CDC25B小分子抑制剂的发现及评价
摘要
CDC25蛋白酪氨酸酯酶(cell division cycle 25,CDC25)属于高保守的双位点磷酸酯酶,它通过对CDK(cyclin-dependent kinase)复合物某些磷酸化位点去磷酸化来活化它,促使细胞过渡到下一个周期,从而参与了细胞周期调控,同时它是损伤修复通路中的重要组成部分,对于遗传信息的稳定起着重要作用。在许多癌症的组织中,CDC25是异常高表达的,而且有一些证据表明,这种高表达可能对于肿瘤的发展起着促进作用。而如果下调CDC25的表达的话,会引起细胞周期阻滞,从而抑制细胞增殖。所以CDC25现在已经被当作了一个潜在的药物作用靶点。
我们从大肠杆菌系统中成功表达并纯化得到重组的CDC25B,并建了CDC25B高通量筛选模型,对国家新药筛选中心的48000个化合物进行了筛选,发现了两个新颖的CDC25B的抑制剂—LGH00031和LGH00045。其中LGH00031是不可逆的抑制剂,LGH00045是有较好选择性的、可逆的、混合型的抑制剂。在细胞水平对它们进行评价发现,它们能抑制细胞增殖,引起细胞的G2/M期阻滞,同时抑制了CDK1的15位酪氨酸的去磷酸化。对LGH00031的作用机制进行了研究,发现了LGH00031在体外是通过与DTT反应产生ROS带来的这种抑制作用,在细胞中LGH00031能够诱导产生ROS,ROS的清除剂—谷胱甘肽的前体NAC能够削弱LGH00031在细胞中的作用。推测LGH00031在细胞中带来的CDC25B的抑制是通过与细胞中的二氢硫辛酸反应产生ROS而引起的。
这两个高活性的新型小分子CDC25B抑制剂的发现、相关抑制性质的研究以及在细胞水平的生物活性评价,为其进一步结构优化和药理学、药效学研究打下坚实的基础,这对于进一步理解选择性所依存的分子机制及寻找高效特异的PTPs抑制剂有着重要的意义,同时也为更加广泛地研究VHR的生理及病理功能提供了有力的工具。
另外工作是有关烟酸保护胰岛β细胞的研究,发现烟酸能够保护链脲佐菌素(STZ)诱导的糖尿病大鼠的β细胞,这个作用可能有烟酸的抗氧化应激作用有关。
Cell division cycle 25(CDC25)phosphatases regulate key transitions between cell cycle phases during normal cell division by dephosphorylating and activating the CDK(cyclin-dependent kinase)complexes.And in the event of DNA damage they are the targets of the checkpoint machinery that ensures genetic stability.Since then,CDC25 was reported to be overexpressed in primary tissue samples from various human cancers.Some evidences suggested that the overexpression of CDC25 may contribute to cancer.And down-regulation of CDC25 caused cell cycle arrest and inhibition of cell proliferation.These in turn make them ideal targets for a new anticancer therapy.
We purified recombinant CDC25B expressed in Escherichia coli. High-through screening(HTS)assay was set up to find out novel inhibitors of CDC25B from 48000 pure chemicals collected from different sources with wide structural diversity of National Center for Drug Screening.LGH00031 and LGH00045 were discovered and characterized.LGH00031 was thought to be an irreversible inhibitor,and LGH00045 was a reversible and mixed-type inhibitor of CDC25 with relative good selection.The studies in cellular levels showed that both of the inhibitors caused a G2/M phase arrest which in turn resulted in the inhibition of cell proliferaton,and inhibited dephosphorylation of Tyr15 of CDK1. The mechanism of the inhibition of LGH00031 against CDC25B was also studied here.The result showed that in vitro the inhibition was caused by ROS which was generated by the reaction of LGH00031 and DTT.In cultured cells,LGH00031 increased the generation of ROS,and NAC,a precursor of glutathione,impaired the action of LGH00031 in cells.The generation of ROS by the reaction of LGH00031 and dihydrolipoic acid was conjectured,which may be the reason that LGH00031 inhibited CDC25B in the absence of DTT in cultured cells.
The discovery and characterization of the novel potent and selective inhibitor as well as its evaluation in cellular levels will be ground work for further structure modification,cell-based and animal-based pharmacological and efficacy studies in the future.It might be extremely powerful tools to exploit the physiological and pathological function of CDC25B.
Another part of work is the study on the protection of isletsβcells by nicotinic acid.Nicotinic acid could protect isletβcells of streptozocin induced diabetic rats.This protection may be relative to the anti-oxidative stress of nicotinic acid.
我们从大肠杆菌系统中成功表达并纯化得到重组的CDC25B,并建了CDC25B高通量筛选模型,对国家新药筛选中心的48000个化合物进行了筛选,发现了两个新颖的CDC25B的抑制剂—LGH00031和LGH00045。其中LGH00031是不可逆的抑制剂,LGH00045是有较好选择性的、可逆的、混合型的抑制剂。在细胞水平对它们进行评价发现,它们能抑制细胞增殖,引起细胞的G2/M期阻滞,同时抑制了CDK1的15位酪氨酸的去磷酸化。对LGH00031的作用机制进行了研究,发现了LGH00031在体外是通过与DTT反应产生ROS带来的这种抑制作用,在细胞中LGH00031能够诱导产生ROS,ROS的清除剂—谷胱甘肽的前体NAC能够削弱LGH00031在细胞中的作用。推测LGH00031在细胞中带来的CDC25B的抑制是通过与细胞中的二氢硫辛酸反应产生ROS而引起的。
这两个高活性的新型小分子CDC25B抑制剂的发现、相关抑制性质的研究以及在细胞水平的生物活性评价,为其进一步结构优化和药理学、药效学研究打下坚实的基础,这对于进一步理解选择性所依存的分子机制及寻找高效特异的PTPs抑制剂有着重要的意义,同时也为更加广泛地研究VHR的生理及病理功能提供了有力的工具。
另外工作是有关烟酸保护胰岛β细胞的研究,发现烟酸能够保护链脲佐菌素(STZ)诱导的糖尿病大鼠的β细胞,这个作用可能有烟酸的抗氧化应激作用有关。
Cell division cycle 25(CDC25)phosphatases regulate key transitions between cell cycle phases during normal cell division by dephosphorylating and activating the CDK(cyclin-dependent kinase)complexes.And in the event of DNA damage they are the targets of the checkpoint machinery that ensures genetic stability.Since then,CDC25 was reported to be overexpressed in primary tissue samples from various human cancers.Some evidences suggested that the overexpression of CDC25 may contribute to cancer.And down-regulation of CDC25 caused cell cycle arrest and inhibition of cell proliferation.These in turn make them ideal targets for a new anticancer therapy.
We purified recombinant CDC25B expressed in Escherichia coli. High-through screening(HTS)assay was set up to find out novel inhibitors of CDC25B from 48000 pure chemicals collected from different sources with wide structural diversity of National Center for Drug Screening.LGH00031 and LGH00045 were discovered and characterized.LGH00031 was thought to be an irreversible inhibitor,and LGH00045 was a reversible and mixed-type inhibitor of CDC25 with relative good selection.The studies in cellular levels showed that both of the inhibitors caused a G2/M phase arrest which in turn resulted in the inhibition of cell proliferaton,and inhibited dephosphorylation of Tyr15 of CDK1. The mechanism of the inhibition of LGH00031 against CDC25B was also studied here.The result showed that in vitro the inhibition was caused by ROS which was generated by the reaction of LGH00031 and DTT.In cultured cells,LGH00031 increased the generation of ROS,and NAC,a precursor of glutathione,impaired the action of LGH00031 in cells.The generation of ROS by the reaction of LGH00031 and dihydrolipoic acid was conjectured,which may be the reason that LGH00031 inhibited CDC25B in the absence of DTT in cultured cells.
The discovery and characterization of the novel potent and selective inhibitor as well as its evaluation in cellular levels will be ground work for further structure modification,cell-based and animal-based pharmacological and efficacy studies in the future.It might be extremely powerful tools to exploit the physiological and pathological function of CDC25B.
Another part of work is the study on the protection of isletsβcells by nicotinic acid.Nicotinic acid could protect isletβcells of streptozocin induced diabetic rats.This protection may be relative to the anti-oxidative stress of nicotinic acid.
引文
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