ABSTRACT
Cilostazol is a phosphodiesterase III inhibitor characterized by poor solubility. This limitation can be overcome by using a drug carrier capable of delivering the drug to the target site. Cyclodextrins are essential as drug carriers because of their outstanding complexation abilities and their capacity to improve drug bioavailability. This study comprises two stages: The first involves verifying different cyclodextrins and their complexation abilities towards cilostazol. This was accomplished using molecular docking simulations (MDS) and density functional theory (DFT). Both techniques indicate that the largest Sulfobutyl Ether-ß-Cyclodextrin forms the most stable complex with cilostazol. Additionally, other important parameters of the complex are described, including binding sites, dominant interactions, and thermodynamic parameters such as complexation enthalpy, Gibbs free energy, and Gibbs free energy of solvation. The second stage involves a binding study between cilostazol and Phosphodiesterse3 (PDE3). This study was conducted using molecular docking simulations, and the most important energetic parameters are detailed. This is the first such report, and we believe that the results of our predictions will pave the way for future drug development efforts using cyclodextrin-cilostazol complexes as potential therapeutics.
Subject(s)
Cilostazol , Cyclodextrins , Molecular Docking Simulation , Phosphodiesterase 3 Inhibitors , Thermodynamics , Cilostazol/chemistry , Phosphodiesterase 3 Inhibitors/chemistry , Phosphodiesterase 3 Inhibitors/pharmacology , Cyclodextrins/chemistry , Binding Sites , Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Protein Binding , HumansABSTRACT
Molecular glues are a class of small molecular drugs that mediate protein-protein interactions, that induce either the degradation or stabilization of target protein. A structurally diverse group of chemicals, including 17-ß-estradiol (E2), anagrelide, nauclefine, and DNMDP, induces apoptosis by forming complexes with phosphodiesterase 3A (PDE3A) and Schlafen 12 protein (SLFN12). They do so by binding to the PDE3A enzymatic pocket that allows the compound-bound PDE3A to recruit and stabilize SLFN12, which in turn blocks protein translation, leading to apoptosis. In this work, we report the high-resolution cryo-electron microscopy structure of PDE3A-SLFN12 complexes isolated from cultured HeLa cells pre-treated with either anagrelide, or nauclefine, or DNMDP. The PDE3A-SLFN12 complexes exhibit a butterfly-like shape, forming a heterotetramer with these small molecules, which are packed in a shallow pocket in the catalytic domain of PDE3A. The resulting small molecule-modified interface binds to the short helix (E552-I558) of SLFN12 through hydrophobic interactions, thus "gluing" the two proteins together. Based on the complex structure, we designed and synthesized analogs of anagrelide, a known drug used for the treatment of thrombocytosis, to enhance their interactions with SLFN12, and achieved superior efficacy in inducing apoptosis in cultured cells as well as in tumor xenografts.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Drug Design , Intracellular Signaling Peptides and Proteins/chemistry , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Binding Sites , Cryoelectron Microscopy , HeLa Cells , Humans , Hydrophobic and Hydrophilic Interactions , Indoles/chemistry , Mice , Multiprotein Complexes , Naphthyridines/chemistry , Pyridazines/chemistry , Quinazolines/chemistry , Quinazolines/pharmacology , Quinazolines/therapeutic use , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.
Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Intracellular Signaling Peptides and Proteins/chemistry , Pyridazines/chemistry , Adenosine Monophosphate/chemistry , Calorimetry, Differential Scanning , Catalytic Domain , Cell Survival/drug effects , Cell Survival/genetics , Cryoelectron Microscopy , Cyclic Nucleotide Phosphodiesterases, Type 3/genetics , Endoribonucleases/chemistry , HEK293 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Kinetics , Mass Spectrometry , Multienzyme Complexes/ultrastructure , Mutation , Protein Binding , Protein Conformation, alpha-Helical , Protein Multimerization , Pyridazines/pharmacology , Recombinant Proteins , Tetrahydroisoquinolines/chemistryABSTRACT
Spired by the chemical structure of Cilostazol, a selective phosphodiesterase 3A (PDE3A) inhibitor, several novel hybrid compounds of nucleobases (uracil, 6-azauracil, 2-thiuracil, adenine, guanine, theophylline and theobromine) and tetrazole were designed and successfully synthesized and their inhibitory effects on PDE3A as well as their cytotoxicity on HeLa and MCF-7 cancerous cell lines were studied. Obtained results show the linear correlation between the inhibitory effect of synthesized compounds and their cytotoxicity. In some cases, the PDE3A inhibitory effects of synthesized compounds are higher than the Cilostazol. Besides, compared to a standard anticancer drug methotrexate, some of the synthesized compounds showed the higher cytotoxicity against the HeLa and MCF-7 cancerous cell lines.
Subject(s)
Antineoplastic Agents/chemical synthesis , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Nucleotides/chemistry , Phosphodiesterase 3 Inhibitors/chemistry , Tetrazoles/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Binding Sites , Cell Line, Tumor , Cell Survival/drug effects , Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Humans , Kinetics , Molecular Docking Simulation , Phosphodiesterase 3 Inhibitors/metabolism , Quinolones/chemistry , Structure-Activity RelationshipABSTRACT
Cytotoxic molecules can kill cancer cells by disrupting critical cellular processes or by inducing novel activities. 6-(4-(Diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) is a small molecule that kills cancer cells by generation of novel activity. DNMDP induces complex formation between phosphodiesterase 3A (PDE3A) and schlafen family member 12 (SLFN12) and specifically kills cancer cells expressing elevated levels of these two proteins. Here, we examined the characteristics and covariates of the cancer cell response to DNMDP. On average, the sensitivity of human cancer cell lines to DNMDP is correlated with PDE3A expression levels. However, DNMDP could also bind the related protein, PDE3B, and PDE3B supported DNMDP sensitivity in the absence of PDE3A expression. Although inhibition of PDE3A catalytic activity did not account for DNMDP sensitivity, we found that expression of the catalytic domain of PDE3A in cancer cells lacking PDE3A is sufficient to confer sensitivity to DNMDP, and substitutions in the PDE3A active site abolish compound binding. Moreover, a genome-wide CRISPR screen identified the aryl hydrocarbon receptor-interacting protein (AIP), a co-chaperone protein, as required for response to DNMDP. We determined that AIP is also required for PDE3A-SLFN12 complex formation. Our results provide mechanistic insights into how DNMDP induces PDE3A-SLFN12 complex formation, thereby killing cancer cells with high levels of PDE3A and SLFN12 expression.
Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Neoplasms/pathology , Base Sequence , Biomarkers, Tumor/metabolism , CRISPR-Cas Systems/genetics , Catalytic Domain , Cell Death/drug effects , Cell Line, Tumor , Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Frameshift Mutation/genetics , Genome , Heterozygote , Humans , Protein Binding/drug effects , Pyridazines/pharmacologyABSTRACT
Considerable evidence suggests that as breast cancer progresses, genetic and epigenetic mechanisms contribute to the emergence of self-renewing cells (CSC), which may also arise as a consequence of metastasis. Although the molecular pathways that trigger stemness and metastasis are known, key molecular and mechanistic gaps in our understanding of these processes remain unclear. Here, we first screened the inflammation-associated stemness gene phosphodiesterase 3A (PDE3A) using a medium-throughput siRNA library, which was overexpressed in breast tumors and significantly correlated with clinical progression. PDE3A induced the inflammatory nuclear factor NFκB signaling pathway by suppressing cAMP/PKA, which promotes the expression of the stem cell marker OCT4. In addition, PDE3A also promoted the translocation of CCDC88A from the cytoplasm to nuclei, thereby boosting the invasion-metastasis cascade in breast cancer. Most importantly, the PDE3A-selective inhibitor cilostazol dramatically suppressed breast tumor growth and reduced metastasis to the lungs in xenograft breast cancer models, with minimum toxicity. Taken together, we show that PDE3A could predispose patients with breast cancer to metastases by acting as a mediator of cancer stemness. PDE3A is a potential therapeutic target for advanced breast cancer.
Subject(s)
Breast Neoplasms/drug therapy , Cilostazol/pharmacology , Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Neoplastic Stem Cells/drug effects , Animals , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Breast Neoplasms/secondary , Cell Proliferation , Cyclic Nucleotide Phosphodiesterases, Type 3/genetics , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Female , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Microfilament Proteins/metabolism , Middle Aged , Neoplastic Stem Cells/enzymology , Neoplastic Stem Cells/metabolism , Phosphodiesterase 3 Inhibitors/pharmacology , Prognosis , Protein Transport , Signal Transduction , Tumor Cells, Cultured , Vesicular Transport Proteins/metabolism , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Phosphodiesterase type-1 (PDE1) hydrolyzes cAMP and cGMP and is constitutively expressed in the heart, although cardiac effects from its acute inhibition in vivo are largely unknown. Existing data are limited to rodents expressing mostly the cGMP-favoring PDE1A isoform. Human heart predominantly expresses PDE1C with balanced selectivity for cAMP and cGMP. Here, we determined the acute effects of PDE1 inhibition in PDE1C-expressing mammals, dogs, and rabbits, in normal and failing hearts, and explored its regulatory pathways. METHODS: Conscious dogs chronically instrumented for pressure-volume relations were studied before and after tachypacing-induced heart failure (HF). A selective PDE1 inhibitor (ITI-214) was administered orally or intravenously±dobutamine. Pressure-volume analysis in anesthetized rabbits tested the role of ß-adrenergic and adenosine receptor signaling on ITI-214 effects. Sarcomere and calcium dynamics were studied in rabbit left ventricular myocytes. RESULTS: In normal and HF dogs, ITI-214 increased load-independent contractility, improved relaxation, and reduced systemic arterial resistance, raising cardiac output without altering systolic blood pressure. Heart rate increased, but less so in HF dogs. ITI-214 effects were additive to ß-adrenergic receptor agonism (dobutamine). Dobutamine but not ITI-214 increased plasma cAMP. ITI-214 induced similar cardiovascular effects in rabbits, whereas mice displayed only mild vasodilation and no contractility effects. In rabbits, ß-adrenergic receptor blockade (esmolol) prevented ITI-214-mediated chronotropy, but inotropy and vasodilation remained unchanged. By contrast, adenosine A2B-receptor blockade (MRS-1754) suppressed ITI-214 cardiovascular effects. Adding fixed-rate atrial pacing did not alter the findings. ITI-214 alone did not affect sarcomere or whole-cell calcium dynamics, whereas ß-adrenergic receptor agonism (isoproterenol) or PDE3 inhibition (cilostamide) increased both. Unlike cilostamide, which further enhanced shortening and peak calcium when combined with isoproterenol, ITI-214 had no impact on these responses. Both PDE1 and PDE3 inhibitors increased shortening and accelerated calcium decay when combined with forskolin, yet only cilostamide increased calcium transients. CONCLUSIONS: PDE1 inhibition by ITI-214 in vivo confers acute inotropic, lusitropic, and arterial vasodilatory effects in PDE1C-expressing mammals with and without HF. The effects appear related to cAMP signaling that is different from that provided via ß-adrenergic receptors or PDE3 modulation. ITI-214, which has completed phase I trials, may provide a novel therapy for HF.
Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 1/metabolism , Myocytes, Cardiac/physiology , Animals , Calcium/metabolism , Cyclic AMP/blood , Cyclic Nucleotide Phosphodiesterases, Type 1/antagonists & inhibitors , Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Dobutamine/therapeutic use , Dogs , Heart Failure/drug therapy , Heart Failure/etiology , Heart Rate/drug effects , Hemodynamics/drug effects , Heterocyclic Compounds, 4 or More Rings/pharmacology , Heterocyclic Compounds, 4 or More Rings/therapeutic use , Humans , Male , Mice , Mice, Inbred C57BL , Myocardial Contraction/drug effects , Myocytes, Cardiac/drug effects , Rabbits , Receptors, Adrenergic, beta-2/chemistry , Receptors, Adrenergic, beta-2/metabolism , Signal Transduction/drug effectsABSTRACT
The enzyme acid sphingomyelinase-like phosphodiesterase 3B (SMPDL3B) was shown to act as a negative regulator of innate immune signaling, affecting cellular lipid composition and membrane fluidity. Furthermore, several reports identified this enzyme as an off target of the therapeutic antibody rituximab, with implications in kidney disorders. However, structural information for this protein is lacking. Here we present the high resolution crystal structure of murine SMPDL3B, which reveals a substrate binding site strikingly different from its paralogs. The active site is located in a narrow boot-shaped cavity. We identify a unique loop near the active site that appears to impose size constraints on incoming substrates. A structure in complex with phosphocholine indicates that the protein recognizes this head group via an aromatic box, a typical choline-binding motif. Although a potential substrate for SMPDL3B is sphingomyelin, we identify other possible substrates such as CDP-choline, ATP, and ADP. Functional experiments employing structure-guided mutagenesis in macrophages highlight amino acid residues potentially involved in recognition of endogenous substrates. Our study is an important step toward elucidating the specific function of this poorly characterized enzyme.
Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/genetics , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , Animals , Choline/chemistry , Choline/genetics , Choline/metabolism , Crystallography, X-Ray , Cyclic Nucleotide Phosphodiesterases, Type 3/genetics , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Mice , Protein Domains , Protein Structure, Secondary , Sf9 Cells , Sphingomyelins/chemistry , Sphingomyelins/genetics , Sphingomyelins/metabolism , Spodoptera , Substrate SpecificityABSTRACT
The MeOH extract of agarwood showed inhibitory activity against phosphodiesterase (PDE) 3A. Fractionation of the extract led to the isolation of two new 2-(2-phenylethyl)chromones, 6,8-dihydroxy-2-[2-(4'-methoxyphenyl)ethyl]chromone (6), and 6,7-dihydroxy-2-(2-phenylethyl)chromone (8), together with six known compounds. All isolated compounds were tested for their PDE 3A inhibitory activity using fluorescence polarization method. Compound 7 showed PDE 3A inhibitory activity with IC50 of 4.83 µM.
Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Flavonoids/chemistry , Phosphodiesterase 3 Inhibitors/chemistry , Plant Extracts/chemistry , Thymelaeaceae/chemistry , Flavonoids/isolation & purification , Molecular StructureABSTRACT
Cell signaling relies extensively on dynamic pools of redox-inactive metal ions such as sodium, potassium, calcium and zinc, but their redox-active transition metal counterparts such as copper and iron have been studied primarily as static enzyme cofactors. Here we report that copper is an endogenous regulator of lipolysis, the breakdown of fat, which is an essential process in maintaining body weight and energy stores. Using a mouse model of genetic copper misregulation, in combination with pharmacological alterations in copper status and imaging studies in a 3T3-L1 white adipocyte model, we found that copper regulates lipolysis at the level of the second messenger, cyclic AMP (cAMP), by altering the activity of the cAMP-degrading phosphodiesterase PDE3B. Biochemical studies of the copper-PDE3B interaction establish copper-dependent inhibition of enzyme activity and identify a key conserved cysteine residue in a PDE3-specific loop that is essential for the observed copper-dependent lipolytic phenotype.
Subject(s)
Copper/pharmacology , Cyclic AMP/metabolism , Lipolysis/drug effects , Phosphodiesterase 3 Inhibitors/pharmacology , 3T3-L1 Cells , Animals , Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Dose-Response Relationship, Drug , Mice , Molecular Structure , Structure-Activity RelationshipABSTRACT
The classical phosphodiesterase 3A (PDE3A) inhibitors provide relaxation of the vasculature system via increasing the cellular level of cyclic adenosine monophosphate (cAMP) and proved to be useful in the management of congestive heart failure. Consequently, the present paper deals with the development of novel pyrazole derivatives tethered with substituted 1,3,5-triazine derivatives in search for novel PDE3 inhibitors. The synthesis of designed inhibitors was realized in a multi-step reaction and the structures were ascertained with the help of various spectroscopic techniques. Subsequently, these analogs were tested for their inhibitory activities against PDE3 enzymes, where they exhibited considerable inhibition, revealing 9g as the most promising inhibitor of the class. In a docking study, the morpholine fragment of compound 9g was efficiently engulfed in the small pocket of the active site lined by Gly940 and Pro941. The substituted aromatic ring of the core scaffold was found to be positioned deep in the cavity bordered by Tyr829, Asn830, Leu850, Glu851, and Thr893. Moreover, it considerably improved the contractility of cardiac muscles without altering the heart beat frequency in experimental subjects.
Subject(s)
Cardiotonic Agents/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Phosphodiesterase 3 Inhibitors/chemistry , Triazines/chemistry , Animals , Cardiotonic Agents/chemical synthesis , Cardiotonic Agents/pharmacology , Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Heart Rate/drug effects , Male , Molecular Docking Simulation , Myocardial Contraction/drug effects , Phosphodiesterase 3 Inhibitors/chemical synthesis , Phosphodiesterase 3 Inhibitors/pharmacology , Rats, Wistar , Triazines/chemical synthesis , Triazines/pharmacologyABSTRACT
A series of novel 2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(4-phenyl)thiazole derivatives, 6(a-o) were designed, synthesized and evaluated for inhibitory activity against human PDE3A and PDE3B. In PDE3 assay, entire set of targeted analogs showed considerable inhibition of PDE3A (IC50=0.24 ± 0.06-16.42 ± 0.14 µM) over PDE3B (IC50=2.34 ± 0.13-28.02 ± 0.03 µM). Among the synthesized derivatives, compound 6d exhibited most potent inhibition of PDE3A with IC50=0.24 ± 0.06 µM than PDE3B (IC50=2.34 ± 0.13 µM). This compound was further subjected for evaluation of cardiotonic activity (contractile and chronotropic effects) in comparison with Vesnarinone. Results showed that, it selectively modulates the force of contraction (63%± 5) rather than frequency rate (23% ± 2) at 100 µM. Docking study of above compound was also carried out in the active site of PDE3 protein model to give proof to the mechanism of action of designed inhibitor. Further, in sub-acute toxicity experiment in Swiss-albino mice, it was found to be non-toxic up to 100mg/kg dose for 28days.
Subject(s)
Cardiotonic Agents/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Drug Design , Phosphodiesterase 3 Inhibitors/chemistry , Pyrazoles/chemistry , Thiazoles/chemistry , Animals , Binding Sites , Cardiotonic Agents/chemical synthesis , Cardiotonic Agents/metabolism , Catalytic Domain , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Drug Evaluation, Preclinical , Humans , Male , Molecular Docking Simulation , Phosphodiesterase 3 Inhibitors/chemical synthesis , Phosphodiesterase 3 Inhibitors/metabolism , Protein Binding , Pyrazoles/metabolism , Rats , Rats, Wistar , Thiazoles/chemical synthesis , Thiazoles/metabolism , Toxicity TestsABSTRACT
Using computational bioactivity prediction models we identified phosphodiesterase 3B (PDE3B) and cathepsin L as macromolecular targets of de novo designed compounds. By disclosing the most potent cathepsin L activator known to date, small molecule repurposing by target panel prediction represents a feasible route towards innovative leads for chemical biology and molecular medicine.
Subject(s)
Cathepsin L/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Phosphodiesterase 3 Inhibitors/chemistry , Protease Inhibitors/chemistry , Cathepsin L/antagonists & inhibitors , Computer Simulation , Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Phosphodiesterase 3 Inhibitors/metabolism , Protease Inhibitors/metabolism , Protein Binding , Structure-Activity RelationshipABSTRACT
Activation of the cyclic AMP (cAMP) pathway reduces bladder contractility. However, the role of phosphodiesterase (PDE) families in regulating this function is poorly understood. Here, we compared the contractile function of the cAMP hydrolyzing PDEs in neonatal rat bladder smooth myocytes. RT-PCR and Western blotting analysis revealed that several isoforms of PDE1-4 were expressed in neonatal rat bladder. While 8-methoxymethyl-3-isobutyl-1-methylxanthine (a PDE1 inhibitor) and BAY-60-7550 (a PDE2 inhibitor) had no effect on the carbachol-enhanced phasic contractions of bladder strips, cilostamide (Cil, a PDE3 inhibitor) and Ro-20-1724 (Ro, a PDE4 inhibitor) significantly reduced these contractions. This inhibitory effect of Ro was blunted by the PKA inhibitor H-89, while the inhibitory effect of Cil was strongly attenuated by the PKG inhibitor KT 5823. Application of Ro in single bladder smooth myocytes resulted in an increase in Ca(2+) spark frequency but a decrease both in Ca(2+) transients and in sarcoplasmic reticulum (SR) Ca(2+) content. In contrast, Cil had no effect on these events. Furthermore, Ro-induced inhibition of the phasic contractions was significantly blocked by ryanodine and iberiotoxin. Taken together, PDE3 and PDE4 are the main PDE isoforms in maintaining the phasic contractions of bladder smooth myocytes, with PDE4 being functionally more active than PDE3. However, their roles are mediated through different mechanisms.
Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Muscle Cells/metabolism , Animals , Calcium/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 4/chemistry , Female , Ions/chemistry , Ions/metabolism , Male , Muscle Cells/cytology , Muscle Cells/drug effects , Muscle Contraction/drug effects , Peptides/pharmacology , Phosphodiesterase 3 Inhibitors/pharmacology , Phosphodiesterase 4 Inhibitors/pharmacology , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Quinolones/pharmacology , Rats , Rats, Sprague-Dawley , Ryanodine/pharmacology , Sarcoplasmic Reticulum/metabolism , Signal Transduction/drug effects , Urinary Bladder/cytologyABSTRACT
The phosphodiesterase-4 (PDE4) enzyme is a promising therapeutic target for several diseases. Our previous studies found resveratrol and moracin M to be natural PDE4 inhibitors. In the present study, three natural resveratrol analogs [pterostilbene, (E)-2',3,5',5-tetrahydroxystilbene (THSB), and oxyresveratrol] are structurally related to resveratrol and moracin M, but their inhibition and mechanism against PDE4 are still unclear. A combined method consisting of molecular docking, molecular dynamics (MD) simulations, binding free energy, and bioassay was performed to better understand their inhibitory mechanism. The binding pattern of pterostilbene demonstrates that it involves hydrophobic/aromatic interactions with Phe340 and Phe372, and forms hydrogen bond(s) with His160 and Gln369 in the active site pocket. The present work also reveals that oxyresveratrol and THSB can bind to PDE4D and exhibits less negative predicted binding free energies than pterostilbene, which was qualitatively validated by bioassay (IC50=96.6, 36.1, and 27.0µM, respectively). Additionally, a linear correlation (R(2)=0.953) is achieved for five PDE4D/ligand complexes between the predicted binding free energies and the experimental counterparts approximately estimated from their IC50 values (≈RT ln IC50). Our results imply that hydrophobic/aromatic forces are the primary factors in explaining the mechanism of inhibition by the three products. Results of the study help to understand the inhibitory mechanism of the three natural products, and thus help the discovery of novel PDE4 inhibitors from resveratrol, moracin M, and other natural products.
Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Phosphodiesterase 4 Inhibitors/chemistry , Plant Extracts/chemistry , Stilbenes/chemistry , Binding Sites , Biological Assay , Cyclic Nucleotide Phosphodiesterases, Type 4 , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Kinetics , Molecular Docking Simulation , Molecular Dynamics Simulation , Plant Extracts/isolation & purification , Protein Binding , Protein Structure, Tertiary , Resveratrol , Stilbenes/isolation & purification , Structure-Activity Relationship , ThermodynamicsABSTRACT
Obesity is linked to various diseases, including insulin resistance, diabetes, and cardiovascular disorders. The idea of inducing white adipose tissue (WAT) to assume characteristics of brown adipose tissue (BAT), and thus gearing it to fat burning instead of storage, is receiving serious consideration as potential treatment for obesity and related disorders. Phosphodiesterase 3B (PDE3B) links insulin- and cAMP-signaling networks in tissues associated with energy metabolism, including WAT. We used C57BL/6 PDE3B knockout (KO) mice to elucidate mechanisms involved in the formation of BAT in epididymal WAT (EWAT) depots. Examination of gene expression profiles in PDE3B KO EWAT revealed increased expression of several genes that block white and promote brown adipogenesis, such as C-terminal binding protein, bone morphogenetic protein 7, and PR domain containing 16, but a clear BAT-like phenotype was not completely induced. However, acute treatment of PDE3B KO mice with the ß3-adrenergic agonist, CL316243, markedly increased the expression of cyclooxygenase-2, which catalyzes prostaglandin synthesis and is thought to be important in the formation of BAT in WAT and the elongation of very long-chain fatty acids 3, which is linked to BAT recruitment upon cold exposure, causing a clear shift toward fat burning and the induction of BAT in KO EWAT. These data provide insight into the mechanisms of BAT formation in mouse EWAT, suggesting that, in a C57BL/6 background, an increase in cAMP, caused by ablation of PDE3B and administration of CL316243, may promote differentiation of prostaglandin-responsive progenitor cells in the EWAT stromal vascular fraction into functional brown adipocytes.
Subject(s)
Adipogenesis , Adipose Tissue, Brown/metabolism , Adipose Tissue, White/metabolism , Adult Stem Cells/cytology , Cyclic AMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Adipogenesis/drug effects , Adipose Tissue, Brown/cytology , Adipose Tissue, Brown/drug effects , Adipose Tissue, White/cytology , Adipose Tissue, White/drug effects , Adrenergic beta-3 Receptor Agonists/pharmacology , Adult Stem Cells/drug effects , Adult Stem Cells/metabolism , Animals , Biomarkers/metabolism , Crosses, Genetic , Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 3/genetics , Cyclooxygenase 2/biosynthesis , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Dioxoles/pharmacology , Enzyme Induction/drug effects , Epididymis , Gene Expression Profiling , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Phosphodiesterase Inhibitors/pharmacology , Quinolones/pharmacologyABSTRACT
Analogues with the scaffolds of 3-cyano-4-alkoxyphenyl-6-bromoaryl-2-pyridone and 2-amino-3-cyano-4-alkoxyphenyl-6-bromoarylpyridine were synthesized. Cyclization of the 2-amino derivatives with formic acid and formamide gave the corresponding pyrido[2,3-d]pyrimidin-4(3H)-one and the pyrido[2,3-d]-pyrimidin-4-amine derivatives, respectively. Active phosphodiesterase 3 (PDE3) inhibitors were identified from each of the four aforementioned scaffolds. This is the first report that pyrido[2,3-d]pyrimidin-4(3H)-one and pyrido[2,3-d]pyrimidin-4-amine derivatives can inhibit PDE3. The analogues with the pyridone and pyrido[2,3-d]pyrimidin-4(3H)-one scaffolds inhibited both cAMP and cyclic guanosine monophosphate (cGMP) hydrolysis by PDE3, while the amine containing scaffolds were more selective for cGMP hydrolysis. This observation may set the base for substrate-selective pharmacological modulation of this important class of drug targets and with less side effects, particularly tachcardia. The dual inhibitors of PDE3 were more potent inhibitor towards the growth of HT-29 cancer cell lines.
Subject(s)
Cyclic GMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Phosphodiesterase 3 Inhibitors/chemistry , Pyridines/chemistry , Pyrimidines/chemistry , Binding Sites , Catalytic Domain , Cell Proliferation/drug effects , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Cyclization , HT29 Cells , Humans , Hydrolysis , Molecular Docking Simulation , Phosphodiesterase 3 Inhibitors/metabolism , Phosphodiesterase 3 Inhibitors/toxicity , Protein Binding , Structure-Activity Relationship , Substrate SpecificityABSTRACT
BACKGROUND AND PURPOSE: PDE3 and/or PDE4 control ventricular effects of catecholamines in several species but their relative effects in failing human ventricle are unknown. We investigated whether the PDE3-selective inhibitor cilostamide (0.3-1 µM) or PDE4 inhibitor rolipram (1-10 µM) modified the positive inotropic and lusitropic effects of catecholamines in human failing myocardium. EXPERIMENTAL APPROACH: Right and left ventricular trabeculae from freshly explanted hearts of 5 non-ß-blocker-treated and 15 metoprolol-treated patients with terminal heart failure were paced to contract at 1 Hz. The effects of (-)-noradrenaline, mediated through ß1 adrenoceptors (ß2 adrenoceptors blocked with ICI118551), and (-)-adrenaline, mediated through ß2 adrenoceptors (ß1 adrenoceptors blocked with CGP20712A), were assessed in the absence and presence of PDE inhibitors. Catecholamine potencies were estimated from -logEC50s. KEY RESULTS: Cilostamide did not significantly potentiate the inotropic effects of the catecholamines in non-ß-blocker-treated patients. Cilostamide caused greater potentiation (P = 0.037) of the positive inotropic effects of (-)-adrenaline (0.78 ± 0.12 log units) than (-)-noradrenaline (0.47 ± 0.12 log units) in metoprolol-treated patients. Lusitropic effects of the catecholamines were also potentiated by cilostamide. Rolipram did not affect the inotropic and lusitropic potencies of (-)-noradrenaline or (-)-adrenaline on right and left ventricular trabeculae from metoprolol-treated patients. CONCLUSIONS AND IMPLICATIONS: Metoprolol induces a control by PDE3 of ventricular effects mediated through both ß1 and ß2 adrenoceptors, thereby further reducing sympathetic cardiostimulation in patients with terminal heart failure. Concurrent therapy with a PDE3 blocker and metoprolol could conceivably facilitate cardiostimulation evoked by adrenaline through ß2 adrenoceptors. PDE4 does not appear to reduce inotropic and lusitropic effects of catecholamines in failing human ventricle.
Subject(s)
Adrenergic beta-1 Receptor Antagonists/adverse effects , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Heart Failure/drug therapy , Heart Ventricles/drug effects , Metoprolol/adverse effects , Receptors, Adrenergic, beta-1/metabolism , Receptors, Adrenergic, beta-2/metabolism , Adrenergic alpha-Agonists/chemistry , Adrenergic alpha-Agonists/pharmacology , Adrenergic beta-1 Receptor Antagonists/pharmacology , Adrenergic beta-1 Receptor Antagonists/therapeutic use , Adrenergic beta-2 Receptor Antagonists/pharmacology , Adrenergic beta-Agonists/chemistry , Adrenergic beta-Agonists/pharmacology , Anti-Arrhythmia Agents/adverse effects , Anti-Arrhythmia Agents/therapeutic use , Cardiotonic Agents/pharmacology , Cardiotonic Agents/therapeutic use , Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 4/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Drug Resistance/drug effects , Epinephrine/agonists , Epinephrine/pharmacology , Heart Failure/metabolism , Heart Failure/physiopathology , Heart Failure/surgery , Heart Transplantation , Heart Ventricles/metabolism , Heart Ventricles/physiopathology , Humans , In Vitro Techniques , Metoprolol/therapeutic use , Middle Aged , Myocardial Contraction/drug effects , Norepinephrine/agonists , Norepinephrine/pharmacology , Phosphodiesterase 3 Inhibitors/pharmacology , Phosphodiesterase 4 Inhibitors/pharmacology , Receptors, Adrenergic, beta-1/chemistry , Receptors, Adrenergic, beta-2/chemistryABSTRACT
Insulin resistance, hyperlipidemia, and cardiovascular complications are common dysregulations of metabolic syndrome. Transplant patients treated with immunosuppressant drugs such as cyclosporine A (CsA), an inhibitor of calcineurin phosphatase, frequently develop similar metabolic complications. Although calcineurin is known to mediate insulin sensitivity by regulating ß-cell growth and adipokine gene transcription, its role in lipid homeostasis is poorly understood. Here, we examined lipid homeostasis in mice lacking calcineurin Aß (CnAß(-/-)). We show that mice lacking calcineurin Aß are hyperlipidemic and develop age-dependent insulin resistance. Hyperlipidemia found in CnAß(-/-) mice is, in part, due to increased lipolysis in adipose tissues, a process mediated by ß-adrenergic G-protein-coupled receptor signaling pathways. CnAß(-/-) mice also exhibit additional pathophysiological phenotypes caused by the potentiated GPCR signaling pathways. A cell autonomous mechanism with sustained cAMP/PKA activation is found in CnAß(-/-) mice or upon CsA treatment to inhibit calcineurin. Increased PKA activation and cAMP accumulation in CnAß(-/-) mice, however, are sensitive to phosphodiesterase inhibitor. Indeed, we show that calcineurin regulates degradation of phosphodiesterase 3B, in addition to phosphodiesterase 4D. These results establish a role for calcineurin in lipid homeostasis. These data also indicate that potentiated cAMP signaling pathway may provide an alternative molecular pathogenesis for the metabolic complications elicited by CsA in transplant patients.
Subject(s)
Calcineurin/deficiency , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Hyperlipidemias/enzymology , Signal Transduction , Aging/drug effects , Aging/pathology , Amino Acid Sequence , Animals , COS Cells , Calcineurin/metabolism , Chlorocebus aethiops , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 3/chemistry , Cyclosporine/pharmacology , Embryo, Mammalian/cytology , Enzyme Activation/drug effects , Fibroblasts/drug effects , Fibroblasts/enzymology , Hyperlipidemias/pathology , Insulin Resistance , Lipid Metabolism/drug effects , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Phosphodiesterase Inhibitors/pharmacology , Receptors, Adrenergic, beta/metabolism , Signal Transduction/drug effects , Triglycerides/biosynthesisABSTRACT
Acrodysostosis is characterized by nasal hypoplasia, peripheral dysostosis, variable short stature, and intellectual impairment. Recently, mutations in PRKAR1A were reported in patients with acrodysostosis and hormone resistance. Subsequently, mutations in a phosphodiesterase gene (PDE4D) were identified in seven sporadic cases. We sequenced PDE4D in seven acrodysostosis patients from five families. Missense mutations were identified in all cases. Families showed de novo inheritance except one family with three affected children whose father was subsequently found to have subtle features of acrodysostosis. There were no recurrent mutations. Short stature and endocrine resistance are rare in this series; however, cognitive involvement and obesity were frequent. This last finding is relevant given PDE4D is insulin responsive and potentially involved in lipolysis. PDE4D encodes a cyclic AMP regulator and places PDE4D-related acrodysostosis within the same family of diseases as pseudohypoparathyroidism, pseudopseudohypoparathyroidism, PRKAR1A-related acrodysostosis and brachydactyly-mental retardation syndrome; all characterized by cognitive impairment and short distal extremities.