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