CaltechAUTHORS
Published January 4, 2022 | Version Supplemental Material + Accepted Version + Submitted + Published
Journal Article Open

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

Creators

  • 1. ROR icon California Institute of Technology
  • 2. ROR icon University of Southern California
  • 3. ROR icon Leiden University
  • 4. ROR icon Howard Hughes Medical Institute
  • 5. ROR icon University of Bristol

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.

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.

Attached Files

Published - elife-74648-v2.pdf

Accepted Version - elife-74648-v1.pdf

Submitted - 2021.10.04.463082v4.full.pdf

Supplemental Material - elife-74648-supp1-v2.xlsx

Supplemental Material - elife-74648-supp2-v2.xlsx

Supplemental Material - elife-74648-transrepform1-v2.docx

Files

2021.10.04.463082v4.full.pdf

Files (29.0 MB)

Name Size Download all
md5:902d44d8080732afa0d7a5d053f86292
3.2 MB Preview Download
md5:cedbfa4c20766fdff5fd5b10cffa0dc7
11.3 kB Download
md5:4163db7b249b6378792472fd437afd9c
11.4 kB Download
md5:9bdc3cca5dc58766c2edb4db7e853dc5
113.0 kB Download
md5:7bd48bcf963b4102bf9cc2b0f48d9070
17.2 MB Preview Download
md5:99559284b803eb2be1a0325a61c10320
8.5 MB Preview Download

Additional details

Identifiers

PMCID
PMC8820738
Eprint ID
111334
Resolver ID
CaltechAUTHORS:20211008-224629724

Related works

Describes
https://www.rcsb.org/structure/7S7T (URL)
https://www.rcsb.org/structure/7S7U (URL)
https://www.rcsb.org/structure/7S7V (URL)
10.1101/2021.10.04.463082 (DOI)

Funding

California Tobacco-Related Disease Research Program
27FT-0022
California Tobacco-Related Disease Research Program
27IP-0057
California Tobacco-Related Disease Research Program
T29IR0455
NIH
GM-123582
NIH
DA043829
NIH
DA049140
NIH
GM7616
Howard Hughes Medical Institute (HHMI)
Engineering and Physical Sciences Research Council (EPSRC)
EP/N024117/1
Leiden University
LISF L18020-1-45

Dates

Created
2021-10-11
Created from EPrint's datestamp field
Updated
2022-02-15
Created from EPrint's last_modified field

Caltech Custom Metadata

Caltech groups
Jacobs Institute for Molecular Engineering for Medicine, Division of Biology and Biological Engineering (BBE)