Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands.

Laura Luebbert,Philip M Borden,Charlene H Kim,Anand K Muthusamy,Chengcheng Fan,Dennis A Dougherty,Annet Em Blom,Bruce N Cohen,Henry A Lester,Hailey J Knox,Douglas C Rees,Loren L Looger,Timothy Gallagher,Hugo Rego Campello,Jonathan S Marvin,Jonathan H Wang,Amol V Shivange,Zack Blumenfeld,Aaron L Nichols

doi:10.7554/elife.74648

Abstract

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.

Highlights

Smoking cessation is an important goal to help decrease the burden, both individual and societal, of tobacco-related disease
The difference in plasma membrane (PM) and endoplasmic reticulum (ER) detection of 10-fluorocytisine again shows decreased membrane permeability into HeLa cells compared to other drugs we have examined with other iDrugSnFRs
Varenicline, dianicline, and the cytisine analogs studied here have calculated membrane partition coefficients some 3 to 6 orders of magnitude lower. These values and their order vary according to the algorithm, partially because of uncertainties in predicting pKa (Pienko, et al, 2016); here we provide values calculated by Chemicalize: 10

Summary

INTRODUCTION

Smoking cessation is an important goal to help decrease the burden, both individual and societal, of tobacco-related disease. Prior work suggests that partial agonists with lower efficacy than nicotine could serve as effective smoking-cessation drugs (Rose, et al, 1994), and efforts continue in that direction (Rollema & Hurst, 2018) Estimates of LogDpH7.4 are inexact, 81 extrapolated, or rely on algorithmic calculations whose results differ over two log units for individual molecules (Pienko, Grudzien, Taciak, & Mazurek, 2016) These estimates have unknown applicability to biological membranes at the LogDpH7.4 values < 0 that characterize 84 varenicline, dianicline, and the cytisine analogs. Nicotine itself (logDpH7.4 0.99) enters the endoplasmic reticulum (ER), binds to nascent nAChRs, 87 becomes a pharmacological chaperone for the nAChRs, and eventually causes selective upregulation of these receptors on the plasma membrane (PM) (Henderson & Lester, 2015) For this reason, it is especially important to understand permeation into the ER. We hypothesized that a family of newly developed iDrugSnFRs would enable quantifiable fluorescence signals that compare the differences in permeation among these compounds

RESULTS

Discussion

Methods

References for Main Article and Appendices