High Biochemical Selectivity of Tadalafil, Sildenafil and Vardenafil for Human Phosphodiesterase 5A1 (PDE5) Over PDE11A4 Suggest

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Original Research High biochemical selectivity of tadalafil, sildenafil and vardenafil for human phosphodiesterase 5A1 (PDE5) over PDE11A4 suggests the absence of PDE11A4 cross-reaction in patients

JL Weeks II1, R Zoraghi1, A Beasley1, KR Sekhar2, SH Francis1 and JD Corbin1*

1Department of Molecular Physiology and Biophysics, Vanderbilt University, School of Medicine, Nashville, Tennessee, USA; and 2Vanderbilt Center for Radiation Oncology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

The physiological role of phosphodiesterase (PDE)11 is unknown and its biochemical characteristics are poorly understood. We have expressed human His-tagged PDE11A4 and purified the enzyme to apparent homogeneity. PDE11A4 displays Km values of 0.97 lMforcGMPand2.4lMfor cAMP, and maximal velocities were 4- to 10-fold higher for cAMP than for cGMP. Given the homology between PDE11 and PDE5, we have compared the biochemical potencies of tadalafil (Cialist, Lilly-ICOS), vardenafil (Levitrat, Bayer-GSK), and sildenafil (Viagrat, Pfizer Inc.) for PDE11A4 and PDE5A1. PDE5A1/PDE11A4 selectivities are 40-, 9300-, and 1000-fold for tadalafil, vardenafil, and sildenafil, respectively. This suggests that none of these three compounds is likely to crossreact with PDE11A4 in patients. International Journal of Impotence Research (2005) 17, 5–9. doi:10.1038/sj.ijir.3901283 Published online 11 November 2004

Keywords: PDE11; PDE5; vardenafil; sildenafil; tadalafil; PDE5 inhibitors

Introduction dampening of cellular CN signaling; it is therefore of great interest to determine the biochemical characteristics that provide for interaction of PDEs with The cyclic nucleotides (CNs), cAMP and cGMP, are CNs and related ligands.1–5 second messengers that are of particular import in The mammalian PDE superfamily currently con- cellular signaling. The CNs are involved in the sists of 11 members that preferentially hydrolyze regulation of most physiological processes and have cAMP (PDE4, PDE7, PDE8) or cGMP (PDE5, PDE6, multiple intracellular targets in mammals including PDE9) or both cAMP and cGMP (PDE1, PDE2, CN-gated channels, guanine nucleotide exchange PDE3, PDE10, PDE11).5 PDE11 is the most recently factors, CN-specific phosphodiesterases (PDE), CN- described member of this superfamily, and its binding PDEs, and CN-dependent protein kinases. biochemical characteristics and physiological roles Cellular CN levels are modulated by changes in are poorly understood. Research focused on the PDE the rate of CN synthesis by adenylyl or guanylyl superfamily has proved to be of great consequence cyclases and the rate of CN breakdown by PDEs. The in the treatment of numerous human maladies and PDEs are a superfamily of metallophosphohydro- disease. Specific inhibitors of members of the PDE lases that catalyze the hydrolysis of the CNs to their superfamily are currently being investigated for respective 50 noncyclic forms. Hydrolysis of CNs by treatment of asthma, acute ischemic stroke, cancer, PDEs provides the major cellular mechanism for the systemic inflammation, pulmonary hypertension, and many others.5,6 PDE inhibitors have been approved for clinical use in the treatment of intermittent claudication, congestive heart failure, *Correspondence: JD Corbin, Department of Molecular chronic obstructive pulmonary disease and erectile Physiology and Biophysics, Light Hall Room 702, 21st & 6 Garland, Vanderbilt University School of Medicine, dysfunction. Given the therapeutic implications of Nashville, TN 37232-0615, USA. PDEs, there is strong precedence for investigations E-mail: [email protected] of poorly understood members such as PDE11. Received 21 July 2004; revised 5 October 2004; accepted 5 Four human PDE11 variants (PDE11A1–A4) have October 2004 been reported (Figure 1); these arise from a single Selectivity of PDE5 inhibitors for PDE5 over PDE11 JL Weeks et al 6 also been reported to cause similar side effects such as myalgia,16 and a recent study concluded that there was no effect of tadalafil on spermatogenesis in men.14 Herein, we present biochemical evidence comparing the potencies of tadalafil, vardenafil, and sildenafil for PDE11A4, and the fold selectivity of these inhibitors for PDE5A1 over PDE11A4.

Methods and Results

Expression and purification of recombinant human PDE11A4

The cDNA clone encoding human PDE11A4 was a Figure 1 PDE11 splice variants in humans. All PDE11 variants generous gift from Tanabe Seiyaku Co., Ltd (Saitama, consist of an N-terminal regulatory domain (R domain) and a conserved C-terminal catalytic domain (C domain). The amino- Japan). To generate an expression plasmid for full- acid lengths of each PDE11A variant are shown in the far right length PDE11A4, this cDNA was amplified by High of the figure. PDE11A4 contains two complete GAF domains, Fidelityt PCR System (Roche Molecular Biochem- which have been noted as GAF a and GAF b in keeping icals) using the forward primer, PDE11A4For (50- with the nomenclature used for the two GAFs in PDEs 2, 5, and 6. GTAGTCGACATGGCAGCCTCCCGCCTGGAC-30)and PDE11A2 and PDE11A3 each contain one complete GAF b 0 domain and an incomplete GAF a domain, while PDE11A1 the reverse primer, PDE11A4Rev (5 -GTAGTCTCGAG contains one incomplete GAF b domain. In addition, the TTAGTTCCTGTCTTCCTTG-30), containing an SalI N-terminal region of PDE11A4 contains two consensus phosphor- and XhoI restriction site (underlined) and the 117 162 ylation sites (Ser and Ser ) for PKA and PKG. original start and termination codons (bolded). The resulting PCR fragment of 2825 bp was cloned into pCR-2.1 TOPOt (Invitrogen) and verified by DNA sequencing. The insert was then excised by diges- gene and hydrolyze both cAMP and cGMP.7–11 All tion with SalI and XhoI and cloned into the pFast- PDE11 variants contain a conserved C-terminal Bact HTc vector (Invitrogen), which was linearized catalytic domain and an N-terminal regulatory by digestion with SalI and XhoI and then ligated domain of varying lengths. By mRNA analysis, after the addition of the fragment. This yielded PDE11 variants demonstrate tissue-specific expres- the expression plasmid for full-length His-tagged sion with PDE11A1 being present in skeletal human PDE11A4 termed His-hPDE11A4. This muscle, PDE11A3 in testis, and PDE11A4 in the expression plasmid was then used to generate a prostate gland.7,9,10 Of the four PDE11A variants, recombinant baculovirus according to the protocol PDE11A4 contains the most extensive regulatory in the Bac-to-Bac Baculovirus Expression System domain structure, suggesting the possibility that this manual (Invitrogen). For large-scale protein produc- variant is both highly regulated and plays an tion, Sf9 (Pharmingen) cells were grown in spinner important physiological role. flasks in Grace’s insect media with 10% fetal bovine The N-terminal regulatory region of PDE11A4 serum, 0.06 mg/ml gentamycin, and 100 mM 3-iso- contains two GAF domains (cGMP-binding PDEs- butyl-1-methylxanthine to a density of 1 Â 106 cells/ anabaena adenylyl cyclase-Fhla transcription factor) ml, and then infected for 4 days at 271C with the as well as two consensus phosphorylation sites for optimal amount of virus. Cells were harvested by protein kinase A (PKA) and protein kinase G (PKG), low-speed centrifugation and suspended in ice-cold although functional effects of either region have not lysis buffer (20 mM Tris-HCl, pH ¼ 8.0, 100 mM been demonstrated.10 The amino-acid sequence of NaCl) containing a mixture of protease inhibitors the catalytic domain of PDE11 is approximately 51% (Protease Complete tablets, Roche Molecular Bio- identical with that of the catalytic domain of PDE5,9 chemicals). Cells were then lysed at 41Cby2Â 4-s suggesting the possibility that commercially avail- bursts using an Ultraturrax homogenizer (Tekmar). able PDE5 inhibitors might crossreact with PDE11. Cell lysate was clarified by centrifugation at 41C for An initial study reported a relatively low selectivity 20 min at 10 000 r.p.m. in a Beckman JA-20 rotor. A (five-fold) of tadalafil for PDE5 over PDE11A1.12 It measure of 1 ml of Ni-NTA agarose beads (Qiagent) has been suggested that PDE11 inhibition could was then poured into a 0.8 Â 0.9 cm2 column account for the back pain and myalgia reported by (Pharmacia Biotech) kept at 41C and the column some men taking tadalafil, as well as for alterations was washed with 100 ml of ice-cold lysis buffer. The in spermatogenesis seen in dogs given tadalafil cell lysate supernatant was then passed over the daily.13–15 Interestingly, other PDE5 inhibitors have column twice and the column was washed exten-

International Journal of Impotence Research Selectivity of PDE5 inhibitors for PDE5 over PDE11 JL Weeks et al 7 sively with lysis buffer containing a stepwise a single band that comigrated with the 100 kDa band gradient of imidazole from 0.8–20 mM. Protein was on SDS-PAGE (Figure 2). The polyclonal antibody eluted from the column with lysis buffer containing generated against the N-terminal regions of 100 mM imidiazole and individual fractions were PDE11A3 and PDE11A4 was a generous gift from collected. PDE activity assays were performed as Tanabe Seiyaku Co., Ltd (Saitama, Japan) and was described17 on eluted fractions and fractions con- used at dilutions from 1:1000 to 1:5000. taining PDE activity were pooled together and dialyzed against 700 volumes of 10 mM potassium phosphate buffer, pH 6.8, containing 25 mM b-mercaptoethanol (KPM) for 48 h. After dialysis, Kinetic analysis of human PDE11A4 the enzyme was quick-frozen using liquid nitrogen in an aqueous solution containing 10% sucrose and 150 mM NaCl and stored at À801C until use. Studies of PDE catalytic activity were performed Human PDE5A1 was expressed and purified as using [3H]cGMP or [3H]cAMP as substrate by the described.17 modified method17 in a final volume of 100 ml. Relative protein concentrations were determined Determination of the Michaelis–Menten constant 18 by the Bradford method using bovine serum (Km value) and maximal velocities were determined albumin (BSA, Sigma) as a standard. Using this as follows: purified PDE11A4 protein (0.7 nM final purification scheme, PDE11A4 was purified to protein concentration) was diluted in KPM-BSA apparent homogeneity as assessed by SDS-PAGE (KPM containing 1 mg/ml BSA) and 10 ml was added followed by Coomassie Blue staining. The enzyme to 90 ml of PDE assay mixture (50 mM Tris-HCl, pH migrated with a molecular weight of approximately 7.5, 10 mM MgCl2, cGMP or cAMP and 0.33 mg/ml 100 kDa (Figure 2), which was consistent with the BSA) supplemented with concentrations of predicted molecular weight of 105 kDa based on [3H]cAMP or [3H]cGMP from 1 to 50 mM (1:20 ratio amino-acid composition and with that of partially of labeled to unlabeled CN). Unlabeled cGMP purified PDE11A4 reported previously.10 Western and cAMP were from Sigma and [3H]cAMP and blotting with a polyclonal antibody against the N- [3H]cGMP were from Amersham Biosciences. The terminal region of PDE11A3 and PDE11A4 revealed reaction mixtures were incubated at 301C for 10 min and terminated by the addition of 20 ml of stop mix (143 mM EDTA, 60 mM theophylline, 30 mM cAMP, 30 mM cGMP, and 286 mM Tris-HCl, pH 7.5) and the tubes were transferred to an ice-water bath. Next, snake venom nucleotidase (200 mg, Sigma) was added to each tube and reactions were incubated for 10 min at 301C, after which the tubes were transferred to an ice-water bath and 1 ml of ice-cold dilution solution (0.1 mM adenosine, 0.1 mM gua- nosine, and 15 mM EDTA) was added. The reaction mixtures were then applied to 2.1 mL QAE–Sepha- dex (Pharmacia) anion-exchange columns that had been equilibrated with 0.02 M ammonium formate, pH 7.5, and the flow-through was collected. The columns were then washed with 2 ml of 0.02 M ammonium formate, pH 7.5, and this flow-through was also collected and combined with the first flow- through. The combined eluates were then placed in scintillation fluid and counted in a scintillation counter. Counts per minute were converted to pmol of CN hydrolyzed/min/ml of enzyme. The nonlinear regression function of GraphPad Prism was then used to analyze the data and determine the Km and Figure 2 SDS-PAGE and immunoblot of purified human maximal velocities. PDE11A4. Lanes 1 and 2 are a 10% SDS-PAGE gel stained The K values of purified human PDE11A4 were with Coomassie Brilliant Blue, and lane 3 is an immunoblot. m 2.470.96 mM for cAMP and 0.9770.08 mM for cGMP, Recombinant human His-tagged PDE11A4 was purified to 10 apparent homogeneity on SDS-PAGE as described in Methods which agreed well with those reported previously. and Results and migrated with an apparent molecular weight of Error values are standard error of the mean of values 100 kDa. Lane 1 shows low-molecular-weight standards with from at least two experiments, each of which was molecular weight (in kDa) shown to the left. Lane 2 contains 1 mg of purified PDE11A4. Western blot analysis of purified human performed in triplicate. In all experiments per- PDE11A4 (lane 3) revealed a single band that comigrated with the formed, maximal velocities for PDE11A4 were four- purified protein shown in lane 2. to 10-fold higher for cAMP than for cGMP (Figure 3).

International Journal of Impotence Research Selectivity of PDE5 inhibitors for PDE5 over PDE11 JL Weeks et al 8

Figure 3 A representative experiment demonstrating the determination of Km and maximal velocity values for cAMP and cGMP of purified human PDE11A4. Km values and maximal velocities were determined as described in Methods and Results. Average Km values for purified PDE11A4 were determined to be 0.97 mM for cGMP and 2.4 mM for cAMP and maximal velocities were 4- to 10-fold higher for cAMP than for cGMP.

Biochemical potency of CNs and CN analogs Table 1 IC50 values of cyclic nucleotides and inhibitors for a for inhibition of PDE11A4 and PDE5A1 PDE11A4

Compound IC50 (nM) The IC values (inhibitor concentration at 50% 50 7 inhibition) for cAMP, cGMP, sildenafil, tadalafil, Tadalafil 73 3.1 cGMP 560722 vardenafil, and dipyridamole were determined for Dipyridamole 8407220 PDE11A4 using the PDE catalytic activity assay Vardenafil 8407160 with cGMP as substrate. The assay was performed as cAMP 1200763 described above, using a final concentration of Sildenafil 38007750

labeled and unlabeled cGMP of 100 nM (80 nM a [3H]cGMP and 20 nM cGMP). This concentration of Error values are standard error of the mean of values from at least cGMP was approximately 10 times below the two experiments, each of which was performed in triplicate. apparent Km for cGMP and ensured that the IC50 19–21 value approximated the Ki value. Sildenafil was purified from Viagrat tablets as described.22 Tada- nafil and sildenafil was also reported for PDE5A1, 17 lafil was synthesized in-house as described. where the IC50 of vardenafil was markedly lower 17 Vardenafil was a generous gift from Bayer Inc. than that of sildenafil. The IC50 values for cGMP Dipyridamole was obtained from Sigma. All drugs and cAMP were 560 and 1200 nM, respectively, were diluted serially in Milliporet water from their which were in reasonable agreement with Km values respective stock solutions. CN stock solutions were for these nucleotides. The PDE inhibitor dipyrida- prepared in Milliporet water. Stock dipyridamole, mole had an IC50 of 840 nM. The IC50 value for vardenafil, and tadalafil were prepared in 100% dipyridamole and Km values for cAMP and cGMP DMSO and stock sildenafil was prepared in 100% agreed well with those reported previously,10 and ethanol. The highest concentrations of either DMSO verified the structural integrity of the PDE11A4 or ethanol used in the assay did not affect catalytic preparation. activity (not shown). Reactions were started by the The IC50 values of tadalafil, vardenafil, and addition of 10 ml of PDE11A4 (final concentration sildenafil for PDE11A4 and PDE5A1 were then was 2.1 nM) diluted in KPM-BSA and the catalytic compared and fold selectivity values were calcu- assay was performed as described above. The IC50 lated (Table 2). IC50 values for sildenafil, vardenafil, values were calculated using GraphPad Prism soft- and tadalafil for PDE5 have been previously ware. The concentrations of inhibitors were selected reported by our laboratory.17 Of the three PDE5 so that nearly 100% inhibition was attained in each inhibitors, vardenafil was the most selective for experiment. PDE5, with a 9300-fold selectivity ratio over Table 1 shows the IC50 of each of the three PDE5 PDE11A4. Sildenafil and tadalafil demonstrated inhibitors for purified recombinant human selectivity ratios of 1000- and 40-fold, respectively. PDE11A4. Error values are standard error of the The 40-fold selectivity ratio of tadalafil for PDE5 mean and represent at least two experiments, each over PDE11 reported here is significantly higher performed in triplicate. Of the commercially avail- than that reported previously using PDE11A1, able PDE5 inhibitors, tadalafil was the most potent instead of PDE11A4.12 This discrepancy could be for PDE11A4 with an IC50 value of 73 nM. Vardenafil due to differences in the kinetic properties of and sildenafil demonstrated a 4.5-fold difference in PDE11A1 and PDE11A4 that may arise from differ- potency for PDE11A4, with IC50 values of 840 and ences in N-terminal regulatory domains between 3800 nM, respectively. A difference between varde- these variants.10–11

International Journal of Impotence Research Selectivity of PDE5 inhibitors for PDE5 over PDE11 JL Weeks et al 9 Table 2 Selectivity of PDE5 inhibitors for PDE5 over PDE11A4 cular Biology and Molecular Medicine. The Humana Press, Inc.: Towata, in press. Compound IC50 (nM) Fold selectivity for PDE5 2 Charbonneau H. Structure–function relationships among cyclic nucleotide phosphodiesterases. In: Beavo JA, Houslay PDE5A1 PDE11A4 MD (eds). Cyclic Nucleotide Phosphodiesterases: Structure, Regulation, Drug Action. Wiley: New York, 1990 pp 267–296. 3 Burns F, Zhao AZ, Beavo JA. Cyclic nucleotide phosphodies- Vardenafil 0.09170.031 8407160 9300 terases: gene complexity, regulation by phosphorylation, Sildenafil 3.771.4 38007750 1000 physiological implications [Review]. Adv Pharmacol (New Tadalafil 1.870.4 7373.1 40 York) 1996; 36: 29–48. 4 Francis SH, Corbin JD. Cyclic nucleotide-dependent protein kinases: intracellular receptors for cAMP and cGMP action. Crit Rev Clin Lab Sci 1999; 36: 275–328. 5 Francis SH, Turko IV, Corbin JD. Cyclic nucleotide phosphodiesterases: relating structure and function. Prog Nucleic Acid Concluding remarks Res Mol Biol 2001; 65: 1–52. 6 Lin CS et al. Phosphodiesterases as therapeutic targets. Urology 2003; 61: 685–691. With the expanding use of PDE5 inhibitors in the 7 Hetman JM et al. Cloning and characterization of two splice treatment of erectile dysfunction and other maladies variants of human phosphodiesterase 11A. Proc Natl Acad Sci USA 2000; 97: 12891–12895. such as pulmonary hypertension, there is increased 8 Fawcett L et al. Molecular cloning and characterization of interest in understanding potential cross-reaction of a distinct human phosphodiesterase gene family: PDE11A. these drugs with other proteins. Cross-reaction with Proc Natl Acad Sci USA 2000; 97: 3702–3707. proteins other than PDE5 would suggest the poten- 9 Yuasa K et al. Genomic organization of the human phospho- tial for side effects or safety issues. PDE11 is an diesterase PDE11A gene. Evolutionary relatedness with other PDEs containing GAF domains. Eur J Biochem 2001; 268: obvious candidate for such a role since it is closely 9 168–178. related to PDE5. We now present biochemical 10 Yuasa K et al. Isolation and characterization of two novel potencies of sildenafil, vardenafil, and tadalafil for phosphodiesterase PDE11A variants showing unique struc- purified recombinant human PDE11A4 and estab- ture and tissue-specific expression. J Biol Chem 2000; 275: 31469–31479. lish the following order of potency for PDE11A4: 11 Yuasa K et al. Identification of rat cyclic nucleotide phospho- tadalafilbvardenafil4sildenafil. Comparison of the diesterase 11A (PDE11A): comparison of rat and human fold selectivity for PDE5 over PDE11A4 for sildenafil PDE11A splicing variants. Eur J Biochem 2001; 268: (1000-fold selectivity) and vardenafil (9000-fold 4440–4448. selectivity) reveals that these drugs are very unlikely 12 Gbekor E et al. Selectivity of sildenafil and other phosphodiesterase type 5 (PDE5) inhibitors against all human to crossreact with PDE11A4 in patients taking the phosphodiesterase families. Eur Urol 2002; 1(Suppl 1): 63. prescribed dosage of these medications. Tadalafil 13 Gresser U, Gleiter CH. Erectile dysfunction: comparison of demonstrated a 40-fold selectivity for PDE5 over efficacy and side effects of the PDE5-inhibitors sildenafil, PDE11A4, making it also unlikely that tadalafil vardenafil and tadalafil-review of the literature. Eur J Med Res 2002; 7: 435. would crossreact with PDE11A4 in patients. 14 Hellstrom W et al. Tadalafil has no detrimental effect on human spermatogenesis or reproductive hormones. J Urol 2003; 170: 887–891. 15 Pomara G, Morelli G. Re: tadalafil has no detrimental effect on Acknowledgements human spermatogenesis or reproductive hormones. J Urol 2004; 171(Part 1): 2390–2391. 16 Cheitlin MD et al. Use of sildenafil (Viagra) in patients with We thank Drs Jun Kotera and Kenji Omori of Tanabe cardiovascular disease. J Am Coll Cardiol 1999; 33: 273–282. Seiyaku Co., Ltd (Saitama, Japan) for the generous 17 Blount MA et al. Binding of tritiated sildenafil, tadalafil, or gifts of PDE11A3/A4 polyclonal antibody as well as vardenafil to PDE5 catalyic site displays potency, specificity, heterogeneity, and cGMP stimulation. Mol Pharmacol 2004; the cDNA encoding human PDE11A4. We also thank 66: 144–152. Bayer Inc. for kindly providing purified vardenafil 18 Bradford MM. A rapid and sensitive method for the quantita- (Levitrat, Bayer-GSK). This work was supported by tion of microgram quantities of protein utilizing the principle NIH Grants DK58277 and DK40029, NIH Training of protein-dye binding. Anal Biochem 1976; 72: 248–254. 19 Ballard SA et al. Effects of sildenafil on the relaxation of Grant 5T32HL07752, and AHA Postdoctoral Grant human corpus cavernosum tissue in vitro and on the activities 032525B. of cyclic nucleotide phosphodiesterase isozymes. J Urol 1998; 159: 2164–2171. 20 Corbin J et al. [3H]sildenafil binding to phosphodiesterase-5 is specific, kinetically heterogenous, and stimulated by cGMP. References Mol Pharmacol 2003; 63: 1364–1372. 21 Bush K. Screening and characterization of enzyme inhibitors as drug candidates. Drug Metab Rev 1983; 14: 689–708. 1 Weeks JL et al. Radioligand binding to the catalytic sites or 22 Francis S et al. Single step isolation of sildenafil from allosteric sites of PDE5 and PDE11. In: Lugnier C (ed). commercially available Viagra tablets. Int J Impot Res 2003; Phosphodiesterase Methods and Protocols: Methods in Mole- 15: 369–372.

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