BUILDING TOOLS FOR IMPROVED MODULATION OF THE HUMAN GABAA RECEPTOR, A CENTRAL NERVOUS SYSTEM TARGET FOR THE TREATMENT OF ANXIETY
Author ORCID Identifier
Year of Publication
Doctor of Philosophy (PhD)
Dr. Joseph Chappell
Dr. Robert Lodder
In the U.S., anxiety is recognized as an increasing range of mentally and physically debilitating psychiatric health disorders with significant economic repercussions. Over the last 20 years, several novel anti-anxiety therapies have entered the drug development pipeline, but none have made it to market.
The work in this dissertation focused on structurally modifying valerenic acid (VA), a structurally unique carboxylated sesquiterpene acid found in Valeriana officinalis. VA is putatively reported to have allosteric modulatory activity of the human GABAA receptor, a ligand-gated ion channel responsible for attenuating neurotransmissions. Structural modeling of VA’s GABAA receptor interaction suggests that constraining the isobutenyl group relative to the 5,6 membered ring system of VA could improve its binding specificity and affinity to the GABAA receptor. In planta, valerena-1,10-diene (VLD) is synthesized from farnesyl pyrophosphate (FPP) valerenadiene synthase (VDS), a sesquiterpene synthase. VLD is then carboxylated at one of its isobutenyl terminal methyl groups to yield VA. Our first objective was to engineer the VDS enzyme for altered product specificity and a more chemically constrained VLD scaffold.
Using computational homology modelling and phylogenetic sequence analysis of characterized sesquiterpene synthases, amino acid residues in or near the active site and potentially impinging on catalytic specificity of VDS were identified. Residues were mutated via site-directed mutagenesis and mutants evaluated in vivo and in vitro. While wild type VDS’ products were 66 % VLD, 5 % allo-aromadendrene, and 29 % bicyclogermacrene (BCG), mutant Y535F yielded solely BCG. VDS with alanine or serine substituted for asparagine at position 455 lost all its ability to produce any of the wild type products and instead yielded a suite of seven new products dominated by germacrene-D-ol (≥ 40 %).
To install a carboxylic acid functional group onto the sesquiterpene hydrocarbon scaffolds, we focused on the development of a host platform harboring an endomembrane system suitable for the expression of eukaryotic cytochrome P450 enzymes (P450s). As an example, plasmid co-expression of VDS, Lactuca sativa germacrene-A oxidase, and Artemisia annua cytochrome P450 oxidoreductase yielded an average 2mg/L of VA.
For biological evaluation of sesquiterpene analogs, HEK293 cells transiently transfected with the human GABAA receptor subunit genes ⍺1, β3, and 𝛾2L, as well as a HEK293 cell line stably expressing the same GABA subunit genes, were optimized for sensing changes in membrane potential using a fluorescent bioassay. Effective concentration of test compounds and absolute magnitude of membrane depolarization in the transiently transfected cells gave the greatest responsiveness as determined for 𝛾𝛾-aminobutyric acid (EC50 = 808 ± 206 nM, Emax = 13,309 ± 953 AFU’s), clonazepam (EC50 = 15 ± 8 nM, Emax = 4,211 ± 334 AFU’s), and VA (EC50 = 2,397 ± 341 nM, Emax = 5,935 ± 104 AFU’s). Abscisic acid, gibberellic acid, and cyclopartheniol demonstrated little to no detectable activity for modulating the human GABAA receptor.
Digital Object Identifier (DOI)
This study was supported by the University of Kentucky College of Pharmacy from 2015 to 2021.
Zinck, Garrett Edward, "BUILDING TOOLS FOR IMPROVED MODULATION OF THE HUMAN GABAA RECEPTOR, A CENTRAL NERVOUS SYSTEM TARGET FOR THE TREATMENT OF ANXIETY" (2022). Theses and Dissertations--Pharmacy. 134.
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