The contributions of various functional groups to the pharmacophore of the N-type calcium-channel blocker, ω-conotoxin GVIA (GVIA), have been investigated using structural and in-vitro functional studies of analogues substituted at one or two positions with non-native residues. In most cases the structure of the analogue was shown to be native-like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) were employed to monitor N-type calcium-channel activity. The data provide a more detailed picture of the roles in GVIA structure and activity of the crucial Lys2 and Tyr13, as well as all other positively charged residues. Tyr22, the hydroxyproline residues and the C-terminal amido moiety, many of which were identified as being important for activity in an alanine scan [Lew et al. (1997) J. Biol. Chem. 272, 1201412023]. Substitutions of Lys2 with nonstandard amino acids and arginine quantified the roles of the length and charge of the Lys side chain. The orientation of the Tyr13 side chain and its hydroxyl moiety was shown to be important by substitution with D-Tyr and the D-form and L-form of the constrained analogue 7-hydroxy- 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid [Tic(OH)]. The roles of the Hyp10 and Hyp21 hydroxyl groups, investigated by proline substitutions, appear to be more structural (as monitored by NMR) than functional, although small decreases in potency were observed in some assays. The reversibility of the channel blockade was also studied, and several analogues with faster wash-out characteristics than native GVIA were identified. Rapid reversibility (as in the case of ω-conotoxin MVIIA) may be beneficial for therapeutic applications. Disubstituted analogues revealed some interesting cooperative effects, which were not predicted from single-residue substitutions. A disubstituted chimera of GVIA and ω-conotoxin MVIIA was more potent than either native molecule. The more detailed description of the GVIA pharmacophore obtained here provides a better basis for the future design of truncated peptide and peptidomimetic analogues.
- Calcium channel
- Peptide synthesis