A structure-function analysis of the marine toxin neodysiherbaine and its synthetic analogs on kainate receptors
Laura Leanne Lash
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Kainate receptor antagonists show efficacy in multiple animal models of neuropathologies, including pain and epilepsy. These receptors are selectively targeted by a number of natural and synthetic ligands. The goals of this project were to identify compounds with unique specificity for kainate receptors and to gain insight into structural determinants of affinity and activity of the marine toxin dysiherbaine, DH. DH is a high-affinity kainate receptor agonist and the most potent convulsant excitatory amino acid identified to date. The structure of DH is unique relative to other kainate receptor agonists because it has a glutamate backbone attached to a tetra-substituted hydrofuropyran ring system with two functional groups, at the C8 and C9 positions, that are important determinants for selectivity. Here, four groups of structural analogs were screened. Among the structural analogs characterized in the current study, 2,4-epi-neoDH, displayed a novel pharmacological profile at kainate receptors. In radioligand binding assays, 2,4-epi-neoDH showed micromolar binding affinity to GluR5(Q)-2a and GluR6a subunits. Electrophysiology recordings revealed it acted as an antagonist at these receptors, without detectable activity at AMPA receptors. This molecule represents the first antagonist for GluR5 and GluR6 receptors without concurrent activity at AMPA receptors. Our characterization of the series of structural analogs revealed they exhibit diverse binding affinities and a range of seizure-inducing potencies. It was determined that their seizure-inducing potency correlates with their binding affinity for GluR5 receptors, suggesting they induce seizure behavior through direct activation of GluR5 kainate receptors in the brain. Additionally, the C8 hydroxyl group of the naturally occurring analog, neodysiherbaine, was identified as a critical determinant for divergent affinity for GluR6 versus GluR7 subunits. Site-directed mutagenesis revealed two amino acid residues in the GluR6 binding cavity that are important for the selectivity. Our studies reveal insight into the structural requirements of DH and analogs for their diverse activity at kainate receptors. These studies offer a foundation for design of selective kainate receptor ligands and provide pharmacological tools for studying structure and function of these receptors.