Novel Xanomeline-Containing Bitopic Ligands of Muscarinic Acetylcholine Receptors: Design, Synthesis and FRET Investigation

Abstract

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.

Designing drugs and chemical probes with the dualsteric approach

“…In more cases, dualsteric modulators are designed by combining the orthosteric and allosteric ligand with a linker. 50,56,57,59,62,63,65,66,69,70,73,75,77–86,88,89,91,92,96,97,99,100,102–104,106–109,111,114–118,120–123,126,127,132,133,136,138,140,143,145,148–150,153,154,156,160,161,163,165,166,169,170,178–182,185,186,188,195,228 This is similar with the fragment-based drug discovery (FBDD). 229 The core fragments of drug binding is first identified and then the fragments are grown, merged, or linked into one complete molecule.…”

“…After a manual filtering, 133 research articles focusing on identifications of novel dualsteric modulators were found. Modulators targeting GPCR 50,53,56–137 or kinase 138–157 account for more than three quarters (Fig. 5).…”

“…GPCR itself has accommodated 62.4% targets of reports. 50,53,56–137 GPCR is a class of 7-transmembrane (7TM) proteins decorated on the cell membrane for transduction amplification of exterior signals into cells. 189 They are vital in many physiological processes and are therefore popular drug targets for a series of diseases like cancers and neurological disorders.…”

“…Alkyl 50,56,59,61–66,72,73,75,77–79,81–85,89,97,99,100,103,106–109,111,114,116–118,120–123,126,127,132,133,140,145,160,165,170,181,182 and PEG 56,69,138,145,148,149,160,161,163,182,195 are two main components reported, probably due to their simple chemical structures. However, the simplicity has also led to several drawbacks.…”