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

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.

Selective Serotonin Reuptake Inhibitors Within Cells: Temporal Resolution in Cytoplasm, Endoplasmic Reticulum, and Membrane

“…Localization of the biosensor resembled previously described iDrugSnFR _PM and _ER constructs (Bera et al, 2019;Shivange et al, 2019;Muthusamy et al, 2022;Nichols et al, 2022) and the localization pattern of iEscSnFR and iFluoxSnFR in primary hippocampal culture imaging (Fig. 3).…”

“…We used several methods. Analogous to recent work on the subcellular pharmacokinetics of other neuropsychiatric drugs (Bera et al, 2019; Shivange et al, 2019; Muthusamy et al, 2022; Nichols et al, 2022), we developed and applied new intensity-based drug sensing fluorescent reporters (“iDrugSnFRs”) for SSRIs, targeted to the plasma membrane (PM), cytoplasm, and endoplasmic reticulum (ER). We detected fluorescence changes resulting from ∼ 1 s solution changes, at cultured neurons and HeLa cells.…”

Three Mutations Convert the Selectivity of a Protein Sensor from Nicotinic Agonists to S-methadone for Use in Cells, Organelles, and Biofluids

“…In the initial round of mutations, most sites yielded no improvement, except for a W436F mutation spatially near Y65 and Y357 (Figure S3). We previously reported nicotine and varenicline making cation-π interactions with Y65 and Y357 in iNicSnFR3a (PDB: 7S7T, 7S7U, respectively) 27 . Each nicotinic ligand bears a protonated nitrogen lying midway on the axis of the aromatic centroids of Y65 and Y357 ( Figure 3a ).…”

Three Mutations Convert the Selectivity of a Protein Sensor from Nicotinic Agonists to S-Methadone for Use in Cells, Organelles, and Biofluids

“…We previously reported nicotine and varenicline making cation−π interactions with Y65 and Y357 in iNicSnFR3a (PDB:7S7T and 7S7U, respectively). 27 Each nicotinic ligand bears a protonated nitrogen lying midway on the axis of the aromatic centroids of Y65 and Y357 (Figure 3a). In the subsequent round, second-shell mutation N11V created additional volume next to F12, in the second shell.…”