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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

Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee (M.A.B., R.Z., E.P.B., J.D.C., S.H.F.); and Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina (H.K.)

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: PDE51-321 contained GAF-B domain, C domain, and the sequence between GAF-A and -B; PDE51-419 contained GAF-B and C domain; PDE51-465 contained the C domain and the C-terminal portion of GAF-B; and PDE51-534 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.

Address correspondence to: Dr. Sharron H. Francis, Department of Molecular Physiology and Biophysics, 702 Light Hall, Vanderbilt University School of Medicine, Nashville, TN 37232-0615. E-mail: sharron.francis{at}vanderbilt.edu

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