Structure-Activity Relationship Of Potent Photo-Switchable Neuromuscular Inhibitors

Muscular nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels located in the plasma membrane of the post-junctional motor endplate in skeletal muscles. They are responsible for triggering the electrical signal that leads to muscle contraction. Here, we have developed a family of photoisomerizable nAChR inhibitors, using the azobenzene scaffold. Biochemical assays showed that these novel compounds have higher affinity for muscular nAChR (up to 60-fold) than for either α7 or α4β2 nAChRs. Thus, we proceeded to characterize the action of these compounds on the activity of the embryonic muscular nAChR expressed in Xenopus oocytes. To do so, we recorded currents evoked by acetylcholine using the Two-Electrode Voltage Clamp technique. We observed that all but one of the compounds were effective inhibitors of the receptor's activity. As reported elsewhere, azobenzene-based molecules can interchange between cis- or trans-isomers, displaying remarkable difference in their pharmacology. To test whether the isomers had different activity, we irradiated the drugs with either near UV (335-365 nm) or blue (400-450 nm) light while recording currents. In all cases, we observed that the trans-isomers were the most potent form of these molecules. The main difference among these compounds is the “molecular volume” of the functional group. The molecules, except for one, were effective nAChR inhibitors at the nanomolar to micromolar range, decreasing their potency as their size decrease. The trans-isomer of the smallest derivative had the opposite behavior, acting as an agonist drug, with its cis-isomer being unable to activate the receptors. In terms of structure-activity relationship parameters, these observations led us to infer that the “molecular volume” of the compound is a critical determinant of both the character and potency of these novel azobenzene-based inhibitors. Funding: MINECO (SAF2015-64948-C2-1-R; FPU16/01704; FPUEST17/00233), CSIC (PIE201580E109), Nutting Foundation Grant (CAVG).