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. 2022 Nov 20;12(22):e4551.
doi: 10.21769/BioProtoc.4551.

Fluorescence Screens for Identifying Central Nervous System-Acting Drug-Biosensor Pairs for Subcellular and Supracellular Pharmacokinetics

Zoe G Beatty  1 , Anand K Muthusamy  2 , Elizabeth K Unger  3 , Dennis A Dougherty  2 , Lin Tian  3 , Loren L Looger  4 , Amol V Shivange  1 , Kallol Bera  1 , Henry A Lester  1 , Aaron L Nichols  1
Affiliations

Fluorescence Screens for Identifying Central Nervous System-Acting Drug-Biosensor Pairs for Subcellular and Supracellular Pharmacokinetics

Zoe G Beatty et al. Bio Protoc. .

Abstract

Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)-acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the plasma membrane and in organelles. We describe screening and validation protocols for identifying hit pairs comprising a drug and biosensor, with each screen including 13-18 candidate biosensors and 44-84 candidate drugs. After a favorable hit pair is identified and validated via these protocols, the biosensor is then optimized, as described in other papers, for sensitivity and selectivity to the drug. We also show sample hit pair data that may lead to future intensity-based drug-sensing fluorescent reporters (iDrugSnFRs). These protocols will assist scientists to use fluorescence responses as criteria in identifying favorable fluorescent biosensor variants for CNS-acting drugs that presently have no corresponding biosensor partner. This protocol was validated in: eLife (2022), DOI: 10.7554/eLife.74648 Graphical abstract.

Keywords: Biosensor; CNS-acting drugs; Directed evolution; Fluorescence screening; Pharmacokinetics; iDrugSnFR.

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Conflict of interest statement

Competing interests Anand Muthusamy, Henry Lester, Loren Looger, and Jonathan Marvin have filed a patent application that includes opioid biosensors. Lin Tian is a co-founder of Seven Biosciences.

Figures

Figure 1.
Figure 1.. Fast protein liquid chromatography (FPLC) setup.
Figure 2.
Figure 2.. Drug plate design example of a drug–biosensor fluorescence screen.
Figure 3.
Figure 3.. Eppendorf epMotion setup for single concentration drug–biosensor fluorescence screen.
Figure 4.
Figure 4.. Results map from the 2017 single concentration drug–biosensor fluorescence screen.
Figure 5.
Figure 5.. Results map from the 2018 single concentration drug–biosensor fluorescence screen.
Figure 6.
Figure 6.. Results map from the 2019 single concentration drug–biosensor fluorescence screen results map.
Figure 7.
Figure 7.. Multiple concentration drug–biosensor fluorescence validations for 5HT3 antagonists.
Figure 8.
Figure 8.. Multiple concentration drug–biosensor fluorescence validations for CB 1 /CB 2 ligands.
See this image and copyright information in PMC

References

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