Receptor dynamics and kinetic discrimination contribute to selectivity of β1-adrenoceptor antagonists

Human {beta}1-adrenoceptor (h[beta]1AR)-selective antagonists are widely used to suppress cardiac adrenergic signalling in cardiovascular disease with limited but still clinically relevant inhibition of h[beta]2AR functions in airway and vascular smooth muscle. However, achieving strong subtype selectivity remains challenging because the orthosteric binding sites (OBS) of h[beta]1AR and h[beta]2AR are highly conserved. Here we studied the molecular mechanisms for three highly subtype selective h[beta]1AR antagonists, CGP20712A, NDD713 and NDD825. Three cryo-EM structures were therefore determined for each of the ligands bound to an inactive state of turkey t[beta]1AR in complex with an engineered nanobody fiducial (Nb60.5) to facilitate image processing. There were striking differences in how each antagonist bound to the receptor compared to what was observed in previous antagonist-bound t[beta]1AR and h[beta]2AR structures; these included non-canonical binding of the oxypropan-2-olamine (secondary amine and [beta]-hydroxyl) cores of CGP20712A and NDD825 and the opening of a cleft between transmembrane helices H4 and H5 adjacent to the OBS. Mutagenesis of residues in the extended OBS that differ between h[beta]1AR and h[beta]2AR only partially explained the subtype selectivity. Subsequent kinetic analyses showed that all three ligands associated more rapidly with h[beta]1AR than h[beta]2AR, whereas dissociation effects were ligand dependent. Reciprocal receptor chimeras identified the h[beta]1AR extracellular vestibule as a key factor for fast ligand association. MD simulations further revealed that the cleft opening between H4 and H5 where CGP20712A and NDD825 insert is energetically feasible in h[beta]1AR, but is energetically inaccessible in h[beta]2AR. Together, these results establish that h[beta]1AR selectivity for CGP20712A and NDD825 arises from coordinated effects of OBS recognition, the ligand-entry pathway and receptor dynamics.