Identification of Amino Acid Residues Responsible for the Selectivity of Tadalafil Binding to Two Closely Related Phosphodiesterases, PDE5 and PDE6
Identification of Amino Acid Residues Responsible for the Selectivity of Tadalafil Binding to Two Closely Related Phosphodiesterases, PDE5 and PDE6
复制标题
DOI：
10.1074/jbc.m112.389189
复制
发表时间：
2012-11-30
影响因子：
4.8
通讯作者：
Cote, Rick H.
中科院分区：
文献类型：
--
作者：
Cahill, Karyn B.;Quade, Jonathan H.;Cote, Rick H.
I类环核苷酸磷酸二酯酶（PDE）的11个家族对于环核苷酸信号传导的调节至关重要。PDE5（在调节血管平滑肌收缩中很重要）和PDE6（负责调节脊椎动物光感受器中的视觉转导）在结构上相似，但有几个功能差异，其结构基础知之甚少。使用进化痕迹分析和结构同源性建模结合定点诱变，我们已经测试了这一假设，类特定的差异PDE5和PDE6占两个酶家族的生化和药理学差异。将PDE5选择性抑制剂他达拉非（Cialis（R））结合位点的M环区域中的人PDE5残基替换为相应的类别特异性锥型PDE6残基（P773 E、I778 V、E780 L、F787 W、E796 V、D803 P、L804 M、N806 D、I813 L、S815 K），可将他达拉非结合亲和力降低至PDE6的特征水平。这些突变不能改变伐地那非（Levitra（R））对活性位点的亲和力。类特异性差异的PDE5与锥PDE6，有助于加速催化效率的PDE6被确定，但需要异源表达全长PDE5构建体。将PDE6残基引入到PDE5蛋白序列的背景中通常导致催化活性的丧失和蛋白溶解度的降低，这支持了PDE6的多个结构元件在异源表达期间极易发生错误折叠的观点。这项工作验证了使用PDE5作为模板，以确定PDE5和PDE6之间的功能差异，这将加速开发下一代PDE5选择性抑制剂的努力，减少PDE6抑制引起的不良副作用。
The 11 families of the Class I cyclic nucleotide phosphodiesterases (PDEs) are critical for regulation of cyclic nucleotide signaling. PDE5 (important in regulating vascular smooth muscle contraction) and PDE6 (responsible for regulating visual transduction in vertebrate photoreceptors) are structurally similar but have several functional differences whose structural basis is poorly understood. Using evolutionary trace analysis and structural homology modeling in conjunction with site-directed mutagenesis, we have tested the hypothesis that class-specific differences between PDE5 and PDE6 account for the biochemical and pharmacological differences in the two enzyme families. Replacing human PDE5 residues in the M-loop region of the binding site for the PDE5-selective inhibitor tadalafil (Cialis (R)) with the corresponding class-specific cone PDE6 residues (P773E, I778V, E780L, F787W, E796V, D803P, L804M, N806D, I813L, S815K) reduces tadalafil binding affinity to levels characteristic of PDE6. These mutations fail to alter vardenafil (Levitra (R)) affinity for the active site. Class-specific differences in PDE5 versus cone PDE6 that contribute to the accelerated catalytic efficiency of PDE6 were identified but required heterologous expression of full-length PDE5 constructs. Introduction of PDE6 residues into the background of the PDE5 protein sequence often led to loss of catalytic activity and reduced protein solubility, supporting the idea that multiple structural elements of PDE6 are highly susceptible to misfolding during heterologous expression. This work validates the use of PDE5 as a template to identify functional differences between PDE5 and PDE6 that will accelerate efforts to develop the next generation of PDE5-selective inhibitors with fewer adverse side effects resulting from PDE6 inhibition.