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

Abstract

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-HT 1A receptor where mutagenesis studies map three spatially distinct binding regions which correspond to the binding sites of thè`s mall, one site-®lling'' ligands 5-HT, propranolol and 8-OH-DPAT, respectively. The models of the 5-HT 1A ligandreceptor complexes provide a frame for the discussion of other ligand-receptor interactions, including a 1 and b 2 adrenoceptors, D 1 and D 2 dopamine, and 5-HT 1D and 5-HT 2A 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 re¯ected by the architecture of known high af®nity ligands which crosslink two or three``one site-®lling'' fragments around a basic amino group. Typical ligands reported in the CipslineyMDDR portfolio illustrate this point despite the occasional dif®culty of attributing the individual ligand fragments to a speci®c 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-speci®c 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.

G protein-coupled receptors:In silicodrug discovery in 3D

“…A binding pocket was easily identified on the extracellular side of the structure, allowing the 5-HT amine moiety to interact with Asp-116 in TM3 and its hydroxyl moiety to interact with Ser-199 in TM5; both in good agreement with experimental data (41,42). Fig.…”

Proteochemometrics Modeling of the Interaction of Amine G-Protein Coupled Receptors with a Diverse Set of Ligands

“…Thus, according to the so-called three-binding site hypothesis, amine ligands bind at three spatially distinct binding regions, all of which are supposed to be located between the seven helix transmembrane domains of the amine GPCRs. Moreover, depending on ligand size and shape of the ligand, the binding is proposed to more or less overlap these three binding regions (Jacoby et al, 1999). Obviously, as the hypothesis is based on 3D modeling, it is restricted to the correctness of the docking procedures.…”

“…The picture emerging from these studies is also not entirely clear and sometimes even quite contradictory; e.g., modeling and mutagenesis studies suggest the placement of the binding pocket of pirenzepine in alternative binding pockets of the muscarinic receptors (Fanelli et al, 1995;Bourdon et al, 1997) and it is not an easy tasks to resolve such ambiguities using 3D modeling. It has also been suggested that antagonist ligands for amine receptors may bind to alternative binding pockets in the receptors (Schwartz 1994;Jacoby et al, 1999). Thus, according to the so-called three-binding site hypothesis, amine ligands bind at three spatially distinct binding regions, all of which are supposed to be located between the seven helix transmembrane domains of the amine GPCRs.…”

“…The physicochemical description of the amine GPCRs was derived from the amino acid sequences of their seven transmembrane (TM) regions. Restricting the analysis to the TM helices seemed reasonable because 3D modeling and mutagenesis studies have indicated that the amine binding sites of amine GPCRs are situated within the dihedral cleft located between these helices (Bikker et al, 1998;Jacoby et al, 1999). The amino acid sequences of seven transmembrane (TM1-TM7) regions were first aligned, using the alignments retrieved from the GPCRDB database (Horn et al, 1998).…”

A Novel Chemogenomics Knowledge-Based Ligand Design Strategy—Application to G Protein-Coupled Receptors

“…For the monoamine GPCRs, a three-bindingsite hypothesis [2] was recently demonstrated by combining: 1) analyses of the architectures of known monoamine GPCR ligands; 2) analyses of molecular models of the ligandreceptor interactions; and 3) structural bioinformatics analyses of the sequence similarities of the three distinct binding regions of ''one-site-filling'' ligand fragments within the monoamine GPCR family. For the 5HT 1A receptor, which provided a frame for the discussion of other related ligand-GPCR interactions, 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 ( Figure 1).…”