Regulation, interaction, and inhibitor discovery of phosphodiesterases

描述(申请人提供)：环核苷酸磷酸二酯酶(PDE)控制细胞内“第二信使”cAMP和cGMP的浓度，它们调节许多新陈代谢过程。人类基因组编码21个PDE基因，分属11个家族，通过RNA剪接表达100多种蛋白质亚型。PDEs的选择性抑制剂已被研究为各种人类疾病的治疗剂。一个例子是PDE5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。对我们上一次拨款提案的研究已经发表了14篇原始研究论文，并对PDE的底物专一性和抑制剂选择性的机制提供了洞察力。然而，目前还没有同时具有调节域和催化域的全长或片段结构，因此PDE催化是如何由它们的调节域调控的尚不清楚。PDE如何与其他信号通路中的蛋白质进行交叉对话也是一个谜。这一建议旨在了解PDE活性的调节结构域、PDE与其他蛋白质的相互作用以及PDE抑制剂的发现。具体目标包括：(1)近全长PDE5A1及其与底物和抑制剂的络合物的结构和生化研究；(2)PDE8A及其与抑制剂和结合蛋白I？B的络合物的结构；(3)PDE1A与钙调蛋白的络合物的结构和动力学研究；(4)基于结构的PDE4抑制剂的发现，用于治疗炎症疾病。特异性靶点I、II和III不仅揭示了PDE5、PDE8和PDE1的近全长结构信息，而且有助于深入了解这些PDE家族的催化机制。PDE1-钙调蛋白和PDE8-I？B的复杂结构将阐明PDE家族在信号通路中的潜在作用。特定的目的IV可能导致发现一类新的PDE4抑制剂用于治疗人类疾病。 与公共健康相关：磷酸二酯酶(PDE)是唯一一种降解细胞第二信使cAMP和cGMP的酶，在生理过程中发挥重要作用。这一建议旨在(1)近全长PDE1、5和8的结构和生化研究以及它们与钙调蛋白和IKB的相互作用；(2)基于结构的PDE4选择性抑制剂的发现。这些研究不仅将对PDE催化的调节以及与其他信号通路中蛋白质的相互作用提供洞察力，而且还将导致发现用于治疗人类疾病的PDE4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.