A drug to photocontrol endogenous muscarinic acetylcholine receptors in vivo

Muscarinic acetylcholine receptors (mAChRs) are class A GPCRs 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. “Dualsteric ligands” are a new strategy for the selective modulation of mAChRs: they can bind simultaneously to both the orthosteric and the allosteric sites of such receptors, incorporating a) iperoxo, an oxotremorine- related unselective orthosteric superagonist, b) a polymethylene spacer, and c) a moiety targeting the allosteric site.[1] Inspired by this strategy, in the course of our ongoing development of photoswitchable ligands for the optical control of (neuro)biological functions,[2] 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 two pharmacophoric moieties with light, thus potentially modulating affinity and efficacy of our compounds as a function of their photoisomerization state. One of the ligands turned out to be a potent activator of M2 receptors in the dark or under illumination with visible light (trans isomer), but inactive under UV illumination (cis isomer). We have evaluated in vitro photoresponses using a calcium imaging assay in genetically unmodified receptors overexpressed in mammalian cells. As stimulation of M2 mAChRs decreases heart rate (directly in atria) and force of contraction (indirectly in ventricles),3 we have studied the applications of this compound as a photopharmacological tool to remotely control cardiac function in vivo in wildtype frog tadpoles and in rats. References 1. Disingrini T et al. J Med Chem 2006; 49(1):366-372; Antony J et al. FASEB J 2009; 23(2):442-50; Matera C et al. Eur J Med Chem 2014; 75:222-232. 2. Izquierdo-Serra M et al. Nat Commun 2016; 7:12221; Pittolo S et al. Nat Chem Biol 2014; 10(10):813-815. 3. Dhein S, van Koppen CJ, Brodde OE. Pharmacol Res 2001; 44:161–82.