Structure−Activity Studies of Conantokins as HumanN-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.

A New Mechanism for Metal Ion-assisted Interchain Helix Assembly in a Naturally Occurring Peptide Mediated by Optimally Spaced γ-Carboxyglutamic Acid Residues

“…In structure-activity studies of conantokins with human NMDARs, con-G inhibitory activity was observed to be unaffected by the presence of physiologically relevant levels (1.5 mM) of CaCl 2 . 44 However, this concentration of Ca 2þ is sub-saturating with regard to con-G, as is the Ca 2þ composition of the ocean habitat of the cone snail, and the physiological milieu of potential prey. Selfassociation, in vivo, of a fraction of the con-G pool may result in effects not directly related to receptor action including alterations to transport properties and resistance to proteolysis.…”

Conus Venom Peptide Pharmacology

“…As opposed to the vast majority of conopeptides, conantokins do not contain cysteines but instead have a high content of ␥-carboxyglutamic acid residues that when correctly spaced, induce ␣-helicity in the presence of divalent cations. Structure-activity studies of conantokins (Nielsen et al, 1999b) have revealed a number of important residues for binding, and a docking model showed that Con-G can fit into the agonist binding cleft of the NR2 subunit (see Fig. 8) (Wittekindt et al, 2001), revealing interactions between Glu386/Asp689 and Asn662 from NR2 subunit and Asn17/Lys15 and Glu2 of Con-G, respectively.…”

Inhibition of the Norepinephrine Transporter by the Venom Peptide χ-MrIA

“…H␣ chemical shifts compared with random coil values (25), are a sensitive measure of backbone conformation (26 -28) and can provide an indication whether the overall global fold of a series of a peptide is maintained (29). For a series of structurally related peptides, secondary H␣ chemical shifts can be used to identify the location but not the nature of local changes in conformation (29). Secondary H␣ chemical shifts were used in the first instance to compare -MrIA with its alanine-substituted analogs (Fig.…”