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Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

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. doi:10.7554/eLife.74648. (In Press) 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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.7554/eLife.74648DOIArticle
https://doi.org/10.1101/2021.10.04.463082DOIDiscussion Paper
ORCID:
AuthorORCID
Nichols, Aaron L.0000-0001-9341-0049
Blumenfeld, Zack0000-0002-4627-5582
Fan, Chengcheng0000-0003-4213-5758
Luebbert, Laura0000-0003-1379-2927
Blom, Annet E. M.0000-0002-7441-4893
Marvin, Jonathan S.0000-0003-2294-4515
Borden, Philip M.0000-0003-1653-7067
Muthusamy, Anand K.0000-0003-1041-914X
Shivange, Amol V.0000-0002-4169-2969
Knox, Hailey J.0000-0003-0608-2855
Rego Campello, Hugo0000-0001-8588-0198
Dougherty, Dennis A.0000-0003-1464-2461
Looger, Loren L.0000-0002-7531-1757
Gallagher, Timothy0000-0002-3544-327X
Rees, Douglas C.0000-0003-4073-1185
Lester, Henry A.0000-0002-5470-5255
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. Received: October 12, 2021; Accepted: January 3, 2022; Accepted Manuscript published: January 4, 2022 (version 1). 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. Funding: California Tobacco-Related Disease Research Program (TRDRP) (27FT-0022), Aaron L. Nichols. California Tobacco-Related Disease Research Program (TRDRP) (27IP-0057), Henry A. Lester. California Tobacco-Related Disease Research Program (TRDRP) (T29IR0455), Dennis A. Dougherty. NIH (GM-123582, DA043829), Henry A. Lester. NIH (DA049140, GM7616), Anand K. Muthusamy. Howard Hughes Medical Institute (Loren L. Looger, Jonathan S. Marvin, Douglas C. Rees). UK Engineering and Physical Sciences Research Council (No. EP/N024117/1), Timothy Gallagher. Nichols, Blumenfeld, Fan, Luebbert, et al, Leiden University International Studies Fund (LISF L18020-1-45), Laura Luebbert. The authors declare no financial or non-financial competing interests. 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.
Group:Jacobs Institute for Molecular Engineering for Medicine
Funders:
Funding AgencyGrant Number
California Tobacco-Related Disease Research Program27FT-0022
California Tobacco-Related Disease Research Program27IP-0057
California Tobacco-Related Disease Research ProgramT29IR0455
NIHGM-123582
NIHDA043829
NIHDA049140
NIHGM7616
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Engineering and Physical Sciences Research Council (EPSRC)EP/N024117/1
Leiden UniversityLISF L18020-1-45
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:11 Jan 2022 23:22

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