Regulation, interaction, and inhibitor discovery of phosphodiesterases

描述（由申请人提供）：环核苷酸磷酸二酯酶（PDE）控制调节许多代谢过程的“第二信使”cAMP和cGMP的细胞浓度。人类基因组编码21个PDE基因，分为11个家族，通过RNA剪接表达超过100种蛋白质亚型。PDE的选择性抑制剂已被研究作为各种人类疾病的治疗剂。一个例子是PDE 5选择性抑制剂西地那非（伟哥），它是一种治疗勃起功能障碍和肺动脉高压的药物。我们最后一次资助申请的研究导致了14篇原创研究论文的发表，并提供了对PDE底物特异性和抑制剂选择性机制的深入了解。然而，没有全长或片段的结构与调节和催化结构域是可用的，所以它仍然是未知的PDE催化是如何调节其调节结构域。偏微分方程如何与其他信号通路中的蛋白质发生串扰也是一个谜。该提案旨在了解PDE活性的调节，PDE与其他蛋白质的相互作用，PDE抑制剂的发现。具体目标包括：（1）近全长PDE 5A 1及其与底物和抑制剂的复合物的结构和生物化学研究;（2）PDE 8A及其与抑制剂和结合蛋白I的复合物的结构和生物化学研究; B？（3）PDE 1A与钙调蛋白复合物的结构和动力学研究;（4）基于结构的发现用于治疗炎性疾病的PDE 4抑制剂。具体目标I、II和III不仅将揭示近全长PDE 5、PDE 8和PDE 1的结构信息，还将提供对这些PDE家族的催化机制的深入了解。PDE 1-钙调素和PDE 8-I的复合物结构？B将阐明PDE家族在信号通路中的潜在作用。具体目标IV可能导致发现用于治疗人类疾病的新型PDE 4抑制剂。 公共卫生相关性：磷酸二酯酶（PDE）是唯一降解细胞内第二信使cAMP和cGMP的酶，在生理过程中起重要作用。该提案旨在（1）近全长PDE 1，5和8的结构和生物化学研究及其与钙调蛋白和IkB的相互作用，以及（2）基于结构的PDE 4选择性抑制剂的发现。这些研究不仅将提供对PDE催化的调节以及与其他信号通路中蛋白质的相互作用的深入了解，而且还将导致发现用于治疗人类疾病的PDE 4抑制剂。

DESCRIPTION (provided by applicant): Cyclic nucleotide phosphodiesterases (PDEs) control cellular concentration of "second messengers" cAMP and cGMP that modulate many metabolic processes. Human genome encodes 21 PDE genes that are categorized into 11 families and express over 100 isoforms of proteins via RNA splicing. Selective inhibitors of PDEs have been studied as therapeutic agents for various human diseases. An example is PDE5-selective inhibitor sildenafil (Viagra) that is a drug for treatment of erectile dysfunction and pulmonary hypertension. The studies of our last grant proposal have led to publications of 14 original research papers, and provided insight into the mechanisms of substrate specificity and inhibitor selectivity of PDEs. However, no structures of full-length or fragments with both regulatory and catalytic domains are available so that it remains unknown how the PDE catalysis is regulated by their regulatory domains. It is also a puzzle how PDEs cross-talk to proteins in other signaling pathways. This proposal aims at understanding regulation of PDE activities by their regulatory domains, PDE interaction with other proteins, and PDE inhibitor discovery. The specific aims include: (1) structural and biochemical studies of the near-full length PDE5A1 and its complexes with substrate and inhibitors, (2) structures of PDE8A and its complexes with inhibitors and with the binding protein I?B¿, (3) structural and kinetic studies on PDE1A in complex with calmodulin, and (4) structure-based discovery of PDE4 inhibitors for treatment of inflammatory diseases. Specific aims I, II, and III will not only reveal the structural information of near full-length PDE5, PDE8 and PDE1, but also provide insight into the catalytic mechanisms of these PDE families. The complex structures of PDE1-calmodulin and PDE8-I?B¿ will shed light on the potential roles of the PDE families in the signaling pathways. Specific aim IV may lead to discovery of a new category of PDE4 inhibitors for treatment of human diseases. PUBLIC HEALTH RELEVANCE: Phosphodiesterases (PDEs) are sole enzymes degrading cellular second messengers cAMP and cGMP that play important roles in physiological processes. This proposal aims at (1) structural and biochemical studies of near-full length PDE1, 5, and 8 and their interactions with calmodulin and IkB and (2) structure-based discovery of PDE4 selective inhibitors. These studies will not only provide insight into the regulation of PDE catalysis and interactions with proteins in other signaling pathways, but also lead to discovery of PDE4 inhibitors for treatment of human diseases.