Copyright 2004 The American Society for Pharmacology and Experimental Therapeutics Mol Pharmacol 66:144–152, 2004 Printed in U.S.A. Binding of Tritiated Sildenafil, Tadalafil, or Vardenafil to thePhosphodiesterase-5 Catalytic Site Displays Potency,Specificity, Heterogeneity, and cGMP Stimulation Mitsi A. Blount, Alfreda Beasley, Roya Zoraghi, Konjeti R. Sekhar, Emmanuel P. Bessay,Sharron H. Francis, and Jackie D. Corbin Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee Received January 21, 2004; accepted April 9, 2004 This article is available online at ABSTRACT
Sildenafil, tadalafil, and vardenafil each competitively inhibit
competing against one another indicated that each occupies cGMP hydrolysis by phosphodiesterase-5 (PDE5), thereby fos- the same site on PDE5. Studies of sildenafil and vardenafil tering cGMP accumulation and relaxation of vascular smooth analogs demonstrated that higher potency of vardenafil is muscle. Biochemical potencies (affinities) of these compounds caused by differences in its double ring. Exchange-dissociation for PDE5 determined by IC , K (isotherm), K studies revealed two binding components for each inhibitor.
rate), and K (1⁄ ), respectively, were the following: silde- Excess unlabeled inhibitor did not significantly affect 3H inhib- nafil (3.7 ⫾ 1.4, 4.8 ⫾ 0.80, 3.7 ⫾ 0.29, and 11.7 ⫾ 0.70 nM), itor dissociation after infinite dilution, suggesting the absence tadalafil (1.8 ⫾ 0.40, 2.4 ⫾ 0.60, 1.9 ⫾ 0.37, and 2.7 ⫾ 0.25 nM); of subunit-subunit cooperativity. cGMP addition increased and vardenafil (0.091 ⫾ 0.031, 0.38 ⫾ 0.07, 0.27 ⫾ 0.01, and binding affinity of [3H]tadalafil or [3H]vardenafil, an effect pre- 0.42 ⫾ 0.10 nM). Thus, absolute potency values were similar for sumably mediated by cGMP binding to PDE5 allosteric sites, each inhibitor, and relative potencies were vardenafil ⬎⬎ implying that either inhibitor potentiates its own binding to tadalafil ⬎ sildenafil. Binding of each 3H inhibitor to PDE5 was PDE5 in intact cells by elevating cGMP. Without inhibitor specific as determined by effects of unlabeled compounds. 3H present, cGMP accumulation would stimulate cGMP degrada- Inhibitors did not bind to isolated PDE5 regulatory domain.
tion, but with inhibitor present, this negative feedback process Close correlation of EC values using all three 3H inhibitors would be blocked.
Phosphodiesterase-5 (PDE5) is 1 of 11 mammalian PDE domains (a and b) because of their presence in cGMP-binding families known to date (Francis et al., 2001). PDE5 is a cyclic nucleotide PDEs, Anabaena adenylyl cyclase, and the cGMP-specific PDE and is abundant in most smooth muscle bacterial transcription factor FhlA (Thomas et al., 1990a; tissues as well as in platelets, gastrointestinal epithelial McAllister-Lucas et al., 1993; Aravind and Ponting, 1997).
cells, and Purkinje cells of the cerebellum (Francis et al., Isolated GAF a monomer binds cGMP with high affinity, but 2001; Shimizu-Albergine et al., 2003). The enzyme was first cGMP binding to GAF b has yet to be demonstrated (Liu et identified, purified, and cloned in this laboratory (Lincoln et al., 2002). Allosteric binding of cGMP to PDE5 regulatory al., 1976; Francis et al., 1980; Thomas et al., 1990a; McAllis- domain increases affinity of the catalytic site for cGMP, ter-Lucas et al., 1993). PDE5 is a homodimer, and each thereby stimulating the rate of cGMP hydrolysis (Thomas et monomer is a chimeric protein that is composed of a regula- al., 1990b; Corbin and Francis, 1999; Okada and Asakawa, tory domain and a catalytic domain (Corbin and Francis, 2002; Corbin et al., 2003; Mullershausen et al., 2003; Ry- 1999). The catalytic domain catalyzes the breakdown of balkin et al., 2003). cGMP binding to the regulatory domain cGMP to 5⬘-GMP, and the regulatory domain contains allo- also stimulates phosphorylation of PDE5 at Ser-92 (bovine) steric cGMP-binding sites and a phosphorylation site (Corbin by cGMP-dependent protein kinase in vitro and in vivo and Francis, 1999). Two tandem homologous repeats of ⬃110 (Thomas et al., 1990b; Wyatt et al., 1998; Mullershausen et amino acids each in the regulatory domain are termed GAF al., 2001; Murthy, 2001; Rybalkin et al., 2002). It is presumedthat cGMP binding to the regulatory domain produces a This work was supported by National Institutes of Health Research grants conformational change in PDE5 that exposes Ser-92. The DK40029 and DK58277, National Institutes of Health Training grant 5T32HL-07751, and the Bayer Pharmaceuticals Corporation.
resulting phosphorylation of PDE5 increases affinity of the ABBREVIATIONS: PDE, cyclic nucleotide phosphodiesterase; GAF, mammalian cGMP-binding phosphodiesterase, Anabaena adenylyl cyclases,
Escherichia coli FhlA; IBMX, 3-isobutyl-1-methylxanthine; KPM, 10 mM potassium phosphate, pH 6.8, containing 15 mM ␤-mercaptoethanol.
3H Inhibitor Binding to PDE5
regulatory domain for cGMP and increases catalytic activity 2003; Francis et al., 2003). All three 3H inhibitors were resolved in as well (Corbin et al., 2000). These effects suggest that PDE5 single peaks and coeluted with purified unlabeled inhibitors, sug- is critically involved in negative feedback regulation of cel- gesting that the 3H inhibitors were unaltered after storage. Even so, lular cGMP levels.
it cannot be completely ruled out that the curvilinearity observed in Several compounds that potently inhibit PDE5 have been the dissociation of 3H inhibitors in Fig. 5 could be caused by slightstructural heterogeneity of the inhibitors.
synthesized recently, and three of these are now in clinical Isolated Regulatory Domain of PDE5. Residues Met1 to Glu539
use for treatment of male erectile dysfunction. After sexual of human PDE5 were amplified from the hPDE5 cDNA (courtesy of arousal, these inhibitors enhance accumulation of cGMP in Tanabe Research Laboratories Inc., San Diego, CA). Using the forward the smooth muscle of the arteries supplying the penis and the sinusoids of the penile corpus cavernosum. Sildenafil (Vi- CCCAGCT-3⬘) and the reverse primer RZGlu539rev (5⬘-GATGAT- agra; Pfizer, New York, NY) was the first compound of this class to be marketed for the treatment of male erectile dys- EcoRI and NotI sites (underlined) and a stop codon (bold italic). The function. It also shows promise in the clinical treatment of resulting PCR fragment (1649 base pairs) was cloned into pCR 2.1-Topo ailments related to smooth muscle tissues, such as pulmo- (Invitrogen, Carlsbad, CA) and verified by sequencing. The fragment nary hypertension (Weimann et al., 2000). Newer PDE5 in- was excised by digestion with EcoRI and NotI and was inserted intobaculovirus transfer pAcHLT-A (BD PharMingen, San Diego, CA) di- hibitors that have the same therapeutic mechanism as silde- gested with the same enzymes. The resulting plasmid was cotrans- nafil, such as tadalafil (Cialis; Lilly-ICOS, Bothell, WA), and fected with the BaculoGold baculovirus DNA (BD PharMingen) into Sf9 vardenafil (Levitra; Bayer Corporation, West Haven, CT), cells according to the manufacturer's instructions. The transfected cells have also been approved for use in many countries. The were incubated at 27°C for 5 days. Afterward, 100 ␮l of collected culture availability of these high-affinity inhibitors provides signifi- medium was used to infect 2 ⫻ 107 freshly prepared Sf9 cells for viral cant new tools for studies of the PDE5 catalytic domain. This amplification. The recombinant baculovirus was amplified two more laboratory recently examined some characteristics of the cat- times to obtain a high titer stock solution by infecting freshly seeded Sf9 alytic domain and its regulation by investigating [3H]silde- cells. The infected cells were incubated at 27°C for 4 days before protein nafil binding to the enzyme (Corbin et al., 2003). The struc- was harvested. Purification was carried out using nickel/nitrilotriacetic tures of tadalafil and vardenafil differ significantly from that acid agarose as described previously (Corbin et al., 2003).
of sildenafil, and these three compounds have differing in- PDE Assays. PDE activity was determined using a modified
method (Martins et al., 1982) as described previously (Gopal et al., hibitory potencies. Molecular contacts of the three inhibitors 2001) with 0.4 ␮M [3H]cGMP as substrate.
within the catalytic site of the PDE5 have recently been [3H]cGMP-Binding Assay. The procedure was modified slightly
revealed by X-ray crystallography (Sung et al., 2003). In from that described previously (Corbin et al., 2000). PDE5 or PDE5 addition to [3H]sildenafil, we have synthesized or acquired (80 ␮l) isolated regulatory domain (4 nM final protein concentration [3H]tadalafil and [3H]vardenafil. The availability of these in reaction mixture) was added to 2 ml of a mixture of 0.2 ␮M compounds has allowed a thorough analysis of the interac- [3H]cGMP, 10 mM potassium phosphate, pH 6.8, 25 mM 2-mercap- tion of these agents with PDE5, which is reported herein.
toethanol, and 0.2 mg/ml Type II-AS histone (Sigma). After 45 min at These radiolabeled inhibitors have also permitted the most 4°C, the sample was filtered onto premoistened Millipore filters (pore comprehensive, head-to-head comparison of potencies of size, 0.45 ␮m), which were then rinsed with 3 ml of 10 mM potassium these agents to bind to PDE5 using several approaches.
phosphate, pH 6.8, and 25 mM ␤-mercaptoethanol, dried, andcounted.
Moreover, some novel features of the inhibitors and of PDE5 3H Inhibitor Membrane Filtration-Binding Assay. Full-
are uncovered using these approaches.
length bovine His-tagged PDE5 (80 ␮l) was added to 2 ml of a bindingreaction mixture that contained 0.2 mg/ml histone IIA-S, various Materials and Methods
concentrations of 3H inhibitor, and buffer that consisted of 10 mMpotassium phosphate, pH 6.8, and 25 mM ␤-mercaptoethanol (KPM).
Materials. [3H]cGMP and DEAE-Sephacel were purchased from
Sticking of 3H inhibitor to the sides of the test tube occurred when 3H Amersham Biosciences Inc. (Piscataway, NJ). 3-Isobutyl-1-methyl- inhibitor was added in the absence of or before addition of histone.
xanthine (IBMX), histone type II-AS, Crotalus atrox snake venom, Histone also increased retention of PDE5 on the Millipore mem- 5⬘-GMP, and cGMP were obtained from Sigma Chemical Co. (St.
branes. Binding reaction mixture containing the enzyme was incu- Louis, MO). His-tagged, full-length recombinant bovine PDE5 was bated on ice or in a 30°C water bath for 45 min. Millipore nitrocel- isolated from infected Sf9 cells using nickel/nitrilotriacetic acid aga- lulose membranes (0.45 ␮m) were placed under house vacuum and rose (QIAGEN, Valencia, CA) as described previously (Corbin et al., prewetted with 1 ml of ice-cold 10 mM potassium phosphate, pH 6.8, 2003). Native bovine lung PDE5 was obtained and purified using that contained 0.1% Triton X-100. Next, 200 ␮l of 25% Triton X-100 Blue Sepharose described in an earlier report (Francis and Corbin, at room temperature in KPM was added to the reaction tube. The 1988; Thomas et al., 1990a). Sildenafil was purified from Viagra entire contents of the tube were applied to the prewetted filter. The tablets by following the method established previously in this labo- reaction tube was then washed with 3 ml of cold 0.1% Triton X-100 ratory (Corbin et al., 2003). Purified sildenafil was submitted to in 10 mM potassium phosphate, pH 6.8, and the wash was also Amersham Biosciences for radiolabeling with tritium. Tadalafil was applied to the filter. Filter membranes were removed, dried, and synthesized according to Daugan (2000). After confirming the com- transferred to 6-ml scintillation vials. Nonaqueous scintillant (5 ml) pound structure by mass spectrometry, tadalafil was submitted to was added to the tubes, which were then placed in a scintillation Amersham Biosciences for radiolabeling with tritium. High-perfor- mance liquid chromatography results from Amersham indicated that Statistical Analyses. All values are given as mean ⫾ standard
[3H]sildenafil was ⬎98% pure, whereas the [3H]tadalafil preparation error of mean (S.E.M.) as determined by GraphPad Prism graphics was ⬎99% pure. Vardenafil, [3H]vardenafil, demethyl-vardenafil, software (GraphPad Software Inc., San Diego, CA). The software and methyl-sildenafil were provided by Bayer AG (Wuppertal, Ger- uses the following equation: S.E.M. ⫽ standard deviation/n1/2, where many). All three 3H inhibitors that had been stored for more than a standard deviation is determined as [兺(y ⫺ y )2/(n ⫺ 1)]1 year were subjected to Sephadex G-25 chromatography, which ad- S.E.M. values reported fit within a 95% confidence interval, which sorbs PDE inhibitors and provides high resolution (Corbin et al., quantifies the precision of the mean.

Blount et al.
recombinant bovine PDE5 (Fig. 2). A 240-fold excess of un-labeled Inhibition of PDE5 Catalytic Activity. The concentra-
[3H]tadalafil or [3H]vardenafil binding. Addition of cAMP or tion of inhibitor that produces 50% inhibition of PDE5 cata- 5⬘-GMP at 375,000-fold excess did not affect binding of either lytic activity (IC ) was determined for each of the inhibitors inhibitor. At 375,000-fold excess, cGMP reduced binding of (sildenafil, tadalafil, and vardenafil) using 0.4 ␮M [3H]cGMP either 3H inhibitor by 40 to 60%. A 2400-fold excess of rolip- as substrate (Fig. 1). The IC values were the following: ram (a PDE4-specific inhibitor) or cilostamide (a PDE3-spe- sildenafil, 3.7 ⫾ 1.4 nM (n ⫽ 4); tadalafil, 1.8 ⫾ 0.4 nM (n ⫽ cific inhibitor) did not affect 3H inhibitor binding. IBMX, a 7); and vardenafil, 0.091 ⫾ 0.031 nM (n ⫽ 5). Similar values general, albeit weak, PDE inhibitor had a substantial inhibitory were obtained when using native bovine PDE5 (data not effect at 100,000-fold excess. The data suggested that binding of shown). These values agreed with the range of published IC50 all three inhibitors is specific for the catalytic domain of PDE5 values [sildenafil, 1–9 nM (Ballard et al., 1998; Turko et al., and that all three inhibitors compete for the same site.
1999; Corbin and Francis, 2002); tadalafil, 1–7 nM (Corbin et Lack of Binding of Each of the 3H Inhibitors to an
al., 2002; Gresser and Gleiter, 2002); and vardenafil, 0.1–0.8 nM (Saenz de Tejada et al., 2001; Gresser and Gleiter, 2002; [3H]cGMP bound to the isolated regulatory domain of PDE5 Corbin et al., 2002)].
nearly stoichiometrically, none of the 3H inhibitors bound to Stoichiometry of 3H Inhibitor Binding to PDE5. The
this domain using the same assay conditions and concentra- binding stoichiometry was determined for each inhibitor by tion used in the studies of binding to full-length PDE5 (data dividing maximum binding (B , picomoles of 3H inhibitor not shown). Addition of a ⬃5-fold excess (0.96 ␮M) of unla- binding per milliliter of PDE5) obtained from GraphPad beled sildenafil, tadalafil, or vardenafil, which was in the Prism graphics, by PDE5 enzyme concentration (picomoles of range of 1000 times the K of each inhibitor for the catalytic PDE5 subunit per milliliter of PDE5). PDE5 protein concen- domain, did not lower [3H]cGMP binding to the regulatory tration was determined by amino acid analysis. Stoichiome- domain (data not shown). In contrast, a 2500-fold (0.5 mM) try was corrected for 75% recovery of 3H inhibitor binding to excess of unlabeled cGMP, which was also approximately PDE5 using the vacuum filtration method as determined 1000 times the K of this ligand for the catalytic domain, previously (Corbin et al., 2003). [3H]Tadalafil bound to PDE5 abolished [3H]cGMP binding to the regulatory domain. To- with a stoichiometry of 0.68 ⫾ 0.10 mol/subunit (n ⫽ 7), gether, these results indicated that inhibitor is specific for which was similar to the [3H]vardenafil stoichiometry of the PDE5 catalytic domain and does not bind to the regula- 0.41 ⫾ 0.05 mol/subunit (n ⫽ 8). These values compared well tory domain under the conditions of the assays.
with the stoichiometry previously reported for [3H]sildenafil Potencies (Affinities) for Binding of 3H Inhibitors to
of 0.61 ⫾ 0.13 mol/subunit (Corbin et al., 2003). The [3H]sil- denafil binding stoichiometry was duplicated using the same ([3H]sildenafil, [3H]tadalafil, or [3H]vardenafil) binding to enzyme preparation used to determine the [3H]tadalafil and PDE5 is shown in Fig. 3. K values, obtained by using non- [3H]vardenafil stoichiometry values calculated above.
linear regression analysis with GraphPad Prism software, Specificity for 3H Inhibitor Binding to PDE5. The
were as follows: sildenafil, 4.8 ⫾ 0.8 nM (n ⫽ 3); tadalafil, specificity of [3H]sildenafil binding to the catalytic domain of 2.4 ⫾ 0.6 nM (n ⫽ 4); and vardenafil, 0.38 ⫾ 0.07 nM (n ⫽ 5).
PDE5 was presented in our previous report (Corbin et al.,2003). The specificities of [3H]tadalafil and [3H]vardenafilbinding to PDE5 were determined by testing the effects ofvarious unlabeled compounds using 4 nM 3H inhibitor and Fig. 2. Effects of nucleotides and inhibitors on binding of 3H inhibitors to
PDE5. PDE5 (0.7 nM final concentration in assay) was incubated in 2
ml of binding reaction mixture with 4 nM 3H inhibitor and the follow-
Fig. 1. Potency of inhibition of PDE catalytic activity by PDE5 inhibitors.
ing concentrations of competing compounds: unlabeled vardenafil PDE5 (10 ␮l; 0.113 nM final concentration in assay) was added to the (Var) ⫽ 0.96 ␮M, unlabeled sildenafil (Sild) ⫽ 0.96 ␮M, unlabeled PDE assay reaction mixture containing increasing concentrations of tadalafil (Tad) ⫽ 0.96 ␮M, cAMP (cA) ⫽ 1.5 mM, 5⬘-GMP (5⬘G) ⫽ 1.5 PDE5 inhibitors. PDE activity was determined in a 15-min incubation as mM, cGMP (cG) ⫽ 1.5 mM, rolipram (Roli) ⫽ 9.6 ␮M, cilostamide described under Materials and Methods using 0.4 ␮M (final concentra- (Cilo) ⫽ 9.6 ␮M, and IBMX ⫽ 0.4 mM. All were filtered as described tion) [3H]cGMP as substrate. Data represent a typical experiment per- under Materials and Methods. Data represent three experiments, each formed in triplicate.
performed in triplicate.
3H Inhibitor Binding to PDE5
These values agreed well with the IC Heterogeneity of the PDE5 Catalytic Domain Re-
vealed by 3H Inhibitor Dissociation Kinetics. Exchange-
Potencies for sildenafil, tadalafil, and vardenafil were also dissociation kinetics of each of the 3H inhibitors from PDE5 determined by competition studies. For example, Fig. 4 were examined. PDE5 was first saturated with 3H inhibitor shows the effect of increasing concentrations of unlabeled (30 nM), and aliquots were removed to determine 3H inhibi- vardenafil on binding of 3 nM [3H]tadalafil. The EC tor binding at 0 time. Unlabeled inhibitor (⬃33,000-fold ex- was calculated from GraphPad Prism graphics software us- cess) was then added to the reaction mixture, and aliquots ing a sigmoidal dose-response curve. Because EC were removed for filtration at various times to follow the time were determined using a 3H inhibitor concentration at the course of dissociation (exchange) of the radiolabeled inhibitor approximate K value for PDE5, the Cheng and Prusoff/ from the enzyme. Under these conditions, the enzyme re- Chou equation (Cheng and Prusoff, 1973; Chou, 1974) could mained saturated at all times with inhibitor. All three inhib- be applied to calculate the K itors exhibited nonlinear dissociation kinetics indicative of values by two (Table 1). It can be seen that 1⁄2 EC the presence of at least two rate components (Fig. 5A). In Fig.
general agreement with the K or IC for each inhibitor, and 5B, the x-axis was changed to emphasize the earlier time the order of potency for the inhibitors was retained. The points. Assuming the presence of two components, when the values for unlabeled inhibitor in competition line of the slower component was extrapolated to the y-axis, with either [3H]vardenafil, [3H]sildenafil, or [3H]tadalafil the calculated percentages of the two components were dif- were similar. This suggested that the inhibitors compete for ferent for each inhibitor. Sildenafil, as reported previously, the same site on PDE5.
exhibited two equal components. The dissociation behavior of Potencies of Sildenafil and Vardenafil Analogs. Vard-
[3H]tadalafil revealed 60% high-affinity (slow) and 40% low- enafil has a ⬃40-fold higher affinity for PDE5 over sildenafil affinity (fast) components. [3H]Vardenafil dissociation exhib- values shown here. To determine which of ited 85% high-affinity and 15% low-affinity components. The the distinguishing molecular features of the two compounds overall rate of dissociation of [3H]vardenafil was much slower determines this difference in potency, two analogs were than that of the other two inhibitors. After estimation of the synthesized. The first, demethyl-vardenafil, contained the for dissociation, the K of each inhibitor was calculated [5,1-f][1,2]triazine ring of vardenafil and the appended from the following equation: K ⫽ 6.93 ⫻ 10⫺7 M 䡠 s/t , methyl group of sildenafil. The second analog, methyl- where M ⫽ molar, s ⫽ seconds, and t sildenafil, contained the pyrazolo[4,3-d]pyrimidine ring of seconds. (Limbird, 1995). All exchange-dissociation experi- sildenafil and the appended ethyl group of vardenafil. The ments were performed three times with each 3H inhibitor.
of each analog for PDE5 was determined using 0.4 ␮M [3H]cGMP as substrate. These experiments yielded IC50values of 0.14 ⫾ 0.02 nM for demethyl-vardenafil and 8.90 ⫾ 1.7 nM for methyl-sildenafil (Table 2). The EC each of the analogs was determined using 0.5 nM [3H]vard-enafil. EC values were 0.88 ⫾ 0.19 nM for demethyl- vardenafil and 72 ⫾ 13 nM for methyl-sildenafil. K calculated was in general agreement with the IC analog (Table 2). The results indicated that the higher biochem-ical potency of vardenafil over sildenafil is caused by differenceswithin the double rings of the two compounds.
Fig. 4. Determination of EC
for vardenafil. Increasing concentrations of unlabeled vardenafil were included in 2 ml of binding reaction mixturethat contained 3 nM [3H]tadalafil. PDE5 was then added (80 ␮l; 0.035 nMfinal concentration in assay). Filtration was performed as outlined underMaterials and Methods. Data represent three experiments, each per-formed in triplicate.
values for PDE5 inhibitors Increasing concentrations of unlabeled inhibitor were added to 2 ml of bindingreaction mixture that contained either 0.5 nM 关3H兴vardenafil, 4 nM 关3H兴sildenafil, or3 nM 关3H兴tadalafil. Filtration was performed as outlined under Materials and Meth-ods. Based on Student's t tests, the three KD values for each unlabeled inhibitor werenot significantly different from each other.
Fig. 3. Affinity of PDE5 for binding 3H inhibitors. PDE5 (80 ␮l; 0.26 nM
final concentration in assay) was incubated with increasing concentra-
tions of 3H inhibitors in 2 ml of binding reaction mixture containing 10 M cGMP for 20 min on ice and then filtered as described under Mate- rials and Methods. Data represent a typical experiment performed in

Blount et al.
The resulting K values for the two [3H]sildenafil compo- ature from 4° to 30°C had no effect or perhaps slightly inhib- nents were 14.7 ⫾ 2.3 and 0.7 ⫾ 0.06 nM, for the two ited sildenafil and tadalafil binding (data not shown).
[3H]tadalafil components were 9.3 ⫾ 2.67 and 0.6 ⫾ 0.00 nM, However, the increase in temperature increased vardenafil and for the two [3H]vardenafil components were 6.0 ⫾ 0.00 binding in the presence of cGMP, as is discussed below.
and 0.1 ⫾ 0.01 nM. The geometric mean K values for each The effect of increasing cGMP concentrations on [3H]vard- inhibitor (n ⫽ 3) were the following: sildenafil, 3.1 nM; enafil binding was carried out using 0.5 nM [3H]vardenafil at tadalafil, 1.7 nM; and vardenafil, 0.32 nM. Each average K both 4° and 30°C (Fig. 7). At 4°C, [3H]vardenafil showed a determined by this method was similar to IC , K obtained 2.5-fold increase in binding at low levels of cGMP (1–50 ␮M), from isotherm, or K obtained from 1⁄ although this effect waned at higher cGMP concentrations.
values and average K values determined from Repeating the experiment at 30°C with increasing cGMP dissociation rates of the respective inhibitors suggested that produced a ⬃3.5-fold stimulation of [3H]vardenafil binding to interaction of the inhibitor with both kinetic components PDE5 at 30°C. The cGMP effect remained constant at mod- contributes to inhibition of PDE5 catalytic activity.
erate concentrations and waned slightly at very high cGMP In addition to the exchange-dissociation method used above, [3H]tadalafil or [3H]vardenafil dissociation from When binding using increasing concentrations of [3H]vard- PDE5 was examined by infinite dilution. Dissociation of the enafil was performed at 30°C in the presence of constant 10 respective radiolabeled inhibitor was determined in the ab- ␮M cGMP, the labeled compound bound to PDE5 with a sence and presence of excess unlabeled inhibitor after equi- slightly higher affinity than at 4°C (0.42 ⫾ 0.06 nM, n ⫽ 3, librium binding and 80-fold dilution of the binding reaction.
versus 0.59 ⫾ 0.02 nM, n ⫽ 3). [3H]Vardenafil binding to The pattern of [3H]tadalafil dissociation (Fig. 6A) revealed PDE5 in the absence of cGMP at 30°C yielded a lower KD two components either in the presence or absence of a 5000- than that found for [3H]vardenafil binding at 4°C (0.74 ⫾ fold excess of unlabeled tadalafil during dissociation. The 0.10 nM, n ⫽ 3, versus 2.19 ⫾ 0.62 nM, n ⫽ 3) (Fig. 8, A and lack of an effect of unlabeled tadalafil on the dissociation of [3H]tadalafil from PDE5 suggested that even though PDE5 is The addition of 10 ␮M cGMP to increasing concentrations dimeric, the catalytic domain in each of the respective mono- of [3H]vardenafil at 4°C decreased the K (0.74 ⫾ 0.10 nM, n mers of the enzyme may not kinetically influence each other ⫽ 3, to 0.59 ⫾ 0.02 nM, n ⫽ 3) while increasing the B to a large degree. Likewise, the dissociation of [3H]vardenafil PDE5 (5.63 ⫾ 0.38 to 6.58 ⫾ 0.10 pmol/ml) (Fig. 8A). At 30°C, after infinite dilution was not different from that in the cGMP caused a 3.6-fold decrease in K from 2.19 ⫾ 0.62 nM presence of excess vardenafil, again suggesting that the (n ⫽ 3) to 0.42 ⫾ 0.06 nM (n ⫽ 3), whereas the B PDE5 catalytic domains of the two monomers function inde- significantly change (4.93 ⫾ 0.71 versus 5.25 ⫾ 0.22 pmol/ml) pendently (Fig. 6B).
Effect of cGMP on 3H Inhibitor Binding. We recently
As shown in Fig. 9, cGMP also stimulated binding of 3 nM reported that cGMP stimulates [3H]sildenafil binding to the [3H]tadalafil at 4°C, and the effect was maximal at ⬃25 ␮M PDE5 catalytic domain at 4°C (Corbin et al., 2003). In addi- cGMP. The stimulatory effect waned at higher cGMP concen- tion to determining whether the same cGMP effect occurred trations in a manner similar to the cGMP effect on vardenafil with [3H]vardenafil and [3H]tadalafil, we also investigated if binding at 4°C. The addition of 10 ␮M cGMP to increasing cGMP stimulates 3H inhibitor binding at 30°C, which ap- concentrations of [3H]tadalafil at 4°C decreased K proaches physiological temperature. Increasing the temper- from 3.7 ⫾ 0.39 nM (n ⫽ 3) to 1.74 ⫾ 0.05 nM (n ⫽ 3), values for PDE5 inhibitor analogs Structures of analogs are shown with differences encircled. IC50 values were determined by adding PDE5 (10 ␮l; 0.11 nM final concentration) to PDE assay reaction mixturecontaining increasing concentrations of the analogs. PDE activity was determined in a 15-min incubation as described under Materials and Methods using 0.4 ␮M (finalconcentration) 关3H兴cGMP as substrate. EC50 values were determined by adding increasing concentrations of unlabeled inhibitor analog to 2 ml of binding reaction mixturethat contained 0.5 nM 关3H兴vardenafil. Filtration was performed as outlined under Materials and Methods. Student's t tests indicate that IC50 and KD values formethyl-sildenafil were significantly different (p ⬍ 0.05) from the IC50 and KD values for demethyl-vardenafil, vardenafil, and sildenafil.
3.7 ⫾ 1.4 (n⫽4) 0.17 ⫾ 0.04 (n⫽7) 0.14 ⫾ 0.02 (n⫽4) 8.90 ⫾ 1.7 (n⫽3) 2.47 ⫾ 0.3 (n⫽3) 1.0 ⫾ 0.26 (n⫽5) 0.44 ⫾ 0.10 (n⫽4) 36 ⫾ 6.5 (n⫽3) 3H Inhibitor Binding to PDE5
whereas the B was 4.97 ⫾ 0.14 and 5.95 ⫾ 0.19 pmol/ml, The isolated regulatory domain of PDE5 did not bind 3H respectively (Fig. 10).
inhibitor using the same binding assay used for PDE5 ho- The combined results suggested that [3H]vardenafil, but loenzyme even though the regulatory domain bound not [3H]sildenafil or [3H]tadalafil, binds to PDE5 with higher [3H]cGMP nearly stoichiometrically. In addition, unlabeled affinity at 30°C than at 4°C. The affinities of all three inhib- sildenafil, tadalafil, or vardenafil did not compete with itors are increased by the presence of cGMP, whereas maxi- [3H]cGMP for binding to the regulatory domain, confirming mum binding of each inhibitor is increased only slightly by that these inhibitors do not bind to the regulatory domain.
values determined by binding isotherms, EC , or ex- change-dissociation agreed with IC of each inhibitor, again supporting the conclusion that the PDE5-specific inhibitorsinteract exclusively with the catalytic site of PDE5. Because [3H]Sildenafil binding to PDE5 is specific for the catalytic cGMP-binding sites in the PDE5 regulatory and catalytic site of PDE5 (Corbin et al., 2003). The present report dem- domains are evolutionarily and biochemically distinct, this onstrates that [3H]tadalafil and [3H]vardenafil are also spe- result was not surprising.
cific for binding to the catalytic site. Binding of each of the This laboratory has used membrane vacuum filtration to three 3H inhibitors was inhibited by catalytic site-selective measure [3H]cGMP binding (Francis and Corbin, 1988), 65Zn agents and by unlabeled sildenafil, tadalafil, or vardenafil, binding (Francis et al., 1994), and [3H]sildenafil binding suggesting that binding of each inhibitor is restricted to the (Corbin et al., 2003). This assay was modified slightly for catalytic domain and that all three inhibitors also bind to the specific [3H]tadalafil and [3H]vardenafil binding to PDE5. All same catalytic site. The stoichiometry of each 3H inhibitor three 3H inhibitor binding assays produced high recoveries binding approached 1 mol/PDE5 subunit, which was consistent and yielded nearly 1 mol/subunit binding. Radiolabeled roli- with inhibitor binding specifically to the catalytic site and also pram binding to PDE4 has been reported (Schneider et al., was indicative of one catalytic site per PDE5 monomer.
1986; Torphy et al., 1992); however, the stoichiometry of Fig. 5. Exchange-dissociation of 3H inhibitor from PDE5. PDE5 (0.35 nM
final concentration in assay) was added to 4.5 ml of binding reaction
mixture containing 3H inhibitor (30 nM final concentration). Then, to
Fig. 6. Dissociation of 3H inhibitors from PDE5 after infinite dilution.
determine the zero time point, a 520-␮l aliquot of this mixture was PDE5 (76 ␮l, 0.32 nM final concentration in assay) was added to 360 ␮l filtered as described under Materials and Methods. Next, 30 ␮l of a 1 mM of binding reaction mixture containing a final concentration of 2 nM solution of the corresponding unlabeled inhibitor was added to the re- [3H]tadalafil (A) or 0.5 nM [3H]vardenafil (B). After incubating for 1 h on maining incubating binding reaction mixture at 4°C. Aliquots were re- ice, 35 ml of 0.2 mg/ml histone AII-S in the absence or presence of 10 ␮M moved and filtered by the same procedure at the indicated time points. A of the respective unlabeled inhibitor was added to dilute the binding represents a longer time course, whereas B shows a shorter time course reaction mixture 80-fold. Filtration was performed at the indicated time to emphasize curvilinear kinetics. Data represent three experiments, points by the procedure outlined under Materials and Methods. Data each performed in triplicate.
represent three experiments, each performed in triplicate.
Blount et al.
binding in those studies was less than 0.01 mol/subunit using which of these structural differences of the compounds deter- mines potency, two analogs were synthesized: demethyl- values of sildenafil, tadalafil, and vardenafil deter- vardenafil (analog of vardenafil containing the appended mined here in head-to-head assays using bovine PDE5 were methyl group of sildenafil) and methyl-sildenafil (analog of in the same range as IC values reported in the literature sildenafil containing the appended ethyl group of vardenafil).
using human PDE5 (Table 3) (Corbin and Francis, 2002; Demethyl-vardenafil and vardenafil had almost identical Corbin et al., 2002). Therefore, results are similar using values, whereas methyl-sildenafil had 52-times higher recombinant PDE5, native PDE5, or PDE5 from different which was similar to the IC of sildenafil. K mammalian species. Whereas IC is the classic method of experiments using both analogs also determining potency (affinity) of PDE inhibitors, measure- indicated that methyl-sildenafil had much lower potency ment of binding strength, or K , is a more direct method of than either of the other two analogs. From these results, the determining potency and it also provides a measure of stoi- higher biochemical potency of vardenafil compared with sil- chiometry of ligand binding. This report determined the po- denafil is caused by differences within the double rings of the tencies for sildenafil, tadalafil, and vardenafil using four two compounds. The crystal structure of the PDE5 catalytic separate head-to-head methods. IC measurements yielded domain containing either sildenafil or vardenafil was re- a potency ratio of 1:2:41, K (binding isotherm) yielded a ported recently (Sung et al., 2003); however, the resolution of ratio of 1:2:13, K (1⁄ ) yielded a ratio of 1:5:26, and K the crystal structure was not sufficient to identify distinct (exchange-dissociation) yielded a ratio of 1:2:14 for sildenafil, interactions of either of these two inhibitors with the enzyme.
tadalafil, and vardenafil, respectively (Table 3). This inves- The difference in the double ring of vardenafil, compared tigation represents the most comprehensive examination of with sildenafil, may possibly allow for a stronger interaction the absolute and relative potencies of these drugs.
between the compound and one or more of the amino acids Dissociation rates of inhibitors from PDE5 correlated with (e.g., Tyr-612, Val-782, Phe-820, Leu-785, and Gln-817) that potencies determined by IC or isotherm K , i.e., the slower the rate, the higher the potency. However, the faster disso-ciation rate of tadalafil from PDE5 compared with that ofvardenafil may be unexpected in view of the longer lastingclinical effects of tadalafil. These clinical differences oftadalafil may be caused by pharmacokinetic considerationssuch as slower intestinal absorption or slower degradation bythe liver, rather than by different biochemical properties.
In comparing the distinctive chemical structures of silde- nafil and vardenafil, two major differences are evident: 1) a methyl group is appended to the piperazine ring of sildenafil,whereas the same ring in vardenafil has an appended ethylgroup, and 2) a nitrogen atom is present in the 7-position ofthe double ring of sildenafil, but it is not present in the ringof vardenafil, although vardenafil contains a nitrogen atomin the 5-position, which is absent in sildenafil. To resolve Fig. 8. Effect of temperature on cGMP stimulation of [3H]vardenafil
binding using varying concentrations of [3H]vardenafil. PDE5 (80 ␮l, 0.07
Fig. 7. Effect of cGMP on [3H]vardenafil binding at 4° and 30°C. PDE5
nM final concentration in assay) was added to 2 ml of binding reaction (80 ␮l, 0.07 nM final concentration in assay) was added to 2 ml of binding mixture containing 0.05 to 6.4 nM [3H]vardenafil in the absence and reaction mixture containing 0.5 nM [3H]vardenafil and 0 to 350 ␮M presence of 10 ␮M cGMP and incubated for 45 min on ice (A) or for 20 min cGMP and incubated for 45 min on ice or 20 min in a 30°C water bath.
in a 30°C water bath (B). Binding was performed as outlined under Filtration was performed as outlined under Materials and Methods.
Materials and Methods. Units indicate picomoles of inhibitor per millili- Units indicate picomoles of inhibitor per milliliter. Data represent three ter of PDE5 added to the reaction. Data represent three experiments, experiments, each performed in triplicate.
each performed in triplicate.
3H Inhibitor Binding to PDE5
could be important for binding of the double ring of the sildenafil or that provides an indirect contact resulting from inhibitor to human PDE5 (Sung et al., 2003). In addition, the change in the electron distribution in the double ring.
position of the nitrogen atom in the vardenafil double ring Exchange-dissociation experiments using each of the three may impart a change in an atom or group of this molecule 3H inhibitors revealed curvilinear dissociation kinetics, sug- that provides contact with a residue that is not contacted by gesting the presence of two or more catalytic site compo-nents. There was an apparent link between inhibitor potencyand percentage of high-affinity (slow) component of binding.
This could mean that 1) the three 3H inhibitors selecteddifferently for binding to two preexisting populations ofPDE5 having different affinities; 2) the inhibitors had differ-ent potencies for promoting conversion of one population intoanother; or 3) a combination of both mechanisms. Dissocia-tion of 3H inhibitor induced by infinite dilution also displayedheterogeneous kinetics. One possible explanation for thepresence of two or more components of 3H inhibitor dissoci-ation is that PDE5 exists in different conformations (Franciset al., 1998). PDE2 (Manganiello et al., 1990) and PDE4 (Laliberte et al., 2000) also demonstrated kinetic heteroge-neity, which was interpreted to represent different enzymeconformations. The present report is the first to extensivelydemonstrate that the PDE5 catalytic site exhibits more thanone kinetic state, but whether or not it was caused by the Fig. 9. Effect of cGMP on [3H]tadalafil binding. PDE5 (80 ␮l, 0.07 nM
presence of different PDE5 conformations remains to be final concentration) was added to 2 ml of binding reaction mixture con- proved. It cannot be ruled out that PDE5 undergoes partial taining 3 nM [3H]tadalafil and 0 to 350 ␮M cGMP. The mixtures were modification during preparation, which could explain the then incubated for 45 min on ice. Filtration was performed as outlinedunder Materials and Methods. Units indicate picomoles of inhibitor per heterogeneity observed, although the presence of two compo- milliliter of PDE5 added to the reaction. Data represent three experi- nents is observed in different preparations of recombinant ments, each performed in triplicate.
PDE5 and native PDE5. Regardless, caution must now beused in interpreting binding isotherm K values that assume the presence of a single component in the calculation (Corbinet al., 2003).
Cooperativity of inhibitor binding to PDE5 might occur if binding of inhibitors to the catalytic site of one of the twosubunits affects binding to the other subunit. However, in-hibitor dissociation after infinite dilution in the absence andpresence of excess unlabeled inhibitor indicated that this isnot the case, at least under the conditions used for the ex-periment.
Whereas the molecular mechanism for stimulation of PDE5 catalytic activity by cGMP binding to the regulatorydomain is unknown, it is suggested that cGMP binding tothis domain relieves PDE5 of an autoinhibitory constraint, atwhich point filling of the catalytic site at subsaturating sub-strate levels of cGMP is facilitated, increasing catalytic ac-tivity. This negative feedback mechanism promotes rapid Fig. 10. Effect of cGMP on [3H]tadalafil binding using varying concen-
degradation of cGMP within the cell. This negative feedback trations of [3H]tadalafil. PDE5 (80 ␮l, 0.07 nM final concentration inassay) was added to 2 ml of binding reaction mixture containing 0.05 to could be problematic for individuals with erectile dysfunction 6.4 nM [3H]tadalafil in the absence and presence of 10 ␮M cGMP, and the who are unable to maintain the high level of cGMP in the mixture was incubated for 45 min on ice. Filtration was performed as corpus cavernosum for the extended time that is required to outlined under Materials and Methods. Units indicate picomoles of in- achieve and maintain penile erection. This potential defi- hibitor per milliliter of PDE5 added to the reaction. Data represent threeexperiments, each performed in triplicate.
ciency is apparently overcome by the presence of nonhydro- TABLE 3Head-to-head comparison of PDE5-specific inhibitor potencies (affinities)Student's t tests indicated that KD values for each unlabeled inhibitors were significantly different from each other with the exception of the KD value of sildenafil obtainedfrom 1/2 EC50, which was significantly different (p ⬍ 0.05) from all other KD and IC50 values for sildenafil. The IC50 value for vardenafil was significantly different (p ⬍ 0.05)from all KD values for vardenafil.
D from Exchange-Dissociation Average IC50 from Literature Blount et al.
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Breastfeeding is Best! It seems that every year in the summer just before remember being quite shocked to see an old picture of Canadians celebrate World Breastfeeding Week there one of my children bottle feeding when I ‘remembered' is media coverage of something that undermines him as exclusively breastfed. breastfeeding. This year we seem to have gotten an


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