Target-directed evolution of novel modulators of the dopamine transporter in Lobelia cardinalis hairy root cultures

Dennis T. Rogers, Francois Pomerleau, Zachary Kelley, Dustin Brown, Bert Lynn, Greg A. Gerhardt, John Littleton

Research output: Contribution to journalArticlepeer-review

Abstract

The dopamine transporter (DAT) is targeted in substance use disorders (SUDs), and “non-classical”” DAT inhibitors with low abuse potential are therapeutic candidates. Lobinaline, from Lobelia cardinalis, is an atypical DAT inhibitor lead. Chemical synthesis of lobinaline is challenging; thus, “target-directed evolution” was used for lead optimization. A target protein is expressed in plant cells, and a mutant cell population is selected under conditions where target protein functional inhibition confers a survival advantage. Surviving mutants are “mined” for the targeted activity. Applied to a mutant L. cardinalis cell population expressing the human DAT, we identified 20 mutants overproducing DAT inhibitors. Microanalysis prioritized novel lobinaline derivatives, and we first investigated the more water-soluble lobinaline N-oxide. It inhibited rat synaptosomal [3H]DA uptake with an IC50 similar to lobinaline. Against repeated DA microinjections into the rat striatum, lobinaline produced transient DA clearance reductions. In contrast, lobinaline N-oxide prolongingly increased DA peak amplitudes, particularly in the ventral striatum. Lobinaline N-oxide also produced complex changes in post-peak DA clearance inconsistent with simple DAT inhibition. This unusual DAT interaction may prove therapeutically useful for treating SUDs. This study demonstrates the value of target-directed evolution of plant cells for optimizing lead compounds difficult to synthesize chemically.

Original languageEnglish
Pages (from-to)28-35
Number of pages8
JournalJournal of Biotechnology
Volume342
DOIs
StatePublished - Dec 10 2021

Bibliographical note

Publisher Copyright:
© 2021 Elsevier B.V.

Keywords

  • Activation tagging mutagenesis
  • Chrono-amperometry
  • Directed evolution
  • Dopamine transport inhibitors
  • Lobinaline
  • Substance use disorders

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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