WO2003003009A1 - Utilisation de chelates a ions metalliques dans la validation de molecules biologiques utilisees comme cibles medicamenteuses dans des modeles animaux experimentaux - Google Patents

Utilisation de chelates a ions metalliques dans la validation de molecules biologiques utilisees comme cibles medicamenteuses dans des modeles animaux experimentaux Download PDF

Info

Publication number
WO2003003009A1
WO2003003009A1 PCT/DK2002/000456 DK0200456W WO03003009A1 WO 2003003009 A1 WO2003003009 A1 WO 2003003009A1 DK 0200456 W DK0200456 W DK 0200456W WO 03003009 A1 WO03003009 A1 WO 03003009A1
Authority
WO
WIPO (PCT)
Prior art keywords
vii
metal
ion
lll
iii
Prior art date
Application number
PCT/DK2002/000456
Other languages
English (en)
Inventor
Øystein RIST
Thomas Högberg
Birgitte Holst Lange
Thue W. Schwartz
Christian E. Elling
Original Assignee
7Tm Pharma A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/DK2001/000867 external-priority patent/WO2002054077A2/fr
Application filed by 7Tm Pharma A/S filed Critical 7Tm Pharma A/S
Publication of WO2003003009A1 publication Critical patent/WO2003003009A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure

Definitions

  • the present invention relates to the use of chemical compounds or selections of chemical compounds (libraries) for in vivo methods for testing or validating the physiological importance and/or the therapeutic or pharmacological potential of biological target molecules, notably proteins such as, e.g., receptors and especially 7TM receptors in test animals expressing the biological target molecule with, notably, a silent, engineered metal-ion site.
  • biological target molecules notably proteins such as, e.g., receptors and especially 7TM receptors in test animals expressing the biological target molecule with, notably, a silent, engineered metal-ion site.
  • the present invention also relates to the use of specific metal-ion binding sites of a generic nature in specific biological target molecules such as, e.g. transmembrane proteins wherein the metal-ion binding site is capable of forming a complex with a metal ion.
  • a test animal suitable for use in the present invention is normally a genetically modified animal. At any given time during the development of the test animal or in adult life, it is then possible to turn the biological target molecule (such as, e .g., the receptor) on or off - depending on the engineered site with a pharmacological tool, i.e. a metal-ion chelate formed between a metal-ion and a metal-ion chelator.
  • a pharmacological tool i.e. a metal-ion chelate formed between a metal-ion and a metal-ion chelator.
  • the thus developed pharmacologically controlled "knock-out" methods are useful in the evaluation of biological target molecules such as, e.g., proteins as drug targets as well as in the characterization of the physiological role of orphan receptors.
  • the present invention also relates to chemical compounds or libraries suitable for use in methods for improving the in vivo pharmacokinetic behaviour of metal-ion chelates (e.g. the absorption pattern, the plasma half-life, the distribution, the metabolism and/or the elimination of the metal-ion chelates).
  • metal-ion chelates e.g. the absorption pattern, the plasma half-life, the distribution, the metabolism and/or the elimination of the metal-ion chelates.
  • it is advantageous e.g. to increase the time period during which the metal-ion chelate is in the circulatory system and/or localised at the target.
  • the invention relates to metal-ion chelating compounds, which are designed to be suitable for use in a target validation process according to the invention and to libraries of at least two or more of such metal-ion chelating compounds.
  • the drug targets known today are only a small fraction of the huge number of potential drug targets that are currently becoming available through the characterization of the humane genome. Even for subtypes of well-known receptors (e.g. monoamine receptor and neuropeptide receptor subtypes) we do not know the physiological function and/or the pharmacological potential thereof. Accordingly, it is a difficult task to evaluate new drug targets since our knowledge of their physiological role normally is very limited.
  • well-known receptors e.g. monoamine receptor and neuropeptide receptor subtypes
  • the subject of the present invention is methods for target validation, i.e. for validating the function of a specific target or for validating the effect of specific substances acting on that target.
  • the present invention is also directed to how to design and develop chemical compounds suitable for use in such methods.
  • One technology which has been employed for evaluating drug targets in general, has been various forms of gene knock-out methods. Using such methods it is possible to specifically delete the gene for a particular biological target molecule from the genome of a test animal - usually a mouse. It is also possible by transgenic techniques to over- express a particular receptor gene in a tissue specific manner. From the phenotype of such animals the usefulness of a particular receptor as a drug target is then evaluated. In many cases the phenotype of a receptor knock-out animal corresponds to what would be expected based on the effect of known drugs, acting through the receptors in question. However, the method is not always working as expected. For example, deletion of certain genes causes unforeseen problems in being embryonic lethal or inducing developmental malformations.
  • the transgenic animals develop compensatory mechanisms, which impair the interpretation of the phenotype.
  • the deletion of one gene may lead to the up-regulation of other redundant regulatory systems that masks the effect of the gene knockout, i.e. the suspected effect of a putative or antagonistic drug in the animal model is masked.
  • Conditional pharmacological knock-out is a recently developed technique by which the expression is turned off in an inducible manner, both in respect of tissue and time.
  • the crucial difference from the classical knockout method is that two different genetic modifications have to be introduced, which accordingly require development of two different transgenic animals.
  • One of the genetic modifications is introduction of an artificial gene fragment, which codes for a recombinase enzyme.
  • the promoter of this gene element obtains cell type specificity. Whereas the time dependence is achieved by use of an inducible promoter, that require an inducer (e.g. tetracycline) in order to become active.
  • the other genetic modification includes introduction of a silent gene fragment, e.g. loxP, upstream of the relevant gene, which addresses the recombinase enzyme very specifically.
  • the recombination event destroys the adjacent gene and turn off the expression.
  • the main disadvantages are:
  • inhibition of the gene product is not a direct consequence of the administration of the inducer, as is seen with administration of ordinary drug that target the protein directly. In contrast it takes a while - often several days - from administration of the inducer to the inhibition of the protein expression of the protein level is obtained.
  • transgenic animals have to be developed, one expressing the transactivator gene and the other expressing the silent gene upstream of the relevant gene.
  • the antisense technique is based on a specific base-pair interaction between the target gene, frequently at messenger RNA level, and the antisense probe. This hybrid is subsequently degraded by endogenous double strand specific ribonucleases, hence, no translation or protein expression will occur. It is also possible to address the chromatin DNA with the antisense probe forming triple helices and thereby stop the transcription. In vivo the gene is administered either by transgenic expression of the gene under an inverted promoter, by viruses or by direct injection of the nucleotide fragment. Often recognised disadvantages of the antisense techniques include:
  • ZFPs zinc finger DNA-binding proteins
  • the ZFP's have two different domains: a DNA binding domain and a functional domain, the later may have either activating or repressing abilities.
  • substitution of amino acids in the DNA binding domain it is possible to generate ZFP that recognize and bind to specific DNA sequences, and thereby turn off or on the adjacent gene (Rebar, E. J. & Pabo, C. O (1994); Science 263:671).
  • the present invention aims at fulfilling this need.
  • the present invention provides a novel target validation method using test animals, which express biological target molecules such as, e.g., proteins like receptors with engineered metal-ion sites.
  • biological target molecules such as, e.g., proteins like receptors with engineered metal-ion sites.
  • metal-ion sites may be constructed as silent "switches", i.e. in a way that allows the natural ligand to bind normally in the absence of the metal ion.
  • the activity of the biological target molecule is inhibited or stimulated. If the wild-type protein in an animal is replaced with a protein holding a silent metal ion switch, the animal should develop normally and no compensatory mechanisms would be up-regulated in the absence of the metal ion.
  • a metal ion e.g. in the form of a metal-ion chelate is administrated to the test animal the switch is turned on or off and the physiological and/or pharmacological impact on the animal can be monitored.
  • the present invention relates to chemical compounds for use in a target validation process for testing or validation the physiological importance and/or the therapeutic potential of a biological target molecule.
  • test compound in vitro testing of a test compound for its ability to bind to the introduced silent metal ion site in the silent metal ion engineered biological target molecule or for any change in the activity of the biological target molecule
  • iii) optionally, chemically optimising the test compound and/or the biological target molecule to create secondary interaction(s), especially by forming covalent or slowly reversible bonds with -SH, OH or NH 2 groups in the vicinity of the metal ion site in the silent metal ion engineered biological target molecule or by forming ionic interactions with charged groups such as Asp, Glu, Lys, and Arg.
  • secondary interaction residues present in e.g. Cys, Ser, Thr, Tyr, His, Asp, Glu, Lys, and Arg can be introduced by engineering into the biological target in the vicinity of the silent metal ion binding site,
  • test compound optionally, chemically optimising the test compound to improve the pharmacokinetic and/or biopharmaceutical properties of the test compound, vi) preparing a genetically modified test animal containing the silent metal ion site engineered biological target molecule,
  • the present invention also relates to the use of specific metal-ion sites in specific biological target molecules in a target validation process
  • the invention relates to the use of test compounds of formula I, to libraries comprising such compounds and to the use of such libraries in a target validation process.
  • the present invention relates to the use of chemical compounds for use in methods for in vivo target validation, i.e. methods to determine the effect of a specific agonist or antagonist for a specific biological target molecule.
  • metal-ion chelates are used Elling et al. (PNAS 96, 1999, pp. 12322-12327) and Hoist et al. (Mol. Pharmacol. 2000, 58: 263-270) have indicated a use of metal-ion chelators for such purposes but considered the affinity of the metal ion sites to be too low.
  • the present invention provides means for establishing a suitable affinity and a suitable pharmacokinetic behaviour.
  • the present invention is based on a technology which makes it possible to genetically modify animals in such a manner that they express a silent metal-ion site in a potential drug target, i.e. a metal-ion site is created in which the mutations do not significantly affect the binding and action of the endogenous ligand for the biological target molecule such as, e.g., a receptor.
  • a metal-ion site engineered biological target molecules e.g. a receptor
  • classical gene-replacement technology i.e.
  • the animals will develop normally without compensatory mechanisms, which otherwise frequently impair the interpretation of the phenotypes of the animals in classical gene knock-out technology.
  • an appropriate metal-ion chelating agent which then will act as an antagonist (or agonist) and turn off (or on) the function of the metal-ion site engineered biological target molecules such as, e.g. a receptor.
  • the technology of the present invention it is possible to increase the affinity of metal-ion chelates significantly and make them more stable, which will make it considerably more easy to reach therapeutic and efficient concentrations of the metal-ion chelates in the animals and also to increase the "therapeutic window" due to the higher degree of selectivity of the small organic molecule ligands (i.e. the metal ion chelates which also are denoted test compounds) caused by the establishment of more than one molecular interaction point.
  • Establishment of just a single or a few additional secondary chemical interaction(s), e.g. a charge-charge interaction will increase the affinity and selectivity of the metal-ion chelate significantly and thereby making the whole process usable.
  • a specific covalent bond is introduced between the metal-ion chelate and a residue in the biological target molecule.
  • a target validation process is performed by establishing an interaction between a silent metal ion site in a biological target molecule and a chelate.
  • the chelate suitable for use according to the present invention may be a test compound i.e. a small organic compound itself or it may be a transport form or a depot form of the test compound provided that the function of the test compound is not significantly impaired, i.e. it is able to interact with the silent metal ion site and possible secondary interaction site(s) on the biological target molecule.
  • the test compound itself may be very effective in establishing an interaction between the target molecule and itself, an improvement of its pharmacokinetic properties or its biopharmaceutical properties may be required in order to e.g. make sure that the test compound reaches the site of action or is maintained in the circulatory system long enough to give rise to a physiological effect.
  • a target validation process employing chemical compounds or libraries according to the invention includes the following main steps: 1. a process in which a silent metal-ion site is introduced into a biological target molecule
  • test compound in vitro test in which a test compound is tested for its ability to bind to the engineered metal ion site in the biological target molecule 3.
  • a chemical optimisation step in which the test compound and/or the biological target molecule is modified in such a manner that secondary interactions with chemical groups in the vicinity of the metal ion site in the biological target molecule are created 4.
  • step(s) 2 (and 3) repetition of step(s) 2 (and 3) until a suitable binding affinity is obtained, 5.
  • chemical optimisation of the test compound in order to improve the pharmacokinetic and/or biopharmaceutical properties of the test compound,
  • step 3 mentioned above is necessary in the target validation process according to the invention.
  • step 3 may be irrelevant and can thus be excluded.
  • a "chemical compound” or a “test compound” is intended to indicate a small organic molecule ligand or a small organic compound, which is capable of interacting with a biological target molecule, in particular with a protein, in such a way as to modify the biological activity thereof.
  • the term includes in its meaning metal ion chelates of the formulas shown below.
  • the term includes in its meaning metal ion chelates of the formulas shown below as well as chemical derivatives thereof constructed to interact with other part(s) of the biological target molecule than the metal ion binding site.
  • a test compound may also be an organic compound, which in its structure includes a metal atom via a covalent binding.
  • a "metal ion chelator” is intended to indicate a compound capable of forming a complex with a metal atom or ion, and contains at least two interactions between the metal centre and the chelator. Such a compound will generally contain two heteroatoms such as N, O, S, Se or P with which the metal atom or ion is capable of forming a complex.
  • a "metal ion chelate” is intended to indicate a complex of a metal ion chelator and a metal atom or ion.
  • metal ion is intended to indicate a charged or neutral element. Such elements belong to the groups denoted main group metals, light metals, transition metals, semi-metals or lanthanides (according to the periodic system).
  • metal ion includes in its meaning metal atoms as well as metal ions.
  • a "library” is intended to indicate a collection of chemical compounds having a common basic structural element.
  • the number of compounds in a library is three or more. All the chemical compounds contained in a library according to the invention have the same common basic structural element or scaffold.
  • the number of compounds in a library is generally in a range from about 3 to about 250 such as, e.g. from about 3 to about 100, from about 3 to about 50, from about 3 to about 30, from about 3 to about 25, from about 3 to about 20, from about 3 to about 15 or from about 3 to about 10 such as at least 3, 4, 5, 6, 7, 8, 9 or 10 compounds. More generally the number of compounds in a library is in a range of from about 3 to about 10,000 compounds such as, e.g.
  • libraries based on focused structures contain from about 3 to about 500 compounds such as, e.g. from about 3 to about 100 compound, whereas chemical diverse randomized libraries contain from about 500 to about 10,000 compounds such as, e.g. 750 to about 10,000 compounds, from about 1 ,000 to about 10,000 compounds.
  • a "metal-ion binding site" is intended to indicate a part of a biological target molecule, which comprises an atom or atoms capable of complexing with a metal atom or ion. Such an atom will typically be a heteroatom, in particular N, O, S, Se or P. With respect to proteins a metal-ion binding site is typically an amino acid residue of the protein, which comprises an atom capable of forming a complex with a metal ion. These amino acid residues are typically, but not restricted to, histidine, cysteine, glutamate and aspartate.
  • a “receptor-ligand” is intended to include any substance that either inhibits or stimulates the activity of a biological target molecule such as, e.g. a protein or that competes for a receptor in a binding assay.
  • An "agonist” is defined as a ligand increasing the functional activity of a biological target molecule (e.g. signal transduction through a receptor).
  • An "antagonist” is defined as a ligand decreasing the functional activity of a biological target molecule either by inhibiting the action of an agonist or by its own intrinsic activity.
  • An "inverse agonist” (also termed “negative antagonist”) is defined as a receptor-ligand decreasing the basal functional activity of a biological target molecule.
  • a “biological target molecule” is intended to include proteins such as, e.g., membrane proteins, nucleic acids, carbohydrates, nucleoproteins, glycoproteins and glycolipids.
  • proteins such as, e.g., membrane proteins, nucleic acids, carbohydrates, nucleoproteins, glycoproteins and glycolipids.
  • the biological target molecule normally has been manipulated to contain a metal-ion binding site. However, in some cases, the biological target molecule may be in its wild-type form.
  • a "protein” is intended to include any protein, polypeptide or oligopeptide with a discernible biological activity in any unicellular or multicellular organism, including bacteria, fungi, plants, insects, animals or mammals, including humans.
  • the protein may suitably be a drug target, i.e. any protein which activity is important for the development or amelioration of a disease state, or any protein which level of activity may be altered (i.e. up- or down-regulated) due to the influence of a biologically active substance such as a small organic chemical compound.
  • a "membrane protein” is intended to include but is not limited to any protein anchored in a cell membrane and mediating cellular signalling from the cell exterior to the cell interior.
  • Important classes of membrane proteins include receptors such as tyrosine kinase receptors, G-protein coupled receptors, adhesion molecules, ligand- or voltage-gated ion channels, or enzymes.
  • the term is intended to include membrane proteins whose function is not known, such as orphan receptors. In recent years, largely as part of the human genome project, large numbers of receptor-like proteins have been cloned and sequenced, but their function is as yet not known.
  • the present invention may be of use in elucidating the function of the presumed receptor proteins by making it possible to develop methods of identifying ligand for orphan receptors based on compounds developed from metal ion chelates that bind to mutated orphan receptors into which artificial metal ion binding sites have been introduced.
  • Signal transduction is defined as the process by which extracellular information is communicated to a cell by a pathway initiated by binding of a ligand to a membrane protein, leading to a series of conformational changes resulting in a physiological change in the cell in the form of a cellular signal.
  • a “functional group” is intended to indicate any chemical entity which is a component part of the test compound and which is capable of interacting with an amino acid residue or a side chain of an amino acid residue of the biological target molecule such as, e.g. a membrane protein.
  • a functional group is also intended to indicate any chemical entity which is a component part of the biological target molecule and which is capable of interacting with other parts of the biological target molecule or with a part of the test compound. Examples of such functional groups include, but are not limited to, ionic groups involved in ionic interactions such as e.g.
  • ammonium ion or carboxylate ion hydrogen bond donor or acceptor groups such as amino, amide, carboxy, sulphonate, etc.
  • hydrophobic groups involved in hydrophobic interactions, pi-stacking, ion-dipole interactions, dipole-dipole interactions, edge-on aromatic interactions, dispersion and induction forces, metal complex interactions and the like.
  • a "wild-type" membrane protein is understood to be a membrane protein in its native, non-mutated form, in this case not comprising an introduced metal-ion binding site or residues introduced for secondary interactions.
  • the term "in the vicinity of” is intended to include an amino acid residue or any other residue or functional group located in the area defining the binding site of the metal ion chelate and at such a distance from the metal ion binding amino acid residue that it is possible, by attaching suitable functional groups to the test compound, to generate an interaction between said functional group or groups and said amino acid residue, another residue or functional group.
  • a "silent metal ion site” ⁇ s intended to mean a metal-ion site engineered into a biological target molecule in such a manner, that it does not affect or significantly does not affect the function of the biological target molecule, i.e. in the case of a receptor, for example the binding and function of the endogenous ligand.
  • a silent metal ion site may be evaluated in an in vitro model employing transiently transfected cultured cell systems measuring, for example binding affinities and functional responses.
  • a metal ion site is considered to be a silent metal ion site if it does not change the structure and function of the biological target molecule at all or only change its structure and function to a limited extent.
  • a metal-ion site will be considered to be silent if no or only limited change in the cellular surface expression of the receptor occurs and/or if no or only limited change in binding affinity of the endogenous ligand - being a hormone, a transmitter or another chemical messenger - is observed and no or only limited change is observed in the ability of the endogenous ligand to stimulate signal transduction through the receptor.
  • transfection of tissue culture cells with expression plasmids ensuring a suitable, controllable gene expression in a cell type where the biological target molecule is expressed in a cellular context being as close as possible to the cellular contexts in which the biological target molecule normally is expressed in vivo.
  • a “genetically modified animal” means an animal in which a chromosome or a part thereof has been modified so that a specific gene or gene sequence has been deleted, exchanged with another or a further gene or gene sequence has been inserted.
  • a genetically modified animal specifically refers to an animal in which a gene coding for a biological target molecule has been introduced in a modified form which makes it suitable for target validation though treatment of the animal with a receptor- ligand, which specifically affects the function of the modified gene product. It is also possible to introduce a gene modification in the test animal in its embryonic state. Preferably the gene is introduced into the animal by selectively replacing the gene coding for the endogenous biological target molecule, however this is not a requirement.
  • test animal is intended to embrace a genetically modified animal as well as a non- genetically modified animal.
  • Suitable test animals for use in methods according to the invention are mammals such as, e.g., rodents, mice, rats, rabbits, guinea pigs, monkeys, dogs, domestic animals such as, e.g. pigs, cows, horses racing animals such as, e.g., horses, dogs.
  • a “spacer” or a “linker” is intended to embrace bifunctional chemical substances which on the one hand are able to react or interact with the test compound and on the other hand with a carrier.
  • a “carrier” is intended to embrace compounds that provides the test compounds with specific properties, e.g. with respect to the physiological, pharmacokinetic and/or biopharmaceutic behaviour of the test compound.
  • the carrier is normally directly or indirectly linked to the test compound through establishment of a covalent or a non- covalent bound.
  • a spacer between the test compound and the carrier is normally included.
  • the biological target molecules include but are not restricted to proteins, nucleoproteins, glycoproteins, nucleic acids, carbohydrates, and glycolipids.
  • the biological target molecule normally has been genetically manipulated to contain a metal- ion binding site, but in some cases the biological target molecule may perse contain a metal-ion binding site. Irrespective of whether the metal-ion binding site has been artificially introduced or it is a natural site, the metal ion site is preferably silent.
  • the biological target molecule is a protein, which may be for example a membrane receptor, a protein involved in signal transduction, a scaffolding protein, a nuclear receptor, a steroid receptor, a transcription factor, an enzyme, and an allosteric regulator protein, or it may be a growth factor, a hormone, a neuropeptide or an immunoglobuiin.
  • Proteins as drug targets may be for example a membrane receptor, a protein involved in signal transduction, a scaffolding protein, a nuclear receptor, a steroid receptor, a transcription factor, an enzyme, and an allosteric regulator protein, or it may be a growth factor, a hormone, a neuropeptide or an immunoglobuiin. Proteins as drug targets
  • Most drug compounds act by binding to and altering the function of proteins.
  • These can be intracellular proteins such as, for example enzymes and transcription factors, or they can be extracellular proteins, for example enzymes, or they can be membrane proteins.
  • Membrane proteins constitute a numerous and varied group whose function is either structural, for example being involved in cell adhesion processes, or the membrane proteins are involved in intercellular communication and communication between the cell exterior and the interior by transducing chemical signals across cell membranes, or they facilitate or mediate transport of compounds across the lipid membrane.
  • Membrane proteins are for instance receptors and ion channels to which specific chemical messengers termed ligands bind resulting in the generation of a signal, which gives rise to a specific intracellular response (this process is known as signal transduction).
  • Membrane proteins can, for example also be enzymes, which are associated to the membrane for functional purposes, e.g. proximity to their substrates. Most membrane proteins are anchored in the cell membrane by a sequence of amino acid residues, which are predominantly hydrophobic to form hydrophobic interactions with the lipid bilayer of the cell membrane. Such membrane proteins are also known as integral membrane proteins. In most cases, the integral membrane proteins extend through the cell membrane into the interior of the cell, thus comprising an extracellular domain, one or more transmembrane domains and an intracellular domain.
  • GPCR G protein coupled receptors
  • the biological target molecule is a membrane protein, which suitably is an integral membrane protein, which is to say a membrane protein anchored in the cell membrane.
  • the membrane protein is preferably of a type comprising at least one transmembrane domain.
  • interesting membrane proteins for the present purpose are mainly found in classes comprising 1-14 transmembrane domains.
  • 1TM - membrane proteins of interest comprising one transmembrane domain include but are not restricted to receptors such as tyrosine kinase receptors, e.g. a growth factor receptor such as the growth hormone, insulin, epidermal growth factor, transforming growth factor, erythropoietin, colony-stimulating factor, platelet-derived growth factor receptor or nerve growth factor receptor (TrkA or TrkB).
  • receptors such as tyrosine kinase receptors, e.g. a growth factor receptor such as the growth hormone, insulin, epidermal growth factor, transforming growth factor, erythropoietin, colony-stimulating factor, platelet-derived growth factor receptor or nerve growth factor receptor (TrkA or TrkB).
  • 2TM - membrane proteins of interest comprising two transmembrane domains include but are not restricted to, e.g., purinergic ion channels.
  • 4, 5TM - membrane proteins of interest comprising 3, 4 or 5 transmembrane domains includes but are not restricted to e.g. ligand-gated ion channels, such as nicotinic acetylcholine receptors, GABA receptors, or glutamate receptors (NMDA or AMPA).
  • ligand-gated ion channels such as nicotinic acetylcholine receptors, GABA receptors, or glutamate receptors (NMDA or AMPA).
  • 6TM - membrane proteins of interest comprising 6 transmembrane domains include but are not restricted to e.g., voltage-gated ion channels, such as potassium, sodium, chloride or calcium channels.
  • the membrane protein of interest comprising a G-protein coupled receptor, such as the receptor for (- in brachet the receptor subtypes are mentioned): acetylcholine (m1-5), adenosine (A1-3) and other purines and purimidines (P2U and P2Y1-12), adrenalin and noradrenalin ( ⁇ 1A-D, ⁇ 2A-D and ⁇ 1-3), amylin, adrenomedullin, anaphylatoxin chemotactic factor, angiotensin (AT1A, -1B and -2), apelin, bombesin, bradykinin (land 2), C3a, C5a, calcitonin, calcitonin gene related peptide, CD97, conopressin, corticotropin releasing factor (CRFIand -2), calcium, cannabinoid (CBIand -2), chemokines (CCR1-11 , CXCR1-6, CX3CR and X
  • SUBST4TUTE SHEET (RULE 26) (US27, US28, UL33, UL78, ORF74, U12, U51); and 7TM proteins coded for in the human genome but for which no endogenous ligand has yet been assigned such as mas-proto- oncogene, EBI (I and II), lactrophilin, brain specific angiogenesis inhibitor (BAI1-3), EMR1 , RDC1 receptor, GPR12 receptor or GPR3 receptor, and 7TM proteins coded for in the human genome but for which no endogenous ligand has yet been assigned.
  • transporter proteins such as, e.g., i) Na + cotransporters, including Na?CI " transporters, such as, e.g., GABA transporters, monoamine transporters, neutral amino acids transporters, kreatinin transporters and nucleoside transporters, and Na + ,K + coupled transporter such as, e.g., glutamate transporters, neutral amino acids transporters, and inositol transporters, and Na + ,glucose cotransporters, and Na?K?CI " cotransporters, ii) H+ coupled transporter including oligopeptide transporters and multi drug transporters, iii) antiporters, including Na + /H + - exchangers, anion exchangers such as, e.g., HCO 3 7CI " exchangers and Na + /Ca +
  • transporters such as, e.g., GABA transporters, monoamine transporters, neutral amino acids transporters, kreatinin transporters and nucleoside transport
  • the membrane protein may also be a cell adhesion molecule, including but not restricted to for example NCAM, VCAM, ICAM or LFA-1.
  • the membrane protein may be an enzyme such as adenylyl cyclase.
  • the biological target molecules are 7 transmembrane domain receptors (7TM receptors) also known as G-protein coupled receptors (GPCRs).
  • 7TM receptors 7 transmembrane domain receptors
  • GPCRs G-protein coupled receptors
  • ligands acting through 7TMs includes a wide variety of chemical messengers such as ions (e.g. calcium ions), amino acids (glutamate, -amino butyric acid), monoamines (serotonin, histamine, dopamine, adrenalin, noradrenalin, acetylcholine, cathecolamine, etc.), lipid messengers (prostaglandins, thromboxane, anandamide, etc.), purines (adenosine, ATP), neuropeptides (tachykinin, neuropeptide Y, enkephalins, cholecystokinin, vasoactive intestinal polypeptide, etc.), peptide hormones (angiotensin, bradykinin, glucagon, calcitonin, parathyroid hormone, etc.), chemokines (interleukin-8, RANTES, etc.), glycoprotein hormones (LH, FSH, TSH, choriogonadotropin
  • the G-protein consists of three subunits, a -subunit that binds and hydrolyses GTP, and a ? -subunit.
  • GDP When GDP is bound, the ⁇ subunit associates with the ⁇ y subunit to form an inactive heterotrimer that binds to the receptor.
  • a signal is transduced by a changed receptor conformation that activates the G- protein.
  • monoamine agonists appear to bind in a pocket relatively deep between TM-III, TM-V and TM-VI, while peptide agonists mainly appear to bind to the exterior parts of the receptors and the extracellular ends of the TMs (Strader et al., (1991) J. Biol. Chem. 266: 5-8; Strader et al., (1994) Ann. Rev. Biochem. 63: 101-132; Schwartz et al. Curr. Pharmaceut. Design.
  • ligands can be developed independent on the chemical nature of the endogenous ligand, for example non-peptide agonists or antagonists for peptide receptors.
  • non-peptide antagonists for peptide receptors often bind at different sites from the peptide agonists of the receptors.
  • non-peptide antagonists may bind in the pocket between TM-III, TM-V, TM-VI and TM-VII corresponding to the site where agonists and antagonists for monoamine receptors bind (Hoist et al. (1998) Mol. Pharmacol. 53:166-175).
  • the 7TM receptor superfamily is composed many hundreds of receptors that may be further divided into smaller sub-families of receptors.
  • the largest of these smaller sub-families of 7TM receptors is composed of the rhodopsin- like receptors (also termed the family A receptors), which are named after the light- sensing molecule from our eye.
  • the receptors are integral membrane proteins characterized by seven transmembrane (7TM) segments traversing the membrane in an antiparallel way, with the N-terminal on the extracellular side of the membrane and the C- terminal on the intracellular side.
  • the polypeptide adopts a helical secondary structure.
  • the lengths, and the beginning center and ends relative to the lipid bilayer membrane of these helices may be deduced from solved three-dimensional structures of the receptor proteins (Palczewski K. et al., Science, 289(5480), 2000, pp. 739-45).
  • the helical lengths, and the beginning, center and ends relative to the lipid bilayer membrane of each of the seven helices may be dissected by sequence analysis (J.M. Baldwin, EMBO J. 12(4), 1993, pp. 1693-703; J.M. Baldwin et al., J. Mol Biol, 272(1), 1997, pp. 144-64).
  • a useful tool in the identification and engineering of metal-ion sites is the generic numbering system for residues of 7TM receptors.
  • the largest family of 7TM receptors is composed of the rhodopsin-like receptors, which are named after the light-sensing molecule from our eye.
  • a number of residues, termed key residues, especially within each of the transmembrane segments are highly but not totally conserved. These residues may be used to direct an alignment of the primary protein sequences within the transmembrane segments together with other standard principles and techniques, for example hydrophobicity plots, well-known to persons skilled in the art.
  • transmembrane segments that are very conserved, and these may be used to further direct an alignment of the transmembrane segments. These are particularly useful when a given key residue in a transmembrane segment has been substituted through evolution by another aminoacid of a dissimilar physiochemical nature.
  • the transmembrane segments are generically numbered.
  • Aspartate (Asp) in the rhodopsin-like family is given the generic number 10, i.e.
  • Fig. 1 a schematic depiction of the structure of rhodopsin-like 7TMs is shown with one or two conserved, key residues highlighted in each TM: Asnl:18; Aspll:10; Cyslll:01 and Arglll:26; TrplV:10; ProV:16; ProVI:15; ProVII:17.
  • residues involved in for example metal-ion binding sites can be described in this generic numbering system.
  • a tri-dentate metal-ion site constructed in the tachykinin NK1 receptor (Elling et al., (1995) Nature 374, 74-77) and subsequently transferred to the kappa-opioid receptor (Thirstrup et al., (1996) J. Biol. Chem. 271 , 7875- 7878) and to the viral chemokine receptor ORF74 (Rosenkilde et al., J. Biol. Chem. 1999 Jan 8; 274(2), 956-61) can be described to be located between residues V:01 , V:05, and Vl:24 in all of these receptors although the specific numbering of the residues is very different in each of the receptors.
  • an analogous system may be developed for the other families of 7TM receptors, for example the family B class of receptors, composed of for example the glucagon receptor, the glucagon-like peptide (GLP) receptor-1 , the gastric inhibitory peptide receptor (GIP), the corticotropin releasing factor (CRF) receptor-1 , vasoactive intestinal peptide (VIP) receptor, pituitary adenylate cyclase-activating polypeptide (PACAP) receptor etc. (J.W. Tarns et al., Receptors Channels 1998;5(2), pp. 79-90).
  • GLP gastric inhibitory peptide receptor
  • CRF corticotropin releasing factor
  • VIP vasoactive intestinal peptide
  • PACAP pituitary adenylate cyclase-activating polypeptide
  • the transmembrane segments are generically numbered.
  • Ser serine
  • His histidine
  • Cyslll cysteine
  • Pro highly conserved proline
  • Orphan 7TM receptors - one embodiment of the invention is directed to a method of assessing the physiological function and pharmacological potential of orphan 7TM receptors by the introduction of metal-ion sites in the orphan receptor.
  • Orphan 7TM receptors Today there are several hundreds of such orphan 7TM receptors. Based on characterization of their expression pattern in different tissues or expression during development or under particular physiological or patho-physiological conditions and based on the fact that the orphan receptors sequence-wise appear to belong to either established sub-families of 7TM receptors or together with other orphans in new families, it is believed that the majority of the orphan receptors are in fact important entities.
  • Orphan 7TMs are "the next generation of drug targets" or "A neglected opportunity for pioneer drug discovery” (Wilson et al. Br. J.Pharmacol. (1998) 125: 1387-92; Stadel et al. Trends Pharmacol. Sci.; (1997) 18: 430-37; Howard A et al.; Trends Pharmacol. Sci. (2001) 22: 132-140).
  • the problem is that it is very difficult to characterize orphan receptors and find their endogenous ligands, since no assays are available for these receptors due to the lack of specific ligands - a "catch 22" situation.
  • the present invention provides a method of validating also the use of orphan receptors as drug targets through "pharmacological knock-out" technology.
  • metal ion binding sites in orphan receptors at locations where it is or will become known from work on multiple other 7TM receptors with known ligands and with binding and functional assays that binding of metal ions and metal ion chelates will act as either agonists or more common as antagonists, then it will be possible to make a potentially silent metal-ion site to be used in vivo as described for biological target molecules in general.
  • metal-ion sites can be built into proteins by introduction of metal- ion chelating residues at appropriate sites.
  • sites are constructed at strategic sites in the biological target molecule with the purpose to serve as anchor sites for test compounds in a target validation process.
  • Mutagenesis - the nucleotide sequence encoding the target protein of interest may be subjected to site-directed mutagenesis in order to introduce the amino acid residue, which includes the metal-ion binding site or a residue, which is to serve as target for secondary site chemical interaction.
  • Site-directed mutagenesis may be performed according to well- known techniques e.g. as described by Ho et al. Gene (1989) 77: 51-59.
  • the mutation is introduced into the coding sequence of the target molecule by the use of a set of overlapping oligonucleotide primer both of which encode the mutation of choice and through polymerisation using a high-fidelity DNA polymerase such as e.g.
  • a site-directed mutation event is subsequently confirmed through DNA sequence analysis throughout the genetic segment generated by PCR.
  • this may involve the introduction of one or more amino acid residues capable of binding metal-ions including but not restricted to, for example His, Asp, Cys or Glu residues.
  • any amino acid could be introduced.
  • a Cys residue is introduced as target for a bridging metal-ion site between the ligand and this residue or as a target for generation of a covalent bond.
  • the mutated target molecule will initially be tested with respect to the ability to still constitute a functional, although altered, molecule through the use of an activity assay suitable in the specific case.
  • mutations in proteins may obviously occasionally alter the structure and affect the function of the protein, this is by far always the case. For example, only a very small fraction (less than ten) of the many hundred Cys mutations performed in rhodopsin as the basis for site directed spin-labelling experiments and in for example the dopamine and other 7TM receptors as the basis for Cys accessibility scanning experiments have impaired the function of these molecules.
  • Lac-permease almost all residues have been mutated and only a few of these substitutions directly affect the function of the protein.
  • the method of the invention may suitably include a step of determining the location of, for example the metal ion binding amino acid residue(s) in a mutated protein and determining the location of at least one other amino acid residue in the vicinity of the metal ion binding amino acid residue, based on either the actual three-dimensional structure of the specific biological target molecule in question (e.g. by conventional X-ray crystallographic or NMR methods) or based on molecular models based on the primary structure of the specific molecule together with the three-dimensional structure of the class of molecules to which the specific molecule belongs (e.g. established by sequence homology searches in DNA or amino acid sequence databases).
  • the metal-ion binding site may suitably be introduced to serve as an anchoring, primary binding site for the test compound, which can thereby be targeted to affect a site in the biological target molecule having one or more of the following properties (the metal-ion site may be placed either within or close to this site):
  • a site where the biological target molecule binds to another biological target molecule for example a regulatory protein
  • a site which will control the activity of the biological target molecule in a positive or negative fashion i.e. up-regulating or down regulating the activity of the biological target molecule, for example by an allosteric mechanism
  • a site where the binding of the test compound will directly or indirectly interfere with the binding of the substrate or natural ligand or the binding of an allosteric modulatory factor for the biological target molecule iv) a site where the binding of the test compound may interfere with the intra-molecular interaction of domains within the biological target molecule, for example the interaction of a regulatory domain with a catalytic domain
  • Metal-ion site engineering in 7TM proteins Metal-ions play a fundamental role in biology - In natural proteins they are involved in functional purposes such as electron transfer or catalysis or in structural purposes stabilizing the three-dimensional structure of the protein. It is well known that also several integral membrane proteins include binding sites for metal ions. Regardless of whether the metal-ions play a functional or structural role, the specific properties exclusive to metals are utilized. In the proteins, the particular properties of the metal-ions may be fine-tuned by the amino acids defining the binding site to the application. An important general consideration is that metal-ions in fact offer the strongest binding interaction when viewed on a per atom basis compared to other ligands (I.D. Kuntz et al., Proc. Natl. Acad. Sci.
  • metal-ion binding in proteins is one of the most well characterised forms of receptor-ligand-protein interactions known.
  • characterising a metal ion-binding site in a membrane protein using, for example, molecular models and site directed mutagenesis can yield information about the structure of the membrane protein and importantly where the "ligand" (metal ion) binds (e.g. Elling et al. Fold. Des. 2(4), 1997, pp. S76-80).
  • the forces that control metal-ion binding - Amino acid residues that function as effective metal binding residues are typically those that contain electron-donation atoms (S, O or N) (J.P. Glusker, Adv. Protein Chem., 42, 1991 , pp. 1-76). Although this group includes amino acids such as Ser, Lys, Arg and Tyr the strongest interactions typically involve Asp, Glu, Cys and His. Binding of a metal-ion to a ligand (a residue or an organic compound) can be considered in terms of Lewis acid base theory (J.P. Glusker, Adv. Protein Chem., 42, 1991 , pp. 1-76).
  • an acid is any species that can accept a pair of electrons
  • a base is any species that can donate a pair of electrons. Consequently a metal acts as a Lewis acid when accepting an electron pair, and the ligand acts as a Lewis base when donating an electron pair.
  • the nature of this electron transfer depends on the atoms involved, i.e. on the polarizability. On the basis of polarizability, metal ions may be classified as being hard/soft, hard meaning difficult to polarize. Important examples of cations such as Zn 2+ , Cu 2+ , Fe 2+ , Fe 3+ and Ni 2+ are classified as being 'borderline' (J.P. Glusker, Adv.
  • Residues useful in metal-ion binding site engineering include for example aspartate, glutamate, histidine or cysteine. Aspartate and glutamate residues may carry one negative charge. Each oxygen atom has two lone pairs disposed at 120° to its C-O bond and in the plane of the carboxyl group. D.W. Christianson et al. (Am. Chem. Soc, 110(16), 1988, pp.
  • One carboxyl group can bind one or two metal ions, e.g. leucine aminopeptidase (Straeter et al., Biochemistry, 34(45), 1996, pp. pp. 14792-14800). Histidine residues are involved in binding of metal-ions in a variety of enzymes and are the most abundant ligands in zinc binding sites (I. L. Alberts et al..Protein Sci., 7(8), 1998, pp. 1700-1716). The imidazole ring exists in two tautomeric forms in which the proton is either on the N ⁇ or N ⁇ nitrogen. The ratio is approximately 80% Ne-H and 20% H ⁇ -H (Fig. 3) (W.F. Reynolds et al., J. Am.
  • Coordination geometry The most common structures of metal coordination spheres are octahedral, tetrahedral and square planar geometry's (F.A. Cotton et al, Basic Inorganic Chemistry (John Wiley & Sos, Inc.), 1995) (Fig. 4).
  • the coordination number and geometry depend on the nature of the metal and the size of the ligands (metal ion chelators, protein sidechains, water etc.), and is usually as high as possible.
  • the surrounding ligand atoms arrange in a geometry that minimizes the repulsive energy between them (J.P. Glusker, Adv. Protein Chem., 42, 1991 , pp. 1-76).
  • Zinc(ll) (Zn(ll)) and copper(ll) (Cu(ll)) are examples of two metal ions useful in metal-ion binding site engineering. They are both well-investigated transition metals. Zinc is involved in many hydrolytic enzymes, such as carboxypeptidase and carbonic anhydrase, which utilize zinc in the active site (L. Stryer, Annu. Rev. Biochem., 37, 1968, pp. 25-50). Copper is also found in a number structures, e.g. azurin (E.T. Adam et al., J. Mol. Biol., 123(1), 1978, pp. 35-47).
  • Zn(II) are most often found in a tetrahedral and octahedral geometry (D.W. Christianson, Adv. Protein Chem., 42, 1991 , pp. 281-355).
  • the mean zinc-ligand distance in protein coordination spheres is approximately 2 A (2.05A for histidine as a ligand) (P. Chakrabarti, Protein Eng., 4(1), 1990, pp. 57-63).
  • Cu(ll) are most often found in a square planar or a distorted octahedral geometry (see Fig. 4) (J.P. Glusker, Adv. Protein Chem., 42, 1991 , pp. 1-76).
  • Cu 2+ The coordination octahedron of Cu 2+ is found not to contain six bonds of equal lengths, but has four short bonds (-2.0A) and two long bonds (-2.4A) (in trans) (Jahn and Teller, Proc. R. Soc London, Ser. A 161 , 1937, 220-235).
  • Cu 2+ usually bind its ligands (N & O) stronger than Zn 2+ (Irving and Williams, Nature, 162, 1948, pp. 746) giving Cu 2+ a different binding.
  • metal-ion sites are introduced in 7TM receptors.
  • Engineering of artificial metal ion binding sites into membrane proteins has been employed to explore the structure and function of these proteins.
  • CE. Elling et al., Nature 374, 1995, pp. 74-77 have reported how the binding site for a proto-type antagonist for the tachykinin NK-1 receptor could be converted into a metal ion-binding site by systematic substitution of residues in the binding pocket with His residues.
  • a tridentate zinc-site was constructed, composed of two histidine residues located in an / and 1+4 position at the exterior end of TM-V (V:01 and V:05) and a single His residue located in TM-VI (Vl:24).
  • SUBST-ttUT-E-SHEET (RULE 26) exchanged with a metal-ion site through specific substitutions in the binding pocket for the agonists (OE. Elling et al, PNAS 96, 1999, pp. 12322-12327).
  • This metal-ion binding site could be addressed also with metal-ions in complex with metal-ion chelators, i.e. small organic compounds binding metal-ions.
  • the activating metal-ion site has successfully been transferred to another 7TM receptor, the tachykinin NK1 receptor (B. Hoist et al., Mol. Pharmacol. 2000, 58: 263-270).
  • metal-ion chelators or chelates will at best bind with affinities in the single digit micromolar range and therefore inhibit the function of the biological target molecule with similar or lower potency. Such low affinities and potencies will prohibit the use of the technology due to the fact that administration of metal-ions and metal-ion chelators in such concentrations will have multiple disturbing side-effects due to similar, non-specific, low affinity interaction of the test compounds with other - usually unknown - biological target molecules.
  • Metal-ion binding sites are constructed by mutating one or more amino acid residue in the biological target molecule into residues, which can bind metal-ions. These are usually His, Cys, Asp, or Glu residues but could also be Trp, Tyr, Ser, Thr, Lys, Arg, Asn, Gin and Met. Additionally an engineered site may utilize electron donating groups from the polypeptide backbone. It should be noted that also non-natural amino acids, which can bind metal-ions could be used, provided that a suitable method is employed to introduce these or a precursor for these - which can be chemically modified to become a metal-ion binding residue - into the biological target molecule.
  • a metal-ion site includes one, two, three or four amino acid residues although more residues also can occur.
  • the metal-ion binding site includes two residues, which allow for the metal-ion to bind also the metal-ion chelator.
  • the metal-ion binding residue(s) are already present in the biological target molecule in a suitable location and consequently only a single extra metal-ion binding residue needs to be introduced in the spatial vicinity through mutational substitution of the residue found in the wild-type of the biological target molecule.
  • the metal-ion binding atom of the amino acid residues need to be located or be able to move into a location which satisfies the geometrical criteria for making a metal-ion binding site with the particular metal-ion used, which will be known to a person skilled in the art (I. Lalbert, 1998, Protein Science, 7: 1700-1716, B. Lvallee, 1990, Biochemistry, 29(24):5647-5659), Hellinga HW et al., J. Mol. Biol., 222(3), 1991 , pp. 763-85).
  • metal-ion binding sites When engineering a metal-ion binding site in a 7TM receptor a general classification of the site may be performed based on the general placement of the introduced amino acid sidechains chelating the metal-ion.
  • the metal-ion binding sites may be described as being either intra-helical, i.e. the residues composing the site are located on the same transmembrane helix; as being inter- helical, i.e. the residues composing the site are located on at least two transmembrane helices; or generally as involving residues within transmembrane segments and/or loops and turns of the protein.
  • residues defining an intra-helical metal-ion binding site are located on the same face of the helix, for example in an /, i+4 manner
  • residues defining inter-helical residues are located on opposing faces of the involved helices and the site may be composed of one or more residues from each helix.
  • one or more of the employed metal-ion binding residues will be a sulphur containing residue such as Cys which binds for example Ru(ll), Pd(ll) and Pt(ll) particularly well.
  • an important part of the present invention is to increase the affinity of the test compound through the establishment of just a single or a few secondary chemical interaction(s) besides the anchoring binding of the metal-ion part of the complex.
  • the anchoring metal-ion sites are in a preferred embodiment of the invention built into the biological target molecules at sites where knowledge from the known three-dimensional structure or from models of the three-dimensional structure indicate, that a suitable chemical moiety is present in the vicinity of the engineered metal-ion site.
  • a metal-ion site can be build in the vicinity of the charged residue, and chemical modifications of a suitable metal-ion chelate can be performed in order to establish a charge-charge interaction with the supposedly charged residue in the biological target molecule.
  • sites for engineering of metal-ion sites can be chosen in order to establish other types of suitable, secondary site chemical interactions through appropriate chemical modifications of the test compound.
  • the secondary chemical interaction includes the binding of second metal-ion between the test compound and the secondary site residue.
  • the secondary chemical interaction is a covalent bond, for example established with a thiol-containing residue, for example a Cys, or an amine- containing residue, for example a Lys.
  • a thiol-containing residue for example a Cys
  • an amine- containing residue for example a Lys.
  • chemically reactive groups with suitable chemical reactivity can be introduced into the test compound in order for these to selectively react with the intended, particular secondary site residue in the biological target molecule - for example a Cys - and not with such residues in general in the biological target molecule or in the test animal in general, due to the close proximity in which the reactive group on the test compound is brought through the binding of the test compound to the metal-ion site in the biological target molecule.
  • a metal-ion binding site is introduced into the biological target molecule, but also one or more residue(s), which can form a suitable secondary site chemical interaction - as indicated above - are introduced. Also in this case, it is required that this extra substitution is made as a relatively silent substitution fulfilling the criteria, which were discussed in relation to the silent metal-ion binding site.
  • Any type of natural or non-natural amino acid residue could be introduced with the purpose of establishing secondary site interactions.
  • the residue introduced for the establishment of a secondary site interaction is a Cys residue due to the fact, that this residue can be introduced in a relatively silent manner at multiple sites in biological target molecules such as for example membrane proteins such as 7TM receptors and transporters.
  • a Cys residue is particularly suited for establishing secondary site interactions such as a metal-ion bridge or a covalent bond.
  • metal-ion binding sites in 7TMs may be found in the helices or in the loops.
  • the binding site may be intrehelical, i.e. within the same helix or interhelical, i.e. involving two or more helices.
  • single positions in the different transmembrane segments of a 7TM are positions which are suitable metal-ion binding sites:
  • transmembrane segment-I Single Positions: transmembrane segment-I:
  • a very important step in the target validation process suitable for the use described herein is a selection of a suitable metal-ion chelate for administration to the test animals (and to the control animals).
  • a metal-ion chelator may be employed and in such cases the chelator may be administered together with a suitable metal ion (e.g. in the form of a suitable salt, complex etc) or the metal-ion chelate may be formed in situ after administration by means of metal ions present in the body.
  • the metal ion chelate (or a transport or depot form thereof) has ideally the following properties:
  • test compounds e.g. items 3, 4, 5 and 6 above
  • parameters which are relevant in connection with a pharmacokinetic and/or biopharmaceutical selection of the test compounds e.g. items 1, 2, 5 and 6 above.
  • Chemical compounds, which are suitable for use in target validation processes involving biological target molecules having a metal-ion site are any compound that is capable of forming a complex with a metal ion.
  • complexes of interest are chelates comprising three major parts: the functionalised chelator, the metal ion (central metal or coordinated metal), and displaceable ligands bound to the metal.
  • the metal ion central metal or coordinated metal
  • displaceable ligands bound to the metal.
  • Functional groups in a ligand attached to the metal ion are the ligand's coordinating groups.
  • a ligand attached directly through only one coordinating atom (or using only one coordination site on the metal) is called a monodentate ligand.
  • a ligand that may be attached through more than one atom is multidentate, the number of actual coordinating sites being indicated by the terms bidentate, tridentate, tetradentate and so forth.
  • Multidentate ligands attached to a central metal by more than one coordinating atom are called chelating ligands, or chelators.
  • a chemical compound for use in the present context is at least bidentate, i.e. it is a so-called metal-ion chelator.
  • a chemical compound for use in a library according to the invention has at least two heteroatoms, similar or different, selected from the group consisting of nitrogen (N), oxygen (O), sulphur (S) and phosphorous (P).
  • displaceable ligands it is very important to find the right balance between too tight binding to the metal-ion and too weak binding to achieve a useful complex.
  • An example is the fact that chloride binds tightly to the metal-ion, whereas dimethylsulfoxide has a much weaker interaction.
  • the interaction ability of a displaceable ligand is usually correlated with the acidity of the corresponding acid, and a very useful leaving group is triflate, which is the corresponding base of the highly acidic trifluorometyl sulphonic acid.
  • the metal-ion chelator complex is able to reach and specifically bind to the modified receptor in the genetically modified animal as an intact complex.
  • metal-ion binding molecules there are several metal-ion binding molecules to compete with the chelator for the metal ion.
  • different metal-ion binding proteins such as albumin and ⁇ -macroglobulin are very abundant. They are binding the metal ion with an affinity in the micromolar range, but with a considerable capacity.
  • inert complexes which do not freely exchange the metal-ion with other metal-ion binding molecules in the body fluid, and which have a high affinity and selectivity for the genetically modified receptor, are used.
  • the inertness of metal ion in a complex is increased with the number of the period (in the periodic table), e.g. complexes with group 8, iron (Fe) has a V/z measured in seconds, ruthenium (Ru) has a TV2 measured in hours and osmium (Os) is almost unable to engage in complex reactions.
  • transition metals in period 5 of the periodic table are used as the preferred metal-ions in the test compounds.
  • metal-ions in period 4 have a faster exchange rate, such metal-ions can also be employed.
  • the affinity and selectivity of the metal-ion chelate for the metal-ion site in the engineered biological target molecule should have a reasonable high affinity and therefore it often needs to be improved.
  • Such a chemical optimisation can be performed either in a random fashion or in a more targeted fashion, utilising structural information on the target protein.
  • the optimisation is based on a collection of test compounds, i.e. based on selected libraries of test compounds.
  • the present invention aims at providing chemical compounds and collections of chemical compounds (libraries), which are suitable for use in optimising the primary and secondary interactions with a biological target molecule (i.e. optimising the interaction between the metal-ion chelate and the metal-ion binding site in the biological target molecule and, furthermore, optimising the secondary interactions of the metal-ion chelate with suitable functional groups in the vicinity of the metal-ion binding site).
  • a library according to the invention contains normally three or more chemical compounds.
  • a library based on structural information from biological target molecules contains at least 3 and often at the most 100 compounds.
  • the nature of the complex can be altered depending on the metal-ion binding site in the biological target molecule. It is usually advantageous in the described target validation process to have inert complexes. Preferably, complexes capable of forming an irreversible binding with the biological targets should be made. Furthermore, the chelator with its metal should be compatible with the route of administration and time course of the experiment.
  • the types of ligands can roughly be divided into three groups; (i) the pure ⁇ -donating ligands, e.g. amines or thiols, (ii) ligands with additional 77-back bonding to an electron deficient 77 systems, e.g. pyridines, and (iii) ligands with additional 77-back bonding to an electron rich 77 systems, e.g. thiophenes.
  • the pure ⁇ -donating ligands e.g. amines or thiols
  • ligands with additional 77-back bonding to an electron deficient 77 systems e.g. pyridines
  • ligands with additional 77-back bonding to an electron rich 77 systems e.g. thiophenes.
  • ligands with back bonding capabilities is favorable, and this is further improved with electron deficient 77 systems.
  • this strengthens the binding between the chelator and the metal, and secondly, removing electron density from the metal allows the metal site in the target to form stronger interactions with the metal.
  • Another aspect to be considered is the size of the metal centre. If the metal is among the heavier metals, a larger heteroatom may be of value, e.g. sulphur atoms instead of nitrogen atoms.
  • chelators containing e.g. pyridine moieties are of interest, as well as crown ether types like tetraazacyclononane or cyclam. Possible chelators advantageous for the task are 2,2'- bipyridine, 8-hydroxyquinoline, 8-mercaptoquinoline, and 2-(2-pyridyl)thiophenol.
  • Zinc and copper have proven useful for complexing to bipyridyls, but both are forming labile complexes, where interchange of ligands is fast. They may be useful for the purpose in certain cases, but metals forming more stabile complexes are preferable. Metals represented by technetium, ruthenium, rhodium, palladium, osmium, platinum etc. are known to form more inert complexes. The various metals have different electronic properties, thus different metals will show different preferences for various electronic environments, both in the metal site in the target, as well as from the chelator. Also the oxidation level is important for complexation reactions. Thus in a preferred embodiment of the invention specific oxidation states of metal ions are used which create the most stable complexes, for example Ru(ll), Rh(lll), Pd(ll), Pt (II).
  • the interaction between the metal and the metal site in the protein target can be modulated.
  • Histidine with its imidazole moiety
  • cysteine with its thiol group
  • Cysteine is a pure ⁇ -donor
  • histidine also includes additional 77-back bonding capabilities.
  • Other residues that may be used in the metal sites are glutamates and aspartates.
  • the soft metal ions e.g. Pd(ll), Ru(ll), Rh(lll) and Pt(ll) have a high tendency to form inert interaction with the metal-ion binding site modified biological target molecule, dependent on which residues that form the metal ion site.
  • test compounds for use according to the invention may be chemically optimised by introducing functional groups, which are able to establish an interaction with specific chemical groups located in the vicinity of the metal-ion site in the biological target molecule.
  • the bonds formed may be of, but not limited to, one or several of the following types:
  • charge-charge interaction introduction of charged groups such as ammonium, phosphonium or sulphonium groups, or ionisable groups such as amino or carboxy groups.
  • aromatic-aromatic interactions introduction of an aromatic group in the test compound.
  • hydrophobic interactions introduction of hydrophobic groups.
  • the functionality can be introduced in a targeted manner, targeting either a natural residue in the protein target, or targeting an engineered residue, optimised for the interaction.
  • Further functional groups may be introduced to increase affinity, selectivity, or physicochemical properties.
  • the reactive groups used for forming secondary interactions should be compatible with the route of administration and time course of the experiment.
  • the listed groups provide a proper range of reactivity that will be used for the specific biological target proteins and experiments.
  • the aim of the above-mentioned manipulation or chemical modification of the test compounds is to improve the properties of a test compound with respect to:
  • test compound may also be changed with respect to its pharmacokinetic and/or biopharmaceutical properties cf. the discussion below.
  • test compound which has been optimised for binding affinity and selectivity for the biological target molecule will often not have the appropriate pharmacokinetic etc. properties to be useful in the in vivo setting of the genetically modified animal.
  • test compound since the test compound is only to be used in animal experiments and not directly in human beings, there is a rather large degree of freedom in respect of what can be done to improve, for example, the pharmacokinetic properties of the test compound as compared to what can be done with a compound which is going to be used for the treatment of human patients.
  • the test compound which has been optimised for binding affinity and selectivity for the biological target molecule is further optimised for appropriate properties of absorption, distribution, metabolism, and excretion either through further organic chemical modifications or through association of the test- compound with a molecule providing the desired pharmacokinetic properties.
  • a change of the pharmacokinetic properties of a test compound may include a change in the absorption rate, the plasma half-life, the distribution, the metabolism and/or the elimination of the test compound.
  • a change in the biopharmaceutical properties of a test compound may include a change in the water-solubility (e.g. by salt or complex formation), in the lipid solubility (e.g. by formation of a salt or a complex) and/or in the particle size of the test compound.
  • the formulation technique chosen depends on which properties of the test compound, that are desirable. Whether a test compound is presented in dissolved form or not depends on the available administration route. Thus, if the test compound is administered orally, it can be presented in dissolved or non-dissolved form. However, it is contemplated that the test compound must dissolve before it can be absorbed and enter the systemic circulation.
  • Test compounds may by themselves have an undesirable plasma half-life.
  • the test compound may be chemically modified in such a manner that it is linked to a larger molecule (carrier) optionally via a spacer.
  • carrier e.g. macromolecular carriers of natural, synthetic or semisynthetic origin like e.g. polysaccharides (e.g.
  • test compound may either through a covalent or a non-covalent bond be directly linked to the macromolecular carrier or it may be indirectly linked to the macromolecular carrier via a spacer.
  • a spacer may serve the following purposes:
  • the spacer includes chemically functional groups, which make it possible to react at the one end with the carrier and at the other end with the test compound. Thus, the spacer is necessary in order to connect the carrier and the test substance.
  • the spacer enables a distance between the carrier and the test substance so that a possible interaction from the carrier on the biological target molecule is eliminated or significantly reduced.
  • a suitable spacer may be a short chain peptide, a poly- or oligoethyleneglycol, or a short chain polysaccharide in which one or more hydroxy groups optionally have been substituted with e.g. amino, sulphate, amide, ester or ether groups.
  • a test compound may also be modified in order to improve the localization of the test compound after administration to a test animal.
  • incorporation of specific chemical groups or association of the test compound with certain specific molecules, peptides, proteins or other macromolecules provide the test compound with the property of accumulating or localizing to specific sites within the body, e.g. to the brain (by passage of the blood brain barrier), to tumor-associated tissue or the like.
  • the test compound can also be modified to pass through biological membranes and thereby become accessible to intracellular biological target molecules.
  • the distribution of the test compound may be changed by incorporating carriers, which mainly are hydrophilic or lipophilic of nature.
  • the carriers may be an integral part of the test compound.
  • pharmaceutically acceptable excipients normally used in localised drug delivery may be included in the dosage form.
  • test compound or a transport form thereof
  • controlled release formulation techniques well-known for at person skilled in the art of pharmaceutical formulation can be employed, e.g. in order to prepare a controlled release composition containing the test compound and from which the test compound is only slowly released over a time period of, e.g., 2-20 hours or even longer.
  • Controlled release formulation techniques are known for many kinds of dosage forms including oral, topical, parenteral, rectal, ocular and nasal dosage forms.
  • the test compounds normally fulfil certain criteria with respect to molecular weight (at the most 3000 such as, e.g., at the most 2000, at the most 1500, at the most 1000, at the most 750, at the most 500), number of hydrogen bond donors (at the most 15 such as, e.g. at the most 13, 12, 11 , 10, 8, 7, 6 or at the most 5) and number of hydrogen bond acceptors (at the most 15 such as, e.g. at the most 13, 12, 11 , 10, 8, 7, 6 or at the most 5).
  • the molecular weight, number of hydrogen bond donors and/or number of hydrogen bond acceptors of a test compound of a library of the invention have other values than the above-mentioned.
  • Chemical compounds, which are suitable for use in target validation processes involving biological target molecules having a metal-ion site are any compounds that are capable of forming a complex with a metal ion.
  • a chemical compound for use according to the invention has at least two heteroatoms, similar or different, selected from the group consisting of nitrogen (N), oxygen (O), sulphur (S), selenium (Se) and phosphorous (P).
  • Suitable metal ion chelators may have any log K value.
  • a log K value from about 1 to about 50 is suitable such as, e.g. a log K value in a range of from about 3 to about 40, such as, e.g., from about 3 to about 30, from about 3 to about 26, from about 3 to about 18, from about 3 to about 15, from about 3 to about 12, from about 4 to about 10, from about 4 to about 8.
  • the log K value may be from about 4.5 to about 7, from about 5 to about 6.5 such as from about 5.5 to about 6.5.
  • K is an individual complex constant (also denoted equilibrium or stability constant). The constant's subscript 1 , 2, 3 etc. indicates which coordination step the constant is valid for, i.e.
  • K-i is the complex constant for the coordination of the first ligand
  • K 2 is for the second ligand and so forth
  • log K can be determined as described in W.A.E. McBryde, "A Critical Review of Equilibrium Data for Protons and Metal Complexes of 1 ,10-Phenanthroline, 2,2'-bipyridyl and related Compounds.” Pergamon Press, Oxford, 1978.
  • Test compounds which have been found to be useful in the present methods, are typically compounds comprising a heteroalkyl, heteroalkenyl, heteroalkynyl moiety or a heterocyclyl moiety for chelating the metal ion.
  • heteroalkyl is understood to indicate a branched or straight-chain chemical entity of 1-15 carbon atoms containing at least one heteroatom.
  • heteroalkenyl is intended to indicate a branched or straight-chain chemical entity of 2-15 carbon atoms containing at least one double bond and at least one heteroatom.
  • heteroalkynyl is intended to indicate a branched or straight-chain chemical entity of 2-15 carbon atoms containing at least one triple bond and at least one heteroatom.
  • heterocyclyl is intended to indicate a cyclic unsaturated (heteroalkenyl), aromatic (“heteroaryl”) or saturated (“heterocycloalkyl”) group comprising at least one heteroatom.
  • Preferred “heterocyclyl” groups comprise 5- or 6- membered rings with 1-4 heteroatoms or fused 5- or 6-membered rings comprising 1-4 heteroatoms.
  • the heteroatom is typically N, O, S, Se or P, normally N, O or S.
  • the heteroatom is either an integrated part of the cyclic, branched or straight-chain chemical entity or it may be present as a substituent on the chemical entity such as, e.g., a thiophenol, phenol, hydroxyl, thiol, amine, carboxy, etc.
  • heteroaryl groups are indolyl, dihydroindolyl, furanyl, benzofuranyl, pyridinyl, pyrimidinyl, quinolinyl, triazolyl, imidazolyl, thiazolyl, tetrazolyl and benzimidazolyl.
  • the heterocycloalkyl group generally includes 2-20 such as 3-20 carbon atoms, and 1-4 heteroatoms.
  • Particularly useful chemical compounds of the present invention are those having at least two heteroatoms connected according to the general formula I abbreviated as Che-R1
  • F is N, O, S, Se or P
  • G is N, O, S, Se or P
  • X, Y and Z which are the same or different, are straight or branched C C 12 alkyl, CrC 12 alkenyl, C C 12 alkynyl, C C 12 cyclyl, aryl, C C 12 heteroalkyl, C C 12 heteroalkenyl, heteroalkynyl, C C 12 heterocyclyl, heteroaryl;
  • R 1 may be present anywhere on the X, Y and/or Z moiety and it may be present on X, Y and/or Z up to as many times as possible, i.e. if X is -CH 2 -CH 2 -, then R 1 may be present on the first and/or second carbon atom one or several times; R 1 could optionally be hydrogen;
  • X may together with Y and/or Z fuse to form a cyclic ring system;
  • Y may together with X and/or Z fuse to form a cyclic ring system;
  • X, Y and Z may together fuse to form a cyclic ring system;
  • R 1 corresponds to a structure -A-B-C, wherein the element A is a coupling or connecting moiety, B is a spacer moiety and C is a functional group; -B- may be substituted one or more times with a further C, which may be the same or different, and
  • -B- is absent or selected from the group consisting of:
  • alkyl straight or branched alkyl, alkenyl (straight or branched), alkynyl (straight or branched), aryl, cycloalkyl, heteroaryl, heterocycloalkyl, alkyloxyalkyl, alkylaminoalkyl,
  • -C is absent or selected from the group consisting of:
  • R" and/or R' has the same meaning as given for B above optionally substituted with one or more C;
  • A may be absent and then -R 1 is -B-C or -C, and B may be substituted one or more times with C, which may be the same or different; the total number of atoms (X+F+Y+G+Z) excluding hydrogen atoms is at the most 25;
  • the size of a ring is at the most 14 atoms, preferably 5 or 6 atoms.
  • X, Y and/or Z may fuse to form one or more rings.
  • X-F-Y may be part of a heterocyclyl ring system:
  • X-F-Y and Y-G-Z may be part of heterocyclyl ring systems:
  • X-F-Y-G-Z may also be part of heterocyclyl ring systems:
  • X-F-Y and X-F-Y-G-Z may be part of heterocyclyl ring systems:
  • X-F-Y and Y-G-Z and X-F-Y-G-Z may be part of heterocyclyl ring systems:
  • alkyl is intended to indicate a branched or straight-chain, saturated chemical group containing 1-15 such as, e.g. 1-12, 1-10, preferably 1-8, in particular 1-6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, sec. butyl, tert. butyl, pentyl, isopentyl, hexyl, isohexyl, heptyl etc.
  • 1-15 such as, e.g. 1-12, 1-10, preferably 1-8, in particular 1-6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, sec. butyl, tert. butyl, pentyl, isopentyl, hexyl, isohexyl, heptyl etc.
  • alkenyl is intended to indicate an unsaturated alkyl group having one or more double bonds.
  • alkynyl is intended to indicate an unsaturated alkyl group having one or more triple bonds.
  • cycloalkyl is intended to denote a cyclic, saturated alkyl group of 3-7 carbon atoms.
  • cycloalkenyl is intended to denote a cyclic, unsaturated alkyl group of 5-7 carbon atoms having one or more double bonds.
  • aryl is intended to denote an aromatic (unsaturated), typically 6-membered, ring, which may be a single ring (e.g. phenyl) or fused with other 5- or 6-membered rings (e.g. naphthyl or anthracyl).
  • alkoxy is intended to indicate the group alkyl-O-.
  • amino is intended to indicate the group -NR"R'" where R" and R'" which are the same or different, have the same meaning as R " in formula I.
  • R" and R' are hydrogen
  • a secondary amino group either but not both R" and R'" is hydrogen
  • neither of R" and R'" is hydrogen.
  • R" and R'" may also be fused to form a ring.
  • esters is intended to indicate the group COO-R", where R" is as indicated above except hydrogen, -OCOR , or a sulphonic acid ester or a phosphonic acid ester.
  • R is as indicated above except hydrogen, -OCOR , or a sulphonic acid ester or a phosphonic acid ester.
  • X, Y and/or Z groups may be present adjacent to the F and/or G groups.
  • the present invention relates to libraries containing three or more test compounds.
  • a library based on structural information from biological target molecules contains from about 3 to about 20 compounds and typically there are from about 20 to about 100 or from about 100 to about 10,000 compounds in more diverse libraries suitable for probing the vicinity of the metal-ion binding site for secondary interactions in the biological target molecule.
  • the aim of using a library is in a relatively easy and fast manner to identify the most suitable compound among a number of compounds.
  • a library i.e. a collection of test compounds.
  • the testing of the library is performed in suitable in vitro test (i.e. binding affinity test signal transduction test etc.) and then a proper selection or choice of compound(s) can be made for the in vivo testing.
  • suitable in vitro test i.e. binding affinity test signal transduction test etc.
  • the library has a relatively small size (e.g. up to 10 compounds), which makes it possible to perform in vivo testing without any in vitro testing.
  • a library according to the invention may contain chemical compounds having a specific characteristic feature in common or it may contain chemical compounds representing a broad diversity of chemical functional groups and/or chemical structures.
  • the chemical compounds of a library may also have a basic common structural element such as, e.g., 2.2'-bipyridine.
  • a library of the present invention may, e.g., contain
  • Test compounds having the same chemical functional group, C 1. Test compounds having the same or almost same spacer moiety, B, in order to establish a distance from the heteroatom containing skeleton to the chemical functional group; in such a library, the chemical functional group may be the same or different. 3. Test compounds which in principle are prepared by the same method and/or which have the same kind of attachment, A, to the basic common structural element.
  • Test compounds which are capable of establishing a non-covalent interaction.
  • Test compound which are capable of establishing an irreversible or slowly reversible interaction. 6. Different test compounds which are chelated with the same metal ion. 7. The same test compound that is chelated with different metal ions.
  • Libraries containing chemical compounds of the following general formulas are of specific interest in the present context.
  • the individual compounds mentioned in the following as a part of a library may of course also be employed as separate compounds according to the present invention. Libraries are especially useful in step 2 and/or 7 of the target validation process (cf. page 7-8 herein).
  • F and/or G have the same meaning as indicated above, i.e. F and/or G are heteroatoms.
  • Q is a structural element containing a heteroatom
  • L — L' represent the heteroatoms
  • M represents a metal ion
  • L* and/or L * ' are leaving groups such as, e.g. H 2 O, DMSO, Cl " , triflate etc..
  • a circle indicates a cyclic alkyl, alkenyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl or heteroaryl ring having from 3-7 atoms in the ring.
  • R 1 has the same meaning as indicated above and, when more than one R 1 is present they may be the same or different. If no specific position is given for the radical, the radical may be placed anywhere in the cyclic system and there may also be as many radicals as there is positions possible in the structure. Other symbols employed in the formulas below have the same meaning as given under formula I above. In the formulas below, the structure of the compounds are given in different structure levels. First it is given in a very general form and then in more and more specific forms. It should be noted that the invention is not limited to structures given below; these only serve to illustrate the idea of the invention and representative structures suitable for use according to the invention.
  • a library according to the present invention comprises test compounds, which have one of the following structures.
  • Y' is the remainder of the group Y which also includes Y ' being absent, i.e. G being directly linked to the ring.
  • the coordinating atom F is included in a 5- or 6-membered aromatic, unsaturated or saturated heterocycle containing between one and three heteroatoms and the coordinating atom G is either included in a 5- or 6-membered aromatic, unsaturated or saturated ring or an open chain.
  • F is N, O or S
  • G is N, O or S:
  • the coordinating atom F is appended to an aromatic, unsaturated or saturated 5- or 6-memebered ring.
  • F is N, O or S
  • G is N, O or S.
  • the coordinating atom G is included in a 5-or 6- membered aromatic, unsaturated or saturated heterocycle containing between one and four heteroatoms and the coordinating atom F contained within an aromatic, unsaturated or saturated 5- or 6-memebered heterocycle containing between one and four heteroatoms.
  • F is N, O or S
  • G is N, O or S.
  • the coordinating atom G is included in a 5- or 6-membered aromatic, unsaturated or saturated heterocycle containing between one and three heteroatoms and the coordinating atom F appended to an annelated aromatic, unsaturated or saturated 5- or 6-memebered ring.
  • X-F can optionally be included in a fused ring as indicated by the dashed line.
  • F is N, O or S; and G is N, O or S.
  • annelated derivatives may be substituted with one or more R 1 moieties.
  • a library of the present invention may contain mono-, di-, tri-, tetra-, pentasubstituted derivatives.
  • Suitable heterocyclic coordinating rings could be appended with coordinating moieties G to produce other chelating scaffolds containing one or more R1 groups.
  • Typical coordinating scaffolds of this type are imine moieties appended to coordinating heterocycles.
  • the coordinating groups e.g. thiol and imine, may be attached to a ring moiety containing one or more R1 groups.
  • Suitable open-chain chelating scaffolds are hydroxamic acids or 1 ,2-diamine coordinating moieties.
  • Chelator scaffolds containing one or more R1 groups of particular value are:
  • 2-pyridyl systems may also be connected to other six-membered nitrogen containing rings having one nitrogen adjacent to the connecting bond, such as
  • Non-pyridyl six-membered nitrogen containing aromatic rings may also be coupled to another non-pyridyl six-membered nitrogen containing ring where both ring systems having one nitrogen adjacent to the connecting bond, form useful scaffolds
  • biheterocyclyl derivatives may be substituted with one or more R 1 moieties.
  • a library of the present invention may contain mono-, di-, tri-, tetra-, pentasubstituted biheterocyclyl derivatives.
  • the biheterocyclyl system may be symmetric or asymmetric and they may be symmetricly or asymmetricly substituted with R1 groups.
  • the 5-membered ring may also be annelated with e.g. a benzene ring.
  • 2,2'-bipyridine is given as an example on a common basic structural element for chemical compounds in a library of the invention, i.e. the 2,2'-bipyridine here functions as the chelator skeleton.
  • a suitable library according to the present invention is a library, that has 2,2'- bipyridine as the same basic structural element.
  • this basic structural element is 2,2'- bipyridine
  • libraries according to this invention will be exemplified by the use of 2,2 ' -bipyridines with no intention to exclude other chelating scaffolds including the general Che-R 1 / Che-A-B-C, wherein Che constitutes the different chelating scaffolds optionally substituted further with one or more, the same or different, R 1 or more specifically A-B-C groups.
  • a library of the present invention may contain mono-, di-, tri-, tetra-, penta-, hexa- or heptasubstituted bipyridines.
  • the di-, tetra- and/or hexasubstituted bipyridines may be symmetric or asymmetric substituted bipyridines. Normally, up to 4 or at the most 5 substituents are present on the 2,2'- bipyridine skeleton.
  • the position 3' is preferably substituted with a hydrogen atom.
  • test compounds suitable for use in a target validation process may advantageously form a covalent bond as a secondary interaction.
  • Che-R 1 or more specifically Che-A-B-C wherein Che constitutes the different chelating scaffolds derived from Formula I and described above is optionally substituted further with one or more, the same or different, R 1 or more specifically A-B-C groups.
  • B is optionally substituted further with one or two, the same or different, C, the following structures for C are important in order for the (optimised) test compounds to establish a covalent (irreversible or slowly reversible) bond with e.g. an amino acid side chain in the biological target molecule.
  • the reactivity of the C group can be modified depending on the biological system to be investigated, e.g. less reactive groups are used in in vivo settings whereas more reactive moieties might also be useful in for example in vitro cell systems.
  • Various reactive groups can be appended as "C" to the metal chelator to ascertain an irreversible or a slowly reversible binding to an additional amino acid side chain in the biological target molecule containing a compatible reactive functional group such as, but not limited thereto, -SH, -OH, or NH 2 in the vicinity of the metal-ion binding site.
  • a compatible reactive functional group such as, but not limited thereto, -SH, -OH, or NH 2 in the vicinity of the metal-ion binding site.
  • Such reactive functional groups could be endogenous or mutationally introduced amino acid side-chains.
  • Neighbouring groups on B capable of providing anchimeric assistance might modify the reactivity of these reactive groups C.
  • the reactive groups can be acylating reagents of suitable reactivity as shown below that could be appended to the metal chelator (Che as described in Formula I and the more specific examples detailed thereafter) via a spacer group (-A-B-) attached at the point indicated in the formulas with examples on -C.
  • Lg denotes a suitable leaving group.
  • Michael acceptors can be appended to the metal-ion chelator.
  • Some specific examples of a conjugated aldehyde, ketone or a conjugated carboxylic acid derivative are indicated, but the double bond could also be attached to other suitable electron withdrawing groups (W) including cyano, carboxamide, nitro, sufonyl, sulfoxide and pyridine.
  • the electron withdrawing groups can also be incorporated in a ring structure as specifically exemplified by the N-maleimide and quinone derivatives.
  • alkylating groups with varying reactivity will be useful; e.g.
  • Che-A-B-CH 2 -Lg wherein Lg being Br, Cl, F or other suitable leaving groups, for forming a covalent bond with a reactive functional group such as -SH, -OH, or NH 2 in the vicinity of the metal-ion binding site.
  • Additional useful reactive groups can be selected from
  • Reactive groups capable of reacting with -SH moieties could be selected from the following
  • Cationic C groups such as amines, guanidines, amidines, ammonium, sulphonium or phosphonium ions might be useful to provide strong ionic interactions with negatively charged amino acids in the biological target molecule.
  • Anionic C groups such as -COOH, -SO 3 H, -PO(OH)NH 2 , tetrazoles or enoles, might be useful to provide strong ionic interactions with positively charged amino acids in the biological target molecule.
  • test compounds as elements in a library for covalent attachment to a biological target molecule.
  • the individual compounds mentioned in the following as a part of a library may of course also be employed as separate compounds according to the present invention.
  • Some of the structures given below are based on 2,2'-bipyridine substituted with an alkyl or alkenyl chain.
  • the 2,2'-bipyridine structure is given as an example of a Che group and should not in any way limit the invention thereto.
  • the alkyl or alkenyl chain has the function of separating e.g.
  • the thiol group from the 2,2'-bipyridine structural element in such a manner that the two heteroatoms of the 2,2'-bipyridine moiety are able to interact with the metal ion in question and subsequently with the engineered metal-ion site in the biological target molecule and the thiol group is capable of interacting with e.g. a Cys residue present in the vicinity of the metal-ion site of the biological target molecule.
  • the length of the alkyl or alkenyl chain may be from Ci to C 10 such as, e.g. from Ci to C 5 ; in other words, a library of the invention is not limited to the structures given below. Some of the examples are also useful as intermediates in the preparation of other reactive C groups mentioned above.
  • the bipyridine structural element may of course have one or more further substituents such as one or more, the same or different, R?
  • carboxylic acid esters useful as test compounds and as intermediates for preparing other test compounds may be obtained by treatment of 4-methyl-2,2'-bipyridyl with LDA (lithium diisopropyl amide) followed by a reaction with ethyl 3-bromopropionate (R" being ethyl).
  • LDA lithium diisopropyl amide
  • R" ethyl 3-bromopropionate
  • the ester can be reduced with e.g. LAH (lithium aluminium hydride) to form the corresponding alcohol useful as intermediate in the preparation of other test compounds.
  • LAH lithium aluminium hydride
  • test compounds for use in a library and for use as intermediates for preparation of other test compounds of the present invention are exemplified by the following structures, wherein Lg' is a leaving group such as - but not limited thereto - triflate, mesylate, halogen except fluorine etc.
  • Test compounds containing an aldehyde or imine group are also suitable for use in a library according to the present invention.
  • the aldehydes are also useful as intermediates.
  • the aldehyde can be obtained by reduction of the appropriate ester, prepared from 3- methyl-2,2'-bipyridyl treated with LDA (lithium diisopropyl amide) followed by reaction with ethyl bromoacetate, with DIBAL (diisobutylaluminium hydride).
  • the ester may be reduced with LAH (lithium aluminiumhydride) to form the alcohol, followed by an oxidation to the aldehyde using e.g. Swern conditions with dimethyl sulfoxide and dicyclohexylcarbodiimide.
  • Test compounds containing a trifluoromethyl group can be synthesised from the corresponding aldehyde dy reaction with TMS-CF 3 and TBAF, followed by Swern oxidation (Sammakia et al., J. Org. Chem., 2000, 65, 974-978.).
  • the conjugated Michael acceptor shown below may be obtained by reacting 4-formyl-2,2'- bipyridyl with vinyl magnesium bromide followed by rearrangement with acid to produce the allylic alcohol that is oxidized with manganese dioxide.
  • the B-linker can be designed via coupling of Che-COOH with diamines or amino acids as illustrated for a couple of Michael acceptors as C-moieties.
  • thiol compounds illustrated below may be obtained from the corresponding alkyl halide by reaction with NaSH.
  • the following compounds may be obtained from the corresponding mercaptans.
  • the unsymmetrical disulfide is made by treatment of the mercaptan with diethyl azodicarboxylate to give an adduct that is reacted with another mercaptan R'-SH.
  • R' is substituted or unsubstituted alkyl or aryl.
  • Test compounds designed for establishment of a non-covalent interaction such as, e.g., an ionic interaction may comprise test compounds of the following formulas:
  • libraries may also be designed such as, e.g., libraries of macrocyclic test compounds:
  • Metal atoms or ions forming the complex with the heteroalkyl or heterocyclyl moiety in the test compounds may be selected from metal atoms or ions which have been tested for or are used for pharmaceutical purposes.
  • Such metal atoms or ions belong to the groups denoted light metals, transition metals, post-transition metals or semi-metals (according to the periodic system).
  • the metal ion is selected from the group consisting of aluminium, antimony, arsenic, astatine, barium, beryllium, bismuth, boron, cadmium, calcium, cerium, caesium, chromium, cobalt, copper, dysprosium, erbium, europium, gadolinium, gallium, germanium, gold, hafnium, holmium, indium, iridium, iron, lanthanum, lead, lutetium, magnesium, manganese, mercury, molybdenum, neodymium, nickel, niobium, osmium, palladium, platinum, polonium, praseodymium, promethium, rhenium, rhodium, rubidium, ruthenium, sama
  • a particularly favourable test compound is a chelate between any of the test compounds of the formulas mentioned above and any of the metal atoms or ions mentioned above.
  • chelates between any of the test compounds and any atom or ion of Ru, Pt, Tc, Rh, Pd, Cu, Zn, Co and/or Ni are of interest in a target validation process according to the invention.
  • a library of the invention suitable for use in a target validation process is therefore a library comprising either the non-chelated test compounds or the chelated test compounds.
  • chelated test compound libraries comprise metals such as Pd and Pt forming more or less covalently bond complexes whereas non-chelated test compound libraries utilise endogenously available ions such as Zn or Cu in test preparations or animals which could have been spiked with additional non-toxic amounts of such metal ions.
  • the chelator is complexed with a metal ion before subjecting the test compound to the metal-ion binding site in the biological target molecule.
  • the preparation of such complexes is well-known to persons skilled in the art. However, there may be situations where the complex is formed relatively easy and then the complex may be formed in situ immediately before or at the same time as the testing is performed.
  • the test animals are normally animals, which have been genetically modified to express a silent metal ion site in a biological target molecule, which for example could be a metal-ion site engineered version of their own endogenous biologically target molecule or it could for example be a the human version of this molecule.
  • the advantages by using animals, which have been genetically modified, are that the animal is otherwise normal but express a biological target molecule which can be activated or inactivated by a specific drug-like substance, i.e. a test compound which has been optimised for selective interaction with the modified biological target molecule (Jackson I. J. and Abbott C. M.. (2000) "Mouse genetics and transgenics" Practical approach series.).
  • test compounds which form covalent bonds to the biological target molecule can be used as test compounds e.g. as part of all kinds of covalent or non-covalent complexes formed with other compounds or artificial or biological macromolecules which infer desired characteristics to the test compound for example in respect of bioavailability and/or pharmacokinetics.
  • administration routes may be employed including the oral, parenteral, topical, rectal, vaginal, ocular, nasal etc. route.
  • a convenient administration route is the oral and the parenteral route.
  • Dosage forms suitable for the oral route include solutions, dispersions, mixtures, emulsions, suspensions, tablets, capsules, sachets, powders, feeding powder, feeding mixture or drinking water, lotions, plasters, implants, etc.
  • the dosage forms may be in the form of a single unit or it may presented in the form of multiple units, e.g. in capsules containing a multiplicity of individual units.
  • the units may be in the form of pelletizied feed for animal feeding.
  • suitable dosage forms normally includes the use of one or more pharmaceutically acceptable excipients such as, e.g., fillers, binders, disintegrants, coating materials, solvents, emulsifiers, suspending agents, preservatives, stabilising agents, pH adjusting agents etc. all agents well-known to a person skilled in the art of pharmaceutical formulation.
  • pharmaceutically acceptable excipients such as, e.g., fillers, binders, disintegrants, coating materials, solvents, emulsifiers, suspending agents, preservatives, stabilising agents, pH adjusting agents etc. all agents well-known to a person skilled in the art of pharmaceutical formulation.
  • the dosage forms may be prepared in accordance with standard textbooks such as, e.g. Remington's Pharmaceutical Sciences.
  • the dose of a test compound depends on the specific test animal used. Normally, a daily dose is within the range of from about 1 ///kg to about 1g/kg depending on the particular test compound in question. A person skilled in the art will know to find a suitable dose range. Often the dose range lies between 0.1 and 100 mg/kg.
  • the dose may be given once daily or in separate doses during the day, e.g. two, three, four, five or six times daily or the test animal may have the dose at feeding time such as, e.g., together or via the feed. If the composition is in the form of a controlled release composition or in the form of an implant, the composition may be given less frequent such as, e.g. every day, every second day, every week, every month etc.
  • the animals After administration of the test substances to the genetically modified animals (and often also to control animals, i.e. animals which have not been genetically modified) the animals are monitored with respect to any biochemical, physiological, pharmacological and/or behavioural change compared to the control animal.
  • Chemical compounds or selections of chemical compounds described herein are suitable for use in a target validation process. Details on a suitable target validation process is given in the following items:
  • a target validation process for testing or validation the physiological importance and/or the therapeutic of a biological target molecule comprising
  • test compound and/or the biological target molecule optionally, chemically optimising the test compound and/or the biological target molecule to create secondary interaction(s) with chemical groups in the vicinity of the metal ion site in the silent metal ion engineered biological target molecule,
  • test compound optionally, chemically optimising the test compound to improve the pharmacokinetic and/or biopharmaceutical properties of the test compound
  • a target validation process according to item 1 , wherein the biological target molecule is selected from the group consisting of proteins, polypeptides, oligopeptides, nucleic acids, carbohydrates, nucleoproteins, glycoproteins, glycolipids, lipoproteins and derivatives thereof.
  • a target validation process wherein the biological target molecule is a protein selected from the group consisting of membrane receptors, signal transduction proteins, scaffolding proteins, nuclear receptors, steroid receptors, intracellular receptors, transcription factors, enzymes, allosteric enzyme regulator proteins, growth factors, hormones, neuropeptides or immunoglobulins.
  • the biological target molecule is a protein selected from the group consisting of membrane receptors, signal transduction proteins, scaffolding proteins, nuclear receptors, steroid receptors, intracellular receptors, transcription factors, enzymes, allosteric enzyme regulator proteins, growth factors, hormones, neuropeptides or immunoglobulins.
  • a target validation process according to item 4 wherein the biological target molecule is a membrane protein and the silent metal ion site in the biological target molecule is introduced in a ligand binding crevice of the membrane protein. 6. A target validation process according to item 4, wherein the membrane protein is an integral membrane protein.
  • the membrane protein is a receptor such as a tyrosine kinase receptor, e.g. a growth factor receptor such as the growth hormone, insulin, epidermal growth factor, transforming growth factor, erythropoietin, colony-stimulating factor, platelet-derived growth factor receptor or nerve growth factor receptor (TrkA or TrkB).
  • a tyrosine kinase receptor e.g. a growth factor receptor such as the growth hormone, insulin, epidermal growth factor, transforming growth factor, erythropoietin, colony-stimulating factor, platelet-derived growth factor receptor or nerve growth factor receptor (TrkA or TrkB).
  • the membrane protein is a ligand-gated ion channel, such as a nicotinic acetylcholine receptor, GABA receptor, or glutamate receptor (NMDA or AMPA).
  • a ligand-gated ion channel such as a nicotinic acetylcholine receptor, GABA receptor, or glutamate receptor (NMDA or AMPA).
  • a target validation process wherein the membrane protein is a 7TM receptor, a G-protein coupled receptor, such as the receptor for (- in brachet the receptor subtypes are mentioned): acetylcholine (m1-5), adenosine (A1-3) and other purines and purimidines (P2U and P2Y1-12), adrenalin and noradrenalin ( ⁇ 1A-D, ⁇ 2A-D and ⁇ 1-3), amylin, adrenomedullin, anaphylatoxin chemotactic factor, angiotensin (AT1A, - 1 B and -2), apelin, bombesin, bradykinin (land 2), C3a, C5a, calcitonin, calcitonin gene related peptide, CD97, conopressin, corticotropin releasing factor (CRFIand -2), calcium, cannabinoid (CBIand -2), chemokines (CCR1-11 , C
  • a target validation process wherein the membrane protein is a transporter protein, such as, e.g. i) Na + cotransporters, including Na + ,CI " transporters, such as, e.g., GABA transporters, monoamine transporters, neutral amino acids transporters, kreatinin transporters and nucleoside transporters, and Na?K + coupled transporter such as, e.g., glutamate transporters, neutral amino acids transporters, and inositol transporters, and Na + ,glucose cotransporters, and Na + ,K + ,CI " cotransporters, ii) H+ coupled transporter including oligopeptide transporters and multi drug transporters, iii) antiporters, including Na + /H + - exchangers, anion exchangers such as, e.g., HCO 3 7CI " exchangers and Na + /Ca + exchangers, iv) ion-transporting ATPases including Na + ,
  • ATPase H + ,K + ATPase and Ca 2+ ATPase and v) transporters from the ABC (ATP Binding Cassette) transporter family, including multidrug resistance related proteins and cystic fibrosis transmembrane regulators, and multidrug resistance proteins such as, e.g., P- glycoproteins, lung resistance related proteins and breast cancer resistance proteins.
  • ABC ATP Binding Cassette
  • a target validation process according to item 7, wherein the membrane protein is a multidrug resistance protein, e.g. a P-glycoprotein, multidrug resistance associated protein, drug resistance associated protein, lung resistance related protein, breast cancer resistance protein, adenosine triphosphate-binding cassette protein, Bmr, QacA or EmrAB/TolC pump.
  • the membrane protein is a cell adhesion molecule, e.g. NCAM, VCAM or ICAM.
  • a target validation process wherein the silent metal ion site is constructed in a biological target molecule by mutating one or more amino acid residues into one or more amino acid residues capable of binding a metal ion.
  • a target validation process according to items 18 or 19, wherein silent metal ion site comprises at least two such as, e.g., at least three, at least four, at least five such as, e.g., two, three or four amino acid residues capable of binding a metal ion.
  • a target validation process according to item 1 , wherein the silent metal ion site is constructed in a biological target molecule by mutating one or more non-natural amino acid residues into residues which in themselves are capable of binding a metal ion or which chemically can be modified to become a metal ion binding residue.
  • a target validation process according to any of the preceding items, wherein the silent metal ion site in the silent metal ion site engineered biological target molecule - when tested in an in vitro cell expression system- results in at the most a 20 fold such as, e.g., at the most 10, at the most 8, at the most 5, at the most 4, at the most 3 fold decrease in surface expression and/or affinity.
  • a target validation process according to any of the preceding items, wherein the silent metal ion site in the silent metal ion site engineered biological target molecule - when tested in vivo - does not alter or significantly alter the function of the endogenous biological target molecule.
  • test compound is has a log K value in a range of from about 1 to about 50.
  • test compound forms a chelate with a metal ion selected from the group consisting Ni, Pd, Pt, Ru and Zn including all possible oxidation steps such as, e.g., Pt(0), Pt (II), Pt (IV), Pd (0), Pd(ll), Pd(IV), Rh , Ru(0), Ru(ll), Ru(lll), Ru(IV), Ru(VI) and Ru(VIII).
  • a metal ion selected from the group consisting Ni, Pd, Pt, Ru and Zn including all possible oxidation steps such as, e.g., Pt(0), Pt (II), Pt (IV), Pd (0), Pd(ll), Pd(IV), Rh , Ru(0), Ru(ll), Ru(lll), Ru(IV), Ru(VI) and Ru(VIII).
  • test compound forms a chelate with a metal ion selected from the group consisting of Co, Cu Zn and Ni including the various oxidation steps.
  • test compound has at least two heteroatoms, similar or different, selected from the group consisting of nitrogen (N), oxygen (O), sulphur (S), selenium (Se) and phosphorous (P).
  • step ii) or item 1 comprises
  • test compound which comprises a moiety for chelating a metal ion under conditions permitting non-covalent binding of the test compound to the metal ion site of the metal ion site engineered biological target molecule
  • irreversible or slowly reversible covalent bonds ii) irreversible or slowly reversible covalent bonds, ii) charge-charge interaction (introduction of charged groups such as ammonium, phosphonium or sulphonium groups, or ionisable groups such as amino or carboxy groups), iii) hydrogen bond interactions (introduction of e.g.
  • 35. A target validation process according to item 34, wherein the covalent bond is established with a thiol-containing amino acid residue (e.g. Cys) or an amine-containing amino acid residue (e.g. Lys).
  • a target validation process according to any of the preceding items, wherein a residue, which can form a secondary chemical interaction with the test compound, is introduced into the biological target molecule.
  • test compound is optimised for appropriate properties with respect to in vivo absorption, distribution metabolism and excretion, or with respect to biopharmaceutical properties such as, e.g., water solubility, lipid solubility or particle size.
  • test compound is linked to a carrier.
  • a target validation process wherein the carrier is a protein, an oligopeptide, a peptide, a polysaccharide, an oligosaccharide, a polyethylene glycol, a poly lactic acid, a poly glycolic acid, a poly(lactic-glycolic) acid, an acrylic acid polymer, an ethyl-vinyl acetate polymer, hyaluronic acid, gelatine, an antibody, a fragment of an antibody or the like.
  • the carrier is a protein, an oligopeptide, a peptide, a polysaccharide, an oligosaccharide, a polyethylene glycol, a poly lactic acid, a poly glycolic acid, a poly(lactic-glycolic) acid, an acrylic acid polymer, an ethyl-vinyl acetate polymer, hyaluronic acid, gelatine, an antibody, a fragment of an antibody or the like.
  • a target validation process according to any of items 37-41 , wherein the link between the test compound and the carrier is established through formation of a complex or through a covalent binding.
  • a target validation process according to any of items 37-41 wherein the link between the test compound and the carrier is established through a spacer.
  • 44. A target validation process, wherein the genetically modified animal is an animal, which has a metal-ion site biological target molecule.
  • a target validation process wherein the genetically modified animal is an animal into which a metal ion site engineered biological molecule has been introduced.
  • a target validation process wherein the genetically modified animal is an animal, which expresses a metal ion site engineered biological molecule.
  • a target validation process according to any of the preceding items, wherein the genetically modified animal containing a metal ion site engineered biological target molecule is obtained by employment of a double replacement method.
  • test compound optionally chemically optimised
  • biochemical, physiological and/or behaviour parameters compared are monitored.
  • a target validation process according to any of the preceding items, wherein the metal ion is one that binds to an amino acid residue containing a S, O, N, Se and/or P atom or with an aromatic amino acid residue.
  • Figure 1 shows the generic nomenclature of 7TM receptors exemplified by family A 7TM receptors. One or two conserved key residues are highlighted in each TM: Asnl:18; Aspll:10; Cyslll:01 and Arglll:26; TrplV:10; ProV:16; ProVI:15; ProVII:17.
  • Figure 2 shows the syn, anti and direct coordination of the metal-ion in carboxylate groups. The percentage of each is shown in brackets, based on the analyses of 67 compounds from Cambridge Structural Database (Carrell et al, 1988).
  • Figure 3 shows the two tautomeric forms of the neutral imidazole side chain of histidine. Without metal, the NH-epsilon-2 form is predominant (80%), whereas the NH-delta-1 form predominates upon metal-binding (75%).
  • Figure 4 shows the geometric shapes and coordination numbers of metal complexes. The most common motifs are italicised.
  • Figure 5 relates to the Identification of naturally occurring metal-ion binding site in the 7TM leukotriene LTB4 receptor in Example 1.1.
  • the figure shows a whole cell competition binding experiment with COS-7 cells expressing the wild type and mutant variants of the leukotriene LTB4 receptor using [ 3 H]-LTB4 as the radioligand.
  • Panel A Affinity of Cu(ll), 2,2'-bipyridine and the complex therof in the wild type LTB4 receptor.
  • Panel B Affinity of Cu(biprydine) in mutant forms of the LTB4 receptor in which the metal- ion binding is severely imparired.
  • Panel C Helical wheel diagram illustrating the transmembrane segments of the LTB4 receptor.
  • the two cysteine residues within the transmembrane segment III which have been identified as critical for metal-ion chelator complex binding, Cys93 and Cys97 are indicated in dark gray.
  • Figure 6 relates to identification of a naturally occurring metal-ion binding site in the 7TM Galanin receptor-1 in Example I.2.
  • Panels A-F Mutational analysis and identification of putative Zn(ll) chelating residues in the Gal-R1.
  • Panels G Model of the Gal-R1 receptor with putative Zn(ll) chelating residues shown in the transmembrane segments.
  • Figure 7 relates to identification of naturally occurring metal-ion chelator binding site in the 12TM dopamine transporter in Example I.3.
  • the figure shows a competition analysis of uptake of [ 3 H]-dopamine in whole COS-7 cells expressing the dopamine transporter.
  • Panel A Uptake of [ 3 H]-dopamine by the wild-type dopamine transporter in the presence of free metal zinc-ion and zinc in complex with the chelator 2,2'-bipyridine.
  • Panel B Dopamine uptake analysis in a mutant form of the dopamine transporter,
  • Figure 8 shows the binding of various metal-ion complexes to a library of inter-helical metal-ion sites engineered into the tachykinin NK1 receptor as described in Example 11.1.
  • COS-7 cells expressing various engineered forms of the NK1 receptor were analyzed by competition binding using [ 125 l]-Substance P as radioligand.
  • IC 50 values for the zinc and copper metal-ions and complexes thereof with the chelators, 2,2'-bipyridine and phenanthroline are presented in the table. N indicated the number of experiments performed.
  • Panel B Data obtained using the chelator cyclam are presented for the NK1 mutant in which an inter-helical metal-ion site has been generated through the introduction of the
  • Panel C A helical diagram representing the four sets of inter-helical metal-ion sites which appear in Panel A are indicated.
  • Figure 9 relates to re-engineering of a metal-ion chelator binding site in the 12TM dopamine transporter as described in Example 11.2.
  • Dopamine uptake was analysed in COS-7 cells expressing the wild type and mutant forms of the dopamine transporter in competition with the metal-ion chelator complex, zinc(ll)-2,2'-bipyridine.
  • the two panels show two forms of re-engineered dopamine transporters in which the ability to bind the metal-ion chelator complexes have been reconstituted following the elimination of the His193 interaction point.
  • Figure 10 relates to fluorescence measurement of the relative strength of a selection of chelators to chelate Zn(ll) in competition with FluoZin-3 as described in Example II.3.
  • Figure 11 shows the structure-activity relationship of metal-ion complexes in the leukotriene LTB4 receptor as described in Example III.1. Competition binding analysis in COS-7 cells expressing the LTB4 receptor. Binding of [ 3 H]-LTB4 was analysed in the presence of various copper-ion chelator complexes.
  • Figure 12 shows the structure-activity relationship of antagonist metal-ion complexes in the metal-ion site engineered tachykinin NK1 receptor as described in Example III.2 Binding of [ 125 l]-Substance P was analysed in COS-7 cells expressing NK1 receptor which have been engineered to bind the zinc metal-ion. Ligand binding is presented in competiton with the zinc metal-ion, the zinc-1 ,10-phenanthroline complex and with other zinc-chelator xomplexes as indicated.
  • Figure 13 relates to engineering o agonistic metal-ion bindng sites in the Beta-2 adrenergic receptor, demonstrating the importance of the specific amino acids defining the site as described in Example 111.3.1.
  • Panel A Agonistic metal-ion binding sites probed with either Cu(ll)-(2,2'-bipyridine)3 or Cu(ll)-(1 ,10-phenanthroline)3 demonstrating the importance of the specific amino acids composing the site in defining the potency of the sites.
  • Panel B Histogram showing the observed efficacy using copper-complexes of 2,2'- bipyridine or 1 ,10-phenanthroline on selected engineered agonistic metal-ion sites in the Beta-2 adrenergic receptor demonstrating the importance of the observed efficacy on the specific amino acids composing the site. See also figure 20.
  • Figure 14 shows the structure-activity relation ship of agonistic metal-ion complexes in the metal-ion site engineered beta-2-adrenergic receptor as described in Example III.3.2
  • Panel A Washing experiment demonstrating the reversibility of the stimulatory action of the metal-ion complexes.
  • Panel B Dose-response analysis of selected copper-chelator complexes on the
  • Figure 15 shows the structure-activity relation ship of agonistic metal- ion complexes in the metal-ion site engineered Beta-2-adrenergic receptor.
  • the effect of Cu(ll)-chelator complexes on stimulation of accumulation of intracellular cAMP was analyzed in COS-7 cells expressing the beta2-adrenoceptor.
  • Panel C Testing a library of Cu(ll)-2,2'-Bipyridine complexes at 10 micromolar on the [[F289C]-Beta-2 Adrenergic receptor for their efficacy in stimulating cAMP. For a list of compounds see the list below.
  • Figure 16 shows the structure-activity relationship of antagonistic metal-ion complexes in a soluble protein, the enzyme factor Vila as described in Example III.4.
  • the figure shows a comparison of selected metal-ion complexes on the binding of [3H]-LTB4 and the inhibition of the enzymatic activity of the active form of Factor VII (FVIIa) in COS-7 cells expressing respectively the LTB4 receptor (Panel B) and the FVIIa (Panles A and C).
  • FVIIa Factor VII
  • Figure 17 shows a structure-based optimization of metal-ion chelators for secondary interactions in the CXCR4 receptor and other biological targets as described in Example III.5 Helical wheel diagram for the CXCR4 receptor.
  • the Asp171 residue present in the transmembrane segment IV, and which is considered a major attachment site for the binding of the cyclam chelator is shown in white on black. Positions, which in combination are proposed to constitute putative metal-ion binding sites, are high-lighted in pairs and in black on dark gray.
  • Figure 18 relates to affinity optimization of metal-ion chelators in the LTB-4 leukotriene receptor as described in Example 111.6.
  • Figure 19 relates to probing different metal-ions in an engineered Bis-His TM-V Kappa opioids.
  • Figure 20 shows the dependency of amino acids defining the metal-ion site, metal-ion or metal-ion chelator on the observed efficacy in agonist metal-ion binding sites in the beta-2 adrenergic receptor.
  • FIG. 7 The figure illustrates the establishment of increased affinity in a silent metal-ion site engineered receptor through second-site interaction obtained by side-chain modification of a stable metal-ion chelator complex to be used in a genetically modified animal.
  • Signal transduction is determined as accumulation of [3H] Inositol triphosphat in COS-7 cells expressing either the wild-type RASSL receptor (called RO2 in the figure) or the metal-site engineered RASSL receptor (called CysVII:06 in the figure).
  • the metal ion site is located between position TM lll:08 (a natural Asp residue) and Vll:06 (an engineered Cys residue).
  • the receptors are stimulated with a constant dose of the non-peptide agonist ICI 199,441 to a sub-maximal level and inhibitory dose- response experiments are performed with preformed stable Pd(ll) complex with either 4,4- dimethyl-bipyridine (chemical structure shown to the upper right and dose-response experiments in wild-type receptor and metal-ion site engineered receptor are shown in the panel to the upper left) or in complex with compound 433 (chemical structure shown to the lower right and dose-response experiments in wild-type receptor and metal-ion site engineered receptor are shown in the panel to the lower left) - in both cases acetate (AcO) was used as leaving group.
  • 4,4- dimethyl-bipyridine chemical structure shown to the upper right and dose-response experiments in wild-type receptor and metal-ion site engineered receptor are shown in the panel to the upper left
  • compound 433 chemical structure shown to the lower right and dose-response experiments in wild-type receptor and metal-
  • Figures 22 and 23 show the structure-activity relationship of antagonist metal-ion complexes in the metal-ion site engineered RASSL receptor. Accumulation of [3H] Inositol triphosphat was measured in COS-7 cells expressing RASSL receptor, which have been engineered to bind the zinc metal-ion. The metal ion site is located between TM 111:08 and Vll:06. The receptor is stimulated with the non-peptide agonist IC1 199,441 to a sub- maximal level and inhibited with the zinc-1 ,10-phenanthroline complex and with the 5'- chloro substituted phenenthroline analog.
  • Figure 24 shows an antagonist of different metal-ions in complex with 5-chloro1 ,10 phenanthroline in the metal-ion site engineered RASSL receptor.
  • Accumulation of [3H] Inositol triphosphat was measured in COS-7 cells expressing RASSL receptor, which have been engineered to bind the zinc metal-ion.
  • the metal ion site is located between TM 111:08 and Vll:06.
  • the receptor is stimulated with the non-peptide agonist IC1 199,441 to a sub-maximal level and inhibited with the 5-chloro-1 ,10-phenanthroline in complex with Zn(ll) and in complex with Pd(ll).
  • Figure 25 shows that Pd(ll)-5-chloro1 ,10phenanthroline act as an antagonist in the metal- ion site engineered RASSL receptor but not in the wild type RASSL receptor.
  • Accumulation of [3H] Inositol triphosphat was measured in COS-7 cells expressing wild type RASSL receptor and the RASSL receptor, which have been engineered to bind the metal-ion.
  • the metal ion site is located between TM lll:08 and Vll:06.
  • the receptors are stimulated with the non-peptide agonist ICI 199,441 to a sub-maximal level and inhibited with the Pd(ll)-5-chloro-1 ,10-phenanthroline.
  • Figure 26 shows pre-incubation with Pd(ll)-5-chloro1 ,10phenanthroline before stimulation with the non-peptide agonist ICM 99.441 in the metal-ion site engineered RASSL receptor. Accumulation of [3H] Inositol triphosphat was measured in COS-7 cells expressing the RASSL receptor, which have been engineered to bind the metal-ions. The metal ion site is located between TM lll:08 and Vll:06. The receptor is pre-incubated with the Pd(II)-5- chlorophenanthrolin for 30 min and after repeated washing it is stimulated with the non- peptide agonist ICI 199,441.
  • Pd(II)-5- chlorophenanthrolin for 30 min and after repeated washing it is stimulated with the non- peptide agonist ICI 199,441.
  • Formula 1 may be constructed by well-known synthetic steps involving coupling reactions, including Stille-, Suzuki-, Negishi-, Ullmann-couplings (C-C bond formations), condensation reactions, including heterocyclic ring-forming reactions, elimination reactions, cycloaddition reactions, and/or substitution reactions known from the common literature, as illustrated with some typical but non-limiting reaction schemes.
  • coupling reactions including Stille-, Suzuki-, Negishi-, Ullmann-couplings (C-C bond formations), condensation reactions, including heterocyclic ring-forming reactions, elimination reactions, cycloaddition reactions, and/or substitution reactions known from the common literature, as illustrated with some typical but non-limiting reaction schemes.
  • the usual considerations regarding which functional groups that are compatible with the different types of chemistries should always be taken into account when selecting synthetic routes, order of introduction of functional groups and their interconversions, etc, which accordingly will differ on a case by case basis but are evident for the skilled person.
  • Scheme II illustrates the C-C-bond forming reaction in the 2,2'-bipyridine series.
  • Coupling of functionalised heterocyclic ring systems such as chloropyridines with trialkyl tin pyridines can be performed by the Stille coupling method, and exemplified in Scheme V.
  • chelator systems may be formed and manipulated.
  • a chelator which have one of the coordinating atom(s) outside the ring system is 2-(2- pyridyl)thiophenol (See Scheme XV).
  • the construction may follow different routes, i.e. the coordinating atoms may be introduced at various stages, protected or unprotected, schematically illustrated in Scheme XV.
  • 3-Aminomethyl-2,2'-bipyridine 3-hydroxymethyl-2,2'-bipyridine (0.19mmol, 36 mg) was dissolved in dry THF (5 ml), with triethylamine (0.3 ml) at ambient temperature before PS- tosyl chloride (200 mg) was added. The resulting suspension was shaken for 3h before the resin was removed by filtration and washed sequentially with dimethylformamide (2 x 5 ml), tetrahydrofuran (2 x 5 ml) and dichloromethane (2 x 5 ml). To the resin was added dry dichloromethane (5 ml) then through this suspension was passed a stream of ammonia gas for a period of 10 min.
  • Methanethiosulfonic acid S- ⁇ 4-[4-([2,2']bipyridinyl-5-carbonyl)-piperazin-1-yl]-4-oxo-butyl ⁇ ester The thiol from Example 8 (0.135mmol, 50 mg) was dissolved in dichloromethane (0.5 ml) under nitrogen atmosphere before methanesulphonyl chloride (0.149mmol, 11.5 ⁇ ) was added. The solution was stirred for 2 h before a further portion of methanesulphonyl chloride (0.149mmol, 11.5 //I) was added and the solution stirred for a further 16 h. The volatiles were removed in vacuo.
  • 5-Thiomethyl-2,2'-bipyridine 5-Hydroxymethyl-2,2'-bipyridine (2.0 g, 10.7 mmol) was added thionyl chloride (40 ml, 0.55 mol), and the reaction was heated to 70 °C for 2 h. The reaction was allowed to reach room temperature, and concentrated to give 5- chloromethyl-2,2'-bipyridine dihydrochloride. The crude 5-chloromethyl-2,2'-bipyridine dihydrochloride (137 mg, 0.67 mmol) in MeOH (3 ml) was added thiourea (140 mg, 1.84 mmol), and the reaction mixture was heated to reflux.
  • 5-Amino-2,2 -bipyridine 5-Nitro-2,2'-bipyridine (0.641 mol, 129 mg) was dissolved in MeOH/THF (5 ml+5 ml). To the solution was added Pd/C (5 %, 50 mg) and the reaction mixture was set under an H 2 -atmosphere and stirred for 24 h at room temperature. The reaction mixture was filtered through Celite, and the filtrate was evaporated in vacuo. The residue was purified by column chromatography (neutral AI 2 O 3 , 5 % EtOH in DCM), to yield the desired product. Yield: quantitative.
  • 4-(4-Aminobutyl)-4'-methyl-2,2'-bipyridyl 4-(3-Cyanopropyl)-4'-methyl-2,2'-bipyridyl (125 mg, ca. 0.5 mmol) was dissolved in 96 % ethanol (5 ml) and catalytic amount of Raney nickel was added. The reaction was stirred over night under 1 atmosphere of hydrogen. Evaporated and purified by chromatography (alumina, DCM:MeOH:NH 4 OH 95:5:0.5). Yield: 70 mg (58 %).
  • 6-Chloro- fe/t-butylnicotinate (12.7 mmol, 2.7g) was dissolved in dry m-xylene (150 ml) whereupon Me 3 SnSnMe 3 (15.26 mmol, 5.0 g) was added together with PdCI 2 (PPh 3 ) 2 (1.5 mmol, 1.0g). The reaction solution was heated to 130C under an N 2 atmosphere for 4h.
  • N-(8-Hydroxy-quinolin-5-yl)-acetamide N-(8-Hydroxy-quinolin-5-yl)-acetamide.
  • 5-Amino-8-hydroxyquinoline (1 mmol, 0.233g) was stirred in ether at ambient temperature before acetic anhydride (10 mmol, 1 ml), followed by sodium acetate (10 mmol, 1.36g) was added.
  • the resulting mixture was heated to 40 °C for 16 h before being diluted with ether (100ml) poured onto a saturated solution of ammonium chloride (50 ml).
  • the organics were separated and washed with sodium bicarbonate (50 ml), water (3 x 50 ml), brine (50 ml), dried over sodium sulphate and concentrated in vacuo.
  • N-(8-Hydroxy-quinolin-5-yl)-4-trifluoromethyl-benzamide 5-Amino-8-hydroxyquinoline (0.15mmol, 25 mg) was dissolved in dry dichloromethane (5 ml) before the sequential addition of dimethyl formamide (0.2 ml), ⁇ /, ⁇ /,-dimethylaminopyridine (1 crystal), PS- carbodiimide (750mg) and 1-hydroxybenzotriazole monohydrate (0.6mmol, 81 mg).
  • 2-(2-Pyridyl)fluorobenzene 2-Fluorophenylboronic acid (3.0 g, 21.4 mmol) was dissolved in DME (40 ml). 2-Bromopyridine (1.64 ml, 17.2 mmol) was added followed by 2M K 2 CO 3 (20 ml). The mixture was degassed by bubbling nitrogen gas through for 34 min. Bis- (triphenylphosphine)palladium chloride (1.2 g, 1.72 mmol) was added and the mixture was heated to 80°C over night. The mixture was cooled to room temperature and filtered through celite.
  • 2-(2-Pyridyl)thiophenol S-ferf-Butyl-2-(2-pyridyl)thiophenol (200 mg, 0.82 mmol) was dissolved in 37 % HCl (4 ml) and the mixture was heated to 110 °C over night. The mixture was cooled to room temperature. H 2 O (10 ml) was added and the mixture was extracted with EtOAc (20 ml). pH of the aqueous phase was adjusted to 7 and the mixture was extracted with EtOAc (50 ml). The organic phase was dried over MgSO 4 , filtered and evaporated. Purification by column chromatography (SiO2, EtOAc:Heptane 1 :1).
  • 2-(2-Pyridyl)pyrazine 2-Chloropyrazine (100 mg, 0.87 mmol) was dissolved in m-xylen (2 ml), 2-tri-n-butylstannylpyridin (354 mg, 0.96 mmol) was added followed by bis- (triphenylphosphine)palladium chloride (1.2 mg, 0.0017 mmol). The mixture was heated to 130 °C over night under nitrogen. The mixture was allowed to cool to room temperature. The crude mixture was purified by column chromatography (SiO2; EtOAc:Heptane 1 :1).
  • N-Hydroxy-pyridine-2-carboxamidine Sodium (0.53 g 23 mmol) was dissolved in MeOH (15 ml), hydroxylamine hydrochloride (1.53 g 22 mmol) dissolved in MeOH (15 ml) was added, and stirred in ice bath for 1 hour. After filtration the solution was added 2- cyanopyridin (1.93 ml, 20mmol),and stirred at R.T. over night. The reaction mixture was reduced in vacuo. After cooling on ice the product precipitate. Filtered and washed with diethyl ether. Yield: 2.1 g (73 %).

Abstract

L'invention concerne l'utilisation de composés chimiques ou de sélections de composés chimiques (bibliothèques) représentés par la formule générale (I) dans des méthodes in vivo de contrôle ou de validation de l'importance physiologique et/ou du potentiel thérapeutique ou pharmacologique de molécules biologiques cibles, et notamment de protéines telles que des récepteurs, et en particulier des récepteurs 7TM, dans des animaux expérimentaux exprimant la molécule biologique cible avec, notamment, un site d'ions métalliques silencieux manipulé. L'invention concerne également l'utilisation de sites de liaison d'ions métalliques spécifiques de nature générique dans des molécules biologiques cibles spécifiques telles que les protéines transmembranaires dans lesquelles le site de liaison d'ions métalliques peut former un complexe avec un ion métallique. L'invention concerne en outre des composés chimiques ou des bibliothèques, destinés à être utilisés dans des méthodes visant à améliorer le comportement pharmacocinétique in vivo des chélates à ions métalliques (par exemple les caractéristiques d'absorption, la demi-vie plasmatique, la distribution, le métabolisme et/ou l'élimination des chélates à ions métalliques). Afin d'améliorer l'efficacité de l'effet des chélates à ions métalliques sur la molécule biologique cible après l'administration du chélate à ions métalliques in vivo à un animal expérimental, il est avantageux, par exemple, d'améliorer la durée pendant laquelle le chélate à ions métalliques se trouve dans le système circulatoire et/ou localisé au niveau de la cible. L'invention concerne enfin des composés de chélation à ions métalliques destinés à être utilisés dans un processus de validation de cible selon l'invention et des bibliothèques d'au moins deux ou plusieurs de ces composés de chélation à ions métalliques.
PCT/DK2002/000456 2001-06-29 2002-06-28 Utilisation de chelates a ions metalliques dans la validation de molecules biologiques utilisees comme cibles medicamenteuses dans des modeles animaux experimentaux WO2003003009A1 (fr)

Applications Claiming Priority (14)

Application Number Priority Date Filing Date Title
US30193101P 2001-06-29 2001-06-29
DKPA200101031 2001-06-29
DKPA200101030 2001-06-29
DKPA200101028 2001-06-29
DKPA200101026 2001-06-29
US60/301,931 2001-06-29
DKPA200101027 2001-06-29
DKPA200101031 2001-06-29
DKPA200101028 2001-06-29
DKPA200101027 2001-06-29
DKPA200101026 2001-06-29
DKPA200101030 2001-06-29
PCT/DK2001/000867 WO2002054077A2 (fr) 2000-12-29 2001-12-21 Validation de molecules biologiques en tant que cibles de medicaments au moyen de chelates d'ions metalliques chez des modeles d'animaux de laboratoire
DKPCT/DK01/00867 2001-12-21

Publications (1)

Publication Number Publication Date
WO2003003009A1 true WO2003003009A1 (fr) 2003-01-09

Family

ID=27561825

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2002/000456 WO2003003009A1 (fr) 2001-06-29 2002-06-28 Utilisation de chelates a ions metalliques dans la validation de molecules biologiques utilisees comme cibles medicamenteuses dans des modeles animaux experimentaux

Country Status (1)

Country Link
WO (1) WO2003003009A1 (fr)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006124748A3 (fr) * 2005-05-13 2007-03-01 Lexicon Genetics Inc Composes polycycliques et procedes pour les utiliser
CN1306848C (zh) * 2004-04-30 2007-03-21 中兴通讯股份有限公司 使用智能网方式实现双模手机呼叫的***和方法
US7425556B2 (en) 2005-12-20 2008-09-16 Astrazeneca Ab Compounds and uses thereof
US7465795B2 (en) 2005-12-20 2008-12-16 Astrazeneca Ab Compounds and uses thereof
FR2921062A1 (fr) * 2007-09-17 2009-03-20 Commissariat Energie Atomique Composes utiles comme ligands et notamment comme chromophores organiques de complexation des lanthanides et leurs applications
JP2011219577A (ja) * 2010-04-07 2011-11-04 Jsr Corp 新規化合物および新規色素
CN102533247A (zh) * 2010-12-12 2012-07-04 陈文通 一种含镝荧光晶体及其制备方法
CN102924529A (zh) * 2012-11-22 2013-02-13 中山大学 一种环金属化铱-偶氮配合物及其制备方法和应用
CN103254127A (zh) * 2013-05-28 2013-08-21 北京哈三联科技有限责任公司 甘氨酸重摄取抑制剂及其应用
CN109734709A (zh) * 2019-02-12 2019-05-10 温州医科大学 一种小分子荧光探针及其制备方法与应用
US10647661B2 (en) 2017-07-11 2020-05-12 Vertex Pharmaceuticals Incorporated Carboxamides as modulators of sodium channels
US10752594B2 (en) 2013-03-14 2020-08-25 Sumitomo Dainippon Pharma Oncology, Inc. JAK1 and ALK2 inhibitors and methods for their use
US10894797B2 (en) 2018-09-18 2021-01-19 Nikang Therapeutics, Inc. Fused tricyclic ring derivatives as SRC homology-2 phosphatase inhibitors
CN112920254A (zh) * 2020-02-14 2021-06-08 西南交通大学 高效抗凝的功能分子、螯合物及其应用、仿生功能材料及其制备方法
US11040038B2 (en) 2018-07-26 2021-06-22 Sumitomo Dainippon Pharma Oncology, Inc. Methods for treating diseases associated with abnormal ACVR1 expression and ACVR1 inhibitors for use in the same

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998005961A1 (fr) * 1996-08-01 1998-02-12 Isis Pharmaceuticals, Inc. Composes contenant une pluralite de substituants azotes
WO1999010016A1 (fr) * 1997-08-21 1999-03-04 Resolution Pharmaceuticals Inc. Banque de composes de combinaison
WO2001006260A1 (fr) * 1999-07-19 2001-01-25 California Institute Of Technology Detection de molecules biologiques a l'aide de substrats lies a un agent sensibilisateur
WO2001050127A2 (fr) * 1999-12-30 2001-07-12 7Tm Pharma Methode permettant d'identifier des ligands de molecules biologiques cibles

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998005961A1 (fr) * 1996-08-01 1998-02-12 Isis Pharmaceuticals, Inc. Composes contenant une pluralite de substituants azotes
WO1999010016A1 (fr) * 1997-08-21 1999-03-04 Resolution Pharmaceuticals Inc. Banque de composes de combinaison
WO2001006260A1 (fr) * 1999-07-19 2001-01-25 California Institute Of Technology Detection de molecules biologiques a l'aide de substrats lies a un agent sensibilisateur
WO2001050127A2 (fr) * 1999-12-30 2001-07-12 7Tm Pharma Methode permettant d'identifier des ligands de molecules biologiques cibles

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ELLING CHRISTIAN E ET AL: "Connectivity and orientation of the seven helical bundle in the tachykinin NK-1 receptor probed by zinc site engineering", EMBO JOURNAL, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 15, no. 22, 1996, pages 6213 - 6219, XP002174723, ISSN: 0261-4189 *
ELLING CHRISTIAN E ET AL: "Conversion of agonist site to metal-ion chelator site in the beta2-adrenergic receptor", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 96, no. 22, 26 October 1999 (1999-10-26), pages 12322 - 12327, XP002174721, ISSN: 0027-8424 *
ELLING CHRISTIAN E ET AL: "Disulfide bridge engineering in the tachykinin NK1 receptor", BIOCHEMISTRY, AMERICAN CHEMICAL SOCIETY. EASTON, PA, US, vol. 39, no. 4, 1 February 2000 (2000-02-01), pages 667 - 675, XP002174724, ISSN: 0006-2960 *
NORREGAARD LENE ET AL: "Delineation of an endogenous zinc-binding site in the human dopamine transporter", EMBO JOURNAL, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 17, no. 15, 3 August 1998 (1998-08-03), pages 4266 - 4273, XP002174722, ISSN: 0261-4189 *
SZURDOKI F ET AL: "A combinatorial approach to discover new chelators for optical metal ion sensing.", ANALYTICAL CHEMISTRY, vol. 72, no. 21, 1 November 2000 (2000-11-01), pages 5250 - 5257, XP002902763 *
WANG F ET AL: "A Convenient Set of Bidentate Pyridine Ligands for Combinatorial Synthesis", TETRAHEDRON LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 40, no. 26, 25 June 1999 (1999-06-25), pages 4779 - 4782, XP004168642, ISSN: 0040-4039 *

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1306848C (zh) * 2004-04-30 2007-03-21 中兴通讯股份有限公司 使用智能网方式实现双模手机呼叫的***和方法
WO2006124748A3 (fr) * 2005-05-13 2007-03-01 Lexicon Genetics Inc Composes polycycliques et procedes pour les utiliser
US7425556B2 (en) 2005-12-20 2008-09-16 Astrazeneca Ab Compounds and uses thereof
US7465795B2 (en) 2005-12-20 2008-12-16 Astrazeneca Ab Compounds and uses thereof
US9040689B2 (en) 2007-09-17 2015-05-26 Commissariat A L'energie Atomique Compounds useful as ligands and particularly as organic chromophores for complexing lanthanides and applications thereof
FR2921062A1 (fr) * 2007-09-17 2009-03-20 Commissariat Energie Atomique Composes utiles comme ligands et notamment comme chromophores organiques de complexation des lanthanides et leurs applications
WO2009037277A1 (fr) * 2007-09-17 2009-03-26 Commissariat A L'energie Atomique Composes utiles comme ligands et notamment comme chromophores organiques de complexation des lanthanides et leurs applications
JP2011219577A (ja) * 2010-04-07 2011-11-04 Jsr Corp 新規化合物および新規色素
CN102533247A (zh) * 2010-12-12 2012-07-04 陈文通 一种含镝荧光晶体及其制备方法
CN102924529B (zh) * 2012-11-22 2015-12-02 中山大学 一种环金属化铱-偶氮配合物及其制备方法和应用
CN102924529A (zh) * 2012-11-22 2013-02-13 中山大学 一种环金属化铱-偶氮配合物及其制备方法和应用
US10752594B2 (en) 2013-03-14 2020-08-25 Sumitomo Dainippon Pharma Oncology, Inc. JAK1 and ALK2 inhibitors and methods for their use
CN103254127B (zh) * 2013-05-28 2015-08-19 北京哈三联科技有限责任公司 甘氨酸重摄取抑制剂及其应用
CN103254127A (zh) * 2013-05-28 2013-08-21 北京哈三联科技有限责任公司 甘氨酸重摄取抑制剂及其应用
US11603351B2 (en) 2017-07-11 2023-03-14 Vertex Pharmaceuticals Incorporated Carboxamides as modulators of sodium channels
US10647661B2 (en) 2017-07-11 2020-05-12 Vertex Pharmaceuticals Incorporated Carboxamides as modulators of sodium channels
US11040038B2 (en) 2018-07-26 2021-06-22 Sumitomo Dainippon Pharma Oncology, Inc. Methods for treating diseases associated with abnormal ACVR1 expression and ACVR1 inhibitors for use in the same
US10894797B2 (en) 2018-09-18 2021-01-19 Nikang Therapeutics, Inc. Fused tricyclic ring derivatives as SRC homology-2 phosphatase inhibitors
US11034705B2 (en) 2018-09-18 2021-06-15 Nikang Therapeutics, Inc. Fused tricyclic ring derivatives as Src homology-2 phosphate inhibitors
US11459340B2 (en) 2018-09-18 2022-10-04 Nikang Therapeutics, Inc. Tri-substituted heteroaryl derivatives as Src homology-2 phosphatase inhibitors
US11518772B2 (en) 2018-09-18 2022-12-06 Nikang Therapeutics, Inc. Fused tricyclic ring derivatives as Src homology-2 phosphate inhibitors
CN109734709A (zh) * 2019-02-12 2019-05-10 温州医科大学 一种小分子荧光探针及其制备方法与应用
CN112920254A (zh) * 2020-02-14 2021-06-08 西南交通大学 高效抗凝的功能分子、螯合物及其应用、仿生功能材料及其制备方法
CN112920254B (zh) * 2020-02-14 2023-06-13 西南交通大学 抗凝的功能分子、螯合物及其应用、仿生功能材料及其制备方法

Similar Documents

Publication Publication Date Title
WO2003003009A1 (fr) Utilisation de chelates a ions metalliques dans la validation de molecules biologiques utilisees comme cibles medicamenteuses dans des modeles animaux experimentaux
Davoren et al. Discovery of the potent and selective M1 PAM-agonist N-[(3 R, 4 S)-3-hydroxytetrahydro-2 H-pyran-4-yl]-5-methyl-4-[4-(1, 3-thiazol-4-yl) benzyl] pyridine-2-carboxamide (PF-06767832): Evaluation of efficacy and cholinergic side effects
WO2003003008A1 (fr) Bibliotheques chimiques utiles aux procedes de decouvertes de medicaments
Fleming et al. Nitrile-containing pharmaceuticals: efficacious roles of the nitrile pharmacophore
Chen et al. Discovery of Sodium R-(+)-4-{2-[5-(2-Fluoro-3-methoxyphenyl)-3-(2-fluoro-6-[trifluoromethyl] benzyl)-4-methyl-2, 6-dioxo-3, 6-dihydro-2 H-pyrimidin-1-yl]-1-phenylethylamino} butyrate (Elagolix), a Potent and Orally Available Nonpeptide Antagonist of the Human Gonadotropin-Releasing Hormone Receptor
Melancon et al. Allosteric modulation of seven transmembrane spanning receptors: theory, practice, and opportunities for central nervous system drug discovery
US8980908B2 (en) Non-peptidyl, potent, and selective mu opioid receptor antagonists and their use in treating opioid addiction and opioid induced constipation
CN105693645B (zh) 作为离子通道调节剂的稠合杂环化合物
WO2010083384A9 (fr) Antagonistes non peptidiques, puissants et sélectifs des récepteurs opioïdes mu
US7045527B2 (en) Piperidine derivatives
JP5775819B2 (ja) 新規なキメラリガンド開口型イオンチャネルおよびその使用方法
WO2004033431A2 (fr) Hydroxypyrazoles et methodes prophylactiques ou de traitement de troubles lies au metabolisme
US20070254903A1 (en) Novel Spiroindoline or Spiroisoquinoline Compounds, Methods of Use and Compositions Thereof
US20020061599A1 (en) Method of identifying ligands of biological target molecules
Ma et al. Discovery of potent inhibitors against P-glycoprotein-mediated multidrug resistance aided by late-stage functionalization of a 2-(4-(pyridin-2-yl) phenoxy) pyridine analogue
JP2017520588A (ja) Ｒａｌ ＧＴＰアーゼを標的とする抗癌化合物及びそれを使用する方法
Kim et al. Discovery of β-arrestin biased ligands of 5-HT7R
Zhang et al. 6-Benzhydryl-4-amino-quinolin-2-ones as potent cannabinoid type 1 (CB1) receptor inverse agonists and chemical modifications for peripheral selectivity
Ishida et al. Discovery and SAR studies of orally active somatostatin receptor subtype-2 (SSTR2) agonists for the treatment of acromegaly
TW200914020A (en) Substituted piperazinyl pyrazines and pyridines as 5-HT7 receptor antagonists
WO2003055477A1 (fr) Methode de traitement de troubles lies au recepteur de la melanocortine (mc), faisant intervenir un chelate et/ou un chelateur
Tan et al. Toward deciphering the code to aminergic G protein-coupled receptor drug design
Zhang et al. Structure-based discovery of MDM2/4 dual inhibitors that exert antitumor activities against MDM4-overexpressing cancer cells
CN108602807A (zh) 杂环磺酰胺衍生物及含有其的药物
KR20050103184A (ko) 융합된 헤테로사이클릭 화합물들

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ CZ DE DE DK DK DM DZ EC EE EE ES FI FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2004112774

Country of ref document: RU

Kind code of ref document: A

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP