Emmanuel P. Bessay

A 46-Amino Acid Segment in Phosphodiesterase-5 GAF-B Domain Provides for High Vardenafil Potency over Sildenafil and Tadalafil and Is Involved in Phosphodiesterase-5 Dimerization

Phosphodiesterase-5 (PDE5) contains a catalytic domain (C domain) that hydrolyzes cGMP and a regulatory domain (R domain) that contains two mammalian cGMP-binding phosphodiesterase, Anabaena adenylyl cyclases, Escherichia coli FhlAs (GAFs) (A and B) and a phosphorylation site for cyclic nucleotide-dependent protein kinases (cNPKs). Binding of cGMP to GAF-A increases cNPK phosphorylation of PDE5 and improves catalytic site affinity for cGMP or inhibitors. GAF-B contributes to dimerization of PDE5, inhibition of cGMP binding to GAF-A, and sequestration of the phosphorylation site. To probe potential PDE5 R domain effects on catalytic site affinity for certain inhibitors, four N-terminal truncation mutants were generated: PDE5Δ1-321 contained GAF-B domain, C domain, and the sequence between GAF-A and -B; PDE5Δ1-419 contained GAF-B and C domain; PDE5Δ1-465 contained the C domain and the C-terminal portion of GAF-B; and PDE5Δ1-534 contained only C domain. Truncated proteins with a complete GAF-B were dimers, but those lacking the N-terminal 46 amino acids of GAF-B were monomers, indicating that these residues are vital for GAF-B-mediated PDE5 dimerization. Km values of the mutants for cGMP were similar to that of full-length PDE5. All PDE5 constructs had similar affinities for 3-isobutyl-1-methylxanthine, sildenafil, tadalafil, and UK-122764, but mutants containing a complete GAF-B had 7- to 18-fold higher affinity for vardenafil-based compounds compared with those lacking a complete GAF-B. This indicated that the N-terminal 46 amino acids in GAF-B are required for high vardenafil potency. This is the first evidence that PDE5 R domain, and GAF-B in particular, influences affinity and selectivity of the catalytic site for certain classes of inhibitors.

Phosphorylation Increases Affinity of the Phosphodiesterase-5 Catalytic Site for Tadalafil

Phosphodiesterase-5 (PDE5) is phosphorylated at a single serine residue by cyclic nucleotide-dependent protein kinases. To test for a direct effect of phosphorylation on the PDE5 catalytic site, independent of cGMP binding to the allosteric sites of the enzyme, binding of the catalytic site-specific substrate analog [3H]tadalafil to PDE5 was measured. Phosphorylation increased [3H]tadalafil binding 3-fold, whereas cGMP caused a 1.6-fold increase. Combination of both treatments caused more than 4-fold increase in [3H]tadalafil binding, and effects were additive only at submaximal stimulation. Consistent with the increase in affinity, phosphorylation slowed the [3H]tadalafil exchange-dissociation rate from PDE5 more than 6-fold. Finally, phosphorylation increased affinity for hydrolysis of a catalytic site-specific cGMP analog, 2′-O-anthraniloyl-cGMP, by ∼3-fold. The combined results showed that phosphorylation activates PDE5 catalytic site independently of cGMP binding to the allosteric sites. The results suggested that phosphorylation acts in concert with allosteric cGMP binding to stimulate the PDE5 catalytic site, which should promote negative feedback regulation of the cGMP pathway in intact cells. By increasing the affinity of the catalytic site, phosphorylation should also consequently increase the potency and duration of PDE5 inhibitor action.

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

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 site-specific 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 of [3H]sildenafil (Viagra), [3H]vardenafil (Levitra), or [3H]tadalafil (Cialis) from full-length PDE5 or isolated catalytic domain revealed two kinetic components (slow and fast). Extended preincubation of full-length PDE5, but not isolated catalytic domain, with 3H inhibitors converted the biphasic pattern to a single slow (high-affinity) 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.

Metal ion stimulators of PDE5 cause similar conformational changes in the enzyme as does cGMP or sildenafil

Purified PDE5 preparations exhibited variable proportions of two mobility forms (Bands 2 and 3) by native PAGE. Treatment of recombinant or native PDE5 with either cGMP or a substrate analog such as sildenafil, each of which is known to produce stimulatory effects on enzyme functions, caused a similar native PAGE band-shift to the lower mobility form (shift of Band 2 to Band 3). Incubation of PDE5 with Mg(++) or Mn(++), which is known to stimulate activity, caused a similar shift of the enzyme from Band 2 to Band 3 as did cGMP or sildenafil, but incubation with EDTA caused a time- and concentration-dependent shift to higher mobility (shift of Bands 2 and 3 to Band 1). A slow time course of the EDTA-induced band-shift suggested removal of a pre-bound metal ion (Me(++)) with affinity of ~0.1 nM, which was similar to the previously determined affinity of PDE5 for Zn(++). The EDTA-treated enzyme (Band 1) could be shifted to Bands 2 and 3 by addition of cGMP, sildenafil, or Me(++); however, the cGMP- or sildenafil-induced shift was inhibited and the Me(++)-induced shift was facilitated by treatment with EDTA. Results suggested that Me(++) removal from PDE5 produces a unique apoenzyme form (Band 1, more globular, negatively charged, or both) of PDE5 that can be partially converted to forms (Band 2, less globular or negatively charged, or both; and Band 3, more elongated/positively charged, or both) by addition of Me(++), substrate, or substrate analog. It is concluded that Me(++) causes conversion of PDE5 to similar conformational forms as caused by substrate or inhibitor binding to the catalytic site.

Negative feedback control of the nitric oxide/cGMP pathway in smooth muscle

Two major intracellular receptors for cGMP in smooth muscle are cGMP-dependent protein kinase (PKG) and phosphodiesterase-5 (PDE5), the latter of which contains both hydrolytic and allosteric sites for cGMP. We have shown that either one or both of these enzymes are involved in at least eight events leading to negative feedback control after nitric oxide-induced cGMP elevation: 1) increased hydrolysis of cGMP due to substrate elevation; 2) activation of PKG, which phosphorylates and activates PDE5; 3) enhanced phosphorylation of PDE5 by PKG due to exposure of the PDE5 phosphorylation site by elevated PDE5 hydrolytic site occupancy; 4) enhanced phosphorylation of PDE5 by PKG due to exposure of the PDE5 phosphorylation site by elevated PDE5 allosteric site occupancy; 5) increased sequestration of cGMP by PDE5 allosteric sites; 6) stimulation of PDE5 allosteric site occupancy by increased hydrolytic site occupancy; 7) stimulation of PDE5 allosteric site occupancy by increased phosphorylation of PDE5; and 8) conversion of PDE5 to a more active conformer by increased hydrolytic site occupancy. In concert, these events are predicted to result in increased cGMP breakdown and sequestration, producing negative feedback after cGMP elevation. The events could be explained by the presence of two interconvertible forms of PDE5: a less active conformer and a more active conformer that has increased cGMP hydrolytic activity, cGMP allosteric binding, and phosphorylation. By native PAGE (no SDS), cGMP or phosphorylation causes a gel shift consistent with a conformational change, and hydrolytic site-specific inhibitors such as sildenafil, vardenafil, and tadalafil cause a similar shift. Combined studies of xray crystal structures and site-directed mutagenesis identify three key amino acids (human Tyr-612, Gln-817, and Phe-820) in the PDE5 catalytic site that contribute importantly to high affinity of cGMP as well as potency and selectivity for PDE5 inhibitors. Binding of each of these ligands can initiate several of the cGMP feedback processes mediated by PDE5.