Multiple Affinity States of cGMP-Specific Phosphodiesterase for Sildenafil Inhibition Defined by cGMP-Dependent and cGMP-Independent Mechanisms

Abstract

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.

Phosphodiesterase Type 5 Inhibitors Synergize Vincristine in Killing Castration-Resistant Prostate Cancer Through Amplifying Mitotic Arrest Signaling

“…The data questioned the functional role of cGMP. Because it is reported that sildenafil sensitivity of PDE5 can be regulated by cGMP-independent mechanisms ( 32 ), the role of PDE5 and its dependence on cGMP needs further elucidation.…”

Activation of PDE2 and PDE5 by specific GAF ligands: delayed activation of PDE5

“…A recent study described a new ‘super‐high’ sensitivity state for sildenafil inhibition (Rybalkina et al ., 2010) raising the question whether the different sensitivity states are related to the two conformations described above. As the two sensitivity states were observed only in the non‐stimulated enzyme whereas the two conformations described in our study were observed at non‐stimulating (0.03 µM cGMP) and stimulating cGMP concentrations (100 µM), the sildenafil sensitivity states appear to be distinct from the two activity states.…”

CO metabolism, sensing, and signaling

“…Remarkably, CO and exogenous compounds such as 3-(5 0 -hydroxymethyl-2 0 -furyl)-1-benzylindazole (YC-1) and 3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyradazolo [3,4-b]pyridine (BAY 41-2272) synergistically activate sGC to levels approximately similar to those induced by NO [88,89]. Such sGC induction leads to the increase of the cGMP level and to the activation of the cGMP-dependent protein kinase (PKG) phospho-transferase activity, thus affecting several cellular functions based on ion channel, phosphodiesterase, and protein kinase actions [77,80,[90][91][92][93][94][95].…”