title: Structure-activity studies of conantokins as human N-methyl-D-aspartate receptor modulators.
abstract: The activities of conantokin-G (con-G), conantokin-T (con-T), and several novel analogues have been studied using polyamine enhancement of [3H]MK-801 binding to human glutamate-N-methyl-D-aspartate (NMDA) receptors, and their structures have been examined using CD and 1H NMR spectroscopy. The potencies of con-G[A7], con-G, and con-T as noncompetitive inhibitors of spermine-enhanced [3H]MK-801 binding to NMDA receptor obtained from human brain tissue are similar to those obtained using rat brain tissue. The secondary structure and activity of con-G are found to be highly sensitive to amino acid substitution and modification. NMR chemical shift data indicate that con-G, con-G[D8, D17], and con-G[A7] have similar conformations in the presence of Ca2+. This consists of a helix for residues 2-16, which is kinked in the vicinity of Gla10. This is confirmed by 3D structure calculations on con-G[A7]. Restraining this helix in a linear form (i.e., con-G[A7,E10-K13]) results in a minor reduction in potency. Incorporation of a 7-10 salt-bridge replacement (con-G[K7-E10]) prevents helix formation in aqueous solution and produces a peptide with low potency. Peptides with the Leu5-Tyr5 substitution also have low potencies (con-G[Y5,A7] and con-G[Y5,K7]) indicating that Leu5 in con-G is important for full antagonist behavior. We have also shown that the Gla-Ala7 substitution increases potency, whereas the Gla-Lys7 substitution has no effect. Con-G and con-G[K7] both exhibit selectivity between NMDA subtypes from mid-frontal and superior temporal gyri, but not between sensorimotor and mid-frontal gyri. Asn8 and/or Asn17 appear to be important for the ability of con-G to function as an inhibitor of polyamine-stimulated [3H]MK-801 binding, but not in maintaining secondary structure. The presence of Ca2+ does not increase the potencies of con-G and con-T for NMDA receptors but does stabilize the helical structures of con-G, con-G[D8,D17], and, to a lesser extent, con-G[A7]. The NMR data support the existence of at least two independent Ca2+-chelating sites in con-G, one involving Gla7 and possibly Gla3 and the other likely to involve Gla10 and/or Gla14.
authors.affiliation: Centre for Drug Design and Development, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: K J
authors.initials: KJ
authors.lastname: Nielsen
authors.forename: N
authors.initials: N
authors.lastname: Skjaerbaek
authors.forename: M
authors.initials: M
authors.lastname: Dooley
authors.forename: D A
authors.initials: DA
authors.lastname: Adams
authors.forename: M
authors.initials: M
authors.lastname: Mortensen
authors.forename: P R
authors.initials: PR
authors.lastname: Dodd
authors.forename: D J
authors.initials: DJ
authors.lastname: Craik
authors.forename: P F
authors.initials: PF
authors.lastname: Alewood
authors.forename: R J
authors.initials: RJ
authors.lastname: Lewis
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D020916:Conotoxins; D016291:Dizocilpine Maleate / Q000378:metabolism; D018691:Excitatory Amino Acid Antagonists / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000737:chemistry* / Q000494:pharmacology*; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology*; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013329:Structure-Activity Relationship