Characterization of phosphodiesterase catalytic sites by means of cyclic nucleotide derivatives

Couchie, Dominique; Petridis, Georg; Jastorff, Bernd; Erneux, Christophé

doi:10.1111/j.1432-1033.1983.tb07778.x

Cited by 18 publications

(6 citation statements)

References 25 publications

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“…To quantify the role of this position in PDE11, N7 was replaced with a carbon (7-deaza-cGMP). This compound had 16-fold lower affinity for the PDE11 catalytic site (K i ϭ 5.4 Ϯ 2.0 M) than did cGMP; the extent of the change in affinity with this modification was similar to that found previously with certain other PDEs (Couchie et al, 1983).…”

supporting

confidence: 53%

“…In addition, these results revealed considerable spatial tolerance for substituents at this position and in the region extending from N1 and N 2 . Realization of the importance of contact with N7 of CNs in some PDE catalytic sites is longstanding (Couchie et al, 1983). To quantify the role of this position in PDE11, N7 was replaced with a carbon (7-deaza-cGMP).…”

mentioning

confidence: 99%

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

Weeks

Corbin²,

Francis³

2009

J Pharmacol Exp Ther

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Poor understanding of the topography of cyclic nucleotide (CN) phosphodiesterase (PDE) catalytic sites compromises development of potent, selective inhibitors for therapeutic use. In the X-ray crystal structures of the catalytic domains of some PDEs, an invariant glutamine hydrogen bonds with groups at C6 and N1 or N7 on catalytic products or analogous positions of some inhibitors, inferring similar bonds with CNs (Nature 425: 98 -102, 2003; J Mol Biol 337:355-365, 2004; Mol Cell 15:279 -286, 2004). A site-directed mutant (Q869A) lacking this invariant Gln in cGMP-/cAMP-hydrolyzing PDE11 had unaltered catalytic activity and affinity for sildenafil; but cGMP/cAMP or tadalafil affinity was reduced ϳ50-or 140-fold, respectively, and calculated free energy of binding suggested one hydrogen bond for each. A cGMP analog lacking the C6 oxygen had ϳ80-fold weakened affinity, modifications at N 2 , N7, or 2Ј-OH diminished affinity ϳ16-fold, and analogs with groups appended at N1 had only 2-to 6-fold weakened affinity. Analogs with C8 substitutions were ineffective inhibitors, suggesting that cGMP binds in the anti conformation. Calculated decline in free energy of binding was consistent with that for one hydrogen bond only in the analog lacking binding potential at C6. In conclusion, Gln-869 interacts strongly with cGMP/cAMP and tadalafil, but not with sildenafil; interactions with CN analogs suggest a hydrogen bond only between Gln-869 and the C6 substituent. The results define interactions between the PDE11 catalytic site and substrates/inhibitors and advance potential for inhibitor design.There are 11 human cyclic nucleotide phosphodiesterase (PDE) families. 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). To date, there are no potent, specific inhibitors for the PDE11 family. Tadalafil (Cialis; Lilly-ICOS Co., Indianapolis, IN), which potently inhibits PDE5, also inhibits PDE11 albeit with significantly lower affinity (Weeks et al., 2007); sildenafil (Viagra) and vardenafil (Levitra) are weak inhibitors of PDE11. Most characteristics of the PDE11 catalytic site are unknown.Models of CNs bound in PDE catalytic sites have been based on X-ray crystal structures of isolated C domains in complex with low-affinity 5Ј-nucleotide catalytic product or substrate-analog inhibitors because a cocrystal of a ...

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supporting

confidence: 53%

mentioning

confidence: 99%

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

Weeks

Corbin²,

Francis³

2009

J Pharmacol Exp Ther

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“…In summary, based on the findings reported here, on results of cyclic nucleotide analog studies (9,12,24), and on modeling using the NKXD motif of GTP-binding proteins, the cGMPbinding site of PDEs represents a new family of cyclic nucleotide-binding sites which contain critical Asn, Lys, and Asp residues.…”

mentioning

confidence: 56%

“…Asparagine 276 -Hydrogen bonding from the side chain of asparagine to the N-7 guanine ring nitrogen is absolutely conserved in the NKXD motif of GTP-binding proteins (23). cGMP analog studies examining the importance of the N-7 position of the allosteric cGMP-binding sites of PDEs suggest interaction at the N-7 ring nitrogen (11,24). These data taken together suggest the possibility of an interaction between the N-7 ring nitrogen of cGMP and Asn 276 of the putative NKX n D motif of cGB-PDE (Fig.…”

mentioning

confidence: 99%

Identification of Key Amino Acids in a Conserved cGMP-binding Site of cGMP-binding Phosphodiesterases

Turko

Haik

McAllister-Lucas

et al. 1996

Journal of Biological Chemistry

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, and Asp 289 in cGMP binding. These residues could be presented as a putative NKX n D motif, and their functions were predicted based on analogy with the canonical NKXD motif in GTP-binding proteins. No marked differences in catalytic functions such as specific activity, K m for cGMP, and IC 50 for zaprinast or 3-isobutyl-1-methylxanthine were found among wild-type and mutant cGB-PDEs. This suggested that cGMP binding to site a does not influence the catalytic properties of cGB-PDE.3Ј:5Ј-Cyclic nucleotide phosphodiesterases (PDEs) 1 catalyze the hydrolysis of 3Ј:5Ј-cyclic nucleotides to the corresponding nucleoside 5Ј-monophosphates. On the basis of their regulatory features and substrate specificities, the multiple PDEs can be classified into at least seven major families, three of which exhibit cGMP-binding sites that are distinct from sites of cyclic nucleotide hydrolysis (1). Comparison of their amino acid sequences reveals a conserved segment located toward the N terminus, which has been proposed to constitute an allosteric cGMP-binding region. This region consists of two similar blocks of conserved residues: block a ϭ NKX 5 or 7 FX 3 DE and block b ϭ N(K/R)X 4 or 10 FX 3 DE. These internally homologous repeats were postulated to be contained in two cGMP-binding sites, a and b, which have different affinities for cGMP (2).Recently, studies of site-directed mutagenesis of the cGMPbinding, cGMP-specific PDE (cGB-PDE; PDE 5A) (3) provided evidence for the importance of Asp 289 in block a and Asp 478 in block b for cGMP binding. These two residues are the first amino acid residues to be identified as critical for cGMP binding in allosteric sites of cGMP-binding PDEs. [ 32 P]cGMP dissociation kinetics of each of the mutant cGB-PDEs identified site a and site b as the high affinity site and low affinity site, respectively.Since the discovery of cyclic nucleotides, the characterization of cAMP and cGMP binding to their receptors has been the subject of intense investigations. As a result, the cyclic nucleotide-binding sites of catabolite gene activator protein (CAP) (4), cAMP-and cGMP-dependent protein kinases (5-7), and the cGMP-gated cation channels (8) have been described in detail. The major interactions for all of these proteins are similar: glutamic acid bonds with the ribose 2Ј-OH, while arginine forms an ion pair with the charged phosphate oxygen. The cGMP-binding sites of cGMP-binding PDEs do not exhibit any sequence similarity with CAP-related proteins (9), and the cyclic nucleotide-binding characteristics between these two families of proteins are quite different. In order to probe the structural elements that contribute to cGMP binding and to develop a better understanding of the functions of these elements in the cGMP-binding sites of PDEs, five mutants in the invariant N 276 KX 7 FX 3 DE 290 sequence of site a of cGB-PDE were generated. N276A, K277A, K277R, D289A, and E290A mutants were expressed in insect High Five cells, and then purified. These mutations were selected in part because of the res...

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“…The Ca2+/calmodulin-sensitive phosphodiesterase was isolated from bovine brain as described earlier (Couchie et al, 1983). The dog thyroid cyclic AMP-specific phosphodiesterase was obtained by DEAE-cellulose chromatography of a crude supernatant fraction (Miot et al, 1983).…”

Section: Introductionmentioning

confidence: 99%

Modulation of cyclic AMP action in the dog thyroid by its agonist and antagonist Sp- and Rp-adenosine 3′,5′-monophosphorothioate

Erneux¹,

Sande²,

Jastorff³

et al. 1986

Biochemical Journal

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The diastereoisomeric forms of adenosine 3',5'-monophosphorothioate, Sp-cAMPS and Rp-cAMPS, have been shown to mimic and to inhibit activation of protein kinase type I and type II by cyclic AMP. In the present work, Sp-cAMPS mimicked thyrotropin (TSH) action on thyroid hormone secretion and protein iodination in dog thyroid slices, whereas Rp-cAMPS antagonized those effects. The phosphorothioates have been tested as inhibitors or activators of the three major phosphodiesterases: the Ca2+/calmodulin-sensitive form, the cyclic GMP-stimulated form and the cyclic AMP-specific enzyme. At 100,M Sp-cAMPS inhibited the three enzyme activities. In contrast, Rp-cAMPS failed to stimulate activity of the three enzymes. From a comparison of the biological properties of Sp-and Rp-cAMPS and 3-isobutyl-1-methyl xanthine, it is suggested that one site of action of the phosphorothioates is on the cyclic AMP-dependent protein kinases, i.e. the effects of Sp-cAMPS and Rp-cAMPS observed in intact cell can be ascribed to the agonistic and antagonistic effects on the cyclic AMP-dependent protein kinases. However, partial inhibition of phosphodiesterase activities by the phosphorothioates cannot be excluded.

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Characterization of phosphodiesterase catalytic sites by means of cyclic nucleotide derivatives

Cited by 18 publications

References 25 publications

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

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

Identification of Key Amino Acids in a Conserved cGMP-binding Site of cGMP-binding Phosphodiesterases

Modulation of cyclic AMP action in the dog thyroid by its agonist and antagonist Sp- and Rp-adenosine 3′,5′-monophosphorothioate

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