Highlights d Simulations identify conformational changes in the a4b2 nAChR caused by nicotine d Nicotine induces structural changes in the extracellular and transmembrane domains d Nonequilibrium simulations reveal a binding pocket to ion channel communication d The results suggest a general signal transduction mechanism for the Cys-loop family Authors

Efficient access to C(10) of (À)-cytisine via C-H activation provides access to enantiomerically pure nicotinic acetylcholine receptor ligands that target the highaffinity nicotine a4b2 subtype with enhanced selectivity. These C(10) cytisine variants retain a partial agonist profile at the a4b2 subtype but, critically, display negligible activity at the a7 receptor subtype. Using computational methods, Gallagher and colleagues link receptor selectivity to key protein residues associated with, as well as beyond, the immediate ligand binding site.

Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug sensing fluorescent reporters ('iDrugSnFRs') for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by > 30 fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.

Nicotinic acetylcholine receptors (nAChR) are crucial for communication between synapses in the central nervous system. As such they are also implicated in several neuropsychiatric and addictive diseases. Cytisine is a partial agonist of some nAChRs and has been used for smoking cessation. Previous studies have established a binding model for several agonists to several nAChR subtypes. Here, we evaluate the extent to which this model applies to cytisine at the α4β2 nAChR, a subtype known to play a prominent role in nicotine addiction. Along with the commonly seen cation-π interaction and two hydrogen bonds, we find that cytisine makes a second cation-π interaction at the agonist binding site. We also evaluated a series of C(10)-substituted cytisine derivatives, using two-electrode voltage-clamp electrophysiology and non-canonical amino acid mutagenesis. Double mutant-cycle analyses revealed C(10) substitution generally strengthens the newly established second cation-π interaction while it weakens the hydrogen bond typically seen to LeuE in the complementary subunit. The results suggest a model for how cytisine derivatives substituted at C(10) (as well as C(9)/C(10)) adjust their binding orientation in response to pyridone ring-substitution.

Nicotinic partial agonists provide a partial aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug sensing fluorescent reporters (iDrugSnFRs) for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives – 10-fluorocytisine and 9-bromo-10-ethylcytisine. Development of the series was aided and explained by the first atomic-scale structural studies on liganded periplasmic binding protein-based biosensors. Members of the series detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and primary mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by > 30 fold. The new nicotinic iDrugSnFRs, in combination with previously described nicotine and varenicline sensors, provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.