Conantokins Derived from the Asprella Clade Impart ConRl-B, an NMDA Receptor Antagonist with a Unique Selectivity Profile for NR2B Subunits

Using molecular phylogeny has accelerated the discovery of peptidic ligands targeted to ion channels and receptors. One clade of venomous cone snails, Asprella , appears to be significantly enriched in conantokins, antagonists of N-Methyl D-Asparate receptors (NMDARs). Here, we describe the characterization of two novel conantokins from Conus rolani , including conantokin con Rl -B that has shown an unprecedented selectivity for blocking NMDARs that contain NR2B subunits. Con Rl -B shares only some sequence similarity to the most studied NR2B-selective conantokin, con G . The divergence between con Rl -B and con G in the second inter-Gla loop was used to design analogs for structure-activity studies; the presence of Pro10 was found to be key to the high potency of con Rl -B for NR2B, whereas the ε-amino group of Lys8 contributed to discrimination in blocking NR2B- and NR2A-containing NMDARs. In contrast to previous findings from Tyr5 substitutions in other conantokins, con Rl -B [L5Y] showed potencies on the four NR2 NMDA receptor subtypes that were similar to those of the native con Rl -B. When delivered into the brain, con Rl -B was active in suppressing seizures in the model of epilepsy in mice, consistent with NR2B-containing NMDA receptors being potential targets for antiepileptic drugs. Circular dichroism experiments confirmed that the helical conformation of con Rl -B is stabilized by divalent metal ions. Given the clinical applications of NMDA antagonists, con Rl -B provides a potentially important pharmacological tool for understanding the differential roles of NMDA receptor subtypes in the nervous system. This work shows the effectiveness of coupling molecular phylogeny, chemical synthesis and pharmacology for discovering new bioactive natural products.