Substrate specificity and inhibitor selectivity of PDE

描述（由申请人提供）：环核苷酸磷酸二酯酶（PDEs）是控制细胞“第二信使”腺苷或鸟苷3',5'-环单磷酸（cAMP或cGMP）浓度的关键酶。人类基因组编码21个PDE基因和60多个PDE亚型，分为11个家族。所有pde都包含一个保守的催化结构域，但每个家族都具有单独的底物特异性和选择性抑制剂。选择性PDEs抑制剂作为治疗多种疾病的药物已被广泛研究。例如，PDEs抑制剂西地那非（伟哥（tm））是一种治疗勃起功能障碍的药物，PDE3抑制剂西洛唑（Pletal(tm)）是一种治疗间歇性跛行的药物。PDE抑制剂广泛的医学应用引起了学术界和产业界的高度关注。然而，PDEs的相似活性位点如何区分不同的底物和抑制剂一直是一个谜。我们假设底物特异性和抑制剂选择性是由活性位点残基的化学性质和PDE活性位点的构象共同决定的。本课题选择cAMP特异性PDE4和cGMP特异性PDE5、PDE9作为靶标体系，运用晶体学和蛋白质工程的方法研究底物特异性和抑制剂选择性。PDE4、PDES和PDE9与底物、底物类似物和选择性抑制剂配合物的结构将被确定。候选残基将通过单个或多个突变在PDE4和PDE5之间切换，以进一步说明底物特异性。本提案中的结构，连同上次资助期的结构，将揭示用于确定底物特异性的关键残基和元件，并确定有助于抑制剂选择性结合的潜在子口袋。由于抑制剂的选择性是药物副作用的关键问题，PDEs与抑制剂复合物的结构将为设计家族或亚家族选择性抑制剂提供模板，最终提高药物治疗疾病的效率。

DESCRIPTION (provided by applicant): Cyclic nucleotide phosphodiesterases (PDEs) are the key enzymes that control the cellular concentration of "second messengers" adenosine or guanosine 3', 5'-cyclic monophosphate (cAMP or cGMP). The human genome encodes 21 PDE genes and over 60 PDE isoforms categorized into 11 families. All PDEs contain a conserved catalytic domain, but each family possesses individual substrate specificity and selective inhibitors. Selective inhibitors of PDEs have been widely studied as therapeutic agents for various diseases. For example, PDEs inhibitor sildenafil (VIAGRA(tm)) is a drug for erectile dysfunction and PDE3 inhibitor cilostazole (Pletal(tm)) is a drug for intermittent claudication. The wide medical applications of PDE inhibitors have attracted great attention from both academic and industrial research groups. However, it has been mysteries how the similar active sites of PDEs distinguish the different substrates and inhibitors. We hypothesize that the substrate specificity and inhibitor selectivity are determined by both the chemical nature of active site residues and the conformations of the PDE active sites. This proposal chooses cAMP specific PDE4 and cGMP specific PDE5 and PDE9 as the target systems to study the substrate specificity and inhibitor selectivity with approaches of crystallography and protein engineering. The structures of PDE4, PDES and PDE9 in complex with substrate, substrate analogues, and selective inhibitors will be determined. The candidate residues will be switched between PDE4 and PDE5 by a single or multiple mutations for further illustration of the substrate specificity. The structures in this proposal, together with those from the last funding period, will reveal key residues and elements for determination of the substrate specificity and identify potential subpockets contributing to the selective binding of the inhibitors. Since the inhibitor selectivity is a key issue for side effects of drugs, the structures of PDEs in complex with inhibitors will provide templates for design of family- or subfamily-selective inhibitors and ultimately improve the drugs efficiency for treatment of the diseases.