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Tyrosine-612 in PDE5 contributes to higher affinity for vardenafil over sildenafil

A Corrigendum to this article was published on 04 July 2006


Despite close structural similarity, vardenafil (Levitra®) is 32-fold more potent than sildenafil (Viagra®) to inhibit cGMP-binding cGMP-specific PDE (PDE5); this is due to differences between their heterocyclic rings. In co-crystals with PDE5, one of the rings of vardenafil or sildenafil interacts with Tyr612, a catalytic site AA, via (1) a hydrogen bond with a water molecule and (2) hydrophobic bonds. For mutant PDE5Y612F, which ablates hydrogen-bonding potential, vardenafil or sildenafil inhibition was strengthened (2.2- or 3.0-fold, respectively), implying that the Tyr612 hydroxyl is a negative determinant for these inhibitors. For mutant PDE5Y612A, which ablates both hydrogen bonding and hydrophobic-bonding potential, vardenafil inhibition was weakened much more than sildenafil inhibition (122- and 26-fold, respectively), suggesting that hydrophobic bonds involving Tyr612 are stronger for vardenafil than for sildenafil.


cGMP-binding cGMP-specific phosphodiesterase (PDE5) is an important component of the cGMP-hydrolyzing activity in many tissues, including the smooth muscle of penile arteries and corpus cavernosum. PDE5 is the target of sildenafil (Viagra™), vardenafil (Levitra™), and tadalafil (Cialis™), which are used for treatment of erectile dysfunction and other maladies associated with vascular disease.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 These inhibitors directly compete with the cGMP substrate for the PDE5 catalytic site, are highly potent, and share some structural features of cGMP and many nonspecific, low-potency PDE inhibitors such as 3-isobutyl-1-methylxanthine (IBMX) (Figure 1).4, 9, 15, 16, 17 All of these compounds have a heterocyclic core ring structure that is likely to be essential for competition with cGMP, which contains a guanine heterocyclic ring. Biochemical potency (IC50) of the inhibitors to block PDE5 catalytic activity is also powerfully impacted by unique structural features in each that account for the difference in IC50 for weak inhibitors, for example, IBMX, and potent inhibitors such as sildenafil, vardenafil, and tadalafil.

Figure 1

Molecular structures of sildenafil and vardenafil compared with those for IBMX and cGMP. Circled components denote differences in nitrogen and carbon distribution in the pyrazolopyrimidinone and imidazotrizinone rings of sildenafil and vardenafil, respectively, and in the methyl versus ethyl groups appended to the piperazine moiety in the two inhibitors.

The molecular basis for the potency difference between vardenafil and sildenafil is enigmatic since X-ray crystallographic structures of PDE5 isolated catalytic domain in complex with each did not reveal any difference in binding mode.18 However, contribution of contacts to inhibitor potency cannot be fully quantified from crystal structures. The atomic structures of sildenafil and vardenafil share structural similarities that include a heterocyclic pyrazolopyrimidinone and imidazotriazinone ring system, respectively (Figure 1), which differ in (1) presence of either a carbon or nitrogen at two positions (circled in Figure 1) in the five-member pyrazole/imidazole ring and (2) a substituent (methyl or ethyl) of the piperazine moiety (circled in Figure 1). Differences in the five-member rings could also alter electron distribution in the heterocyclic ring and affect the electronegativity of the nitrogen that is common to the five-member rings (Figure 1).

Tyr612, a conserved AA in the PDE5 catalytic site, interacts with the five-member ring in either sildenafil or vardenafil (Figure 2) via (1) a hydrogen bond of the nitrogen (indicated as position 8 in Figure 2) with a water that, in turn, forms hydrogen bonds with both the hydroxyl of Tyr612 and a second water that is coordinated to the catalytic site Zn2+, and (2) hydrophobic interactions with the aromatic side chain of Tyr612.18 We previously demonstrated that substitution of alanine for Tyr612 (PDE5Y612A) causes a dramatic decrease in affinity of PDE5 for the substrate, cGMP.19 Affinity of PDE5Y612A for either zaprinast, UK-122764, or sildenafil is also decreased; compared to PDE5WT which has IC50 values of 100–300 nM, 20 nM, and 4 nM, respectively, for the inhibitors, IC50 values of PDEY612A for these inhibitors are decreased 7-, 21-, and 25-fold, respectively.20 Therefore, we hypothesize that difference in interactions of sildenafil and vardenafil with Tyr612 through either hydrophobic contacts, hydrogen bonding, or both types of contacts accounts for a portion of the difference in potencies. Contributions of these interactions to the potencies of sildenafil and vardenafil have been investigated using site-directed mutagenesis of human PDE5A1 to replace Tyr612 with either phenylalanine (PDE5Y612F) or alanine (PDE5Y612A). If the electronegativity of the nitrogen indicated as position 8 in sildenafil and vardenafil structures (Figure 2) differs, then potency of hydrogen-bonding interactions involving the water bridge that connect this atom to Tyr612 may also differ. Such a finding could also suggest different electron distribution in the five-member rings of the inhibitors; this might also contribute to different potency of interactions with Tyr612. Therefore, electrochemical characteristics (pKa) of this nitrogen in the respective pyrazole and imidazole rings in the inhibitors have been experimentally determined. His-tagged PDE5 proteins were expressed in Sf9 cells, purified, and characterized for allosteric cGMP-binding properties, kinetics of catalytic activity (Km and Vmax), and inhibitor potencies (IC50). Results of these studies provide insight into the mechanism of discrimination between vardenafil and sildenafil binding to the PDE5 catalytic site.

Figure 2

Schematic depiction of interactions of Tyr612 with the pyrazole or imidazole moiety of sildenafil or vardenafil. Dotted lines depict the chain of hydrogen bonds between the nitrogen common to both five-member rings (pyrazole/imidazole moieties) of the heterocyclic ring with a water that is also hydrogen-bonded to the phenolic hydroxyl of Tyr612 and another water that forms a water bridge to the catalytic site Zn2+. The black arc indicates hydrophobic interaction between the aromatic side chain of Tyr612 and the pyrazole/imidazole ring of the respective inhibitors. Positions in the heterocyclic rings of each inhibitor are numbered 1–9 in keeping with the numbers of these positions in the guanine ring of cGMP. Experimentally determined pKa values for the nitrogen in the five-member ring that is common to both sildenafil and vardenafil are noted.

Materials and methods


[3H]cGMP was purchased from Amersham Biosciences Inc. (Piscataway, NJ, USA). IBMX, Crotalus atrox snake venom, cGMP, and histone type II-AS were from Sigma Chemical Co. (St Louis, MO, USA). Sildenafil was purified as described earlier.21

Generation of wild-type and mutant hPDE5A1

Human cDNA coding for full-length PDE5A1 (courtesy of Dr K Omori, Tanabe-Seiyaku Pharmaceutical Co. Ltd., Saitama, Japan) was used as template to generate full-length PDE5 by introduction of start and stop codons at appropriate loci. The resulting PCR fragment was cloned into pCR 2.1-TOPO® vector (Invitrogen), verified by sequencing, and ligated into the EcoRI and NotI unique sites of baculovirus expression vector pAcHLT-A (Pharmingen), which contains a His6 sequence preceding the coding region. This step resulted in plasmid pAcA-PDE5 (M1–N875), which generated N-terminally His-tagged recombinant hPDE5A1. The QuikChange site-directed mutagenesis kit (Stratagene) was used to make point mutations (Y612 to F and Y612 to A) in the pAcA-PDE5 expression vector according to the manufacturer's protocol using the following pairs of mutagenic oligonucleotides (altered bases underlined): (1) for Y612F, 5′-IndexTermGAAGAATGTTGCCTTCCATAATTGGAGACATG-3′ and 5′-IndexTermCATGTCTCCAATTATGGAAGGCAACATTCTTC-3′; (2) for Y612A, 5′-IndexTermGAAGAATGTTGCCGCTCATAATTGGAGAC-3′ and 5′-IndexTermGTCTCCAATTATGAGCGGCAACATTCTTC-3′. The presence of the desired mutation was verified by sequencing the entire DNA segment.

Expression of hPDE5A1 proteins

Sf9 cells (BD Pharmingen) were cotransfected with BaculoGold linear baculovirus DNA (BD Pharmingen) and one of the hPDE5A1 constructs (PDE5WT (M1–N875), PDE5Y612F (M1–N875)Y612F, and PDE5Y612A (M1–N875)Y612A) in the pAcHLT-A baculovirus expression vector by the calcium phosphate method according to the protocol from BD Pharmingen. At 5 days postinfection, the cotransfection supernatant was collected, amplified three times in Sf9 cells, and then used directly as virus stock for expression without additional purification of recombinant viruses. Sf9 cells grown at 27°C in complete Grace's insect medium with 10% fetal bovine serum and 10 μg/ml gentamicin (Sigma) in T-175 flasks (Corning) were infected with 100 μl viral stock/flask and harvested 92 h postinfection.

Purification of hPDE5A1 proteins

All purification steps were carried out at 4°C. Cell pellet from each T-175 flask (2 × 107 cells) was suspended in 3 ml lysis buffer (20 mM Tris-HCl, pH 8, 100 mM NaCl) containing protease inhibitors as recommended (Complete™; Roche Molecular Biochemicals) and homogenized in 10- to 20-ml aliquots by 2 × 6-s bursts in an Ultra Turrex microhomogenizer (Tekmar) with 20-s recovery between bursts. Homogenate was centrifuged (20 min, 10 000 r.p.m. in a Beckman JA-20 rotor). Supernatant was applied to a Ni-NTA agarose (Qiagen) column (1 × 2 cm) equilibrated with lysis buffer. Column was washed with 100 ml lysis buffer followed by sequential washes with lysis buffer containing a stepwise gradient of imidazole (0.8–20 mM). Lysis buffer containing 100 mM imidazole was soaked into the resin, and column was plugged for 2 h before collection of ten 1-ml elutions. Elutions containing PDE5 were pooled, dialyzed versus 2000 volumes of 10 mM potassium phosphate, pH 6.8, 25 mM β-mercaptoethanol (KPM) to remove imidazole, flash-frozen in KPM containing 0.15 M NaCl and 10% sucrose, and stored at −70°C. Enzyme activity in frozen samples was stable over 10 months.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Purity and integrity of proteins were assessed using SDS-PAGE. Protein samples containing 10% SDS, 2 M β-mercaptoethanol, and 0.1% bromphenol blue were boiled for 4 min and subjected to 12% SDS-PAGE before visualizing by Coomassie Brilliant Blue staining.

cGMP binding

To measure cGMP binding, Millipore filter binding assays were conducted in a total volume of 50 μl containing 10 mM potassium phosphate buffer, pH 6.8, 1 mM EDTA, 0.5 mg/ml histone type II-AS, 30 mM DL-dithiothreitol, 0.2 mM sildenafil, and either 3 μ M (stoichoimetry determination) or 0.05–4 μ M (binding-affinity determination) [3H]cGMP. Addition of enzyme initiated the reaction. Following 60 min incubation (4°C), 1 ml cold KP buffer (10 mM potassium phosphate, pH 6.8) was added; samples were filtered immediately onto premoistened Millipore HAWP filters (pore size 0.45 μm). Filters were then washed twice with 2 ml cold KP buffer, dried, and counted. Bound protein counts were corrected by subtraction of nonspecific binding (+1 mM unlabeled cGMP). Blanks lacking protein were run for each [3H]cGMP concentration.

Catalytic activity of hPDE5A1 proteins

PDE activity was determined using a modification of the procedure described previously;22 reaction mixture contained 50 mM Tris/HCl, pH 7.5, 10 mM MgCl2, 0.3 mg/ml bovine serum albumin, 0.5 μ M cGMP, [3H]cGMP (100 000 c.p.m./tube), and one of the PDE5 proteins in a total volume of 100 μl. Incubation time was 10 min at 30°C. Less than 10% of [3H]cGMP was hydrolyzed during the reaction. Apparent Km and Vmax values were determined by nonlinear analysis of data using the one-site binding model (hyperbola) in Prism Graphpad Software.

Biochemical potency (IC50) for sildenafil or vardenafil inhibition of PDE5 catalytic activity was determined using a range of inhibitor concentrations (1–1 000 000 pM). IC50 values were determined using the nonlinear regression analysis of the sigmoidal dose response Prism Graphpad Software (variable slope). All values represent three measurements, each in triplicate. Gibbs free energy change (ΔG) of binding upon formation of enzyme–inhibitor complex was calculated from the IC50 of each inhibitor for the respective proteins using Equation (1):

Contribution of a substituted AA side chain to the free energy of binding in the enzyme–inhibitor complex was calculated from differences in the ΔG values for PDE5WT and PDE5mutant using Equation (2):

ΔΔG is the change in free energy of binding in enzyme–inhibitor complexes attributable to the substituted group,23, 24 R is the ideal gas constant (1.98 × 10−3 kcal/degree/mol), and T is the temperature at which the assay was done (303°K).

Attempts were made to confirm IC50 values by measuring direct binding affinity (KD) of [3H]sildenafil and [3H]vardenafil to PDE5. However, because of poor binding affinity of PDE5Y612A, exceptionally high concentrations of the tritiated PDE5 inhibitors were required for determination of KD. At concentrations of PDE5 inhibitor of >20 nM, the blank was excessive and the low signal to noise ratio precluded determination of binding affinity.

pKa measurements

Measurement of the pKa of the respective nitrogens at the position indicated number 8 in sildenafil and vardenafil (Figure 2) was performed on an SGA-profiler (SIRIUS UK: analyzing software: Profiler SGA 3.0) in two replicates by titrating the compounds from acidic to basic buffer and from basic to acidic buffer and following UV changes.


Experimental determination of pKa for the nitrogen that is common to both five-member rings in sildenafil and vardenafil

Measurements of pKa for the nitrogen common to both the pyrazole of sildenafil and the imidazole of vardenafil were performed as described in Materials and methods. The mean of four measurements was calculated. For vardenafil, two pKa values were found; the pKa of the nitrogen located at position 8 (Figure 2) was 3.6. For sildenafil, only the pKa for the nitrogen at position 1 was detected. A pKa for the nitrogen located at position 8 in the pyrazole ring of sildenafil was not observed. As the measurements could not be performed below pH 2, the pKa of this nitrogen in sildenafil was inferred to be <2.

Expression and purification of hPDE5 proteins

Recombinant His-tagged wild-type human PDE5A (PDE5WT) and two mutants (PDE5Y612F and PDE5Y612A) were expressed, purified to near homogeneity (>98%), and detected on SDS-PAGE (12% (w/v) gel) as described in Materials and methods (Figure 3). Each migrated as a 99-kDa band that correlated well with the predicted molecular weight based on AA composition of human PDE5. Identities of the recombinant proteins were verified by Western blot analysis using a polyclonal PDE5-specific antibody (not shown). Protein yields were 2.7 mg/l infected cell media.

Figure 3

SDS-PAGE of purified human PDE5A1 wild-type and mutant proteins. In all, 10 μg of wild-type or mutant human PDE5A1 proteins that had been expressed in Sf9 cells and purified to apparent homogeneity through the Ni-NTA chromatography step was applied to each lane. MW is the molecular mass of standards. Proteins were visualized by staining the gel with Coomassie Brilliant Blue dye.

Structural integrity of PDE5 mutants

Overall structural integrity of the PDE5 proteins was assessed using the Millipore filtration assay to measure the stoichiometry and affinity of [3H]cGMP binding to the allosteric cGMP-binding sites in the regulatory domain of each PDE5WT, PDE5Y612F, and PDE5Y612A bound cGMP with comparable affinities (KD=190–210 nM) and stoichiometry (0.50–0.55 mol bound [3H]cGMP/PDE5 monomer) (data not shown). These values compared well to those previously reported for native and non-His-tagged recombinant bovine PDE5,25, 26 indicating that overall structures of the mutant proteins were preserved, and that differences in kinetic parameters were not due to nonspecific conformational effects.

Kinetic analysis of catalytic activity of PDE5 proteins

Specific enzyme activity of each purified PDE5 construct was measured as described under Materials and methods. Km and Vmax for cGMP were determined by nonlinear regression analysis of data (Prism Graphpad Software) (Table 1). PDE5WT had Km of 2.9±0.8 μ M and Vmax of 1.0±0.2 μmol/min/mg for cGMP. To examine components of the interaction of Tyr612 with substrate and inhibitors, this residue was replaced by either phenylalanine (PDE5Y612F) or alanine (PDE5Y612A). PDE5Y612F showed a slight change in affinity for cGMP (Km=6.1±0.8 μ M) (Table 1), and no change in Vmax (1.0±0.2 μmol/min/mg). PDE5Y612A exhibited profound loss in affinity for cGMP as substrate, with 15-fold increase in Km (42±2.1 μ M) (Table 1), whereas specific enzyme activity (Vmax=0.50±0.03 μmol/min/mg) was slightly lower than that of PDE5WT. These results agreed with our previous finding that substitution of alanine for Tyr612 mainly affects substrate-binding affinity, not maximum catalysis, and suggested that the main interaction between cGMP and Tyr612 involves hydrophobic contacts.

Table 1 Effect of PDE5 Tyr612 mutations on affinity for cGMP as substrate, as well as on potency and selectivity of inhibition by sildenafil and vardenafil

IC50 of sildenafil and vardenafil for PDE5 proteins

IC50 values (Figure 4, Table 1) were determined as described in Materials and methods using 0.5 μ M [3H]cGMP, which is a significantly lower concentration than the Km for each protein. Under these conditions, IC50 approaches Ki. Vardenafil inhibited human PDE5A1 with 32-fold greater potency than did sildenafil, with IC50 values for these two inhibitors of 0.12±0.02 nM and 3.9±0.4 nM, respectively (Table 1). The contribution of the hydrogen-bonding potential between the hydroxyl of Tyr612 and the water bridge with the nitrogen that is common to the five-member rings of sildenafil and vardenafil was tested using PDE5Y612F; this substitution retained the aromatic side chain of tyrosine, but lacked the hydrogen-bonding potential. PDE5Y612F mutant had increased sensitivity to inhibition by both inhibitors compared to PDE5WT (Figure 4, Table 1). The IC50 of sildenafil or vardenafil for PDE5Y612F (1.3±0.3 or 0.06±0.01 nM, respectively) was 3.0- or 2.2-fold lower than that for PDE5WT (Table 1). Furthermore, the difference in biochemical potency of vardenafil compared to sildenafil was narrowed slightly from 32-fold to 24-fold. The fact that this Tyr612 to Phe mutation, which increased hydrophobicity, strengthened affinity for both inhibitors suggested that higher affinity of vardenafil in PDE5WT is not due exclusively to greater vardenafil hydrophobic interactions.

Figure 4

Potency of sildenafil or vardenafil inhibition of wild-type or mutant PDE5 proteins. Experiments to determine IC50 values were performed as described in Materials and methods. Data shown are representative of three experiments and values are mean±s.e.m. of triplicate determinations. (a) Sildenafil concentration curve; (b) vardenafil concentration curve.

Potency of each inhibitor was significantly less for PDE5Y612A compared to PDE5WT, but the effect was greater for vardenafil than for sildenafil. Sildenafil inhibited PDE5Y612A with an IC50=98.3±4.7 nM (26-fold lower potency (or affinity)). The effect of this same mutation on vardenafil potency was 5 times greater than that on sildenafil; vardenafil inhibited PDE5Y612A mutant with an IC50=15±1.6 nM, that is, 122-fold lower potency compared to PDE5WT. Therefore, this mutation decreased vardenafil potency from 32-fold greater than that of sildenfail to 4.7-fold greater than that of sildenafil (Table 1).

Contribution of Tyr612 to sildenafil and vardenafil potency

Contributions of the side chain components of Tyr612 were quantified by calculating the change in free energy of binding (ΔΔG) of sildenafil or vardenafil in the mutants following the standard procedure used for assessing potential bonding interactions of ligands with receptor proteins.24 The ΔΔG value for PDE5Y612F inhibition by sildenafil and vardenafil for each inhibitor (−0.7 and −0.5 kcal/mol, respectively) indicated a modest enhancement of binding affinity. This suggested that the tyrosine hydroxyl could be a slightly negative determinant for both inhibitors. In contrast, ΔΔG values for PDE5Y612A inhibition by sildenafil or vardenafil (2.7 and 2.9 kcal/mol, respectively) were in the range expected for an important role for interaction of the aromatic ring of Tyr612 with both inhibitors in PDE5WT.


Previous reports showed that the IC50 for sildenafil inhibition of PDE5 is 4 nM versus a value of 0.1 nM for vardenafil,17, 27 and that differences in the heterocyclic ring systems of these inhibitors define the higher potency of vardenafil (Figure 1).28, 29 Recently solved X-ray crystal structures of isolated PDE5 catalytic domain revealed that vardenafil and sildenafil have similar binding modes. Among several contacts, this binding involves interactions of the five-member ring of each of the inhibitors through (1) hydrophobic interactions with Tyr612 and (2) a hydrogen-bond interaction between the shared nitrogen (at position 8 in Figure 2) of the pyrazole/imidazole moieties of sildenafil or vardenafil with a water molecule that also forms a hydrogen-bond bridge with both the hydroxyl of Tyr612 and another water molecule that coordinates to the catalytic site Zn2+ (Figure 2).18

The present biochemical study has determined that Tyr612 in PDE5, which is critical for binding the cGMP substrate, contributes a component of the binding affinity (potency) for both sildenafil and vardenafil as well as to higher affinity of PDE5 for vardenafil over sildenafil. This study also quantifies this contribution. Based on mutation of Tyr612 to Ala, the contribution to vardenafil affinity is 5-fold greater than that for sildenafil affinity. This appears to primarily involve hydrophobic interactions between the aromatic side chain of Tyr612 and the five-member ring structures of the inhibitors. Hydrophobic interaction of Tyr612 with vardenafil may be more optimal than that with sildenafil.

Lower binding affinity of sildenafil compared to vardenafil for PDE5 catalytic site could have several possible molecular mechanisms. Sildenafil binding could impose a slight strain on the binding pocket; this could occur in part through less than ideal interactions between the Tyr612 side chain and the pyrazole group. More optimal interaction when vardenafil is bound could contribute to greater potency of vardenafil.

In sum, the results herein establish that Tyr612 in PDE5 is important for binding both sildenafil and vardenafil although it is significantly more favorable for vardenafil. The hydrophobicity of this tyrosine is critical for potency of both inhibitors. From the crystal structure, the Tyr612 hydroxyl is indicated to form a bond with a water bridge between the nitrogen at position 8 and the catalytic site Zn2+. However, the present studies indicate that this hydroxyl group is not essential for binding of either sildenafil or vardenafil. The different electronegativity found at position 8 of the two inhibitors could also play a role in affinity and selectivity, but the data do not suggest a mechanism for either property.



3′,5′-cyclic nucleotide phosphodiesterase


cGMP-binding cGMP-specific PDE


10 mM potassium phosphate

pH 6.8:

1 mM EDTA, and 25 mM β-mercaptoethanol






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Corbin, J., Francis, S. & Zoraghi, R. Tyrosine-612 in PDE5 contributes to higher affinity for vardenafil over sildenafil. Int J Impot Res 18, 251–257 (2006).

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  • phosphodiesterases (PDEs)
  • GAF
  • allosteric cGMP-binding sites
  • noncatalytic cGMP-binding sites
  • vardenafil
  • sildenafil
  • PDE inhibitors

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