WO2003059943A2 - Peptides specifiques a la conformation, de liaison de la proteine kinase, procedes et produits associes - Google Patents

Peptides specifiques a la conformation, de liaison de la proteine kinase, procedes et produits associes Download PDF

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WO2003059943A2
WO2003059943A2 PCT/EP2003/000343 EP0300343W WO03059943A2 WO 2003059943 A2 WO2003059943 A2 WO 2003059943A2 EP 0300343 W EP0300343 W EP 0300343W WO 03059943 A2 WO03059943 A2 WO 03059943A2
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peptide
peptides
amino acid
protein
protein kinase
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PCT/EP2003/000343
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WO2003059943A3 (fr
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Syed Salman Ashraf
Lawrence Michael Ballas
Paul T. Hamilton
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Karo Bio Ab
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate

Definitions

  • the invention relates to methods of identifying chemical entities which bind to protein kinases in a conformation-specific manner, the entities per se, and the uses thereof.
  • Protein Kinase C is of particular interest.
  • PKC enzymes Within the family of PKC enzymes exist classes of enzymes which require different dependencies for activation (5,25). Classically,. 'these have been defined by phosphatidylserine (PS) dependence, calcium levels and the production of diacylglycerol (DG or DAG) in response to receptor(s) activation (26). However, other important signaling lipids can activate specific PKC enzymes (27-29, 51) and are the products of other signaling enzymes. Phospholipase A2, phosphat ⁇ dylmos ⁇ tol-3 kinase, and phospholipase D can be activated m response to receptor ligand stimulation.
  • PS phosphatidylserine
  • DG or DAG diacylglycerol
  • Phospholipase A2 phosphat ⁇ dylmos ⁇ tol-3 kinase
  • phospholipase D can be activated m response to receptor ligand stimulation.
  • PKC beta I and II can be inhibited 80 to >1000 fold better than the other PKC enzymes by a macrocyclic bis ( ⁇ ndolyl)male ⁇ m ⁇ de and this compound shows good activity in animal models preventing diabetic complications (33) .
  • PKC beta II is elevated n endothelial cells m diabetics and apparently leads to many of the abnormalities associated with diabetic complications of the retina, kidneys and central nervous system.
  • Another compound, rottle ⁇ n has been reported to inhibit PKC delta more potently than the other PKC enzymes although it can also inhibit Ca 2+ /calmodulin dependent kinase III (34-35) .
  • Staurosporin may be the best known structural class of kinase inhibitor. Analogs of staurosporin, show selectivity toward PKC enzymes, and in some cases favor the Ca 2+ dependent PKC enzymes over the other classes (36), but are not specific for any particular PKC enzyme (however, see reference 32) .
  • the regulatory subunit of PKC In its inactive conformation, the regulatory subunit of PKC is folded over the catalytic subunit, and/or various ligands can alter the conformation of PKC so that association with membranes or other proteins can be accomplished. Protein conformation may be altered significantly depending on the activator. Conformational activation will most likely be different for PKC enzymes activated by PS and DAG compared to AA or PI345P. In addition, conformational changes may also occur upon protein/protein interactions independent of lipid. Conformational changes do occur upon specific phosphorylation of the enzyme either through autophosphorylations on serine or threonine residues or phosphorylations on tyrosine by specific tyrosine kinase enzymes (30) .
  • the conformational possibilities of the different PKC enzymes are many and varied. Conformational changes most likely result in specific subcellular localization and specific substrate interactions. Additionally, many of the potential interacting sites on PKC may not be detectable with classical drug discovery activity r t screens. For instance, an activity assay probing for the ATP binding site will not discover sites for protein/protein interactions . It would be desirable to obtain a chemical entity, such as a peptide, which bound to a protein kinase, such as PKC, in a conformation-specific manner. The entity could preferentially bind the inactive conformation, or an active conformation. In the latter case, it could bind substantially all active conformations, or just a particular one (e.g., the Ca 2+ /PS/DAG-elicited conformation of PKC ⁇ ).
  • This entity could also be specific to a particular protein kinase, such as protein kinase C if specific for protein kinase C, it could be isoform-specific (e.g., PKC ⁇ , PKC ⁇ ll) as well.
  • protein kinase C if specific for protein kinase C, it could be isoform-specific (e.g., PKC ⁇ , PKC ⁇ ll) as well.
  • PKC ⁇ has been implicated in prostate cancer, and PKC ⁇ ll in diabetes.
  • PKC may be activated in response to an increase in DAG and negate the affect of the therapeutic treatment.
  • Ara-C a treatment for cancer, is an example where DAG levels increase in response to the drug subsequently increasing the activity of PKC (44). The increase in activity can negate the effect of Ara-C as a therapeutic treatment.
  • Recent evidence indicates that PKC location within cells is well controlled (18). Although found in the cytoplasm of most quiescent cells, activation of a PKC occurs with translocation to the membrane.
  • RACKS Receptor Activated C. Kinase Substrates
  • PKC alpha as a target for drug intera ction : PKC alpha is a lipid dependent, calcium dependent protein kinase C enzyme whose functions are not well understood. PKC alpha has been found in all tissues. PKC alpha is found within specific subcellular compartments of cells to accommodate and participate in cellular function. The enzyme has been shown to be associated with other specific enzymes within cells; specifically phospholipase D and the caveolae complex (49, 50) (however, these are not the only associations of PKC alpha with intracellular compartments) . The association of PKC alpha in protein complexes must be coordinated in a way that will allow a specific signal to be directed and to be completed. Protein/protein interactions must be designed to allow this dynamic process to occur. Activation can be sustained in the presence of protein/protein interaction, specifically as reported with enzyme interaction with RACKS. Sustained activation of the enzyme leads to a continuous signal for that specific PKC enzyme and maintenance of a disease process.
  • PKC alpha has been shown to be associated with protein such as phospholipase D and caveolae (49, 50). PKC alpha can activate phospholipase D (PLD) in a manner independent of enzyme activity.
  • PLD phospholipase D
  • PKC alpha to PLD.
  • the enzymes association with the caveolae allows signaling process to be directed as external signals are received by the cell (51) .
  • Microlocalization of PKC alpha can serve multiple purposes. Different receptor activation may generate different activators for the same enzyme. Depending on the intracellular signals generated activation or inhibition can occur assuring a smooth flow of metabolic responses.
  • PKC alpha can bind to an activate PLD in an ATP independent manner.
  • PLD is involved with vesicle movement to the plasma membrane and exocytosis (49) .
  • PKC alpha is also associated with the caveolae (50) and can inhibit vesicle uptake when activated.
  • endocytosis and exocytosis are well controlled and dependent on the conformational interaction of PKC alpha and the enzymes and proteins it binds with. Disruption of these effects at multiple sites on the PKC alpha protein lends it as a good target for therapeutic intervention.
  • sites of therapeutic intervention are ATP, protein substrate, phospholipid (PS, Pi's, PA, PG, AA) , phorboid, calcium, and pleckstrin homology domains and RACKS and other binding proteins.
  • phage display random peptide libraries with affinity to a wide variety of targets: Strepavidin, protein A, goat polyclonal antibodies to mouse immunoglobulins (52) , a number of monoclonal mouse antibodies, calmodulin (53), SH3 domains (54), cytoplasmic dynein, ubiquitin, glutathione S-transferase, gene promoter DNA's, immobilized metal ions, bacterial enzymes, estrogen receptors and protein kinase C beta II (unpublished data) .
  • Phage display random libraries have also been used to identify the specificity of SH3 domains from Abl, cortactin, Crk, Fyn, Lyn, p53BP2, P13K, PLC (gamma) and Yes (62-63).
  • peptides or analogs thereof may be used to modulate or inhibit the activity of protein kinases, e.g. PKC, especially PKC alpha, and may be used as a therapeutic agent for diseases in which such kinases play a central role, e.g. PKC alpha in prostate cancer.
  • protein kinases e.g. PKC, especially PKC alpha
  • Peptoids and other peptidomimetics and analogues of these peptoids, may be used for similar purposes.
  • FIG. 1 Surface Plasmon Resonance (SPR) analysis of the interaction of PKC ⁇ peptides with PKC ⁇ .
  • the sensorgrams show the binding of PKC ⁇ in TBST (left) or PKC ⁇ in activation buffer with phosphatidyl serine (PS) and diacylglycerol (DAG) (right) to peptides 1029, 1270, and 1271.
  • the sensorgrams (from Biacore 2000) were obtained with approximately 50 Resonance Units of immobilized peptide (as ligand) and 200 nM of PKC ⁇ (as analyte) in TBST or PS/DAG.
  • Figure 4 Determination of binding affinity of peptides 1270, 1271 arid 1272 to PKC ⁇ in activation buffer containing PS and DAG. The experiment was done in triplicates with the mean fluorescence polarization (mP) plotted against PKC ⁇ concentration.
  • mP mean fluorescence polarization
  • a receptor is a component, usually macromolecular, of an organism with which a chemical agent interacts in some specific fashion to cause an action which leads to an observable biological effect.
  • the term is also applied to non-naturally occurring polypeptides which comprise a domain substantially identical in amino acid sequence to such a component, or a domain thereof, and which are able, when expressed in a cell to mediate a biological response by that cell to some chemical agent.
  • antibodies are not considered receptors.
  • the term "receptor" includes both surface and intracellular receptors.
  • receptors proteins embedded in the phospholipid bilayer of cell membranes.
  • the binding of an agonist (activator) to the receptor can cause an allosteric change at an intracellular site, altering the receptor's interaction with other bio olecules .
  • the physiological response is initiated by the interaction with this "second messenger" (the agonist is the "first messenger") or "effector” molecule.
  • the second messenger of one receptor may be the activator of another.
  • Enzymatic receptors include both activators (which cause enzymes in an inactive conformation to assume an active conformation) and substrates (which, after binding to the receptor, are chemically modified) .
  • Enzymatic receptors include protein kinases; plasma membrane-bound proteins that act by phosphorylatmg target proteins. Some phosphorylate tyrosine residues and others phosphorylate serine or threonme residues. These proteins typically comprise an extracellular, ligand-binding domain, and an intracellular catalytic (kinase) domain.
  • a related family of receptors lack the intracellular kinase domain but, in response to agonist, activate independent membrane-embedded or cytosolic protein kinases.
  • Hormones, growth factors, neurotransr ⁇ itters and many other biomolecules normally act through interaction with specific cellular receptors. Drugs may activate or block particular receptors to achieve a desired pharmaceutical effect.
  • Cell surface receptors mediate the transduction of an "external” signal (the binding of a ligand to the receptor) into an "internal” signal (the modulation of a pathway n the cytoplasm or nucleus involved m the growth, metabolism or apotosis of the cell) .
  • transduction is accomplished by the following signaling cascade: • An agonist (the ligand) binds to a specific protein (the receptor) on the cell surface.
  • the transducing protein activates, within the cell, production of so-called "second messenger molecules.”
  • Cyclic nucleotide regulated protein kinase (PKA & PKG) family 2.AGC Group II Diacylglycerol-activated/phospholipid-dependent protein kinase C (PKC) family 3.AGC Group III
  • ERK (MAP) kinase family 3.CMGC Group III Glycogen synthase kinase 3 (GSK3) family
  • Trk/Ror family 20 Trk/Ror family 20.
  • At least 11 isoforms of PKC have been recognized, on the basis of primary structure, tissue distribution, subcellular localization, made of action in vitro, response to extracellular signals, and substrate specificity.
  • Novel PKC members are fully activated by the combination of a phorboid and a phospholipid, but not either alone. They do not require Ca 2+ for such activation. They include PKC ⁇ , PKC-, PKC ⁇ , and PKC ⁇ .
  • PKCs are sometimes referred to as typical PKCs.
  • Atypical PKC members are fully activated by phospholipid even the absence of Ca 2+ and phorboids, and include PKC ⁇ and PKC ⁇
  • isoforms are differentiated primarily by ammo acid sequence.
  • the following are sources of sequence data for human
  • a "human PKC ⁇ ” may be defined as a polypeptide which (1) has classical PKC activity, (2) has higher percentage sequence identity with the above-identified human PKC ⁇ sequence than with the other above identified human PKC sequences, and (3) is at least 90% identical to the above- identified PKC ⁇ sequence.
  • Human PKC ⁇ is about 80% identical to PKC ⁇ l and PKC ⁇ ll.
  • KPCA_BOVINP0 409 (-97%) Forms of "human PKC ⁇ " which occur naturally in humans are called "wild type human PKC ⁇ ".
  • Other human PKC isoforms are analogously defined, e.g., a human PCK ⁇ is a polypeptide which (1) has novel PKC activity, (2) has higher percentage sequence identity to the above-identifled human PKC ⁇ sequence than to the other above-identifled human PKC sequences, and (3) is at least 90% identical to the above-identifled PKC ⁇ sequence.
  • Human PKC ⁇ s only about 83% identical to mouse PKC ⁇ . For methods of determining percentage ammo acid identity, see the definition of "substantially identical", below.
  • All PKCs are characterized by an N-termmal regulatory region and a C-termmal catalytic domain.
  • the binding of PS was stereospecific; replacement of Sn-1, 2-phosphatidyl-L-serine with the enantiomer sn-2, 3-phosphatidyl-D-serine reduced binding 2-fold, and replacement of the physiological sn-1, 2- diacylglycerol with the enantiomer sn-2, 3-diacylglycerol abolished detectable (Ka of 20 M "1 ) ClB binding.
  • the ClB domain bound phosphatidyl serine (PS) /PMA membranes 15-fold better than it bound phosphatidylglycerol (PG)/PMA membranes.
  • PKG also contains a hydrophobic binding site for alcohols. Effects vary with chain length; n-butyl alcohol inhibits phorbol ester activity, while n-octanol enhances it. See Slater, et al., J. Biol. Chem., 277:6167-73 (1997) .
  • PKCs may also be activated by free fatty acids, such as arachidonic acid, oleic acid, and other cis-unsaturated fatty acids. This activation is independent of both phosphatidylserine (PS) and calcium (even when the PKC isozenzyme in question is one whose activation by DAG is calcium-dependent) . DAG/phorbol ester and oleic acid do not inhibit each other's interaction with PKC, suggesting that they bind PKC at different sites. DAG interacts with membrane-recruited PKC, while free fatty acids preferentially activate cytosolic PKC. See generally Khan, et al., Cellular Signalling, 7:171-84 (1995).
  • the PKC activating environment may comprise a free fatty acid, such as arachidonic acid or oleic acid.
  • phosphatide phosphoglyce ⁇ de
  • R" is - CH 2 CH 2 N(CH): in phosphatidylcholme, -CH : CH 2 NH ; in phosphatidylethanolamine, and a sugar m phosphatidylinositol .
  • indole alkaloids indole alkaloids
  • indolactams such as the teleocidins, lyngbyatoxin, and indolactam V;
  • glycerol backbone is constrained into a five membered lactone ring (DAC-lactones) , and branched alkyl groups incorporated, one obtain activators with nanomolar affinity for PKC. Analogous changes in diacylglycerols also have beneficial outcomes.
  • the lipophilicity of a functional group may be quantified by its octanol/water portion coefficient, log P. Nacvo, et al., Biorg. Med. Chem. Lett., 10:653-5 (2000) studied the effect of small, medium and large acyl groups on PKC ⁇ affinity.
  • the log P value for lead DAG-lactone 4_ was 5.9, and Nacro et al . concluded that a log P value between 5 and 6 is desirable. (The explored log P values between 2.1 and 14.6, and low log P seemed more detrimental than high log P.) They concluded that the optimal length of each acyl chain was 7 or 8 carbons. Their work also shows that constraining the glycerol backbone resulted in a 10-fold change in affinity between compounds 4 . and _9b, whose log P values were also identical .
  • representative members include phorbol 12-myristate 13-acetate (PMA) , phorbol 12-retinoate 13-acetate, ingenol 3-tetradecanoate, synaptolepis factor ki, pimelea factor P 2r daphnopsis factor R 6 , mezere , simplex, pimelea factor s 2 , gnidimacnn, and des- (RingA) -phorbol 12-myristate 13-acetate, all of which have a hydroxymethyl or 1-hydroxyethyl group.
  • PMA phorbol 12-myristate 13-acetate
  • phorbol 12-retinoate 13-acetate ingenol 3-tetradecanoate
  • synaptolepis factor ki pimelea factor
  • P 2r daphnopsis factor R 6 mezere
  • simplex simplex
  • pimelea factor s 2 gnidimacnn
  • Phorbol is a polycyclic alcohol (4 , 9, 12-beta, 13,20- pentahydroxy-1, 6-tigliadien-3-on) occurring naturally in croton oil, and is the parent alcohol of the "phorbol esters".
  • the four rings are conventionally labeled as shown in the depiction of compound 1 of Scheme 1 in ender, et al., Organic Lett., 1:1009-12 (1999).
  • A is a five-membered ring with one double bond
  • B and C are six- membered (B containing a double bond)
  • D is three- membered.
  • the same figure also shows the conventional numbering of the phorbol carbons.
  • diacylglycerol (DAG) is a native activator of PKC.
  • DAG is a diester of glycerol, not phorbol.
  • Phorbol esters, diacylglycerols and phospholipids all include fatty acid groups.
  • the fatty acids may be designated by "the number of carbon atoms: number of double bonds", and optionally the locations of cis/trans isomerism.
  • suitable fatty acids include those with designations 4:0, 6:0, 8:0, 10:0, 12:0, 14:0, 16:0,
  • a typical activating environment would comprise cyclic AMP.
  • cyclic AMP a typical activating environment
  • it could be an environment in which cyclic AMP is produced, e g., one comprising activated adenylate cyclase and AMP.
  • a typical activating environment would comprise insulin.
  • a typical activating environment for EGF receptor would comprise EGF; for PDGR receptor, PDGF; for FGF receptor, FGF; and for HGF receptor, HGF.
  • a typical activating environment would comprise an inflammatory cytokine.
  • Examples would be TNF ⁇ and IL-1.
  • a target receptor When a target receptor is in an unliganded state, it has a particular conformation, i.e., a particular 3-D structure. When the receptor is complexed to a ligand, the receptor's conformation changes. If the ligand is a pharmacological agonist, the new conformation is one which interacts with other components of a biological signal transduction pathway, e.g.; transcription factors, to elicit a biological response in the target tissue. If the ligand is a pharmacological antagonist, the new conformation is one m which the receptor cannot be activated by one or more agonists which otherwise could activate that receptor.
  • a biological signal transduction pathway e.g.; transcription factors
  • ligands may cause a receptor to assume the same conformation, or different conformations. If the ligands cause different physiological responses, it is likely that they are eliciting different receptor conformations . Screening for protein kinase binding
  • PKC alpha Due to the apparently great amount of conformational flexibility associated with the PKC enzymes, particular, PKC alpha, we performed our molecular recognition discovery process employing two independent methods. The first proceeded in the absence of any ligands of the enzyme. Probing PKC alpha (or other PKs, esp PKCs) with phage display libraries would identify any sites on the protein that can interact with other proteins or ligand molecules in its inactive state. The second method was performed m the presence of one or more combinations of activation lipids (PS and other anionic phospholipids and DAG) , calcium, ATP, and/or phosphate acceptor protein. These conditions change the protein from an enzymatically inactive protein into a conformational relevant form of the enzyme.
  • activation lipids PS and other anionic phospholipids and DAG
  • activators are added and present during the phage selection process. Preferably, different combinations of activators are employed to explore as much of the PKC protein as possible.
  • activators of substrates probes only for those sites not protected by these ligands will be discovered Conformational changes initiated by the ligands will expose cryptic sites that are likely to include sites for macromolecular interaction as well as binding sites for peptides that can serve as PKC conformational probes.
  • the methodology comprises: (a) contacting a protein kinase the presence and absence of a kmase-activatmg environment, with a library (esp. a combinatorial library) of peptides in such manner that peptide which bind the protein kinase may be distinguished from peptides which do not so bind, and
  • a library esp. a combinatorial library
  • the method comprises:
  • PKC alpha is immobilized on a microtiter plate, either directly or biotinylated then captured on strepavidin.
  • Phage displaying peptides are added to certain wells and allowed to bind to PKC alpha in the absence of ligands.
  • Phage displaying peptides are added to other wells and allowed to bind to PKC alpha in the presence of ligands.
  • PKC specific ligands include anionic phospholipids, phorboids (including phorbol eters and DAG) , Ca 2+ , ATP, peptide or protein substrate, and RACKS peptide sequence (s).
  • Peptides are analyzed for clustering (similarity) or as being unique.
  • Peptides are synthesized and labeled with a suitable label, such as a fluorescent label (e.g., fluorescein) an enzymatic label (e.g., alkaline phosphatase) , or biotin.
  • a suitable label such as a fluorescent label (e.g., fluorescein) an enzymatic label (e.g., alkaline phosphatase) , or biotin.
  • binding peptides or those which have a desired minimum binding affinity (e.g., K d ⁇ 10 M) may be aligned so as to identify "clusters".
  • Each cluster will usually correspond to a single binding site on the kinase.
  • the characteristics of the cluster may be used to design additional peptides which so bind the kinase, as well as peptide mimetics etc.
  • the assay to screen for modulators can be provided m a number of different ways, e.g. FRET (Fluorescence Resonance Energy Transfer) , FP (Fluorescence Polarization), SPR (Surface Plasmon Resonance), TOP-TRF (Target-On-Plate Time Resolved Fluorescence) , or POP-TRF (Peptide-On-Plate Time Resolved Fluorescence) .
  • FRET Fluorescence Resonance Energy Transfer
  • FP Fluorescence Polarization
  • SPR Surface Plasmon Resonance
  • TOP-TRF Tiget-On-Plate Time Resolved Fluorescence
  • POP-TRF Peptide-On-Plate Time Resolved Fluorescence
  • these peptides can also be used as diagnostic probes (once the peptides are conjugated with a reporter molecule) to measure activated PKC ⁇ levels in vivo, as in cells or tissues, or in vi tro, to quantitate PKC ⁇ activity (as in assay kits) .
  • This new technology provides a general format for studying activation of protein kinases (although this invention describes the results with PKC ⁇ , the approach could be used for any other kinase, especially a PKC, that undergoes a conformational change upon activation) .
  • This technique allows peptides to be used as surrogate ligands for various functional sites on PKC ⁇ which are otherwise difficult or impossible to target.
  • the peptides that we have identified target a site on PKC ⁇ other than the substrate binding site i.e. they do not inhibit the ability of PKC ⁇ to phosphorylate its substrate.
  • the substrate-binding site peptides can then serve as surrogate ligands to identify compounds that bind to the PKC ⁇ substrate binding site or as peptide or peptidomimetic inhibitor of PKC ⁇ phosphorylation function or as a surrogate ligands to identify other compounds that target PKC ⁇ phosphorylation active site.
  • a molecule B binds a target Tl substantially more strongly than a target T2, or that a molecule Bl binds a target T substantially more strongly than an alternative molecule B2 binds the same target T, it means that the difference in binding is detectable and is manifest to a useful degree in the relevant context, e.g., screening, diagnosis, purification, or therapy.
  • combinatorial library refers to a library in which the individual members are either systematic or random combinations of a limited set of basic elements, the properties of each member being dependent on the choice and location of the elements incorporated into it .
  • the members of the library are at least capable of being screened simultaneously. Randomization may be complete or partial; some positions may be randomized and others predetermined, and at random positions, the choices may be limited in a predetermined manner.
  • the members of a combinatorial library may be oligomers or polymers of some kind, in which the variation occurs through the choice of monomeric building block at one or more positions of the oligomer or polymer, and possibly in terms of the connecting linkage, or the length of the oligomer or polymer, too.
  • the members may be nonoligomeric molecules with a standard core structure, like the 1, 4-benzodiazepine structure, with the variation being introduced by the choice of substituents at particular variable sites on the core structure.
  • the members may be nonoligomeric molecules assembled like a jigsaw puzzle, but wherein each piece has both one or more variable moieties (contributing to library diversity) and one or more constant moieties (providing the functionalities for coupling the piece in question to other pieces) .
  • the scintillant (which emits light when a beta particle passes close by) is conjugated to an analyte binding molecule (ABM) .
  • ABS analyte binding molecule
  • the analyte is allowed to compete with a short range beta particle-emitting radiolabeled analyte analogue for binding to the ABM. If the analyte analogue binds, the beta particles emitted by its label come close enough to stimulate the scintillant.
  • Fluorescence Resonance Energy Transfer A method for detection of complex formation, such as ligand-receptor binding, that relies upon the through-space interactions between two fluorescent groups.
  • a fluorescent molecule has a specific wavelength for excitation and another wavelength for emission. Pairs of fluorophores are selected that have an overlapping emission and excitation wavelength. Paired fluorophores are detected by a through-space interaction referred to as resonance energy transfer.
  • resonance energy transfer When a donor fluorophore is excited by light, it would normally emit light at a higher wavelength; however, during FRET energy is transferred from the donor to the acceptor fluorophore allowing the excited donor to relax to the ground-state without emission of a photon.
  • Ethylasparagine Hydroxylysine; allo-Hydroxylysine; 3- Hydroxyproline;
  • the peptides of the present invention include peptides whose sequences are disclosed in this specification, or sequences differing from the above solely by no more than one nonconservative substitution and/or one or more conservative substitutions, preferably no more than a single conservative substitution.
  • the substitutions may be of non- genetically encoded (exotic) amino acids, in which case the resulting peptide is nonbiogenic.
  • a peptoid library composed for peptoids related to one or more lead peptides, may be synthesized and screened.
  • a peptoid library may comprise true peptides, too.
  • the side chains attached to the core main chain atoms of the monomers linked by the pseudopeptide bonds and/or (2) the side chains (e.g., the -R of an -NRCO- ) of the pseudopeptide bonds.
  • the monomeric units which are not amino acid residues are of the structure -NR1-CR2-CO-, where at least one of Rl and R2 are not hydrogen.
  • the R group will usually be any of the side chains characterizing the amino acids of peptides, as previously discussed.
  • Step (c) may be carried out by reference to a template database, see Wilson, et al. Tetrahedron, 49:3655-63 (1993) .
  • the templates will typically allow the mounting of 2-8 pharmacophores, and have a relatively rigid structure. For the latter reason, aromatic structures, such as benzene, biphenyl, phenanthrene and benzodiazepine, are preferred.
  • aromatic structures such as benzene, biphenyl, phenanthrene and benzodiazepine.
  • orthogonal protection techniques see Tuchscherer, et al., Tetrahedron, 17:3559-75 (1993).
  • Homologues are compounds which differ by an increase or decrease in the number of methylene groups in an alkyl moiety.
  • Classical isosteres are those which meet Erlenmeyer's definition: "atoms, ions or molecules in which the peripheral layers of electrons can be considered to be identical".
  • Nonclassical isosteric pairs include -CO- and -S0 2 -, -
  • the mutated sequence may correspond to a receptor (including both endogenous and chimeric receptors) , a ligand for a receptor (including the ligand-like moiety of a hybrid protein) , or a component of a signal producing system.
  • the latter may be, for example, a DNA-binding or transactivation domain of a transcription factor, a reporter (or fragment thereof) , or a "downstream" protein component of the signal producing system.
  • a target-binding member of the screened library may also be mutated.
  • desirable mutants may be further mutated.
  • the invention also contemplates mutation of nucleic acids, for example, the target DNA operator for the DNA-binding domain of a transcription factor.
  • the mutant protein is
  • the mutant protein may also be a hybrid (chimera) of at least one domain of each of two more reference proteins (or domains) , as hereafter discussed. It may be, for example, a hybrid of a domain from a protein A, and a domain from protein B. These domains may be identical to the original domains, or mutants thereof.
  • percentage amino acid identity is determined by aligning the mutant and reference sequences according to a rigorous dynamic programming algorithm which globally aligns their sequences to maximize their similarity, the similarity being scored as the sum of scores for each aligned pair according to an unbiased PAM250 matrix, and a penalty for each internal gap of -12 for the first null of the gap and -4 for each additional null of the same gap.
  • the percentage identity is the number of matches expressed as a percentage of the adjusted (i.e., counting inserted nulls) length of the reference sequence.
  • BLAST is a local alignment method.
  • nucleotide sequences which are substantially identical exceed the minimum identity of 50% e.g., are at least 51%, 66%, 75%, 80%, 85%, 90%, 95% or 99% identical in sequence.
  • insertions or deletions are limited to the termini. More preferably, there are no indels; the modifications are just conservative substitutions.
  • a conservative substitution is a substitution of one amino acid for another of the same exchange group, the exchange groups being defined as follows I Gly, Pro, Ser, Ala (Cys) (and any nonbiogenic, neutral amino acid with a hydrophobicity not exceeding that of the aforementioned a.a.'s) II Arg, Lys, His (and any nonbiogenic, positively- charged amino acids)
  • substitutions are defined herein as being substitutions within supergroup I/II/III or within supergroup IV/V, but not within a single one of groups I-V. They also include replacement of any other amino acid with alanine. If a substitution is not conservative, it preferably is semi-conservative.
  • Non-conservative substitutions are substitutions which are not conservative. They include “semi-conservative substitutions” as a subset.
  • a protein is nearly conservatively identical to a reference protein (peptide) if it differs from the latter, if at all, solely by one or more conservative modifications and/or a single nonconservative substitution.
  • the core sequence of a reference protein is the largest single fragment which retains at least 10% of a particular specific binding activity, if one is specified, or otherwise of at least one specific binding activity of the referent. If the referent has more than one specific binding activity, it may have more than one core sequence, and these may overlap or not.
  • a peptide of the present invention may have a particular similarity relationship (e.g., markedly identical) to a reference protein (peptide)
  • preferred peptides are those which comprise a sequence having that relationship to a core sequence of the reference protein (peptide) , but with internal insertions or deletions m either sequence excluded. Even more preferred peptides are those whose entire sequence has that relationship, with the same exclusion, to a core sequence of that reference protein (peptide) .
  • Biokeys of the present invention include not only the listed (reference) peptides, but also other peptides which are markedly identical. Preferably, the degree of identity (similarity) is higher than merely markedly identical . Where this specification sets forth a consensus sequence for a particular class of peptides then any peptide comprising said consensus is a preferred peptide according to this invention.
  • Non-naturally occurring means that it does not occur, as a unitary molecule, in non-genetically engineered cells or viruses. It may be biologically produced in genetically engineered cells, or genetically engineered virus-transfected cells, and it may be a segment of a larger, naturally occurring protein.
  • a peptide preferably is not naturally occurring, it more preferably is not conservatively identical to any naturally occurring peptide.
  • (b) is an insertion or deletion at the terminus, rather than internally, or, if internal, is at a domain boundary, or a loop or turn, rather than in an alpha helix or beta strand;
  • (e) is a substitution of one amino acid for another of similar size, charge, and/or hydrophobicity, and does not destroy a disulfide bond or other crosslink; and (f) is at a site which is subject to substantial variation among a family of homologous proteins to which the protein of interest belongs.
  • Surface residues may be identified experimentally by various labeling techniques, or by 3-D structure mapping techniques like X-ray diffraction and NMR. A 3-D model of a homologous protein can be helpful.
  • Residues forming the binding site may be identified by (1) comparing the effects of labeling the surface residues before and after complexing the protein to its target, (2) labeling the binding site directly with affinity ligands, (3) fragmenting the protein and testing the fragments for binding activity, and (4) systematic mutagenesis (e.g., alanine-scanning mutagenesis) to determine which mutants destroy binding. If the binding site of a homologous protein is known, the binding site may be postulated by analogy.
  • Protein libraries may be constructed and screened that a large family (e.g., 10 8 ) of related mutants may be evaluated simultaneously.
  • the small organic compound combinatorial library (“compound library”, for short) is a combinatorial library whose members are suitable for use as drugs if, indeed, they have the ability to mediate a biological activity of the target protein.
  • Peptides have certain disadvantages as drugs. These include susceptibility to degradation by serum proteases, and difficulty in penetrating cell membranes.
  • all or most of the compounds of the compound library avoid, or at least do not suffer to the same degree, one or more of the pharmaceutical disadvantages of peptides.
  • Benzodiazepines The design of a library may be illustrated by the example of the benzodiazepines .
  • Benzodiazepine drugs including chlordiazepoxide, diazepam and oxazepam, have been used on anti-anxiety drugs.
  • Derivatives of benzodiazepines have widespread biological activities; derivatives have been reported to act not only as anxiolytics, but also as anticonvulsants, cholecystokinin (CCK) receptor subtype A or B, kappa opioid receptor, platelet activating factor, and HIV transactivator Tat antagonists, and GPIIblla, reverse transcriptase and ras farnesyltransferase inhibitors.
  • CCK cholecystokinin
  • the benzodiazepine structure has been disjoined into a 2-aminobenzophenone, an amino acid, and an alkylating agent. See Bunin, et al., Proc. Nat. Acad. Sci. USA, 91:4708 (1994). Since only a few 2-aminobenzophenone derivatives are commercially available, it was later disjoined into 2- aminoarylstannane, an acid chloride, an amino acid, and an alkylating agent. Bunin, et al., Meth. Enzymol., 267:448 (1996).
  • the arylstannane may be considered the core structure upon which the other moieties are substituted, or all four may be considered equals which are conjoined to make each library member.
  • a basic library synthesis plan and member structure is shown in Figure 1 of Fowlkes, et al., U.S. Serial No. 08/740,671, incorporated by reference in its entirety.
  • the acid chloride building block introduces variability at the R 1 site.
  • the R 2 site is introduced by the amino acid, and the R 3 site by the alkylating agent.
  • the R 4 site is inherent in the arylstannane.
  • Bunin, et al. generated a 1, 4- benzodiazepine library of 11,200 different derivatives prepared from 20 acid chlorides, 35 amino acids, and 16 alkylating agents.
  • variable elements included both aliphatic and aromatic groups.
  • aliphatic groups both acyclic and cyclic (mono- or poly-) structures, substituted or not, were tested. (although all of the acyclic groups were linear, it would have been feasible to introduce a branched aliphatic) .
  • the aromatic groups featured either single and multiple rings, fused or not, substituted or not, and with heteroatoms or not.
  • the secondary substitutents included - NH 2 , -OH, -OMe, -CN, -Cl, -F, and -COOH.
  • spacer moieties such as -0-, -S-, -00-, -CS-, -NH-, and - NR-, could have been incorporated.
  • Bunm et al. suggest that instead of using a 1, 4- benzodiazepine as a core structure, one may instead use a 1, 4-benzod ⁇ azepine-2, 5-dione structure.
  • combinatorial nonoligomeric compound libraries known or suggested in the art have been based on carbamates, mercaptoacylated pyrrolidines, phenolic agents, aminimides, -acylamino ethers (made from amino alcohols, aromatic hydroxy acids, and carboxylic acids), N-alkylamino ethers (made from aromatic hydroxy acids, amino alcohols and aldehydes) 1, 4-piperazines, and 1, 4-piperazine-6-ones .
  • DeWitt, et al., Proc. Nat. Acad. Sci. (USA), 90:6909-13 (1993) describes the simultaneous but separate, synthesis of 40 discrete hydantoins and 40 discrete benzodiazepines. They carry out their synthesis on a solid support (inside a gas dispersion tube) , in an array format, as opposed to other conventional simultaneous synthesis techniques (e.g., in a well, or on a pin) .
  • the hydantoins were synthesized by first simultaneously deprotecting and then treating each of five amino acid resins with each of eight isocyanates.
  • the benzodiazepines were synthesized by treating each of five deprotected amino acid resins with each of eight 2-amino benzophenone imines. Chen, et al., J. Am. Chem. Soc, 116:2661-62 (1994) described the preparation of a pilot (9 member) combinatorial library of formate esters. A polymer bead- bound aldehyde preparation was "split" into three aliquots, each reacted with one of three different ylide reagents. The reaction products were combined, and then divided into three new aliquots, each of which was reacted with a different Michael donor.
  • the library is preferably synthesized so that the individual members remain identifiable so that, if a member is shown to be active, it is not necessary to analyze it.
  • Several methods of identification have been proposed, including:
  • each member is synthesized only at a particular coordinate on or in a matrix, or in a particular chamber.
  • the present invention is not limited to any particular form of identification.
  • Heteronitrogen and Heterooxygen dikelomorpholines isoxazoles isoxazolines Heteronitrogen and Heterosulfur thiazolidines
  • the peptides of the present invention may be used as surrogate ligands in screening a library for inhibitors of a target protein bound by the peptides.
  • This library is preferably combinatorial in nature and preferably a library of small organic compounds.
  • the target- binding members of the library in question can be used as surrogates for an unknown or unavailable natural binding partner in screening a second combinatorial library (the "complementary library”), which need not be a biopolymeric library, for members which can inhibit the complexing of target protein to its natural binding partner .
  • the complementary library need not be, and preferably is not, a peptide library and it may be of lower overall diversity. It may be screened against all of the surrogate peptides; or only against selected ones. The screenings may be individual or collective. Often, the members of the complementary library will be less specific in their binding to the paratopes of the target protein than are the members of the first library, possibly because their surface area is smaller and offers fewer opportunities for favorable (or unfavorable) interactions with other molecules.
  • a preferred complementary library is a benzodiazepine library.
  • the degree of complex-inhibitory activity of the members of the complementary library may be quantified by means of a labeled surrogate peptide and an insolubilized target protein. Either the amount of labeled surrogate peptide is fixed, and the amount of complementary compound varied, or, more preferably, the amount of labeled surrogate peptide is varied and the amount of complementary compound held constant. The greater the activity of the complementary compound, the less labeled surrogate peptide will be in the solid phase (i.e., complexed to the. target protein) and the more will be in the liquid phase (i.e., uncompleted). The amount of label in either phase is then measured and correlated with the amount of the variable component.
  • false hits i.e., compounds which inhibit the binding of a false surrogate to the target protein, or which inhibit binding of a true surrogate but at the wrong site
  • inhibitory compounds which bind the target protein from those which bind the surrogate peptide by use of either the target protein or surrogate peptide alone, in labeled or immobilized form, as an assay or affinity separation reagent.
  • Certain of the peptides of the present invention bind protein kinases in a conformation-sensitive manner.
  • a panel of "BioKeys” receptor conformation-sensitive receptor binding molecules, typically peptides
  • a “fingerprint” of how a compound of interest interacts with that receptor in its various BioKey-modifled conformations, each element of the fingerprint being a measure of the strength of interaction of the compound with the receptor m the presence of a given BioKey.
  • fingerprints are obtained for a reasonable number of reference compounds with known biological activities, preferably as measured by a "gold standard" (whole animal, or isolated organ or tissue) assay, the similarity of the fingerprint of a new compound to that of the reference compounds may be calculated, and used to predict the bioactivity of the new compound.
  • a "gold standard" whole animal, or isolated organ or tissue
  • the invention has advantages over whole animal-based assay systems in that 1) the same technology can be applied to a variety of different receptors, 2) the system can be used for high throughput screening and compound characterization, and 3) the system gives very distinct patterns for agonists and antagonists of receptor activity using very little protein.
  • the biological activity of a test substance, as mediated by a particular receptor, m a particular organism, and thereof is predicted by:
  • test compound is similarly screened for its ability to alter the binding of the "Biokeys" to the receptor, thereby obtaining a test fingerprint
  • (V) the biological activity of the test substance in one or more target organisms, and m one or more target tissues thereof, is predicted on the basis of the biological activities of the reference substances therein, appropriately weighted by the similarity between the test substance and the reference substance.
  • the Biokey panel of step (I) is preferably obtained by screening the members of a combinatorial library for the ability to bind to (a) the unliganded receptor, and (b) a liganded receptor.
  • a combinatorial library is first screened against (a) , and then either the whole library, or only the unliganded receptor-bmdmg members, are screened against (b) .
  • the whole library is screened against (a) and (b) simultaneously. It is also permissible to screen first against (b) and then against (a) . It will be appreciated that step (II) need only be performed once for a given receptor and that it is not necessary that all reference substances be fingerprinted simultaneously.
  • steps (II) and (III) may be interchanged.
  • similarity may be determined in a qualitative and subjective way, i.e., by "eyeballing" the fingerprints and judging from experience which is more similar, or in a quantitative and objective manner, using the similarity measures set forth infra.
  • the biological activity may be predicted in a qualitative and subjective way, or more quantitatively and objectively, by mathematically weighting each reference substance's activity scores by the calculated similarity of its fingerprint to the fingerprint of the test substance.
  • the reference and test fingerprints are based on in vitro (cell-free) assays.
  • the reference and test fingerprints are based on cellular assays (but not on assays of whole multicellular organisms, or their organs or tissues) . It will be appreciated that both techniques may be used, either sequentially or simultaneously. For example, MB may be used as a first screen and CB as a second screen of the first round positives. Or compounds may be screened by both MB and CB, and compounds earmarked by either screen given further attention. Similarities may be calculated separately from in vitro and cell-based assays, or the results of these two types of assays may be combined into a single fingerprint for each reference or test compound.
  • the oligomers identified as binding molecules by the screening assays of the present invention may also be used as pharmaceuticals or in diagnostic reagents as described below.
  • the preferred animal subject of the present invention is a mammal.
  • mammal an individual belonging to the class Mammalia.
  • the invention is particularly useful in the treatment of human subjects, although it is intended for veterinary uses as well.
  • Preferred nonhuman subjects are of the orders Primata (e.g., apes and monkeys), Artiodactyla or Perissodactyla
  • protection is intended to include “prevention,” “suppression” and “treatment.”
  • prevention involves administration of the protein prior to the induction of the disease (or other adverse clinical condition) .
  • suppression involves administration of the composition prior to the clinical appearance of the disease.
  • Treatment involves administration of the protective composition after the appearance of the disease. Protection, including prevention, need not be absolute .
  • An agent which provides protection to a lesser degree than do competitive agents may still be of value if the other agents are ineffective for a particular individual, if it can be used in combination with other agents to enhance the level of protection, or if it is safer than competitive agents.
  • the drug may provide a curative effect, an ameliorative effect, or both.
  • At least one of the drugs of the present invention may be administered, by any means that achieve their intended purpose, to protect a subject against a disease or other adverse condition.
  • the form of administration may be systemic or topical.
  • administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes.
  • parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes.
  • parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes.
  • parenteral administration can be by bolus injection or by gradual perfusion over time.
  • a typical regimen comprises administration of an effective amount of the drug, administered over a period ranging from a single dose, to dosing over a period of hours, days, weeks, months, or years.
  • the suitable dosage of a drug of the present invention will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the most preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. This will typically involve adjustment of a standard dose, e.g., reduction of the dose if the patient has a low body weight.
  • a drug Prior to use in humans, a drug will first be evaluated for safety and efficacy in laboratory animals.
  • the total dose required for each treatment may be administered by multiple doses or in a single dose.
  • the protein may be administered alone or in conjunction with other therapeutics directed to the disease or directed to other symptoms thereof.
  • the appropriate dosage form will depend on the disease, the protein, and the mode of administration; possibilities include tablets, capsules, lozenges, dental pastes, suppositories, inhalants, solutions, ointments and parenteral depots.
  • the drug may be administered in the form of an expression vector comprising a nucleic acid encoding the peptide, such a vector, after in corporation into the genetic complement of a cell of the patient, directs synthesis of the peptide.
  • Suitable vectors include genetically engineered poxviruses (vaccinia) , adenoviruses, adeno-associated viruses, herpesviruses and lentiviruses which are or have been rendered nonpathogenic.
  • a pharmaceutical composition may contain suitable pharmaceutically acceptable carriers, such as excipients, carriers and/or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. See, e.g., Berker, supra , Goodman, supra, Avery, supra and Ebadi, supra, which are entirely incorporated herein by reference, included all references cited therein. Diagnostic Assays
  • diagnostic assays employ a binding molecule of known binding activity, or a conjugate or derivative thereof, as a diagnostic reagent.
  • the "binding molecule” may be a peptide, peptoid, peptidomimetic or other analogue of the present invention, or an oligonucleotide of the present invention, which binds the analyte or a binding partner of the analyte.
  • the analyte is a target protein.
  • In vitro assays may be diagnostic assays (using a known binding molecule to detect or measure an analyte) or screening assays (determining whether a potential binding molecule in fact binds a target) .
  • the format of these two types of assays is very similar and, while the description below refers to 112 diagnostic assays for analytes, it applies, mutatis mutandis, to the screening of molecules for binding to targets.
  • the in vitro assays of the present invention may be applied to any suitable analyte-containing sample, and may be qualitative or quantitative in nature.
  • the assay In order to detect the presence, or measure the amount, of an analyte, the assay must provide for a signal producing system (SPS) in which there is a detectable difference in the signal produced, depending on whether the analyte is present or absent (or, in a quantitative assay, on the amount of the analyte) .
  • SPS signal producing system
  • This signal is, or is derived from, one or more observable raw signals.
  • the raw signal for a particular state e.g., presence or amount of analyte
  • the signal is a difference in raw signals, depending on the states to be differentiated by the assay.
  • the signal may be direct (increased if the amount of analyte increases) or inverse (decreased raw signal if the amount of analyte increases).
  • the signal may be absolute (in one state, there is no detectable raw signal at all) or relative (a change in the level of the raw signal, or of the rate of change in the level of the raw signal) .
  • the signal may be discrete (yes or no, depending on the level of the raw signal relative to some threshold) or continuous in value.
  • the signal may be simple (based on a single raw signal) or composite (based on a plurality of raw signals) .
  • the detectable raw signal may be one which is visually detectable, or one detectable only with instruments. Possible raw signals include production of colored or luminescent products, alteration of the characteristics (including amplitude or polarization) of absorption or emission of radiation by an assay component or product, and precipitation or agglutination of a component or product.
  • the raw signal may be monitored manually or automatically.
  • a label may be, e.g., a radioisotope, a fluorophore, an enzyme, a co-enzyme, an enzyme substrate, an electron-dense compound, or an agglutinable particle.
  • One diagnostic reagent is a conjugate, direct or indirect, or covalent or noncovalent, of a label with a binding molecule of the invention.
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • Isotopes which are particularly useful for the purpose of the present invention are 3 H, 125 I, 131 I, 3S S, l ⁇ C, and, preferably, 125 I.
  • fluorescent labelling compounds fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, _-phthaldehyde and fluorescamine .
  • fluorescein isothiocyanate fluorescein isothiocyanate
  • rhodamine phycoerythrin
  • phycocyanin allophycocyanin
  • fluorescamine fluorescein isothiocyanate
  • rhodamine phycoerythrin
  • phycocyanin allophycocyanin
  • fluorescamine fluorescamine
  • 15 Eu, or others of the lanthanide series may be attached to the binding protein using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) of ethylenediamine-tetraacetic acid (EDTA) .
  • the binding molecules also can be detectably labeled by coupling to a chemiluminescent compound. The presence of the chemiluminescent compound is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction after a suitable reactant is provided.
  • chemiluminescent labeling compounds are luminol, isolumino, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • Bioluminescent compound may be used to label the binding molecule.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • Enzyme labels such as horseradish peroxidase and alkaline phosphatase, are preferred.
  • the signal producing system must also include a substrate for the enzyme. If the enzymatic reaction product is not itself detectable, the SPS will include one or more additional reactants so that a detectable product appears .
  • Assays may be divided into two basic types, heterogeneous and homogeneous.
  • heterogeneous assays the interaction between the affinity molecule and the analyte does not affect the label, hence, to determine the amount or presence of analyte, bound label must be separated from free label.
  • homogeneous assays the interaction does affect the activity of the label, and therefore analyte levels can be deduced without the need for a separation step.
  • a target-binding molecule of the present invention may be used diagnostically in the same way that a target-binding antibody is used.
  • the sample will normally be a biological fluid, such as blood, urine, lymph, semen, milk, or cerebrospinal fluid, or a fraction or derivative thereof, or a biological tissue, in the form of, e.g., a tissue section or homogenate.
  • the sample conceivably could be (or derived from) a food or beverage, a pharmaceutical or diagnostic composition, soil, or surface or ground water.
  • a biological fluid or tissue it may be taken from a human or other mammal, vertebrate or animal, or from a plant.
  • the preferred sample is blood, or a fraction or derivative thereof.
  • the binding molecule is insolubilized by coupling it to a macromolecular support, and target in the sample is allowed to compete with a known quantity of a labeled or specifically labelable target analogue.
  • the conjugate of the binding molecule to a macromolecular support is another diagnostic agent within the present invention.
  • the "target analogue” is a molecule capable of competing with target for binding to the binding molecule, and the term is intended to include target itself. It may be labeled already, or it may be labeled subsequently by specifically binding the label to a moiety differentiating the target analogue from authentic target.
  • the solid and liquid phases are separated, and the labeled target analogue in one phase is quantified.
  • target analogue in the solid phase i.e., sticking to the binding molecule
  • level of target analyte in the sample In a "sandwich assay", both an insolubilized target- binding molecule, and a labeled target-binding molecule are employed.
  • the target analyte is captured by the insolubilized target-binding molecule and is tagged by the labeled target-binding molecule, forming a tertiary complex.
  • the reagents may be added to the sample in either order, or simultaneously.
  • the target-binding molecules may be the same or different, and only one need be a target-binding molecule according to the present invention (the other may be, e.g., an antibody or a specific binding fragment thereof) .
  • the amount of labeled target-binding molecule in the tertiary complex is directly proportional to the amount of target analyte in the sample.
  • the two embodiments described above are both heterogeneous assays. However, homogeneous assays are conceivable. The key is that the label be affected by whether or not the complex is formed.
  • a label may be conjugated, directly or indirectly
  • the target binding molecule may be conjugated to a solid-phase support to form a solid phase ("capture") diagnostic reagent.
  • Suitable supports include glass ; polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to its target.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Analyte-binding molecules can be used for in vivo imaging.
  • Radio-labelled binding molecule may be administered to the human or animal subject. Administration is typically by injection, e.g., intravenous or arterial or other means of administration in a quantity sufficient to permit subsequent dynamic and/or static imaging using suitable radio-detecting devices.
  • the preferred dosage is the smallest amount capable of providing a diagnostically effective image, and may be determined by means conventional in the art, using known radio-imaging agents as a guide. Typically, the imaging is carried out on the whole body of the subject, or on that portion of the body or organ relevant to the condition or disease under study.
  • the radio-labelled binding molecule has accumulated. The amount of radio-labelled binding molecule accumulated at a given point in time in relevant target organs can then be quantified.
  • a particularly suitable radio-detecting device is a scintillation camera, such as a gamma camera.
  • a scintillation camera is a stationary device that can be used to image distribution of radio-labelled binding molecule.
  • the detection device m the camera senses the radioactive decay, the distribution of which can be recorded.
  • Data produced by the imaging system can be digitized.
  • the digitized information can be analyzed over time discontinuously or continuously.
  • the digitized data can be processed to produce images, called frames, of the pattern of uptake of the radio-labelled binding protein in the target organ at a discrete point in time.
  • quantitative data is obtained by observing changes in distributions of radioactive decay m target organs over time. In other words, a time-activity analysis of the data will illustrate uptake through clearance of the radio-labelled binding molecule by the target organs with time.
  • the radioisotope must be selected with a view to obtaining good quality resolution upon imaging, should be safe for diagnostic use in humans and animals, and should preferably have a short physical half-life so as to decrease the amount of radiation received by the body.
  • the radioisotope used should preferably be pharmacologically inert, and, in the quantities administered, should not have any substantial physiological effect.
  • the binding molecule may be radio-labelled with different isotopes of iodine, for example 123 I, 125 I, or 131 I 03 00343
  • the radio-labelled binding molecule may be prepared by various methods. These include radio-halogenation by the chloramine - T method or the lactoperoxidase method and subsequent purification by HPLC (high pressure liquid chromatography) , for example as described by J. Gutkowska et al in "Endocrinology and Metabolism Clinics of America: (1987) 16. (1):183. Other known method of radio-labelling can be used, such as IODOBEADSTM.
  • radio-labelled binding molecule there are a number of different methods of delivering the radio-labelled binding molecule to the end-user. It may be administered by any means that enables the active agent to reach the agent's site of action in the body of a mammal. If the molecule is digestible when administered orally, parenteral administration, e.g., intravenous, subcutaneous, or intramuscular, would ordinarily be used to optimize absorption. Other Uses
  • Peptides in general, can be used as molecular weight markers for reference in the separation or purification of peptides by electrophoresis or chromatography . In many instances, peptides may need to be denatured to serve as molecular weight markers.
  • a second general utility for peptides is the use of hydrolyzed peptides as a nutrient source. Hydrolyzed peptide are commonly used as a growth media component for culturmg microorganisms, as well as a food ingredient for human consumption. Enzymatic or acid hydrolysis is normally carried out either to completion, resulting in free ammo acids, or partially, to generate both peptides and amino acids. However, unlike acid hydrolysis, enzymatic hydrolysis (proteolysis) does not remove non-ammo acid functional groups that may be present. Peptides may also be used to increase the viscosity of a solution.
  • Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem. Biophys. Res. Commun. 172:993-999.
  • 1-beta-D-arabinofuranosylcytosone is antagonistic to stimulation of apoptosis and Bcl-2alpha down-regulation. J. Biol. Chem. 272:23481-23484.
  • Table 3 Peptide sequences isolated on PKC ⁇ .
  • sequences show the peptides as they are displayed on the phage.
  • Many of the libraries use cloning sites that leave an N-terminal SS or SR and a C-terminal SR, hence conservation of SS or SR is not a result of the selection process.
  • Twenty-two unique sequences were obtained and clustered in three groups, based on location of acidic (D, E) , basic (R, H, K) , and hydrophobic groups (e.g., W, F, Y, M, V, I, L) .
  • Hydrophobic AAs may be divided into aliphatic (V, L, I, M) and aromatic (F, W, Y) subgroups. Ala can be considered an aliphatic hydrophobic AA.
  • Phage affinity selections were carried out in the absence or presence of phospholipid/diacylglycerol (PS/DAG) or phospholipid/phorbol dibutyrate (PS/PDBu) . Phage #1270 was selected twice, under both PS/DAG and PS/PDBu.
  • PS/DAG phospholipid/diacylglycerol
  • PS/PDBu phospholipid/phorbol dibutyrate
  • This table shows the alignment of a segment of clathrin-associated protein (P53677) (partial sequence depicted is SEQ ID NO: 28) with PKC ⁇ peptides 1270 and 1271.
  • any description of a class or range as being useful or preferred in the practice of the invention shall be deemed a description of any subclass (e . g. , a disclosed class with one or more disclosed members omi tted) or subrange contained therein, as well as a separate description of each individual member or value in said class or range.
  • the description of preferred embodiments individually shall be deemed a description of any possible combina tion of such preferred embodiments , except for combinations which are impossible (e . g, mutually exclusive choices for an element of the invention) or which are expressly excluded by this specifica tion . If an embodiment of this invention is disclosed in the prior art, the description of the invention shall be deemed to include the invention as herein disclosed with such embodiment excised.
  • the invention includes but is not limited to the subj ect matter set forth in the appended claims, and presently unclaimed combina tions thereof. It further includes such subj ect matter further limited, if not already such, to that which overcomes one or more of the disclosed deficiencies in the prior art . To the extent that any claims encroach on subject matter disclosed or suggested by the prior art, applicant (s) contemplate the invention (s) corresponding to such claims with the encroaching subject matter excised.

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  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Hospice & Palliative Care (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un procédé d'identification de peptides qui lient des protéines kinases, en particulier une protéine kinase C, d'une manière : (a) active, spécifique à la conformation, (b) inactive, spécifique à la conformation ou (c) active, indépendante à la conformation. les peptides qui lient spécifiquement la PKCα d'une manière active, spécifique à la conformation, ont été identifiés, et peuvent être utilisés pour des diagnostics et à d'autres fins.
PCT/EP2003/000343 2002-01-18 2003-01-15 Peptides specifiques a la conformation, de liaison de la proteine kinase, procedes et produits associes WO2003059943A2 (fr)

Priority Applications (1)

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AU2003205605A AU2003205605A1 (en) 2002-01-18 2003-01-15 Conformation-specific, protein kinase binding peptides and related methods and products

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US34925002P 2002-01-18 2002-01-18
US60/349,250 2002-01-18

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WO2005071112A2 (fr) * 2004-01-26 2005-08-04 Isis Innovation Ltd Analyse moleculaire
FR2869416A1 (fr) * 2004-04-27 2005-10-28 Centre Nat Rech Scient Procede d'identification d'un ligand capable de moduler selectivement une cascade fonctionnelle impliquant une cible et ses applications pour le criblage a haut-debit de molecules d'interet.
WO2008037688A2 (fr) * 2006-09-25 2008-04-03 Novartis Ag Formes cristallines de la kinase pkc alpha, procédés de fabrication de tels cristaux et leurs utilisations
EP1922328A1 (fr) * 2005-08-05 2008-05-21 Pharmagap Inc. Peptides ciblés sur des isoformes c de protéine kinase et utilisation de ceux-ci
US8158586B2 (en) 2005-04-11 2012-04-17 Pharmagap Inc. Inhibitors of protein kinases and uses thereof
KR101611494B1 (ko) 2012-12-20 2016-04-12 한양대학교 산학협력단 ＰＫＣ－δ 검출용 폴리펩티드 및 이의 용도
CN108033995A (zh) * 2017-12-19 2018-05-15 渤海大学 两种来源于大黄鱼肌联蛋白的ace抑制肽

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005071112A3 (fr) * 2004-01-26 2005-12-08 Isis Innovation Analyse moleculaire
WO2005071112A2 (fr) * 2004-01-26 2005-08-04 Isis Innovation Ltd Analyse moleculaire
US9110077B2 (en) 2004-04-27 2015-08-18 Centre National De La Recherche Scientifique Method for identifying a ligand capable of selectively modulating a functional cascade involving a target, and uses thereof for high-throughput screening of molecules of interest
WO2005106481A2 (fr) * 2004-04-27 2005-11-10 Centre National De La Recherche Scientifique Procede d'identification d'un ligand capable de moduler selectivement une cascade fonctionnelle impliquant une cible, et ses applications pour le criblage a haut-debit de molecules d'interet.
WO2005106481A3 (fr) * 2004-04-27 2006-11-02 Centre Nat Rech Scient Procede d'identification d'un ligand capable de moduler selectivement une cascade fonctionnelle impliquant une cible, et ses applications pour le criblage a haut-debit de molecules d'interet.
FR2869416A1 (fr) * 2004-04-27 2005-10-28 Centre Nat Rech Scient Procede d'identification d'un ligand capable de moduler selectivement une cascade fonctionnelle impliquant une cible et ses applications pour le criblage a haut-debit de molecules d'interet.
US8158586B2 (en) 2005-04-11 2012-04-17 Pharmagap Inc. Inhibitors of protein kinases and uses thereof
EP1922328A4 (fr) * 2005-08-05 2010-03-10 Pharmagap Inc Peptides ciblés sur des isoformes c de protéine kinase et utilisation de ceux-ci
EP1922328A1 (fr) * 2005-08-05 2008-05-21 Pharmagap Inc. Peptides ciblés sur des isoformes c de protéine kinase et utilisation de ceux-ci
EP2298792A3 (fr) * 2005-08-05 2011-10-19 Pharmagap Inc. Peptides ciblés pour les isoformes de protéine kinase C et utilisations associées
WO2008037688A3 (fr) * 2006-09-25 2008-05-22 Novartis Ag Formes cristallines de la kinase pkc alpha, procédés de fabrication de tels cristaux et leurs utilisations
EP1911838A1 (fr) * 2006-09-25 2008-04-16 Novartis AG Formes crystallines de la protéine PKC alpha kinase, procédé de manufacture desdits cristaux et leur utilisation
WO2008037688A2 (fr) * 2006-09-25 2008-04-03 Novartis Ag Formes cristallines de la kinase pkc alpha, procédés de fabrication de tels cristaux et leurs utilisations
KR101611494B1 (ko) 2012-12-20 2016-04-12 한양대학교 산학협력단 ＰＫＣ－δ 검출용 폴리펩티드 및 이의 용도
CN108033995A (zh) * 2017-12-19 2018-05-15 渤海大学 两种来源于大黄鱼肌联蛋白的ace抑制肽
CN108033995B (zh) * 2017-12-19 2020-12-29 渤海大学 两种来源于大黄鱼肌联蛋白的ace抑制肽

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