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  1. Evaluation Summary:

    Nichols et al. developed and characterized the first fluorescent sensors for several nicotinic receptor partial agonists relevant to smoking cessation. It is potentially a major advance for the field. They leveraged crystallography to understand the mechanism by which the ligands enhance fluorescence, then characterized top sensors for sensitivity, selectivity, and kinetics, and their utility in plasma membrane and ER sensing in neurons and cell lines. The tools developed by this team will enable investigators to track nicotinic receptor partial agonists in different subcellular compartments with relatively fast time resolution.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

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  2. Reviewer #1 (Public Review):

    Overall the findings are novel in that these are the first sensors for a panel of 4 nicotinic receptor partial agonists and their characterization is thorough; the experimental results support the conclusions. The main strengths are in that organellar partitioning is likely an essential aspect of the mechanism of nicotine addiction, and is therefore also essential to understand in its treatment. We lacked tools to follow the anti-addiction candidates in cells. This study provides these tools and shows enough cellular characterization to suggest they will be useful. A perceived weakness is that the current compounds for which sensors were developed are not promising anti-addiction drugs or leads; however, the study provides a template for what appears to be facile development of bespoke sensors for promising lead compounds.

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  3. Reviewer #2 (Public Review):

    This beautifully crafted paper describes what may be a true paradigm shift. Only time will tell. The paper bridges very different fields; the design and use of genetically encoded fluorescent biosensors, pharmacology, and medicinal chemistry. Here we can see for the first time signaling dynamics caused by different compounds at the nicotinic acetylcholine receptor. The compounds act on receptors on many membranes, including intracellular ones in fascinating ways. The current world of drug discovery typically involves cell homogenates and end point assays, crude measurements to be sure. Here we glimpse the subcellular and temporal patterns of activation of the different biosensors which begins to provide a richer, more mechanistic view of the compounds action. While this paper focuses on just one small set of ligands and one target, it is easy to imagine that in the future this sort of approach could become common place in the search for the better drugs our society needs.

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  4. Reviewer #3 (Public Review):

    The manuscript by Nichols and collaborators presents a description of the development and characterization of new biosensors for detection of a set of nicotinic ligands that may play a role in smoking cessation. The sensors can be targeted to the plasma membrane (PM) or endoplasmic reticulum (ER) providing a means to enable cell-based measurements of concentration and kinetics of acetylcholine and nicotinic ligands at these sites. This work is an extension of previous work describing development of sensors (iNicSnFRs) for acetylcholine, nicotine and varenicline, an approved smoking cessation treatment. These sensors combine a periplasmic binding protein (PBP) coupled to a circularly permuted GFP variant such that nicotinic ligand binding to the PBP leads to an increased fluorescence emission from the GFP variant. The authors used both a structural approach to guide sensor development and a site-saturation mutagenesis tactic to evaluate and optimize sensor design leading to a set of iDrugSnFR sensors for four nicotinic partial agonists dianicline, cytisine, - 10-fluorocytisine, and 9-bromo-10-ethylcytisine. These sensors complement previously developed sensors for nicotine and acetylcholine. The approach is rigorous, and a variety of complementary techniques were used to evaluate the novel sensors.

    These novel sensors can provide important information to guide development of improved smoking cessation agents. Previous work has suggested a framework for interpreting the acute effects of nicotine and nicotine dependence, in which acute effects and reward are mediated by plasma membrane nicotinic acetylcholine receptors (nAchR) and dependence may be, in part, based on nicotine effects on trafficking of intracellular AchRs acting via an "inside out" mechanism. Prior work suggests that partial agonists with lower efficacy than nicotine acting at the cell surface could possibly serve as effective smoking-cessation drugs. However, compounds with physiochemical properties that enable CNS exposure may also enter neurons and engage the "inside out" mechanism, limiting efficacy. The novel sensors described in this paper now provide a means to measure concentrations of these nicotinic ligands, and of structurally related compounds, in these compartments with sufficient sensitivity and temporal response to evaluate compound performance. This toolbox of sensors can presumably be expanded to accommodate additional structurally diverse nicotinic agents.

    The approach used in the paper is rigorous, the data are high quality and support the conclusions. The use of a variety of complementary techniques to evaluate the novel sensors is a clear strength. The structural information may be useful to guide future efforts in design of related sensors. The kinetic analysis of sensor response provides clear information to guide use of these sensors in biological experiments, but also raises additional mechanistic questions for future studies.

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