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WO1999025384A2 - Ligands multimeres attaches et leur utilisation dans les interactions recepteur-ligand - Google Patents

Ligands multimeres attaches et leur utilisation dans les interactions recepteur-ligand Download PDF

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Publication number
WO1999025384A2
WO1999025384A2 PCT/US1998/024467 US9824467W WO9925384A2 WO 1999025384 A2 WO1999025384 A2 WO 1999025384A2 US 9824467 W US9824467 W US 9824467W WO 9925384 A2 WO9925384 A2 WO 9925384A2
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ligand
compound
receptor protein
activation
moiety
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PCT/US1998/024467
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English (en)
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WO1999025384A3 (fr
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Richard H. Kramer
Jeffrey W. Karpen
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University Of Miami
University Technology Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol

Definitions

  • the present invention relates to multimeric tethered ligands for receptor proteins which have multiple ligand binding sites. More in particular, the present invention relates to compounds comprising two or more ligands for a receptor protein having two or more ligand binding sites, in which the ligands are coupled to each other by a flexible tether, such as a polymer, having an average length which facilitates simultaneous occupancy of each of the multiple ligand binding sites on the receptor protein by an individual ligand of the multimeric ligand.
  • the multimeric tethered ligands of the invention "therefore, provide extraordinarily potent ligands, either agonists or antagonists, which may be used, for instance, therapeutically to target a desired receptor protein with multiple binding sites.
  • the invention also relates to methods for producing such potent multimeric ligands specific for a receptor with multiple ligand binding sites, including ligands specific for a particular member of a family of receptors for a common ligand, without a priori knowledge of the structure of the particular receptor.
  • a multimeric ligand selective for a particular receptor is obtained by varying the average length of the tether connecting multiple ligands to determine an optimal length for simultaneous binding of individual ligands to multiple binding sites separated by the particular distance between binding sites occurring in that particular receptor protein.
  • Cyclic nucleotide effectors include CNG ion channels, such as those from vertebrate photoreceptors and olfactory neurons (1), and protein kinases activated either by cGMP (2) or by adenosine 3 ',5 '-cyclic monophosphate (cAMP) (3) (PKG and PKA, respectively).
  • CNG ion channels such as those from vertebrate photoreceptors and olfactory neurons (1)
  • protein kinases activated either by cGMP (2) or by adenosine 3 ',5 '-cyclic monophosphate (cAMP) (3) (PKG and PKA, respectively).
  • cAMP adenosine 3 ',5 '-cyclic monophosphate
  • Multivalent receptor binding molecules are known in the art. For' instance, cross- linked antibodies are known to cause individual surface immunoglobulin receptors on B- lymphocytes to form aggregates known as patches, which collect into a cap assembly at one pole of the cell. See, for example, Graziadei, L. et al, Nature 347:396-399 (1990). Capping is thought to be an essential event in antibody-mediated signal transduction across cell membranes.
  • polypeptide growth factors and hormones are known to function as bivalent ligands to effect receptor dimerization.
  • human growth hormone molecules each have two binding sites, enabling the hormone to bind sequentially two separate receptor proteins.
  • receptor dimerization of EGF receptor family tyrosine kinases is mediated by the bivalent behavior of at least one ligand.
  • a differentiation factor encoded by the neu gene is known to simultaneously bind two receptor proteins, creating functional dimers that mediate signal transduction, thereby promoting cell growth. See, for example, Tzahar, E. et al., EMBO J. 16:4938- 4950 (1997).
  • Analogs of small molecule ligands which comprise more than one moiety structurally related to such a ligand are also known.
  • analogs of acetylcholine are known in which two identical moieties structurally related to acetylcholine are connected by a short methylene polymer, for instance, decamethonium bromide in which two trimethylamonium bromide residues are connected by a ten carbon polymeric chain to produce the following structure:
  • tethered ligand has been used to describe a portion of a receptor protein which binds to a ligand binding site of that same receptor protein. For instance, such a receptor is thought to be involved in thrombin inhibition of platelet • adenylate cyclase. Seiler, S. M. et al., Biochem. Biophy. Res. Comm. 752:1296-1302 (1992). A portion of the receptor sequence resembles the carboxy terminus of hirudin, an peptide inhibitor of thrombin, and thrombin evidently recognizes this sequence and cleaves a single peptide bond in the receptor protein that releases a peptide containing the original N-terminus. The resulting new N-terminus apparently serves as a tethered ligand which activates the receptor.
  • tethered ligand also has been used to describe a ligand attached by a polymer to a macromolecule or molecular complex, for instance, liposomes, to target the macromolecule or complex to a receptor for the ligand. Attaching a ligand to such a macromolecule or complex by a flexible tether is known to influence the binding of the ligand to its receptor.
  • bacteriophage particles displaying multiple copies of peptide ligands have been used as multivalent ligands to "stain” and monitor expression of various receptor proteins. Li. M., Nature Biotechnology 75:559-563 (1997).
  • While the above approach may expedite design of high affinity ligands for a single ligand binding site, such as the binding site for FK506 on the FK506 binding protein, by identifying small molecules which bind to different subsites with that single ligand binding site, it does not specifically address design of high affinity ligands for receptors with multiple binding sites for the same ligand, much less ligands which would discriminate among related receptors that bind a common ligand at multiple binding sites, such as cyclic nucleotide effectors.
  • the length may be optimized to provide simultaneous occupancy of multiple binding sites separated by a particular distance which occurs in a particular member of a group of related receptors that bind a common ligand at multiple binding sites
  • a compound which is a multimeric tethered ligand comprising a plurality of ligand moieties tethered to a joint moiety, wherein each ligand moiety comprises a ligand for at least one receptor protein having multiple binding sites for said ligand and each ligand moiety is linked by a tether moiety to that joint moiety.
  • This compound has a structure which may be described by the general formula: J(Cj 1 -T 1 -C L 1 -L 1 )(Cj 2 -T 2 -C L 2 -L 2 )(Cj 3 -T 3 -C L 3 -L 3 )...(Cj x -T x -C L X -L x ) wherein: x is the number of ligand moieties in said compound and is an integer greater than or equal to 2; each of L 1 to L x is one of said ligand moieties and comprises a ligand which is the same as or different from the ligand of any other ligand moiety in said compound, J is said joint moiety selected from the group consisting of a covalent bond, an atom and a molecule; each of Cj 1 to Cj x is a joint coupling moiety selected from the group consisting of a covalent bond, an atom and a molecule, and is covalently coupled to said joint mo
  • each of L 1 and L 2 comprises a ligand for the same receptor protein.
  • a dimeric tethered ligand of the invention may be described by a simpler version of the above formula, as follows: In a more preferred embodiment of this dimeric compound, each of L 1 and L 2 comprises the same ligand.
  • each of the tethers of the invention compounds, T 1 and T 2 is a polymeric molecule selected from the group consisting of polymers containing only C, H and O, polynucleotides and polypeptides.
  • Cj'.T 1 , Cj 2 and T 2 together are a molecule of polyethylene glycol (PEG) having an average molecular weight in the range of about 47 to about 100,000 daltons.
  • PEG molecules having an average molecular weight in the range of about 282 to about 20,000 daltons.
  • tether of the invention molecule may be used to form the tether of the invention molecule, provided that the average (root mean square; "rms") length of the tether is sufficient to span the distance between two ligand binding sites on the target receptor, typically on the order of about 10 to about 100 angstroms (A).
  • rms root mean square
  • A angstroms
  • the ligand moieties, for instance L 1 and L 2 , of the multimeric ligands of the invention may comprise ligands for any ligand binding protein, including such ligands as compounds comprising a purine or a nucleoside or a nucleotide, including cyclic nucleotides and oligonucleotides, compounds comprising an amino acid, such as peptides, polypeptides and proteins which are know to function as hormones, growth factors, cytokines or anti-idiotypic antibodies, for instance.
  • Ligand moieties of the invention may also comprise various small molecule ligands, such as drugs and metabolites.
  • each of L 1 and L 2 is a guanosine 3 ',5 'cyclic monophosphate (cGMP) molecule of a derivative thereof, and each of C 1 - and C L 2 -has the structure: S-CH 2 -C H 2 -S0 - and is covalently coupled to the carbon in the 8 position of the guanosine moiety in the cGMP molecule or derivative thereof.
  • L and L are ligands for a receptor protein selected from the group consisting of a cyclic nucleotide-gated ion channel or a guanosine 3 ',5 '-cyclic monophosphate activated kinase.
  • exemplary compounds of the invention exhibit an affinity for such receptors that is substantially greater than the average affinity of a monomer of the ligand for that receptor.
  • compounds of the invention may exhibit an affinity for a receptor with multiple ligand binding sites that is at least about 10 times, preferably at least about 100 times to at least about 2,000 times, and still more preferably at least about 10,000 times greater than the average affinity of a monomer of the ligand for that same receptor.
  • Other ligands particularly suited for use in the multimeric tethered ligands of the invention include the following ligands or derivatives thereof, which are listed in relation to the receptor(s) to which they bind: 1.
  • Nicotinic acetylcholine receptors Agonists and antagonists of muscle and neuronal nicotinic acetylcholine receptors. Includes ⁇ -bungarotoxin, neuronal bungarotoxin, anatoxin, ABT418, cytisine, epibatidine, nicotine, atropine, (+)-tubocurarine, and other agonists and antagonists (see Lindstrom J.M., (1994) in Handbook of Receptors and Channels:. Ligand and Voltage-Gated Ion Channels (R.A. North, ed) CRC Press, pp. 153-175.
  • Capsaicin receptors Capsaicin, capsazepine, ruthenium red, resiniferatoxin, and their derivatives. (Caterina et al., 1997).
  • Cyclic nucleotide-gated channels Cyclic nucleotides (cAMP, cGMP, cCMP, cMP, cUMP) and 8-substitued derivatives including, but not limited to 8-Br cAMP and cGMP, 8-parachlorothio (8-pCPT) cAMP and cGMP, 8-fluorosceinyf cAMP and cGMP, and 8-n-propylthio cGMP. Also included are phosphorothioate derivatives of cAMP, cGMP, and their 8-substituted derivatives , partially listed above.
  • Both isomers of these phosphorothioate derivatives are included (for example Rp-cAMPS and Rp-cGMPS, Sp-cAMPS, Sp-cGMPS, Rp- and Sp-8-pCPT-cAMPS and Rp- and Sp-8-pCPT-cGMPS.
  • PET ⁇ -phenyl-1, N2-etheneoguanosine- 3',5'-cyclic monophosphate
  • PET forms of 8- substituted derivatives of cAMP and cGMP PET forms of phosphorothioate derivatives of cAMP and cGMP.
  • GABAA and GABAC receptors Ionotropic GABA receptors (GABAA and GABAC receptors). Agonists and antagonists of the GABA, benzodiazepine, and barbiturate binding sites on GABA receptors.
  • GABA site ligands Isoguvacine, muscimol, THIP, piperidine-4-sulphonic acid, bicuculline, SR95531, and their derivatives and related compounds.
  • Benzodiazepine site benzodiazepines including flunitrazem, diazepam, zolpidem, abecarnil, ZK93423, DMCM, Rol94603, flumazenil, ZK93426, CGS8216, cis-4- aminocrotonic acid and related compounds and derivatives.
  • Barbiturate site phenobarbitoal, pentabarbitol, and related compounds and derivatives. Borman and Feigenspan (1995), Bowery, (1997), Johnston (1996), Sieghart (1995), Whiting et al., (1995).
  • NMDA N-methyl D-aspartate
  • Agonists and antagonists of the glycine- binding site including, but not limited to glycine, (+)HA966, D-serine, 5,7- dichlorokynurenate, L689560, MNQX and related compounds and derivatives.
  • AMP A Agonists and antagonists including, but not limited to AMPA, (s)-5- flurowillardine, NBQX, CNQX, LY215490, LY293558, GYK153655, and related compounds and derivatives.
  • Kainate Agonists and antagonists including, but not limited to kainate, quisqualate, 4- methyl glutamic acid, domoic acid, NS 102 and related compounds and derivatives.
  • Glycine receptors Agonists and antagonists including glycine, ⁇ -alanine, taurine, strychnine, picrotoxin, quinoline, cyanotriphenylborate, their derivatives and related compounds. Becker, (1992); Schmeiden and Betz, (1995); Rundstrom et al., (1994);
  • G Ionotropic purinergic receptors (P 2X receptors) ATP, ADP, AMP, and their analogs and derivatives, particularly ATP- ⁇ -S and ⁇ , ⁇ methylene-ATP. Also GTP, GDP, GMP, and their analogs and derivatives, particularly GTP- ⁇ -S. Suramin, an antagonist of P 2x receptors. Also adenosine, guanosine, cytosine, and inosine, and their analogs, derivatives and related compounds. (Brake et al., 1994; Burnstock, 1990; Cusack and Hourani, 1990; Dalziel and Westfall, 1994; Fredholm et al., 1994). H.
  • Ionotropic serotonin receptors (5-HT 3 receptors) Agonists and antagonists including 5-hydroxytryptamine (5-HT, also known as serotonin), 2-Methyl-5-HT, m- CPBG, granisetron, ondansetron, tropisteron, zacopride, ⁇ -methyl-5-HT, and derivatives of above agents. (References in TIPS review book on Receptors and Ion channels , 1997).
  • K ATP channels (ATP, ADP, AMP and other nucleotides and derivatives, also sulfonylurea compounds and derivatives)
  • Ligands include !P 3 , D° 4 and other inositol phosphates, and derivatives.
  • Proteins in this group include "ABC” transporters (e.g. multidrug resistance protein, cyctic fibrosis transmembrane regulator (CFTR) protein, and adenylate cyclase).
  • ABSC cyctic fibrosis transmembrane regulator
  • PLD ligands for this group of proteins include ATP, GTP, CTP, ADP, GDP, AMP, GMP, - cAMP, cGMP, cCMP, NADP, NADPH, cyclic ADP-ribose, and derivatives and analogs of these and other nucleotides.
  • Nucleotides are the most common of these regulatory ligands, so PLD ligands for this group of proteins include ATP, GTP, CTP, ADP, GDP, AMP, GMP, cAMP, cGMP, cCMP, NADP, NADPH, cyclic ADP-ribose, and derivatives and analogs of these and other nucleotides.
  • the following enzymes are examples: aspartate carbamoyl transferase, glycogen phosphorylase, cyclic AMP- and cyclic GMP-dependent proteins kinases, and phosphofructokinase. This last enzyme appears to multiple regulatory sites that bind GDP; the effect of GDP on enzynmatic activity has a Hill coefficient near 2.
  • References for allosteric regulation of enzymes include: Fersht, A. (1985) Enzyme Structure and Mechanism (2 nd edn.), W.H. Freeman and Co., NY; Perutz, M. (1990) Mechanisms of Cooperativity and Allosteric Regulation in Proteins ; Cambridge Univ. Press.
  • a derivative of a ligand in the context of the present invention indicates a moiety of an invention compound which is substantially structurally similar to the ligand and retains an ability to bind specifically to a receptor protein having a binding site that specifically binds to that ligand.
  • the invention provides a method of activating or antagonizing activation of a receptor protein comprising multiple binding sites for a ligand L comprising adding to the receptor protein, under conditions where the ligand L activates or antagonizes activation of the receptor protein, a compound of the invention in which each of said ligand moieties L 1 to L x comprises said ligand L or a derivative thereof, and the average (rms) length of the portion of said compound linking any two' of said ligand moieties L 1 to L x is greater than or equal to the distance between any two of said multiple binding sites, whereby each of at least two of the ligand moieties L 1 to L x binds to one of the multiple binding sites on the receptor protein, thereby activating or antagonizing activation of the receptor protein.
  • the average (root-mean-squared; rms) length of a particular polymer preparation may be determined empirically, as in previous studies which have determined the rms lengths of several specific PEGs (12) of known molecular weights, which, like other flexible polymers, increases with the square root of the number of monomeric units (13). This information then allows estimation of the rms length of various preparations of the same polymer, based on the determination of the average molecular weight of the polymer preparation.
  • Another aspect of the invention is a method of obtaining a compound invention which activates or antagonizes activation of a receptor protein comprising multiple binding sites for a ligand L, said compound having a higher specific activity than said ligand L for activation of said receptor protein, said method comprising: (a) providing a group of compounds of the invention, each compound in the group having each of the ligand moieties L 1 to L x comprising ligand L or a derivative thereof.
  • Each compound in said group differs in the average (rms) length of the portion of the compound (that is, the flexible tether portion) linking any two of said ligand moieties L 1 to L ⁇ the lengths of these portions in the molecules being selected to span the range of possible distances between any two of the multiple binding sites on the receptor protein.
  • the method further comprises (b) adding a fixed amount of each compound in the group separately to the receptor protein, under conditions where the ligand L activates or antagonizes activation of the receptor protein, (c) determining the level of activation or antagonization of activation of the receptor protein produced by that fixed amount of each compound in that group, (d) comparing the level of activation or antagonization of activation of the receptor protein produced by the fixed amount of each compound in the group to the level of activation or antagonization of activation obtained by adding the same fixed amount of the ligand L to the receptor under the same conditions, and (e) selecting the compound in the group producing the highest level of activation or antagonization of activation of the receptor protein that is greater than the level of activation or antagonization of activation of the receptor by the ligand L.
  • Yet another aspect of the invention relates to a method of determining whether a receptor protein activated or antagonized by a ligand L comprises multiple binding sites for the ligand L such that binding of the ligand L to more than one of the multiple binding sites activates or antagonizes activation of the receptor protein to a higher level than does occupation of only one of those multiple binding sites.
  • This method comprises: (a) providing a group of compounds of the invention, each compound in the group having each of the ligand moieties L 1 to L x comprising the ligand L or a derivative thereof, and each compound in the group differing in the average (rms) length of the portion of the compound linking any two of the ligand moieties L 1 to L ⁇
  • the lengths of these portions in the molecules are selected to span the expected range of possible distances between any two of the multiple binding sites on the receptor protein.
  • This method further comprises: (b) adding a fixed amount of each compound in the group separately to the receptor protein, under conditions where the ligand L activates or antagonizes activation of the receptor protein, and (c) determining whether any compound in the group produces a higher level of activation or antagonization of activation of said receptor protein than produced by adding the same fixed amount of said ligand L to said receptor under the same conditions.
  • the receptor protein activated by ligand L comprises multiple binding sites for that ligand L such that binding of said ligand L to more than one of the multiple binding sites activates said receptor protein to a higher level than does occupation of only one of the multiple binding sites. More particularly, as described below, when the rms length of the tether between two ligand moieties comprising ligand L is substantially less than the distance between two binding sites for ligand L on the receptor protein, a dimeric ligand of the invention is expected to exhibit an apparent affinity for the receptor about twice that of the monomeric ligand L alone.
  • the apparent affinity of the dimeric ligand will substantially increase, reaching a maximum level when the rms length is approximately equal to the distance between the two binding sites on the receptor which bind ligand- L.
  • Longer tethers beyond that optimum length approximately equal to the distance between the two binding sites for the ligand L on the receptor results in reduced apparent affinity of the dimeric ligand of the invention.
  • the rms length of the flexible tether of the dimeric ligand of the invention can be optimized so as to selectively bind to a receptor having a particular distance between two binding sites for ligand L.
  • the rms lengths of tethers connecting more than two ligand moieties can be optimized for binding to a protein having more than two binding sites for the same ligand moieties, by a similar optimization of rms tether lengths between a pair of ligand moieties whereby multiple peaks of high affinity are expected at rms tether lengths approximately equal to each distance separating any pair of binding sites on the receptor.
  • the compounds of the invention are useful, for instance, for studying the structure of receptors with multiple ligand binding sites, and for therapeutic modulation of receptor-mediated activities.
  • cyclic nucleotide dependent kinases have been implicated in a wide variety of functions including plasticity of growth and neuronal connections, skeletal muscle contraction, regulation of cardiac rate and output, kidney and liver function, secretion from endocrine and exocrine glands, and many more bodily prociesses.
  • Cyclic AMP-dependent protein kinases have more than 200 known phosphorylation targets in cells. Despite the widespread importance of these molecules in cell signalling processes, selective ligands for these proteins have been lacking.
  • cAMP signaling by targeting membrane receptors, such as the beta- adrenergic receptor (targeted by propanolol), involved in cyclic nucleotide-signaling cascades in a variety to cell types, or enzymes involved in cyclic nucleotide metabolism, such as phosphodiesterase (inhibited, for instance, by theophylline).
  • membrane receptors such as the beta- adrenergic receptor (targeted by propanolol)
  • enzymes involved in cyclic nucleotide metabolism such as phosphodiesterase (inhibited, for instance, by theophylline).
  • phosphodiesterase enzymes involved in cyclic nucleotide metabolism
  • drugs target cGMP production important for regulating vasodilation and kidney function, such as nitroglycerin and sodium nitroprusside.
  • the present cyclic nucleotide multimeric tethered ligands may be used to directly and specifically target either the cyclic nucleotide-dependent kinases or the CNG channels for pharmacological intervention, rather than targeting upstream components of the signaling cascade, thereby providing drugs which may act at lower doses, more effectively, and with fewer side effects.
  • Figure 1 illustrates a preferred compound of the invention, a polymer-linked ligand dimer (PLD).
  • Panel A Structure of a PLD containing 2 cGMP moieties and a polyethylene glycol (PEG) linker, with n ethylene glycol units.
  • Panel B Schematic diagrams of PLDs binding to a channel with 4 ligand binding sites. Illustrated are PLDs with polymers that have an average length that is too short (left), just right (center), or longer than necessary (right), to allow the ligands to span two binding sites on the channel.
  • C eff the effective concentration of the tethered unbound ligand
  • Figure 2 depicts activation of CNG channels by PLDs and monomeric cyclic nucleotides. Dose-response curves of activation of OLF (Panel A) and RET (Panel B) CNG channels by cGMP, 3 different PLDs, and the PLM. Responses from each patch were normalized to the response elicited by 2 mM cGMP.
  • Figure 4 illustrates the identification of optimal PLDs for cGMP-binding proteins according to the method of the invention.
  • Panel A Activation of CNG channels (RET and OLF).
  • Panel B Activation of PKG. Arrows indicate PLDs optimal for activating each protein. Polymer lengths were estimated from previous determinations (12), assuming an increase with the square root of MW (13).
  • the invention relates to multimeric tethered ligands comprising a plurality of ligand moieties tethered to a joint moiety, wherein each ligand moiety comprises a ligand for at least one receptor protein having multiple binding sites for said ligand and each ligand moiety is linked by a tether moiety to that joint moiety.
  • This compound has a structure described by the general formula:
  • a leading approach to drug design involves determining the structure of binding sites on proteins, providing a template for constructing new ligands.
  • combinatorial chemistry utilizes random combinations of chemical groups to generate diverse molecules, which are screened to select effective species.
  • the present inventors devised a third strategy combining elements of these approaches using PLDs in which two ligands are linked with a variable length polymer chain. This strategy can be used to develop extraordinarily potent ligands for proteins with multiple binding sites.
  • Exemplary PLDs containing two guanosine 3', 5' cyclic monophosphate (cGMP) moieties are described below.
  • PLDs are up to 1 ,000-fold more potent than cGMP in activating cyclic nucleotide-gated (CNG) channels and protein kinases.
  • CNG cyclic nucleotide-gated
  • Each protein responds optimally to a PLD with a different average polymer length, indicating that each has a unique spacing of binding sites. Since optimal ligands are selected empirically, the multivalent tethered ligand strategy allows identification of highly potent and specific agents with no prior structural information about target proteins.
  • PLDs The multimeric tethered ligand strategy for developing selective and extraordinarily high affinity ligands for proteins that possess multiple ligand binding sites, such as cyclic nucleotide-binding proteins, is illustrated here using PLDs.
  • PLDs were synthesized by reacting a sulfhydryl derivative of cGMP (9) with a bifunctional vinylsulfone-derivatized PEG (10).
  • Alternative flexible polymers and chemistries for bifunctional coupling of such polymers with various ligands are known in the art.
  • the present reaction produces a "barbell-shaped" molecule that contains two cGMPs connected to both ends of a flexible polymer via a thioether linkage at the 8-position of the purine (see Fig.
  • FIG. 1B schematically illustrates how changing the polymer length of a PLD affect its ability to bind to a protein with multiple ligand binding sites, using the interaction between a CNG channel and 3 distinct PLDs.
  • Each of the four subunits that comprise the CNG channel contains a cyclic nucleotide binding site that is accessible to the cytoplasmic solution.
  • the apparent affinity of the PLD should be about twice that of cGMP itself. This assumes that the polymer has no effect on the ability of the cGMP moieties either to bind, or to activate the channel.
  • the rms length of the PLD exactly matches the distance between the binding sites on the channel, a dramatic increase in the apparent affinity is predicted.
  • the Hill coefficient indicative of the minimum number of individual molecules required for significant activation, was similar for cGMP, the PLM, and 282 PEG-(cGMP) 2 (1.7, 2.0, and 2.0, respectively).
  • a larger PLD, 3400 PEG-(cGMP) 2 had a dramatic effect on the OLF channels.
  • the estimated K ⁇ /2 for this PLD was 12 nM, 260-fold lower than exhibited by cGMP itself.
  • CNG channels all appear to have four nucleotide binding sites, the molecular dimensions may differ, including the distance between binding sites.
  • a series of PLDs was used, containing PEG polymers with molecular weights from 282 to 20,000, corresponding to rms lengths of 15 - 123 A (12, 13, 18) .
  • Figure 4A shows that the PLD optimal for activating the RET CNG channels is shorter than the PLD optimal for activating OLF channels.
  • CNG channels and acetylcholine receptors are similar in molecular weight, the closer spacing of cyclic nucleotide binding sites on CNG channels may indicate that sites on these channels are not located on the perimeter of the protein, but perhaps centered on the cytoplasmic surface of each subunit, as illustrated in Figure IB.
  • PKG is a homodimer, and each subunit has a regulatory (R) domain that contains two heterologous binding sites: one with high and one with low affinity for cyclic nucleotides.
  • R domain of PKG has extensive sequence homology with the R subunit of PKA (7), where structural determinations using X-ray crystallography indicate that the two sites are separated by 26 A (22).
  • the two heterologous sites within a PKG monomer are also likely to be about 26 A apart, while the distance between homologous cGMP-binding sites across two subunits is not known.
  • the present invention also provides PLDs that contain cyclic nucleotides other than cGMP, including derivatives such as Rp-cAMPS and Rp-cGMPS, that act as antagonists for specific cyclic nucleotide-binding proteins.
  • PLDs that contain cyclic nucleotides other than cGMP, including derivatives such as Rp-cAMPS and Rp-cGMPS, that act as antagonists for specific cyclic nucleotide-binding proteins.
  • PLDs appear to be membrane-permeant, as determined in experiments in which extracellular application of 2,000 PEG-(cGMP) 2 results in activation of CNG channels in intact cells (data not shown).
  • specific PLDs may be useful for distinguishing whether physiological responses involving cGMP are mediated either by CNG channels or by PKG.
  • the multivalent tethered ligand approach has several remarkable features.
  • Homotetrameric channels composed solely of a-subunits can also be functionally expressed in Xenopus oocytes, and also contain 4 binding sites [D.T. Liu, G.R. Tibbs, S.A. Siegelbaum, Neuron 16, 983, (1996)].
  • PLDs were synthesized by reacting 8-thio cGMP (9) with bifunctional PEG vinylsulfone (VS-PEG-VS) (Shearwater Polymers, Inc, Huntsville, AB) at 37°C and pH 7.5 for 24-48 hours. PLDs were purified by reverse phase HPLC using a preparative C18 column eluted with a methanol gradient. Reactions were run either with an excess of 8- thio cGMP, to bias formation of PLDs (cGMP-PEG-cGMP), or with an excess of VS- PEG-VS, to bias formation of half-reacted monomers (cGMP-PEG-VS).
  • VS-PEG-VS bifunctional PEG vinylsulfone
  • HPLC analysis showed that PLDs synthesized from a 1,000 MW PEG-(VS)2 contained a range of molecules with 15-33 ethylene glycol units, with a median at 24 units. HPLC fractions were collected to purify less polydispersed products with MW of 800 ⁇ 132 (15-21 units) and 1,200 ⁇ 132 (24-30 units). Similar degrees of polydispersity were exhibited by higher MW PLDs (2,000 - 20,000 MW). The 282 MW PLD exhibited no polydispersity because it was synthesized from a discrete derivatized PEG (6 ethylene glycol units). The two VS couplings added to the PEGs are likely to add a few A to the rms length of all the PLDs.
  • Bovine recombinant PKG type 1 a was obtained from Calbiochem.
  • the PLM was a partial agonist. At saturation, it activated about 30% of the PKG activity elicited by saturating cGMP.

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Abstract

L'invention concerne des ligands multimères attachés, comprenant une pluralité de fractions de ligand attachées à une fraction commune. Chaque fraction de ligand comprend un ligand pour au moins une protéine récepteur dotée de plusieurs sites de liaison correspondant au ligand et, par ailleurs, chaque fraction de ligand est reliée à la fraction commune par une fraction d'attache. L'invention concerne un certain nombre d'exemples de ligands dimères attachés, où l'attache est un polymère simple, à savoir polyéthylène glycol, moyennant quoi on parle pour ce type de ligand de 'dimères liés par polymère'. Par exemple, on décrit des dimères de ce type contenant deux fractions guanosine -3'- 5'-monophosphate cyclique (GMPc), lesquelles sont jusqu'à 1 000 fois plus puissantes que le GMPc dans l'activation des protéines kinases et des canaux contrôlés par nucléotides cycliques. Les différentes protéines réagissent de manière optimale à différents 'dimères liés par polymère' qui n'ont pas la même longueur moyenne de polymère, chaque protéine ayant donc un espacement unique de sites de liaison. Puisque la sélection des ligands optimaux est empirique, l'utilisation de ligands attachés multivalents permet d'identifier des agents très puissants et spécifiques sans disposer préalablement d'informations structurelles sur les protéines cibles.
PCT/US1998/024467 1997-11-17 1998-11-16 Ligands multimeres attaches et leur utilisation dans les interactions recepteur-ligand WO1999025384A2 (fr)

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WO2006029234A1 (fr) * 2004-09-03 2006-03-16 Receptors Llc Recepteurs artificiels combinatoires comprenant des synthons d'attache
US7166463B2 (en) 2001-11-16 2007-01-23 The Regents Of The University Of Colorado Nucleic acids encoding modified olfactory cyclic nucleotide gated ion channels
US7469076B2 (en) 2003-09-03 2008-12-23 Receptors Llc Sensors employing combinatorial artificial receptors
US7504364B2 (en) 2002-03-01 2009-03-17 Receptors Llc Methods of making arrays and artificial receptors
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KR100361933B1 (ko) 1993-09-08 2003-02-14 라 졸라 파마슈티칼 컴파니 화학적으로정의된비중합성결합가플랫폼분자및그것의콘주게이트
AU2001268228A1 (en) 2000-06-08 2001-12-17 La Jolla Pharmaceutical Company Multivalent platform molecules comprising high molecular weight polyethylene oxide

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WO2002056016A3 (fr) * 2001-01-11 2003-02-06 Theravance Inc Procede d'application d'un ligand pour un substrat biologique
US6656694B2 (en) 2001-01-11 2003-12-02 Theravance, Inc. Method for identifying a ligand for a biological substrate
WO2002056016A2 (fr) * 2001-01-11 2002-07-18 Theravance, Inc. Procede d'application d'un ligand pour un substrat biologique
US7166463B2 (en) 2001-11-16 2007-01-23 The Regents Of The University Of Colorado Nucleic acids encoding modified olfactory cyclic nucleotide gated ion channels
US7341836B2 (en) 2001-11-16 2008-03-11 The Regents Of The University Of Colorado Modified cyclic nucleotide gated ion channels
US7964535B2 (en) 2002-03-01 2011-06-21 Receptors Llc Arrays and artificial receptors
US7504364B2 (en) 2002-03-01 2009-03-17 Receptors Llc Methods of making arrays and artificial receptors
US7469076B2 (en) 2003-09-03 2008-12-23 Receptors Llc Sensors employing combinatorial artificial receptors
WO2006028930A2 (fr) * 2004-09-03 2006-03-16 Receptors Llc Recepteurs artificiels combinatoires contenant des elements constitutifs de liaison sur des squelettes
US7504365B2 (en) 2004-09-03 2009-03-17 Receptors Llc Combinatorial artificial receptors including tether building blocks
WO2006028930A3 (fr) * 2004-09-03 2006-05-26 Receptors Llc Recepteurs artificiels combinatoires contenant des elements constitutifs de liaison sur des squelettes
WO2006029234A1 (fr) * 2004-09-03 2006-03-16 Receptors Llc Recepteurs artificiels combinatoires comprenant des synthons d'attache
WO2018010965A1 (fr) 2016-07-11 2018-01-18 Biolog Life Science Institute Forschungslabor Und Biochemica-Vertrieb Gmbh Nouveaux multimères lies a un polymère modifié équatoriellement des monophosphates de guanosine-3', 5'-cyclique
JP2019529523A (ja) * 2016-07-11 2019-10-17 ミレカ メディシンズ ゲゼルシャフト ミット ベシュレンクテル ハフツング ３’，５’−環状グアノシン一リン酸の新規エクアトリアル修飾ポリマー連結マルチマー
US11407781B2 (en) 2016-07-11 2022-08-09 Graybug Vision, Inc. Equatorially modified polymer linked multimers of guanosine-3′, 5′-cyclic monophosphates
JP2022140440A (ja) * 2016-07-11 2022-09-26 グレイバグ ヴィジョン インコーポレイテッド ３’，５’－環状グアノシン一リン酸の新規エクアトリアル修飾ポリマー連結マルチマー
WO2018041942A1 (fr) 2016-08-31 2018-03-08 Biolog Life Science Institute Forschungslabor Und Biochemica-Vertrieb Gmbh Nouveaux multimères liés au polymère monophosphate de guanosine-3', 5'-cycliques

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