Berecki G, Daly N L, Huang Y H, Vink S, Craik D J, Alewood P F, Adams D J
Health Innovations Research Institute, RMIT University, Melbourne, Vic, Australia.
Br J Pharmacol. 2014 Jul;171(13):3313-27. doi: 10.1111/bph.12686.
ω-Conotoxins CVIE and CVIF (CVIE&F) selectively inhibit Cav2.2 channels and are lead molecules in the development of novel analgesics. At physiological membrane potentials, CVIE&F block of Cav2.2 channels is weakly reversible. To improve reversibility, we designed and synthesized arginine CVIE&F analogues in which arginine was substituted for lysine at position 10 ([R10K]CVIE&F), and investigated their serum stability and pharmacological actions on voltage-gated calcium channels (VGCCs).
Changes in peptide structure due to R10K substitution were assessed by NMR. Peptide stability in human serum was analysed by reversed-phase HPLC and MS over a 24 h period. Two-electrode voltage-clamp and whole-cell patch clamp techniques were used to study [R10K]CVIE&F effects on VGCC currents in Xenopus oocytes and rat dorsal root ganglion neurons respectively.
R10K substitution did not change the conserved ω-conotoxin backbone conformations of CVIE&F nor the ω-conotoxin selectivity for recombinant or native Cav2.2 channels, although the inhibitory potency of [R10K]CVIF was better than that of CVIF. At -80 mV, the R10K chemical modification significantly affected ω-conotoxin-channel interaction, resulting in faster onset kinetics than those of CVIE&F. Heterologous and native Cav2.2 channels recovered better from [R10K]CVIE&F block than CVIE&F. In human serum, the ω-conotoxin half-lives were 6-10 h. CVIE&F and [R10K]CVIE&F were more stable than CVID.
R10K substitution in CVIE&F significantly alters the kinetics of ω-conotoxin action and improves reversibility without diminishing conotoxin potency and specificity for the Cav2.2 channel and without diminishing the serum stability. These results may help generate ω-conotoxins with optimized kinetic profiles for target binding.
ω-芋螺毒素CVIE和CVIF（CVIE&F）可选择性抑制Cav2.2通道，是新型镇痛药研发中的先导分子。在生理膜电位下，CVIE&F对Cav2.2通道的阻断作用具有较弱的可逆性。为提高可逆性，我们设计并合成了精氨酸CVIE&F类似物，其中精氨酸取代了第10位的赖氨酸（[R10K]CVIE&F），并研究了它们的血清稳定性以及对电压门控钙通道（VGCCs）的药理作用。
通过核磁共振（NMR）评估因R10K取代导致的肽结构变化。在24小时内，采用反相高效液相色谱（HPLC）和质谱（MS）分析人血清中的肽稳定性。分别使用双电极电压钳和全细胞膜片钳技术研究[R10K]CVIE&F对非洲爪蟾卵母细胞和大鼠背根神经节神经元中VGCC电流的影响。
R10K取代并未改变CVIE&F保守的ω-芋螺毒素主链构象，也未改变其对重组或天然Cav2.2通道的ω-芋螺毒素选择性，尽管[R10K]CVIF的抑制效力优于CVIF。在-80 mV时，R10K化学修饰显著影响ω-芋螺毒素与通道的相互作用，导致起效动力学比CVIE&F更快。异源和天然Cav2.2通道从[R10K]CVIE&F阻断中恢复的情况比CVIE&F更好。在人血清中，ω-芋螺毒素的半衰期为6 - 10小时。CVIE&F和[R10K]CVIE&F比CVID更稳定。
CVIE&F中的R10K取代显著改变了ω-芋螺毒素的作用动力学并提高了可逆性，同时不降低芋螺毒素对Cav2.2通道的效力和特异性，也不降低血清稳定性。这些结果可能有助于生成具有优化动力学特征的用于靶点结合的ω-芋螺毒素。
