A Three Binding Site Hypothesis for the Interaction of Ligands with Monoamine G Protein‐coupled Receptors: Implications for Combinatorial Ligand Design

Three-dimensional models of ligand-receptor complexes based on site-directed mutagenesis experiments of the monoamine G protein-coupled receptors reveal the existence of three distinct drug binding sites inside the receptors. Here, we develop this “three-site” hypothesis and outline its implications for the modular design of ligands for monoamine GPCRs. Molecular models of receptor-ligand complexes are built for the 5-HT1A receptor where mutagenesis studies map three spatially distinct binding regions which correspond to the binding sites of the “small, one site-filling” ligands 5-HT, propranolol and 8-OH-DPAT, respectively. The models of the 5-HT1A ligand-receptor complexes provide a frame for the discussion of other ligand-receptor interactions, including α1 and β2 adrenoceptors, D1 and D2 dopamine, and 5-HT1D and 5-HT2A receptors, where mutagenesis and modelling studies also showed occupation of the corresponding three binding locations. All three binding sites are located within the highly conserved seven helix transmembrane domain of the receptor and overlap partially at the prominent Asp residue in TM3 which constitutes the benchmark anchor site for monoamine ligands. The analysis of the sequence similarity, for each binding site, among the monoamine GPCR superfamily shows that the three loci display different degrees of evolutionary conservation. This result suggests different roles for each of the binding sites in intrinsic receptor functions and provides additional insights for the design of ligand functionality and selectivity. The existence of three distinct binding sites is also reflected by the architecture of known high affinity ligands which crosslink two or three “one site-filling” fragments around a basic amino group. Typical ligands reported in the Cipsline/MDDR portfolio illustrate this point despite the occasional difficulty of attributing the individual ligand fragments to a specific receptor site. The database exploration illustrates the binding site promiscuity of some fragments which is particularly evident for symmetrical ligands and which has implications for 3D QSAR methods dependent on alignments. We propose to generate by deconvolution of known ligands three distinct databases of site-specific bioisosters which should provide keystones for the design of novel recomposed monoamine GPCR ligands. The systematic exploration of the “three site” hypothesis should open novel perspectives for the understanding of ligand recognition for this class of therapeutically important receptors.