Adrenergic receptor (AR) subtypes are G-Protein Coupled Receptors (GPCRs) activated by the same endogenous catecholamines, adrenaline and nor-adrenaline. The two subtypes α1A- and α1B-AR maintain a complex balance in modulating the functions of sympathetic and central nervous systems, whereby chronic activity can be either detrimental or protective for both heart and brain function. Regulation is believed to be mediated through the distinct activation of individual α1-AR subtypes and thus, subtype selective activation or blocking may have major clinical implications. Despite such physiological and pharmacological importance, there are no clinically approved selective α1A- or α1B-AR drugs, most likely due to conservation of sequence and possibly structure of the orthosteric binding site hampering molecular design. This is further exacerbated by a general lack of structural and dynamic knowledge on α1-ARs and the ligands that bind and modulate the activity of these receptors.
In this study, we have used thermostabilized mutants of α1A- and α1B-AR to assess the ligand binding conformations by employing solution-based ligand-observed Nuclear Magnetic Resonance methods. These experiments take advantage of known orientations of a ligand, for example adrenaline, to determine the orientation of novel ligands. We obtained experimental NMR data for the ligand A-61603 (α1A-AR selective agonist) with respect to adrenaline for both α1A- and α1B-AR. These data were compared to the back-calculated spectra obtained from molecular dynamics simulations. The results helped mechanistically explain the selectivity of A-61603 towards α1A-AR. Overall, we have shown that this solution-based methodology provides valuable information on ligand-binding poses inside the highly conserved orthosteric binding site of ARs, dissecting out subtle structural variations across the subtypes and thereby may aid in future subtype selective drug development.