Substrate specificity and inhibitor selectivity of PDE

描述(申请人提供)：环核苷酸磷酸二酯酶(PDE)是控制细胞内“第二信使”腺苷或3‘，5’-环一磷酸(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，分属于11个家族。所有的PDE都含有一个保守的催化结构域，但每个家族都有各自的底物特异性和选择性的抑制剂。PDEs的选择性抑制剂已被广泛研究为各种疾病的治疗药物。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抑制剂西洛他唑(Pletal(Tm))是一种治疗间歇性跛行的药物。PDE抑制剂在医学上的广泛应用引起了学术界和产业界的高度重视。然而，PDE相似的活性部位如何区分不同的底物和抑制剂一直是个谜。我们假设底物的特异性和抑制剂的选择性是由活性中心残基的化学性质和PDE活性中心的构象共同决定的。本方案选择cAMP特异的PDE4和cGMP特异的PDE5和PDE9作为靶系统，用结晶学和蛋白质工程的方法研究底物专一性和抑制剂的选择性。将确定PDE4、PDEs和PDE9与底物、底物类似物和选择性抑制剂的络合物的结构。候选残基将通过一个或多个突变在PDE4和PDE5之间切换，以进一步说明底物的特异性。本提案中的结构，连同上一次供资期间的结构，将揭示用于确定底物专一性的关键残留物和元素，并确定有助于选择性结合抑制剂的潜在亚袋。由于抑制剂的选择性是药物副作用的一个关键问题，PDE与抑制剂形成的复合体的结构将为设计家族或亚家族选择性抑制剂提供模板，最终提高治疗疾病的药物效率。

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.