A 46-amino acid segment in phosphodiesterase-5 GAF-B domain provides for high vardenafil potency over sildenafil and tadalafil and is involved in phosphodiesterase-5 dimerization.
Phosphodiesterase-5 (PDE5) contains a catalytic domain (C domain) that hydrolyzes cGMP and a regulatory domain (R domain) that contains two mammalian cGMP-binding phosphodiesterase, Anabaena adenylyl cyclases, Escherichia coli FhlAs (GAFs) (A and B) and a phosphorylation site for cyclic nucleotide-dependent protein kinases (cNPKs). Binding of cGMP to GAF-A increases cNPK phosphorylation of PDE5 and improves catalytic site affinity for cGMP or inhibitors. GAF-B contributes to dimerization of PDE5, inhibition of cGMP binding to GAF-A, and sequestration of the phosphorylation site. To probe potential PDE5 R domain effects on catalytic site affinity for certain inhibitors, four N-terminal truncation mutants were generated: PDE5Delta1-321 contained GAF-B domain, C domain, and the sequence between GAF-A and -B; PDE5Delta1-419 contained GAF-B and C domain; PDE5Delta1-465 contained the C domain and the C-terminal portion of GAF-B; and PDE5Delta1-534 contained only C domain. Truncated proteins with a complete GAF-B were dimers, but those lacking the N-terminal 46 amino acids of GAF-B were monomers, indicating that these residues are vital for GAF-B-mediated PDE5 dimerization. K(m) values of the mutants for cGMP were similar to that of full-length PDE5. All PDE5 constructs had similar affinities for 3-isobutyl-1-methylxanthine, sildenafil, tadalafil, and UK-122764, but mutants containing a complete GAF-B had 7- to 18-fold higher affinity for vardenafil-based compounds compared with those lacking a complete GAF-B. This indicated that the N-terminal 46 amino acids in GAF-B are required for high vardenafil potency. This is the first evidence that PDE5 R domain, and GAF-B in particular, influences affinity and selectivity of the catalytic site for certain classes of inhibitors.
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CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL GENE-Y GENE-N GENE-N GENE-N GENE-Y CHEMICAL GENE-Y GENE-Y GENE-N GENE-N GENE-N GENE-N GENE-N GENE-N GENE-Y GENE-Y CHEMICAL GENE-Y GENE-N GENE-N GENE-N GENE-N GENE-N GENE-Y GENE-N GENE-Y GENE-N CHEMICAL GENE-N GENE-Y GENE-N GENE-N GENE-Y GENE-N GENE-Y GENE-N GENE-Y GENE-Y CHEMICAL GENE-Y GENE-N CHEMICAL CHEMICAL CHEMICAL CHEMICAL
N amino acids vardenafil adenylyl cyclic nucleotide cGMP cGMP cGMP N cGMP N C sildenafil tadalafil amino acid vardenafil Phosphodiesterase-5 GAF-B GAF-B GAF-B PDE5 amino acids PDE5 PDE5 mammalian cGMP-binding phosphodiesterase GAF-B GAF-B GAF-B PDE5 R domain GAF-B Anabaena adenylyl cyclases Escherichia coli FhlAs cGMP PDE5 (GAFs) (A and B) cyclic nucleotide-dependent protein kinases cNPKs GAF-A cNPK PDE5 GAF-B PDE5 GAF-A 3-isobutyl-1-methylxanthine PDE5 R domain PDE5Delta1-321 GAF-B domain GAF-A and -B PDE5Delta1-419 GAF-B and C domain PDE5Delta1-465 GAF-B PDE5Delta1-534 phosphodiesterase-5 sildenafil phosphodiesterase-5 GAF-B domain tadalafil UK-122764 vardenafil cGMP