Flinn J P, Pallaghy P K, Lew M J, Murphy R, Angus J A, Norton R S
Biomolecular Research Institute, Parkville, Australia.
Eur J Biochem. 1999 Jun;262(2):447-55. doi: 10.1046/j.1432-1327.1999.00383.x.
The contributions of various functional groups to the pharmacophore of the N-type calcium-channel blocker, omega-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, 12014-12023]. 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 omega-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 omega-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.
通过对在一个或两个位置被非天然残基取代的类似物进行结构和体外功能研究，探究了各种官能团对N型钙通道阻滞剂ω-芋螺毒素GVIA（GVIA）药效基团的贡献。在大多数情况下，通过1H NMR光谱显示类似物的结构与天然结构相似。在某些情况下观察到的微小构象变化通过二维NMR进行了表征。采用三种功能测定法（对大鼠离体输精管、右心房和肠系膜动脉进行交感神经刺激）来监测N型钙通道活性。这些数据提供了一幅更详细的图景，展示了关键的Lys2和Tyr13以及所有其他带正电荷的残基、Tyr22、羟脯氨酸残基和C末端酰胺部分在GVIA结构和活性中的作用，其中许多在丙氨酸扫描中被确定对活性很重要[Lew等人（1997年）《生物化学杂志》272，12014 - 12023]。用非标准氨基酸和精氨酸取代Lys2定量了Lys侧链长度和电荷的作用。用d-Tyr以及受限类似物7-羟基-1,2,3,4-四氢异喹啉-3-羧酸[Tic(OH)]的d型和l型取代，表明Tyr13侧链及其羟基部分的取向很重要。通过脯氨酸取代研究的Hyp10和Hyp21羟基的作用，虽然在某些测定中观察到效力略有降低，但似乎更多是结构性的（通过NMR监测）而非功能性的。还研究了通道阻断的可逆性，鉴定出了几种比天然GVIA具有更快洗脱特性的类似物。快速可逆性（如ω-芋螺毒素MVIIA的情况）可能对治疗应用有益。双取代类似物揭示了一些有趣的协同效应，这是单残基取代无法预测的。GVIA和ω-芋螺毒素MVIIA的双取代嵌合体比任何一种天然分子都更有效。此处获得的对GVIA药效基团更详细的描述为未来设计截短肽和拟肽类似物提供了更好的基础。
