Aiming to design partial agonists as well as allosteric modulators for the M muscarinic acetylcholine (MAChR) 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 3 and the orthosteric building block 4-((4,5-dihydroisoxazol-3-yl)oxy)-N,N-dimethylbut-2-yn-1-amine (base of iperoxo) 1 or the endogenous ligand 2-(dimethylamino)ethyl acetate (base of acetylcholine) 2, respectively. The two pharmacophores were linked via alkylene chains of different lengths (C4, C6, C8, and C10). Furthermore, the corresponding structural analogues of 1 and 2 and of modified BQCA 3 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 MAChR. 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.

The muscarinic M 1 acetylcholine receptor is an important drug target for the treatment of various neurological disorders. Designing M 1 receptor-selective drugs has proven challenging, mainly due to the high conservation of the acetylcholine binding site among muscarinic receptor subtypes. Therefore, less conserved and topographically distinct allosteric binding sites have been explored to increase M 1 receptor selectivity. In this line, bitopic ligands, which target orthosteric and allosteric binding sites simultaneously, may provide a promising strategy. Here, we explore the allosteric, M 1 -selective BQCAd scaffold derived from BQCA as a starting point for the design, synthesis, and pharmacological evaluation of a series of novel bitopic ligands in which the orthosteric moieties and linker lengths are systematically varied. Since βarrestin recruitment seems to be favorable to therapeutic implication, all the compounds were investigated by G protein and β-arrestin assays. Some bitopic ligands are partial to full agonists for G protein activation, some activate β-arrestin recruitment, and the degree of β-arrestin recruitment varies according to the respective modification. The allosteric BQCAd scaffold controls the positioning of the orthosteric ammonium group of all ligands, suggesting that this interaction is essential for stimulating G protein activation. However, β-arrestin recruitment is not affected. The novel set of bitopic ligands may constitute a toolbox to study the requirements of β-arrestin recruitment during ligand design for therapeutic usage.

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The cholinergic hypothesis has been reported first being the cause of memory dysfunction in the Alzheimer’s disease. Researchers around the globe have focused their attention on understanding the mechanisms of how this complicated system contributes to processes such as learning, memory, disorientation, linguistic problems, and behavioral issues in the indicated chronic neurodegenerative disease. The present review reports recent updates in hybrid molecule design as a strategy for selectively addressing multiple target proteins involved in Alzheimer’s disease (AD) and the study of their therapeutic relevance. The rationale and the design of the bifunctional compounds will be discussed in order to understand their potential as tools to investigate the role of the cholinergic system in AD.

In the last few years, fluorescence resonance energy transfer (FRET) receptor sensors have contributed to the understanding of GPCR ligand binding and functional activation. FRET sensors based on muscarinic acetylcholine receptors (mAChRs) have been employed to study dual-steric ligands, allowing for the detection of different kinetics and distinguishing between partial, full, and super agonism. Herein, we report the synthesis of the two series of bitopic ligands, 12-Cn and 13-Cn, and their pharmacological investigation at the M1, M2, M4, and M5 FRET-based receptor sensors. The hybrids were prepared by merging the pharmacophoric moieties of the M1/M4-preferring orthosteric agonist Xanomeline 10 and the M1-selective positive allosteric modulator 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) 11. The two pharmacophores were connected through alkylene chains of different lengths (C3, C5, C7, and C9). Analyzing the FRET responses, the tertiary amine compounds 12-C5, 12-C7, and 12-C9 evidenced a selective activation of M1 mAChRs, while the methyl tetrahydropyridinium salts 13-C5, 13-C7, and 13-C9 showed a degree of selectivity for M1 and M4 mAChRs. Moreover, whereas hybrids 12-Cn showed an almost linear response at the M1 subtype, hybrids 13-Cn evidenced a bell-shaped activation response. This different activation pattern suggests that the positive charge anchoring the compound 13-Cn to the orthosteric site ensues a degree of receptor activation depending on the linker length, which induces a graded conformational interference with the binding pocket closure. These bitopic derivatives represent novel pharmacological tools for a better understanding of ligand-receptor interactions at a molecular level.