RESUMO
Cyclic nucleotide phosphodiesterases (PDEs) have been investigated for years as targets for therapeutic intervention in a number of pathophysiological processes. Phosphodiesterase-5 (PDE5), which is highly specific for guanosine 3'-5'-cyclic-monophosphate (cGMP) at both its catalytic site and its allosteric sites, has generated particular interest because it is potently and specifically inhibited by three drugs: sildenafil (Viagra, Pfizer), tadalafil (Cialis, Lilly-ICOS), and vardenafil (Levitra, Bayer GSK). Previously, we have used [(3)H]cGMP to directly study the interaction of cGMP with the allosteric sites of PDE5, but because cGMP binds with relatively low affinity to the catalytic site, it has been difficult to devise a binding assay for this particular binding reaction. This approach using measurement of radiolabeled ligand binding continues to allow us to more precisely define functional features of the enzyme. We now use a similar approach to study the characteristics of high-affinity [(3)H]inhibitor binding to the PDE5 catalytic domain. For these studies, we have prepared [(3)H]sildenafil and [(3)H]tadalafil, two structurally different competitive inhibitors of PDE5. The results demonstrate that radiolabeled ligands can be used as probes for both catalytic site and allosteric site functions of PDE5. We describe herein the methods that we have established for studying the binding of radiolabeled ligands to both types of sites on PDE5. These techniques have also been successfully applied to the study of binding of radiolabeled PDE5 inhibitors to PDE11, suggesting that these methods are applicable to the study of other PDEs, and perhaps other enzyme families.
