Identification of inhibitor binding sites of the cAMP-specific phosphodiesterase 4

Using the technique of site-directed mutagenesis, point mutants of human PDE4A have been developed in order to identify amino acids involved in inhibitor binding. Relevant amino acids were selected according to a peptidic binding site model for PDE4 inhibitors, which suggests interaction with two tryptophan residues, one histidine and one tyrosine residue, as well as one Zn(2+) ion. Mutations were directed at those tryptophan, histidine, and tyrosine residues, which are conserved among the PDE4 subtypes (PDE4A-D) and lie within the high-affinity 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) binding domain of human PDE4A (amino acids 276-681 according to the PDE4A sequence L20965). Truncations to this region do not alter enzyme activity or inhibitor sensitivity. The mutants were expressed in COS1 cells, and the recombinant cyclic nucleotide phosphodiesterase (PDE) forms have been characterized in terms of their catalytic activity and inhibitor sensitivities. Tyrosine residues 432 and 602, as well as histidine 588, were found to be involved in inhibitor binding, but no interaction was detected between tryptophan and PDE inhibitors tested. To test the possibility that other amino acids are of importance for hydrophobic interactions, selected phenylalanine residues were also mutated. We found phenylalanine 613 and 645 to influence inhibitor binding to PDE4. The significant differences in the inhibitor sensitivities of the mutants show that the various inhibitors have different enzyme binding sites. Based on the assumption that the known side effects of PDE4 inhibitors (like emesis and nausea) are caused directly by selective inhibition of different conformation states of PDE4, our results may be a hint to differ between PDE4 inhibitors, which have emetic side effects (like rolipram), and those that do not have side effects (like N-(3,5-dichlorpyrid-4-yl)-[1-(4-fluorbenzyl)-5-hydroxy-indol-3-yl]-glyoxylateamide [AWD12-281]) by the differences of their binding sites and in that context contribute to the development of novel drugs. Furthermore, the identification of amino acid interactions proposed by the peptidic binding site model, which was used for the mutant selection, verifies the PrGen modeling as a useful method for the prediction of inhibitor binding sites in cases where detailed knowledge of the protein structure is not available.     

Selective PDE4 inhibitors as potent anti-inflammatory drugs for the treatment of airway diseases

Phosphodiesterases (PDEs) are responsible for the breakdown of intracellular cyclic nucleotides, from which PDE4 are the major cyclic AMP metabolizing isoenzymes found in inflammatory and immune cells. This generated greatest interest on PDE4 as a potential target to treat lung inflammatory diseases. For example, cigarette smoke-induced neutrophilia in BAL was dose and time dependently reduced by cilomilast. Beside the undesired side effects associated with the first generation of PDE4 inhibitors, the second generation of selective inhibitors such as cilomilast and roflumilast showed clinical efficacy in asthma and chronic obstructive pulmonary diseases trials, thus re-enhancing the interest on these classes of compounds. However, the ability of PDE4 inhibitors to prevent or modulate the airway remodelling remains relatively unexplored. We demonstrated that selective PDE4 inhibitor RP 73-401 reduced matrix metalloproteinase (MMP)-9 activity and TGF-beta1 release during LPS-induced lung injury in mice and that CI-1044 inhibited the production of MMP-1 and MMP-2 from human lung fibroblasts stimulated by pro-inflammatory cytokines. Since inflammatory diseases of the bronchial airways are associated with destruction of normal tissue structure, our data suggest a therapeutic benefit for PDE4 inhibitors in tissue remodelling associated with chronic lung diseases.     

Structures of the four subfamilies of phosphodiesterase-4 provide insight into the selectivity of their inhibitors

PDE4 (phosphodiesterase-4)-selective inhibitors have attracted much attention as potential therapeutics for the treatment of both depression and major inflammatory diseases, but their practical application has been compromised by side effects. A possible cause for the side effects is that current PDE4-selective inhibitors similarly inhibit isoforms from all four PDE4 subfamilies. The development of PDE4 subfamily-selective inhibitors has been hampered by a lack of structural information. In the present study, we rectify this by providing the crystal structures of the catalytic domains of PDE4A, PDE4B and PDE4D in complex with the PDE4 inhibitor NVP {4-[8-(3-nitrophenyl)-[1,7]naphthyridin-6-yl]benzoic acid} as well as the unliganded PDE4C structure. NVP binds in the same conformation to the deep cAMP substrate pocket and interacts with the same residues in each instance. However, detailed structural comparison reveals significant conformational differences. Although the active sites of PDE4B and PDE4D are mostly comparable, PDE4A shows significant displacements of the residues next to the invariant glutamine residue that is critical for substrate and inhibitor binding. PDE4C appears to be more distal from other PDE4 subfamilies, with certain key residues being disordered. Our analyses provide the first structural basis for the development of PDE4 subfamily-selective inhibitors.     

Discovery of novel inhibitors of phosphodiesterase 4 with 1-phenyl-3,4-dihydroisoquinoline scaffold: Structure-based drug design and fragment identification

Currently, it is in urgent need to develop novel selective PDE4 inhibitors with novel structural scaffolds to overcome the adverse effects and improve the efficacy. Novel 1-phenyl-3,4-dihydroisoquinoline amide derivatives were developed as potential PDE4 inhibitors based on the structure-based drug design and fragment identification strategy. A SARs analysis was performed in substituents attached in the C-3 side chain phenyl ring, indicating that the attachment of methoxy group or halogen atom substitution at the ortho-position of the phenyl ring was helpful to enhance both inhibitory activity toward PDE4B and selectivity. Compound 15 with excellent selectivity, exhibited the most potent inhibition in vitro and in vivo, which is a promising lead for development of a new class of selective PDE4 inhibitors.     

Therapeutic potential of phosphodiesterase-4 and -3 inhibitors in Th1-mediated autoimmune diseases

Phosphodiesterase-4 (PDE4) inhibitors have the potential to modulate immune responses from the Th1 toward the Th2 phenotype and are considered candidate therapies for Th1-mediated autoimmune disorders. However, depending on the model and cell types employed, studies of atopic individuals have come to the opposite conclusion, i.e., that PDE inhibitors may be beneficial in asthma. Using in vitro immunopharmacologic techniques we analyzed the effects of PDE4 and PDE3 inhibitors on human immune cells to address these discrepancies and broaden our understanding of their mechanism of action. Our results indicate that PDE inhibitors have complex inhibitory effects within in vivo achievable concentration ranges on Th1-mediated immunity, whereas Th2-mediated responses are mostly unaffected or enhanced. The Th2 skewing of the developing immune response is explained by the effects of PDE inhibitors on several factors contributing to T cell priming: the cytokine milieu; the type of costimulatory signal, i.e., up-regulation of CD86 and down-regulation of CD80; and the Ag avidity. The combination of PDE4 and PDE3 inhibitors expresses synergistic effects and may broaden the therapeutic window. Finally, we observed a differential sensitivity to PDE inhibition in autoreactive vs foreign Ag-specific T cells and cells derived from multiple sclerosis patients vs those derived from healthy donors. This suggests that PDE inhibition weakens the strength of the T cell stimulus and corrects the underlying disease-associated cytokine skew in T cell-mediated autoimmune disorders. These new findings broaden the understanding of the immunomodulatory actions of PDE inhibitors and underscore their promising drug profile for the treatment of autoimmune disorders.     

Phosphodiesterase 4 inhibitors and drugs of abuse: current knowledge and therapeutic opportunities

Background

Long-term exposure to drugs of abuse causes an upregulation of the cAMP-signaling pathway in the nucleus accumbens and other forebrain regions, this common neuroadaptation is thought to underlie aspects of drug tolerance and dependence. Phosphodiesterase 4 (PDE4) is an enzyme that the selective hydrolyzes intracellular cAMP. It is expressed in several brain regions that regulate the reinforcing effects of drugs of abuse.

Objective

Here, we review the current knowledge about central nervous system (CNS) distribution of PDE4 isoforms and the effects of systemic and brain-region specific inhibition of PDE4 on behavioral models of drug addiction.

Methods

A systematic literature search was performed using the Pubmed.

Results

Using behavioral sensitization, conditioned place preference and drug self-administration as behavioral models, a large number of studies have shown that local or systemic administration of PDE4 inhibitors reduce drug intake and/or drug seeking for psychostimulants, alcohol, and opioids in rats or mice.

Conclusions

Preclinical studies suggest that PDE4 could be a therapeutic target for several classes of substance use disorder. We conclude by identifying opportunities for the development of subtype-selective PDE4 inhibitors that may reduce addiction liability and minimize the side effects that limit the clinical potential of non-selective PDE4 inhibitors. Several PDE4 inhibitors have been clinically approved for other diseases. There is a promising possibility to repurpose these PDE4 inhibitors for the treatment of drug addiction as they are safe and well-tolerated in patients.

     

Design and synthesis of boron-containing PDE4 inhibitors using soft-drug strategy for potential dermatologic anti-inflammatory application

PDE4 inhibitors are a validated approach as anti-inflammatory agents but are limited by systemic side effects including emesis. We report a soft-drug strategy incorporating a carboxylic ester group into boron-containing PDE4 inhibitors leading to the discovery of a series of benzoxaborole compounds with good potency (for example IC₅₀ = 47 nM of compound 2) and low emetic activity. These compounds are intended for dermatological use further limiting possible systemic side effects.     

Selective potentiating effect of RS14203 on a serotoninergic pathway in anesthetized rats

The usefulness of selective inhibitors of type 4 phosphodiesterase (PDE4) in the treatment of inflammation and pulmonary diseases is limited by their side effects: nausea and vomiting. We studied the effect of three structurally diverse PDE4 inhibitors on the vagal nerve afferent and efferent fibers in anesthetized rats. The effects of RS14203, (R)-rolipram, and CT-2450 were evaluated on the von Bezold-Jarisch reflex (vagal afferent fibers) and in a model of vagal electrical stimulation (vagal efferent fibers). All three PDE4 inhibitors were administered at 1, 10, or 100 microg/kg (iv) 15 min prior to the induction of bradycardia by an iv injection of 2-methyl-5-HT (von Bezold-Jarisch reflex) or by vagal electrical stimulation. At 100 microg/kg, RS14203 significantly potentiated the 2-methyl-5-HT response. No statistically significant effects were observed with (R)-rolipram or CT-2450 at the doses studied. RS14203, (R)-rolipram, or CT-2450 (1-100 microg/kg iv) did not affect the bradycardia induced by vagal electrical stimulation. Consequently, our results show that RS14203 selectively facilitates serotoninergic neurotransmission in vagal afferent fibers. The emetic action of RS14203 may be mediated by this mechanism.     

Pharmacophore Modeling and Virtual Screening for the Discovery of New type 4 cAMP Phosphodiesterase (PDE4) Inhibitors

Type 4 cAMP phosphodiesterase (PDE4) inhibitors show a broad spectrum of anti-inflammatory effects in almost all kinds of inflamed cells, by an increase in cAMP levels which is a pivotal second messenger responsible for various biological processes. These inhibitors are now considered as the potential drugs for treatment of chronic inflammatory diseases. However, some recently marketed inhibitors e.g., roflumilast, have shown adverse effects such as nausea and emesis, thus restricting its use. In order to identify novel PDE4 inhibitors with improved therapeutic indexes, a highly correlating (r = 0.963930) pharmacophore model (Hypo1) was established on the basis of known PDE4 inhibitors. Validated Hypo1 was used in database screening to identify chemical with required pharmacophoric features. These compounds are further screened by using the rule of five, ADMET and molecular docking. Finally, twelve hits which showed good results with respect to following properties such as estimated activity, calculated drug-like properties and scores were proposed as potential leads to inhibit the PDE4 activity. Therefore, this study will not only assist in the development of new potent hits for PDE4 inhibitors, but also give a better understanding of their interaction with PDE4. On a wider scope, this will be helpful for the rational design of novel potent enzyme inhibitors.     

Phosphodiesterase 4 conformers: preparation of recombinant enzymes and assay for inhibitors.

Cyclic nucleotides are key regulators of many cellular processes. Their immediate action is terminated through the activity of phosphodiesterases, a diverse family of enzymes. This diversity has given rise to drug discovery opportunities, and assay technology is therefore of key importance. Inhibitors of the cyclic-AMP-specific phosphodiesterases (the PDE4 family) are drug candidates for a variety of inflammatory disorders. However, PDE4 inhibitors, besides their immunomodulatory effects, also cause side effects including nausea and emesis. Recently, it has been suggested that PDE4 exists in two different conformations with respect to inhibition by the prototypical compound rolipram. Inhibition of the low-affinity conformer is thought to give rise to anti-inflammatory effects, and inhibition of the high-affinity conformer to side effects. Therefore, a selective inhibitor of the low-affinity conformer may have clinical utility. Methods are described to prepare recombinant forms of PDE4B that allow screening for compounds that could preferentially inhibit the low-affinity conformer. Furthermore, conditions for an efficient, scintillation proximity, microtiter plate-based assay are described, providing a considerable advance over previous assays in terms of throughput and automatability.     

Synthesis and biological evaluation of imidazol-2-one and 2-cyanoiminoimidazole derivatives: novel series of PDE4 inhibitors

This communication describes the synthesis and in vitro PDE4 inhibitory activity of a novel series of imidazol-2-one and 2-cyanoiminoimidazole derivatives. The compounds described were also tested in in vivo models to evaluate their anti-inflammatory activity after topical administration as well as their gastro-intestinal side effects. Several compounds proved to be potent PDE4 inhibitors and some 2-cyanoiminoimidazoles showed less pronounced gastro-intestinal side effects than reference compounds but maintained anti-inflammatory activity after topical administration.     

PDE4D targeting enhances anti-tumor effects of sorafenib in clear cell renal cell carcinoma and attenuates MAPK/ERK signaling in a CRAF-dependent manner

Clear cell renal cell carcinoma (ccRCC) is the most lethal form of kidney cancer and effective treatment regimens are yet to be established. Tyrosine kinase inhibitors (TKI) have widely been used as ccRCC therapeutics, but their efficacy is limited due to accompanying resistance mechanisms. Previous studies have provided substantial evidence for crosstalk between cAMP and the MAPK/ERK signaling pathway. Low levels of intracellular cAMP have been found in several human malignancies and some data suggest that elevation of cAMP expression can be achieved by phosphodiesterase 4 (PDE4) inhibition, resulting in cell growth arrest and/or cell death. The effects of crosstalk between cAMP and the MAPK/ERK pathway on the development progression in ccRCR, however, remain to be fully understood. In this study, we sought to explore the involvement of PDE4 in ccRCC and to assess its potential as a target for therapeutic intervention. We demonstrated that PDE4D is the predominant subtype of PDE4 expressed in healthy and cancerous renal cell lines, particularly in metastatic Caki-1 cells. We generated a CRISPR/Cas9-mediated PDE4D-KO Caki-1 cell model and showed that PDE4D depletion reduced cell proliferation and recovered cAMP expression in these cells. PDE4D-KO and/or PDE4 inhibition with the FDA approved PDE4 inhibitor, roflumilast, also attenuated MAPK/ERK signaling in a CRAF-dependent manner. Most interestingly, we showed that PDE4D-KO enhanced the effectiveness of the TKI, sorafenib, to stunt cell survival. In conclusion, we provide preliminary evidence of PDE4 involvement in ccRCC and suggest a rationale for dual tyrosine kinase/PDE4D targeting in patients with CRAF-dependent MAPK activation.     

Identification of 2,3-disubstituted pyridines as potent,non-emetic PDE4 inhibitors

A series of 2,3-disubstituted pyridines were synthesized as potential non-emetic PDE4 inhibitors. To decrease brain exposure and minimize emesis, we modified the lipophilic moiety of a series of emetic PDE4 inhibitors and found that introduction of a hydroxy group into the pyridine moiety of the side chain led to non-emetic compounds with preserved PDE4 inhibitory activity. Following optimization at the phenoxy group, we identified compound 1 as a potent non-emetic PDE4 inhibitor. Compound 1 showed significant efficacy in an animal model of asthma without inducing emesis.     

The Unrecognized Effects of Phosphodiesterase 4 on Epithelial Cells in Pulmonary Inflammation

Acute pulmonary inflammation is characterized by migration of polymorphonuclear neutrophils (PMNs) into the different compartments of the lung, passing an endothelial and epithelial barrier. Recent studies showed evidence that phosphodiesterase (PDE)4-inhibitors stabilized endothelial cells. PDE4B and PDE4D subtypes play a pivotal role in inflammation, whereas blocking PDE4D is suspected to cause gastrointestinal side effects. We thought to investigate the particular role of the PDE4-inhibitors roflumilast and rolipram on lung epithelium. Acute pulmonary inflammation was induced by inhalation of LPS. PDE4-inhibitors were administered i.p. or nebulized after inflammation. The impact of PDE4-inhibitors on PMN migration was evaluated in vivo and in vitro. Microvascular permeability, cytokine levels, and PDE4B and PDE4D expression were analyzed. In vivo, both PDE4-inhibitors decreased transendothelial and transepithelial migration even when administered after inflammation, whereas roflumilast showed a superior effect compared to rolipram on the epithelium. Both inhibitors decreased TNFα, IL6, and CXCL2/3. CXCL1, the strong PMN chemoattractant secreted by the epithelium, was significantly more reduced by roflumilast. In vitro assays with human epithelium also emphasized the pivotal role of roflumilast on the epithelium. Additionally, LPS-induced stress fibers, an essential requirement for a direct migration of PMNs into the alveolar space, were predominantly reduced by roflumilast. Expression of PDE4B and PDE4D were both increased in the lungs by LPS, PDE4-inhibitors decreased mainly PDE4B. The topical administration of PDE4-inhibitors was also effective in curbing down PMN migration, further highlighting the clinical potential of these compounds. In pulmonary epithelial cells, both subtypes were found coexistent around the nucleus and the cytoplasm. In these epithelial cells, LPS increased PDE4B and, to a lesser extend, PDE4D, whereas the effect of the inhibitors was prominent on the PDE4B subtype. In conclusion, we determined the pivotal role of the PDE4-inhibitor roflumilast on lung epithelium and emphasized its main effect on PDE4B in hyperinflammation.     

Discover natural compounds as potential phosphodiesterase-4B inhibitors via computational approaches

cAMP, intracellular cyclic adenosine monophosphate, is a ubiquitous second messenger that plays a key role in many physiological processes. PDE4B which can reduce the cAMP level by hydrolyzing cAMP to 5′-AMP has become a therapeutic target for the treatment of human diseases such as respiratory disorders, inflammation diseases, neurological and psychiatric disorders. However, the use of currently available PDE4B inhibitors is restricted due to serious side effects caused by targeting PDE4D. Hence, we are attempting to find out subfamily-selective PDE4B inhibitors from natural products, using computer-aided approaches such as virtual screening, docking, and molecular dynamics simulation. Finally, four potential PDE4B-selective inhibitors (ZINC67912770, ZINC67912780, ZINC72320169, and ZINC28882432) were found. Compared to the reference drug (roflumilast), they scored better during the virtual screening process. Binding free energy for them was ?317.51, ?239.44, ?215.52, and ?165.77 kJ/mol, better than ?129.05 kJ/mol of roflumilast. The pharmacophore model of the four candidate inhibitors comprised six features, including one hydrogen bond donor, four hydrogen bond acceptors, and one aromatic ring feature. It is expected that our study will pave the way for the design of potent PDE4B-selective inhibitors of new drugs to treat a wide variety of diseases such as asthma, COPD, psoriasis, depression, etc.     

Highly potent PDE4 inhibitors with therapeutic potential

The hypothesis that the dose-limiting side effects of PDE4 inhibitors could be mediated via the central nervous system prompted us to design and synthesize a hydrophilic piperidine analog to improve the side effect profile of Ariflo 1, which is an orally active second-generation PDE4 inhibitor. During evaluation of various water-soluble piperidine analogs, 2a-b, 11b-14b, and 17a showed therapeutic potential in cross-species comparison studies. The following three findings were obtained: (1) The hydroxamic acid group, a well known metal chelator, caused a marked increase of inhibitory activity. (2) Water-soluble piperidine analogs lacked the configurational isomerism of Ariflo 1 without loss of inhibitory activity. (3) Replacement of the 4-methoxy residue with a difluoromethoxy residue led to an increase of in vivo potency. Structure-activity relationships are presented. Single-dose rat pharmacokinetic data for 11b, 12b, and 17a are also presented.     

Quinazolines as potent and highly selective PDE5 inhibitors as potential therapeutics for male erectile dysfunction

In an effort to minimize side effects associated with low selectivity against PDE isozymes, we have successfully identified a series of 6,7,8-substituted quinzaolines as potent inhibitors of PDE5 with high level of isozyme selectivity, especially against PDE6 and PDE11. PDE5 potency and isozyme selectivity of quinazolines were greatly improved with substitutions both at 6- and 8-position. The synthesis, structure-activity relationships and in vivo efficacy of this novel series of potent PDE5 inhibitors are described.     

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.     

Cross talk between NO and cyclic nucleotide phosphodiesterases in the modulation of signal transduction in blood vessel.

An increase in cAMP and/or cGMP induces vasodilation which could be potentiated by endothelium or NO-donors. Cyclic nucleotide phosphodiesterases (PDE) are differently distributed in vascular tissues. cAMP hydrolyzing PDE isozymes in endothelial cells are represented by PDE2 (cGMP stimulated-PDE) and PDE4 (cGMP insensitive-PDE), whereas in smooth muscle cells PDE3 (cGMP inhibited-PDE) and PDE4 are present. To investigate the role of NO in vasodilation induced by PDE inhibitors, we studied the effects of PDE3- or PDE4-inhibitor alone and their combination on cyclic nucleotide levels, on relaxation of precontracted aorta and on protein kinase implication. Furthermore, the direct effect of dinitrosyl iron complex (DNIC) was studied on purified recombinant PDE4B. The results show that: 1) in endothelial cells PDE4 inhibition may up-regulate basal production of NO, this effect being potentiated by PDE2 inhibition; 2) in smooth muscle cGMP produced by NO inhibits PDE3 and increases cAMP level allowing PDE4 to participate in vascular contraction; 3) protein kinase G mediates the relaxing effects of PDE3 or PDE4 inhibition. 4) DNIC inhibits non competitively PDE4B indicating a direct effect of NO on PDE4 which could explain an additive vasodilatory effect of NO. A direct and a cGMP related cross-talk between NO and cAMP-PDEs, may participate into the vasomodulation mediated by cAMP activation of protein kinase G.     

cAMP phosphodiesterase inhibitors potentiate effects of prostacyclin analogs in hypoxic pulmonary vascular remodeling

We investigated the effects of prostacyclin analogs and isoform-selective phosphodiesterase (PDE) inhibitors, alone and in combination, on pulmonary vascular remodeling in vitro and in vivo. Vascular smooth muscle cells (VSMC) isolated from pulmonary (proximal and distal) and systemic circulations demonstrated subtle variations in expression of PDE isoform mRNA. However, using biochemical assays, we found PDE3 and PDE4 isoforms to be responsible for the majority of cAMP hydrolysis in all VSMC. In growth assays, the prostacyclin analogs cicaprost and iloprost inhibited mitogen-induced proliferation of VSMC in a cAMP-dependent manner. In addition, isoform-selective antagonists of PDEs 1, 3, or 4 inhibited VSMC proliferation, an effect that synergized with the effect of prostacyclin analogs. The inhibitory effects were greater in cells isolated from pulmonary circulation. In an in situ perfused rat lung preparation, administration of prostacyclin analogs or the PDE inhibitors vinpocetine (PDE1), cilostamide (PDE3), or rolipram (PDE4), but not EHNA (PDE2), attenuated acute hypoxic vasoconstriction (HPV). Combinations of agents led to a greater reduction in HPV. Furthermore, during exposure to hypoxia for 13 days, Wistar rats were treated with iloprost, rolipram, cilostamide, or combinations of these agents. Compared with normoxic controls, hypoxic animals developed pulmonary hypertension and distal pulmonary artery muscularization. These parameters were attenuated by iloprost+cilostamide, iloprost+rolipram, and cilostamide+rolipram but were not significantly affected by single agents. Together, these findings provide a greater understanding of the role of cAMP PDEs in VSMC proliferation and provide rationale for combined use of prostacylcin analogs plus PDE3/4 inhibitors in treatment of pulmonary vascular remodeling.