以nNOS和PSD-95相互作用为靶标的解耦联剂筛选模型的建立
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摘要
复合物神经元型一氧化氮合酶(neuronal nitric oxide synthase, nNOS)-突触后密度蛋白-95 (postsynaptic density protein-95, PSD-95)形成的增加在缺血性脑卒中引发的神经损伤中起着重要作用,本研究以nNOS和PSD-95相互作用为靶点,建立nNOS-PSD-95解耦联剂筛选模型。模型采用转染的方式将质粒pCDH-Flag-nNOS和pcDNA3.1-PSD-95分别转染入人胚胎肾-293T (human embryonic kidney-293T, HEK-293T)细胞,通过真核表达的方式富集Flag-nNOS和PSD-95。随后,通过nNOS和PSD-95体外共孵育获得nNOS-PSD-95复合物,以ZL006为阳性药干预nNOS-PSD-95的结合,考察ZL006在该系统的解耦联效率,并对解耦联模型进行优化。结果表明,在该模型中, ZL006显示出较明显的解耦联效果,并通过戊二醛法获得抗体耦联磁珠,从而优化筛选模型,提高了筛选效率,降低了成本。最后运用该模型对另一个已知的nNOS-PSD-95解耦联化合物IC87201及候选ZL006结构类似物进行了解耦联活性评价,发现IC87201在该模型中同样具有解耦联活性,并且在候选化合物中也筛选出了潜在的解耦联化合物。
In ischemic stroke, increased level of neuronal complex of nitric oxide synthase(nNOS)-postsynaptic density protein-95(PSD-95) plays an important role in neuronal damage. We aimed to establish a screening model to identify compounds capable of uncoupling nNOS interaction with PSD-95. In this model, human embryonic kidney-293 T(HEK-293 T) cells were transfected with either pCDH-Flag-nNOS or pcDNA3.1-PSD-95 plasmid to obtain the protein of Flag-nNOS or PSD-95. Incubating Flag-nNOS with PSD-95 causes formation of the nNOS-PSD-95 complex. ZL006, a known uncoupler of nNOS-PSD-95 interaction, can disturb the interaction between Flag-nNOS and PSD-95, serving as a positive control. The method coupling antibodies to magnetic beads with glutaraldehyde was used to decrease the cost and increase the efficiency. To establish that our model is suitable for selecting nNOS-PSD-95 uncouplers, we evaluated the ability of IC87201, another reported uncoupler of nNOS-PSD-95 interaction, and structural analogs of ZL006. IC87201 and one structure analog of ZL006 showed uncoupling effect, supporting that our model can be used to select different types uncoupler blocking nNOS-PSD-95 interaction.
In ischemic stroke, increased level of neuronal complex of nitric oxide synthase(nNOS)-postsynaptic density protein-95(PSD-95) plays an important role in neuronal damage. We aimed to establish a screening model to identify compounds capable of uncoupling nNOS interaction with PSD-95. In this model, human embryonic kidney-293 T(HEK-293 T) cells were transfected with either pCDH-Flag-nNOS or pcDNA3.1-PSD-95 plasmid to obtain the protein of Flag-nNOS or PSD-95. Incubating Flag-nNOS with PSD-95 causes formation of the nNOS-PSD-95 complex. ZL006, a known uncoupler of nNOS-PSD-95 interaction, can disturb the interaction between Flag-nNOS and PSD-95, serving as a positive control. The method coupling antibodies to magnetic beads with glutaraldehyde was used to decrease the cost and increase the efficiency. To establish that our model is suitable for selecting nNOS-PSD-95 uncouplers, we evaluated the ability of IC87201, another reported uncoupler of nNOS-PSD-95 interaction, and structural analogs of ZL006. IC87201 and one structure analog of ZL006 showed uncoupling effect, supporting that our model can be used to select different types uncoupler blocking nNOS-PSD-95 interaction.
引文
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[9]Courtney MJ,Li LL,Lai YY.Mechanisms of NOS1AP action on NMDA receptor-nNOS signaling[J].Front Cell Neurosci,2014,8:252.
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[11]Zhou L,Li F,Xu HB,et al.Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95[J].Nat Med,2010,16:1439-1443.
[12]Chen D,Zhao T,Ni K,et al.Metabolic investigation on ZL006for the discovery of a potent prodrug for the treatment of cerebral ischemia[J].Bioorg Med Chem Lett,2016,26:2152-2155.
[13]Wu S,Yue Y,Tian H,et al.Tramiprosate protects neurons against ischemic stroke by disrupting the interaction between PSD-95 and nNOS[J].Neuropharmacology,2014,83:107-117.
[14]Yu CZ,Li C,Pei DS,et al.Neuroprotection against transient focal cerebral ischemia and oxygen-glucose deprivation by interference with GluR6-PSD-95 protein interaction[J].Neurochem Res,2009,34:2008-2021.
[15]Yang S,He R,Zhang FY,et al.Application of cell co-culture techniques in central nervous system diseases[J].Acta Pharm Sin(药学学报),2016,51:338-346.
[16]Lohse MJ,Susanne N,Carsten H.Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling[J].Pharmacol Rev,2012,64:299-336.
[17]Draczkowski P,Matosiuk D,Jozwiak K.Isothermal titration calorimetry in membrane protein research[J].J Pharm Biomed Anal,2014,87:313-325.
[18]Wang CQ.Basics of Molecular Immunology(分子免疫学基础)[M].Beijing:Peking University Press,1997:13-15.
[19]Tochio H,Mok YK,Zhang Q,et al.Formation of nNOS/PSD-95PDZ dimer requires a preformedβ-finger structure from the nNOS PDZ domain 1[J].J Mol Biol,2000,303:359-370.
[20]Christopherson KS,Hillier BJ,Lim WA,et al.PSD-95 assembles a ternary complex with the N-methyl-D-aspartic acid receptor and a bivalent neuronal NO synthase PDZ domain[J].J Biol Chem,1999,274:27467-27473.
[21]Zhou L,Zhu DY.Neuronal nitric oxide synthase:structure,subcellular localization,regulation,and clinical implications[J].Nitric Oxide,2009,20:223-230.
[22]Bach A,Pedersen SW,Dorr LA,et al.Biochemical investigations of the mechanism of action of small molecules ZL006 and IC87201 as potential inhibitors of the nNOS-PDZ/PSD-95-PDZinteractions[J].Sci Rep,2015,5:12157.
[23]Doucet MV,Levine H,Dev KK,et al.Small-molecule inhibitors at the PSD-95/nNOS interface have antidepressant-like properties in mice[J].Neuropsychopharmacology,2013,38:1575-1584.
[24]Lee WH,Xu Z,Ashpole NM,et al.Small molecule inhibitors of PSD-95-nNOS protein-protein interactions as novel analgesics[J].Neuropharmacology,2015,97:464-475.
[25]Sattler R,Xiong Z,Lu WY,et al.Specific coupling of NMDAreceptor activation to nitric oxide neurotoxicity by PSD-95protein[J].Science,1999,284:1845-1848.
[26]Hu W,Guan LS,Dang XB,et al.Small-molecule inhibitors at the PSD-95/nNOS interface attenuate MPP+-induced neuronal injury through Sirt3 mediated inhibition of mitochondrial dysfunction[J].Neurochem Int,2014,79:57-64.
[2]Flynn RW,Macwalter RS,Doney AS.The cost of cerebral ischaemia[J].Neuropharmacology,2008,55:250-256.
[3]Gállego J,Mu?oz R,Martínezvila E.Emergent cerebrovascular disease risk factor weighting:is transient ischemic attack an imminent threat?[J].Cerebrovasc Dis,2010,27:88-96.
[4]Sevick LK,Ghali S,Hill MD,et al.Systematic review of the cost and cost-effectiveness of rapid endovascular therapy for acute ischemic stroke[J].Stroke,2017,48:2519-2526.
[5]Arundine M,Tymianski M.Molecular mechanisms of glutamatedependent neurodegeneration in ischemia and traumatic brain injury[J].Cell Mol Life Sci,2004,61:657-668.
[6]Aarts M,Liu YT,Liu LD,et al.Treatment of ischemic brain damage by perturbing NMDA receptor-PSD-95 protein interactions[J].Science,2002,298:846-850.
[7]Cui H,Hayashi A,Sun HS,et al.PDZ protein interactions underlying NMDA receptor-mediated excitotoxicity and neuroprotection by PSD-95 inhibitors[J].J Neurosci,2007,27:9901-9915.
[8]Cao J,Viholainen JI,Dart C,et al.The PSD-95-nNOS interface:a target for inhibition of excitotoxic p38 stress-activated protein kinase activation and cell death[J].J Cell Biol,2005,168:117-126.
[9]Courtney MJ,Li LL,Lai YY.Mechanisms of NOS1AP action on NMDA receptor-nNOS signaling[J].Front Cell Neurosci,2014,8:252.
[10]Takagi N,Logan R,Teves L,et al.Altered interaction between PSD-95 and the NMDA receptor following transient global ischemia[J].J Neurochem,2010,74:169-178.
[11]Zhou L,Li F,Xu HB,et al.Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95[J].Nat Med,2010,16:1439-1443.
[12]Chen D,Zhao T,Ni K,et al.Metabolic investigation on ZL006for the discovery of a potent prodrug for the treatment of cerebral ischemia[J].Bioorg Med Chem Lett,2016,26:2152-2155.
[13]Wu S,Yue Y,Tian H,et al.Tramiprosate protects neurons against ischemic stroke by disrupting the interaction between PSD-95 and nNOS[J].Neuropharmacology,2014,83:107-117.
[14]Yu CZ,Li C,Pei DS,et al.Neuroprotection against transient focal cerebral ischemia and oxygen-glucose deprivation by interference with GluR6-PSD-95 protein interaction[J].Neurochem Res,2009,34:2008-2021.
[15]Yang S,He R,Zhang FY,et al.Application of cell co-culture techniques in central nervous system diseases[J].Acta Pharm Sin(药学学报),2016,51:338-346.
[16]Lohse MJ,Susanne N,Carsten H.Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling[J].Pharmacol Rev,2012,64:299-336.
[17]Draczkowski P,Matosiuk D,Jozwiak K.Isothermal titration calorimetry in membrane protein research[J].J Pharm Biomed Anal,2014,87:313-325.
[18]Wang CQ.Basics of Molecular Immunology(分子免疫学基础)[M].Beijing:Peking University Press,1997:13-15.
[19]Tochio H,Mok YK,Zhang Q,et al.Formation of nNOS/PSD-95PDZ dimer requires a preformedβ-finger structure from the nNOS PDZ domain 1[J].J Mol Biol,2000,303:359-370.
[20]Christopherson KS,Hillier BJ,Lim WA,et al.PSD-95 assembles a ternary complex with the N-methyl-D-aspartic acid receptor and a bivalent neuronal NO synthase PDZ domain[J].J Biol Chem,1999,274:27467-27473.
[21]Zhou L,Zhu DY.Neuronal nitric oxide synthase:structure,subcellular localization,regulation,and clinical implications[J].Nitric Oxide,2009,20:223-230.
[22]Bach A,Pedersen SW,Dorr LA,et al.Biochemical investigations of the mechanism of action of small molecules ZL006 and IC87201 as potential inhibitors of the nNOS-PDZ/PSD-95-PDZinteractions[J].Sci Rep,2015,5:12157.
[23]Doucet MV,Levine H,Dev KK,et al.Small-molecule inhibitors at the PSD-95/nNOS interface have antidepressant-like properties in mice[J].Neuropsychopharmacology,2013,38:1575-1584.
[24]Lee WH,Xu Z,Ashpole NM,et al.Small molecule inhibitors of PSD-95-nNOS protein-protein interactions as novel analgesics[J].Neuropharmacology,2015,97:464-475.
[25]Sattler R,Xiong Z,Lu WY,et al.Specific coupling of NMDAreceptor activation to nitric oxide neurotoxicity by PSD-95protein[J].Science,1999,284:1845-1848.
[26]Hu W,Guan LS,Dang XB,et al.Small-molecule inhibitors at the PSD-95/nNOS interface attenuate MPP+-induced neuronal injury through Sirt3 mediated inhibition of mitochondrial dysfunction[J].Neurochem Int,2014,79:57-64.