Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases.

Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca(2+) levels and activates the CamKKß-AMPK pathway via phospholipase C and the ryanodine receptor Ca(2+)-release channel. As a consequence, resveratrol increases NAD(+) and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging.

Inhibition of phosphodiesterase PDE8B reduces activation of primordial follicles in mouse ovaries.

In the ovaries, cyclic adenosine 3',5'-monophosphate (cAMP) is a second messenger supporting the generation of steroids. Phosphodiesterases (PDEs) are regulators of intracellular cAMP, and therefore, potential regulators of ovarian function. Interestingly, the family of PDE genes are differentially expressed in human oocytes and granulosa cells from primordial and primary follicles, suggesting diverse roles. In this study, we addressed the functions of PDE3B and PDE8B in primordial follicle regulation using inhibitors of PDE3B and PDE8B in murine ovary primary in vitro cultures. Inhibition of PDE8B in ovarian cultures prevented primordial follicle activation, while inhibition of PDE3B had no effect on follicle distribution in the ovary, under the tested conditions. As cAMP levels may increase steroid levels, we assessed the protein levels of the steroidogenic acute regulatory protein (StAR) and aromatase enzymes, and found that inhibition of PDE3B reduced StAR protein levels, whereas inhibition of PDE8 did not alter StAR expression in our murine ovary culture system conditions. Our results showed that ketotifen-induced inhibition of PDE8B can decrease primordial follicle activation, whereas we observed no effect of follicle distribution, when PDE3B was inhibited. Expression of the StaR enzyme was not altered when PDE8B was inhibited, which might reflect not sufficient inhibition by ketotifen to induce StAR alterations, or redundant mechanisms.

Clinical Implication of Phosphodiesterase-4-Inhibition.

The pleiotropic function of 3',5'-cyclic adenosine monophosphate (cAMP)-dependent pathways in health and disease led to the development of pharmacological phosphodiesterase inhibitors (PDE-I) to attenuate cAMP degradation. While there are many isotypes of PDE, a predominant role of PDE4 is to regulate fundamental functions, including endothelial and epithelial barrier stability, modulation of inflammatory responses and cognitive and/or mood functions. This makes the use of PDE4-I an interesting tool for various therapeutic approaches. However, due to the presence of PDE4 in many tissues, there is a significant danger for serious side effects. Based on this, the aim of this review is to provide a comprehensive overview of the approaches and effects of PDE4-I for different therapeutic applications. In summary, despite many obstacles to use of PDE4-I for different therapeutic approaches, the current data warrant future research to utilize the therapeutic potential of phosphodiesterase 4 inhibition.

Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease.

Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric-oxide- and natriuretic-peptide-coupled signalling, stimulating phosphorylation changes by protein kinase G. Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation. Furthermore, although PDE5A regulates nitric-oxide-generated cGMP, nitric oxide signalling is often depressed by heart disease. PDEs controlling natriuretic-peptide-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A (refs 7, 8) is expressed in the mammalian heart, including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates natriuretic-peptide- rather than nitric-oxide-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neurohormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

Phosphodiesterases Expression during Murine Cardiac Development.

3'-5' cyclic nucleotide phosphodiesterases (PDEs) are a large family of enzymes playing a fundamental role in the control of intracellular levels of cAMP and cGMP. Emerging evidence suggested an important role of phosphodiesterases in heart formation, but little is known about the expression of phosphodiesterases during cardiac development. In the present study, the pattern of expression and enzymatic activity of phosphodiesterases was investigated at different stages of heart formation. C57BL/6 mice were mated and embryos were collected from 14.5 to 18.5 days of development. Data obtained by qRT-PCR and Western blot analysis showed that seven different isoforms are expressed during heart development, and PDE1C, PDE2A, PDE4D, PDE5A and PDE8A are modulated from E14.5 to E18.5. In heart homogenates, the total cAMP and cGMP hydrolytic activity is constant at the evaluated times, and PDE4 accounts for the majority of the cAMP hydrolyzing ability and PDE2A accounts for cGMP hydrolysis. This study showed that a subset of PDEs is expressed in developing mice heart and some of them are modulated to maintain constant nucleotide phosphodiesterase activity in embryonic and fetal heart.

The Involvement of PDE4 in the Protective Effects of Melatonin on Cigarette-Smoke-Induced Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a significant disease threatening human health. Currently, roflumilast, a phosphodiesterase (PDE)4 inhibitor, is recommended as a therapeutic agent for COPD. In this study, we investigated the therapeutic effects of melatonin against COPD, focusing on determining whether it is a PDE4 inhibitor via in vivo and in vitro experiment using cigarette smoke (CS) and cigarette smoke condensate (CSC), respectively. In the in vivo experiments, melatonin treatment reduced inflammatory responses, including inflammatory cell counts. Melatonin treatment also suppressed the CS-exposure-induced upregulation of cytokine and matrix metalloproteinase (MMP)-9, reduced the PDE4B expression, and elevated cAMP levels. In addition, these effects were synergistic, as melatonin and roflumilast cotreatment eventually ameliorated the CS-exposure-induced worsening of lung function. In the CSC-stimulated NCI-H292 cells, melatonin inhibited elevation in the levels of inflammatory cytokines, MMP-9, and PDE4, and elevated cAMP levels. Furthermore, melatonin and roflumilast cotreatment was more effective on inflammatory responses than only melatonin or roflumilast treatment. Our results indicate that melatonin relieves inflammatory response and loss of lung function in COPD, which is associated with decreased PDE4 expression. Therefore, we suggest that melatonin is a putative candidate for the treatment of COPD.

Nanodomain Regulation of Cardiac Cyclic Nucleotide Signaling by Phosphodiesterases.

Cyclic nucleotide phosphodiesterases (PDEs) form an 11-member superfamily comprising 100 different isoforms that regulate the second messengers cyclic adenosine or guanosine 3',5'-monophosphate (cAMP or cGMP). These PDE isoforms differ with respect to substrate selectivity and their localized control of cAMP and cGMP within nanodomains that target specific cellular pools and synthesis pathways for the cyclic nucleotides. Seven PDE family members are physiologically relevant to regulating cardiac function, disease remodeling of the heart, or both: PDE1 and PDE2, both dual-substrate (cAMP and cGMP) esterases; PDE3, PDE4, and PDE8, which principally hydrolyze cAMP; and PDE5A and PDE9A, which target cGMP. New insights regarding the different roles of PDEs in health and disease and their local signaling control are broadening the potential therapeutic utility for PDE-selective inhibitors. In this review, we discuss these PDEs, focusing on the different mechanisms by which they control cardiac function in health and disease by regulating intracellular nanodomains.

Design, synthesis and evaluation of pyrazolopyrimidinone derivatives as novel PDE9A inhibitors for treatment of Alzheimer's disease.

Phosphodiesterase-9 (PDE9) is a promising target for the treatment of Alzheimer's disease (AD). To discover efficient PDE9 inhibitors with good metabolic stability and solubility, a series of novel pyrazolopyrimidinone derivatives have been designed with the assistance of molecular docking and dynamics simulations. All the fourteen synthesized compounds gave excellent inhibition ratio against PDE9 at 10 nM. Compound 1k with the IC50 of 2.0 nM against PDE9, showed good metabolic stability (t1/2 of 57 min) in the RLM as well as good solubility (195 mg/L). The analysis on binding modes of targeted compounds may provide insight for further structural modification.

Analyses of PDE-regulated phosphoproteomes reveal unique and specific cAMP-signaling modules in T cells.

Specific functions for different cyclic nucleotide phosphodiesterases (PDEs) have not yet been identified in most cell types. Conventional approaches to study PDE function typically rely on measurements of global cAMP, general increases in cAMP-dependent protein kinase (PKA), or the activity of exchange protein activated by cAMP (EPAC). Although newer approaches using subcellularly targeted FRET reporter sensors have helped define more compartmentalized regulation of cAMP, PKA, and EPAC, they have limited ability to link this regulation to downstream effector molecules and biological functions. To address this problem, we have begun to use an unbiased mass spectrometry-based approach coupled with treatment using PDE isozyme-selective inhibitors to characterize the phosphoproteomes of the functional pools of cAMP/PKA/EPAC that are regulated by specific cAMP-PDEs (the PDE-regulated phosphoproteomes). In Jurkat cells we find multiple, distinct PDE-regulated phosphoproteomes that can be defined by their responses to different PDE inhibitors. We also find that little phosphorylation occurs unless at least two different PDEs are concurrently inhibited in these cells. Moreover, bioinformatics analyses of these phosphoproteomes provide insight into the unique functional roles, mechanisms of action, and synergistic relationships among the different PDEs that coordinate cAMP-signaling cascades in these cells. The data strongly suggest that the phosphorylation of many different substrates contributes to cAMP-dependent regulation of these cells. The findings further suggest that the approach of using selective, inhibitor-dependent phosphoproteome analysis can provide a generalized methodology for understanding the roles of different PDEs in the regulation of cyclic nucleotide signaling.

Imidazole Derivatives as Promising Agents for the Treatment of Chagas Disease.

More than 100 years after being first described, Chagas disease remains endemic in 21 Latin American countries and has spread to other continents. Indeed, this disease, which is caused by the protozoan parasite Trypanosoma cruzi, is no longer just a problem for the American continents but has become a global health threat. Current therapies, i.e., nifurtimox and benznidazole (Bz), are far from being adequate, due to their undesirable effects and their lack of efficacy in the chronic phases of the disease. In this work, we present an in-depth phenotypic evaluation in T. cruzi of a new class of imidazole compounds, which were discovered in a previous phenotypic screen against different trypanosomatids and were designed as potential inhibitors of cAMP phosphodiesterases (PDEs). The confirmation of several activities similar or superior to that of Bz prompted a synthesis program of hit optimization and extended structure-activity relationship aimed at improving drug-like properties such as aqueous solubility, which resulted in additional hits with 50% inhibitory concentration (IC50) values similar to that of Bz. The cellular effects of one representative hit, compound 9, on bloodstream trypomastigotes were further investigated. Transmission electron microscopy revealed cellular changes, after just 2 h of incubation with the IC50 concentration, that were consistent with induced autophagy and osmotic stress, mechanisms previously linked to cAMP signaling. Compound 9 induced highly significant increases in both cellular and medium cAMP levels, confirming that inhibition of T. cruzi PDE(s) is part of its mechanism of action. The potent and selective activity of this imidazole-based PDE inhibitor class against T. cruzi constitutes a successful repurposing of research into inhibitors of mammalian PDEs.

The Novel Phosphodiesterase 9A Inhibitor BI 409306 Increases Cyclic Guanosine Monophosphate Levels in the Brain, Promotes Synaptic Plasticity, and Enhances Memory Function in Rodents.

N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) is an established cellular model underlying learning and memory, and involves intracellular signaling mediated by the second messenger cyclic guanosine monophosphate (cGMP). As phosphodiesterase (PDE)9A selectively hydrolyses cGMP in areas of the brain related to cognition, PDE9A inhibitors may improve cognitive function by enhancing NMDA receptor-dependent LTP. This study aimed to pharmacologically characterize BI 409306, a novel PDE9A inhibitor, using in vitro assays and in vivo determination of cGMP levels in the brain. Further, the effects of BI 409306 on synaptic plasticity evaluated by LTP in ex vivo hippocampal slices and on cognitive performance in rodents were also investigated. In vitro assays demonstrated that BI 409306 is a potent and selective inhibitor of human and rat PDE9A with mean concentrations at half-maximal inhibition (IC50) of 65 and 168 nM. BI 409306 increased cGMP levels in rat prefrontal cortex and cerebrospinal fluid and attenuated a reduction in mouse striatum cGMP induced by the NMDA-receptor antagonist MK-801. In ex vivo rat brain slices, BI 409306 enhanced LTP induced by both weak and strong tetanic stimulation. Treatment of mice with BI 409306 reversed MK-801-induced working memory deficits in a T-maze spontaneous-alternation task and improved long-term memory in an object recognition task. These findings suggest that BI 409306 is a potent and selective inhibitor of PDE9A. BI 409306 shows target engagement by increasing cGMP levels in brain, facilitates synaptic plasticity as demonstrated by enhancement of hippocampal LTP, and improves episodic and working memory function in rodents. SIGNIFICANCE STATEMENT: This preclinical study demonstrates that BI 409306 is a potent and selective PDE9A inhibitor in rodents. Treatment with BI 409306 increased brain cGMP levels, promoted long-term potentiation, and improved episodic and working memory performance in rodents. These findings support a role for PDE9A in synaptic plasticity and cognition. The potential benefits of BI 409306 are currently being investigated in clinical trials.

Discovery of novel PDE9A inhibitors with antioxidant activities for treatment of Alzheimer's disease.

Phosphodiesterase-9 (PDE9) is a promising target for treatment of Alzheimer's disease (AD). To discover multifunctional anti-AD agents with capability of PDE9 inhibition and antioxidant activity, a series of novel pyrazolopyrimidinone derivatives, coupling with the pharmacophore of antioxidants such as ferulic and lipolic acids have been designed with the assistance of molecular docking and dynamics simulations. Twelve out of 14 synthesised compounds inhibited PDE9A with IC50 below 200 nM, and showed good antioxidant capacities in the ORAC assay. Compound 1h, the most promising multifunctional anti-AD agent, had IC50 of 56 nM against PDE9A and good antioxidant ability (ORAC (trolox) = 3.3). The selectivity of 1h over other PDEs was acceptable. In addition, 1h showed no cytotoxicity to human neuroblastoma SH-SY5Y cells. The analysis on structure-activity relationship (SAR) and binding modes of the compounds may provide insight into further modification.

Design and synthesis of novel annulated thienopyrimidines as phosphodiesterase 5 (PDE5) inhibitors.

Novel cycloalkene-fused thienopyrimidine analogues with enhanced phosphodiesterase 5 (PDE5) inhibitory properties are presented. The structure of the reported scaffold was modulated through variation of the terminal cycloalkene ring size, as well as by varying the substituents at position 4 through the attachment of different groups including aniline, benzylamine, cyclohexylethylamine, methyl/acetyl/aryl piperazines, and aryl hydrazones. Compound 15Y with a benzylamine substituent and cycloheptene as terminal ring showed the highest PDE5 inhibitory activity with an IC50 value as low as 190 nM and with good selectivity versus PDE7 and PDE9.

C33(S), a novel PDE9A inhibitor, protects against rat cardiac hypertrophy through upregulating cGMP signaling.

Phosphodiesterase-9A (PDE9A) expression is upregulated during cardiac hypertrophy and heart failure. Accumulating evidence suggests that PDE9A might be a promising therapeutic target for heart diseases. The present study sought to investigate the effects and underlying mechanisms of C33(S), a novel selective PDE9A inhibitor, on cardiac hypertrophy in vitro and in vivo. Treatment of neonatal rat cardiomyocytes (NRCMs) with PE (100 µmol/L) or ISO (1 µmol/L) induced cardiac hypertrophy characterized by significantly increased cell surface areas and increased expression of fetal genes (ANF and BNP). Furthermore, PE or ISO significantly increased the expression of PDE9A in the cells; whereas knockdown of PDE9A significantly alleviated PE-induced hypertrophic responses. Moreover, pretreatment with PDE9A inhibitor C33(S) (50 and 500 nmol/L) or PF-7943 (2 µmol/L) also alleviated the cardiac hypertrophic responses in PE-treated NRCMs. Abdominal aortic constriction (AAC)-induced cardiac hypertrophy and ISO-induced heart failure were established in SD rats. In ISO-treated rats, oral administration of C33(S) (9, 3, and 1 mg·kg-1·d-1, for 3 consecutive weeks) significantly increased fractional shortening (43.55%±3.98%, 54.79%±1.95%, 43.98%±7.96% vs 32.18%±6.28%), ejection fraction (72.97%±4.64%, 84.29%±1.56%, 73.41%±9.37% vs 49.17%±4.20%) and cardiac output (60.01±9.11, 69.40±11.63, 58.08±8.47 mL/min vs 48.97±2.11 mL/min) but decreased the left ventricular internal diameter, suggesting that the transition to heart failure was postponed by C33(S). We further revealed that C33(S) significantly elevated intracellular cGMP levels, phosphorylation of phospholamban (PLB) and expression of SERCA2a in PE-treated NRCMs in vitro and in ISO-induced heart failure model in vivo. Our results demonstrate that C33(S) effectively protects against cardiac hypertrophy and postpones the transition to heart failure, suggesting that it is a promising agent in the treatment of cardiac diseases.

A phase I, randomized, proof-of-clinical-mechanism study assessing the pharmacokinetics and pharmacodynamics of the oral PDE9A inhibitor BI 409306 in healthy male volunteers.

OBJECTIVE: Cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase (PDE) inhibitors are hypothesized to improve cognition in schizophrenia and Alzheimer disease by increasing cGMP levels in certain brain regions. This phase I, randomized, parallel-group, double-blind, placebo-controlled study provides proof-of-mechanism evidence for BI 409306, a novel, oral PDE9A inhibitor. METHODS: In healthy males, exposure of BI 409306 (25-, 50-, 100-, and 200-mg single dose) and placebo was assessed in plasma and cerebrospinal fluid (CSF). Effect of BI 409306 on CSF cGMP levels was evaluated, and adverse events (AEs) were monitored. RESULTS: In all enrolled subjects (N = 20), plasma BI 409306 concentration increased rapidly (median tmax : 0.75-1.25 hr) followed by rapid increases in CSF (median tmax : 1.5-2.0 hr). Maximum CSF cGMP concentrations were achieved within 2 to 5 hr, declining to baseline levels 10 to 14 hr after dosing. Dose-dependent increases in plasma and CSF exposure and CSF cGMP were shown. BI 409306 was safe and well tolerated. Most AEs were mild to moderate in intensity and study procedure-related. CONCLUSIONS: BI 409306 increased rapidly in plasma and was subsequently detected in CSF, resulting in dose-dependent increases in cGMP levels in CSF. Results indicate BI 409306 efficiently crosses the blood-CSF barrier, with an acceptable level of AEs.

Influence of nitric oxide and phosphodiesterases during in vitro maturation of bovine oocytes on meiotic resumption and embryo production.

This study aimed to examine the effects of nitric oxide (NO) and different phosphodiesterase (PDE) families on meiosis resumption, nucleotides levels and embryo production. Experiment I, COCs were matured in vitro with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) associated or not with the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), meiotic resumption and nucleotides levels were assessed. SNAP delayed germinal vesicle breakdown (GVBD) (53.4 ± 1.2 versus 78.4 ± 2.4% for controls, P 0.05). Cyclic GMP levels were higher in SNAP (3.94 ± 0.18, P 0.05). Embryo development did not differ from the control for SNAP and cilostamide groups (38.7 ± 5.8, 37.9 ± 6.2 and 40.5 ± 5.8%, P > 0.05), but SNAP + cilostamide decreased embryo production (25.7 ± 6.9%, P < 0.05). In conclusion, SNAP was confirmed to delay meiosis resumption by the NO/sGC/cGMP pathway, by increasing cGMP, but not cAMP. Inhibiting different PDEs to further increase nucleotides in association with SNAP did not show any additive effects on meiosis resumption, indicating that other pathways are involved. Moreover, SNAP + cilostamide affected the meiosis progression and decreased embryo development.

First-in-human study assessing safety, tolerability and pharmacokinetics of BI 409306, a selective phosphodiesterase 9A inhibitor, in healthy males.

AIMS: The aim of the present study was to investigate the safety, tolerability, dose proportionality and relative bioavailability of tablet and oral solution formulations of BI 409306 in healthy male subjects, and to compare the safety and pharmacokinetics in subjects who were extensive metabolizers (EMs) or poor metabolizers (PMs) of cytochrome P450 (CYP)-2C19. METHODS: The present randomized, double-blind, placebo-controlled, single-centre study evaluated single rising doses of BI 409306 (0.5-500 mg) administered as a tablet or oral solution to EMs or PMs. RESULTS: Of 80 enrolled subjects (mean age 36.7 years), 79 (CYP2C19 EMs, 71; CYP2C19 PMs, eight) received treatment and completed the study. Adverse events (AEs) were mild to moderate in intensity. Overall, 17/71 (23.9%) EMs and 6/8 (75.0%) PMs experienced 28 and eight AEs, respectively, of which, 25 and seven AEs, respectively, were considered to be drug related. The most frequently reported AEs were nervous system and eye disorders; all occurred shortly (20-30 min) after administration and mostly resolved within 1-2 h. No serious AEs occurred. BI 409306 systemic absorption and elimination were rapid; peak plasma concentration (Cmax ) was reached <1 h after drug administration, and the half-life ranged from 0.99 h to 2.71 h. Both the tablet and oral solution resulted in similar exposures. In PMs, at dose levels of 10 mg and 100 mg, Cmax was 2.2-2.3-fold higher, and the area under the plasma concentration-time curve over the time interval 0 extrapolated to infinity was 4.1-5.0-fold higher compared with EMs. CONCLUSIONS: In healthy male subjects, BI 409306 was generally safe and well tolerated, with rapid absorption and elimination. Systemic exposure was higher in CYP2C19 PMs than EMs at the same dose level.

Synthesis and evaluation of analogs of the phenylpyridazinone NPD-001 as potent trypanosomal TbrPDEB1 phosphodiesterase inhibitors and in vitro trypanocidals.

Trypanosomal phosphodiesterases B1 and B2 (TbrPDEB1 and TbrPDEB2) play an important role in the life cycle of Trypanosoma brucei, the causative parasite of human African trypanosomiasis (HAT), also known as African sleeping sickness. Knock down of both enzymes leads to cell cycle arrest and is lethal to the parasite. Recently, we reported the phenylpyridazinone, NPD-001, with low nanomolar IC50 values on both TbrPDEB1 (IC50: 4nM) and TbrPDEB2 (IC50: 3nM) (J. Infect. Dis.2012, 206, 229). In this study, we now report on the first structure activity relationships of a series of phenylpyridazinone analogs as TbrPDEB1 inhibitors. A selection of compounds was also shown to be anti-parasitic. Importantly, a good correlation between TbrPDEB1 IC50 and EC50 against the whole parasite was observed. Preliminary analysis of the SAR of selected compounds on TbrPDEB1 and human PDEs shows large differences which shows the potential for obtaining parasite selective PDE inhibitors. The results of these studies support the pharmacological validation of the Trypanosome PDEB family as novel therapeutic approach for HAT and provide as well valuable information for the design of potent TbrPDEB1 inhibitors that could be used for the treatment of this disease.

Apremilast reversed carfilzomib-induced cardiotoxicity through inhibition of oxidative stress, NF-κB and MAPK signaling in rats.

Carfilzomib (CFZ), is a potent, selective second generation proteasome inhibitor, used for the treatment of multiple myeloma. The aim of the present study was to investigate the possible protective effect of apremilast (AP) on the CFZ -induced cardiotoxicity. Rats were randomly divided into four groups: Group 1, served as the control group, received normal saline. Group 2, served as the toxic group, received CFZ (4 mg/kg, intraperitoneally [i.p.]). Groups 3 and 4, served as treatment groups, and received CFZ with concomitant oral administration of AP in doses of 10 and 20 mg/kg/day, respectively. In the present study, administration of CFZ resulted in a significant increase in serum aspartate transaminase (AST), lactate dehydrogenase (LDH), creatine kinase (CK) and creatine kinase-MB (CK-MB), which were reversed by treatment with AP. CFZ resulted in a significant increase in heart malondialdehyde (MDA) contents and decrease in cardiac glutathione (GSH) level and catalase (CAT) enzyme activity which were significantly reversed by treatment with AP. Induction of cardiotoxicity by CFZ significantly increased caspase-3 enzyme activity which were reversed by treatment with AP. RT-PCR analysis revealed an increased mRNA expression of NF-κB, ERK and JNK which were reversed by treatment with AP in cardiac tissues. Western blot analysis revealed an increased expression of caspase-3 and NF-κB p65 and a decrease expression of inhibitory kappa B-alpha (Iκbα) with CFZ, which were reversed by treatment with AP. In conclusion, apremilast showed protective effect against CFZ-induced cardiotoxicity.

Structural Asymmetry of Phosphodiesterase-9A and a Unique Pocket for Selective Binding of a Potent Enantiomeric Inhibitor.

Phosphodiesterase-9 (PDE9) inhibitors have been studied as potential therapeutics for treatment of central nervous system diseases and diabetes. Here, we report the discovery of a new category of PDE9 inhibitors by rational design on the basis of the crystal structures. The best compound, (S)-6-((1-(4-chlorophenyl)ethyl)amino)-1-cyclopentyl-1,5,6,7-tetrahydro-4H-pyrazolo[3,4-day]pyrimidin-4-one [(S)-C33], has an IC50 value of 11 nM against PDE9 and the racemic C33 has bioavailability of 56.5% in the rat pharmacokinetic model. The crystal structures of PDE9 in the complex with racemic C33, (R)-C33, and (S)-C33 reveal subtle conformational asymmetry of two M-loops in the PDE9 dimer and different conformations of two C33 enantiomers. The structures also identified a small hydrophobic pocket that interacts with the tyrosyl tail of (S)-C33 but not with (R)-C33, and is thus possibly useful for improvement of selectivity of PDE9 inhibitors. The asymmetry of the M-loop and the different interactions of the C33 enantiomers imply the necessity to consider the whole PDE9 dimer in the design of inhibitors.