P2X receptors are a family of extracellular ATP-gated trimeric cation channels that is widely distributed in human tissues. Quite some drug candidates targeting P2X receptors have entered into preclinical or main phas...P2X receptors are a family of extracellular ATP-gated trimeric cation channels that is widely distributed in human tissues. Quite some drug candidates targeting P2X receptors have entered into preclinical or main phases of clinical trials, but many of them failed due to low subtype-selectivity or species differences in pharmacological activities between human and experimental animals. Here, we identified the distinct inhibitory efficacies of NF110, a competitive inhibitor, between the rat(rP2X3) and human(hP2X3) P2X3 receptors. We demonstrated that this difference is determined by two amino acids located in the dorsal fin(DF) domain of P2X3 receptors. As revealed by mutagenesis, metadynamics, and covalent modification, NF110-mediated rP2X3 inhibition may be through a filling in the cavity formed by the DF,left flipper(LF) and lower body(LB) to partially, rather than fully, occupy the ATP-binding pocket.Moreover, substitution of residues located in the DF and/or LF domains of the rP2X2 receptor, a NF110-insensitive subtype, with the equivalent amino acids of rP2X3, bestowed the sensitivity of rP2X2 to NF110. The critical roles of the DF and LF domains in channel gating of P2X and low conservativity in residue sequences of those two domains raise the possibility that small molecules differentially interacting with the residues of the DF and LF domains of different P2X receptors may modulate channel's activity in a subtype-selective manner. However, the possible species-specificity of P2X inhibitors/modulators makes it more complex when interpreting the preclinical data into clinical researches.Nevertheless, our data provide new insights into the subtype-selectivity of competitive inhibitors and their distinct potencies in the human and experimental animals, both of which are extremely important in the drug discovery of P2X receptors.展开更多
目的：采用实时荧光定量聚合酶链式反应（polymerase chain reaction，PCR）技术，研究P2X受体在大鼠神经胶质瘤和嗜铬细胞瘤及原代培养皮层神经元和星形胶质细胞上的表达差异。方法：取新生1-2 d SD大鼠大脑皮层，分离纯化神经元和星...目的：采用实时荧光定量聚合酶链式反应（polymerase chain reaction，PCR）技术，研究P2X受体在大鼠神经胶质瘤和嗜铬细胞瘤及原代培养皮层神经元和星形胶质细胞上的表达差异。方法：取新生1-2 d SD大鼠大脑皮层，分离纯化神经元和星形胶质细胞，并采用实时荧光定量PCR技术，比较P2X受体在大鼠胶质瘤C6细胞、大鼠肾上腺嗜铬细胞瘤PC-12细胞、星形胶质细胞和皮层神经元上的表达差异。结果：C6细胞P2X2、P2X3和P2X5表达水平显著高于星形胶质细胞，P2X4、P2X6和P2X7表达水平显著低于星形胶质细胞，PC-12细胞P2X1、P2X2、P2X3和P2X6表达水平显著高于皮层神经元，P2X5和P2X7表达水平则显著低于皮层神经元。此外，还发现P2X2、P2X5和P2X6在C6和PC-12细胞上的表达水平存在显著差异。结论：大鼠胶质瘤和嗜铬细胞瘤细胞表达多种P2X受体，且与原代培养细胞存在表达差异，提示核苷酸介导的信号传递系统可能作为潜在的肿瘤治疗靶点。展开更多
基金supported by the National Natural Science Foundation of China(No.81070884)Distinguished Professor Awardfrom Education Department of Jiangsu Province,China(No.SR21500111)
基金supported by the National Natural Science Foundation of China(31570832)the National Program on Key Basic Research Project of China(2014CB9103000/02)+4 种基金Science and Technology Department of Hunan Province(2018RS3086)National Postdoctoral Program for Innovative Talents(BX201700306)China Postdoctoral Science Foundation(2018M632127)the Hunan Provincial Natural Science Foundation(2017JJ2198,2018JJ1012)Guangxi Funds for Distinguished Experts
文摘P2X receptors are a family of extracellular ATP-gated trimeric cation channels that is widely distributed in human tissues. Quite some drug candidates targeting P2X receptors have entered into preclinical or main phases of clinical trials, but many of them failed due to low subtype-selectivity or species differences in pharmacological activities between human and experimental animals. Here, we identified the distinct inhibitory efficacies of NF110, a competitive inhibitor, between the rat(rP2X3) and human(hP2X3) P2X3 receptors. We demonstrated that this difference is determined by two amino acids located in the dorsal fin(DF) domain of P2X3 receptors. As revealed by mutagenesis, metadynamics, and covalent modification, NF110-mediated rP2X3 inhibition may be through a filling in the cavity formed by the DF,left flipper(LF) and lower body(LB) to partially, rather than fully, occupy the ATP-binding pocket.Moreover, substitution of residues located in the DF and/or LF domains of the rP2X2 receptor, a NF110-insensitive subtype, with the equivalent amino acids of rP2X3, bestowed the sensitivity of rP2X2 to NF110. The critical roles of the DF and LF domains in channel gating of P2X and low conservativity in residue sequences of those two domains raise the possibility that small molecules differentially interacting with the residues of the DF and LF domains of different P2X receptors may modulate channel's activity in a subtype-selective manner. However, the possible species-specificity of P2X inhibitors/modulators makes it more complex when interpreting the preclinical data into clinical researches.Nevertheless, our data provide new insights into the subtype-selectivity of competitive inhibitors and their distinct potencies in the human and experimental animals, both of which are extremely important in the drug discovery of P2X receptors.
文摘目的：采用实时荧光定量聚合酶链式反应（polymerase chain reaction，PCR）技术，研究P2X受体在大鼠神经胶质瘤和嗜铬细胞瘤及原代培养皮层神经元和星形胶质细胞上的表达差异。方法：取新生1-2 d SD大鼠大脑皮层，分离纯化神经元和星形胶质细胞，并采用实时荧光定量PCR技术，比较P2X受体在大鼠胶质瘤C6细胞、大鼠肾上腺嗜铬细胞瘤PC-12细胞、星形胶质细胞和皮层神经元上的表达差异。结果：C6细胞P2X2、P2X3和P2X5表达水平显著高于星形胶质细胞，P2X4、P2X6和P2X7表达水平显著低于星形胶质细胞，PC-12细胞P2X1、P2X2、P2X3和P2X6表达水平显著高于皮层神经元，P2X5和P2X7表达水平则显著低于皮层神经元。此外，还发现P2X2、P2X5和P2X6在C6和PC-12细胞上的表达水平存在显著差异。结论：大鼠胶质瘤和嗜铬细胞瘤细胞表达多种P2X受体，且与原代培养细胞存在表达差异，提示核苷酸介导的信号传递系统可能作为潜在的肿瘤治疗靶点。
