Regulation, interaction, and inhibitor discovery of phosphodiesterases

描述（由申请人提供）：环核苷酸磷酸二酯酶（PDEs）控制“第二信使”cAMP和cGMP的细胞浓度，调节许多代谢过程。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100多种蛋白质同种异构体。PDEs的选择性抑制剂已被研究作为各种人类疾病的治疗剂。pde5选择性抑制剂西地那非（伟哥）就是一个例子，它是一种治疗勃起功能障碍和肺动脉高压的药物。我们上一次申请的研究已经发表了14篇原创研究论文，并对PDEs的底物特异性和抑制剂选择性的机制提供了深入的了解。然而，没有全长结构或片段同时具有调控和催化结构域，因此PDE催化如何受到其调控结构域的调节仍然未知。PDEs如何与其他信号通路中的蛋白质相互作用也是一个谜。本研究旨在了解PDE活性的调控结构域、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复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¿，(3)PDE1A与钙调素复合物的结构和动力学研究，(4)基于结构的PDE4抑制剂治疗炎症性疾病的发现。具体目标I、II和III不仅可以揭示PDE5、PDE8和PDE1近全长的结构信息，还可以深入了解这些PDE家族的催化机制。pde1 -钙调素和PDE8-I？B¿将阐明PDE家族在信号通路中的潜在作用。特异性目的IV可能导致发现一类新的PDE4抑制剂用于治疗人类疾病。

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.