Roles of key functional groups in omega-conotoxin GVIA - Synthesis, structure and functional assay of selected peptide analogues

The contributions of various functional groups to the pharmacophore of the N-type calcium-channel blocker, omega-conotoxin GVIA (GVIA), have been inve stigated using structural and in-vitro functional studies of analogues subs tituted at one or two positions with non-native residues. In most cases the structure of the analogue was shown to be native-like by H-1 NMR spectrosc opy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Three functional assays (sympathetic nerve stimula tion of rat isolated vas deferens, right atrium and mesenteric artery) were employed to monitor N-type calcium-channel activity. The data provide a mo re detailed picture of the roles in GVIA structure and activity of the cruc ial Lys2 and Tyr13, as well as all other positively charged residues, Tyr22 , the hydroxyproline residues and the C-terminal amido moiety, many of whic h were identified as being important for activity in an alanine scan [Lew e t al. (1997) J. Biol. Chem. 272, 12014-12023]. Substitutions of Lys2 with n onstandard 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 i ts 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-tetrah ydroisoquinoline-3-carboxylic acid [Tic(OH)]. The roles of the Hyp10 and Hy p21 hydroxyl groups, investigated by proline substitutions, appear to be mo re structural (as monitored by NMR) than functional, although small decreas es in potency were observed in some assays. The reversibility of the channe l blockade was also studied, and several analogues with faster wash-out cha racteristics than native GVIA were identified. Rapid reversibility (as in t he case of omega-conotoxin MVIIA) may be beneficial for therapeutic applica tions. Disubstituted analogues revealed some interesting cooperative effect s, which were not predicted from single-residue substitutions. A disubstitu ted chimera of GVIA and omega-conotoxin MVIIA was more potent than either n ative molecule. The more detailed description of the GVIA pharmacophore obt ained here provides a better basis for the future design of truncated pepti de and peptidomimetic analogues.