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 [(3)H]tadalafil to PDE5 was measured. Phosphorylation increased [(3)H]tadalafil binding 3-fold, whereas cGMP caused a 1.6-fold increase. Combination of both treatments caused more than 4-fold increase in [(3)H]tadalafil binding, and effects were additive only at submaximal stimulation. Consistent with the increase in affinity, phosphorylation slowed the [(3)H]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 approximately 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.

Phosphorylation of phosphodiesterase-5 is promoted by a conformational change induced by sildenafil, vardenafil, or tadalafil.

Phosphodiesterase-5 (PDE5) inhibitors (sildenafil, vardenafil, or tadalafil) or phosphorylation by cyclic nucleotide-dependent protein kinase causes an apparent conformational change in PDE5, as indicated by a shift in migration on non-denaturing PAGE gels and an altered pattern of tryptic digestion. Combination of cGMP and a PDE5 inhibitor or phosphorylation does not cause a further gel shift or change in tryptic digest. Phosphorylation of PDE5 is stimulated by inhibitors, and combination of cGMP and inhibitor does not cause further phosphorylation. Dephosphorylation of PDE5 by either purified phosphoprotein phosphatase-1 or -2A catalytic subunit or by a crude phosphatase mixture is not affected by cGMP or inhibitors, suggesting that phosphorylation itself maintains conformational exposure of the phosphorylation site. The combined results imply that cGMP binding to the catalytic site initiates negative feedback control of many cellular cGMP signaling pathways by directly stimulating phosphorylation and activation of PDE5; by exploiting this molecular mechanism, PDE5 inhibitors stimulate their own potencies.

Radiolabeled ligand binding to the catalytic or allosteric sites of PDE5 and PDE11.

Cyclic nucleotide phosphodiesterases (PDEs) have been investigated for years as targets for therapeutic intervention in a number of pathophysiological processes. Phosphodiesterase-5 (PDE5), which is highly specific for guanosine 3'-5'-cyclic-monophosphate (cGMP) at both its catalytic site and its allosteric sites, has generated particular interest because it is potently and specifically inhibited by three drugs: sildenafil (Viagra, Pfizer), tadalafil (Cialis, Lilly-ICOS), and vardenafil (Levitra, Bayer GSK). Previously, we have used [(3)H]cGMP to directly study the interaction of cGMP with the allosteric sites of PDE5, but because cGMP binds with relatively low affinity to the catalytic site, it has been difficult to devise a binding assay for this particular binding reaction. This approach using measurement of radiolabeled ligand binding continues to allow us to more precisely define functional features of the enzyme. We now use a similar approach to study the characteristics of high-affinity [(3)H]inhibitor binding to the PDE5 catalytic domain. For these studies, we have prepared [(3)H]sildenafil and [(3)H]tadalafil, two structurally different competitive inhibitors of PDE5. The results demonstrate that radiolabeled ligands can be used as probes for both catalytic site and allosteric site functions of PDE5. We describe herein the methods that we have established for studying the binding of radiolabeled ligands to both types of sites on PDE5. These techniques have also been successfully applied to the study of binding of radiolabeled PDE5 inhibitors to PDE11, suggesting that these methods are applicable to the study of other PDEs, and perhaps other enzyme families.

Vardenafil: structural basis for higher potency over sildenafil in inhibiting cGMP-specific phosphodiesterase-5 (PDE5).

Phosphodiesterase-5 (PDE5) inhibitors act by competing with the substrate, cGMP, for the catalytic site of the enzyme. Two commercialized PDE5 inhibitors, sildenafil and vardenafil, are being used to treat erectile dysfunction. These two compounds differ in the heterocyclic ring system used to mimic the purine ring of cGMP. They also differ in the substituent (ethyl/methyl) of a piperazine side chain. Although these are the only two structural differences, vardenafil has more than 20-fold greater potency than sildenafil for inhibiting purified PDE5. The molecular structural basis for the difference in potency of the two compounds was investigated by synthesizing an analog of sildenafil ("methyl-sildenafil") that contained the sildenafil ring system but with the appended ethyl group found in vardenafil, and an analog of vardenafil ("demethyl-vardenafil") that contained the vardenafil ring system but with the appended methyl group found in sildenafil. The IC50 of methyl-sildenafil for inhibiting PDE5 indicated that it was 64 times less potent than demethyl-vardenafil, which was similar to the finding that, based on IC50, sildenafil was 40 times less potent than vardenafil. Similarly, the EC50 of methyl-sildenafil for inhibiting [3H]vardenafil binding to PDE5 indicated that it was 84 times less potent than demethyl-vardenafil, while the EC50 for sildenafil indicated that it was 31 times less potent than vardenafil. It is concluded that the methyl/ethyl appended group on the piperazine moiety plays very little role in the difference in potency between sildenafil and vardenafil for inhibiting PDE5, whereas the differences in the ring systems play a critical role in higher potency of vardenafil over sildenafil.

Sildenafil citrate does not affect cardiac contractility in human or dog heart.

OBJECTIVE: This study evaluated whether sildenafil citrate, an oral treatment for erectile dysfunction and a selective inhibitor of phosphodiesterase type 5 (PDE5) with modest vasodilating properties, affects cardiac contractility in vitro. RESEARCH DESIGN AND METHODS: Slices of freshly obtained human (n = 2) or dog (n = 3) atrial appendage were suspended in organ baths containing Krebs-Ringer bicarbonate buffer (pH 7.4, 37 degrees C) bubbled continuously with 95% O2 and 5% CO2, and isometric tension was recorded using a Gould physiograph. Contractions were elicited by 1-Hz electric pacing. After 15 min of equilibration, 1 microM sildenafil was added to the bath, followed 15 min later (human and dog) by 5 microM epinephrine, an inotropic agent, and 10 min later (dog) by 88 microM 3-isobutyl-1-methylxanthine (IBMX), a nonselective PDE inhibitor. In a separate experiment, cyclic guanosine monophosphate levels and PDE, protein kinase G, and protein kinase A activities were determined. RESULTS: Addition of 1 microM sildenafil to isolated dog or human atrial tissue had no significant effect on force of cardiac contraction, whereas epinephrine produced a robust increase in contractile force in the same muscle strip. The addition of IBMX produced a marked stimulation of contractile force in dog atrial tissue. Very low amounts of PDE5 were found in extracts of human heart, consistent with its known primary location in the smooth muscle of systemic vasculature. CONCLUSIONS: Sildenafil is unlikely to directly produce inotropic effects on cardiac muscle in patients being treated for erectile dysfunction.

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 [(3)H]sildenafil (Viagra), [(3)H]vardenafil (Levitra), or [(3)H]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 (3)H 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.

High lung PDE5: a strong basis for treating pulmonary hypertension with PDE5 inhibitors.

[3H]Vardenafil (Levitra) or [3H]tadalafil (Cialis) binding was used to quantify PDE5 in rat lung and heart tissue. Each radioligand bound to purified recombinant phosphodiesterase-5 (PDE5) or to PDE5 in crude extracts with strong affinity, high specificity, slow dissociation, and good stoichiometry. PDE5, the only 3H inhibitor-binding protein detected in extracts, was 15 times higher in lung than in heart extracts, and the level measured by PDE5 catalytic activity agreed with that determined by 3H inhibitor binding. High level of PDE5 in lung approximated that in penile corpus cavernosum, the tissue targeted by PDE5 inhibitors. PDE5 was the predominant cGMP-PDE in lung, and on a molar basis was five times higher than cGMP-dependent protein kinase (PKG), which phosphorylates PDE5 in vivo. The PDE5 level was one-half that of PKG in heart. Thus, abundance of PDE5 in lung vascular smooth muscle provides a strong molecular basis for PDE5 inhibitor treatment of pulmonary hypertension.

Binding of tritiated sildenafil, tadalafil, or vardenafil to the phosphodiesterase-5 catalytic site displays potency, specificity, heterogeneity, and cGMP stimulation.

Sildenafil, tadalafil, and vardenafil each competitively inhibit cGMP hydrolysis by phosphodiesterase-5 (PDE5), thereby fostering cGMP accumulation and relaxation of vascular smooth muscle. Biochemical potencies (affinities) of these compounds for PDE5 determined by IC(50), K(D) (isotherm), K(D) (dissociation rate), and K(D) ((1/2) EC(50)), respectively, were the following: sildenafil (3.7 +/- 1.4, 4.8 +/- 0.80, 3.7 +/- 0.29, and 11.7 +/- 0.70 nM), tadalafil (1.8 +/- 0.40, 2.4 +/- 0.60, 1.9 +/- 0.37, and 2.7 +/- 0.25 nM); and vardenafil (0.091 +/- 0.031, 0.38 +/- 0.07, 0.27 +/- 0.01, and 0.42 +/- 0.10 nM). Thus, absolute potency values were similar for each inhibitor, and relative potencies were vardenafil >> tadalafil > sildenafil. Binding of each (3)H inhibitor to PDE5 was specific as determined by effects of unlabeled compounds. (3)H Inhibitors did not bind to isolated PDE5 regulatory domain. Close correlation of EC(50) values using all three (3)H inhibitors competing against one another indicated that each occupies the same site on PDE5. Studies of sildenafil and vardenafil analogs demonstrated that higher potency of vardenafil is caused by differences in its double ring. Exchange-dissociation studies revealed two binding components for each inhibitor. Excess unlabeled inhibitor did not significantly affect (3)H inhibitor dissociation after infinite dilution, suggesting the absence of subunit-subunit cooperativity. cGMP addition increased binding affinity of [(3)H]tadalafil or [(3)H]vardenafil, an effect presumably mediated by cGMP binding to PDE5 allosteric sites, implying that either inhibitor potentiates its own binding to PDE5 in intact cells by elevating cGMP. Without inhibitor present, cGMP accumulation would stimulate cGMP degradation, but with inhibitor present, this negative feedback process would be blocked.

[3H]sildenafil binding to phosphodiesterase-5 is specific, kinetically heterogeneous, and stimulated by cGMP.

Sildenafil (Viagra) potentiates penile erection by acting as a nonhydrolyzable analog of cGMP and competing with this nucleotide for catalysis by phosphodiesterase-5 (PDE5), but the characteristics of direct binding of radiolabeled sildenafil to PDE5 have not been determined. [3H]Sildenafil binding to PDE5 was retained when filtered through nitrocellulose or glass-fiber membranes. Binding was inhibited by excess sildenafil, 2-(2-methylpyridin-4-yl)methyl-4-(3,4,5-trimethoxyphenyl)-8-(pyrimidin-2-yl)methoxy-1,2-dihydro-1-oxo-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride (T-0156), 3-isobutyl-1-methylxanthine, EDTA, or cGMP, but not by cAMP or 5'-GMP. PDE5 was the only [3H]sildenafil binding protein detected in human lung extract. Using purified recombinant PDE5, [3H]sildenafil exchange dissociation yielded two components with t1/2 values of 1 and 14 min and corresponding calculated KD values of 12 and 0.83 nM, respectively. This implied the existence of two conformers of the PDE5 catalytic site. [3H]Sildenafil binding isotherm of PDE5 indicated KD was 8.3 to 13.3 nM, and low cGMP decreased the KD to 4.8 nM but only slightly increased Bmax to a maximum of 0.61 mol/mol-subunit. Results suggest that these effects occur via cGMP binding to the allosteric cGMP binding sites of PDE5. Results imply that by inhibiting PDE5 and thereby increasing cGMP, sildenafil accentuates its own binding affinity for PDE5, which further elevates cGMP. The data also indicate that after physiological elevation, cGMP may directly stimulate the catalytic site by binding to the allosteric cGMP-binding sites of PDE5, thus causing negative feedback on this pathway.