Phosphodiesterase 4D Inhibitors Research Articles

Auditory N1 event-related potential amplitude is predictive of serum concentration of BPN14770 in fragile x syndrome.

Fragile X syndrome (FXS) is a rare neurodevelopmental disorder caused by a CGG repeat expansion ≥ 200 repeats in 5' untranslated region of the FMR1 gene, leading to intellectual disability and cognitive difficulties, including in the domain of communication. A recent phase 2a clinical trial testing BPN14770, a phosphodiesterase 4D inhibitor, showed improved cognition in 30 adult males with FXS on drug relative to placebo. The initial study found significant improvements in clinical measures assessing cognition, language, and daily functioning in addition to marginal improvements in electroencephalography (EEG) results for the amplitude of the N1 event-related potential (ERP) component. EEG results suggest BPN14770 improved neural hyperexcitability in FXS. The current study investigated the relationship between BPN14770 pharmacokinetics (PK) and the amplitude of the N1 ERP component from the initial data. Consistent with the original group-level finding in period 1 of the study, participants who received BPN14770 in the period 1 showed a significant correlation between N1 amplitude and serum concentration of BPN14770. These findings strengthen the validity of the original result, indicating that BPN14770 improves cognitive performance by modulating neural hyperexcitability. This study represents the first report of significant correlation between a reliably abnormal EEG marker and serum concentration of a novel pharmaceutical in FXS.

Abstract PO4-23-10: Restoring the efficacy of standard of care in treatment-refractory ER+ breast cancer by re-activation of cAMP-ROS-DNA damage axis

Abstract Estrogen receptor (ER)-positive (ER+) breast cancer has the highest incidence rate and accounts for around 75% of all cases. Endocrine therapy has been the mainstay therapy for the treatment of both early and late-stage ER+ breast cancer for decades. Although most ER+ breast cancer patients initially respond well to endocrine therapy, resistance is common. The addition of CDK4/6 inhibitors to endocrine therapy led to significant improvements in clinical outcome, and they are now considered one of the standard of care (SOC) therapies. However, a significant proportion of patients still suffer from disease relapse upon prolonged use of endocrine therapy in combination with CDK4/6 inhibitors, representing a major clinical challenge that reduces the long-term benefit and patient survival. Therefore, elucidating the mechanisms of sensitivity and resistance to SOC therapy and identifying novel actionable targets are urgently needed. Here, we show that endocrine therapies and CDK4/6 inhibitors cause toxic PARP1 trapping and generation of a functional BRCAness phenotype by downregulating key DNA repair proteins that ultimately result in increased histone parylation and reduced H3K9 acetylation, leading to transcriptional blockage and cell death. Mechanistically, we found that SOC therapy downregulates phosphodiesterase 4D (PDE4D), resulting in increased cAMP levels, PKA-dependent phosphorylation of mitochondrial COXIV-I, generation of mitochondrial reactive oxygen species (ROS), and DNA damage. Importantly, we identified PDE4D as a novel ER target gene that in turn stimulates ER activity in a feedforward loop in endocrine-responsive models and regulates BRCA1 expression. However, during SOC resistance, an ER-to-EGFR switch induces PDE4D overexpression via c-Jun. Inhibition of PDE4D using BPN14770, the first-in-class PDE4D allosteric inhibitor that has successfully completed Phase II trials in Fragile X syndrome, or its upstream EGFR in combination with SOC therapies in drug-resistant settings, including multiple acquired SOC resistant cell line models, primary cultures and organoids of endocrine resistant ER+ PDXs reinstates PARP1 trapping and BRCAness, leading to drug sensitization in vitro and in vivo. Notably, we demonstrated that high PDE4D mRNA and protein expression is associated with dramatically worse disease-free survival and overall survival in endocrine therapy-treated ER+ breast cancer. Considering the availability of potent and non-toxic PDE4D inhibitors, clinically approved EGFR and PARP1 inhibitors, our findings have great translational potential. Citation Format: Ozge Saatci, Metin Cetin, Meral Uner, Unal Metin Tokat, Ioulia Chatzistamou, Pelin Gulizar Ersan, Elodie Montaudon, Aytekin Akyol, Sercan Aksoy, Aysegul Uner, Elisabetta Marangoni, Sajish Mathew, Ozgur Sahin. Restoring the efficacy of standard of care in treatment-refractory ER+ breast cancer by re-activation of cAMP-ROS-DNA damage axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-23-10.

Amelioration of functional and histopathological consequences after spinal cord injury through phosphodiesterase 4D (PDE4D) inhibition

Spinal cord injury (SCI) is a life-changing event that severely impacts the patient's quality of life. Modulating neuroinflammation, which exacerbates the primary injury, and stimulating neuro-regenerative repair mechanisms are key strategies to improve functional recovery. Cyclic adenosine monophosphate (cAMP) is a second messenger crucially involved in both processes. Following SCI, intracellular levels of cAMP are known to decrease over time. Therefore, preventing cAMP degradation represents a promising strategy to suppress inflammation while stimulating regeneration. Intracellular cAMP levels are controlled by its hydrolyzing enzymes phosphodiesterases (PDEs). The PDE4 family is most abundantly expressed in the central nervous system (CNS) and its inhibition has been shown to be therapeutically relevant for managing SCI pathology. Unfortunately, the use of full PDE4 inhibitors at therapeutic doses is associated with severe emetic side effects, hampering their translation toward clinical applications. Therefore, in this study, we evaluated the effect of inhibiting specific PDE4 subtypes (PDE4B and PDE4D) on inflammatory and regenerative processes following SCI, as inhibitors selective for these subtypes have been demonstrated to be well-tolerated. We reveal that administration of the PDE4D inhibitor Gebr32a, even when starting 2 dpi, but not the PDE4B inhibitor A33, improved functional as well as histopathological outcomes after SCI, comparable to results obtained with the full PDE4 inhibitor roflumilast. Furthermore, using a luminescent human iPSC-derived neurospheroid model, we show that PDE4D inhibition stabilizes neural viability by preventing apoptosis and stimulating neuronal differentiation. These findings strongly suggest that specific PDE4D inhibition offers a novel therapeutic approach for SCI.

Abstract 563: Targeting PDE4D reinstates toxic PARP trapping via cAMP-ROS-DNA damage axis and restores the efficacy of standard of care in treatment-refractory ER+ breast cancer

Abstract Endocrine therapies, modulating ER level and/or activity, and the CDK4/6 inhibitors are among the standard-of-care (SOC) therapies used in estrogen receptor-positive (ER+) breast cancer. However, most ER+ breast cancer patients eventually develop resistance to SOC, representing a major clinical challenge that reduces clinical outcome. Therefore, elucidating the common mechanisms of sensitivity and resistance to SOC therapies, and identifying novel actionable targets are urgently needed. Here, we demonstrated that cell death induced by endocrine therapies and CDK4/6 inhibitors involves toxic PARP1 trapping and generation of a functional BRCAness (i.e., BRCA1/2 deficiency) phenotype, leading to transcriptional blockage. We showed that this is achieved by activation of the cAMP/reactive oxygen species (ROS)/DNA damage axis upon downregulation of phosphodiesterase 4D (PDE4D). Importantly, we identified PDE4D as a novel ER target gene that in turn stimulates ER activity in a feedforward loop in endocrine-responsive models. However, during SOC resistance, an ER-to-EGFR switch induces PDE4D overexpression via c-Jun. Inhibition of PDE4D using BPN14770, the first-in-class PDE4D allosteric inhibitor that has successfully completed Phase II trials in Fragile X syndrome, reinstates PARP1 trapping and BRCAness, leading to drug sensitization in vitro and in vivo. Furthermore, inhibition of EGFR or PARP1 recapitulates the effects of PDE4D targeting and overcomes SOC resistance irrespective of the BRCA1/2 status in cell line models, primary cultures and organoids of endocrine resistant ER+ PDXs. Notably, we demonstrated that high PDE4D mRNA and protein expression is associated with dramatically worse disease-free survival and overall survival in endocrine therapy-treated ER+ breast cancer. Given the availability of potent and non-toxic PDE4D inhibitors, and clinically approved EGFR and PARP1 inhibitors, our findings have great translational potential to improve the clinical outcome of refractory ER+ breast cancers. Citation Format: Ozge Saatci, Metin Cetin, Meral Uner, Unal M. Tokat, Ioulia Chatzistamou, Elodie Montaudon, Aytekin Akyol, Sercan Aksoy, Aysegul Uner, Elisabetta Marangoni, Mathew Sajish, Ozgur Sahin. Targeting PDE4D reinstates toxic PARP trapping via cAMP-ROS-DNA damage axis and restores the efficacy of standard of care in treatment-refractory ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 563.

Robust Quantification of Phosphodiesterase-4D in Monkey Brain with PET and 11C-Labeled Radioligands That Avoid Radiometabolite Contamination.

Phosphodiesterase-4D (PDE4D) has emerged as a significant target for treating neuropsychiatric disorders, but no PET radioligand currently exists for robustly quantifying human brain PDE4D to assist biomedical research and drug discovery. A prior candidate PDE4D PET radioligand, namely [11C]T1650, failed in humans because of poor time stability of brain PDE4D-specific signal (indexed by total volume of distribution), likely due to radiometabolites accumulating in brain. Its nitro group was considered to be a source of the brain radiometabolites. Methods: We selected 5 high-affinity and selective PDE4D inhibitors, absent of a nitro group, from our prior structure-activity relationship study for evaluation as PET radioligands. Results: All 5 radioligands were labeled with 11C (half-time, 20.4 min) in useful yields and with high molar activity. All displayed sizable PDE4D-specific signals in rhesus monkey brain. Notably, [11C]JMJ-81 and [11C]JMJ-129 exhibited excellent time stability of signal (total volume of distribution). Furthermore, as an example, [11C]JMJ-81 was found to be free of radiometabolites in ex vivo monkey brain, affirming that this radioligand can provide robust quantification of brain PDE4D with PET. Conclusion: Given their high similarity in structures and metabolic profiles, both [11C]JMJ-81 and [11C]JMJ-129 warrant further evaluation in human subjects. [11C]JMJ-129 shows a higher PDE4D specific-to-nonspecific binding ratio and will be the first to be evaluated.

Abstract A008: Toxic PARP trapping upon cAMP-induced DNA damage reinstates the efficacy of endocrine therapy and CDK4/6 inhibitors in treatment-refractory ER+ breast cancer

Abstract Estrogen receptor (ER)-positive (ER+) breast cancer has the highest incidence rate and accounts for around 75% of all cases. Endocrine therapy and CDK4/6 inhibitors are the standard of care (SOC) in ER+ breast cancer; however, resistance is common, and it greatly reduces patient survival. Therefore, elucidating the mechanisms of sensitivity and resistance to SOC therapy and identifying actionable targets are urgently needed. Here, we show that SOC therapy causes DNA damage and toxic PARP1 trapping upon generation of a functional BRCAness (i.e., BRCA1/2 deficiency) phenotype, leading to increased histone parylation and reduced H3K9 acetylation, resulting in transcriptional blockage and cell death. Mechanistically, SOC therapy downregulates phosphodiesterase 4D (PDE4D), a novel ER target gene in a feedforward loop with ER, resulting in increased cAMP, PKA-dependent phosphorylation of mitochondrial COXIV-I, ROS generation and DNA damage. However, during SOC resistance, an ER-to-EGFR switch induces PDE4D overexpression via c-Jun-mediated transcription. Importantly, we demonstrated that high PDE4D mRNA and protein expression is associated with dramatically worse disease-free survival and overall survival in endocrine therapy-treated ER+ breast cancer. Notably, combining SOC with inhibitors of PDE4D, EGFR or PARP1 overcomes SOC resistance irrespective of the BRCA1/2 status in multiple acquired SOC resistant cell line models, primary cultures and organoids of endocrine resistant ER+ PDXs in vitro and in vivo, providing actionable targets for restoring SOC efficacy. Considering the availability of potent and non-toxic PDE4D inhibitors, clinically approved EGFR and PARP1 inhibitors, our findings have great translational potential. Citation Format: Ozge Saatci, Mein Cetin, Meral Uner, Unal Metin Tokat, Ioulia Chatzistamou, Pelin Gulizar Ersan, Elodie Montaudon, Aytekin Akyol, Sercan Aksoy, Aysegul Uner, Elisabetta Marangoni, Mathew Sajish, Ozgur Sahin. Toxic PARP trapping upon cAMP-induced DNA damage reinstates the efficacy of endocrine therapy and CDK4/6 inhibitors in treatment-refractory ER+ breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A008.

Ablation of specific long PDE4D isoforms increases neurite elongation and conveys protection against amyloid-β pathology

Inhibition of phosphodiesterase 4D (PDE4D) enzymes has been investigated as therapeutic strategy to treat memory problems in Alzheimer’s disease (AD). Although PDE4D inhibitors are effective in enhancing memory processes in rodents and humans, severe side effects may hamper their clinical use. PDE4D enzymes comprise different isoforms, which, when targeted specifically, can increase treatment efficacy and safety. The function of PDE4D isoforms in AD and in molecular memory processes per se has remained unresolved. Here, we report the upregulation of specific PDE4D isoforms in transgenic AD mice and hippocampal neurons exposed to amyloid-β. Furthermore, by means of pharmacological inhibition and CRISPR-Cas9 knockdown, we show that the long-form PDE4D3, -D5, -D7, and -D9 isoforms regulate neuronal plasticity and convey resilience against amyloid-β in vitro. These results indicate that isoform-specific, next to non-selective, PDE4D inhibition is efficient in promoting neuroplasticity in an AD context. Therapeutic effects of non-selective PDE4D inhibitors are likely achieved through actions on long isoforms. Future research should identify which long PDE4D isoforms should be specifically targeted in vivo to both improve treatment efficacy and reduce side effects.

Scouting Different Phosphodiesterase 4 Inhibitor Chemotypes in Silico To Guide the Design of Anti-inflammatory/Antioxidant Agents.

During the last years, we developed a large library of new selective phosphodiesterase 4D inhibitors, maintaining the catechol portion of the well-known PDE4 inhibitor Rolipram, featuring different substitutions in place of the lactam group of this reference compound. Based on the X-ray analysis of PDE4 inhibitors (PDE4Is) previously synthesized by us and of naphthyridine- and naphthyridinone-containing derivatives exhibiting PDE4 inhibitory ability described in the literature, we designed and synthesized new compounds 1-3. All of them were screened in silico as putative PDE4Is, via molecular docking studies to exploit structural variation at the catechol group to gain further contacts especially with the flat aromatic residues (Phe506 and Phe538) of enzyme. Subsequent in silico prediction of ADMET properties and in vitro biological assays on platelets and endothelial cells are in good agreement with our previous data concerning the antioxidant/anti-inflammatory activity exhibited by our previous PDE4Is and similarly to other well-known PDE4Is.

An Empirical Quantitative Structure-Activity Relationship Equation Assists the Discovery of High-Affinity Phosphodiesterase 4D Inhibitors as Leads to PET Radioligands.

A positron emission tomography (PET) radioligand for imaging phosphodiesterase 4D (PDE4D) would benefit drug discovery and the investigation of neuropsychiatric disorders. The most promising radioligand to date, namely, [11C]T1650, has shown unstable quantification in humans. Structural elaboration of [11C]T1650 was therefore deemed necessary. High target affinity in the low nM range is usually required for successful PET radioligands. In our PDE4D PET radioligand development, we formulated and optimized an empirical equation (log[IC50 (nM)] = P1 + P2 + P3 + P4) that well described the relationship between binding affinity and empirically derived values (P1-P4) for the individual fragments in four subregions commonly composing each inhibitor (R2 = 0.988, n = 62). This equation was used to predict compounds that would have high inhibitory potency. Fourteen new compounds were obtained with IC50 of 0.3-10 nM. Finally, eight compounds were judged to be worthy of future radiolabeling and evaluation as PDE4D PET radioligands.

PDE4D binds and interacts with YAP to cooperatively promote HCC progression

The role of cAMP in the development of hepatocellular carcinoma (HCC) is controversial and the biological function of cAMP-hydrolysing enzyme phosphodiesterase 4D (PDE4D) in HCC remains unclear. In this study, we observed markedly higher PDE4D expression in HCC patients with poor survival. PDE4D bound to yes-associated protein (YAP), and PDE4D expression positively correlated with YAP expression in HCC. Overexpression of PDE4D increased YAP dephosphorylation and activity and promoted HCC cell growth in vitro and in vivo, which was attenuated by the YAP inhibitor verteporfin. In contrast, silencing PDE4D reduced YAP expression and HCC cell growth. Notably, forced expression of YAP promoted PDE4D and YAP target gene expression and cell growth, which were abrogated by the PDE4D inhibitor roflumilast. Mechanistically, silencing of YAP caused PDE4D downregulation and HCC cell apoptosis via extracellular signal-regulated kinase (ERK) activation. Roflumilast activated cAMP-PKA signaling and induced cAMP-PKA-dependent YAP phosphorylation at serine 127, resulting in YAP degradation and suppression of HCC growth, which were reversed by the PKA inhibitor PKI. Additionally, transfection of the YAP-S127A mutant reversed roflumilast-mediated suppression of YAP and cell growth. Taken together, our findings indicate that PDE4D binds to and interacts with YAP to promote HCC progression. Targeting the PDE4D-YAP interaction with roflumilast may be an effective strategy for HCC treatment.

Selective inhibition of phosphodiesterase 4D increases tau phosphorylation at Ser214 residue.

Tau is a protein that normally participates in the assembly and stability of microtubules. However, it can form intraneuronal hyperphosphorylated aggregates that are hallmarks of Alzheimer's disease and other neurodegenerative disorders known as tauopathies. Tau can be phosphorylated by multiple kinases at several sites. Among such kinases, the cAMP-dependent protein kinase A (PKA) phosphorylates tau at Ser214 (pTAU-S214), an event that was shown to reduce the pathological assembly of the protein. Given that the neuronal cAMP/PKA-activated cascade is involved in synaptic plasticity and memory, and that cAMP-enhancing strategies demonstrated promising therapeutic potential for the treatment of cognitive deficits, we investigated the impact of cAMP on pTAU-S214 in N2a cells and rat hippocampal slices. Our results confirm that the activation of adenylyl cyclase increases pTAU-S214 in both model systems and, more interestingly, this effect is mimicked by GEBR-7b, a phosphodiesterase 4D inhibitor with proven pro-cognitive efficacy in rodents.

Phosphodiesterase PDE4D Is Decreased in Frontal Cortex of Aged Rats and Positively Correlated With Working Memory Performance and Inversely Correlated With PKA Phosphorylation of Tau.

Age is the largest risk factor for Alzheimer’s disease (AD) and contributes to cognitive impairment in otherwise healthy individuals. Thus, it is critical that we better understand the risk aging presents to vulnerable regions of the brain and carefully design therapeutics to address those effects. In this study we examined age-related changes in cAMP-regulatory protein, phosphodiesterase 4D (PDE4D). Inhibition of PDE4D is currently under investigation as a therapeutic target for AD based on memory-enhancing effects in rodent hippocampus. Therefore, it is important to understand the role of PDE4D in brain regions particularly vulnerable to disease such as the frontal association cortex (FC), where cAMP signaling can impair working memory via opening of potassium channels. We found that PDE4D protein level was decreased in the FC of both moderately and extremely aged rats, and that PDE4D level was correlated with performance on a FC-dependent working memory task. In extremely aged rats, PDE4D was also inversely correlated with levels of phosphorylated tau at serine 214 (S214), a site phosphorylated by protein kinase A. In vitro studies of the PDE4D inhibitor, GEBR-7b, further illustrated that inhibition of PDE4D activity enhanced phosphorylation of tau. pS214-tau phosphorylation is associated with early AD tau pathology, promotes tau dissociation from microtubules and primes subsequent tau hyperphosphorylation at other critical AD-related sites. Age-related loss of PDE4D may thus contribute to the specific vulnerability of the FC to degeneration in AD, and play a critical role in normal cAMP regulation, cautioning against the use of pan-PDE4D inhibitors as therapeutics.

Abstract 2121: Overcoming tamoxifen resistance and inhibiting metastatic recurrence in estrogen receptor-positive breast cancer

Abstract Estrogen receptor (ER)-positive breast cancer accounts for almost 75% of all breast cancers. Tamoxifen has been used for over 40 years to treat early, locally advanced and metastatic ER-positive breast cancer; however, patients develop resistance over time. Furthermore, metastatic recurrence, the major cause of death, is common in ER-positive breast cancer patients whose cancer progressed on tamoxifen and other hormone therapies. In this line, we and others reported epithelial-mesenchymal (EMT)-like changes upon acquisition of tamoxifen resistance hinting towards the co-occurrence of resistance and metastatic abilities of the cells. However, little is known about common molecular mediators of drug resistance and metastatic recurrence. Therefore, we addresses the question of how we concurrently overcome tamoxifen resistance and prevent lethal metastatic recurrence whereby eliminating the mortality associated with metastatic breast cancer. Combining whole-transcriptome sequencing and downstream pathway analysis, we identified cyclic AMP (cAMP) signaling to be the most significantly altered pathway in acquired tamoxifen resistant breast cancer cells. We found that PDE4D (Phosphodiesterase 4D), which hydrolyzes cAMP, was significantly overexpressed in both MCF-7 and T47D tamoxifen-resistant (TamR) cells. We demonstrated that PDE4D inhibition overcomes tamoxifen resistance in cell lines and tumor xenografts via cAMP induced endoplasmic reticulum stress and cell death. Importantly, our TamR cells have partial EMT and enrichment of TGF-beta signaling, and PDE4D inhibition in combination with tamoxifen inhibited cell migration in vitro and blocked TGF-beta induced EMT. RNA-Seq experiment also identified one of the highly oncogenic long non-coding RNAs (lncRNA) to be upregulated in TamR cells, and its targeting inhibits PDE4D and leads to tamoxifen sensitization. We showed that higher expressions of both PDE4D and the candidate lncRNA predict worse survival in tamoxifen-treated breast cancer patients. Currently, we are testing the tamoxifen sensitizer and metastasis blocker functions of PDE4D and our candidate lncRNA using patient-derived xenograft (PDX) models of metastatic ER-positive breast cancer. Overall, our results suggest that targeting PDE4D, or its upstream regulatory lncRNA, can simultaneously overcome tamoxifen resistance and prevent metastatic recurrence in ER-positive breast cancer. This is then expected to dramatically reduce mortality rates among ER-positive breast cancer patients in future. Citation Format: Pelin Gulizar Ersan, Ozge Saatci, Oguzhan Tarman, Rasmi Mishra, Nevin Belder, Unal Metin Tokat, Yasser Riazalhosseini, Ozgur Sahin. Overcoming tamoxifen resistance and inhibiting metastatic recurrence in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2121.

A selective phosphodiesterase 4D inhibitor BPN14770 reverses beta amyloid‐induced memory impairment in humanized PDE4D mice

Beta amyloid peptides (Aβ) are known risk factors involved in cognitive impairment in Alzheimer's disease (AD). Type 4 phosphodiesterase (PDE4) inhibitors such as rolipram could upregulate intracellular cAMP level, which further activate downstream target cAMP‐responsive‐ element‐binding protein (CREB), brain‐derived neurotrophic factor (BDNF) and nerve growth factor (VGF) signaling. Although conventional PDE4 inhibitors have benefits on memory enhancement in preclinical and clinical studies, they were prevented to be developed as a novel class of memory‐enhancing drugs due to their adverse effects such as GI irritation. BPN14770, a novel allosteric inhibitor of PDE4D, exhibits high selectivity on PDE4D based on its close interaction with a single amino acid present at primate PDE4D. The present study used the humanized PDE4D (hPDE4D) mice, which provide a unique and powerful genetic tool for assessing the PDE4D target engagement, to investigate the effects of BPN14770 on memory impairment induced by Ab1‐42. Results suggested that BPN14770‐treated mice exhibited significant learning and memory enhancement, as shown in Morris water maze and Y maze tests. The morphological analyses suggested that both the number of dendrites and the total length were significantly increased after treatment hPDE4D mice with BPN14770 for two weeks. The subsequent neurochemical assay indicated that neuroprotective proteins such as pCREB, BDNF and VGF in Ab‐treated hPDE4D mice were upregulated after treatment with BPN14770. These findings provide evidence that BPN14770 may be a potential therapeutic drug for treating cognitive deficits in Alzheimer's disease and its underlying mechanism may be involved in the cAMP‐pCREB‐BDNF/VGF signaling.Support or Funding InformationThis project was supported by R44 MH091791 to Drs. Mark Gurney and Ying Xu.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Male-Specific cAMP Signaling in the Hippocampus Controls Spatial Memory Deficits in a Mouse Model of Autism and Intellectual Disability

BackgroundThe prevalence of neurodevelopmental disorders is biased toward male individuals, with male-to-female ratios of 2:1 in intellectual disability and 4:1 in autism spectrum disorder. However, the molecular mechanisms of such bias remain unknown. While characterizing a mouse model for loss of the signaling scaffold coiled-coil and C2 domain-containing protein 1A (CC2D1A), which is mutated in intellectual disability and autism spectrum disorder, we identified biochemical and behavioral differences between male and female mice, and explored whether CC2D1A controls male-specific intracellular signaling. MethodsCC2D1A is known to regulate phosphodiesterase 4D (PDE4D), which regulates cyclic adenosine monophosphate (cAMP) signaling. We tested for activation of PDE4D and downstream signaling molecules in the hippocampus of Cc2d1a-deficient mice. We then performed behavioral studies in female mice to analyze learning and memory, and then targeted PDE4D activation with a PDE4D inhibitor to define how changes in cAMP levels affect behavior in male and female mice. ResultsWe found that in Cc2d1a-deficient male mice PDE4D is hyperactive, leading to a reduction in cAMP response element binding protein signaling, but this molecular deficit is not present in female mice. Cc2d1a-deficient male mice show a deficit in spatial memory, which is not present in Cc2d1a-deficient female mice. Restoring PDE4D activity using an inhibitor rescues cognitive deficits in male mice but has no effect on female mice. ConclusionsOur findings show that CC2D1A regulates cAMP intracellular signaling in a male-specific manner in the hippocampus, leading to male-specific cognitive deficits. We propose that male-specific signaling mechanisms are involved in establishing sex bias in neurodevelopmental disorders.

Synthesis and evaluation of clioquinol-rolipram/roflumilast hybrids as multitarget-directed ligands for the treatment of Alzheimer's disease

Considering the importance of PDE4D inhibition and the modulation of biometals in Alzheimer's disease (AD) therapeutics, we have designed, synthesized and evaluated a series of new clioquinol-rolipram/roflumilast hybrids as multitarget-directed ligands for the treatment of AD. In vitro studies demonstrated that some of the molecules processed remarkable inhibitory activity against phosphodiesterase 4D (PDE4D), strong intracellular antioxidant capacity, potent inhibition of metal-induced aggregation of Aβ, and potential blood-brain barrier permeability. Compound 7a demonstrated significant improvement in cognitive and spatial memory in an Aβ25-35-induce mouse model in Morris water-maze test (MWM). These results indicate that compound 7a is a promising multifunctional candidate that is worthy of further study.

Compound D159687, a phosphodiesterase 4D inhibitor, induces weight and fat mass loss in aged mice without changing lean mass, physical and cognitive function

AimsTherapies that recapitulate the health benefits of caloric restriction in older adults are needed. Phosphodiesterase 4 inhibitors demonstrate such promise. We examined their effects on body weight and composition, physical and cognitive function in aged mice using Compound D159687 (D159687). MethodsNineteen 18-months old mice were randomized to receive either control (DMSO) or D159687 for seven weeks. We assessed food intake, body weight and body composition over time and performed once the following tests: treadmill, inverted grip strength, rotarod, spontaneous Y maze tests and skeletal muscle mitochondrial biogenesis. ResultsFour of the D159687 treated mice died in the first week. Necropsy suggests acute lung injury. D159687 treated mice weighed more than control mice at baseline. After controlling for baseline weight, D159687 treated mice lost 4.2 grams(g) more weight than control mice, mainly from fat mass loss (p value < 0.001). Muscle mass was unchanged between the two mice groups. D159587 mice ate significantly more food than the control mice. We found no difference between the two groups in the results of treadmill, rotarod and spontaneous Y maze tests and in mitochondrial biogenesis. ConclusionCompound D159687 induced weight loss, predominantly fat mass loss and increased food intake in aged mice. The caloric restriction and lean mass preservation potential of PDE4D inhibitors deserve further verification. Findings may have major therapeutic implications when translated to the older adult population. Although physical and cognitive parameters were unchanged in this study, further studies would be needed to verify these results. The high death rate in the D159687 treated mice may have been due to the technical aspects of oral gavage.

Reactivation of cAMP Pathway by PDE4D Inhibition Represents a Novel Druggable Axis for Overcoming Tamoxifen Resistance in ER-positive Breast Cancer.

Purpose: Tamoxifen remains an important hormonal therapy for ER-positive breast cancer; however, development of resistance is a major obstacle in clinics. Here, we aimed to identify novel mechanisms of tamoxifen resistance and provide actionable drug targets overcoming resistance.Experimental Design: Whole-transcriptome sequencing, downstream pathway analysis, and drug repositioning approaches were used to identify novel modulators [here: phosphodiesterase 4D (PDE4D)] of tamoxifen resistance. Clinical data involving tamoxifen-treated patients with ER-positive breast cancer were used to assess the impact of PDE4D in tamoxifen resistance. Tamoxifen sensitization role of PDE4D was tested in vitro and in vivo Cytobiology, biochemistry, and functional genomics tools were used to elucidate the mechanisms of PDE4D-mediated tamoxifen resistance.Results: PDE4D, which hydrolyzes cyclic AMP (cAMP), was significantly overexpressed in both MCF-7 and T47D tamoxifen-resistant (TamR) cells. Higher PDE4D expression predicted worse survival in tamoxifen-treated patients with breast cancer (n = 469, P = 0.0036 for DMFS; n = 561, P = 0.0229 for RFS) and remained an independent prognostic factor for RFS in multivariate analysis (n = 132, P = 0.049). Inhibition of PDE4D by either siRNAs or pharmacologic inhibitors (dipyridamole and Gebr-7b) restored tamoxifen sensitivity. Sensitization to tamoxifen is achieved via cAMP-mediated induction of unfolded protein response/ER stress pathway leading to activation of p38/JNK signaling and apoptosis. Remarkably, acetylsalicylic acid (aspirin) was predicted to be a tamoxifen sensitizer using a drug repositioning approach and was shown to reverse resistance by targeting PDE4D/cAMP/ER stress axis. Finally, combining PDE4D inhibitors and tamoxifen suppressed tumor growth better than individual groups in vivoConclusions: PDE4D plays a pivotal role in acquired tamoxifen resistance via blocking cAMP/ER stress/p38-JNK signaling and apoptosis. Clin Cancer Res; 24(8); 1987-2001. ©2018 AACR.

CAMP/PKA/CREB/GLT1 signaling involved in the antidepressant-like effects of phosphodiesterase 4D inhibitor (GEBR-7b) in rats.

ObjectivesGEBR-7b, a potential phosphodiesterase 4D inhibitor, has been shown to have memory-enhancing effects in rodents. However, it is still unknown whether GEBR-7b also has the antidepressant-like effects in rats. Herein, we examined the potential of GEBR-7b to attenuate depression-like behaviors in the rat model of depression induced by chronic unpredictable stress (CUS). Next, we also investigated the alterations of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA) catalytic subunit (PKAca), cAMP response element-binding (CREB), and glutamate transporter 1 (GLT1) levels produced by GEBR-7b in the rats model of depression.MethodsEffects of GEBR-7b on CUS (35 days)-induced depression-like behaviors were examined by measuring immobility time in the forced swimming test (FST). Hippocampal cAMP levels were examined by enzyme-linked immunosorbent assay, whereas PKAca, phosphorylation of CREB (pCREB), CREB, and GLT1 in the hippocampus of rats were subjected to Western blot analysis.ResultsCUS exposure caused a depression-like behavior evidenced by the increased immobility time in FST. Depression-like behavior induced by CUS was accompanied by a significant increased GLT, decreased cAMP, PKAca, pCREB activities in hippocampus. However, repeated GEBR-7b administration significantly reversed CUS-induced depression-like behavior and changes of cAMP/PKA/CREB/GLT1 signaling. No alteration was observed in locomotor activity in open field test.ConclusionThese findings indicate that GEBR-7b reversed the depression-like behaviors induced by CUS in rats, which is at least in part mediated by modulating cAMP, PKAca, pCREB, and GLT1 levels in the hippocampus of rats, supporting its neuroprotective potential against behavioral and biochemical dysfunctions induced by CUS.

Catecholic amides as potential selective phosphodiesterase 4D inhibitors: Design, synthesis, pharmacological evaluation and structure–activity relationships

In this study, a series of catechol-based amides (8a–n) with different amide linkers linking the catecholic moiety to the terminal phenyl ring was designed and synthesized as potent phosphodiesterase (PDE) 4D inhibitors. The inhibitory activities of these compounds were evaluated against the core catalytic domains of human PDE4 (PDE4CAT), full-length PDE4B1 and PDE4D7 enzymes, and other PDE family members. The results indicated the majority of compounds 8a–n displayed moderate to good inhibitory activities against PDE4CAT. Among these compounds, compound 8j with a short amide linker (CONHCH2) displayed comparable PDE4CAT inhibitory activity (IC50=410nM) with rolipram. More interestingly, compound 8g, a potent and selective PDE4D inhibitor (IC50=94nM), exhibited a 10-fold selectivity over the PDE4B subtypes and an over 1000-fold selectivity against other PDE family members. Docking simulations suggested that 8g forms three extra H-bonds with the NH of residue Asn487 and two water molecules.