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

  1. Aaron L Nichols
  2. Zack Blumenfeld
  3. Chengcheng Fan
  4. Laura Luebbert
  5. Annet EM Blom
  6. Bruce N Cohen
  7. Jonathan S Marvin
  8. Philip M. Borden
  9. Charlene H Kim
  10. Anand K Muthusamy
  11. Amol V Shivange
  12. Hailey J Knox
  13. Hugo Rego Campello
  14. Jonathan H Wang
  15. Dennis A Dougherty
  16. Loren L. Looger
  17. Timothy Gallagher
  18. Douglas C Rees
  19. Henry A. Lester  Is a corresponding author
  1. California Institute of Technology, United States
  2. Howard Hughes Medical Institute, United States
  3. University of Bristol, United Kingdom
  4. 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.

The following data sets were generated

Article and author information

Author details

  1. Aaron L Nichols

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9341-0049
  2. Zack Blumenfeld

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4627-5582
  3. Chengcheng Fan

    Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4213-5758
  4. Laura Luebbert

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1379-2927
  5. Annet EM Blom

    Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Bruce N Cohen

    Division of Biology and Biological Engineering, California Institute of Technology, Claremont, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Jonathan S Marvin

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Philip M. Borden

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Charlene H Kim

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Anand K Muthusamy

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Amol V Shivange

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4169-2969
  12. Hailey J Knox

    Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Hugo Rego Campello

    School of Chemistry, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  14. Jonathan H Wang

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
  15. Dennis A Dougherty

    Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
  16. Loren L. Looger

    Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  17. Timothy Gallagher

    School of Chemistry, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  18. Douglas C Rees

    Division of Chemistry and Chemical Engineering, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4073-1185
  19. Henry A. Lester

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
    For correspondence
    lester@caltech.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5470-5255

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.

Reviewing Editor

  1. Gary Yellen, Harvard Medical School, United States

Publication history

  1. Preprint posted: October 4, 2021 (view preprint)
  2. Received: October 12, 2021
  3. Accepted: January 3, 2022
  4. Accepted Manuscript published: January 4, 2022 (version 1)
  5. Version of Record published: February 7, 2022 (version 2)
  6. Version of Record updated: March 4, 2022 (version 3)

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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  1. Aaron L Nichols
  2. Zack Blumenfeld
  3. Chengcheng Fan
  4. Laura Luebbert
  5. Annet EM Blom
  6. Bruce N Cohen
  7. Jonathan S Marvin
  8. Philip M. Borden
  9. Charlene H Kim
  10. Anand K Muthusamy
  11. Amol V Shivange
  12. Hailey J Knox
  13. Hugo Rego Campello
  14. Jonathan H Wang
  15. Dennis A Dougherty
  16. Loren L. Looger
  17. Timothy Gallagher
  18. Douglas C Rees
  19. Henry A. Lester
(2022)
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

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