Optical control of muscarinic acetylcholine receptors using photoswitchable bitopic ligands

Muscarinic acetylcholine receptors (mAChRs) are class A GPCRs characterized by a widespread tissue distribution and involved in the control of numerous central and peripheral physiological responses. The high sequence homology of the different subtypes (M1–M5) in the transmembrane region hampers the development of subtype selective orthosteric agonists. On the other hand, the allosteric site, located in the extracellular loop, is less conserved, thus muscarinic allosteric agents are commonly endowed with a more pronounced subtype‐selectivity. Recently, a new strategy was developed towards the selective modulation of mAChRs, i.e. the development of dualsteric ligands, which are molecules that can bind simultaneously to both the orthosteric and the allosteric sites of such receptors. The most interesting bitopic ligands emerging from this investigation were hybrid derivatives incorporating (i) iperoxo, an oxotremorine‐related unselective orthosteric superagonist, (ii) a polymethylene spacer, and (iii) a moiety targeting the allosteric site. Inspired by this strategy, in the course of our ongoing development of photoswitchable ligands for the optical control of (neuro) biological functions, we designed and synthesized a new set of light‐regulated muscarinic bitopic ligands by replacing the polymethylene spacer chain with an azobenzene linker to serve as molecular photoswitch. This modification enabled the remote control of the mutual position between the 2 pharmacophoric moieties with light, thus potentially modulating affinity and efficacy of our compounds as a function of their photoisomerization state. One of our ligands (P‐Azo‐Iper) turned out to be a potent activator of M2 receptors under UV illumination (cis isomer), but inactive after relaxation in the dark or under illumination with blue light or white light (trans isomer). All compounds were investigated in binding and enzymatic experiments. Their cellular responses were evaluated in vitro and in vivo in the Xenopus tropicalis heart.