Active site amino acids of cAMP phosphodiesterase 3A

细胞内cAMP和cGMP的增加会对所有的血小板功能产生强烈的抑制作用。环GMP抑制cAMP磷酸二酯酶(CGI-PDE，PDE3A)，使cAMP水解酶活性降低，细胞内cAMP浓度降低。抑制剂是一种有效的抗血小板药物。目前，已证实有效的两种抗血小板药物是阿司匹林，它抑制血栓素A2(TXA2)的环氧合酶依赖的合成，以及氯吡格雷或噻氯匹定，它阻断ADP抑制刺激的腺苷环化酶的能力。对照试验表明，阿司匹林和噻氯匹定均可用于一级预防的二级预防。NO是一种有效的血小板抑制物，通过刺激鸟苷环化酶而升高cGMP，而cGMP通过抑制PDE2A而升高cAMP。当细胞内cAMP增加时，通过阻断血小板内的所有激活途径，PDE3抑制剂有可能调节冠状动脉再闭塞。PDE抑制剂已经在动物模型和患者的冠状动脉支架中显示出希望。近期的目标是确定PDE活性部位的关键氨基酸。这些信息可以被制药公司用来设计临床上有用的抑制剂，而副作用比目前的药物少。我们假设，有不同的氨基酸促进催化反应，水解cAMP，形成底物结合部位，并结合必要的金属阳离子。我们进一步推测，尽管cGMP的抑制剂结合部位可能与底物结合部位重叠，但有不同的氨基酸与该核苷酸相互作用。我们将合成新的cGMP烷化亲和试剂(8-BDP-TcGMP、2-BDB-TcGMP、2-BDB-TEA-3‘5’MP和cGMPS-BDB)，测量它们的掺入量，并测试它们在不同浓度下调节cAMP水解的能力以及cAMP、cGMP和AMP对抗抑制作用的丧失的能力。(2)对新型cGMP亲和剂标记的修饰多肽和cAMP亲和类似物标记的修饰多肽进行鉴定、分离和测序。这些残基在修饰后的酶中的功能将通过动力学、底物和金属结合来评估。(3)根据特异性目标2的结果，我们将设计和生产定点突变体，以验证我们的假设，即负责与PDE3活性部位cGMP结合的氨基酸不同于与AMP相互作用的氨基酸。我们还将继续突变保守的氨基酸，并评估它们对酶动力学、Km(cAMP结合)和KI(cGMP结合)的影响，以及锌和锰等金属的影响。(4)由于在结构数据库中没有与其他水解酶同源的结晶学结构域。在分子水平上对PDE3A的详细描述对于理解血小板在止血中的作用以及合理设计抑制剂是重要的。

An increase of intracellular cAMP and cGMP produces potent inhibition of all platelet functions. Cyclic GMP inhibits cyclic AMP phosphodiesterase (cGI-PDE, PDE3A) and hydrolyzes cAMP, lowering its intracellular concentration. Inhibitors function as potent anti-platelet agents. Currently, the two anti-platelet agents with proven efficacy are aspirin, which inhibits cyclooxygenase-dependent synthesis of thromboxane A2 )TXA2), and clopidogrel or ticlopidine, which blocks away the ability of ADP to inhibit stimulated adenyl cyclase adenyl cyclase. Controlled trials show that both aspirin and ticlopidine are indicated in the secondary prevention of primary prevention. NO, a potent inhibitor of platelets by stimulating guanylate cyclase, elevates cGMP, which, by inhibiting PDE2A, elevates cAMP. By blocking all activating pathways in platelets when cAMP is increased intracellularly, the potential of PDE3 inhibitors is to modulate coronary artery reocclussion. PDE inhibitors have shown promise in animal models and in coronary stents in patients. The immediate goal is to identify critical amino acids in the active site of PDE. This information can be used by pharmaceutical companies to design clinically useful inhibitors with fewer side effects than the current ones. We hypothesize that there are distinct amino acids that facilitate the catalytic reaction, which hydrolyzes cAMP, that form the substrate binding site and that bind essential metal cations. We further postulate that although the inhibitor binding site for cGMP may overlap with the substrate binding site, there are distinct amino acids which interact with this nucleotide. The specific aims of this grant to test this hypothesis are as follows (1) We will synthesize new cGMP-alkylating affinity reagents (8-BDP-TcGMP, 2-BDB-TcGMP, 2-BDB-TeA-3'5'MP and cGMPS-BDB), measure their incorporation, and test their ability to various concentrations to modulate hydrolysis of cAMP and the ability of cAMP, cGMP and AMP to protect against loss of inhibition. (2) We will identify, isolate and sequence the modified peptides labeled by these new cGMP affinity agents and those labeled by the cAMP affinity analog. The function of these residues will be evaluated in the modified enzyme by kinetics, substrate and metal binding. (3) Based on the results of Specific Aim 2, we will design and produce site-directed mutants to test our hypothesis that the amino acids responsible for cGMP binding in the active site of PDE3 differ from those interact with AMP. We will also continue to mutate conserved amino acids and evaluate their effect on enzyme kinetics, Km (cAMP binding), and Ki (cGMP binding), as well as the influence of metals such as Zn++ and Mn++. (4) Since no crystallographic domain based on homologies to other hydrolases in the structural database. A detailed description of PDE3A at the molecular level is important in understanding the role of platelets in hemostasis as well as in the rational design of inhibitors.