Microscopy Using Fluorescent Drug Biosensors for "Inside-Out Pharmacology"

Neuropharmacology offers many tools for connecting molecular to acute behavioral phenomena but has few tools to explain effects of chronic drugs. In the biophysically based “inside-out” approach to neuropharmacology, drugs bind to their nascent targets within the endoplasmic reticulum (ER) (i.e. pharmacological chaperoning). Now we visualize, quantify, and time the first steps: drugs entering the cell and entering organelles. Our fluorescent biosensors are built on an OpuBC-GFP fusion scaffold. OpuBC is a bacterial periplasmic binding protein with two key features: a cation-π box, favorable for binding amines well represented in drugs, and a binding-induced “Venus fly-trap” conformational change. The suitably mutated OpuBC domain is connected, at the hinge regions, to a circularly permuted “superfolder” GFP (cpGFP). Aided by structural information, we use directed evolution to create a family of drug-sensing biosensors meeting the criterion of ΔF/F0 > 1 at 1 μM drug. Our proof of principle study concerns nicotine entering the ER, as measured by an “intensity-based nicotine-sensing fluorescent reporter” (iNicSnFR). Previous data demonstrated that exposure to nicotine causes changes in number and stoichiometry of nicotinic receptors by chaperoning within the ER; however nicotine itself entering the ER had not yet been measured. In live cell imaging of an iNicSnFR targeted to the ER, we found that nicotine enters the ER within 10 s of application at concentrations experienced by a cigarette smoker. Moreover, we found that varenicline, a smoking cessation drug, enters the ER almost as rapidly as nicotine, helping to explain varenicline's biochemical and behavioral effects. We are currently developing other “iDrugSnFRs” for antidepressants, antipsychotics, and opioids. These tools to study subcellular pharmacokinetics will help to clarify chronic effects of several families of neural drugs. Support: DA037161, GM123582, NARSAD, California TRDRP, and HHMI