title: Binding of tritiated sildenafil, tadalafil, or vardenafil to the phosphodiesterase-5 catalytic site displays potency, specificity, heterogeneity, and cGMP stimulation.
abstract: 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.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0615, USA.
authors.forename: Mitsi A
authors.initials: MA
authors.lastname: Blount
authors.forename: Alfreda
authors.initials: A
authors.lastname: Beasley
authors.forename: Roya
authors.initials: R
authors.lastname: Zoraghi
authors.forename: Konjeti R
authors.initials: KR
authors.lastname: Sekhar
authors.forename: Emmanuel P
authors.initials: EP
authors.lastname: Bessay
authors.forename: Sharron H
authors.initials: SH
authors.lastname: Francis
authors.forename: Jackie D
authors.initials: JD
authors.lastname: Corbin
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases; D001665:Binding Sites; D002243:Carbolines / Q000737:chemistry / Q000494:pharmacology*; D020134:Catalytic Domain; D006152:Cyclic GMP / Q000378:metabolism*; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010879:Piperazines / Q000737:chemistry / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D011687:Purines; D000068677:Sildenafil Citrate; D013450:Sulfones; D000068581:Tadalafil; D014227:Triazines; D014316:Tritium; D000069058:Vardenafil Dihydrochloride
chemical_list: D002243:Carbolines; D007093:Imidazoles; D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D013450:Sulfones; D014227:Triazines; D014316:Tritium; D000069058:Vardenafil Dihydrochloride; D000068581:Tadalafil; D000068677:Sildenafil Citrate; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; D006152:Cyclic GMP