α-芋螺毒素TxIB异构体对大鼠和人类α6/α3β2β3乙酰胆碱受体的拮抗活性研究
|
摘要
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 antagonistic activities of three isomers of α-conotoxin TxIB on rat and human α6/α3β2β3nicotinic acetylcholine receptors(nAChRs). METHODS Three disulfide bond isomers were synthesized using Fmoc chemistry,which were identified by ultra performance liquid chromatography(UPLC) and confirmed by MALDI-TOF mass spectrometry. Rat and human α6/α3β2β3 nAChRs were expressed in oocytes of Xenopus laevis,which were used to test the antagonistic abilities of the 3 isomers. RESULTS The three isomers of α-conotoxin TxIB were synthesized successfully. The retention time of each isomer of α-conotoxin TxIB was different each other significantly. The observed molecular masses of three isomers were the same,which were consistent with their theoretical molecular mass. Their hydrophilicity orders were globular > ribbon > bead. Both rat and human α6/α3β2β3 nAChRs were expressed in oocytes well. Inhibition of three isomers of α-conotoxin TxIB on rat and human α6/α3β2β3 nAChRs were evaluated respectively. Among the three isomers of TxIB,the activity of the globular isomer was the most potent one,which had almost same activity at rat and human α6/α3β2β3 nAChRs with corresponding IC_(50) of 28.2 and 32.0 nmol·L~(-1) respectively. However,the other two isomers,ribbon and bead isomers displayed little antagonistic effect on both rat and human α6/α3β2β3 nAChRs only with an IC_(50) of > 10 μmol·L~(-1). CONCLUSION The synthesized globular isomer of α-conotoxin TxIB in this work has a high selectivity and potent antagonistic activity on rat and human α6/α3β2β3 nAChRs,which would be helpful for its new drug development.
OBJECTIVE To investigate antagonistic activities of three isomers of α-conotoxin TxIB on rat and human α6/α3β2β3nicotinic acetylcholine receptors(nAChRs). METHODS Three disulfide bond isomers were synthesized using Fmoc chemistry,which were identified by ultra performance liquid chromatography(UPLC) and confirmed by MALDI-TOF mass spectrometry. Rat and human α6/α3β2β3 nAChRs were expressed in oocytes of Xenopus laevis,which were used to test the antagonistic abilities of the 3 isomers. RESULTS The three isomers of α-conotoxin TxIB were synthesized successfully. The retention time of each isomer of α-conotoxin TxIB was different each other significantly. The observed molecular masses of three isomers were the same,which were consistent with their theoretical molecular mass. Their hydrophilicity orders were globular > ribbon > bead. Both rat and human α6/α3β2β3 nAChRs were expressed in oocytes well. Inhibition of three isomers of α-conotoxin TxIB on rat and human α6/α3β2β3 nAChRs were evaluated respectively. Among the three isomers of TxIB,the activity of the globular isomer was the most potent one,which had almost same activity at rat and human α6/α3β2β3 nAChRs with corresponding IC_(50) of 28.2 and 32.0 nmol·L~(-1) respectively. However,the other two isomers,ribbon and bead isomers displayed little antagonistic effect on both rat and human α6/α3β2β3 nAChRs only with an IC_(50) of > 10 μmol·L~(-1). CONCLUSION The synthesized globular isomer of α-conotoxin TxIB in this work has a high selectivity and potent antagonistic activity on rat and human α6/α3β2β3 nAChRs,which would be helpful for its new drug development.
引文
[1]DAVIS J,JONES A,LEWIS R J.Remarkable inter-and intraspecies complexity of conotoxins revealed by LC/MS[J].Peptides,2009,30(7):1222-1227.
[2]LAVERGNE V,HARLIWONG I,JONES A,et al.Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks[J].Proc Natl Acad Sci USA,2015,112(29):3782-3791.
[3]KAAS Q,YU R,JIN A H,et al.Cono Server:updated content,knowledge,and discovery tools in the conopeptide database[J].Nucleic Acids Res,2012,40(database issue):325-330.
[4]LEWIS R J,DUTERTRE S,VETTER I,et al.Conus venom peptide pharmacology[J].Pharmacol Rev,2012,64(2):259-298.
[5]SANFORD M.Intrathecal ziconotide:a review of its use in patients with chronic pain refractory to other systemic or intrathecal analgesics[J].CNS Drugs,2013,27(11):989-1002.
[6]GOTTI C,CLEMENTI F.Neuronal nicotinic receptors:from structure to pathology[J].Prog Neurobiol,2004,74(6):363-396.
[7]LEBBE E K,PEIGNEUR S,MAITI M,et al.Discovery of a new subclass of alpha-conotoxins in the venom of Conus australis[J].Toxicon,2014,91:145-154.
[8]LUO S,ZHANGSUN D,WU Y,et al.Characterization of a novel alpha-conotoxin from conus textile that selectively targets alpha6/alpha3beta2beta3 nicotinic acetylcholine receptors[J].JBiol Chem,2013,288(2):894-902.
[9]MCINTOSH J M,AZAM L,STAHELI S,et al.Analogs of alpha-conotoxin MII are selective for alpha6-containing nicotinic acetylcholine receptors[J].Mol Pharmacol,2004,65(4):944-952.
[10]FANG L C,SHEN L Z,YU J P,et al.Optimization of receptors expressed on Xenopus laevis oocytes[J].Biotechno Bull(生物技术通报),2013,29(6):167-171.
[11]HALAI R,CLARK R J,NEVIN S T,et al.Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues crucial for activity at the alpha9alpha10 nicotinic acetylcholine receptor[J].J Biol Chem,2009,284(30):20275-20284.
[12]VINCLER M,WITTENAUER S,PARKER R,et al.Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors[J].Proc Natl Acad Sci USA,2006,103(47):17880-17884.
[13]AZAM L,MCINTOSH J M.Molecular basis for the differential sensitivity of rat and human alpha9alpha10 n AChRs to alphaconotoxin Rg IA[J].J Neurochem,2012,122(6):1137-1144.
[14]Poly Novo.Metabolic discontinues clinical trial programme for neuropathic pain drug,ACV1[EB/OL].[2007-08-14].http://www.evaluategroup.com/Universal/View.aspx?type=Story&id=135083
[15]YANG K C,JIN G Z,WU J.Mysterious alpha6-containing n AChRs:function,pharmacology,and pathophysiology[J].Acta Pharmacol Sin,2009,30(6):740-751.
[16]KLINK R,DE KERCHOVE D'EXAERDE A,ZOLI M,et al.Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei[J].J Neurosci,2001,21(5):1452-1463.
[17]AZAM L,WINZER-SERHAN U H,CHEN Y,et al.Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons[J].J Comp Neurol,2002,444(3):260-274.
[18]CHAMPTIAUX N,GOTTI C,CORDERO-ERAUSQUIN M,et al.Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice[J].J Neurosci,2003,23(21):7820-7829.
[19]PONS S,FATTORE L,COSSU G,et al.Crucial role of alpha4and alpha6 nicotinic acetylcholine receptor subunits from ventral tegmental area in systemic nicotine self-administration[J].JNeurosci,2008,28(47):12318-12327.
[20]LE NOVERE N,ZOLI M,CHANGEUX J P.Neuronal nicotinic receptor alpha 6 subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain[J].Eur J Neurosci,1996,8(11):2428-2439.
[21]VAILATI S,HANKE W,BEJAN A,et al.Functional alpha6-containing nicotinic receptors are present in chick retina[J].Mol Pharmacol,1999,56(1):11-19.
[22]CHAMPTIAUX N,HAN Z Y,BESSIS A,et al.Distribution and pharmacology of alpha 6-containing nicotinic acetylcholine receptors analyzed with mutant mice[J].J Neurosci,2002,22(4):1208-1217.
[23]QUIK M,POLONSKAYA Y,KULAK J M,et al.Vulnerability of 125I-alpha-conotoxin MII binding sites to nigrostriatal damage in monkey[J].J Neurosci,2001,21(15):5494-5500.
[24]QUIK M,BORDIA T,FORNO L,et al.Loss of alpha-conotoxin MII-and A85380-sensitive nicotinic receptors in Parkinson's disease striatum[J].J Neurochem,2004,88(3):668-679.
[25]JACKSON K J,MCINTOSH J M,BRUNZELL D H,et al.The role of alpha6-containing nicotinic acetylcholine receptors in nicotine reward and with drawal[J].J Pharmacol Exp Ther,2009,331(2):547-554.
[26]SANJAKDAR S S,MALDOON P P,MARKS M J,et al.Differential roles of alpha6beta2*and alpha4beta2*neuronal nicotinic receptors in nicotine-and cocaine-conditioned reward in mice[J].Neuropsychopharmacology,2015,40(2):350-360.
[27]SCHILATY N D,HEDGES D M,JANG E Y,et al.Acute ethanol inhibits dopamine release in the nucleus accumbens via alpha6 nicotinic acetylcholine receptors[J].J Pharmacol Exp T-her,2014,349(3):559-567.
[2]LAVERGNE V,HARLIWONG I,JONES A,et al.Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks[J].Proc Natl Acad Sci USA,2015,112(29):3782-3791.
[3]KAAS Q,YU R,JIN A H,et al.Cono Server:updated content,knowledge,and discovery tools in the conopeptide database[J].Nucleic Acids Res,2012,40(database issue):325-330.
[4]LEWIS R J,DUTERTRE S,VETTER I,et al.Conus venom peptide pharmacology[J].Pharmacol Rev,2012,64(2):259-298.
[5]SANFORD M.Intrathecal ziconotide:a review of its use in patients with chronic pain refractory to other systemic or intrathecal analgesics[J].CNS Drugs,2013,27(11):989-1002.
[6]GOTTI C,CLEMENTI F.Neuronal nicotinic receptors:from structure to pathology[J].Prog Neurobiol,2004,74(6):363-396.
[7]LEBBE E K,PEIGNEUR S,MAITI M,et al.Discovery of a new subclass of alpha-conotoxins in the venom of Conus australis[J].Toxicon,2014,91:145-154.
[8]LUO S,ZHANGSUN D,WU Y,et al.Characterization of a novel alpha-conotoxin from conus textile that selectively targets alpha6/alpha3beta2beta3 nicotinic acetylcholine receptors[J].JBiol Chem,2013,288(2):894-902.
[9]MCINTOSH J M,AZAM L,STAHELI S,et al.Analogs of alpha-conotoxin MII are selective for alpha6-containing nicotinic acetylcholine receptors[J].Mol Pharmacol,2004,65(4):944-952.
[10]FANG L C,SHEN L Z,YU J P,et al.Optimization of receptors expressed on Xenopus laevis oocytes[J].Biotechno Bull(生物技术通报),2013,29(6):167-171.
[11]HALAI R,CLARK R J,NEVIN S T,et al.Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues crucial for activity at the alpha9alpha10 nicotinic acetylcholine receptor[J].J Biol Chem,2009,284(30):20275-20284.
[12]VINCLER M,WITTENAUER S,PARKER R,et al.Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors[J].Proc Natl Acad Sci USA,2006,103(47):17880-17884.
[13]AZAM L,MCINTOSH J M.Molecular basis for the differential sensitivity of rat and human alpha9alpha10 n AChRs to alphaconotoxin Rg IA[J].J Neurochem,2012,122(6):1137-1144.
[14]Poly Novo.Metabolic discontinues clinical trial programme for neuropathic pain drug,ACV1[EB/OL].[2007-08-14].http://www.evaluategroup.com/Universal/View.aspx?type=Story&id=135083
[15]YANG K C,JIN G Z,WU J.Mysterious alpha6-containing n AChRs:function,pharmacology,and pathophysiology[J].Acta Pharmacol Sin,2009,30(6):740-751.
[16]KLINK R,DE KERCHOVE D'EXAERDE A,ZOLI M,et al.Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei[J].J Neurosci,2001,21(5):1452-1463.
[17]AZAM L,WINZER-SERHAN U H,CHEN Y,et al.Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons[J].J Comp Neurol,2002,444(3):260-274.
[18]CHAMPTIAUX N,GOTTI C,CORDERO-ERAUSQUIN M,et al.Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice[J].J Neurosci,2003,23(21):7820-7829.
[19]PONS S,FATTORE L,COSSU G,et al.Crucial role of alpha4and alpha6 nicotinic acetylcholine receptor subunits from ventral tegmental area in systemic nicotine self-administration[J].JNeurosci,2008,28(47):12318-12327.
[20]LE NOVERE N,ZOLI M,CHANGEUX J P.Neuronal nicotinic receptor alpha 6 subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain[J].Eur J Neurosci,1996,8(11):2428-2439.
[21]VAILATI S,HANKE W,BEJAN A,et al.Functional alpha6-containing nicotinic receptors are present in chick retina[J].Mol Pharmacol,1999,56(1):11-19.
[22]CHAMPTIAUX N,HAN Z Y,BESSIS A,et al.Distribution and pharmacology of alpha 6-containing nicotinic acetylcholine receptors analyzed with mutant mice[J].J Neurosci,2002,22(4):1208-1217.
[23]QUIK M,POLONSKAYA Y,KULAK J M,et al.Vulnerability of 125I-alpha-conotoxin MII binding sites to nigrostriatal damage in monkey[J].J Neurosci,2001,21(15):5494-5500.
[24]QUIK M,BORDIA T,FORNO L,et al.Loss of alpha-conotoxin MII-and A85380-sensitive nicotinic receptors in Parkinson's disease striatum[J].J Neurochem,2004,88(3):668-679.
[25]JACKSON K J,MCINTOSH J M,BRUNZELL D H,et al.The role of alpha6-containing nicotinic acetylcholine receptors in nicotine reward and with drawal[J].J Pharmacol Exp Ther,2009,331(2):547-554.
[26]SANJAKDAR S S,MALDOON P P,MARKS M J,et al.Differential roles of alpha6beta2*and alpha4beta2*neuronal nicotinic receptors in nicotine-and cocaine-conditioned reward in mice[J].Neuropsychopharmacology,2015,40(2):350-360.
[27]SCHILATY N D,HEDGES D M,JANG E Y,et al.Acute ethanol inhibits dopamine release in the nucleus accumbens via alpha6 nicotinic acetylcholine receptors[J].J Pharmacol Exp T-her,2014,349(3):559-567.