Fluorescence Activation Mechanism and Imaging of Drug Permeation with New Sensors for Smoking-Cessation Ligands

Abstract

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.

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

“…Although only three mutations were required to generate iS-methadoneSnFR from iNicSnFR3a/3b, advantageous mutations were rare: ∼1% of all mutations screened were accepted as improvements. Docking S-methadone into recently reported structures of liganded iNicSnFR3a 22 showed that the N-methyl groups of S-methadone lie 4.6 and 5.5 Å from the aromatic groups of Y357 and Y65, respectively (slightly greater than the distance from the beta carbons of varenicline to these two groups). In the initial round of mutations, most sites yielded no improvement, except for a W436F mutation spatially near Y65 and Y357 ( Figure S3 ).…”

“…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) 22 . Each nicotinic ligand bears a protonated nitrogen lying midway on the axis of the aromatic centroids of Y65 and Y357 ( Figure 3a ).…”

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

“…To generate iDrugSnFRs for SSRIs, we screened several SSRIs against a panel of biosensors that included our previously published Opu-BC based biosensors (Bera et al, 2019; Borden et al, 2019; Shivange et al, 2019; Unger et al, 2020; Nichols et al, 2022) as well as intermediate constructs from their development process. This screen is described in a protocol under review at Bio-Protocol.…”

“…We examined the subcellular pharmacokinetics of the SSRIs in primary mouse hippocampal neurons transduced with AAV vectors encoding the appropriately targeted iDrugSnFRs. The SSRI iDrugSnFRs were targeted to the PM (iDrugSnFR_PM), the endoplasmic reticulum (iDrugSnFR_ER), or the cytoplasm (iDrugSnFR_cyto) as previously described (Bera et al, 2019; Shivange et al, 2019; Nichols et al, 2022). Spinning-disk confocal microscopy showed targeting to the intended organelle or compartment (Fig.…”

“…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.…”