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目的研究α-芋螺毒素TxIB 3个二硫键异构体对大鼠和人类α6/α3β2β3乙酰胆碱受体(nAChRs)的拮抗活性。方法采用Fmoc固相合成法,分别合成TxIB的3个二硫键异构体。利用非洲爪蟾卵母细胞分别表达大鼠和人类的α6/α3β2β3n AChRs,分别测定3个异构体对它们的拮抗活性。结果成功合成了TxIB的3个二硫键异构体,并通过超高效液相色谱(UPLC)与基质辅助激光解吸电离飞行时间(MALDI-TOF)质谱进行了鉴定,且为正确。α-芋螺毒素TxIB的3个二硫键异构体在UPLC的洗脱出峰时间差异明显,其亲水性顺序分别为球状异构体(globular)>带状异构体(ribbon)>珠子状异构体(bead)。质谱鉴定它们的相对分子质量完全一样,且与理论相对分子质量一致。大鼠与人类α6/α3β2β3 nAChRs在非洲爪蟾卵母细胞中获得了有效表达,并用该技术体系测知了TxIB的3个二硫键异构体的拮抗活性。其中,TxIB的球状异构体活性最强,对大鼠与人类α6/α3β2β3 nAChRs的拮抗活性极其相似,其半抑制浓度(IC_(50))分别为28.2与32.0 nmol·L~(-1)。而带状异构体和珠子状异构体对大鼠与人类α6/α3β2β3 nAChRs几乎没有拮抗活性,其IC_(50)>10μmol·L~(-1)。结论本实验人工合成的TxIB球状异构体对大鼠与人类α6/α3β2β3 nAChRs具有高选择性强拮抗活性,这为后续TxIB新药研发提供了研究基础。
Objective To investigate the antagonistic activity of α-conotoxin TxIB 3 disulfide isomers on rat and human α6 / α3β2β3 acetylcholine receptors (nAChRs). Methods Three disulfide isomers of TxIB were synthesized by Fmoc solid phase synthesis. The α6 / α3β2β3n AChRs of rat and human were respectively expressed by Xenopus laevis oocytes, and the antagonistic activities of the three isoforms were measured respectively. Results TxIB successfully synthesized three disulfide isomers and by matrix assisted laser with ultra performance liquid chromatography (UPLC) desorption ionization time of flight (MALDI-TOF) mass spectrometry were identified, and correct. The three disulfide isomers of α-conotoxin TxIB showed significant differences in the peak times of elution from UPLC. The order of their hydrophilicity was globular> ribbon> Beads. Mass spectrometry identification of their relative molecular mass exactly the same, and with the theoretical relative molecular mass. The rat and human α6 / α3β2β3 nAChRs were efficiently expressed in Xenopus laevis oocytes, and the antisense activity of three disulfide isomers of TxIB was detected by this technique. Wherein the spherical TxIB isomers strongest activity, antagonistic activity of rat and human α6 / α3β2β3 nAChRs very similar, half-inhibitory concentration (IC_ (50)) were 28.2 and 32.0 nmol·L ~ (-1) . However, the band isomers and the bead isomers had almost no antagonistic activity on rat and human α6 / α3β2β3 nAChRs with IC 50 (50)> 10μmol·L -1. CONCLUSION: The synthesized spherical isomers of TxIB have highly selective and potent antagonistic activity against α6 / α3β2β3 nAChRs in rats, which provides the basis for further research and development of new TxIB drugs.