title: Stapling mimics noncovalent interactions of γ-carboxyglutamates in conantokins, peptidic antagonists of N-methyl-D-aspartic acid receptors.
abstract: Conantokins are short peptides derived from the venoms of marine cone snails that act as antagonists of the N-methyl-D-aspartate (NMDA) receptor family of excitatory glutamate receptors. These peptides contain γ-carboxyglutamic acid residues typically spaced at i,i+4 and/or i,i+7 intervals, which by chelating divalent cations induce and stabilize helical conformation of the peptide. Introduction of a dicarba bridge (or a staple) can covalently stabilize peptide helicity and improve its pharmacological properties. To test the hypothesis that stapling can effectively replace γ-carboxyglutamic acid residues in stabilizing the helical conformation of conantokins, we designed, synthesized, and characterized several stapled analogs of conantokin G (conG), with varying connectivities in terms of staple length and location along the face of the α-helix. NMR studies confirmed that the ring-closing metathesis reaction yielded a single product with the Z configuration of the olefinic bond. Based on circular dichroism and molecular modeling, the stapled analogs exhibited significantly enhanced helicity compared with the native peptide in a metal-free environment. Stapling i,i+4 was benign with respect to effects on in vitro and in vivo pharmacological properties. One analog, namely conG[11-15,S(i,i+4)S(8)], blocked NR2B-containing NMDA receptors with IC(50) = 0.7 μm and provided significant protection in the 6-Hz psychomotor model of pharmacoresistant epilepsy in mice. Remarkably, unlike native conG, conG[11-15,S(i,i+4)S(8)] produced no behavioral motor toxicity. Our results extend the applications of peptide stapling to helical peptides with extracellular targets and provide a means for engineering conantokins with improved pharmacological properties.
authors.affiliation: Departments of Biology, University of Utah, Salt Lake City, Utah 84112, USA.
authors.forename: Randall J
authors.initials: RJ
authors.lastname: Platt
authors.forename: Tiffany S
authors.initials: TS
authors.lastname: Han
authors.forename: Brad R
authors.initials: BR
authors.lastname: Green
authors.forename: Misty D
authors.initials: MD
authors.lastname: Smith
authors.forename: Jack
authors.initials: J
authors.lastname: Skalicky
authors.forename: Pawel
authors.initials: P
authors.lastname: Gruszczynski
authors.forename: H Steve
authors.initials: HS
authors.lastname: White
authors.forename: Baldomero
authors.initials: B
authors.lastname: Olivera
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Bulaj
authors.forename: Joanna
authors.initials: J
authors.lastname: Gajewiak
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000737:chemistry* / Q000494:pharmacology; D000818:Animals; D020916:Conotoxins / Q000737:chemistry / Q000494:pharmacology; D004827:Epilepsy / Q000188:drug therapy* / Q000378:metabolism; D018691:Excitatory Amino Acid Antagonists / Q000737:chemistry / Q000494:pharmacology; D008297:Male; D051379:Mice; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D020916:Conotoxins; D018691:Excitatory Amino Acid Antagonists; C121001:NR2B NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D015055:1-Carboxyglutamic Acid; C055407:conotoxin GV