Abstract
Within the last years, for several ligands, binding to G protein-coupled receptors or other target proteins, a binding of the ligand in two different orientations is described. One appropriate experimental technique to detect two different binding orientations is the crystallization of the ligand-protein-complex, but crystallization and subsequent X-ray analysis do not belong to the routine methods. By traditional competitive radioligand equilibrium binding assays, it is not possible to detect or to distinguish between two different binding orientations, but there is a possibility to identify two different binding orientations by performing kinetic competitive radioligand-binding assays. To study the limitations of this new technique, the related differential equations were defined and solved numerically for 8 different sets of rate constants, also considering an experimental error up to ~10%. In principal, the kinetic competitive radioligand binding assay is a suitable technique to detect two different ligand binding orientations. However, the present study shows that this is only possible under distinct conditions: (1) the rate constants of dissociation for both binding orientations of the cold ligand should at least be >> 10-fold different to each other and (2) the experimental error should be as small as possible. Although there are some limitations for the experimental usability of this method, it is worthwhile to perform kinetic competitive binding assays, especially if there are hints for two binding orientations of a ligand, e.g. based on molecular modelling studies.
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References
Birnkammer T, Spickenreither A, Brunskole I, Lopuch M, Kagermeier N, Bernhardt G, Dove S, Seifert R, Elz S, Buschauer A (2012) The bivalent ligand approach leads to highly potent and selective acylguanidine-type histamine H2 receptor agonists. J Med Chem 55:1147–1160
Bock A, Chirinda B, Krebs F, Messerer R, Batz J, Muth M, Dallanoce C, Klingenthal D, Trankle C, Hoffmann C, De Amici M, Holzgrabe U, Kostenis E, Mohr K (2014) Dynamic ligand binding dictates partial agonism at a G protein-coupled receptor. Nat Chem Biol 10:18–20
Bruning JB, Parent AA, Gil G, Zhao M, Nowak J, Pace MC, Smith CL, Afonine PV, Adams PD, Katzenellenbogen JA, Nettles KW (2010) Coupling of receptor conformation and ligand orientation determine graded activity. Nat Chem Biol 6:837–843
Copeland RA (2011) Conformational adaptation in drug-target interactions and residence time. Future Med Chem 3:1491–1501
Copeland RA, Pompliano DL, Meek TD (2006) Drug-target residence time and its implications for lead optimization. Nat Rev Drug Discov 5:730–739
Deml KF, Beermann S, Neumann D, Strasser A, Seifert R (2009) Interactions of histamine H1-receptor agonists and antagonists with the human histamine H4-receptor. Mol Pharmacol 76:1019–1030
Elz S, Kramer K, Pertz HH, Detert H, ter Laak AM, Kuhne R, Schunack W (2000) Histaprodifens: synthesis, pharmacological in vitro evaluation, and molecular modeling of a new class of highly active and selective histamine H1-receptor agonists. J Med Chem 43:1071–1084
Guo D, Hillger JM, IJzerman AP, Heitman LH (2014) Drug-target residence time—a case for G protein-coupled receptors. Med Res Rev 34:856–892
Hughes JP, Rees S, Kalindjian SB, Philpott KL (2011) Principles of early drug discovery. Br J Pharmacol 162:1239–1249
Jacoby E, Bouhelal R, Gerspacher M, Seuwen K (2006) The 7 TM G-protein-coupled receptor target family. ChemMedChem 1:761–782
Motulsky HJ, Mahan LC (1984) The kinetics of competitive radioligand binding predicted by the law of mass action. Mol Pharmacol 25:1–9
Newman DJ, Cragg GM (2012) Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod 75:311–335
Nwachukwu JC, Srinivasan S, Bruno NE, Parent AA, Hughes TS, Pollock JA, Gjyshi O, Cavett V, Nowak J, Garcia-Ordonez RD, Houtman R, Griffin PR, Kojetin DJ, Katzenellenbogen JA, Conkright MD, Nettles KW (2014) Resveratrol modulates the inflammatory response via an estrogen receptor-signal integration network. elife 3:e02057
Overington JP, Al-Lazikani B, Hopkins AL (2006) How many drug targets are there? Nat Rev Drug Discov 5:993–996
Pawson AJ, Sharman JL, Benson HE, Faccenda E, Alexander SP, Buneman OP, Davenport AP, McGrath JC, Peters JA, Southan C, Spedding M, Yu W, Harmar AJ, Nc I (2014) The IUPHAR/BPS guide to PHARMACOLOGY: an expert-driven knowledgebase of drug targets and their ligands. Nucleic Acids Res 42:D1098–D1106
Seifert R, Strasser A, Schneider EH, Neumann D, Dove S, Buschauer A (2013) Molecular and cellular analysis of human histamine receptor subtypes. Trends Pharmacol Sci 34:33–58
Sencanski M, Dosen-Micovic L (2014) In silico study of the structurally similar ORL1 receptor agonist and antagonist pairs reveal possible mechanism of receptor activation. Protein J 33:231–242
Shim J, Coop A, MacKerell AD Jr (2013) Molecular details of the activation of the mu opioid receptor. J Phys Chem B 117:7907–7917
Strasser A, Wittmann HJ (2010) 3D-QSAR CoMFA study to predict orientation of suprahistaprodifens and phenoprodifens in the binding-pocket of four histamine H1-receptor species. Molecular informatics 29:333–341
Strasser A, Striegl B, Wittmann HJ, Seifert R (2008) Pharmacological profile of histaprodifens at four recombinant H1-receptor species isoforms. J Pharmacol Exp Ther 324:60–71
Strasser A, Wittmann HJ, Kunze M, Elz S, Seifert R (2009) Molecular basis for the selective interaction of synthetic agonists with the human histamine H1-receptor compared with the Guinea pig H1-receptor. Mol Pharmacol 75:454–465
Strasser A, Wittmann H-J, Buschauer A, Schneider EH, Seifert R (2013) Species-dependent activities of GPCR ligands: lessons from histamine receptor orthologs. Trends Pharmacol Sci 34:13–32
Tummino PJ, Copeland RA (2008) Residence time of receptor-ligand complexes and its effect on biological function. Biochemistry 47:5481–5492
Vauquelin G (2016) Effects of target binding kinetics on in vivo drug efficacy: koff , kon and rebinding. Br J Pharmacol 173:2319–2334
Wittmann HJ, Seifert R, Strasser A (2011) Influence of the N-terminus and the E2-loop onto the binding kinetics of the antagonist mepyramine and the partial agonist phenoprodifen to H(1)R. Biochem Pharmacol 82:1910–1918
Zanatta G, Della Flora Nunes G, Bezerra EM, da Costa RF, Martins A, Caetano EW, Freire VN, Gottfried C (2016) Two binding geometries for risperidone in dopamine D3 receptors: insights on the fast-off mechanism through docking, quantum biochemistry, and molecular dynamics simulations. ACS Chem Neurosci 7:1331–1347
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Wittmann, HJ., Strasser, A. Competitive association binding kinetic assays: a new tool to detect two different binding orientations of a ligand to its target protein under distinct conditions?. Naunyn-Schmiedeberg's Arch Pharmacol 390, 595–612 (2017). https://doi.org/10.1007/s00210-017-1362-7
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DOI: https://doi.org/10.1007/s00210-017-1362-7