Department of Pharmacology, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195-7280, USA.
Mol Pharmacol. 2010 Apr;77(4):670-7. doi: 10.1124/mol.109.062299. Epub 2010 Jan 19.
cGMP-specific phosphodiesterase (PDE5) has become a target for drug development for the treatment of a number of physiological dysfunctions, affected by changes in the cGMP/cGMP-dependent protein kinase (PKG) signaling pathway. PDE5 has two highly homologous regulatory domains, GAF-A and GAF-B. We showed previously that PDE5 could be converted from a low-activity (nonactivated) state to a high-activity state upon cGMP binding to the GAF-A domain with higher sensitivities toward sildenafil (EMBO J 22:469-478, 2003). Here we investigated whether sildenafil sensitivity of PDE5 could be modified by cGMP-independent mechanisms. Individually expressed recombinant GAF-A and GAF-B proteins were tested for their ability to modulate full-length recombinant PDE5 affinity to sildenafil. The GAF-A domain protein had the most dramatic effect on the affinity of the nonactivated recombinant PDE5 for sildenafil, revealing much higher sensitivity to sildenafil inhibition. The apparent affinity for sildenafil increased from the nanomolar range to the picomolar range, providing evidence for the presence of a "super-high" sensitivity state of PDE5 for sildenafil inhibition. In human platelet, higher sensitivity of PDE5 for sildenafil inhibition has been detected after blocking cGMP-binding sites of the GAF-A domain. Thus, our data demonstrate that high sensitivity of PDE5 for sildenafil can be obtained not only through cGMP-induced activation of PDE, but also through cGMP-independent modulation of PDE5 in the nonactivated state, possibly through protein-protein interaction. Furthermore, data suggest that nonactivated PDE5 with "super-high" affinities for sildenafil inhibition may be responsible for therapeutic effects of long-term treatments with low doses of PDE5 inhibitors.
环鸟苷酸（cGMP）特异性磷酸二酯酶（PDE5）已成为治疗多种生理功能障碍的药物开发靶点，这些功能障碍受 cGMP/cGMP 依赖性蛋白激酶（PKG）信号通路变化的影响。PDE5 有两个高度同源的调节结构域，GAF-A 和 GAF-B。我们之前曾表明，当 cGMP 结合到 GAF-A 结构域时，PDE5 可以从低活性（非激活）状态转变为高活性状态，对西地那非的敏感性更高（EMBO J 22:469-478, 2003）。在这里，我们研究了 cGMP 非依赖性机制是否可以改变 PDE5 对西地那非的敏感性。单独表达的重组 GAF-A 和 GAF-B 蛋白被测试其调节全长重组 PDE5 对西地那非亲和力的能力。GAF-A 结构域蛋白对非激活型重组 PDE5 对西地那非亲和力的影响最为显著，表明对西地那非抑制的敏感性更高。对西地那非的表观亲和力从纳摩尔范围增加到皮摩尔范围，为 PDE5 对西地那非抑制存在“超高”敏感性状态提供了证据。在人血小板中，阻断 GAF-A 结构域的 cGMP 结合位点后，检测到 PDE5 对西地那非抑制的敏感性更高。因此，我们的数据表明，PDE5 对西地那非的高敏感性不仅可以通过 cGMP 诱导的 PDE 激活获得，还可以通过非激活状态下 cGMP 非依赖性调节 PDE5 获得，可能通过蛋白质-蛋白质相互作用。此外，数据表明，对西地那非抑制具有“超高”亲和力的非激活型 PDE5 可能是长期低剂量 PDE5 抑制剂治疗的治疗效果的原因。
