nucleic acid sequence complex, is identified.
  • the nucleotide sequence of the D nucleic acid sequence which binds to the L nucleic acid sequence is determined.
  • the D nucleic acid sequence is synthesized using L nucleotides, resulting in the production of an L nucleic acid sequence which binds a D nucleic acid.
  • D amino acid peptides such as D amino acid peptides identified and produced by the methods described herein, including but not limited to synthetic amino acid peptides which bind the SH3 domain, synthetic D amino acid peptides corresponding to all or a portion of the SH3 domain and, more generally, synthetic D amino acid peptides which bind a domain of an intracellular signaling protein.
  • oligonucleotides (RNA, DNA) of non-natural handedness such as oligonucleotides identified and produced by the methods described herein are the subject of this invention.
  • the present invention also relates to a process for producing a derivative of a macromolecule of non-natural handedness that binds a target macromolecule of natural handedness.
  • the method comprises the steps of identifying the macromolecule of non-natural handedness using the methods described herein and modifying or derivatizing the macromolecule of non-natural handedness to produce a derivative thereof.
  • Derivatives obtainable by the method of the present invention described herein are also encompassed by the present invention.
  • D amino acid peptides and L nucleic acid sequences of the present invention are useful as drugs.
  • D amino acid peptides are not good substrates for naturally- occurring proteases (i.e., resistant to proteolytic degradation) and do not elicit an immune response comparable to that elicited by L amino acid peptides.
  • FIG. 1 is a graphic representation of the identification of a D-peptide ligand through mirror-image phage display.
  • the synthetic enantiomer of a structured biopolymer folds into the mirror-image conformation of the natural molecule; likewise, for a bimolecular complex, the two enantiomers of the original partner molecules also form a complex, with mirror-image symmetry to the original.
  • the present invention is based on the discovery that identification of a macromolecule of natural handedness (e.g., L-peptide, D-single-stranded oligonucleotide) that binds the enantiomer of a chiral biological target molecule, provides for a method of identifying a macromolecule of non-natural handedness which binds the natural form of the target. Such enantiomeric macromolecules are assessed to determine their ability to interfere with the biological activity of the target.
  • the present invention provides an approach to the development of new long-acting therapeutic or diagnostic molecules.
  • the present invention relates to a method of identifying enantiomeric macromolecules, including peptides, polypeptides, proteins, oligonucleotides and sugars, as well as macromolecular complexes (oligonucleotide-protein complexes, protein-lipid complexes) , which are not of the naturally-occurring or wildtype handedness (i.e., chirality) and which are ligands for other chiral molecules (peptides, oligonucleotides and macromolecular complexes) .
  • an enantiomer of a macromolecule of natural handedness is the equivalent of the macromolecule of natural handedness, but is of non-natural handedness.
  • an enantiomer of a naturally occurring target macromolecule is prepared and used to isolate, from a collection of naturally occurring macromolecules, a naturally occurring ligand that interacts with the enantiomer.
  • the enantiomeric form of the isolated naturally occurring ligand will interact with (e.g., bind) the naturally-occurring target macromolecule.
  • the target macromolecule of natural handedness can be any macromolecule having one or more chiral centers.
  • the target macromolecule (chiral targets) can be intracellular or extracellular and includes, but is not limited to, nucleic acids (DNA, RNA) , proteins or a characteristic domain thereof (e.g., peptide), peptides, polypeptides, oligonucleotides, carbohydrates, sugars, oligonucleotide- protein complexes (RNA-protein complex) protein-lipid complexes, and phospholipids, all of which contain chiral centers. Domains, fragments of regions of these macromolecules target macromolecules.
  • the target macromolecules can be of mammalian origin (e.g., human) or non-mammalian origin (e.g., bacterial, fungal, viral, protozoan)
  • target macromolecules which are proteins (polypeptides, peptides) include a intracellular signaling proteins and domains thereof (e.g., the SH3 domains, the SH2 domains, the PH domains) (see Cohen, G.B., et al . , Cell , 80:237-248 (1995)), che okines (e.g., ⁇ -chemokine, ⁇ - chemokine) (See Clore, M. G., e ⁇ al . , FASEB J.
  • cytokines e.g., IL-1, TNF, lymphotoxin-c., IL-lS, IL-6, M-CSF, TGF ⁇
  • enzymes e.g., protein kinase C, phospholipase C, phospholipase D) (See Divecha, N. and Irvine, R. F., Cell , 80 : 269-218 (1995)
  • tyrosine kinases See Divecha, N. and Irvine, R. F., Cell , 80 : 269-218 (1995)
  • polypeptide growth factors, and domains thereof, the growth factor receptors and domains or fragments thereof e.g., growth factors and growth factor receptors in the PDGF family, EGF family, FGF family, IGF family, HFG family, VEGF family, neurotrophin family, Eph family, Class I cytokine family, GH family, IL-3 family, IL-6 family, IL-2 family, Class II cytokine family, TNF family
  • PDGF family EGF family, FGF family, IGF family, HFG family, VEGF family
  • neurotrophin family Eph family, Class I cytokine family, GH family, IL-3 family, IL-6 family, IL-2 family, Class II cytokine family, TNF family
  • protein kinases, protein phosphatases, cyclins and Cdc proteins See Hunter, T.
  • transcription factors and domains thereof e.g., Ets domain, bZIP, rel homology domain, STATs, NF-ATs, TCF, Fos, JAKs
  • Ets domain e.g., Ets domain, bZIP, rel homology domain
  • STATs e.g., STATs, NF-ATs, TCF, Fos, JAKs
  • target macromolecules for use in the present invention include: human CD2, human CD58 (LFA-3), human endothelin, heregulin- ⁇ , human interleukin-1/3 converting enzyme (ICE) , human macrophage inflammatory protein 1 - ⁇ , platet factor 4, human melanoma growth stimulating activity, GRO/melanoma growth stimulating activity, MHC molecules, bacterial muramidase, kringle domains (e.g., plasminogen, apolipoproteins) , ras, ras-GAP, selections (e.g., E-selectin, L-selectin, P- selectin) , Pleckstrin homology domains, stromelysin, thrombin, tissue factor, calmodulin, CD4, collagenase, dihydrofolate reductase, fibronectin, fibronectin type III modules, G-protein subunits, vasopressin, Factor IX GLA domain
  • HIV immunodeficiency virus
  • integrase e.g., HIV protease, integrase, matrix, protein tyrosine phosphatase, reverse transcriptase, nef, tat, rev, envelope, and other HIV proteins, domains, fragments or scaffold mimics thereof
  • nef e.g., HIV protease, integrase, matrix, protein tyrosine phosphatase, reverse transcriptase, nef, tat, rev, envelope, and other HIV proteins, domains, fragments or scaffold mimics thereof
  • NH 2 -terminal SH3 domain GRB2 NH 2 -terminal SH3 domain GRB2
  • target macromolecules which are oligonucleotides (RNA, DNA) include HIV RRE (rev responsive element), HIV Tar, and BCR-ABl fusion DNA sequences.
  • HIV RRE rev responsive element
  • HIV Tar HIV Tar
  • BCR-ABl fusion DNA sequences examples include HIV RRE (rev responsive element), HIV Tar, and BCR-ABl fusion DNA sequences.
  • target macromolecules which are phospholipids include phosphoinositide, phosphoinositidase C, phosphoinositide 3-kinase, phosphatidylinositol, phosphatidylinositol 3-phosphate, phosphatidylinositol (4, 5)bisphosphate, phosphatidylinositol (3,4,5) triphosphate, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, inositol (1,4) bisphosphate, inositol (1,4,5) triphosphate, diacylglycerol, sphingosine, sphingosine phosphate, sphigosine phosphocholine and ceramide (See Divecha, N. and Irvine, R. F. , Cell , 80 : 269 -218 (1995)) .
  • an enantiomer of a naturally occurring macromolecule (e.g., the • enantiomer of the target macromolecule or the enantiomer of the macromolecule of natural handedness identified in the library) is prepared using routine methods.
  • the enantiomer of the naturally occurring macromolecules can be prepared through the use of components of the opposite handedness from that which occurs in nature (e.g., the use of D amino acids for synthesis of mirror image peptides or the use of L-nucleic acids for synthesis of mirror image oligonucleotides) .
  • a D-peptide for use in the present invention can be synthesized chemically and purified using affinity chromatography, as described in
  • Example 1 Other methods for preparing the enantiomers of naturally occurring macromolecules are known in the art.
  • the full enantiomeric form of the target macromolecule or part of the three-dimensional molecular surface of the target macromolecule can be used in the methods of the present invention.
  • the enantiomeric form of a subdomain of the target macromolecule, that by itself attains a conformation that resembles that of the enantiomeric form of this domain in the full macromolecule can be prepared and used in the methods of the present invention (Schumacher, T. , et al. , Science, 271:1854-1857 (1996) ) .
  • continuous or discontinuous fragments of the enantiomeric form of the target macromolecule can be prepared on a peptidic or non-peptidic scaffold in which the composite surface of these fragments resembles part of the molecular surface of the full enantiomeric form of the target macromolecule (Tolman, R.L., et al . , Int . J. Pept . Prot . Res . , 41 :455 (1993) ; Muir, T. W., et al. , Biochem. , 33 : 1101 (1994) ; McConnell, S. J. and Hoess, R.H. , JMB 250:460-470 (1995) ; Ku, J., et al .
  • Identification and production of the macromolecules of non-natural handedness which bind to the target macromolecule can be carried out using any collection of macromolecules of natural handedness.
  • the method described is applicable to all situations in which a biologically encoded library is used to isolate structures that interact with a chiral target (or chiral "bait") (Scott, J.K. and Smith, G.P., Science 249 : 386 (1990) ; Devlin, J.J., et al . , Ibid. 249:404 (1990) ; Cwirla, S.E., et al. , Proc. Natl . Acad. Sci . USA 87:6378 (1990); Cull, M.G., et al .
  • a biologically encoded library such as a phage display library (mirror image phage display) can be used in the methods of the present invention. Because ribonucleotide and deoxyribonucleotides also contain chiral centers which are recognized by nucleases, this approach equally applies to both RNA libraries and DNA libraries (Bock, L.C, et al .
  • RNA e.g., SELEX
  • DNA e.g.,DNA library
  • peptide libraries e.g., in vitro transcription/translation based libraries, mono- and poly ⁇ valent phage libraries and 'peptide on plasmid' libraries
  • Use of a DNA library in the method of the present invention is described in Example 5. Examination of the vast amount of structural space represented in these libraries can yield new ligands for proteins of biological and medical importance. Phages which specifically interact with the D- enantiomer and the L-enantiomer of a naturally-occurring (wildtype) macromolecule have been isolated, as described in the exemplification.
  • the selection process of the macromolecule of natural handedness in the library bound to the enantiomer of the target macromolecule can be performed in an achiral solvent (e.g., water) or chiral solvent. Furthermore, the interaction between the naturally occurring macromolecule and the enantiomer is unlikely to require any additional chiral cofactors.
  • An example of a selection process that can be used is described in the exemplification. Modification of the selection process can be performed using methods known in the art.
  • D amino acid peptides which are ligands for a naturally-occurring L amino acid peptide are identified by the claimed method.
  • Such D amino acid peptides can be produced to correspond precisely to the L amino acid peptide (except the constituent amino acids are D, not L enantiomers) or can be modified, such as by a substitution, deletion or modification of one or more constituent amino acids or addition of one or more D amino acid, to produce derivatives of the D amino acid peptides identified.
  • L nucleic acid sequences which bind to D nucleic acid sequences or L amino acid peptides are identified by the claimed methods.
  • the L nucleic acid sequences are produced to correspond to the D nucleic acid (except that the constituent nucleotides are L nucleotides) or can be modified, such as by substitution, deletion or modification of one or more of the constituent L nucleotides or addition of one or more L nucleotides, to produce derivatives of the L nucleic acid sequence.
  • Methods of producing a derivative of a macromolecule of non-natural handedness which binds a target macromolecule of natural handedness using the methods described herein and modifying the macromolecule of non- natural handedness to produce a derivative thereof- is also encompassed by the present invention.
  • the term "derivative" includes macromolecules of non-natural handedness identified, for example, by the methods described herein, which bind to the target macromolecule and have been modified in such a manner that they differ from the macromolecule of non-natural handedness by the addition, deletion, substitution or alteration of one or more components.
  • the derivative is a modified D- peptide.
  • Derivatives of the D-peptides of the present invention include, for example, D-peptides having modified or altered (e.g., enhanced, decreased) affinity, specificity, membrane permeability, hydrophobicity, lipophilicity, oral bioavailability and/or biological half- life.
  • Strategies for producing derivatives of D-peptides include, for example, modifying the peptide backbone by N- methylation (Ostresh, J.M. , et al . , Proc. Natl . Acad. Sci . , USA, 91:11138-11142 (1994); Drug Discovery Technologies , CR.
  • the sidechains of the D-peptides can be modified by introducing non-natural amino acids and amino acid analogues (Combs, A.P., et al . , J. Am. Chem. Soc , 118 : 287-288 (1996); Rivier, J.E. , et al . , Proc. Natl . Acad. Soc , USA, 93:2031 (1995) ; Munroe, J.E., et al . , Bioorganic & Medicinal Chem. Lett . , 5:2897-2902 (1995)) .
  • the D-peptide contains non-natural amino acids and amino acid analogues.
  • Examples of derivatives of D-amino acid peptides having modified side chains can be described using the following formula: NH 2 -CHR-COOH wherein R is a lower alkyl, which is defined herein as an alkyl group of about 1 to about 50 carbon atoms which can be straight or branched and can include one or more double or triple bonds (e.g., methyl, ethyl) .
  • the lower akyl can be a substituted lower akyl such as a(n) -NH 2 ,-OH, aryl (e.g., phenyl) , heteroaryl (e.g., imidazole, indole) , -COOH (e.g., arginine) group.
  • the lower akyl can be an aryl (e.g., phenyl, naphthyl) , a substituted aryl (e.g., -OH, halogen) , a heteroaryl or a substituted heteroaryl group.
  • aryl e.g., phenyl, naphthyl
  • substituted aryl e.g., -OH, halogen
  • the D-peptides of the present invention can be modified by generating cyclic or polycyclic derivatives wherein, for example, disulfide bonds are replaced to optimize and restrict the conformation of the D-peptide (Katz, B. A., et al., J " . Am. Chem. Soc , 117:8541 (1995); Ladner R. C, TIBTECH, 13:426-430 (1995)) .
  • the D-amino acid peptide identified as described herein can be made cyclic as in the case with cyclosporin, via a peptide bond.
  • derivatives of the D-peptides of the present invention can be coupled to or incorporated within carriers to improve membrane permeability and/or bioavailability using, for example, liposomes and/or lipid derivatives (Eichholtz, T. , et al., J. Biol . Chem. , 268(3) :1982-1986 1982 (1993)) and/or peptidic and non-peptidic polymers (Drug Discovery Technologies, CR. Clark, eds. , John Wiley & Sons, 1990) ; Sheldon, K. , et al . , Proc . Natl . Acad. Sci . , USA, 92:2056 (1995) ) .
  • the D-amino acid peptide identified by the methods described herein can be derivatized by increasing the hydrophobicity of the D-amino acid peptide (e.g., alkylating, for example methylating the D-amino acid peptide backbone) , particularly when the target is an intracellular target.
  • a benzene ring can be added to the side chains to increase hydrophobicity.
  • the D-amino acid peptide can be incorporated into a drug delivery system such as a liposome to increase hydrophobicity of the peptide.
  • the C or N terminus of the D-peptide can be modified with protective groups (See for example, Green, T.H. and Wuts, P.
  • a lipophilic polymer can be bonded to the N or C terminal (e.g., polyethylene glycol ester at the C terminal) .
  • one or more side chains can be bonded to a lipophilic polymer such as a polyalkylene glycol (e.g., polyethylene glycol), or bonded to an ether or ester linkage (e.g., forms an ester with the side chain carboxyl group of aspartic acid or glutamic acid) .
  • the D-peptide can be modified by producing a polymer-D-peptide conjugate wherein the polymer is, for example, monomethoxpolyethylene glycol (PEG) and/or polyoxyethylated glycerol (POG) (see Therapeutic Peptides and Proteins, Marshak, D. and Liu, D. , eds. , Cold Spring Harbor Laboratory (1989)) .
  • PEG monomethoxpolyethylene glycol
  • POG polyoxyethylated glycerol
  • the derivative is an L-nucleic acid.
  • Strategies described herein for modifying D-peptides can also be used to modify RNA and DNA of non-natural handedness identified as described herein in order to optimize their pharmacological properties (e.g., Green, L. S., et al., Chem. Biol. , 2:683 (1995) ; Latham, J. A., et al . , Nucleic Acids Res . , 22:2817 (1994) ; Gold, L., et al . , Ann. .Rev. Biochem. , 64 : 163 (1995)) .
  • Synthetic and biologically encoded libraries have proven to be extremely useful for the identification of ligands and nucleic acid sequences for a large variety of macromolecules.
  • Synthetic peptide libraries composed of (D) -amino acids have been favored over gene-based techniques such as phage display Scott, J.K. and Smith, G.P., Science 249 : 386 (1990) ; Devlin, J.J., et al . , Ibid. 249:404 (1990) ; Cwirla, S.E., et al . , Proc . Natl . Acad. Sci . USA 87:6378 (1990), x peptide on plasmid' Cull, M.G.
  • Cyclosporin A an 11 residue cyclic peptide composed mainly of N-methylated and (D) -amino acids, is a leading im unosuppressant and is generally given orally (Ptachcins, R.J., et al . , Clin. Phar acokinetics, 11 : 101 (1986)) .
  • Proteins composed of (D) -amino acids have a chiral specificity for substrates and inhibitors that is the exact opposite of that of the naturally occurring (L) -amino acid protein (Del Milton, R.C , et al . , Science, 257:1445
  • (D) -amino acid ligands can be obtained by either making the (D) -enantiomer of a natural ligand (Fisher, P.J., et al. , Nature 368:651 (1994)), or by making the 'reverse' (D) -enantiomer (Jameson, B.A., et al .
  • the (D) - and (L) -protein have a chiral specificity for substrates and inhibitors that is the exact opposite, the (D) -enantiomeric form of the phage-displayed peptides that interact with the (D) -protein will interact with the protein of the natural handedness.
  • the L- and D-enantiomers of the chicken c-SRC domain were prepared by bacterial expression and chemical synthesis, respectively.
  • the biotinylated, synthetic, 60- amino acid D-SH3 domain was refolded and purified by affinity chromatography, with a D-amino acid version of a known peptide ligand for the SH3 domain (Yu, H., et al . , Cell , 76:933 (1994)) .
  • bacterially expressed L-SH3 was retained on an affinity column with the L- enantiomer of this peptide, but not with the D-enantiomer, which indicates that the interaction of the SH3 domain with its substrates is stereospecific.
  • a phage library was constructed in which random, 10- residue peptide sequences were expressed at the NH 2 - terminus of the piII protein of the bacteriophage fd (Scott, J.K. and Smith, G.P., Science, 249 : 386 (1990) . Because many natural bioactive peptides, such as the immunosuppressant cyclosporin and the tumor promoter microcystin, are cyclic, the library was designed to include a large number of sequences that have a propensity for disulfide bond formation (Smith, P. and Scott, J.K. , Methods Enzymol . , 217 : 228 (1993)) .
  • the positively charged residues in the ligands for the D-SH3 domain are located in the middle of a stretch of conserved residues, which suggests that the mode of ligand binding is different in the two forms.
  • all ligands for the D-SH3 domain contain a pair of cysteine residues, a property that is not observed for the L-peptides that interact with the L-SH3 domain (Yu, H. , et al., Cell , 76:933 (1994); Rickles, R.J. et al . , EMBO J. , 13:5598 (1994; Sparks, A.B., et al . , J. Biol . Chem . , 269 : 23853 (1994) ; Cheadle, C, et al., ibid. , p. 24034)) .
  • the disulfide bond may increase the affinity of these peptides for the D-SH3 domain by reducing the number of possible conformers.
  • Confirmation that the D-amino acid enantiomers of the peptides expressed by these phage particles interact with the all-L-amino acid SH3 domain is carried out using standard binding and detection methods .
  • a D-peptide denoted Pep-Dl which is the mirror image of one of the phage-displayed peptides that bind to the D-SH3 domain, was synthesized and its interaction with the bacterially expressed L-SH3 domain examined.
  • the ligands that are isolated through this procedure are significantly less or not susceptible to the mechanisms that impair the activity of their biological counterparts.
  • the ligands isolated through the method described herein are significantly less or not susceptible to RNase and DNase activity for nucleotide- based ligands (Ashley, G.W., J. Am. Chem. Soc 114 : 9131 (1992); Urata, H. , et al., J. Am. Chem. Soc , 113:8174 (1991) and proteolysis and activation of an immune response for peptide-based ligands (Gill, T.J., et al .
  • synthetic peptide based strategies have an upper limit in degeneracy that is determined either by peptide solubility limits and detection limits for the assay used (for synthetic combinatorial libraries and other solution-based peptide libraries) , or for solid phase based libraries, by volume considerations.
  • the intermediate amplification steps that are used in biologically encoded library systems allow the identification of ligands in situations where background binding is high (in systems that do not employ amplification steps a specific ligand will only be identified if it constitutes an easily detectable part of the total pool of recovered molecules after a single round of screening) .
  • phage display and other biologically encoded library systems allow for the maturation of affinities through mutation of the encoding DNA through processes such as error-prone PCR.
  • phage libraries can accommodate inserts of significant length as compared to synthetic peptide based libraries. This not only allows the possible isolation of ligands of a different size class, but significantly increases the complexity of short ligands that are contained (as sliding windows) within these inserts.
  • identification of D-amino acid peptide ligands which interact with a specific target as described herein can be used to provide guidelines for the design of biased (D) -amino acid peptide and peptide- based libraries.
  • the libraries can subsequently be used to isolate novel ligands.
  • the residue numbering system is that of the full- length chicken c-SRC protein. Residues 81 to 140 of chicken c-SRC were cloned in other Hind III-Bam HI sites of the plasmid pMMHb (Staley, J.P. and Kim, P.S., Protein Science, 3:1822 (1994)) . In this plasmid, proteins are expressed as a fusion with a modified form of the TrpLE leader sequence in which the methionine residues have been replaced with leucine and the cysteine residues have been replaced with alanine (Staley, J.P.
  • Recombinant protein was purified by resuspension of inclusion bodies in 6 M guanidine-HCl and, 0.2 M tris, pH 8.7 (buffer A) and chromatography on a Ni 2+ column (Ni- 2+ - NTA-agarose; Qiagen) . After elution, dialysis against water, and lyophilization, the fusion protein was dissolved in 70% formic acid and cleaved with CNBr (Stanley, J.P. and Kim P.S., Protein Science, 3:1822 (1994)) .
  • Dialyzed and lyophilized material was subsequently taken up in buffer A, and purified by chromatography on a Ni + column (after cleavage, the isolated SH3 domain flows through the column, whereas uncleaved fusion protein and the cleaved TrpLE leader sequence are retained) .
  • the purity and identity of the SH3 domain were confirmed by high-performance liquid chromatography (HPLC) analysis at neutral pH and by laser desorption mass spectrometry (expected, 6686 daltons,- observed, 6683 daltons) .
  • GGVTTFVALYDYESRTETDLSFKKGERLQIVNNTEGDWWLAHSLTTGQTGYIPSNYVAP S-COOH-terminus (SEQ ID No: 1) , residues 81-140 of chicken c-SRC, was synthesized on HMP resin (ABI/Perkin Elmer) with an ABI 431A peptide synthesizer and ABI fastmoc cycles (Fmoc chemistry with HBTU activation and capping with acetic anhydride) .
  • Protected D-amino acids were obtained from Bachem California, Bachem Bioscience, Advanced Chemtech, and Novabiochem.
  • the side chain enantiomers were used, in which the chirality of the side chain is also inverted relative to naturally occurring L-Thr and L-Ile.
  • the NH 2 -terminus of the peptide was modified with NHS-LC-biotin II (Pierce) .
  • the peptide was lyophilized, dissolved in 6 M guanidine HC1, pH 6.0, and dialyzed against 100 mM NaHPO- and 100 mM NaCl, pH 6.0, with the use of dialysis tubing with a molecular cutoff of 3,500 daltons (D) (Spectra/Por) .
  • This peptide is a derivative of an all (L) -peptide shown to bind to the all (L) -SH3 domain (Yu, H. , et al . , Cell , 76:933-945 (1994)) , with an NH 2 -terminal YGG added to facilitate concentration determination (H. Edelhoch, Biochemistry, 6:1948 (1967)) .
  • the L-peptide with the same NH 2 -terminal YGG served as a control ligand in the experiments.
  • Fractions containing material of the expected mass were pooled and dialyzed against water for 72 hrs, lyophilized and taken up in water at a concentration of 107 ⁇ g/ml.
  • the phage library was designed to provide expression of random peptides as NH 2 -terminal fusions with filamentous phage pill protein. Typically, 3-5 copies were present per phage particle, to permit isolation of low/intermediate affinity ligands.
  • DNA encoding a 10-residue random insert with flanking serine or cysteine residues (S/C-X 10 -S/C) (SEQ ID NO:20) was prepared by PCR of an 85 residue oligonucleotide (Smith, C , "Cloning in Fuse vectors", Division of Biological Sciences, University of Missouri (Edition of February 10, 1992)) using biotinylated primers as described (Smith, CP. and Scott, J.K. , Methods Enzymol . , 217:228 (1993) ) .
  • S/C-X 10 -S/C flanking serine or cysteine residues
  • N A/T/C/G (equimolar mixtures)
  • the library was made by ligation of a random PCR product into Sfi I-cut Fuse 5 vector. After ligation the reaction mixture was extracted with phenol and chloroform, ethanol-precipitated and taken up in 10 mM Tris/lm mM EDTA (pH 8.0) . The ligation product was subsequently transferred into electrocompetent MC1061 cells (Biorad) using a Bio-Rad E__, coli pulser and 0.1 cm cuvettes. After non-restrictive growth for 1 hr aliquots of transformed cells were plated on tetracycline-containing plates to determine the efficiency of transformation, yielding an initial library of 3.6xl0 8 transformants.
  • the transformation mixture was subsequently diluted to a volume of 400 ml LB and 20 /xg/ml tetracycline, and grown for an additional 14 hrs.
  • a phage stock was prepared by two successive polyethylene glycol (PEG) precipitations of the culture supernatant as described.
  • the phage stock was finally resuspended in tris buffered saline/NaN 3 and titered by infection of K91-kan cells (A21) , yielding a total of 2.8X10 11 transforming units.
  • the randomness of the inserts was confirmed by sequencing of individual clones. 4xl0 10 transforming units were subsequently used to infect K91-kan cells to generate an amplified library.
  • phage were purified by three repetitive PEG precipitations yielding 10 ml of the amplified phage library (1.2-10 12 transforming units/ml) in TBS/NaN 3 .
  • the quality of the library was confirmed by selection of phage that expressed inserts that interact with 1) the lectin concanavalin A (Con A) (Oldenburg, K.R. et al., Proc Natl. Acad. Sci. USA, 89:5393 (1992); Scott, J.K. et al., ibid, p. 5398) ; 2) two mouse monoclonal antibodies raised against the mouse MHC class I heavy chain; and 3) a bacterially expressed form of the Src SH3 domain, all screens giving the expected results .
  • Con A the lectin concanavalin A
  • 3) a bacterially expressed form of the Src SH3 domain all screens giving the expected results .
  • the phage display was screened with the D-SH3 domain and a series of peptide sequences, which showed no obvious sequence similarity to the L-SH3-binding sequences, were isolated (see Table 1) .
  • E.I.A./R.I.A. plate (Costar) were coated overnight with 10 ⁇ g streptavidin (Pierce) in 100 ⁇ l 100 mM NaHC0 3 at 4°C After a single wash with water, wells were incubated with 100 ⁇ l (10.7 ⁇ g) of biotinylated (D) -SH3 for 1 hr. at 20oC, blocked for 2 hrs with 30 mg/ml dialyzed bovine serum albumin (BSA) in 100 mM NaHC0 3 , and again incubated with 100 ⁇ l (10.7 ⁇ g) of biotinylated (D) -SH3 for 1 hr.
  • BSA bovine serum albumin
  • Unliganded streptavidin was blocked for 30 minutes by the addition of 8 ⁇ l 5 mM biotin in tris-buffered saline (TBS) .
  • TBS tris-buffered saline
  • Wells were subsequently washed 5 times with phosphate buffered saline (PBS) and 0.1% Tween-20, and incubated overnight with 50 ⁇ l of the phage stock in TBS/NaN 3 and 50 ⁇ l of TBS, 0.1% Tween-20, 1 mg/ml BSA and 0.05% NaN 3 .
  • Wells were subsequently washed by six additions of 200 ⁇ l of TBS, 0.1% Tween-20 and 1 mg/ml BSA with increasing incubation times in the later rounds of the selection procedure.
  • (D) -SH3 bound phage particles were subsequently eluted by the addition of 100 ⁇ l D-SH3 ligand peptide (715 ⁇ M) , sequence D-YGGRELPPLPRF-amide (SEQ ID No: 2) , for 15 minutes at 4°C, at a final concentration of 700 to 1000 ⁇ M peptide. Acid elution of phages in this screen gives no detectable preferential binding to D-SH3 coated wells after four rounds of selection.
  • the eluate was subsequently used to infect K91-kan cells. Briefly 100 ⁇ l eluate was mixed with 100 ⁇ l K91 Terrific Broth cells (prepared as described) an incubated for 20 minutes at room temperature.
  • the mixture was subsequently transferred into an Erlenmeyer flask containing 20 ml LB/0.2 ⁇ g/ml tetracycline. After 1 hr incubation while shaking at 37°C, tetracycline was added to a final concentration of 20 ⁇ g/ml, appropriate dilutions were plated on tetracycline-containing plates (20 ⁇ g/ml) to determine the titer of the eluate and the culture was incubated at 37°C for 12-16 hrs. Phage were isolated from the supernatant by two PEG precipitations and the resulting phage stock was used for titering to determine the yield, and for the subsequent round of selection. In the fourth round of selection, phage were incubated in wells coated with or without the D-SH3 domain to determine the specificity of the capture. The washing conditions and yields of the different rounds of selection were as follows:
  • Peptides in groups I and III contained at least a single arginine residue that may form a salt bridge with aspartic acid99 in the SH3 domain and which interacts with arginine residues in Src-binding poly proline peptides (Feng, S., et al . , Science 266:1241 (1994) ) .
  • CAYGFKLGLIKC (SEQ ID No: 18) fdSRC-3
  • This phage clone has an alanine to arginine substitution directly amino terminal to the insert region.
  • Phage display with the L-SH3 domain was also assessed.
  • the (L) -SH3 domain is used to screen a phage library (A12) for interacting sequences the poly-proline sequences that have been observed by others Rickles, R.J., et al . , EMBO J. , 13:5598 (1994) ; Sparks, A.B., J. Biol . Chem. ,
  • GCN4 leucine zipper short (33 residues) , but low probability
  • c-Src SH-3 domain 60 residues, bind peptides (type II polyPro)
  • GCN4 leucine zipper synthesis straightforward, CD as expected after 5 rounds no difference in recovery +/- zipper Test 59 individual clones, no difference +/- zipper
  • Src SH3 domain Src (L-) SH3 binds to substrate in a stereo-specific manner Src (L-) SH3 selects poly-Pro sequences from library:
  • Pep-D2 corresponds to the fdSRC-1 insert CLSGLRLGLVPC (SEQ ID No: 16) (Table 1) , with the COOH-terminal alanine that is present in all flanking sequences (see Example 2) .
  • the other phage isolates obtained after four rounds of selection expressed one of the following two sequences: CKRFVWRGQALC (SEQ ID No: 13) (10 isolates) and CWYLGYWPGQEC (SEQ ID No: 15) (12 isolates) .
  • the first of these sequences resembles the background sequences that are isolated with a variety of biotinylated ligates (Smith, G. P. and Scott, J.K., Methods in Enzymol. , 217:228 (1993)) and is also similar to a sequence that was isolated previously with a monoclonal antibody against myohemerythrin, although it does not conform to the recognition motif for this antibody (Smith, G. P. and Scott, J.K., Science, 249 : 386 (1990)) .
  • This sequence is therefore likely to bind to some component in the system other than the SH3 domain. Indeed, a D-amino acid version of this sequence fails to bind to the L-SH3 domain, as judged by ELISA and NMR studies.
  • the other sequence that was picked up after four rounds of selection shows limited similarity to the first sequence and has not been examined further.
  • Example 2 An All D Amino Acid Src SH3 Domain Binds to the L Src SH3 Domain
  • the (D) -amino acid peptide denoted Pep-Dl, (D) - RCLSGLRLGLVPCA (SEQ ID NO:11, a representative sequence of group III sequences) which is the mirror image of one of the phage-displayed peptides that binds to the D-Src SH3 domain, was synthesized and its interaction with the bacterially expressed (L) -SH3 domain was examined.
  • Pep-Dl corresponds to the fdSRC-1 insert CLSGLRLGLVPC (SEQ ID No: 16) (Table 1) , with the COOH-terminal alanine that is present in all flanking sequences. The arginine immediately preceding the first cysteine residue was observed in the fdSRC-3 sequence (Table 1) . The presence of arginine and lysine residues close to the NH 2 -terminus of secretory and transmembrane proteins negatively affects protein translocation (Boyd, D. and Beckwith, J. , Cell ,
  • the peptides with and without the NH 2 terminal tyrosine were air- oxidized in 100 mM tris, pH 8.5, for 48 hours at a concentration of 1 mg/ml.
  • Oxidized peptide was purified by reverse-phase HPLC with a C 18 column and a water- acetonitrile gradient in 0.1% trifluoroacetic acid. The identify of the products was confirmed by laser desorption mass spectrometry.
  • Pep-Dl shows no detectable binding activity in this assay (K d >> 800 ⁇ M) , which indicates that the formation of the disulfide is required for efficient binding.
  • the affinity of Pep-Dl for the L-SH3 domain was determined by a competitive enzyme-linked immunosorbent assay (ELISA) . Single wells of a 96-well plate were coated with 5 ⁇ g of the L-SH3 domain (Scott, J.K. and Smith, G.P., Science, 249:386 (1990) ; Smith, CP. and Scott, J.K. ,
  • the K d of the L-peptide YGGRELPPLPRF-amide was determined to be 6.0 ⁇ M by direct tryptophan fluorescence spectroscopy.
  • a solution of the peptide was titrated into 1 ⁇ M SH3 solution in 15 mM NaCl and 10 mM NaHP0 4 , pH 7.2. Tryptophan fluorescence was induced by excitation at 295 nm (5 nm slit width) , and emission was measured at 339 nm (10 nm slit width) , with a Hitachi F-4500 fluorescence spectrometer.
  • the dissociation constant was determined by Scatchard analysis.
  • the ligand-binding site of the SH3 domain for its natural, L-amino acid ligands consists of three pockets that together form a relatively shallow groove on one side of the molecule (Feng, S., et al., Science, 266:1241 (1994) ; Yu, H. , et al . , Science, 258 : 1665 (1992)) .
  • pockets B and C form a hydrophobic surface that accommodates the aliphatic and proline residues in SH3 ligands (Feng, S. et al . , Science, 266:1241 (1994) ; Yu, H. et al . , Science, 258:1665 (1992)) .
  • residues -40 - include residues 94, 97, 112, 115, 117, 119, 120, 131, 132 and 135, the indole resonance of tryptophan 119 , and the side chain amides of asparagine 113 , and asparagine 135 .
  • the resonances of 95, 96, 98, 99, 100, 118 and 134 and the indole resonance of tryptophan 118 were absent in the presence of ligand.
  • Control experiments, using the L- peptide YGGRELPPLPRF amide (SEQ ID NO: 2) resulted in 17 resonances that were shifted by ⁇ 3 0.1 p.p.m. in the ⁇ dimension or ⁇ 3 0.5 p.p.m.
  • Pep-Dl appears to occupy only part of the binding site that is contacted by the polyproline-type ligands for the SH3 domain (Fig. 3) .
  • Residues that form part of pocket B and pocket C (tyrosine 90 and tyrosine 92 ) , or that are adjacent to this pocket (valine 87 and leucine 89 ) are not perturbed upon binding of Pep-Dl (Figs. 2 and 3) .
  • Mutational analysis suggests that, for L-amino acid ligands, interactions at these sites are required for high- affinity binding (Feng, S. et al., Science, 266:1241 (1994) .
  • D-peptide inhibitors of higher affinity could therefore potentially be obtained by the design or selection of analogs of Pep-Dl or Pep-D2 (Table 1) that extend further along the groove, into pocket C of the SH3 domain.
  • Example 4 Use of the D-amino acid peptide ligands to design highly-enriched libraries Random synthetic libraries do not cover enough conformational space to always allow for the isolation of high affinity ligands for a given target.
  • a more promising strategy is the use of libraries that are biased towards structural elements known to interact with a target of interest.
  • the D-amino acid peptide sequences that interact with a given target that are isolated using the strategy described here are used as guidelines for the design of biased (D) -amino acid peptide and peptide-based libraries. These biased libraries may subsequently be used for the isolation of novel ligands.
  • the three classes of (D) -amino acid peptide ligands for the SH3 domain are useful to design (D) -amino acid peptide-based libraries highly enriched for SH3-binding peptides or peptidomimetics. Such libraries are useful to identify peptides which bind the SH3 domain and are particularly useful because they are biased toward (have an enhanced content of) peptides known to bind the SH3 domain. All SH3 domains for which the interaction with (L) -amino acid peptides has been examined bind to ligands with similar structural elements.
  • Synthetic libraries based on the structure of (D) -amino acid ligands for the SH3 domain are also enriched in ligands for other SH3 domains.
  • the biased libraries based on the structure of the (D) -amino acid peptide ligands for the SH3 domain are useful for the isolation of ligands for a variety of SH3 domains.
  • a biased library is constructed, for example, as follows, based on the amino acid sequences of the 3 classes of peptides described in Example 2, which have been shown to bind the (D) -SH3 domain.
  • a chemical peptide library of D-amino acids is prepared in which about 80% of the D-amino acid peptides of the library have the conserved amino acid residues and about 20% of the D-amino acid peptides of the library do not have the conserved amino acid residues.
  • the library which is heavily biased for peptides having the general structure of peptides known to bind (D) -SH3 domain (i.e., 80%) , can be used to isolate other D-amino acid peptides which have the conserved structure and bind to other SH3 domains (i.e., human) .
  • D-amino acid peptides in which the conserved amino acid residue has been altered i.e., from the 20% of the D-amino acid peptides
  • which binds to the SH3 domain with equivalent or greater affinity can be isolated.
  • Vasopressin was prepared using conventional solid phase peptide synthesis.
  • an in vi tro selection was used to isolate single-stranded DNAs (ssDNAs) that bind synthetic D-vasopressin (DVP) .
  • ssDNAs single-stranded DNAs
  • DVP D-vasopressin
  • a starting pool of 10 16 different 96-mers was synthesized on a DNA synthesizer. Each pool molecule contained a central region with 60 random-sequence positions that was flanked by two 18-nt defined regions. Molecules within this starting pool that bind DVP were enriched by affinity chromatography.
  • the affinity resin was prepared by coupling biotinylated DVP to streptavidin agarose.
  • the ssDNA pool was radiolabeled, denatured, renatured in a physiological buffer and passed over this resin. After extensively washing the column, molecules that bind DVP were eluted from the column with a molar excess of DVP. Eluted pool was amplified by PCR using a negative-strand "primer-terminator" , that is, an oligonucleotide bearing a central segment of non-nucleotide material that blocks further extension of the positive strand (Williams, K.P. and Bartel, D.P., Nucleic Acids Res . , 23:4220-4221 (1995)) . Such PCR results in a substantial size difference between the two product strands, facilitating gel-purification of the positive- strand ssDNA pool. This combination of affinity chromatography and PCR constituted one cycle of selection amplification.
  • D-vasopressin aptamers Sequence blocks shared by the two original aptamers are in capital letters. The defined- sequence segments that flanked the random-sequence region are indicated in bold. A segment derived from random- sequence positions that is shared by the two sequences of the final pool is in outline. The 69.1 aptamer is a deletion derivative of 96.4.

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Abstract

L'invention porte sur un procédé d'identification de macromolécules (peptides, oligonucléotides, sucres et complexes macromoléculaires, tels que des complexes protéine/ARN et des complexes lipide/protéine), n'étant pas naturellement chirales (dépourvues de la chiralité d'origine naturelle ou rencontrée dans des molécules de type sauvage) et qui sont des ligands pour d'autres macromolécules chirales.
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WO1997035194A2 (fr) * 1996-03-21 1997-09-25 President And Fellows Of Harvard College Procede de criblage enantiomere et compositions pour ce procede
WO1997043444A1 (fr) * 1996-05-09 1997-11-20 Smithkline Beecham Plc Methode de criblage de composes qui interagissent avec le l-enantiomere d'un arn cible
US6605713B1 (en) 1996-08-30 2003-08-12 Jens Peter Furste Mirror-symmetrical selection and evolution of nucleic acids
WO1998008856A2 (fr) * 1996-08-30 1998-03-05 Fuerste Jens Peter Selection et evolution speculaires d'acides nucleiques
WO1998008856A3 (fr) * 1996-08-30 1998-05-07 Jens Peter Fuerste Selection et evolution speculaires d'acides nucleiques
US6251666B1 (en) 1997-03-31 2001-06-26 Ribozyme Pharmaceuticals, Inc. Nucleic acid catalysts comprising L-nucleotide analogs
US6602858B2 (en) 1997-03-31 2003-08-05 Ribozyme Pharmaceuticals, Inc. Nucleic acid catalysts comprising L-nucleotide analogs
EP1264603A1 (fr) * 2001-06-10 2002-12-11 Noxxon Pharma AG Utilisation de L-polynucleotides pour l'imagerie diagnostique
WO2002100442A3 (fr) * 2001-06-10 2003-04-10 Noxxon Pharma Ag Utilisation de l-polynucleotides et de leurs derives
WO2002100442A2 (fr) * 2001-06-10 2002-12-19 Noxxon Pharma Ag Utilisation de l-polynucleotides et de leurs derives
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WO2004013274A3 (fr) * 2002-08-01 2004-05-13 Noxxon Pharma Ag Acides nucleiques a liaison de ghreline
WO2006058705A1 (fr) * 2004-11-29 2006-06-08 Noxxon Pharma Ag Acide l-nucleique liant la vasopressine
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