Muscarinic receptor agonists are characterized by apparently strict restraints on their tertiary or quaternary amine and their distance to an ester or related center. On the basis of the active state crystal structure of the muscarinic M2 receptor in complex with iperoxo, we explored potential agonists that lacked the highly conserved functionalities of previously known ligands. Using structure-guided pharmacophore design followed by docking, we found two agonists (compounds 3 and 17), out of 19 docked and synthesized compounds, that fit the receptor well and were predicted to form a hydrogen-bond conserved among known agonists. Structural optimization led to compound 28, which was 4-fold more potent than its parent 3. Fortified by the discovery of this new scaffold, we sought a broader range of chemotypes by docking 2.2 million fragments, which revealed another three micromolar agonists unrelated either to 28 or known muscarinics. Even pockets as tightly defined and as deeply studied as that of the muscarinic reveal opportunities for the structure-based design and the discovery of new chemotypes.

Most drugs acting on G-protein-coupled receptors target the orthosteric binding pocket where the native hormone or neurotransmitter binds. There is much interest in finding allosteric ligands for these targets because they modulate physiologic signaling and promise to be more selective than orthosteric ligands. Here we describe a newly developed allosteric modulator of the β₂-adrenergic receptor (β₂AR), AS408, that binds to the membrane-facing surface of transmembrane segments 3 and 5, as revealed by X-ray crystallography. AS408 disrupts a water-mediated polar network involving E122^3.41 and the backbone carbonyls of V206^5.45 and S207^5.46. The AS408 binding site is adjacent to a previously identified molecular switch for β₂AR activation formed by I^3.40, P^5.50 and F^6.44. The structure reveals how AS408 stabilizes the inactive conformation of this switch, thereby acting as a negative allosteric modulator for agonists and positive allosteric modulator for inverse agonists.