Structure−Activity Studies of Conantokins as Human N-Methyl-d-aspartate Receptor Modulators

• Published in: Journal of medicinal chemistry Vol. 42; no. 3; pp. 415 - 426
• Main Authors: Nielsen, Katherine J, Skjærbæk, Niels, Dooley, Michael, Adams, Denise A, Mortensen, Martin, Dodd, Peter R, Craik, David J, Alewood, Paul F, Lewis, Richard J
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 11.02.1999; Amer Chemical Soc
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Biological and medical sciences; Chemistry, Medicinal; Circular Dichroism; Conotoxins; Dizocilpine Maleate - metabolism; Excitatory Amino Acid Antagonists - chemistry; Excitatory Amino Acid Antagonists - pharmacology; Humans; Life Sciences & Biomedicine; Magnetic Resonance Spectroscopy; Medical sciences; Molecular Sequence Data; Mollusk Venoms - chemistry; Mollusk Venoms - pharmacology; Neuropharmacology; Neurotransmitters. Neurotransmission. Receptors; Peptidergic system (neuropeptide, opioid peptide, opiates...). Adenosinergic and purinergic systems; Peptides - chemistry; Peptides - pharmacology; Peptides, Cyclic - chemistry; Peptides, Cyclic - pharmacology; Pharmacology & Pharmacy; Pharmacology. Drug treatments; Protein Structure, Secondary; Receptors, N-Methyl-D-Aspartate - antagonists & inhibitors; Receptors, N-Methyl-D-Aspartate - metabolism; Science & Technology; Structure-Activity Relationship; NMDA RECEPTOR; PEPTIDES; PROTEIN; ANTAGONIST; GAMMA-CARBOXYGLUTAMATE; CHEMICAL-SHIFTS; NMR-SPECTROSCOPY; K+ CHANNEL; COUPLING-CONSTANTS; CONFORMATIONS; Human; Peptides; Central nervous system; In vitro; Structure activity relation; Polypeptide; Affinity; Peptide synthesis; Antagonist; Solid phase; NMDA receptor; Chemical synthesis; Brain (vertebrata)
• ISSN: 0022-2623; 1520-4804
• DOI: 10.1021/jm981052q

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.