Nichols, Aaron L. and Blumenfeld, Zack and Fan, Chengcheng and Luebbert, Laura and Blom, Annet E. M. and Cohen, Bruce N. and Marvin, Jonathan S. and Borden, Philip M. and Kim, Charlene H. and Muthusamy, Anand K. and Shivange, Amol V. and Knox, Hailey J. and Rego Campello, Hugo and Wang, Jonathan H. and Dougherty, Dennis A. and Looger, Loren L. and Gallagher, Timothy and Rees, Douglas C. and Lester, Henry A. (2022) Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands. eLife, 11 . Art. No. e74648. ISSN 2050-084X. PMCID PMC8820738. doi:10.7554/eLife.74648. https://resolver.caltech.edu/CaltechAUTHORS:20211008-224629724
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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.
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| Additional Information: | © 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. Preprinted: 04 October 2021; Received: 12 October 2021; Accepted: 03 January 2022; Published: 04 January 2022. We thank Stefan Petrovic for his stewardship of the isothermal titration calorimeter in the Caltech Center for Molecular Medicine, Jens Kaiser for help with structural studies at the Caltech Molecular Observatory, the Gradinaru lab and Caltech CLOVER Center for help with viral vectors, and Andres Collazo and Giada Spigolon at the Caltech Biological Imaging Facility. We thank Zoe Beatty, Kallol Bera, Eve Fine, Shan Huang, Elaine Lin, Stephen Mayo, Lin Tian, and Elizabeth Unger for advice and guidance. We thank Achieve Life Sciences for a gift of cytisine. California Tobacco-Related Disease Research Program (TRDRP) (27 FT-0022), ALN. California Tobacco-Related Disease Research Program (TRDRP) (27IP-0057), HAL. California Tobacco-Related Disease Research Program (TRDRP) (T29IR0455), DAD. NIH (GM-123582, DA043829), HAL. NIH (DA049140, GM7616), AKM. Howard Hughes Medical Institute (LLL, JSM, DCR). UK Engineering and Physical Sciences Research Council (no. EP/N024117/1), TG. Leiden University International Studies Fund (LISF L18020-1-45), LL. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Author contributions: Aaron L Nichols, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing; Zack Blumenfeld, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – review and editing; Chengcheng Fan, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing; Laura Luebbert, Bruce N Cohen, Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing; Annet EM Blom, Philip M Borden, Conceptualization, Investigation; Jonathan S Marvin, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – original draft, Writing – review and editing; Charlene H Kim, Data curation, Investigation, Methodology, Resources, Writing – review and editing; Anand K Muthusamy, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization; Amol V Shivange, Data curation, Validation; Hailey J Knox, Conceptualization, Methodology, Resources, Writing – review and editing; Hugo Rego Campello, Data curation, Resources, Validation; Jonathan H Wang, Investigation, Methodology, Resources; Dennis A Dougherty, Conceptualization, Data curation, Funding acquisition, Project administration, Resources, Supervision; Loren L Looger, Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review and editing; Timothy Gallagher, Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review and editing; Douglas C Rees, Data curation, Funding acquisition, Project administration, Supervision, Validation, Writing – review and editing; Henry A Lester, Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Software, Supervision, Visualization, Writing – review and editing. Data availability: The plasmids and associated database entries are available from Addgene (as named in our manuscript) with genetic maps. The Protein Data Bank has published the crystallographics and structural data (accession codes 7S7T, 7S7U, 7S7V). Supplementary File 1 gives relevant details. | ||||||||||||||||||||||||||||||||||
| Group: | Jacobs Institute for Molecular Engineering for Medicine | ||||||||||||||||||||||||||||||||||
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| PubMed Central ID: | PMC8820738 | ||||||||||||||||||||||||||||||||||
| DOI: | 10.7554/eLife.74648 | ||||||||||||||||||||||||||||||||||
| Record Number: | CaltechAUTHORS:20211008-224629724 | ||||||||||||||||||||||||||||||||||
| Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20211008-224629724 | ||||||||||||||||||||||||||||||||||
| Official Citation: | Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands. eLife 2022; 11:e74648 DOI: 10.7554/eLife.74648 DOI: 10.7554/eLife.74648 | ||||||||||||||||||||||||||||||||||
| Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | ||||||||||||||||||||||||||||||||||
| ID Code: | 111334 | ||||||||||||||||||||||||||||||||||
| Collection: | CaltechAUTHORS | ||||||||||||||||||||||||||||||||||
| Deposited By: | George Porter | ||||||||||||||||||||||||||||||||||
| Deposited On: | 11 Oct 2021 21:42 | ||||||||||||||||||||||||||||||||||
| Last Modified: | 15 Feb 2022 19:05 |
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