WO1993014781A1 - Nouveaux peptides et procede pour modifier l'activite de proteines allosteriques - Google Patents

Nouveaux peptides et procede pour modifier l'activite de proteines allosteriques Download PDF

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WO1993014781A1
WO1993014781A1 PCT/US1993/000581 US9300581W WO9314781A1 WO 1993014781 A1 WO1993014781 A1 WO 1993014781A1 US 9300581 W US9300581 W US 9300581W WO 9314781 A1 WO9314781 A1 WO 9314781A1
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peptide
protein
peptides
allosteric
activity
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PCT/US1993/000581
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Charles Fredrick Fox
Robert E. Williams
Kanury V.S. Rao
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The Regents Of The University Of California
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    • 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
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
    • 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
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • 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
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to novel peptides and a method for altering the biological activity of allosteric proteins, and more particularly to a method for inhibiting or activating allosteric proteins using effector peptides and to the peptides obtained.
  • Allosteric proteins may be characterized as having flexibility in structure so that interaction of the protein with an allosteric effector molecule at one site affects the three-dimensional conformation of the protein and thus its interactions with other molecules at one or more additional site(s).
  • the other molecules may be substrate molecules or ions, or other ' sites on the allosteric protein involved in intermolecular interactions.
  • the biological activity of a given allosteric protein is a function of precise structural relationships between regions of the protein.
  • certain structural (“allosteric") transitions induced by allosteric effectors and facilitating interactions between regions of the allosteric protein molecule are required to achieve a three-dimensional structure that supports expression of functional activity of the protein, e.g. for the protein to bind a substrate molecule or ion, or support inhibition of functional activity.
  • Allosteric proteins that have enzymatic activity are involved in many important physiological processes in man.
  • allosteric enzymes include receptors that behave as protein kinases.
  • the activity of protein kinases is considered critical in the regulation of cellular functions including regulation of metabolism, cell growth and differentiation.
  • a number of protein kinases have been shown to be highly elevated in particular types of human cancer. It is believed that many cases of cancer arise from the abnormal activity of altered cellular genes, known as oncogenes. These genes, or the amount of expression of their gene products, can be altered as the result of infection by oncogenic viruses or chemical damage by carcinogens.
  • the receptor for epidermal growth factor (EGF)
  • EGF epidermal growth factor
  • an oncogene encoded protein that has tyrosine kinase activity has been shown to be highly elevated in a serious form of cancer known as sguamous cell carcinoma (Kamata et al., Cancer Res. 46:1648-1653 (1986); Cowley et al., Br. J. Cancer 53:223-229 (1986) and Filmus et al., Biochem. Biophys. Res. Commun. 128:898-905 (1985)) .
  • a receptor kinase, homologous to EGF receptor in the intracellular domain, and designated HER-2 has been shown to be overexpressed in other cancers, including certain forms of breast cancer (Slamon et al., Science 235:177-182
  • the Abelson oncogene encodes a protein kinase associated with certain leukemias (Rosenberg et al., Adv.
  • Aspartate transcarbamoylase (ATCase) from E. coli is an allosteric protein that has been used as a model for study of both homotropic (interactions within a subunit of the protein) and heterotropic (interactions between subunits) in proteins (See, Kantrowitz and Lipscomb, TIBS 15:53-59 (1990)).
  • ATCase Aspartate transcarbamoylase
  • contacts are made and/or broken between portions of the polypeptide chain(s) as the protein changes from active to inactive state, or vice versa.
  • the making and breaking of the intramolecular interactions between specific residues in ATCa ⁇ e occur between series of amino acid residues, each on relatively short (less than 12 amino acids) opposing sequences on the enzyme ( Id . )
  • ATCase is one of only a very few proteins for which detailed high resolution structural information on allosteric interactions is available to define the consequences of activity-modifying ligands on structure.
  • extremely detailed x-ray crystallographic and other analytical studies have been performed on species of ATCase mutants modified by specific mutagenesis of the ATCase gene to verify interactions that are functionally important. These studies provide independent experimental evidence of the identity of the short interactive peptide sequences required to support activity of this allosteric protein.
  • a ligand such as a peptide capable of altering the activity of an allosteric protein
  • many new forms of treatment for human disease or dysfunction associated with the activity of that protein may be developed.
  • the platelet-derived growth factor (PDGF) receptor which is implicated in the formation of atherosclerotic lesions can be inhibited to prevent intralumenal smooth muscle cell migration that results in such lesions.
  • Interleukin receptors such as the IL-1 receptor can be inhibited to control inflammation.
  • the T- cell receptor involved in the mechanism of rejection of tissue transplants and pathogenic self-reactivity may be selectively deactivated to prevent rejection of tissue transplants or suppress the self-reactivity associated with a variety of conditions including rheumatoid arthritis,, allergic encephalitis, Hashimoto's thyroiditis, myasthenia gravis and other autoimmune diseases.
  • the insulin receptor may be activated to treat diabetes by enabling the body to use a substitute for insulin that might be delivered orally.
  • the epidermal growth factor (EGF) receptor can be activated to control ulcers and speed healing of corneal wounds and the receptor for PDGF and for other growth factors involved in wound healing can be activated to promote wound healing.
  • Fibroblast growth factor (FGF) receptor may be activated to promote endothelial cell proliferation and the T-cell receptor complex can be activated to promote cellular immunity, resist infection and counter immunity-attacking conditions such as AIDS.
  • the protein In preparation ' for analysis by x-ray crystallography the protein may be crystallized together with substrate molecules or effector ligands so that the structure of the complex formed between the protein and the substrate sites at which the substrate or ligand which binds to it can be determined. Knowledge of the structure of ligand binding sites revealed by such analyses can facilitate synthesis of derivatives of the binding ligand with preferred pharmacological properties.
  • regions of amino acid sequence having hydrophobicity or hydrophilicity may be identified or predicted using hydropathy analysis (Kyte and Doolittle, J. Mol. Biol. 157:105 (1982)) .
  • the information gathered from these procedures may be used to synthesize small peptides having sequences that correspond to the sites in the proteins identified as having a binding or structural function.
  • peptides derived from the binding sites on antigens that recognize antibody have been used to examine antibody/antigen interactions (Houghten, Proc. Natl . Acad. Sci. USA 82:5131-5135 (1985)) .
  • Pierschbacher and Ruoslahti (Nature 309:30-33 (1984)) have synthesized small peptides (four amino acids in length) having cell attachment- inhibiting activity and having the same sequence as a cellular recognition sequence on fibronectin, an extracellular glycoprotein involved in cell attachment.
  • These researchers used sequential proteolytic fragmentation to isolate a domain within the fibronectin molecule that inhibits cell attachment.
  • Synthetic peptides have also been prepared having inhibitory effects on protein kinases. These peptides typically compete with a substrate of the kinase for binding. For example, Yaish et al. (Science 242:933-935
  • inhibitory peptides that correspond to previously identified autoinhibitory domains of several kinases including calcium/calmodulin-dependent protein kinase II, smooth muscle myosin light chain kinase, protein kinase C and the heat-stable inhibitor of cAMP-dependent protein kinase (PKI-tide) .
  • Autoinhibitory domains of protein kinases are believed to inhibit kinase activity by interacting with elements of the catalytic domain including the substrate-binding site. Synthetic peptide analogs of autoinhibitory domains have been shown, to inhibit kinase activity competitively with respect to protein substrate.
  • sequences of autoinhibitory domains of protein kinases often contain basic amino acid residues resembling the natural substrate recognition sequence for kinase, but lacking a phosphate acceptor site, and are thought to be important for interactions with the catalytic domains as "pseudosubstrates" (Hardie, Nature 335:592-593 (1988)) . Because many protein kinases share some of the same basic amino acid determinants for substrate recognition, Smith et al. hypothesized that synthetic peptides based on these "pseudosubstrate” sequences might be recognized by other kinases.
  • a method for the development of effector allosteric modifiers or "ligands” capable of modifying the functional activity of allosteric proteins, without requiring prior knowledge of a substantial amount of the three-dimensional structure of the protein or of specific ligand binding sites and structural relationships, would greatly facilitate the development of therapeutic agents and therapies for human diseases and dysfunctions.
  • a method for producing effector ligands which alter the functional activity of allosteric proteins meets this need.
  • the method is based on the totally unexpected discovery that effector peptides corresponding to portions of the amino acid sequence (the "target sequence") of an allosteric protein can alter the activity of the allosteric protein.
  • effector peptides can either activate or inhibit the functional activity of the protein.
  • the method for producing effector peptides that alter a functional activity of an allosteric protein includes: 1) determining a target sequence of the primary amino ' acid sequence of an allosteric protein, containing at least one site of intramolecular or intermolecular contact within the allosteric protein, the site involved in an allosteric transition resulting in alteration of the expression of a functional activity of the allosteric protein; 2) synthesizing screening peptides of from about 10 to about 20 amino acids in length each of which is substantially identical to a region of the target sequence and which in linear array correspond to substantially all of the target sequence of the primary amino acid sequence determined in step 1) ; and measuring a functional activity of the allosteric protein when reacted with each peptide to identify effector peptides that inhibit or activate a functional activity of the allosteric protein.
  • the invention also provides methods of using the effector peptides of the invention to alter the functional activity of an allosteric protein.
  • the effector peptide is at least 3 amino acids in length.
  • the region of the amino acid sequence of the allosteric protein selected for synthesis of the peptides can contain amino acids capable of forming ⁇ -helical or ⁇ - pleated sheet secondary • structure within the selected portion, and the peptide can then be identical or substantially identical to those amino acids capable of forming such ordered secondary structures.
  • the activity of the allosteric protein can be either increased or decreased by reaction with the effector peptides. This activity can be, for example, enzymatic activity or the binding affinity of the protein for a ligand.
  • EGF epidermal growth factor receptor
  • the allosteric protein can be a receptor protein, enzyme, transport protein, nucleic acid binding protein and extracellular matrix protein. If a receptor protein, the allosteric protein can be epidermal growth factor receptor, insulin receptor, platelet-derived growth, factor receptor, tumor necrosis factor receptor, fibroblast growth factor receptor, erythropoietin receptor, lymphokine receptor and cytokine receptor.
  • the invention includes methods of using the effector peptides of the invention to alter a functional activity of an allosteric protein by reacting the allosteric protein with one or more of the effector peptides.
  • Another aspect of this invention is the effector peptides obtained that are capable of altering the expression of activity of an allosteric protein.
  • These peptides are at least 3 amino acids in length, and preferably are at least 6 amino acids in length and capable of substantially altering,- by inhibiting or activating, the expression of functional activity of an allosteric protein when reacted with the allosteric protein.
  • Each peptide is substantially identical to a portion of a selected target region of the primary amino acid sequence of an allosteric protein, and when taken together in linear array correspond to substantially all of the target sequence.
  • the target sequence contains at least one site of intramolecular or intermolecular contact within the allosteric protein, the site involved in an allosteric transition resulting in an alteration of the expression of functional activity of the allosteric protein.
  • the effector peptides cause inhibition or activation of the functional activity of the allosteric protein when reacted with the protein.
  • Effector peptides of the invention for inhibiting tyrosine kinase activity of human epidermal growth factor receptor are as follows (SEQ ID NO:6) -. V-L-G-S-G-A-F-G-T-V- Y-K-G-L- , T-V-Q-L-I-T-Q-L-M-P, -C-V-Q-I-A-K-G-M-N-Y-L, G- M-N-Y-L-E-D-R-R-L-V-H-R-D-L, V-K-I-T-D-F-G-L-A-K-L-L-G, M- A-L-E-S-I-L-H-R-I-Y-T, Q-S-D-V-W-S-Y-G-V-T-V-W-E-L-M, P-A- S-E-I- ⁇ -S-I-L-E-K, P-I
  • peptides which cause at least 50% inhibition of the tyrosine kinase activity of human epidermal growth factor when the peptide is present at a concentration of about 1 mM (SEQ ID NO:6) : T-V-Q-L-I-T-Q-L-M-P, K-V-K-I-P-V- A-I, I-T-Q-L-M-P-F-G-C-L-L-D, C-L-L-D-Y-V-R-E, W-C-V-Q-I-A- K-G-M-N-Y-L, V-Q-I-A-K-G-M-N-Y-L, G-M-N-Y-L-E-D-R-R-L-V-H- R-D-L, A-A-R-N-V-L-V-K-T-P-Q-H-V-K-I-T, P-I-
  • inhibitory peptides are the following peptides that cause at least 75% inhibition of the tyrosine kinase activity of human epidermal growth factor when the peptide is present at a concentration of about 1 mM (SEQ ID NO:6) : K-V-K-I-P-V-A-I, I-T-Q-L-M-P-F-G- C-L-L-D, W-C-V-Q-I-A-K-G-M-N-Y-L, V-Q-I-A-K-G-M-N-Y-L, G-M- N-Y-L-E-D-R-R-L-V-H-R-D-L,A-A-R-N-V-L-V-K-T-P-Q-H-V-K-I-T, Y-L-V-I-Q-G-D or I-M-V-K-C-W-M-I-D-A-D.
  • inhibitory peptides are- (SEQ ID N0:6) : A-A-R-N-V-L-V-K-T-P-Q-H-V-K-I-T, Y-L-V-I-Q-G-D or V- Q-I-A-K-G-M-N-Y-L that cause at least 85% inhibition of the tyrosine kinase activity of human epidermal growth factor when the peptide is present at a concentration of about 1 mM.
  • the invention also provides effector peptides for stimulating activity of an allosteric protein.
  • Effector peptides for stimulating the tyrosine kinase activity of human epidermal growth factor receptor having the amino acid sequence (SEQ ID NO:6) R-R-H-I-V-R-K-R-T or K-F-R-E-L- I-I-E-F-S-K-M-A-R-D.
  • the peptide having the sequence (SEQ ID NO:6) K-F-R-E-L-I-I-E-F-S-K-M-A-R-D is particularly preferred as a stimulating effector peptide.
  • Still other methods of the invention are for inhibiting the protein tyrosine kinase activity of human epidermal growth factor receptor by reacting an inhibitory effector peptide of the invention with the growth factor, and for stimulating the tyrosine kinase activity of human epidermal growth factor receptor by reacting a stimulatory effector peptide of the invention with the growth factor.
  • Figure 1 shows a comparison of portions of the amino acid sequence of four (4) related receptors, the insulin receptor, INS-R, the platelet-derived growth factor receptor, PDGF-R, the EGF receptor, HER-1 and HER-2, a receptor with sequence nearly identical to EGF receptor in the cytoplasmic kinase domain (boxes indicate regions of sequence homology) .
  • Figure 2 is a table listing the 90 peptides and their derivatives synthesized of which 78 were tested as described in Examples 1 and 2, infra.
  • Figure 3 shows the peptides synthesized to cover various segments of the amino acid sequence of EGF receptor from residue 646 to residue 1000, as described in Example 1, infra (peptides having substantial ⁇ -helical or ⁇ - pleated sheet forming structure are boxed; those having substantial o.-helical structure are boxed and shaded. Some of the peptides extend past the boundaries of the sequence at the end of each line and are to be read contiguously from line to line.)
  • Figure 4 is a bar graph depicting the results, of inhibition tests of certain peptides listed in Figure 2, as described in Examples 1 and 2, infra.
  • Figure 5 is a table summarizing the properties of inhibitors of EGF receptor kinase activity with greater than 34% inhibition occurring at about 1 mM concentrations of peptide, as described in Example 1, infra.
  • Figure 6 is a table summarizing the properties of additional peptide inhibitors for which maximal inhibition was about 50% or less regardless of peptide concentration, as described in Example 1, infra.
  • Figure 7 are graphs depicting the inhibitory effects of peptide numbers 32, 41A, 43 and 21, as described, in Example 1, infra.
  • Figure ' 8 is a graph of the inhibitory effects. of peptides 14 and 44 classified as partial inhibitors, as described in Example l, infra.
  • Figure 9A, B and C are double reciprocal plots of the rate of substrate angiotensin II phosphorylation by EGF receptor incubated with different concentrations of substrate and in the absence or presence of inhibitor peptides, as described in Example 1, infra.
  • Figure 10 is a graph showing the effects of peptide numbers 42 and 26 on substrate phosphorylation by EGF receptor in the presence or absence of EGF, as described in Example 2, infra.
  • Figure 11 is a diagrammatic representation of the possible three dimensional structure of peptide numbers 26 and 42 represented on a peptide wheel, as described in Example 2, infra.
  • the present invention provides a method for rapidly obtaining peptides capable of altering a functional activity of an allosteric protein, as well as the effector peptides produced by this method.
  • effector peptides These peptides capable of alteration of an activity of an allosteric protein are referred to as "effector peptides, " and sequences in the allosteric protein from which these peptides are derived are referred to as "target sequences.”
  • a "functional activity" of an allosteric protein is defined herein as the rate of enzymatic activity if the protein is an enzyme and as the binding affinity of a ligand, e.g. ion molecule or activator, for the protein, expressed as a binding con ⁇ cant, if the protein is not an enzyme.
  • the alteration of the activity can be either positive, i.e. activating, as evidenced by an increase in enzymatic activity or ion transport, or an increase in binding affinity of the protein for a particular ligand, or it can be negative, i.e. inhibiting, such as a decrease in enzymatic activity or ion transport, or in binding affinity for a ligand.
  • the invention provides a method for employing the effector peptides of the invention for inhibiting or activating allosteric proteins.
  • the method of the present invention is believed to be applicable to any allosteric protein -- that is, any protein that is sufficiently flexible that an interaction between one molecule and the protein effects, in some manner, a change within the three-dimensional structure of the protein altering activity at other sites.
  • the method is applicable to proteins comprising at least one binding site for an allosteric modifier, such as ligands, including sites on the same protein, additional proteins, ions or DNA.
  • the protein can, and most commonly does, have more than one subunit.
  • the binding site for the allosteric modifier need not be located on the same subunit for which a portion of the amino acid sequence is determined and to which the peptide corresponds.
  • the protein can be a monomer at some stage and undergo oligomerization in response to some signal or stimulus.
  • An example is EGF receptor, in which activation by EGF may proceed through oligomerization
  • allosteric proteins to which the present invention is applicable there are at least two sites at which other molecules may interact.
  • One of these sites binds an allosteric modifier, and the second site is regulated by binding of the first site to the allosteric modifier and binds to another molecule which is coupled to the expression of activity of the allosteric protein.
  • the interaction of the allosteric modifier and the protein alters the interaction between the protein and the second molecule whether or not the second interaction necessarily occurs subsequent to the first interaction.
  • the interaction of the sites of the allosteric protein with molecules that bind does not have to be sequential.
  • the allosteric protein may have both intracellular and extracellular domains, i.e., in the cytoplasm of the cell, or on the exterior of the cell surface or within the cell membrane, and the regions of intramolecular interaction may occur in either the intracellular or extracellular domain or in both domains.
  • the method of the present invention is applicable to allosteric proteins with a wide range of functions, including, but not limited to: enzymes, transport proteins, nucleic acid binding proteins, and receptors.
  • receptors having enzymatic activity including, but not limited to, epidermal growth factor
  • EGF EGF receptor
  • HER-1 human EGF receptor
  • IN-R insulin receptor
  • PDGF platelet-derived growth factor
  • TNF tumor necrosis factor
  • IL-1 erythropoietin receptor
  • IL-1 interleukin-1
  • the method of the present invention is applicable to the protein tyrosine kinase activity of EGF receptor as set forth in Examples 1 and 2.
  • the screening method of the invention includes A) selection of target sequences in the primary amino acid sequence of an allosteric protein; B) production by synthesis of peptides corresponding to portions of the target sequence; and C) testing of the synthesized peptides for ability to alter a biological activity of the allosteric protein by means other than direct competition for substrate binding.
  • the effector peptides are substantially identical to a selected region of the amino acid sequence encoding the allosteric protein. This region of the allosteric protein is termed the "target sequence.”
  • amino acid sequence of the allosteric protein is known. If it is not already known, the amino acid sequence of the protein or a selected region of the protein is determined. The determination of amino acid sequence is performed by methods well-known in the art. Classically, amino acid sequences of proteins are determined by methods employing sequential degradation, such ' as the Edman degradation employing phenyl isothiocyanate. These methods can be automated and performed in a commercially available device known as a "protein sequenator, " as described in Niall, "Automated Edman Degradation: The Protein Sequenator," Methods Enzvmol. 27, 942-1010 (1973) , incorporated herein by this reference.
  • An important advantage of the method of the present invention is that the entire amino acid sequence need not be known,- knowledge of a region of the sequence will suffice. This portion can be as short as about three amino acids. Moreover, it is not necessary to know the three-dimensional (tertiary or quaternary) structure of the allosteric protein, or to verify the functional or structural relationship of the region to the rest of the protein molecule to apply the method of the invention for producing effector peptides. Obtaining such information most commonly requires use of very expensive and time- consuming X-ray diffraction methods or extensive examination of properties of site-specific mutations in the target sequence.
  • the target sequence may be located in an extracellular or intracellular domain of the protein.
  • the target sequence may be contained within, or itself contain one or more, sequence(s) of ordered structure, such as an ot-helix or a 3-pleated sheet.
  • sequence(s) of ordered structure such as an ot-helix or a 3-pleated sheet.
  • sequence(s) of ordered structure such as an ot-helix or a 3-pleated sheet.
  • sequence(s) of ordered structure such as an ot-helix or a 3-pleated sheet.
  • Additional potential properties of the region of amino ' acid sequence selected for synthesis of peptides include regions bearing net positive charges, e.g. basic amino acid residues, hydrophobic regions and those capable of functioning as binding sites for ATP, substrates, or other interacting ligands or protein molecules.
  • the selected region may also include substrate binding regions of the allosteric protein, however, this is not required. These regions having putative structural significance are most readily predicted by computational analysis. Regions of
  • target sequences for peptide synthesis may thus be based on discrete secondary structural features that may be maintained in allosteric structure rearrangements occurring when the protein is activated or that might participate in allosteric conformational interactions within the protein.
  • a recent analysis of protein subunit and domain interactions (Argos, Protein Engineering 2:101-113 (1988)) has shown that interface interactions do not usually involve long stretches of residues with a given secondary structure, and that less than about 70% of the total interface surface is contributed by single residues in distinct structural units. Consequently, the choice of peptides may be based on a Chou-Fasman secondary structural analysis (Chou & Fas an, Adv. Enzvmol. 47:45-148 (1978); Kyte and Doolittle, J. Mol. Biol. 157:105 (1982)), or on a consensus of secondary structural analyses (e.g. using commercially available computer programs such as Protoplot cm , Intelligenetics, Inc., Mountainview, CA) .
  • predictions of functional or structural significance of certain continuous amino acid sequences may provide guidelines for targeting regions of the amino acid sequence of the allosteric protein for screening
  • the method of the invention does not require confirmation of the predicted functional or structural significance of the selected regions prior to carrying out the generation of peptides and subsequent screening of the peptides for effects on activity of the protein.
  • the method of the invention thus permits generation of a plurality of peptides having overlapping sequence and accounting for the entire sequence of the target region in essentially two steps, and screening of these peptides for effects on activity of the protein, for rapid identification of those peptides capable of altering function.
  • This method permits the efficient production of new effector peptides from regions of a protein not previously identified as possessing a specific function or structural relationship without requiring the complete amino acid sequence or structural analysis of the protein.
  • Such peptides may be used as lead compounds in the design of new therapeutic agents to alter activity of allosteric proteins involved in human diseases or dysfunctions.
  • the effector peptides suitable far altering the biological activity of the allosteric protein are relatively small and substantially identical to a selected region of the allosteric protein whose biological activity is to be altered.
  • sequences of the effector peptides are derived from the selected target region of the allosteric protein suspected of having structural and/or functional activity relative to a functional activity of the protein as described above.
  • a first substep is used in which a first set of peptides of from about 11 to about 20 amino acids in length are synthesized, followed by a second substep of synthesis to produce a second set of peptides of from about 11 to about 20 amino acids in length.
  • Each peptide represents a portion of the target sequence of the allosteric protein.
  • the second set of peptides represent peptides that overlap the sequence gaps of the first set of peptides such that points of discontinuity in amino acid sequence between peptides resulting from the first step of synthesis are included in the second set of peptides and are flanked by at least 5 amino acids on either side.
  • the peptides synthesized in these two substeps are thus partially overlapping, typically for about 5 to ' 7 amino acids, to detect effector sequences that would otherwise be split between two adjacent peptides.
  • extended regions of continuous amino acid sequence that are predicted to form o--helices or ⁇ -pleated sheets are preferably retained within one peptide to the extent possible.
  • This two step synthesis procedure permits the rapid generation of a plurality of peptides together having sequences that encompass every possible peptide of about 11 amino acids in length within the entire target region selected in the allosteric protein in order to assess the activity of all possible peptides six to seven amino acids in length. All of these peptides that are generated by performing both steps of synthesis are tested for effects on activity of the allosteric protein.
  • sequences of the peptides are identical or substantially identical to portions of the sequence of the corresponding region of the protein, i.e., the target sequence.
  • this is not a requirement, and some differences between the sequence of the protein and the corresponding sequence of each peptide can occur as long as the peptide is substantially structurally analogous to the modifier sequence of the protein -- i.e., the peptide assumes a three-dimensional conformation virtually identical to the predicted conformation of the corresponding segment of the protein, despite the occurrence of changes in amino acid sequence between the protein and the peptide.
  • changes can include, but are not limited to, the changes that would result from single basepair changes (i.e., transitions and transversions) in the DNA sequence coding for the modifier sequence.
  • the required degree of substantial structural analogy preferably exists over the entire length of the peptide; however, if the target sequence includes a dominant structural feature that does not encompass the entire length of the target sequence, duplication of that dominant structural feature can suffice to generate substantial structural analogy, even though the other portions of the effector peptide are somewhat divergent in sequence.
  • the existence of substantial structural analogy can often be predicted by consideration of size, charge, and relative hydrophobicity of the amino acids involved. Certain changes in amino acid residues, known in the art as "conservative amino acid substitutions," result in substantial structural analogy in most cases.
  • substitutions ' include, but are not necessarily limited to: glutamic acid (Glu or E) for aspartic acid (Asp or D) and vice versa,- glutamine (Gin or Q) for asparagine (Asn or N) and vice versa; serine (Ser or S) for threonine (Thr or T) and vice versa; and any of isoleucine (lie or I) , valine (Val or V) , and leucine (Leu or L) for any other of these amino acids.
  • the peptides are synthesized by methods well- known in the art.
  • the universally adopted method of choice for synthesis is the solid-phase synthesis protocol developed by Merrifield, as described in Merrifield, J. Am. Chem. Soc. 85: 2149-2154 (1963), incorporated herein by this reference. Variations on this method have enhanced the versatility of the solid-phase synthesis technique. These variations allow the simultaneous synthesis of several peptides of varying sequence and are therefore particularly useful.
  • the first of these variations of the Merrifield method was described by Geysen and coworkers in 1984 in
  • carboxyl-terminal and amino-terminal residues of the synthesized peptides are preferably blocked to avoid the presence of undesirable charges on these residues that might alter the binding of the peptides to the allosteric protein.
  • derivatives or analogs of peptides corresponding to target sequences include, but are not limited to, derivatized peptides in which charged residues such as arginine are modified to balance positive charge so that the peptide ⁇ can penetrate the cell membrane more efficiently and peptides in which the amino-terminus, the carboxyl-terminus, or both are blocked to prevent the introduction of unwanted charges.
  • Other derivatives of modifier sequence peptides can include peptides in which carboxyl, hydroxyl, or sulfhydryl functions are protected or blocked.
  • the term "peptides" embraces generically both underivatized peptides and derivatives or analogs of peptides.
  • the small peptides used in the screening method are generally stable after synthesis, it is not necessary to perform the selection and synthesis steps with the testing steps at the same time or in the same location.
  • the peptides can be stored after synthesis, as a lyophilized powder or in a refrigerated or frozen solution for later reaction with the allosteric protein.
  • each of the synthesized peptides is added to the allosteric protein in a separate reaction mixture and the effect of the peptide on the activity of the protein is determined by assay after incubation.
  • the reaction is carried out as described below.
  • the assay for activity can take the form of an enzymatic assay, such as an assay of kinase or phosphatase activity, if the protein is an enzyme, or a ligand-binding assay, such as an assay of the binding of a hormone to a receptor, if the protein has such activity.
  • peptides that alter the activity of the allosteric protein by at least 50%, at a peptide concentration of 1 mM are chosen for further study in order to determine the most effective peptides.
  • This 50% criterion can be varied for particular allosteric proteins,- in some circumstances, an alteration of 25% of the activity of the allosteric protein at 1 mM peptide can be significant and useful.
  • the protein is reacted with a stoichiometric excess of the peptide in an aqueous medium.
  • the temperature of the incubation is between about 0°C to the minimum temperature at which the allosteric protein is denatured or ceases to display its allosteric behavior, typically between about 0°C to about 40°C, and more typically between about 20°C and about 37°C.
  • the pH of the medium is from about 5 to about 10, typically from about 6 to about 8.5, preferably from about 7 to about 8, and more preferably from about 7.2 to about 7.8.
  • This pH can be maintained by a suitable buffer that does not interact with the protein or the peptide, such as Tris or HEPES; other buffers can also be used.
  • the ionic strength of the solution is typically less than about 0.1.
  • Divalent cations such as Mg 2+ , stabilizers such as bovine serum albumin (BSA) , other salts, antioxidants, or other components can be added to the solution as needed to enhance the stability of the protein or for assay of its activity.
  • the molar concentration of peptide used in the reaction is typically at least 0.1 mM, but generally no greater than 1 mM; more typically, it is at least 0.5 mM.
  • the time of reaction is generally noncritical, and typically ranges from 1 minute to 1 hour.
  • Reactions that are carried out for from between about 1 minute to about l hour at a concentration of peptide of no greater than about 1 mM at a temperature of from about 0°C to the minimum temperature at which the allosteric protein is denatured or ceases to display its allosteric behavior and a pH of from about 5 to about 10 are described herein as reactions under standard conditions.
  • effector peptides obtained from performance of the above-described screening method of the invention, and shown by testing to affect a functional activity of the allosteric protein are used to react with the allosteric protein to alter the expression of its activity.
  • the conditions of reaction will be generally as described above for testing of the synthesized effector proteins.
  • Effector peptides shown to possess inhibiting activity are used to inhibit the allosteric protein, and peptides possessing stimulatory activity are used to activate the protein.
  • An additional application of the method of the invention for altering activity of an allosteric protein is to alter the relative activity of an allosteric protein toward different substrates.
  • EGF receptor can catalyze both autophosphorylation and phosphorylation of tyrosine residues on exogenous peptides or proteins, and the relative inhibition of autophosphorylation and phosphorylation of exogenous peptides can vary as between different effector peptides (Example 1) .
  • an extension of the screening method of the invention is to determine the modification by effector peptides of activity of the allosteric protein toward two or more different substrates, such as different target substrates of a protein kinase, and then select effector peptides that have a differential effect as between the two substrates.
  • This can be done by defining the activity of the allosteric protein as the activity toward the first substrate divided by the activity toward the second substrate, and then selecting effector peptides that maximize the activity thus defined. It may even be possible to find effector peptides that cause inhibition toward one substrate and activation toward another substrate.
  • allosteric proteins are members of protein families that are related in structure and function. For example, about 50 protein tyrosine kinases are known, and these proteins share substantial sequence homology. As shown in Figure 1, the insulin receptor (INS-R) , the platelet derived growth factor receptor (PDGF-R) , and the human EGF receptor HER-1, and HER-2, are highly homologous in primary amino acid sequence in the kinase domain. This homology suggests that effector peptides can be developed using the methods of the invention that affect the activity of more than one of the allosteric proteins in a family by focusing on regions of substantial sequence homology between the family members.
  • IN-R insulin receptor
  • PDGF-R platelet derived growth factor receptor
  • HER-1 human EGF receptor
  • HER-2 human EGF receptor
  • these regions of substantial homology can take the form of a "consensus sequence" that is identical or substantially identical for all members of the family; however, the presence of a consensus sequence is not a requirement for the application of the alteration method to a family of allosteric proteins.
  • Effector peptides that can affect the activity of more than one member of a family of allosteric proteins are produced by identifying the region (or regions) of substantial homology of each allosteric protein belonging to the family of allosteric proteins; synthesizing a plurality of peptides, each peptide substantially identical to a portion of the region of substantial homology between each allosteric protein,- and reacting each peptide synthesized with each of the allosteric proteins under the standard conditions for the modification assay to determine the activity of each of the allosteric proteins subsequent to reaction with each peptide to obtain the peptides that alter the activity of each of the allosteric proteins in the family of allosteric proteins by at least a predetermined fraction, typically 25 to 50% at a concentration of peptide of 1 mM.
  • effector peptides may be synthesized from a target sequence in an allosteric protein that is a member of a family of proteins having sequence homology, where the target sequence does not possess homology to sequences in other members of the family.
  • Such non-homologous effector peptides can be predicted to specifically alter the activity of one or a few members of the family, but not others.
  • EGF epidermal growth factor
  • Both inhibition and activation of this enzymatic activity can be produced by incubation with effector peptides having different amino acid sequences.
  • Full details of the application of this method to EGF receptor are given in Examples 1 (inhibition of EGF receptor protein tyrosine kinase activity) and 2 (activation of EGF receptor protein tyrosine kinase activity) , infra.
  • the following peptides are capable of inhibiting the protein tyrosine kinase activity of human EGF receptor by at least 34% when incubated with the receptor at about 1 mM concentration of the peptide and for which inhibition has been confirmed in dose-response assays (amino acid residues of EGF receptor and peptide numbers are indicated in parentheses) (SEQ ID N0:6) V-L-G-S-G-A-F-G-T-V-Y-K-G-L-W
  • peptides are preferred, being capable of inhibiting the protein tyrosine kinase activity by at least 50% at about 1 mM concentration of the peptide (peptide numbers are indicated in parentheses) (SEQ ID NO: 6) : T-V-Q-L-I-T-Q-L-M-P (14) ; K-V-K-I-P-V-A-I (32); I-T-Q-L-M-P-F-G-C-L-L-D (23) ; C-L-L-D-Y-V-R-E (28) ; W-C-V- Q-I-A-K-G-M-N-Y-L (5) ,- V-Q-I-A-K-G-M-N-Y-L (5A) ; G-M-N-Y-L- E-D-R-R-L-V-H-R-D-L (6) ; A-A-R-N-V-L-K-K-
  • the following peptides are capable of inhibiting the protein tyrosine kinase activity by at least 75% at about 1 mM concentration of the peptide (peptide numbers are indicated in parentheses) (SEQ ID NO:6) : K-V-K-I-P-V-A- I (32); I-T-Q-L-M-P-F-G-C-L-L-D (23); W-C-V-Q-I-A-K-G-M-N- Y-L (5); V-Q-I-A-K-G-M-N-Y-L (5A) ; G-M-N-Y-L-E-D-R-R-L-V-H- R-D-L (6); A-A-R-N-V-L-V-K-T-P-Q-H-V-K-I-T (43 ; Y-L-V-I-Q- G-D (30A) ; and I-M-V-K-C-W
  • the most highly preferred inhibitors (SEQ ID NO:6) : A-A-R-N-V-L-V-K-T-P-Q-H-V-K-I-T (43), Y-L-V-I-Q-G-D (30A) and V-Q-I-A-K-G-M-N-Y-L (5A) , corresponding to amino acid residues 815-830, 954-960 and 795-804, respectively, of EGF receptor, when present at about 1 mM concentration of peptide, produce a greater than 85% inhibition of the protein tyrosine kinase activity of EGF receptor.
  • effector peptides are capable of activating the protein tyrosine kinase activity of EGF receptor: a peptide with a sequence of (SEQ ID NO:6) R-R-H- I-V-R-K-R-T, corresponding to residues 646-654 of the EGF receptor (peptide number 42) , and a peptide with a sequence Of (SEQ ID N0:6) K-F-R-E-L-I-I-E-F-S-K-M-A-R-D, corresponding to residues 936-950 of the EGF receptor (peptide number 26) .
  • effector peptides reactive with the EGF receptor effector peptides reactive with other allosteric enzymes such as ATCase may be produced.
  • allosteric transitions occur between a tense (T) form of enzyme with low affinity substrate binding and low specific activity which is in equilibrium with a relaxed (R) form which has high affinity substrate binding and high specific activity. Conversion from the T to R state is induced by either substrate.
  • T tense
  • R relaxed
  • Several regions of the molecule are engaged in homotypic or heterotypic transitions from T to R state induced by substrate and are good candidates for target sequences for peptide inhibitors or stimulators.
  • sequences include, on the Asp domain, the sequence Lys 164 to Ser 171 in which these residues and Tyr lss and Arg ⁇ e7 are involved in points of homotypic or heterotypic contact engaged in the allosteric transition, the sequence Arg 229 to Tyr 240 in which these residues and Glu 233 , Arg 234 and Glu 239 are engaged in contacts influenced by the allosteric transition, a sequence involving Ser 171 and a sequence involving Asp 271 -Glu 272 .
  • sequences on the CP domain include one in which Gln 133 and His 134 are represented as well as two others containing Glu so and Arg 105 .
  • At least some of these peptides affecting the protein tyrosine kinase activity of EGF receptor may operate by altering the intermolecular interaction between individual monomers of the receptor that result in the oligomerization of EGF receptor. This interaction may occur at small, discrete recognition points on the EGF receptor monomer. Some of these recognition points are likely to occur within the cytoplasmic region of EGF receptor, but others may be extracellular. However, it seems unlikely that all the peptides that inhibit protein tyrosine kinase activity operate by inhibiting receptor oligomerization.. Other mechanisms, such as intramolecular structural alterations or inhibition of intramolecular flexibility required for allosteric signal transmission, may well account for the activity of some of the inhibitory peptides and of the activating peptides.
  • the rapid screening method of the invention shows promise as a general screening method for selecting effector peptides that function as new therapeutic drugs aimed at specific allosteric proteins, or families of proteins. Moreover, once effector peptides of particular sequence are- identified, selective inhibitor and activator reagents may be developed for allosteric proteins using computational chemistry. In addition, the portions of the target sequences in an allosteric protein that correspond to effector peptides identified by the method, may be investigated for heretofore unidentified structural or functional significance.
  • Synthetic human angiotensin II was purchased from Sigma Chemical Co. (St. Louis, MO) .
  • t,-Butyloxycarbonyl (t- BOC) derivatives of amino acids were obtained either from Peninsula Laboratories (Belmont, CA) or from Fisher Scientific Co. (Pittsburgh, PA) .
  • N,N-Diisopropylethylamine and 1,3-diisopropylcarbodiimide were purchased from Aldrich Chemical Co. (Milwaukee, WI) and 4-methylbenzhydrylamine resine from Biosearch (San Rafael, CA) .
  • Trifluoroacetic acid was obtained from Pierce (Rockford, IL) and Fisher Scientific Co. Sheets of 74 ⁇ m pore size nylon mesh used for construction of "teabags" for simultaneous multiple peptide synthesis were obtained from McMaster and Carr, Los Angeles, CA.
  • the amino acid sequence of human epidermal growth factor receptor (EGF receptor) from amino acid residue 646 to residue 1000 is shown in Figure 1. This region is generally considered to encompass the ' protein tyrosine kinase and substrate binding domains (Yarden & Ullrich, Ann. Rev. Biochem. 57:443-478 (1988)).
  • the (SEQ ID NO:6) G-S-G-A-F-G sequence (residues 695-700) , the lysine residue at position 721, and the (SEQ ID NO:6) D-F-G sequence (residues 831-833) are all known to participate in ATP binding (Russ et al. , J. Biol. Chem.
  • the substrate binding domain is thought to be contained in the remaining sequence spanning residues 834 to approximately 1000
  • Figure 2 lists the peptides in order of sequence (Protoplot tm was used to predict structure for the peptides shown in Figure 2) .
  • Figure 3 shows the relationship of the peptides with respect to the amino acid sequence of the EGF receptor, showing the overlaps between peptides. Peptides containing regions with substantial a- helical or ⁇ -pleated sheet structure are boxed; those with substantial ⁇ -helical structure are also shaded.
  • M-SPPS 100 mg (0.5 meq/g) amounts of 4- methylbenzhydrylamine (MBHA) resin were sealed in polypropylene 74- ⁇ m mesh packets having approximate dimensions of 2 cm x 2 cm. These packets were treated concurrently in a common reaction vessel for the standard deprotection, neutralization, and wash procedures. They were separated at the coupling steps where the contents in each packet was reacted with the appropriate amino acid- activator solution in an individual reaction vessel,- coupling was effected with 1,3-diisopropylcarbodiimide. Although individual couplings and deprotections were not monitored, additional dummy packets were processed. At appropriate points during synthesis, a dummy packet was removed, the resin collected and assayed for completeness of either deprotection or coupling by the standard Kaiser ninhydrin test (Stewart and Young, supra) .
  • MBHA 4- methylbenzhydrylamine
  • the protected peptide-resins in the packets were collectively deprotected at the amino-terminus and then acetylated in a solution of N,N-dimethylformamide containing 10% acetic anhydride . and 10% N,N- diisopropylethylamine in dichloromethane (DCM) at room temperature for 1-2 hours.
  • DCM dichloromethane
  • Peptides synthesized by M-SPPS were deprotected and cleaved from the resin by anhydrous hydrogen fluoride in the presence of anisole by Multiple Peptide Systems (San Diego, California) .
  • SPPS peptides were cleaved from the resin using a Multiple Peptide Systems cleavage apparatus. Scavenger was the same as with M-SPPS except for the use of p-cresol and thiocresol in some cases.
  • Peptides were extracted from resin using either glacial acetic acid or 10% acetic acid and lyophilized.
  • Crude peptides were purified by preparative reverse-phase HPLC either on a C-18 (Beckman UltraprepTM, 2.12 x 15 cm) or a C-4 (Vydac, 2.2 x 25 cm) column using an aqueous gradient of 0 to 60% acetonitrile containing 0.1% trifluoroacetic acid. Each peptide was at least 90% pure as analyzed by HPLC; the composition of each peptide was established by amino acid analysis.
  • EGF was purified from male mouse submaxillary glands as described in Savage & Cohen, J. Biol. Chem. 247:7609-7611 (1972) . Briefly, the isolation procedure involved: (l) chromatography in 0.05 N HCl containing 0.15 M NaCl on Bio-GelTM P-10 (Bio-Rad Laboratories, Richmond, Calif.),- and (2) DEAE-cellulose chromatography.
  • the peptides were examined for their inhibitory properties in an EGF-dependent EGF-receptor-catalyzed substrate phosphorylation assay using receptor that had been purified about 500-fold from Triton X-100 extracts of human epidermoid A431 cells by affinity chromatography on Fractogel TSK-immobilized ricin-binding subunit, as described in Ghosh-Dastidar et al. , Proc. Natl. Acad. Sci. USA 81:1654-1658 (1984) .
  • the reaction system was a modification of the procedure described in Pike et al. Proc. Natl. Acad. Sci. USA 79:1443-1447 (1982) .
  • the final 20 ⁇ L reaction system contained: 20 nM EGF receptor or 200 nM EGF; 3 mM angiotensin II as phosphorylation substrate,- concentrations of peptide ranging from 0 to 1 mM as specified; 50 ⁇ M [ ⁇ - 32 P]ATP (1500-3000 cpm/pmoles) ; 5 mM MgCl,; 10 ⁇ g/mL BSA, 0.2% Triton X-100; and 10% glycerol in 10 mM HEPES adjusted to pH 7.4. In each case, control incubations containing all assay components except phosphorylation substrate were included.
  • Reaction systems containing all components except MgCl, and ATP in a total volume of 18 ⁇ L were incubated at 30°C for 3 minutes to allow ligand-receptor complexes to form. Reactions were initiated by the addition of MgCl, and [ ⁇ - 32 P]ATP in 2 ⁇ L and were incubated at 30°C for 3 minutes. Reactions were terminated by mixing 5 ⁇ L aliquots of the reaction mixture with 50 ⁇ L of 5% (w/v) trifluoroacetic acid. Phosphorylated receptor protein was sedimented by a 5-minute centrifugation in a microfuge.
  • EGF-dependent EGF receptor self- phosphorylation activity by the synthetic peptides was determined with 20 nM EGF receptor and 200 nM EGF in the 20- ⁇ L system described for substrate phosphorylation, but with angiotensin II excluded. Reaction systems, complete in 18 ⁇ L, except for MgCl- and ATP, were incubated at 30°C for 3 minutes to allow for ligand-receptor complexes -to form and then for an additional 10 minutes at 0°C prior to addition of MgCl- and [ ⁇ - 32 P]ATP.
  • the distribution of the sequences of the 15 relatively potent peptides along the EGF receptor present several interesting features.
  • two peptides that include regions in EGF receptor known to be involved in ATP binding namely peptides corresponding to EGF sequence positions 693-707 and 827-839 are inhibitors of EGF receptor-catalyzed substrate phosphorylation.
  • a cluster of four inhibitor peptides which inhibit by at least 40% when present at 1 mM concentration correspond to sequences spanning a 49-amino-acid stretch from residue 913 to residue 961. This is closely followed by a second stretch of 28 amino acid residues, residues 973 to 1000, that produced a cluster of three additional inhibitor peptides that inhibit by at least 45% when present at 1 mM concentration.
  • Inhibition by these 15 peptides does not involve inhibition of EGF binding to receptor. Because EGF itself is a stimulator of phosphorylation of angiotensin by the EGF receptor, if the peptides inhibited the binding of EGF to the receptor, that would account for at least some of their inhibitory effects. However, no significant variation in binding of EGF to receptor was observed when 20 nM EGF receptor was incubated with 200 nM [ 125 I]EGF in the . presence or absence of 1 mM of the peptides for 1 hour at 22°C. Accordingly, the peptides did not prevent binding of EGF to the receptor.
  • the effects of peptide inhibitors shown in Figure 7 are representative of the effects of the peptide inhibitors shown in Figure 5 plus peptide 30A representing EGF receptor sequence 954-960 (SEQ ID N0:6), Y-L-V-I-Q-G-D.
  • the plots of inhibitory peptide concentration vs. specific activity continue to decrease as a function of increasing inhibitor concentration throughout the entire range of ⁇ concentrations studies and the shapes of the curves suggest that at very high concentration of peptide inhibitor, complete inhibition will be observed. This behavior is in contrast to qualities of inhibition of peptides presented in Figure 6 and shown in greater detail in Figure 8.
  • Peptide 49 with sequence (SEQ ID NO: 6) P-I-C-T-I-D-V-Y-M-I-M-V-K-C inhibited only that portion of substrate phosphorylation activity induced by EGF. This peptide had no inhibitory effect whatsoever on EGF independent substrate phosphorylation.
  • One other peptide, peptide 27 with sequence (SEQ ID NO:6 " ) F-Y-R-A-L-M-D-E-E-D-M-D also showed this trend with 2.5-fold greater inhibitory effect on EGF-dependent substrate phosphorylation than on EGF- independent substrate phosphorylation.
  • the peptides shown in bold type in Figure 5 are all characterized by the presence of a tyrosine residue raising the possibility that inhibition in response to these peptides might occur through these peptides acting as competing substrates which in their phosphorylated form do not bind to phosphocellulose paper as does phosphorylated angiotensin II.
  • One of these tyrosine containing peptides, peptide 28 with sequence (SEQ ID NO:6) C-L-L-D-Y-V-R-E, and three additional peptides, 32, 43 and 21, which contained no tyrosine residues were tested for competitive vs. noncompetitive inhibition as shown in Figure 9A-C.
  • Figure 9A, 9B and 9C intersection of plots on the ordinate
  • (1/v) axis are indicative of inhibition by peptide with substrate for the substrate binding site.
  • Intersection of plots on the abscissa (x axis) are indicative of a competition which is noncompetitive with respect to substrate.
  • Plots A and B show characteristics of noncompetitive inhibition
  • plot C shows inhibition which may represent a mixture of competitive inhibition at lower inhibitor concentration, e.g. 0.0625 mM, but noncompetitive at higher concentration, e.g. 0.25 mM.
  • Three of these four peptides, 28, 43 and 21 were, characterized by clear noncompetitive inhibition indicating possible direct inhibitor effects through blocking of allosteric properties of the receptor.
  • Peptide 32 was characterized by a mix of competitive inhibitory quality at lower inhibitor concentration and noncompetitive quality at higher inhibitory concentration. The characteristics of inhibition by other peptides shown in Figure 5 were not determined.
  • Peptide 26 may be further optimized by substitution of amino acids at position that may affect structure.
  • Experiments by Moe and Kaiser (Biochemistry 24:1971-1976 (1985)) on calcitonin show that activity of calcitonin which contains an amphiphilic ⁇ .-helix . is maintained, and in some cases even enhanced when sequences of portions of an idealized o;-helix are substituted for portions of the actual sequence of calcitonin.
  • substitution of other amino acids for lysine residues 1 and 11 of peptide 26 and for aspartate and glutamate residues 4, 8 and 15 may provide additional information on those residues that may be pharmacophores required for activity.
  • Figure 7 describes the quality of inhibition representative of these peptides for EGF induced substrate phosphorylation. Maximal inhibition is achieved at inhibitor concentration of 0.25 to 0.50 mM and higher concentrations of inhibitor produced no additional inhibition. This may indicate that these inhibitors induce changes in EGF receptor structure which render the activated form of receptor less functional in substrate phosphorylation, or that these partial inhibitors decrease the concentration of the low activity form of receptor to intermediate levels, or that there are subpopulations of receptor that are insensitive to certain inhibitors. Three additional inhibitors of the seven partial inhibitors also had a quality shared by the two inhibitors for which inhibitory effects are more fully characterized in Figure 8.
  • the peptides used were peptide 42, with a sequence of (SEQ ID NO:6) R-R-H-I-V-R-K-R-T corresponding to residues 646-654 of EGF receptor, and peptide 26, with a sequence of (SEQ ID NO:6) K-F-R-E-L-I-I-E-F-S-K-M-A-R-D, corresponding to residues 936-950 of EGF receptor.
  • EGF was purified from male mouse submaxillary glands as described by Savage and Cohen, supra (Example 1) .
  • the peptides were examined for their activation activity in an. EGF-dependent EGF-receptor-catalyzed substrate phosphorylation assay using receptor that had been purified about 500-fold (Example 1, supra) .
  • the reaction system contained, in a total volume of 20 ⁇ L, 20- 30 nM EGF receptor,- 1.5 mM angiotensin II as a phosphorylation substrate; 10 ⁇ g/mL BSA, 50 ⁇ M [ ⁇ - 32 P]ATP
  • Reaction systems containing all components except MgCl, and ATP, were first incubated at 30°C for 3 minutes. Reactions were then initiated by the addition of
  • Phosphorylated substrate bound to the paper was quantified by Cerenkov counting.
  • EGF receptor and peptide or buffer were incubated for 20-30 minutes at room temperature; then [ 12S I_ EGF (to measure total binding) or a mixture of [ 125 I_ EGF and 2 ⁇ M unlabeled EGF (to measure nonspecific binding) was added.
  • the volume of each sample was adjusted to 20 ⁇ L with 10 mM HEPES, pH 7.4, and incubations were for 1 hour at room temperature.
  • Pellets were washed with 500 ⁇ L of 20% polyethylene glycol in 10 mM HEPES, pH 7.4, vortexed, and centrifuged; supernatant fractions were aspirated. Radioactivity in the pellets was determined by gamma counting.
  • Figure 10A and B shows the effects" of peptides 42 (10A) and 26 (10B) on substrate phosphorylation by EGF receptor in the presence or absence of EGF. Units are turnover numbers and represent moles of substrate phosphorylated per min per mole of substrate under standard assay conditions.
  • Peptide 42 increased substrate phosphorylation by the intrinsic protein tyrosine kinase activity of EGF receptor in the presence or absence of EGF. Maximal stimulation was observed at 0.5 mM with EGF absent, but at 0.25 mM with EGF present. Peptide 42 slightly decreased the stimulatory effect of EGF on the tyrosine kinase activity of EGF receptor at higher concentrations. EGF acted synergistically with peptide 42 to stimulate the tyrosine kinase activity of the EGF receptor.
  • Peptide 42 was nearly as effective as EGF in stimulating substrate phosphorylation by EGF receptor and more than tripled the activity of EGF receptor activated by
  • Peptide 26 is a powerful activator of EGF receptor catalyzed substrate phosphorylation with full response at concentrations less than 0.1 mM ( Figure 10B) . Peptide 26 is itself a three-fold more effective activator than EGF. Peptide 26 also acts synergistically with EGF, with the activity induced by combinations of the two being greater than the sum of activation induced by each acting separately. Peptide 26 also had inhibitory quality at concentrations much higher than required to achieve full activation.
  • the predicted structure for peptide 26 may explain its highly effective properties as an activator relative to those of peptide 42.
  • Figure 11 shows the possible three dimensional structure of. peptides 26 and 42 represented on a peptide wheel which presents the amino acids in the positions they would be likely to assume if the peptide has strong alpha helix forming characteristics, which ' is the case for peptide 26, but not for peptide 42 (see structural predictions in Figure 2) .
  • the five highly charged residues of peptide 26 are clustered on one side of the wheel proceeding clockwise from a positively charged group to a group of three negatively charged residues to a positively charged group.
  • the uncharged residues are clustered on the opposing side of predicted structure of the peptide. If this peptide is highly structured as predicted, amino acids in the sequence would enjoy far less rotational mobility around interatomic bonds than would groups of peptide 42, which is far less effective as an activator and for which much higher concentrations are required for maximal activity.
  • activators and inhibitors described in examples 1 and 2 are based on sequences within the intracellular (cytoplasmic) domain of human EGF receptor,- these activators and inhibitors may be expected to act on sites in that domain. This may prove advantageous because the inhibitors may be even more efficacious for that reason if applied within the context of delivery mechanisms that can be targeted to specific target cells, e.g. tumor cells.
  • the present invention provides a method for altering the functional activity of any allosteric protein using synthetic peptides without any knowledge of its detailed three-dimensional structure or even a complete knowledge of its primary amino acid sequence.
  • the peptides used are stable and easy to synthesize in batches containing a number of peptides with different sequences, making it possible to test a large number of candidate peptides simultaneously.
  • the method can be used to either activate or inhibit the allosteric protein whose activity is affected.
  • the invention can be used to affect the activity of a wide variety of allosteric proteins, including: receptors, such as the EGF receptor, the insulin receptor, and the T-cell receptor complex,- transport proteins, such as hemoglobin, and oncogene-rela ed protein kinases.
  • This method promises to permit totally new treatments for such diseases as cancer, AIDS, diabetes, and arthritis, and to speed wound healing and prevent transplant rejection, among other applications. These treatments would operate by utilizing the body's natural defenses and would act in conjunction with current drug treatments.
  • the abnormally expressed kinases present in many types of cancers could be inhibited, which would slow the growth of the cancer cells and increase their susceptibility to anti-cancer therapies such as radiation or chemotherapy. This could allow smaller doses of anti-cancer drugs or smaller quantities of radiation to be administered.
  • AIDS could be treated by activating the T-cell receptor complex to counteract the efficiency of the HIV virus. This treatment could be performed in conjunction with already-available AIDS drugs such as AZT.
  • Diabetes could be treated by activating the insulin receptor to allow the body to use a scarce supply of insulin more efficiently. This could reduce the need for injections of insulin and provide more precise control of blood sugar levels, preventing some of the complications associated with diabetes, such as eye damage and circulatory impairment.
  • the invention can also be applied to target sequences of the extracellular domains of allosteric proteins to identify inhibitors or activators which bind to an extracellular site for applications such as corneal wound healing.
  • the peptides of the invention can be used as an agent which causes a sheep's wool coat to be shed as a substitute for sheep shearing.

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Abstract

L'invention se rapporte à un procédé qui permet de produire rapidement des peptides effecteurs modifiant l'activité fonctionnelle d'une protéine allostérique et qui utilise à cet effet une région cible dans la séquence d'acides aminés qui code pour la protéine. Ces peptides sont sensiblement identiques en séquence à des parties de la région cible. Le procédé pour modifier l'activité fonctionnelle d'une protéine allostérique dépend de l'interaction de ces peptides effecteurs dérivés de la protéine elle-même avec ladite protéine. Ce procédé permet soit d'augmenter soit de diminuer l'activité de la protéine. Ce procédé peut particulièrement s'appliquer au récepteur du facteur de croissance épidermique humain. Font également partie de l'invention des peptides qui permettent soit d'inhiber soit d'activer l'action de la tyrosine kinase protéinique du récepteur du facteur de croissance humain.
PCT/US1993/000581 1992-01-24 1993-01-22 Nouveaux peptides et procede pour modifier l'activite de proteines allosteriques WO1993014781A1 (fr)

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WO1995034817A1 (fr) * 1994-06-14 1995-12-21 Baxter International Inc. Selection cellulaire positive et positive/negative, obtenue par l'action liberatrice de peptides
EP0770876A1 (fr) * 1995-10-25 1997-05-02 Scriptgen Pharmaceuticals, Inc. Méthode de screening pour l'identification de ligands des protéines cibles
US5679582A (en) * 1993-06-21 1997-10-21 Scriptgen Pharmaceuticals, Inc. Screening method for identifying ligands for target proteins
US6232085B1 (en) 1996-05-09 2001-05-15 3-Dimensional Pharmaceuticals, Inc. Method for determining conditions that stabilize proteins
US6331392B1 (en) 1997-03-05 2001-12-18 Anadys Pharmaceutical, Inc. Screen employing fluorescence anisotropy to identify compounds with affinity for nucleic acids
US6337183B1 (en) 1995-09-08 2002-01-08 Scriptgen Pharmaceuticals, Inc. Screen for compounds with affinity for nucleic acids
WO2002014503A2 (fr) * 2000-08-14 2002-02-21 Corixa Corporation Compositions et methodes de traitement et de diagnostic de cancers associes a her-2/neu
US6569631B1 (en) 1998-11-12 2003-05-27 3-Dimensional Pharmaceuticals, Inc. Microplate thermal shift assay for ligand development using 5-(4″dimethylaminophenyl)-2-(4′-phenyl)oxazole derivative fluorescent dyes
US7122321B2 (en) 1997-11-12 2006-10-17 Johnson & Johnson Pharmaceutical Research & Development, L.L.C. High throughput method for functionally classifying proteins identified using a genomics approach
US7432341B2 (en) 2002-10-24 2008-10-07 Valo Hsj, Limited Partnership Cytokine receptor modulators and method of modulating cytokine receptor activity
US7625558B2 (en) 1998-03-04 2009-12-01 The Trustees Of The University Of Pennsylvania Compositions and methods of treating tumors
US8618054B2 (en) 2004-05-05 2013-12-31 Valorisation-Rechereche Société en Commandite Interleukin-1 receptor antagonists, compositions, and methods of treatment
CN108570096A (zh) * 2017-03-14 2018-09-25 北京伟峰益民科技有限公司 一种多肽或其衍生物及其在制备治疗肿瘤的药物中的应用

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Cited By (25)

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US5679582A (en) * 1993-06-21 1997-10-21 Scriptgen Pharmaceuticals, Inc. Screening method for identifying ligands for target proteins
AU697627B2 (en) * 1994-06-14 1998-10-15 Nexell Therapeutics Inc. Positive and positive/negative cell selection mediated by peptide release
US5968753A (en) * 1994-06-14 1999-10-19 Nexell Therapeutics, Inc. Positive and positive/negative cell selection mediated by peptide release
US6017719A (en) * 1994-06-14 2000-01-25 Nexell Therapeutics, Inc. Positive and positive/negative cell selection mediated by peptide release
WO1995034817A1 (fr) * 1994-06-14 1995-12-21 Baxter International Inc. Selection cellulaire positive et positive/negative, obtenue par l'action liberatrice de peptides
US6337183B1 (en) 1995-09-08 2002-01-08 Scriptgen Pharmaceuticals, Inc. Screen for compounds with affinity for nucleic acids
US6503721B2 (en) 1995-09-08 2003-01-07 Anadys Pharmaceuticals, Inc. Screen for compounds with affinity for nucleic acids
EP0770876A1 (fr) * 1995-10-25 1997-05-02 Scriptgen Pharmaceuticals, Inc. Méthode de screening pour l'identification de ligands des protéines cibles
US6232085B1 (en) 1996-05-09 2001-05-15 3-Dimensional Pharmaceuticals, Inc. Method for determining conditions that stabilize proteins
US6303322B1 (en) 1996-05-09 2001-10-16 3-Dimensional Pharmaceuticals, Inc. Method for identifying lead compounds
US6291191B1 (en) 1996-05-09 2001-09-18 3-Dimensional Pharmaceuticals, Inc. Microplate thermal shift assay for ligand development and multi-variable protein chemistry optimization
US6291192B1 (en) 1996-05-09 2001-09-18 3-Dimensional Pharmaceuticals Inc. Method for identifying conditions that facilitate recombinant protein folding
US6268158B1 (en) 1996-05-09 2001-07-31 3-Dimensional Pharmaceuticals, Inc. Method for determining conditions that facilitate protein crystallization
US6331392B1 (en) 1997-03-05 2001-12-18 Anadys Pharmaceutical, Inc. Screen employing fluorescence anisotropy to identify compounds with affinity for nucleic acids
US6569628B2 (en) 1997-03-05 2003-05-27 Anadys Pharmaceuticals, Inc. Screen employing fluorescence anisotropy to identify compounds with affinity for nucleic acids
US7122321B2 (en) 1997-11-12 2006-10-17 Johnson & Johnson Pharmaceutical Research & Development, L.L.C. High throughput method for functionally classifying proteins identified using a genomics approach
US7625558B2 (en) 1998-03-04 2009-12-01 The Trustees Of The University Of Pennsylvania Compositions and methods of treating tumors
US6569631B1 (en) 1998-11-12 2003-05-27 3-Dimensional Pharmaceuticals, Inc. Microplate thermal shift assay for ligand development using 5-(4″dimethylaminophenyl)-2-(4′-phenyl)oxazole derivative fluorescent dyes
WO2002014503A2 (fr) * 2000-08-14 2002-02-21 Corixa Corporation Compositions et methodes de traitement et de diagnostic de cancers associes a her-2/neu
JP2004522412A (ja) * 2000-08-14 2004-07-29 コリクサ コーポレイション Ｈｅｒ−２／ｎｅｕ−関連悪性腫瘍の治療および診断のための組成物および方法
WO2002014503A3 (fr) * 2000-08-14 2003-09-18 Corixa Corp Compositions et methodes de traitement et de diagnostic de cancers associes a her-2/neu
US7432341B2 (en) 2002-10-24 2008-10-07 Valo Hsj, Limited Partnership Cytokine receptor modulators and method of modulating cytokine receptor activity
US8618054B2 (en) 2004-05-05 2013-12-31 Valorisation-Rechereche Société en Commandite Interleukin-1 receptor antagonists, compositions, and methods of treatment
CN108570096A (zh) * 2017-03-14 2018-09-25 北京伟峰益民科技有限公司 一种多肽或其衍生物及其在制备治疗肿瘤的药物中的应用
CN108570096B (zh) * 2017-03-14 2021-07-02 北京伟峰益民科技有限公司 一种多肽或其衍生物及其在制备治疗肿瘤的药物中的应用

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