Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands
- California Institute of Technology, United States
- Howard Hughes Medical Institute, United States
- University of Bristol, United Kingdom
- Howard Hughes Medical Institute, California Institute of Technology, United States
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.
Data availability
Plasmids containing our sensors have been deposited in Addgene (as named in our manuscript) with genetic maps. They are currently viewable and are available on request.The Protein Data Bank has published the crystallographics and structural data (accession codes 7S7T, 7S7U, 7S7V). Supplemntary Table 1 gives relevant details.
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Crystal structure of iNicSnFR3a Fluorescent Nicotine SensorProtein Data Bank, 7S7V.
Article and author information
Author details
Funding
Tobacco-Related Disease Research Program (Postdoctoral Training Fellowship (27FT-0022))
- Aaron L Nichols
Leiden University International Studies Fund ((LISF L18020-1-45))
- Laura Luebbert
National Institute on Drug Abuse (Exploratory/Developmental Grants (R21) (DA049140))
- Anand K Muthusamy
National Institute of General Medical Sciences (Predoctoral Training in Biology and Chemistry (T32) (GM7616))
- Anand K Muthusamy
Tobacco-Related Disease Research Program (High Impact Pilot Award (27IP-0057))
- Henry A. Lester
Tobacco-Related Disease Research Program (High Impact Research Project Award (T29IR0455))
- Dennis A Dougherty
National Institute of General Medical Sciences (Research Project (GM-123582)
- Henry A. Lester
National Institute on Drug Abuse (Exploratory/Developmental Grants (R21,DA043829))
- Henry A. Lester
Howard Hughes Medical Institute
- Jonathan S Marvin
Howard Hughes Medical Institute
- Loren L. Looger
Howard Hughes Medical Institute
- Douglas C Rees
UK Engineering and Physical Sciences Research Council (No. EP/N024117/1)
- Timothy Gallagher
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: General of primary mouse hippocampal culture was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to the approved institutional animal care and use committee (IACUC) protocols (IA19-1386) of California Institute of Technology. Dissections were performed after euthanasia of the pregnant mouse and every effort was made to minimize suffering.
Copyright
© 2022, Nichols et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands
eLife 11:e74648.
https://doi.org/10.7554/eLife.74648
Further reading
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Chronic back pain (CBP) is a global health concern with significant societal and economic burden. While various predictors of back pain chronicity have been proposed, including demographic and psychosocial factors, neuroimaging studies have pointed to brain characteristics as predictors of CBP. However, large-scale, multisite validation of these predictors is currently lacking. In two independent longitudinal studies, we examined white matter diffusion imaging data and pain characteristics in patients with subacute back pain (SBP) over 6- and 12-month periods. Diffusion data from individuals with CBP and healthy controls (HC) were analyzed for comparison. Whole-brain tract-based spatial statistics analyses revealed that a cluster in the right superior longitudinal fasciculus (SLF) tract had larger fractional anisotropy (FA) values in patients who recovered (SBPr) compared to those with persistent pain (SBPp), and predicted changes in pain severity. The SLF FA values accurately classified patients at baseline and follow-up in a third publicly available dataset (Area under the Receiver Operating Curve ~0.70). Notably, patients who recovered had FA values larger than those of HC suggesting a potential role of SLF integrity in resilience to CBP. Structural connectivity-based models also classified SBPp and SBPr patients from the three data sets (validation accuracy 67%). Our results validate the right SLF as a robust predictor of CBP development, with potential for clinical translation. Cognitive and behavioral processes dependent on the right SLF, such as proprioception and visuospatial attention, should be analyzed in subacute stages as they could prove important for back pain chronicity.
