Hybridization into a Bitopic Ligand Increased Muscarinic Receptor Activation for Isopilocarpine but Not for Pilocarpine Derivatives

Pilocarpine (1), a secondary metabolite of several Pilocarpus species, is a therapeutically used partial agonist of muscarinic acetylcholine receptors (mAChRs). The available pharmacological data and structure–activity relationships do not provide comparable data for all five receptor subtypes. In this study, pilocarpine (1), its epimer isopilocarpine (2), racemic analogues pilosinine (3) and desmethyl pilosinine (4), and the respective hybrid ligands with a naphmethonium fragment (5-C6 to 8-C6) were synthesized and analyzed in mini-G nano-BRET assays at the five mAChRs. In line with earlier studies, pilocarpine was the most active compound among the orthosteric ligands 1–4. Computational docking of pilocarpine and isopilocarpine to the active M2 receptor suggests that the trans-configuration of isopilocarpine leads to a loss of the hydrogen bond from the lactone carbonyl to N6.52, explaining the lower activity of isopilocarpine. Hybrid formation of pilocarpine (1) and isopilocarpine (2) led to an inverted activity rank, with the trans-configured isopilocarpine hybrid (6-C6) being more active. The hydrogen bond of interest is formed by the isopilocarpine hybrid (6-C6) but not by the pilocarpine hybrid (5-C6). Hybridization thus leads to a modified binding mode of the orthosteric moiety, as the binding mode of the hybrid is dominated by the high-affinity allosteric moiety

FRET Studies of Quinolone-Based Bitopic Ligands and Their Structural Analogues at the Muscarinic M₁ Receptor

Aiming to design partial agonists as well as allosteric modulators for the M₁ muscarinic acetylcholine (M₁AChR) receptor, two different series of bipharmacophoric ligands and their structural analogues were designed and synthesized. The hybrids were composed of the benzyl quinolone carboxylic acid (BQCA)-derived subtype selective allosteric modulator <b>3</b> and the orthosteric building block 4-((4,5-dihydroisoxazol-3-yl)­oxy)-N,N-dimethylbut-2-yn-1-amine (base of iperoxo) <b>1</b> or the endogenous ligand 2-(dimethylamino)­ethyl acetate (base of acetylcholine) <b>2</b>, respectively. The two pharmacophores were linked <i>via</i> alkylene chains of different lengths (C4, C6, C8, and C10). Furthermore, the corresponding structural analogues of <b>1</b> and <b>2</b> and of modified BQCA <b>3</b> with varying alkyl chain length between C2 and C10 were investigated. Fluorescence resonance energy transfer (FRET) measurements in a living single cell system were investigated in order to understand how these compounds interact with a G protein-coupled receptor (GPCR) on a molecular level and how the single moieties contribute to ligand receptor interaction. The characterization of the modified orthosteric ligands indicated that a linker attached to an orthoster rapidly attenuates the receptor response. Linker length elongation increases the receptor response of bitopic ligands, until reaching a maximum, followed by a gradual decrease. The optimal linker length was found to be six methylene groups at the M₁AChR. A new conformational change is described that is not of inverse agonistic origin for long linker bitopic ligands and was further investigated by exceptional fragment-based screening approaches