WO2003003008A1 - Bibliotheques chimiques utiles aux procedes de decouvertes de medicaments - Google Patents

Bibliotheques chimiques utiles aux procedes de decouvertes de medicaments Download PDF

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WO2003003008A1
WO2003003008A1 PCT/DK2002/000455 DK0200455W WO03003008A1 WO 2003003008 A1 WO2003003008 A1 WO 2003003008A1 DK 0200455 W DK0200455 W DK 0200455W WO 03003008 A1 WO03003008 A1 WO 03003008A1
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metal
ion
lll
compounds
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Thomas Högberg
Øystein RIST
Anders Hjelmencrantz
Peter Moldt
Christian E. Elling
Thue W. Schwartz
Lars Ole Gerlach
Birgitte Holst Lange
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7Tm Pharma A/S
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    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/44—Radicals substituted by doubly-bound oxygen, sulfur, or nitrogen atoms, or by two such atoms singly-bound to the same carbon atom
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
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    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
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    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
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Definitions

  • the present invention relates to chemical libraries of chemical compounds containing at least two heteroatoms such as, e.g. N, O, S, Se and/or P.
  • the chemical compounds are capable of forming complexes with a metal ion, i.e. in one aspect the invention relates to a library of chelators. In those cases where the chemical compounds are present in the form of metal-ion complexes, the invention relates to a library of chelates.
  • the libraries are useful in a drug discovery method disclosed in PCT/EPOO/13389 which relates to a novel method useful for identifying small organic molecule ligands (in the following also denoted "compounds") for binding to specific sites on biological target molecules such as proteins, nucleic acids, carbohydrates, nucleoproteins, glycoproteins and glycolipids.
  • the compounds are capable of interacting with the biological target molecule, in particular with a protein, in such a way as to modify the biological activity thereof.
  • the invention further relates to libraries that are suitable for use in methods of identifying compounds acting as receptor-ligands of biological target molecules such as, e.g., proteins, the method comprises the introduction of metal ion binding sites into the biological target molecules:
  • the method includes identifying compounds that bind to orphan receptors.
  • Small organic receptor-ligands identified according to the methods of the present invention find use, for example, as novel therapeutic drug compounds or drug lead compounds, enzyme inhibitors, labelling compounds, diagnostic reagents, affinity reagents e.g. for protein purification etc.
  • novel biologically active compounds such as, e.g., therapeutically or propylactically active drug compounds
  • identify and characterize one or more binding receptor-ligand(s) for a given biological target Many molecular techniques have been developed and are currently being employed for identifying novel receptor-ligands or compounds that bind to the biological target.
  • proteins are used as an example on a biological target molecule. 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 receptor-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 that 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
  • Drug discovery traditionally involves a process where a lead compound first is identified and then subsequently chemically optimised for high affinity and selectivity for the protein target (or another biological target molecule) and optimised for other drug-like properties such as lack of toxic effects and desirable pharmacokinetics.
  • Recent drug development has focused on screening of large libraries of chemical compounds in order to identify lead compounds, which are capable of either up-regulating (called agonists) or down-regulating the activity of the protein target (called antagonists), as required.
  • Screening has usually been performed in a "shot-gun” fashion by setting up an assay for screening large numbers of compounds, e.g. large files of compounds or compounds in combinatorial libraries, in order to identify compounds with the desired activity.
  • the subsequent chemical optimization of the lead compounds obtained from such screening procedures has been performed very much in a trial-and-error fashion and has been quite cumbersome and resource-demanding, involving procedures such as described by E. Sun and F.E. Cohen, Gene 1993 137(1), 127-32, or J. Kuhlmann, Int J Clin Pharmacol Ther.
  • a major disadvantage of the drug discovery process is that it is difficult to identify active compounds with sufficient selectivity and specificity for a given target protein or in many cases it is even difficult at all to identify suitable lead compounds, for example for interfering with protein-protein interactions.
  • the lead compound is gradually improved in affinity for the target. Also this process in to a large degree done by trial-and-error, although the medicinal chemist usually is guided by a gradually increasing knowledge in the structure activity relationship (SAR) of the compounds, i.e. the observation of which modification at which site in the compound that increase or decrease the activity of the compound.
  • SAR structure activity relationship
  • the SAR can provide a great deal of information regarding the nature of receptor-ligand-receptor interactions, but no detailed information about the location and actual chemical nature of the binding site in the target protein is provided.
  • a number of closely related chemical structures are used to direct the orientation of the receptor-ligand within the putative binding cavity and to determine what part of the receptor-ligand is involved in binding to the receptor.
  • This technique has its limitations due to the fact that changing the structure of the receptor-ligand may result in a actual change in the binding site of the receptor (Mattos et al. Struct. Biol., 1995 1:55-58) , a fact which obviously still would be un-know to the medicinal chemist.
  • the lack of knowledge of the precise molecular interaction with the receptor of the lead compounds found by chemical screening has prevented a rational chemical approach to the optimisation of the lead compound.
  • Determination of the three-dimensional structure of the target protein either alone or even better in complex with the receptor-ligand by X-ray crystallography provides high- resolution and very high quality information about the molecular recognition of the compound in the target protein structure.
  • the target is a soluble protein
  • Subsequent X-ray analysis of complexes of these improved compounds and the target protein can then lead to the synthesis of a new series of further improved compounds, new compound-target crystallisations and so on until the desired affinity has been obtained.
  • a general problem of the site-directed mutagenesis method is that it is not clear whether the substitution of a residue affects the binding of a receptor-ligand directly (i.e. the residue is directly involved in receptor-ligand binding) or indirectly (i.e. the residue is only involved in the structure of the receptor).
  • Another problem of Ala substitution is false negative results because the procedure basically creates another "hole" in the presumed binding pocket through removal of the side chain on the residue replaced by Ala. The effect of Ala substitution is highly dependent on the relative contribution to the binding energy of the replaced residue.
  • An alternative to Ala substitution is steric hindrance mutagenesis where for example a larger side chain, e.g. Trp, is introduced in a presumed binding pocket as described by Hoist et al., Mol Pharmacol. 53(1), 1998, pp. 166-175.
  • the present invention deals with libraries of chemical compounds useful in methods involving a chemical "anchor? Such methods make use of a metal-ion binding site in the target biological molecule as well a metal binding site in a chemical compound.
  • the metal- ion binding site in the biological target molecule such as, e.g., a target protein may be a natural metal-ion binding site or it may be a metal-ion binding site that has been introduced into the protein by artificial means such as, e.g., engineering means. Background of the invention
  • metal-ion binding sites serve either structural purposes, for example stabilizing the three-dimensional structure of the protein, or they serve functional purposes, where the metal-ion may for example be part of the active site of an enzyme. It is well known that also several integral membrane proteins include binding sites for metal ions. The coordination of metal ions to metal ion binding sites is well characterized in numerous high-resolution X-ray and NMR structures of soluble proteins; for example, distances from the chelating atoms to the metal ion as well as the preferred conformation of the chelating side chains are known (e.g. J.P. Glusker, Adv. Protein Chem. 42, 1991 , pp. 3-76; P. Chakrabarty, Protein Eng.
  • 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 present invention provides libraries of chemical compounds that are suitable for use in a molecular approach for rapidly and selectively identifying small organic molecule receptor-ligands, i.e. compounds that are capable of interacting with and binding to specific sites on biological target molecules. It has been possible to construct libraries with the aim of designing compounds specifically directed against predetermined epitopes on the biological target molecules.
  • the compounds contained in a library of the invention are initially constructed to be bi-functional, i.e. having both a metal-ion binding moiety, which conveys them with the ability to bind to either a natural or an artificially constructed metal- ion binding site as well as a variable moiety, which is varied chemically to probe for interactions with specific parts of the biological target molecule located spatially adjacent to the metal-ion binding site.
  • Compounds may subsequently be further modified to bind to the unmodified biological target molecule without help of the bridging metal-ion.
  • the methods involving the use of a library according to the invention may be performed easily and quickly and lead to unambiguous results.
  • the compounds identified by the methods described herein or in PCT/EPOO/13389 may themselves be employed for various applications or may be further derivatised or modified to provide novel compounds.
  • the present invention further relates to the use of specific metal-ion binding sites in biological target molecules.
  • the libraries of the present invention are applicable in drug discovery processes involving any biological target molecule that has or can be manipulated to have a metal-ion binding site.
  • any biological target molecule that has or can be manipulated to have a metal-ion binding site.
  • proteins are used as examples of biological target molecules.
  • Parts of the present invention utilise the finding that many proteins in their natural form possess a metal-ion binding site, which may or may not have been recognized previously.
  • the invention especially utilises the possibility to mutate proteins, for example a receptor, an enzyme or a transcriptional regulator in such a way, that they comprise a metal-ion binding site.
  • the metal-ion site is then used as an anchor- point for the initial parts of the medicinal chemistry drug-discovery process, during which test compounds can be synthesized, which due to their specific interaction with the metal- ion binding site can be deliberately directed towards interaction with specific, functionally interesting parts of the biological target molecule.
  • test compounds are subsequently structurally optimised for interaction with spatially neighbouring parts of the proteins (that is, interaction with the side chains or backbone of one or more neighbouring amino acid residues). These compounds can then be utilized as leads or starting points for the construction of receptor-ligands binding to the wild-type protein.
  • optimised compounds By selecting the binding site for a test compound at will and thereby selecting the binding site for the optimised compound (such as a drug candidate) in a protein, it is for example possible to:
  • metal-ion chelators certain small organic compounds which bind metal ions
  • metal-ion chelators are also able to bind to metal ion binding sites in various proteins, including membrane proteins for example receptors, in such a way that the metal ion acts as a bridge between the small organic compound and the protein.
  • the present invention has made it possible to use libraries of chemical compounds to predetermine or identify and localise the exact binding site and binding mode of such metal-ion chelates, contrary to what has been known in the art for test compounds in general.
  • the metal-ion binding portion of the test compounds may subsequently be removed or altered to no longer posses metal-ion binding properties, and the test compounds, as well as chemical derivatives thereof may be constructed to interact with side chains of other amino acids in the vicinity of the artificial metal ion binding site, and tested for binding to the wild-type protein which does not include a metal ion binding site. Accordingly, relatively small chemical libraries may be made, the compounds in which may be designed to interact with the specific amino acid residues found in the wild-type protein at or spatially surrounding the location where the metal ion site had initially been engineered.
  • the present invention is based on the general principle, applicable to any biological target molecule including a protein, of introducing metal ion binding sites at any position in e.g. the protein where a test compound binding to the protein is likely to exert an effect on the biological activity of the protein.
  • This may for example be 1) at a site where the test compound will interfere with the binding to another protein, for example a regulatory protein, or to a domain of the same protein; 2) at a site where the binding of the test compound will interfere with the cellular targeting of the protein; 3) at a site where the binding of the test compound will directly or indirectly interfere with the binding of substrate or the binding of an allosteric modulatory factor for the protein; 4) at a site where the binding of the test compound may interfere with the intra-molecular interaction of domains within the protein, for example the interaction of a regulatory domain with a catalytic domain; 5) at a site where binding of the test compound will interfere with the folding of the protein, for example the folding of the protein into its active conformation; or 6) at a site which will interfere with the activity of the protein, for example by an allosteric mechanism.
  • the present invention relates to the use of a chemical library comprising three or more of a chemical compound in a drug discovery process for identification of a small organic compound that is able to bind to a biological target molecule.
  • Such a drug discovery process comprises mutating a biological target molecule in such a way that at least one amino acid residue capable of binding a metal ion is introduced into the biological target molecule so as to obtain a metal ion binding site as an anchor point in the mutated biological target molecule.
  • the mutated biological target molecule may furthermore be contacted with a library of test compounds which comprises a moiety including at least two heteroatoms for chelating a metal ion, under conditions permitting non-covalent binding of the test compound to the introduced metal ion binding site of the mutated biological target molecule, and then followed by detection of any change in the activity of the mutated biological target molecule or determation of the binding affinity of the test compound to the mutated biological target molecule.
  • the present invention relates also to the use of a chemical library in a drug discovery process for identification of a small organic compound that is able to bind to a biological target molecule, which has at least one metal ion binding site.
  • the drug discovery process comprises
  • test compound which comprises a moiety including at least two heteroatoms for chelating a metal ion, under conditions permitting non-covalent binding of the test compound to the metal ion binding site of the biological target molecule, and (b) detecting any change in the activity of the biological target molecule or determining the binding affinity of the test compound to the biological target molecule.
  • a very important class of biological target molecules amenable to testing according to the present invention are proteins such as membrane proteins, which includes proteins that are involved in intercellular communication and other biological processes of profound importance for cellular activity.
  • the present invention relates to a chemical library suitable for use in a method of identifying a metal-ion binding site in a protein. Such a method comprises
  • test compound which comprises a moiety including at least two heteroatoms for chelating a metal ion, under conditions permitting non- covalent binding of the test compound to the protein, and detecting any change in the activity of the protein or determining the binding affinity of the test compound to the protein, and
  • the invention relates to chemical libraries for use in a method of mapping a metal-ion binding site of a protein. This method comprises
  • step (a) contacting the protein with a library of test compounds, which compounds comprise a moiety including at least two heteroatoms for chelating a metal ion, under conditions permitting non-covalent binding of the test compound to the protein, and detecting any change in the activity of the protein or determining the binding affinity of the test compound to the protein, and (b) determining, based on the primary structure of the specific protein in question and the generic three-dimensional model of the class of proteins to which the specific protein of step (a) belongs, at least one metal ion binding amino acid residue located in the membrane protein to identify the metal ion binding site of said membrane protein.
  • the invention relates to chemical libraries comprising test compounds in chelated or non-chelated form and to a chemical library comprising metal ions suitable for chelating test compounds.
  • the metal ions are generally presented in salt form or in the form of complexes or solvates.
  • Essential parts of the present invention relates to chemical libraries for use in methods of identifying compounds that are capable of binding to specific sites on biological target molecules. In a typical form of this process the following steps are involved:
  • the biological target molecule already has a suitable metal-ion site, which may or may not previously have been recognized.
  • metal-ion sites are introduced, for example through mutagenesis, at specific sites in the biological target molecule expected to be useful as anchor points for the development of compounds affecting the function of the target molecule in a desired way.
  • a number of such sites are introduced and one or more are selected for further use.
  • libraries of basic bi- or multi-functional compounds are being constructed in which the compounds have both a anchoring metal-ion binding moiety, which conveys them with the ability to bind to the metal-ion binding site in the biological target molecule, as well as a variable moiety, which is varied chemically to probe for improved interactions with specific parts of the biological target molecule located spatially adjacent to the metal- ion binding site.
  • these libraries are constructed based on structural knowledge of the chemical target moiety in the biological target molecule.
  • a more broad screening of larger libraries of compounds is performed without detailed knowledge of the structure of the biological target molecule surrounding the anchoring metal-ion site.
  • the present invention is directed to chemical libraries for use in methods directly or indirectly involved in the above-mentioned drug discovery process. Furthermore, it is directed to the use of chemical libraries for selecting a chemical compound from a library.
  • 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 of from about 3 to about 10,000 compounds such as, e.g. from about 5 to about 5,000, from about 10 to about 2,500, from about 3 to about 1 ,000, from about 3 to about 750, from about 3 to about 500, from about 3 to about 250, from about 3 to about 100, from about 3 to about 75, from about 3 to about 50, from about 3 to about 25, from about 3 to about 10 compounds.
  • 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 "chemical compound” or a “test compound” is intended to indicate a small organic molecule of low molecular weight 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 chemical 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 chemical 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 "ligand” is intended to indicate a functional group or a structural element that binds or coordinates a metal ion.
  • 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 "metal-ion binding site” is intended to indicate a part of a biological target molecule that comprises atoms in relative positions in such a way that they are capable of complexing with a metal atom or ion. Such atoms will typically be heteroatoms, 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 binds to a biological target molecule and thereby inhibiting or stimulating its activity.
  • 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 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 may have 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 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 chemical 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 chemical compound.
  • Functional groups may be involved in interactions such as, e.g., ionic interactions, ion-dipole interactions, dipole-dipole interactions, hydrogen bond interactions, hydrophobic interactions, pi-stacking interactions, edge-on aromatic interactions, dispersion and induction forces or metal complex interactions.
  • 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
  • the term "in the vicinity of” is intended to include an amino acid residue or any other residue or functional group located in the space defined by 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 chemical compound, to generate an interaction between said functional group or groups and said amino acid residue, another residue or functional group.
  • 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 contains or has been manipulated to contain a metal-ion binding site.
  • 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 transciption factor, an enzyme, and an allosteric regulator protein, or it may be a growth factor, a hormone, a neuropeptide or an immunoglobuiin.
  • 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 receptor-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. A large fraction of current drugs act on membrane proteins and among these the majority are targeted towards the G protein coupled receptors (GPCR) with their seven transmembrane segments, also called 7TM receptors.
  • 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. receptor-ligand-gated ion channels, such as nicotinic acetylcholine receptors, GABA receptors, or glutamate receptors (NMDA or AMPA).
  • receptor-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.
  • membrane proteins of interest comprising 7 transmembrane domains include but are not restricted to G-protein coupled receptors, such as receptors 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 (CCR1-11-11), ace
  • 8 to 14TM - Membrane proteins of interest comprising 8 to 14 transmembrane domains include but are not restricted to e.g., transporter proteins, such as a GABA, monoamine or nucleoside transporter.
  • transporter proteins such as a GABA, monoamine or nucleoside transporter.
  • the membrane protein may also be 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.
  • 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 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 also known as G-protein coupled receptors (GPCRs).
  • GPCRs G-protein coupled receptors
  • This family of receptors constitutes the largest super-family of proteins in the human body and a large number of current drugs are directed towards 7TM receptors, for example: antihistamines (for allergy and gastric ulcer), beta-blockers (for cardiovascular diseases), opioids (for pain), and angiotensin antagonists (for hypertension).
  • antihistamines for allergy and gastric ulcer
  • beta-blockers for cardiovascular diseases
  • opioids for pain
  • angiotensin antagonists for hypertension
  • receptor-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, choriogonadotrop
  • the G-protein consists of three subunits, an -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.
  • the receptor When the receptor is activated, a signal is transduced by a changed receptor conformation that activates the G- protein. This leads to the exchange of GDP for GTP on the ⁇ subunit, which subsequently dissociates from the receptor and the ⁇ y dimer, and activates downstream second messenger systems (e.g.
  • the ⁇ subunit will activate the effector system until its intrinsic GTPase activity hydrolyses the bound GTP to GDP, thereby inactivating the ⁇ subunit.
  • the ⁇ y subunit increases the affinity of the o subunit for GDP but may also be directly involved in intracellular signalling events. 7TM receptor-ligand-binding sites - Mutational analysis of 7TMs has demonstrated that functionally similar but chemically very different types of receptor-ligands can apparently bind in several different ways and still lead to the same function.
  • receptor-ligands can be developed independent on the chemical nature of the endogenous receptor-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). It has been found that in the substance P receptor, when the binding site for a non-peptide antagonist has been exchanged for a metal-ion binding site through introduction of His residues, no effect on agonist binding was observed (Elling et al., (1995) Nature 374: 74-77; Elling et al.
  • 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 , 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.
  • the transmembrane segments are generically numbered.
  • TM-I the highly conserved hydroxy function, serine (Ser) is given the generic number 8, i.e. Serl:08 on the basis of its position in the helix; in TM-II the highly conserved histidine (His) is given the generic number 6 i.e. Hisll:06; in TM-III the highly conserved cysteine (Cys) is given the generic number 1 i.e. Cyslll:01 ; in TM-IV the highly conserved proline (Pro) is given the generic number 13 i.e. ProlV:13; in TM-V the highly conserved asparagine (Asn) is given the generic number 14 i.e.
  • Orphan 7TM receptors - one embodiment of the invention is directed to a method of developing assay for 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).
  • receptor-ligands have been discovered for some of the orphan 7TM receptors, which then immediately have been recognized as "real" drug targets, for example: nocioceptin (for pain) (Reinborg et al. Science (1995) 270: 792- 94), orexin (for appetite regulation and regulation of energy homeostasis) (Sakurai et al.
  • the problem is that it is very difficult to characterize orphan receptors and find their endogenous receptor-ligands, since no assays are available for these receptors due to the lack of specific receptor-ligands - a "catch 22" situation.
  • the present invention is aimed at eliminating this problem.
  • Binding of metal-ion chelates can be monitored either through functional assays in cases where agonistic metal ion sites are created, or through receptor-ligand binding assays.
  • many aromatic metal-ion chelators are by themselves fluorescent and can therefore directly be used as tracers in binding assays.
  • radioactive or other measurable indicators can be incorporated into the metal-ion chelator.
  • the biological target molecules of interest may be obtained in a useful form by different ways including but not limited to recombinantly, synthetically or commercially.
  • the biological target molecule being a protein is obtained recombinantly. This can be achieved through cloning of the gene for the protein from genomic or cDNA libraries generally by the use of PCR techniques in accordance with standard techniques (eg. Sambrook et al. Molecular Cloning: A laboratory manual, 2. Ed. Cold Spring Harbor Laboratory, New York 1989), and expression of the gene in a suitable cell.
  • the nucleotide sequence encoding the target protein - and mutant versions thereof (see below) - may be inserted into a suitable expression vector for the purpose of expression and analysis in a host organism.
  • regulatory element ensuring either constitutive or inducible expression of the protein of interest should be present in the vector, including promoter elements.
  • the host organism into which the nucleotide sequence is introduced may be any cell type or cell line, which is capable of producing the target molecule in a suitable form for the test to be performed including but not restricted to e.g. yeast cells and higher eukaryotic cells such as e.g. insect or mammalian cells. Transformation of the cell line of choice may be performed by standard techniques routinely employed in the field as described e.g.. in Wigler et al. Cell (1978) 14: 725 and in accordance with standard techniques (Sambrook et al. Molecular Cloning: A Laboratory Manual, 2. ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).
  • the biological target molecule being a membrane protein is expressed and tested in mammalian cells usually within the membrane and usually in whole cells or in isolated membrane preparations, which is dealt with and described further in the examples presented in "EXPERIMENTAL".
  • suitable mammalian cell lines are the COS (ATCC CRL 1650 and 1651 ), BHK (ATCC CRL 1632, ATCC CCL 10), CHL (ATCC CCL39), CHO (ATCC CCL 61), HEK293 (ATCC CRL 1573) and NIH/3T3 (ATCC CRL 1658) cell lines.
  • a preferred source may be recombinantly produced protein, which subsequently is isolated and purified to a suitable purity and in a form suited for functional testing by various standard protein chemistry methods well known to those skilled in the art.
  • the biological target molecule comprising a natural or an engineered metal-ion binding site is contacted with a test compound for example consisting of a metal-ion in complex with a metal-ion chelator and any change in the biological activity of the biological target molecule is detected or the binding affinity of the test compound is determined.
  • a test compound for example consisting of a metal-ion in complex with a metal-ion chelator and any change in the biological activity of the biological target molecule is detected or the binding affinity of the test compound is determined.
  • the biological target molecule is a membrane protein and the effect of test compounds is monitored on the signal transduction process of the receptor, i.e. its ability to influence intracellular levels of for example cAMP, inositol phosphates, calcium mobilization etc. in response to the natural receptor-ligand.
  • tests are performed as dose-response analysis in which a range of concentration of metal-ion chelator complexes are exposed to the biological target molecule.
  • the binding affinity of the test compound to the biological target molecule is determined, for example in competition binding experiments against a suitable radioactively labelled receptor-ligand for the protein target.
  • the affinity of the test compound can in some cases be determined by use of a chelating agent, which in itself is detectable or which can be labelled with a detectable labelling agent. Structural testing of the biological target molecule
  • the 3D structure of the test compound in complex with the biological target molecule is determined, for example by techniques such as X-ray analysis of crystals of the receptor-ligand-protein complex or, for example by nuclear magnetic resonance (NMR) spectroscopic analysis of complexes in solution - all known to those skilled in the art.
  • NMR nuclear magnetic resonance
  • the amino acid residues located in the vicinity of the metal-ion site and the chemical interaction of the bifunctional test compound with specific residues in the biological target molecule can be determined as control and as basis for the structure-based design of further modifications of the lead test compound and design of new libraries of compounds.
  • the effect of the test compound on the structure of the biological target protein, domains of this and or effect on the interaction of the target protein with other proteins can be determined.
  • naturally occurring metal-ion sites are used as initial attachment sites for metal-ion chelating test compounds in the drug discovery process.
  • such natural metal-ion sites can be identified functionally by studying the effect of either free metal-ions or by the effect of a library of metal-ion chelator complexes on any function of the biological target molecule.
  • Metal-ion sites can also be identified or confirmed by structural means as described above and location of the site can also be identified by careful, controlled mutagenesis, i.e. exchanging of the residues involved in metal-ion binding with residues not having this property.
  • Natural metal-ion sites are interesting drug targets since binding of a drug at or close by a natural metal-ion site often will act as an allosteric agent, i.e. affecting the structure and function of the biological target molecule at a site different from the usual active site, where most receptor-ligands will bind and act (see below).
  • Natural metal-ion sites in proteins in general - Metal-ion sites are known to occur in many biological target molecules including but not restricted to, for example proteins, glycoproteins, RNA, etc. These sites can serve either structural or functional purposes. Some metal-ion sites are known to occur solely from functional data, for example Zn(ll)- sites in receptor-ligand gated ion channels. Or previously unknown metal-ion sites are discovered in the crystal structure of the protein, as for example Zn(ll) sites in rhodopsin.
  • metal-ion site may be targeted by the technology of the present invention, where they are addressed not only by a metal-ion, but by a metal-ion in complex with a metal-ion chelator, which can affect the protein structure and function differently than the free metal-ion.
  • metal ion sites may just be a reflection of the fact that polar, metal-ion binding amino acid residues (for example: His, Cys, Asp etc.) frequently are found in the water-exposed main receptor-ligand-binding crevice of 7TM receptors.
  • these residues are used as initial attachment sites for metal ion chelating test compounds, i.e. lead compounds in the drug discovery process.
  • 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 drug discovery process and thereby target the medicinal chemistry part of the process towards particularly interesting epitopes on the target molecule.
  • 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.
  • Site-directed mutagenesis may be performed according to well-known techniques, e.g. as described in 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. Pfu Polymerase (Stratagene) according to manufacturers specifications.
  • a high-fidelity DNA polymerase such as e.g. Pfu Polymerase (Stratagene) according to manufacturers specifications.
  • the presence of the 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.
  • 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. Mutations will often also be performed in the biological target molecule to confirm or probe for the chemical interaction of test compounds with other residues in the vicinity of the natural or the engineered metal-ion site often as an integrated part of the general drug discovery process.
  • the drug discovery process 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 and/or the function of the substrate or natural receptor-ligand or the binding of an allosteric modulatory factor for the biological target molecule, a site where the binding of the test compound may interfere with the intramolecular interaction of domains within the biological target molecule, for example the interaction of a regulatory domain with a catalytic domain, a site where binding of the test compound will interfere with the folding of the biological target molecule, for example the folding of a protein into its active conformation, a site where the binding of the test compound will interfere with the cellular targeting of the biological target molecule, a site where the binding of the test compound will stabilise a conformation of the biological target molecule, which presents an epitope normally involved in protein- protein interactions in a non-functional form.
  • Allosteric agents will, for example have the possibility of stabilising a conformation of the biological target molecule where major parts of the protein-protein interface are vastly different from the one enabling the normal interaction.
  • Metal-ion site engineering in 7TM proteins 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. 5560-5) showed that Zn(ll) preferentially coordinates with the syn lonepair of the carboxylates (Z form) (Fig. 2). This was also observed by Carrell et al. (J. Am. Chem. Soc, 110(26), 1988, pp.
  • 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% N5-H (Fig. 3) (W.F. Reynolds et al., J. Am. Chem. Soc, 95(2), 1973, pp. 328-331). However, it is the N ⁇ -H form that predominantly binds the metal-ion (P. Chakrabarti, Protein Eng., 4(1), 1990, pp.
  • Steric factors may be responsible for reversing the preference of the two tautomeres when the histidyl is bound to a metal ion. Another factor may be due to the structural properties of histidyl, where the Ne-metal coordinated form may be stabilized by N£-backbone carbonyl hydrogen bonding to a preceding turn in an -helix (P. Chakrabarti, Protein Eng., 4(1), 1990, pp. 57-63). Freeman (Inorganic Chemistry (G.L. Eichhorn, ed.), 1973, pp. 121-166) found that the metal-nitrogen bond of histidyl can be up to 30° from the imidazole plane and R. Candlen (J. Am.
  • 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(ll) 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).
  • 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 site engineering has been included in the design strategy of de novo proteins.
  • a common obstacle in de novo design of a protein is that the fold of the protein often resembles a state in between a molten globule and a native folded protein presumably due to the lack of specific tertiary interactions.
  • Engineering of metal-ion sites have been utilized to introduce positive interactions in the protein fold stabilizing the folded native conformation as opposed to the molten globule state.
  • a by now classic example of the elegant use of metal-ion site engineering was performed by Charlie Craik and coworkers in trypsin (J.N. Higaki, Biochemistry, 29, 1990, pp. 8582-8586).
  • 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).
  • 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 or more residues, which allow for the metal-ion to bind also the metal-ion chelator.
  • residues which allow for the metal-ion to bind also the metal-ion chelator.
  • single 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 the skilled person (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, 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.
  • an important part of a drug discovery process is to increase the affinity of a 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 may be 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 e.g. 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. Secondary chemical interactions include the binding of second metal-ion between the test compound and the secondary site residue.
  • 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, and not with such residues in general in the biological target molecule, 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 binding site in the biological target molecule.
  • metal-ion binding sites serve either structural purposes, for example stabilizing the three-dimensional structure of the protein, or they serve functional purposes, where the metal-ion may for example be part of the active site of an enzyme.
  • metal-ion sites serve either structural purposes, for example stabilizing the three-dimensional structure of the protein, or they serve functional purposes, where the metal-ion may for example be part of the active site of an enzyme.
  • integral membrane proteins include binding sites for metal ions.
  • the coordination of metal ions to metal ion binding sites is well characterized in numerous high-resolution X-ray and NMR structures of soluble proteins; for example, distances from the chelating atoms to the metal ion as well as the preferred conformation of the chelating side chains are known (e.g. J.P. Glusker, Adv. Protein Chem. 42, 1991 , pp. 3-76; P.
  • metal-ion binding in proteins is one of the most well characterised forms of 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).
  • 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 receptor these single positions are positions which are suitable metal-ion binding sites:
  • ransmembrane segment-I ransmembrane segment-I
  • test compound metal ion chelate
  • the aim with a drug discovery process is to identify novel drug compounds with properties suitable for use in therapy or for diagnostic and/or prophylactic purposes.
  • complexes of interest are chelates consisting of 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 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.
  • the nature of the complex can be altered depending on the metal ion binding site in the target protein. It is usually advantageous in the described drug discovery process to have labile complexes. This means that the complex formation is reversible with a relatively fast interchange of ligands, and the metal ion prefers the complexed structure due to a thermodynamic favorable situation.
  • ligands can roughly be divided into three groups; (i) pure ⁇ -donating ligands, e.g. amines or thiols, (ii) ligands with additional 77-bac bonding to an electron deficient ⁇ systems, e.g. pyridines, and (iii) ligands with additional r ⁇ -back bonding to an electron rich rr systems, e.g. thiophenes.
  • ⁇ -donating ligands e.g. amines or thiols
  • ligands with additional 77-bac bonding to an electron deficient ⁇ systems e.g. pyridines
  • ligands with additional r ⁇ -back bonding to an electron rich rr systems e.g. thiophenes.
  • back bonding 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.
  • chelators containing e.g. pyridine are of interest, but also other heteroaromatic systems, like imidazoles, pyrazoles, triazoles, oxazoles, thiazoles, quinolines etc. should be considered.
  • phenolic and thiophenolic functionalities are known to act as chelating groups.
  • possible chelators advantageous for the task are 2,2'-bipyridine, 8-hydroxyquinoline, 8-mercaptoquinoline, and 2-(2-pyridyl)thiophenol.
  • metal ion Both zinc and copper has proven useful for the present purpose, but other metal ions similar to these are applicable. However, for certain targets it may be advantageous to use a different metal to avoid endogenous metal sites or metal species.
  • suitable metal ions may include, but is not limited to, cobalt, nickel, iron, manganese, as well as the heavier metals, like technetium, ruthenium, rhodium, palladium, osmium, platinum etc.
  • the various metals have different electronic properties, thus a different metal will show different preferences for various electronic environments, both in the metal ion site in the target, as well as from the chelator.
  • the interaction between the metal and the metal site in the protein target can be modulated.
  • the affinity between the metal, metal site and chelator is dependent on their individual properties. For some cases, a specific combination may turn out to be too weak for the purpose.
  • the combination of 2,2'-bipyridine-based chelators and zinc seems to form a too weak complex to be useful, i.e. equilibrium constant log K - 6, when the assay requires low metal ion concentrations ( ⁇ 1 ⁇ M).
  • changing the metal to e.g. copper in combination with 2,2'-bipyridine seems to result in a complex of relevant strength, i.e. equilibrium constant log K >8.
  • the resulting complex is of relevant strength, i.e. equilibrium constant log K >8.
  • the chemical optimisation of the test compound can be guided by detailed knowledge of the 3D structure(s) of the biological target molecule, preferentially determined in complex initially with the un-substituted metal-ion chelator and subsequently in complex with the chemically modified metal-ion chelator in which attempts have been made to establish first one secondary interaction and subsequently further secondary or tertiary interactions.
  • biological target molecules such as soluble proteins this can be achieved through for example crystallization and standard X-ray analysis procedures or through, for example NMR analysis of the complex in solution again using standard procedures.
  • mutated membrane protein in a suitable cell, contacting said cell or a portion thereof including the mutated membrane protein with the test compound, and determining any effect on binding in a competitive binding assay using a labelled ligand of the membrane protein, detection of any changes in signal transduction from the membrane protein or using a chelating agent which is in itself detectable or labelled with a detectable labelling agent. If an amino acid residue involved in interaction with such a functional group of the test compound is mutated to one, which is not - this may be detected as a decrease in binding or other activity
  • the interactions probed by flexible spacer arms can be improved by introduction of rigidity in the spacer group.
  • rigidity e.g. chain branching, higher- order bonds or ring systems in the spacer may enhance the affinity of the molecules. This step may be done for each separate secondary interaction, or it may be done in parallel for several functionalities.
  • bonds formed may be of, but not limited to, one of the following types: Ionic interactions, ion-dipole interactions, dipole-dipole interactions, hydrogen bond interactions, hydrophobic interactions, pi-stacking interactions, edge-on aromatic interactions, dispersion and inductions forces or metal complex interactions. More specifically:
  • charge-charge interaction introduction of charged groups such as ammonium, phosphonium or sulphonium groups, or ionisable groups such as amino or carboxy groups.
  • hydrogen bond interactions introduction of e.g. hydroxy, amino, keto amide, sulphonyl or other groups capable of interacting via formation of a hydrogen bond).
  • hydrophobic interactions introduction of hydrophobic groups.
  • a second metal-ion bridge or anchor established through a metal-ion binding moiety on the variable part of the compound and a metal-ion binding residue on the biological target molecule.
  • the functionality can be introduced in a random fashion, or in a more targeted manner, taking advantage of known structural elements in the protein target.
  • introduction of further functional groups on the chelator will lead to further increase in binding affinity, and this process will be repeated until a satisfactory affinity is obtained.
  • two or more functional groups capable of interacting with the protein may be introduced simultaneously.
  • the reactive groups used for forming secondary interactions should be compatible with the experimental set-up.
  • the functional groups selected should be compatible with properties required in a potential drug candidate.
  • the metal-ion chelator lead compound will gradually be optimized for interactions with chemical groups in the biological target molecule spatially surrounding the metal-ion site - i.e. interactions with chemical groups found also in the wild-type target molecule.
  • the test compound will gradually increase its affinity not only for the metal-ion site engineered molecule but also for the wild-type biological target molecule.
  • test compound When an acceptable affinity of a functionalised chelator has been obtained when tested on the wild type biological target molecule, without the metal ion binding site, the test compound will be optimised for interactions with the wild type biological target molecule, i.e. the part involved in the complexation may be transformed into structural elements having interaction with residues in the wild type biological target molecule.
  • This optimisation may be done randomly, or in a targeted fashion, taking advantage of knowledge of the target.
  • a further aspect of the invention is to utilise naturally occurring binding sites, i.e. binding sites already present in the biological target.
  • retaining the chelator may be advantageously for binding, while utilising physiological concentrations of endogenously occurring metal ions.
  • a library according to the invention contains three or more test compounds.
  • a library based on structural information from biological target molecules contains from about 10 to about 100 compounds and typically there are 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.
  • test compounds contained in the libraries normally fulfill 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).
  • 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 drug discovery 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 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), selenium (Se) and phosphorous (P).
  • Chemical compounds which have been found to be useful in libraries of the present invention, 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, pyridyl, pyrimidyl, pyrazoyl, benzothiazoyl, quinolinyl, triazolyl, imidazolyl, thiazolyl, tetrazolyl and benzimidazolyl.
  • the heterocyclyl group generally includes 2-20 carbon atoms, and 1-4 heteroatoms. Particularly useful chemical compounds in libraries 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 1 -C 1 2 alkyl, C C 12 alkenyl, alkynyl, C ⁇ -C ⁇ 2 cyclyl, aryl, C 1 -C 12 heteroalkyl, C C 12 heteroalkenyl, C ⁇ -C 12 heteroalkynyl, C C 12 heterocyclyl, heteroaryl;
  • R1 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 -CH2-CH2-, then R1 may be present on the first and/or second carbon atom one or several times; R1 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
  • a linked to be -A-B-C is selected from the group consisting of:
  • alk includes straight or branched alkyl, straight or branched alkenyl and straight or branched alkynyl; R' is H or lower alk, i.e. C C
  • alkyl straight or branched alkyl, alkenyl (straight or branched), alkylnyl (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;
  • 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 sulfonic acid ester or a phosphonic acid ester.
  • 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 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.
  • 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.
  • 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.
  • the structure of the compounds are given in different structure levels. First in a very general form and then in more and more specific forms.
  • 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 sacffolds 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 containing one or more R1 groups.
  • 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-py ⁇ dyl 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 one or more 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
  • 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.
  • Libraries containing chemical compounds of the following general formulas are of specific interest in the present context.
  • the libraries are denoted i) "A-libraries” in those cases where the compounds have a common connecting element, -A-, and ii) "C-libraries” in those cases where the compounds have a common functional group, -C.
  • the libraries may be an AA-, AA'-, AC-, CC- or CC'-library (A' is different from A but selected from the same group as A mentioned above; the same applies to C and C).
  • the libraries may be an AAA-, AAA'-, AA'A"-, AAC-, AA'C-, ACC-, ACC-, ACC"-, CCC-, CCC- or CCC"- library (or other possible permutations; the same notation is used as above, i.e. A is different from A', and A and A' are different from A").
  • the same notation applies for tetra-, penta-, hexa- or heptasubstitued 2,2-bipyridine libraries.
  • a library according to the invention may be a collection of compounds having e.g. the same A group; it may, however, also be a combination of two individual libraries.
  • the 2,2'-bipyridines of an A-library have a common connecting group attached directly on the ring system and a variable B-C moiety.
  • the functionalisations are made according to well- known chemical reactions with proper considerations of chemical compatibility of the functional groups with respect to the synthetic steps. Some exemplifications will be shown in the following.
  • Che-N(B-C) 2 ; Che-S-B-C ; Che-CO-NH-B-C ; Che-CH CH-B-C ; Che-O-B-C ; Che-NH- CO-B-C ; Che-SO 2 -NH-B-C as exemplified with the Che being 2,2'-bipyridine :
  • the amide library -CONH-B-C can be obtained by reacting a suitably activated carboxylate derivative with appropriate amines as detailed in the Experimental part.
  • the amine library can be obtained by reacting the bipyridyl amines with suitable B-C reagents or sequentially by reaction with a B reagent followed by a C reagent.
  • the alkene library can be obtained by forming the double bond in either direction, i.e. either having the carbonyl moiety on the bipyridyl scaffold or preferably having the carbonyl moiety located on the B moiety as indicated in the example.
  • the thiol library may be obtained by alkylation of the thiol with a B-C reagent or by nucleophilic addition/elimination with a suitable sulphur-containing derivative.
  • the substituent e.g. -CONH-B-C
  • the substituent may be positioned anywhere in the 3, 4 or 5 position on the 2,2'-bipyridine skeleton.
  • An A-library is typically an example of a library that is suitable for use in the randomised approach for optimization.
  • Alkene libraries with different B and C moieties can be obtained by reacting ylides of phosphonium salts or phosphonates such as:
  • 0 means a phenyl group, with appropriate ketone or aldehyde derivatives as detailed in the Experimental part.
  • the B moiety may optionally be part of a ring appended to an exo-cyclic double bond.
  • Alkene/amide (AA') libraries with different B and C moieties can be obtained by reacting ylides of phosphonium salts or phosphonates containing suitably protected carboxylic functions such as:
  • 0 means a phenyl group, with appropriate ketone or aldehyde derivatives, followed by deprotection, activation and coupling with suitable amines as shown for the amide A-library (see Experimental section).
  • the 2,2'-bipyridines of a C-library have a common functional group either directly attached on the 2,2'-biyridine skeleton or at a position at a distance from the skeleton. Irrespective its position, a characteristic feature of a C-library is that the common functional group is not further derivatized or substituted.
  • Examples on C-libraries are libraries containing 2,2'- bipyridines of formula II wherein R 1 is -A-B-C, -A-C, -B-C or -C (and, if present, B may be further substituted with one or more C groups). Examples on such functional end groups are e.g.
  • C-libraries are suitable for use in the targeted optimisation approach.
  • the present invention also relates to symmetric disustituted bipyridines, i.e. the bipyridine skeleton has been substituted in the same position in the two pyridine rings.
  • the substituent may be the same or different and it may represent the same or different functional group.
  • Metal ions forming the complex with the heteroalkyl or heterocyclyl moiety in the chemical compounds may advantageously be selected from metal ions, which have been tested for or are used for pharmaceutical purposes.
  • Such metal 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 typically 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, samarium, scandium, selenium, silicon, silver, strontium, tantalum, technetium, tellurium, terbium, thallium, thorium
  • chemical diverse libraries consisting of various chelating moieties (Che) containing different spacers (A and B moieties) and functionalities (C moities) can be produced and be tested with different metal ions (e.g. Zn, Cu, Ni, Co, Gd, Mn) and bind to different endogenous or engineered binding sites while exhibiting structure-related effects on receptor ligand-induced responses or receptor ligand binding.
  • Che chelating moieties
  • a and B moieties spacers
  • C moities functionalities
  • a library according to the present invention is suitable for use in a drug discovery process such as the processes disclosed in PCT/EPOO/13389.
  • a drug discovery process is described in the following items:
  • a drug discovery process for identification of a small organic compound that is able to bind to a biological target molecule comprising mutating a biological target molecule in such a way that at least one amino acid residue capable of binding a metal ion is introduced into the biological target molecule so as to obtain a metal ion binding site as an anchor point in the mutated biological target molecule.
  • a drug discovery process according to item 1 further comprising
  • test compound which comprises a moiety including at least two heteroatoms for chelating a metal ion, under conditions permitting non-covalent binding of the test compound to the introduced metal ion binding site of the mutated biological target molecule
  • a drug discovery process according to iteml further comprising
  • a drug discovery process for identification of a small organic compound that is able to bind to a biological target molecule which has at least one metal ion binding site comprising
  • a drug discovery process for identification of a small organic compound that is able to bind to a biological target molecule which has at least one metal ion binding site comprising
  • test compounds capable of forming a non-covalent binding to a biological target molecule, and capable of changing the activity of the biological target molecule or the binding affinity of the test compound to the biological target molecule
  • a drug discovery process according to any of items 1-3 or 6-7 further comprising
  • step (e) contacting the biological target molecule in wild-type, non-mutated form with at least one test compound determined to non-covalently bind the mutated biological target molecule in step (a), and
  • a drug discovery process according to any of items 1-3 or 6-7 further comprising
  • step (e) contacting the biological target molecule in wild-type, non-mutated form with two or more members of a library of test compounds, wherein the test compounds in chelated form have been determined to non-covalently bind the mutated biological target molecule in step (a), and
  • a drug discovery process according to items 8 or 9 further comprising
  • a drug discovery process according to any of items 1-7 further comprising
  • step (e) contacting the biological target molecule in wild-type, non-mutated form with at 5 least one test compound determined to non-covalently bind the mutated or non-mutated biological target molecule in step (a) but lacking a metal ion chelated thereto, and
  • step (e) contacting the biological target molecule in wild-type, non-mutated form with two or more members of a library of non-chelated test compounds, wherein the test 5 compounds in chelated form have been determined to non-covalently bind the mutated or non-mutated biological target molecule in step (a), and
  • a drug discovery process according to items 11 or 12 further comprising
  • a drug discovery process according to any of items 8-13 further comprising (a) identification of any binding or interaction between the non-chelated test compound and the wild-type biological target molecule.
  • a drug discovery process according to any of items 1-14, wherein the biological target molecule is a protein.
  • a drug discovery process wherein the protein comprises an amino acid residue and wherein the metal ion binding site in the protein is introduced by amino acid substitution at or in the vicinity of 1) a site where the binding of the test compound will interfere with the binding to another protein, for example a regulatory protein, or to a domain of the same protein;
  • a site where the binding of the test compound may interfere with the intramolecular interaction of domains within the protein, for example the interaction of a regulatory domain with a catalytic domain;
  • a drug discovery process according to any of the preceding items, wherein the metal ion binding amino acid residue in the biological target molecule is introduced by site-directed mutagenesis.
  • a drug discovery process according to any of the preceding items, wherein the mutated biological target molecule is obtained as a recombinant expression product in purified or non-purified form.
  • step (a) in any of items 2-5 comprises the further step of determining, based on the three-dimensional structure of the specific protein in question or the primary structure of the specific protein together with a three-dimensional model of the class of proteins to which the specific protein belongs, the
  • a drug discovery process wherein the binding of the test 10 compound to the mutated or non-mutated protein in step (a) in any of items 2-5 is determined using detection of any changes in the biological activity of the protein, competition with binding of a labelled ligand of the protein, or using a metal ion chelator which is in itself detectable or labelled with a detectable labelling agent.
  • a drug discovery process wherein the amino acid residue capable of binding at least one functional group of the test compound other than the metal ion is detected using site-directed mutagenesis of at least one amino acid residue of the protein potentially involved in interaction with said functional group of the test compound other than the metal ion, followed by expression of the mutated protein in a suitable cell,
  • step (a) of any items 2-5 comprises the further steps of improving the binding affinity of a metal ion chelate to the mutated or non-mutated protein, the method comprising (i) selecting a metal ion chelate with an activity to or a binding affinity to the mutated protein of 50 ⁇ M or better as identified by the method of item 21 ,
  • step (v) screening the derivatives of step (iv) by the method of item 21 , (vi) selecting metal ion chelates having at least a two fold increase in activity or in binding affinity,
  • step (e) in any of items 8-12 comprises the further steps of generating a library of test compounds which are derivatives of a test compound found to interact with the wild-type protein in step (e), each test compound in the library being provided with at least one functional group for direct or indirect interaction with at least one amino acid of the wild-type protein, which functional group differs from at least one functional group of each of the other test compounds, and screening the test compound library for compounds interacting with the wild-type protein.
  • step (e) in any of items 8-12 is performed by detecting any changes in the activity of the protein, detecting an effect on binding in a competitive binding assay using a labelled ligand of the protein, or using a chelating agent which is in itself detectable or labelled with a detectable labelling agent.
  • step (e) in any of items 8-12 comprises the further step of determining - based on the three-dimensional structure of the specific protein in question or the primary structure of the specific protein together with the three-dimensional model of the class of proteins to which the specific protein belongs, and based on the information provided by the method of item 25 of the location of amino acid residues in the vicinity of the metal ion binding residue introduced in the mutated protein - the location of an amino acid residue in the wild-type protein binding at least one functional group of a test compound.
  • a drug discovery process wherein the amino acid residue capable of binding at least one functional group of the test compound is detected using site-directed mutagenesis of at least one amino acid residue of the wild-type protein potentially involved in interaction with said functional group of the test compound, followed by expression of the mutated protein in a suitable cell, contacting said cell or a portion thereof including the mutated protein with the test compound, and determining any effect on binding using detection of any changes in the biological activity of the protein, a competitive binding assay using a labelled ligand of the protein, or using a chelating agent which is in itself detectable or labelled with a detectable labelling agent.
  • a drug discovery process according to any of items 1-14, 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.
  • 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 drug discovery process according to item 32 wherein the protein is a membrane protein.
  • the biological target molecule is a membrane protein and the metal ion binding site in the biological target molecule is introduced in a ligand binding crevice of the membrane protein.
  • a method of identifying a metal ion binding site in a biological target molecule comprising
  • test compound which comprises a moiety including at least two heteroatoms for chelating a metal ion, under conditions permitting non-covalent binding of the test compound to the biological target molecule
  • a method of identifying a metal ion binding site in a protein comprising 0 (a) analysing the nucleotide sequence of the gene coding for the protein in order to deduce the amio acid sequence,
  • test compound is contacted with two 0 or more biological target molecules for identification of possible metal ion binding sites thereof.
  • a method of identifying a metal ion binding site in a protein comprising
  • test compound which comprises a moiety including at least two heteroatoms for chelating a metal ion, under conditions permitting non-covalent binding of the test compound to the protein, and detecting any change in the activity of the protein or determining the binding affinity of the test compound to the protein, and 5 (d) determining, based on the generic three-dimensional model of the class of proteins to which the protein or suspected protein belongs, at least one metal ion binding amino acid residue located in said protein to locate the metal ion binding site of said protein.
  • a method of mapping a metal ion binding site of a protein comprising contacting the protein with a test compound which comprises a moiety including at least two heteroatoms for chelating a metal ion, under conditions permitting non-covalent binding of the test compound to the protein, and detecting any change in the activity of the protein or determining the binding affinity of the test compound to the 15 protein, and
  • step (b) determining, based on the primary structure of the specific protein in question and the generic three-dimensional model of the class of proteins to which the specific protein of step (a) belongs, at least one metal ion binding amino acid residue located in the membrane protein to identify the metal ion binding site of said membrane 0 protein.
  • the chemical compounds identified according to the methods disclosed in 5 PCT/EPOO/13389 and optimized according to the present invention will find use as e.g. drug compounds with abortifacient, acromegalic, alcohol deterrent, amebicidic, anabolic, analeptic, analgesic, anesthetic, antiacne, antiallergic, ophthalmic, anti-Alzheimer's disease, antianginal, antiarrhythmic, antiarthritic, antiasthmatic, antibacterial, antibiotic, anticancer, anticholelithogenic, anticoagulant, anticonvulsant, antidepressant, antidiabetic, 0 antidiarrheal, antiemetic, antiepileptic, antiestrogen, antifungal, antiglaucoma, antihistamine, antihypertensive, antiinflammatory, antilipidemic, antimalarial, antimigraine, antinauseant, antineoplastic, antiobesity, antiparasitic, antipar
  • proteins for the present purpose are proteins, which may be stabilised in an active or inactive conformation by a biologically active substance. In this way, it may be possible to obtain an effect of a test compound of the type described herein irrespective of whether the active site of the protein is known, or whether the structure of the active site has been resolved (e.g. by X-ray crystallisation).
  • proteins are enzymes, receptors, hormones and other signalling molecules, transcriptional factors and regulators, intra- or extracellular structural proteins, in particular actins; adaptins; antibodies; ATPases; cyclins; dehydrogenases; GTP-binding proteins; GTP/GDP-exchange factors; GTPase activating proteins; GTP/GDP dissociation inhibitors; chaperones; histones; histone acetyltransferases & deacetyltransferases; hormones and other signalling proteins and peptides; kinases; lipases; major facilitator superfamily proteins; motorproteins; nucleases; polymerases; isomerases; proteases; protease inhibitors; phosphatases; ubiquitin-system proteins; membrane proteins including receptors, transporters and channels; transcription factors and tubulins; preferably membrane receptors; nuclear receptors, zinc finger proteins; proteases, tyrosine kinases and matrix proteins.
  • Other important proteins
  • Figure 1 shows a schematic depiction of the structure of rhodopsin-like 7TMs with one or two conserved key residues highlighted in each TM: Asnl:18; Aspll:10; Cys 111:01 and Arglll:26; TrpIV: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 most common 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 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
  • 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.
  • Panel H and I Model for chelation of Zn(ll) by GalR1 from the mutational analysis.
  • Figure 7 relates to identification of naturally occurring metal-ion chelator binding site in the 12TM dopamine transporter in Example 1.3. 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, [H193K], in which binding of the metal-ion complex has been eliminated (Noregaard et al. EMBO J. (1998) 17: 4266-4273).
  • Panel C Effect of metal-ion complex formation on the ability to inhibit [ 3 H]-dopamine uptake in the wild-type and [H193K] mutant 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 His 93 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 111.1. 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 of 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 III.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
  • the effect of Cu(ll) and copper-chelator complexes on stimulation of accumulation of intracellular cAMP was analyzed in COS-7 cells expressing the beta2-adrenoceptor.
  • 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 [F289C;N312C] beta2-AR.
  • 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 III.6.
  • TM- 369 was identified as having a 50-fold increased affinity compared to the unsubstituted 2,2'-bipyridine scaffold.
  • 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. 21 is related to Exampel 111.7.
  • 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 R02 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 111:08 (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-
  • Formula I 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.
  • 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.
  • Typical functional group interconversions are exemplified by transforming -COOCH 3 into a -CH 2 -NH 2 moiety as exemplified with the 2,2'. bipyridine system.
  • 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.
  • 5- Amino-2,2 '-bipyridine 5-Nitro-2,2'-bipyridine (0.641 mol, 129 mg) was dissolved in MeOH/THF (5ml+5ml). 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.
  • Triethyl amine (78.8 mmol, 11.0 ml, 5 equiv.) was then added and the reaction mixture was allowed to warm to ambient temperature.
  • DCM 100 ml
  • sat. NaHCO 3 150 ml
  • the organic phase was separated and the aqueous phase was extracted with DCM (2x100 ml).
  • the combined organic phases were dried over MgSO 4 , and the solvent was evaporated in vacuo
  • the crude product was purified by column chromatography (DCM/MeOH/NH 3 , 100/10/1 ). Yield: 44 %.
  • Triphenyl phosphine (1.0 mmol, 0.262 g) was dissolved in dry benzene (7 ml). 4-(Bromomethyl)-2,2 ' -bipyridine (1.06 mmol, 0.265 g) was added, and the reaction solution was refluxed for 2 h. A white precipitate formed. The solvent was removed in vacuo, and DCM (7 ml) was added to the solid residue. Benzaldehyde (1.0 mmol, 0.102 ml) was added and thereafter aqueous NaOH (1.0 mmol, 0.25 ml, 4M).
  • 6-Chloro- terf-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.

Abstract

L'invention concerne l'utilisation de composés ou de sélection de composés chimiques (bibliothèques) de la formule générale I suivante dans des procédés in vivo d'essai ou de validation de l'importance physiologique et/ou du potentiel thérapeutique ou pharmacologique de molécules cibles biologiques, notamment des protéines telles que, par exemple, des récepteurs et, en particulier des récepteurs 7TM dans des animaux de laboratoire exprimant la molécule cible biologique avec, en particulier, un site d'ions métalliques génétiquement modifié, muet. L'invention concerne également l'utilisation de sites de liaison d'ions métalliques spécifiques de nature générique dans des molécules cibles biologiques spécifiques telles que, par exemple, des protéines transmembranaires dans lesquelles le site de liaison d'ions métalliques est capable de former un complexe avec un ion métallique. Elle concerne en outre des composés ou des bibliothèques chimiques pouvant être utilisés dans des procédés destinés à améliorer le comportement pharmacocinétique de chélates d'ions métalliques (par exemple, motif d'absorption, demi-vie plasmatique, distribution, métabolisme et/ou élimination des chélates d'ions métalliques. Afin d'améliorer l'efficacité de l'impact des chélates d'ions métalliques sur la molécule cible biologique après administration du chélate d'ions métalliques in vivo à un animal de laboratoire, il est intéressant, par exemple, d'accroître la durée pendant laquelle le chélate d'ions métalliques est présent dans le système circulatoire et/ou localisé au niveau de la cible. L'invention concerne enfin des composés chélateurs d'ions métalliques, conçus pour être utilisés dans un processus de validation et des bibliothèques d'au moins deux de ces composés chélateurs d'ions métalliques.
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WO2005040121A2 (fr) * 2003-10-16 2005-05-06 Abbott Laboratories Amides inhibant le recepteur de sous-type 1 des vanilloides 'vr1'
WO2007029078A2 (fr) * 2005-09-05 2007-03-15 Ranbaxy Laboratories Limited Derives de succinimide et de glutarimide comme antagonistes de recepteurs adrenergiques
EP1814551A2 (fr) * 2004-09-20 2007-08-08 Xenon Pharmaceuticals Inc. Derives de pyridazine destines a l'inhibition du desaturase-coa-stearoyl de l'humain
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
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US7951805B2 (en) 2004-09-20 2011-05-31 Xenon Pharmaceuticals Inc. Heterocyclic derivatives and their use as mediators of stearoyl-CoA desaturase
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CN110240609A (zh) * 2019-07-15 2019-09-17 济南大学 一种单组分白光配合物及其合成方法和用途
US10517849B2 (en) 2016-10-26 2019-12-31 Constellation Pharmaceuticals, Inc. LSD1 inhibitors and medical uses thereof
US10526287B2 (en) 2015-04-23 2020-01-07 Constellation Pharmaceuticals, Inc. LSD1 inhibitors and uses thereof
US10647661B2 (en) 2017-07-11 2020-05-12 Vertex Pharmaceuticals Incorporated Carboxamides as modulators of sodium channels
US10894797B2 (en) 2018-09-18 2021-01-19 Nikang Therapeutics, Inc. Fused tricyclic ring derivatives as SRC homology-2 phosphatase inhibitors
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WO2004058265A1 (fr) * 2002-12-24 2004-07-15 Biofocus Plc Bibliotheques de composes de derives de pyrazine 2,3-substitues pouvant se fixer aux recepteurs couples a la proteine g
WO2005040121A2 (fr) * 2003-10-16 2005-05-06 Abbott Laboratories Amides inhibant le recepteur de sous-type 1 des vanilloides 'vr1'
WO2005040121A3 (fr) * 2003-10-16 2005-06-23 Abbott Lab Amides inhibant le recepteur de sous-type 1 des vanilloides 'vr1'
US7037927B2 (en) 2003-10-16 2006-05-02 Abbott Laboratories Amides that inhibit vanilloid receptor subtype 1 (VR1) receptor
US7595332B2 (en) 2003-10-16 2009-09-29 Abbott Laboratories Amides that inhibit vanilloid receptor subtype 1 (VR1)
JP2007508387A (ja) * 2003-10-16 2007-04-05 アボット・ラボラトリーズ バニロイド受容体サブタイプ１（ｖｒ１）受容体を阻害するアミド
US7829712B2 (en) 2004-09-20 2010-11-09 Xenon Pharmaceuticals Inc. Pyridazine derivatives for inhibiting human stearoyl-CoA-desaturase
EP1814551A2 (fr) * 2004-09-20 2007-08-08 Xenon Pharmaceuticals Inc. Derives de pyridazine destines a l'inhibition du desaturase-coa-stearoyl de l'humain
JP2008513506A (ja) * 2004-09-20 2008-05-01 ゼノン・ファーマシューティカルズ・インコーポレイテッド ヒトステアロイル−ＣｏＡデサチュラーゼを阻害するためのピリダジン誘導体
US8071603B2 (en) 2004-09-20 2011-12-06 Xenon Pharmaceuticals Inc. Heterocyclic derivatives and their use as stearoyl-CoA desaturase inhibitors
US7919496B2 (en) 2004-09-20 2011-04-05 Xenon Pharmaceuticals Inc. Heterocyclic derivatives for the treatment of diseases mediated by stearoyl-CoA desaturase enzymes
EP2316458A1 (fr) * 2004-09-20 2011-05-04 Xenon Pharmaceuticals Inc. Derives de pyridazine destines a l'inhibition du desaturase-coa-stearoyl de l'humain
US7951805B2 (en) 2004-09-20 2011-05-31 Xenon Pharmaceuticals Inc. Heterocyclic derivatives and their use as mediators of stearoyl-CoA desaturase
US8026360B2 (en) 2004-09-20 2011-09-27 Xenon Pharmaceuticals Inc. Substituted pyridazines as stearoyl-CoA desaturase inhibitors
JP2013014623A (ja) * 2004-09-20 2013-01-24 Xenon Pharmaceuticals Inc ヒトステアロイル−ＣｏＡデサチュラーゼを阻害するためのピリダジン誘導体
US8541457B2 (en) 2005-06-03 2013-09-24 Xenon Pharmaceuticals Inc. Aminothiazole derivatives as human stearoyl-CoA desaturase inhibitors
WO2007029078A3 (fr) * 2005-09-05 2007-07-12 Ranbaxy Lab Ltd Derives de succinimide et de glutarimide comme antagonistes de recepteurs adrenergiques
WO2007029078A2 (fr) * 2005-09-05 2007-03-15 Ranbaxy Laboratories Limited Derives de succinimide et de glutarimide comme antagonistes de recepteurs adrenergiques
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
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