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-directe d mutagenesis experiments of the monoamine G protein-coupled receptors reve al 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 mutagene sis studies map three spatially distinct binding regions which correspond t o the binding sites of the "small, one site-filling" ligands 5-HT, proprano lol and 8-OH-DPAT, respectively. The models of the 5-HT1A ligand-receptor c omplexes provide a frame for the discussion of other ligand-receptor intera ctions, including alpha(1) and beta(2) adrenoceptors, D-1 and D-2 dopamine, and 5-HT1D and 5-HT2A receptors, where mutagenesis and modelling studies a lso showed occupation of the corresponding three binding locations. All thr ee binding sites are located within the highly conserved seven helix transm embrane domain of the receptor and overlap partially at the prominent Asp r esidue in TM3 which constitutes the benchmark anchor site for monoamine lig ands. 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 ar chitecture of known high affinity ligands which crosslink two or three "one site-filling" fragments around a basic amino group. Typical ligands report ed in the Cipsline/MDDR portfolio illustrate this point despite the occasio nal difficulty of attributing the individual ligand fragments to a specific receptor site. The database exploration illustrates the binding site promi scuity of some fragments which is particularly evident for symmetrical liga nds and which has implications for 3D QSAR methods dependent on alignments. We propose to generate by deconvolution of known ligands three distinct da tabases of site-specific bioisosters which should provide keystones for the design of novel recomposed monoamine GPCR ligands. The systematic explorat ion of the "three site" hypothesis should open novel perspectives for the u nderstanding of ligand recognition for this class of therapeutically import ant receptors.