Conversion of Phosphodiesterase-5 (PDE5) Catalytic Site to Higher Affinity by PDE5 Inhibitors

Abstract

Phosphodiesterase-5 (PDE5) specifically hydrolyzes cGMP, thereby contributing to modulation of intracellular levels of this nucleotide. In the present study, preincubation with cGMP increased PDE5 catalytic activity for cGMP degradation, and it converted the PDE5 catalytic site to a form that was more potently inhibited by each of the three PDE5 catalytic sitespecific inhibitors: sildenafil, vardenafil, and tadalafil. These results implied that elevated cGMP initiates a physiological negative feedback on the cGMP pathway by increasing the affinity of the PDE5 catalytic site for cGMP. This increase in catalytic site activity or affinity for inhibitors could be caused by binding of cGMP to either the PDE5 allosteric sites, catalytic site, or both. Whether occupation of the catalytic site alone could mediate the effect was examined using radiolabeled PDE5 inhibitors in the absence of cGMP. Exchange-dissociation H inhibitors converted the biphasic pattern to a single slow (highaffinity) component. Studies of amino-terminally truncated PDE5 established that full-length mammalian GAF-B (cGMP-binding phosphodiesterase, Anabaena adenylyl cyclases, Escherichia coli FhlA) subdomain conjoined with the catalytic domain was sufficient for this conversion. In conclusion, binding of substrate or substrate analogs such as PDE5 inhibitors to the catalytic site converts a fast (low-affinity) inhibitor dissociation component of the PDE5 catalytic site to a slow (high-affinity) inhibitor dissociation component. This effect is predicted to improve the substrate affinity or inhibitory potencies of these compounds in intact cells.

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

“…This method was also applied for two other PDE5 inhibitors (vardenafil and tadalafil). The ratio between fast and slow components varied between PDE5 inhibitors and could be changed by long preincubation with PDE5 inhibitors (Blount et al, 2007). For example, 12-h preincubation with 30 nM tadalafil resulted in detection of only the slow (high-affinity) component for full-length PDE5 and for a construct containing GAF-B and the catalytic domain but not for the catalytic domain alone.…”

“…However, it is not clear why PDE5 retains sildenafil for 12 to 24 h after taking one dose of sildenafil. Studies of sildenafil dissociation patterns showed that only long preincubation with high concentrations of PDE5 inhibitors could substantially slow down dissociation (Blount et al, 2007). In addition, sildenafil as a moderately lipophilic compound is expected to achieve equilibrium between its intracellular and extracellular/plasma concentrations relatively fast.…”

Molecular mechanisms that could contribute to prolonged effectiveness of PDE5 inhibitors to improve erectile function

“…The basal affinity of PDE5 for the inhibitors, which is intrinsically high (K D ¼ 0.1-4 nM), can increase further in response to elevation of cGMP as would occur with sexual stimulation 12 and/or prolonged exposure to the respective drugs. 54 As discussed below, this increased affinity is caused by allosteric binding of cGMP to PDE5, increased phosphorylation of the enzyme and prolonged perturbation of the catalytic site of the enzyme by elevated cGMP and inhibitor. 9,11,12,55 Therefore, a few hours after ingestion of a PDE5 inhibitor either in the presence or absence of sexual arousal, plasma inhibitor concentration would be high and it is predicted that the catalytic site of PDE5 in VSMC would be saturated with inhibitor and that the affinity of PDE5 for the inhibitor would be significantly higher than in the basal state (Figure 4b).…”

Phosphodiesterase Type 5

“…An important correlate is that when a PDE5 inhibitor is present, these same mechanisms result in enhanced binding affinity of the catalytic site to the inhibitor, perhaps enhancing and prolonging its efficacy. 48 It is presumed this also provides a molecular mechanism for rapid cGMP oscillations to occur in response to rapid changes in NO or ANP, though it remains unclear how this could operate given the very high affinities measured for cGMP binding in vitro. It seems likely that another layer of regulation of cGMP binding affinity exists but has yet to be elucidated.…”