Crystal structures of phosphodiesterases 4 and 5 in complex with inhibitor 3-isobutyl-1-methylxanthine suggest a conformation determinant of inhibitor selectivity

Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes controlling cellular concentrations of the second messengers cAMP and cGMP. Crystal structures of the catalytic domains of cGMP-specific PDE5A1 and cAMP-specific PDE4D2 in complex with the nonselective inhibitor 3-isobutyl-1-methylxanthine have been determined at medium resolution. The catalytic domain of PDE5A1 has the same topological folding as that of PDE4D2, but three regions show different tertiary structures, including residues 79-113, 208-224 (H-loop), and 341-364 (M-loop) in PDE4D2 or 535-566, 661-676, and 787-812 in PDE5A1, respectively. Because H- and M-loops are involved in binding of the selective inhibitors, the different conformations of the loops, thus the distinct shapes of the active sites, will be a determinant of inhibitor selectivity in PDEs. IBMX binds to a subpocket that comprises key residues Ile-336, Phe-340, Gln-369, and Phe-372 of PDE4D2 or Val-782, Phe-786, Gln-817, and Phe-820 of PDE5A1. This subpocket may be a common site for binding nonselective inhibitors of PDEs.     

Human hematopoietic prostaglandin D synthase inhibitor complex structures

In mast and Th2 cells, hematopoietic prostaglandin (PG) D synthase (H-PGDS) catalyses the isomerization of PGH(2) in the presence of glutathione (GSH) to produce the allergic and inflammatory mediator PGD(2). We determined the X-ray structures of human H-PGDS inhibitor complexes with 1-amino-4-{4-[4-chloro-6-(2-sulpho-phenylamino)-[1,3,5]triazin-2-ylmethyl]-3-sulpho-phenylamino}-9,10-dioxo-9,10-dihydro-anthracene-2-sulphonic acid (Cibacron Blue) and 1-amino-4-(4-aminosulphonyl) phenyl-anthraquinone-2-sulphonic acid (APAS) at 2.0 ? resolution. When complexed with H-PGDS, Cibacron Blue had an IC(50) value of 40 nM and APAS 2.1 μM. The Cibacron Blue molecule was stabilized by four hydrogen bonds and π-π stacking between the anthraquinone ring and Trp104, the ceiling of the active site H-PGDS pocket. Among the four hydrogen bonds, the Cibacron Blue terminal sulphonic group directly interacted with conserved residues Lys112 and Lys198, which recognize the PGH(2) substrate α-chain. In contrast, the APAS anthraquinone ring was inverted to interact with Trp104, while its benzenesulphonic group penetrated the GSH-bound region at the bottom of the active site. Due to the lack of extended aromatic rings, APAS could not directly hydrogen bond with the two conserved lysine residues, thus decreasing the total number of hydrogen bond from four to one. These factors may contribute to the 50-fold difference in the IC(50) values obtained for the two inhibitors.     

Kinetic and structural studies of phosphodiesterase-8A and implication on the inhibitor selectivity

Cyclic nucleotide phosphodiesterase-8 (PDE8) is a family of cAMP-specific enzymes and plays important roles in many biological processes, including T-cell activation, testosterone production, adrenocortical hyperplasia, and thyroid function. However, no PDE8 selective inhibitors are available for trial treatment of human diseases. Here we report kinetic properties of the highly active PDE8A1 catalytic domain prepared from refolding and its crystal structures in the unliganded and 3-isobutyl-1-methylxanthine (IBMX) bound forms at 1.9 and 2.1 A resolutions, respectively. The PDE8A1 catalytic domain has a K(M) of 1.8 microM, V(max) of 6.1 micromol/min/mg, a k(cat) of 4.0 s(-1) for cAMP, and a K(M) of 1.6 mM, V(max) of 2.5 micromol/min/mg, a k(cat) of 1.6 s(-1) for cGMP, thus indicating that the substrate specificity of PDE8 is dominated by K(M). The structure of the PDE8A1 catalytic domain has similar topology as those of other PDE families but contains two extra helices around Asn685-Thr710. Since this fragment is distant from the active site of the enzyme, its impact on the catalysis is unclear. The PDE8A1 catalytic domain is insensitive to the IBMX inhibition (IC(50) = 700 microM). The unfavorable interaction of IBMX in the PDE8A1-IBMX structure suggests an important role of Tyr748 in the inhibitor binding. Indeed, the mutation of Tyr748 to phenylalanine increases the PDE8A1 sensitivity to several nonselective or family selective PDE inhibitors. Thus, the structural and mutagenesis studies provide not only insight into the enzymatic properties but also guidelines for design of PDE8 selective inhibitors.     

Crystal structures of 4-alpha-glucanotransferase from Thermococcus litoralis and its complex with an inhibitor

Thermococcus litoralis 4-alpha-glucanotransferase (TLGT) belongs to glucoside hydrolase family 57 and catalyzes the disproportionation of amylose and the formation of large cyclic alpha-1,4-glucan (cycloamylose) from linear amylose. We determined the crystal structure of TLGT with and without an inhibitor, acarbose. TLGT is composed of two domains: an N-terminal domain (domain I), which contains a (beta/alpha)7 barrel fold, and a C-terminal domain (domain II), which has a twisted beta-sandwich fold. In the structure of TLGT complexed with acarbose, the inhibitor was bound at the cleft within domain I, indicating that domain I is a catalytic domain of TLGT. The acarbose-bound structure also clarified that Glu123 and Asp214 were the catalytic nucleophile and acid/base catalyst, respectively, and revealed the residues involved in substrate binding. It seemed that TLGT produces large cyclic glucans by preventing the production of small cyclic glucans by steric hindrance, which is achieved by three lids protruding into the active site cleft, as well as an extended active site cleft. Interestingly, domain I of TLGT shares some structural features with the catalytic domain of Golgi alpha-mannosidase from Drosophila melanogaster, which belongs to glucoside hydrolase family 38. Furthermore, the catalytic residue of the two enzymes is located in the same position. These observations suggest that families 57 and 38 evolved from a common ancestor.     

Thiophene inhibitors of PDE4: crystal structures show a second binding mode at the catalytic domain of PDE4D2

PDE4 inhibitors have been identified as therapeutic targets for a variety of conditions, particularly inflammatory diseases. We have serendipitously identified a novel class of phosphodiesterase 4 (PDE4) inhibitor during a study to discover antagonists of the parathyroid hormone receptor. X-ray crystallographic studies of PDE4D2 complexed to four potent inhibitors reveal the atomic details of how they inhibit the enzyme and a notable contrast to another recently reported thiophene-based inhibitor.     

Discovery of triazines as selective PDE4B versus PDE4D inhibitors

In this study we report a series of triazine derivatives that are potent inhibitors of PDE4B. We also provide a series of structure activity relationships that demonstrate the triazine core can be used to generate subtype selective inhibitors of PDE4B versus PDE4D. A high resolution co-crystal structure shows that the inhibitors interact with a C-terminal regulatory helix (CR3) locking the enzyme in an inactive ‘closed’ conformation. The results show that the compounds interact with both catalytic domain and CR3 residues. This provides the first structure-based approach to engineer PDE4B-selective inhibitors.     

Crystal structure of phosphodiesterase 4D and inhibitor complex(1)

Cyclic nucleotide phosphodiesterases (PDEs) regulate physiological processes by degrading intracellular second messengers, adenosine-3′,5′-cyclic phosphate or guanosine-3′,5′-cyclic phosphate. The first crystal structure of PDE4D catalytic domain and a bound inhibitor, zardaverine, was determined. Zardaverine binds to a highly conserved pocket that includes the catalytic metal binding site. Zardaverine fills only a portion of the active site pocket. More selective PDE4 inhibitors including rolipram, cilomilast and roflumilast have additional functional groups that can utilize the remaining empty space for increased binding energy and selectivity. In the crystal structure, the catalytic domain of PDE4D possesses an extensive dimerization interface containing residues that are highly conserved in PDE1, 3, 4, 8 and 9. Mutations of R358D or D322R among these interface residues prohibit dimerization of the PDE4D catalytic domain in solution.     

Exome sequencing identifies PDE4D mutations in acrodysostosis

Acrodysostosis is a dominantly-inherited, multisystem disorder characterized by skeletal, endocrine, and neurological abnormalities. To identify the molecular basis of acrodysostosis, we performed exome sequencing on five genetically independent cases. Three different missense mutations in PDE4D, which encodes cyclic AMP (cAMP)-specific phosphodiesterase 4D, were found to be heterozygous in three of the cases. Two of the mutations were demonstrated to have occurred de novo, providing strong genetic evidence of causation. Two additional cases were heterozygous for de novo missense mutations in PRKAR1A, which encodes the cAMP-dependent regulatory subunit of protein kinase A and which has been recently reported to be the cause of a form of acrodysostosis resistant to multiple hormones. These findings demonstrate that acrodysostosis is genetically heterogeneous and underscore the exquisite sensitivity of many tissues to alterations in cAMP homeostasis.     

2-Phenylquinazolin-4(3H)-one,a class of potent PDE5 inhibitors with high selectivity versus PDE6

In our efforts to minimize the side effects associated with low selectivity against the other PDE isozymes, a novel class of 2-phenylquinazolin-4(3H)-one derivatives were designed and prepared as potent PDE5 inhibitors with high selectivity against PDE6. The syntheses and SAR studies of such molecules were reported.     

Synthesis and profile of SCH351591, a novel PDE4 inhibitor

The syntheses and pharmacological profiles of some 2-trifluoromethyl-8-methoxyquinoline-5-carboxamides are described. SCH351591 is a potent selective inhibitor of phosphodiesterase type 4 (PDE4).     

Structural determinants for inhibitor specificity and selectivity in PDE2A using the wheat germ in vitro translation system

Phosphodiesterases (PDEs) modulate signaling by cyclic nucleotides in diverse processes such as cardiac contractility, platelet aggregation, lipolysis, glycogenolysis, and smooth muscle contraction. Cyclic guanosine monophosphate (cGMP) stimulated human phosphodiesterase 2 (PDE2) is expressed mainly in brain and heart tissues. PDE2A is involved in the regulation of blood pressure and fluid homeostasis by the atrial natriuretic peptide (ANP), making PDE2-type enzymes important targets for drug discovery. The design of more potent and selective inhibitors of PDE2A for the treatment of heart disease would be greatly aided by the identification of active site residues in PDE2A that determine substrate and inhibitor selectivity. The identification of active site residues through traditional mutational studies involves the time-consuming and tedious purification of a large number of mutant proteins from overexpressing cells. Here we report an alternative approach to rapidly produce active site mutants of human PDE2A and identify their enzymatic properties using a wheat germ in vitro translation (IVT, also known as cell-free translation) system. We also present the crystal structure of the catalytic domain of human PDE2A determined at 1.7 A resolution, which provided a framework for the rational design of active site mutants. Using a rapid IVT approach for expression of human PDE2A mutants, we identified the roles of active site residues Asp811, Gln812, Ile826, and Tyr827 in inhibitor and substrate selectivity for PDE2A.     

Structural basis for herbicidal inhibitor selectivity revealed by comparison of crystal structures of plant and mammalian 4-hydroxyphenylpyruvate dioxygenases

A high degree of selectivity toward the target site of the pest organism is a desirable attribute for new safer agrochemicals. To assist in the design of novel herbicides, we determined the crystal structures of the herbicidal target enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD; EC 1.13.11.27) from the plant Arabidopsis thaliana with and without an herbicidal benzoylpyrazole inhibitor that potently inhibits both plant and mammalian HPPDs. We also determined the structure of a mammalian (rat) HPPD in complex with the same nonselective inhibitor. From a screening campaign of over 1000 HPPD inhibitors, six highly plant-selective inhibitors were found. One of these had remarkable (>1600-fold) selectivity toward the plant enzyme and was cocrystallized with Arabidopsis HPPD. Detailed comparisons of the plant and mammalian HPPD-ligand structures suggest a structural basis for the high degree of plant selectivity of certain HPPD inhibitors and point to design strategies to obtain potent and selective inhibitors of plant HPPD as agrochemical leads.     

An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics

The spatiotemporal regulation of cAMP can generate microdomains just beneath the plasma membrane where cAMP increases are larger and more dynamic than those seen globally. Real-time measurements of cAMP using mutant cyclic nucleotide-gated ion channel biosensors, pharmacological tools and RNA interference (RNAi) were employed to demonstrate a subplasmalemmal cAMP signaling module in living cells. Transient cAMP increases were observed upon stimulation of HEK293 cells with prostaglandin E1. However, pretreatment with selective inhibitors of type 4 phosphodiesterases (PDE4), protein kinase A (PKA) or PKA/A-kinase anchoring protein (AKAP) interaction blocked an immediate return of subplasmalemmal cAMP to basal levels. Knockdown of specific membrane-associated AKAPs using RNAi identified gravin (AKAP250) as the central organizer of the PDE4 complex. Co-immunoprecipitation confirmed that gravin maintains a signaling complex that includes PKA and PDE4D. We propose that gravin-associated PDE4D isoforms provide a means to rapidly terminate subplasmalemmal cAMP signals with concomitant effects on localized ion channels or enzyme activities.     

Association between PDE4D polymorphism and ischemic stroke in young population

ObjectiveTo explore the association between the polymorphisms of the phosphodiesterase (PDE) 4D gene (SNP83 and SNP87) and the risk of ischemic stroke (IS) in Chinese young population.MethodsThis study included 393 patients who were divided into IS group and non-IS group. Semiconductor high-throughput sequencing technology and multivariate logistic regression analysis were performed.ResultsIn the case group, the frequency of CC genotype and C allele of the SNP83 gene was significantly higher than that in the control group. There was no significant difference in genotype frequency distribution of SNP87 between the two groups.ConclusionWe found an association between SNP83 and the risk of IS in Chinese young population from northern Henan province. There was not a significant association between SNP87 and IS in Chinese young population.     

Differential expression and function of phosphodiesterase 4 (PDE4) subtypes in human primary CD4+ T cells: predominant role of PDE4D

Type 4 phosphodiesterases (PDE4) are critical regulators in TCR signaling by attenuating the negative constraint of cAMP. In this study, we show that anti-CD3/CD28 stimulation of human primary CD4(+) T cells increases the expression of the PDE4 subtypes PDE4A, PDE4B, and PDE4D in a specific and time-dependent manner. PDE4A and PDE4D mRNAs as well as enzyme activities were up-regulated within 5 days, PDE4B showed a transient up-regulation with highest levels after 24 h. The induction was shown to be independent of different stimulation conditions and was similar in naive and memory T cell subpopulations. To elucidate the functional impact of individual PDE4 subtypes on T cell function, we used PDE4 subtype-specific short-interfering RNAs (siRNAs). Knockdown of either PDE4B or PDE4D inhibited IL-2 release 24 h after stimulation (time point of maximal IL-2 concentrations) to an extent similar to that observed with the panPDE4 inhibitor RP73401 (piclamilast). Substantial amounts of IFN-gamma or IL-5 were measured only at later time points. siRNA targeting PDE4D showed a predominant inhibitory effect on these cytokines measured after 72 h. However, the inhibition of all cytokines was most effective when PDE4 siRNAs were applied in combination. Although the effect of PDE4 inhibition on T cell proliferation is small, the PDE4D-targeting siRNA alone was as effective as the panPDE4 inhibitor, whereas PDE4A or PDE4B siRNAs had hardly an effect. In summary, individual PDE4 subtypes have overall nonredundant, but complementary, time-dependent roles in propagating various T cell functions and PDE4D is the form likely playing a predominant role.