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

Abstract

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: PDE5⌬1-321 contained GAF-B domain, C domain, and the sequence between GAF-A and -B; PDE5⌬1-419 contained GAF-B and C domain; PDE5⌬1-465 contained the C domain and the C-terminal portion of GAF-B; and PDE5⌬1-534 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.

Conversion of Phosphodiesterase-5 (PDE5) Catalytic Site to Higher Affinity by PDE5 Inhibitors

“…All constructs were confirmed by DNA sequencing before cotransfection with BaculoGold linear baculovirus DNA (BD Biosciences PharMingen) according to the protocol from BD Biosciences PharMingen. Proteins were expressed in Sf9 cells, purified to near homogeneity using Ni 2ϩ -NTA resin and characterized as described previously (Blount et al, 2006). Constructs were flash frozen in Tris buffer (20 mM Tris, pH 8, and 50 mM NaCl) containing a final concentration of 20% glycerol.…”

“…The R domain contains several functional subdomains, including a phosphorylation site (Ser-102 in hPDE5), allosteric cGMP binding sites, and dimerization contacts (Thomas et al, 1992;McAllister-Lucas et al, 1995;Zoraghi et al, 2005;Blount et al, 2006). Binding of inhibitors such as 3-isobutyl-1-methylxanthine or sildenafil to the PDE5 catalytic site increases affinity of the allosteric site in the R domain for cGMP (Francis et al, 1980;Corbin and Francis, 1999).…”

Interactions between Cyclic Nucleotide Phosphodiesterase 11 Catalytic Site and Substrates or Tadalafil and Role of a Critical Gln-869 Hydrogen Bond

“…It has been demonstrated using solution biochemistry that many of the kinetic characteristics of an isolated C domain can be quite similar to those of the holoenzyme (Fink et al, 1999;Wang et al, 2006;Weeks et al, 2007). However, other characteristics of PDE holoenzymes can influence contacts and potency of certain inhibitors (e.g., dimerization, phosphorylation, or additional structural features) (Richter and Conti, 2004;Blount et al, 2006). Because these earlier reports demonstrated that significant structural differences can exist between a PDE holoenzyme and its isolated C domain in solution, it seems equally plausible that contacts defined from an X-ray crystal structure of an isolated C domain and certain ligands may not precisely recapitulate those that occur in the holoenzyme.…”

“…Because these earlier reports demonstrated that significant structural differences can exist between a PDE holoenzyme and its isolated C domain in solution, it seems equally plausible that contacts defined from an X-ray crystal structure of an isolated C domain and certain ligands may not precisely recapitulate those that occur in the holoenzyme. Thus, for development of the most accurate insights into the biochemical properties of interaction between PDEs and ligands, it is important to compare results derived from biochemical studies of PDE11 holoenzyme in solution with insights and predictions derived from X-ray crystal structures of other PDEs (Sung et al, 2003;Zhang et al, 2004;Blount et al, 2006).…”

“…Some selectively hydrolyze cAMP or cGMP, whereas others hydrolyze both (Beavo et al, 2006;Conti and Beavo, 2007). Important advances have been gained from X-ray crystal structures of isolated catalytic domains (C domain) of several PDEs, but contacts between catalytic site amino acids of PDE holoenzymes and cyclic nucleotides (CN) or inhibitors are not fully understood for any PDE (Huai et al, 2003;Xu et al, 2004;Blount et al, 2006;Ke and Wang, 2006;Wang et al, 2006Wang et al, , 2007Zoraghi et al, 2007). The PDE11 family is derived from a single gene that produces four splice variants; all hydrolyze cAMP and cGMP, although kinetic characteristics for the variants are different (Fawcett et al, 2000;Hetman et al, 2000;Yuasa et al, 2000;Weeks et al, 2007).…”