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

Abstract

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 ...

Use of a Schizosaccharomyces pombe PKA-repressible reporter to study cGMP metabolising phosphodiesterases

“…PDE5A assays were conducted via a two-step assay (PDE hydrolysis followed by snake venom conversion of 5’GMP to guanosine) as described by Weeks et al [23] using recombinantly-expressed and purified His-tagged human PDE5A in the presence of 100nM cGMP. Parallel assays were carried out with sildenafil as a control.…”

Mammalian Cyclic Nucleotide Phosphodiesterases: Molecular Mechanisms and Physiological Functions

“…The invariant residues that coordinate the two metal ions are established; the invariant glutamine, conserved phenylalanine, and conserved tyrosine play critical roles in binding the substrate(s) in an orientation that facilitates hydrolysis by the catalytic machinery and are also important in binding PDE inhibitors. The impact of other conserved amino acids lining the catalytic site is modest (76,406,439,440). The H-loop that adjoins the metal-binding site in the primary sequence of PDEs and begins with an invariant glycine has been implicated in determining interactions with substrates and inhibitors (63,161,163,183,287,400,403).…”

“…3) The calculated loss in free energy of binding of cN substrate or inhibitors in PDE5 (cGMP-specific) or PDE11 (dual-specificity) following replacement of the glutamine indicates loss of one hydrogen bond (406,439,440). Catalytic rates in those mutants are like those of WT enzymes.…”

A Role for Phosphodiesterase 11A (PDE11A) in the Formation of Social Memories and the Stabilization of Mood

“…The net effect of the relative differences in substrate affinities versus turnover rates is that the 4 PDE11A isoforms hydrolyze cAMP and cGMP comparably well [4, 5]. Site-directed mutagenesis shows that Q869 is required for substrate binding of the PDE11A catalytic domain [40]; however, in absence of a resolved crystal structure the full implications of these results remain to be determined. Such knowledge would benefit designing compounds capable of selectively targeting the PDE11A catalytic domain over its nearest neighbor PDE5A.…”