| During the last decade, emerging technologies such as genomics and proteomics have induced a paradigm shift in pharmaceutical research. Newly identified targets are classified according to structural rather than functional homology. With the aid of combinatorial chemistry and high-throughput assay techniques, new targets can be addressed by screening of large compound collections. In this respect, one of the most important target classes comprises the family of G-Protein Coupled Receptors (GPCRs). In fact, about 60% of currently marketed drugs exert their mode of action through interaction with a specific GPCR. Not surprisingly, the design of new strategies towards GPCR ligands is a major research activity both in academia and in industrial settings. GPCRs are characterised by a common 7-transmembrane helical motif that binds to a specific ligand, most often a low-molecular weight ligand. In this way, they are involved in a broad array of biological functions. Obviously, selective control of these processes is of eminent importance for the development of efficient drugs with a minimum of side effects. The recent finding that bivalent ligands, molecules containing two pharmacological recognition units (pharmacophores) linked covalently through a spacer, are effective ligands, with increased potency when compared to the respective monomeric ligands, opens the way to the future development of potent, selective GPCR mediators. Fundamental understanding, however, of the mode of action of bivalent ligands is imperative to ensure the generation of future pharmaceutics based on this approach. Several hypotheses have been postulated to rationalise the activity of bivalent ligands. The bivalent ligand interacts simultaneously with two neighbouring receptors or uses a secondary binding site in the vicinity of the primary binding site of the ligand. Alternatively, enhanced binding affinity has been assumed to proceed through a univalently bound state, the unbound recognition unit being in the locus of neighbouring binding sites, equivalent to a high local concentration of the free pharmacophore. Finally, the bivalent ligand has been proposed to induce or stabilise receptor dimerisation. Importantly, none of the above hypotheses have been validated, moreover, different modes of action may apply for bivalent ligands binding to GPCRs of different origins. The here proposed research aims at the generation of insight and understanding in the mode of action of bivalent ligands in binding and (in)activation of GPCRs. To this end we will apply combinatorial chemistry to generate libraries of structurally diverse bivalent ligands, in combination with high-throughput assaying and computational modeling. As a target GPCR we have selected the Gonadotropin Releasing Hormone Receptor (GnRHR). For this receptor, a wide array of structurally diverse GnRHR ligands have been reported. The endogenous ligand for the GnRHR, hypothalamic gonadotropin-releasing hormone (GnRH), is a key regulator in mammalian sexual maturation and reproductive functions. The pharmacophoric elements in the reported monomeric ligands ligands present a valuable starting point for the envisaged preparation, through combinatorial techniques, of bivalent GnRHR modulators. The design of the libraries will address a variety of factors such as the nature of the pharmacophore, its (site of) modification, the length (allowing control over the interpharmacophoric distance), flexibility/rigidity, stereochemistry, lipophilicity/hydrophilicity and hydrogen bond forming potential of the linker-scaffold-system joining the two pharmacophores. Determination of the structure activity relationships (SAR) for the most active bivalent ligands using advanced computational modeling techniques will provide insight in the molecular basis of GnRHR binding. In a subsequent optimisation cycle, the in silico properties will be linked to the experimentally obtained activities to guide the design of new libraries. Furthermore, the detailed knowledge derived from these studies will allow us to extend the proposed methodology towards other GPCRs, with the ultimate aim to provide inroads towards the development of future GPCR directed medicins. |
