Multiple Conformations of Phosphodiesterase-5: IMPLICATIONS FOR ENZYME FUNCTION AND DRUG DEVELOPMENT

Phosphodiesterase-5 (PDE5) is the target for sildenafil, vardenafil, and tadalafil, which are drugs for treatment of erectile dysfunction and pulmonary hypertension. We report here the crystal structures of a fully active catalytic domain of unliganded PDE5A1 and its complexes with sildenafil or icarisid II. These structures together with the PDE5A1-isobutyl-1-methylxanthine complex show that the H-loop ( residues 660-683) at the active site of PDE5A1 has four different conformations and migrates 7-35 angstrom upon inhibitor binding. In addition, the conformation of sildenafil reported herein differs significantly from those in the previous structures of chimerically hybridized or almost inactive PDE5. Mutagenesis and kinetic analyses confirm that the H-loop is particularly important for substrate recognition and that invariant Gly(659), which immediately precedes the H-loop, is critical for optimal substrate affinity and catalytic activity

Phosphorylation increases affinity of the phosphodiesterase-5 catalytic site for tadalafil

ABSTRACT Phosphodiesterase-5 (PDE5) is phosphorylated at a single serine residue by cyclic nucleotide-dependent protein kinases. To test for a direct effect of phosphorylation on the PDE5 catalytic site, independent of cGMP binding to the allosteric sites of the enzyme, binding of the catalytic site-specific substrate analog [ 3 H]tadalafil to PDE5 was measured. Phosphorylation increased [ 3 H]tadalafil binding 3-fold, whereas cGMP caused a 1.6-fold increase. Combination of both treatments caused more than 4-fold increase in [ 3 H]tadalafil binding, and effects were additive only at submaximal stimulation. Consistent with the increase in affinity, phosphorylation slowed the [ 3 H]tadalafil exchange-dissociation rate from PDE5 more than 6-fold. Finally, phosphorylation increased affinity for hydrolysis of a catalytic site-specific cGMP analog, 2Ј-O-anthraniloylcGMP, by ϳ3-fold. The combined results showed that phosphorylation activates PDE5 catalytic site independently of cGMP binding to the allosteric sites. The results suggested that phosphorylation acts in concert with allosteric cGMP binding to stimulate the PDE5 catalytic site, which should promote negative feedback regulation of the cGMP pathway in intact cells. By increasing the affinity of the catalytic site, phosphorylation should also consequently increase the potency and duration of PDE5 inhibitor action

Novel inhibitors of the methicillin-resistant <i>Staphylococcus aureus</i> (MRSA)-pyruvate kinase

<p>Novel bisindolyl-cycloalkane indoles resulted from the reaction of aliphatic dialdehydes and indole. As bisindolyl-natural alkaloid compounds have recently been reported as inhibitors of the methicillin-resistant <i>Staphylococcus aureus</i> (MRSA)-pyruvate kinase (PK), we tested our novel compounds as MRSA PK inhibitors and now report first inhibiting activities. We discuss structure–activity relationships of structurally varied compounds. Activity influencing substituents have been characterized and relations to antibacterial activities of the most active compounds have been proved.</p

Cyclic nucleotide specific phosphodiesterases of the kinetoplastida: A unified nomenclature

Fil: Kunz, Stefan. University of Bern; SuizaFil: Beavo, Joseph A.. University of Washington; Estados UnidosFil: D'angelo, Maximiliano A. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Flawia, Mirtha Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Francis, Sharron H.. Vanderbilt University School of Medicine; Estados UnidosFil: Johner, Andrea. University of Bern; SuizaFil: Laxman, Sunil. University of Washington; Estados UnidosFil: Oberholzer, Michael. University of Bern; SuizaFil: Rascon, Ana. Universidad Central de Venezuela; VenezuelaFil: Shakur, Yasmin. Otsuka Maryland Medicinal Laboratories; Estados UnidosFil: Wentzinger, Laurent. University of Bern; SuizaFil: Zoraghi, Roya. Vanderbilt University School of Medicine; Estados UnidosFil: Seebeck, Thomas. University of Bern; Suiz