CRYSTAL STRUCT. OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE

描述：(逐字摘自申请人摘要)环核苷酸 磷酸二酯酶(PDE)催化腺苷3‘，5’-环的水解 单磷酸(CAMP)和3‘，5’-环一磷酸鸟苷(CGMP) 分别生产5‘-AMP和5’-GMP。PDE是一种关键的控制酶 被称为“第二信使”的cAMP的细胞浓度和 调节细胞对多种激素的反应， 神经递质。人类PDE已有10个家系和22个亚型 已确认身份。进一步拼接22个亚型PDE的mRNAs以产生 PDE有60多种异构体。PDE的不同亚型位于不同的 并具有不同底物的特异性。这两个 年，PDE的特性引起了制药公司的极大关注 过去十年。许多选择性PDE抑制剂已被研究为治疗性药物 强心剂、血管扩张剂、抗哮喘、抗血栓药物 化合物、平滑肌松弛药和抗抑郁药。例如，伟哥，一种 PDE5抑制剂，是治疗男性勃起功能障碍的处方药 病人。该建议旨在表征底物的特异性和 用结晶学方法研究缓蚀剂的选择性。具体目标是 PDE及其缓蚀剂络合物的晶体结构测定 包括(1)PDE4B的催化结构域，(2)PDE4B的催化结构域 与抑制剂罗利普兰和碘化cAMP络合，(3)全长 PDE4D及其与抑制剂罗利普兰和布非林的络合物和(4) PDE3的催化结构域及其与西洛胺的络合物。这些结构 在这份提案中将揭示抑制剂结合在活性的细节 站点，并提供对催化机理的洞察。将cGMP对接到活动的 PDE4B的结合位点以及PDE4B-cAMP类似物的结构将会脱落 光对底物的专一性。两种结构的比较 PDE抑制的络合物将揭示抑制的选择性 不同的PDE家族，从而为设计提供了结构基础 选择性药物。这些结构将由多个同构确定 置换、多波长反常衍射或分子置换。 结构模型将使用程序O构建，并由 程序CNS。

DESCRIPTION: (Verbatim from the Applicant's Abstract) Cyclic nucleotide phosphodiesterase (PDE) catalyzes the hydrolysis of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) to produce, respectively, 5'-AMP and 5'-GMP. PDE is a key enzyme to control cellular concentrations of cAMP that is known as "second messenger" and mediates the response of cells to a wide variety of hormones and neurotransmitters. Ten families and twenty two subtypes of human PDE have been identified. The mRNAs of the 22 subtype PDEs are further spliced to generate over 60 isoforms of PDE. The distinct isoforms of PDE are located in different cellular compartments and possess different specificity of substrate. These two features of PDE have attracted great attention from pharmaceutical companies in the past decade. Many selective PDE inhibitors have been studied as therapeutic agents such as cardiotonic agents, vasodilators, antiasthma, atithrombic compounds, smooth muscle relaxants and antidepressants. For example, VIAGRA, an inhibitor of PDE5, is a prescription drug for erectile dysfunction of male patients. This proposal aims at characterization of substrate specificity and inhibitor selectivity by the approach of crystallography. The specific aims are to determine crystal structures of PDEs and their complexes with inhibitors including (1) the catalytic domain of PDE4B, (2) the catalytic domain of PDE4B complexed with the inhibitors rolipram and iodonated cAMP, (3) full length PDE4D and its complexes with the inhibitors rolipram and denbufylline and (4) the catalytic domain of PDE3 and its complex with cilostamide. The structures in this proposal will reveal the details of inhibitor binding at the active site and provide insight into catalytic mechanism. Docking cGMP into the active site of PDE4B, together with the structure of PDE4B-cAMP analog, will shed light on the substrate specificity. Comparison of the structures of PDE-inhibited complexes will shed light on the selectivity of inhibition of different families of PDE, and thus provide a structural basis for design of selective drugs. The structures will be determined by multiple isomorphous replacement, multiwavelength anomalous diffraction, or molecular replacement. The structural models will be built with the program O and refined by the program CNS.