4种ω-芋螺毒素的合成及活性研究

Study on the synthesis and activity of four ω-conotoxins

目的 合成新型ω-芋螺毒素,测定其作用靶标,获得新型镇痛分子并为N-型钙离子通道抑制剂设计提供依据。方法 采用固相法合成线性肽,然后进行折叠、富集和纯化得到纯肽;利用膜片钳技术,测定其对N、P/Q及L-型钙离子通道的抑制活性;利用金鱼测定毒副作用,采用热板法和醋酸扭体法测定活性高毒素的镇痛活性。结果 Ac6.4对N-型钙离子通道抑制活性高,IC50为0.42μmol/L;C6.4、Castu-c2及Di7.5的抑制活性较低,10μmol/L的C6.4、Castu-c2及Di7.5的抑制活性分别为(35.58±12.32)%、(31.09±12.24)%及(14.03±4.84)%。该4种ω-芋螺毒素对P/Q、L-型钙离子通道的抑制活性低。1.0~3.0μg/kg的Ac6.4显著增加小鼠对热板法的痛阈时间,1.0~10.0μg/kg的Ac6.4显著减少小鼠对醋酸扭体次数,但其镇痛活性均低于齐考诺肽(MVIIA)。Ac6.4对金鱼的毒性显著低于ω-芋螺毒素MVIIA。结论 Ac6.4选择性抑制CaV2.2,且具有较高的镇痛活性及较低毒副作用。

Objective In order to provide new analgesics and clues for the design of novel N-type calcium channel(CaV2.2) inhibitors, four novel ω-conotoxins were synthesized and their targets were identified.Methods The linear peptides were synthesized by solid-phase methods, and then were folded, enriched and purified to obtain target peptide. The patch clamp technique was used to determine its inhibitory activity on three types of N-, P/Q-and L-type calcium channels, and goldfishes were used to test the toxicity of the ω-conotoxin with high inhibitory activity, the mice hot-plate pain model and acetic acid writhing model were used to assess the analgesic activity. Results Ac6.4 displayed a high inhibitory activity against CaV2.2, the IC50 of Ac6.4 was 0.42 μmol/L. C6.4, Castu-c2 and Di7.5 displayed a low inhibitory activity against CaV2.2, and the inhibition ratios at 10 μmol/L concentration were(35.58 ± 12.32)%,(31.09 ± 12.24)% and(14.03 ± 4.84)%, respectively.The four ω-conotoxins displayed a low inhibitory activity on P/Q-and L-type calcium channels. 1.0-3.0 μg/kg of Ac6.4 significantly increased the pain threshold time of mice in hot plate model, and 1.0-10.0 μg/kg of Ac6. 4significantly reduced the number of acetic acid writhing of mice, but its analgesic activities were significantly lower than that of MVIIA. In addition, Ac6.4 possessed lower toxicity than MVIIA. Conclusion Ac6.4 selectively targeted on N-type calcium channels, and displayed high analgesic activity and low poisonous side effects.