Molecular Determinants of Ligand Binding Modes in the Histamine H4 Receptor: Linking Ligand-Based Three-Dimensional Quantitative Structure–Activity Relationship (3D-QSAR) Models to in Silico Guided Receptor Mutagenesis Studies

The histamine H4 receptor (H4R) is a G protein-coupled receptor (GPCR) that plays an important role in inflammation. Similar to the homologous histamine H3 receptor (H3R), two acidic residues in the H4R binding pocket, D3.32 and E5.46, act as essential hydrogen bond acceptors of positively ionizable hydrogen bond donors in H4R ligands. Given the symmetric distribution of these complementary pharmacophore features in H4R and its ligands, different alternative ligand binding mode hypotheses have been proposed. The current study focuses on the elucidation of the molecular determinants of H4R–ligand binding modes by combining (3D) quantitative structure–activity relationship (QSAR), protein homology modeling, molecular dynamics simulations, and site-directed mutagenesis studies. We have designed and synthesized a series of clobenpropit (N-(4-chlorobenzyl)-S-[3-(4­(5)-imidazolyl)­propyl]­isothiourea) derivatives to investigate H4R–ligand interactions and ligand binding orientations. Interestingly, our studies indicate that clobenpropit (2) itself can bind to H4R in two distinct binding modes, while the addition of a cyclohexyl group to the clobenpropit isothiourea moiety allows VUF5228 (5) to adopt only one specific binding mode in the H4R binding pocket. Our ligand-steered, experimentally supported protein modeling method gives new insights into ligand recognition by H4R and can be used as a general approach to elucidate the structure of protein–ligand complexes.