PT - JOURNAL ARTICLE AU - Aaron L. Nichols AU - Zack Blumenfeld AU - Chengcheng Fan AU - Laura Luebbert AU - Annet E. M. Blom AU - Bruce N. Cohen AU - Jonathan S. Marvin AU - Philip M. Borden AU - Charlene H. Kim AU - Anand K. Muthusamy AU - Amol V. Shivange AU - Hailey J. Knox AU - Hugo Rego Campello AU - Jonathan H. Wang AU - Dennis A. Dougherty AU - Loren L. Looger AU - Timothy Gallagher AU - Douglas C. Rees AU - Henry A. Lester TI - Fluorescence Activation Mechanism and Imaging of Drug Permeation with New Sensors for Smoking-Cessation Ligands AID - 10.1101/2021.10.04.463082 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.10.04.463082 4099 - http://biorxiv.org/content/early/2021/10/11/2021.10.04.463082.short 4100 - http://biorxiv.org/content/early/2021/10/11/2021.10.04.463082.full AB - 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, 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.Competing Interest StatementThe authors have declared no competing interest.