Phosphodiesterase Research Articles

Discovery of Selective PDE1 Inhibitors with Anti-pulmonary Fibrosis Effects by Targeting the Metal Pocket.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with no ideal drugs. Our previous research demonstrated that phosphodiesterase 1 (PDE1) could be a promising target for the treatment of IPF. However, only a few selective PDE1 inhibitors are available, and the mechanism of recognition between inhibitors and the PDE1 protein is not fully understood. This study carried out a step-by-step optimization of a dihydropyrimidine hit Z94555858. By targeting the metal pocket of PDE1, a lead compound 3f was obtained, exhibiting an IC50 value of 11 nM against PDE1, moderate selectivity over other PDEs, and significant anti-fibrotic effects in bleomycin-induced pulmonary fibrosis rats. The structure-activity relationship study aided by molecular docking revealed that forming halogen bonds with water in the metal pocket greatly enhanced the PDE1 inhibition, providing a novel strategy for further rational design of PDE1 inhibitors.

Abstract 4135700: Effects of Mitochondrial Dysfunction on Extracellular Vesicle Release and Subsequent Disruption of the Lung Vascular Endothelial Barrier

Background: Lung endothelial cell (EC) barrier disruption is a hallmark of acute lung injury (ALI), which is commonly caused by bacterial pneumonia. Alveolar epithelial cells (AEC) are the first line of defence against invading respiratory pathogens in the lungs and can mediate inflammatory signaling to the adjacent EC contributing to barrier disruption. We have previously demonstrated that pneumolysin (PLY), a major Streptococcus pneumoniae virulence factor, induces both mitochondrial dysfunction in lung AEC and release of large extracellular vesicles (EVs). These EVs, which are enriched with mitochondrial cargo, contribute to increased permeability of the lung EC barrier. Here, we investigate strategies to inhibit mitochondrial dysfunction in AEC and examine how these interventions impact EC barrier function. Methods: Human alveolar epithelial cells (A549) or immortalized AEC (Cell Biologics) were pre-treated with mitoTEMPO (mtROS scavenger), P110 (excessive fission inhibitor), or ensifentrine [dual phosphodiesterase (PDE) 3/4 inhibitor] prior to PLY treatment (100-300 ng/ml, 4 hrs). Mitochondrial function was assessed by the JC-1 assay. EVs were isolated from cell media and analyzed for their mitochondrial content (Tom20 and Tim23 expression). To assess their functional role, EVs were added to TNF-α treated human lung microvascular EC, and barrier function was then determined using the ECIS assay to measure transendothelial electrical resistance (TER). Results: PLY-induced mitochondrial dysfunction was reduced in the presence of mitoTempo or P110. EVs from PLY-treated AEC (EV PLY ; enriched mitochondrial content) exacerbated TNF-α-induced EC barrier permeability as indicated by the significant decrease in TER. EV PLY derived from MitoTEMPO or P110-pretreated AEC contained less mitochondrial cargo and resulted in less EC permeability after TNF-α. Compared to mitoTEMPO and P110, ensifentrine exhibited superior protection against mitochondrial dysfunction after PLY exposure. In addition, in the presence of ensifentrine, the quantity of EV release and their extracelllular mitochondrial content were both dramatically reduced. In contrast to control EV PLY , EV PLY from ensifentrine-pretreated AEC demonstrated no barrier disruptive properties. Conclusions: Mitochondrial dysfunction in AEC contributes to vascular endothelial injury in ALI through mechanisms involving the release of barrier-disruptive EVs, which can be effectively targeted by inhibiting PDE3/4.

Abstract 4122957: C-C motif chemokine receptor-2 disruption Synergizes with Phosphodiesterase Type 5 Inhibition in Ameliorating Pulmonary Hypertension in Monocrotaline-treated Rats: Proof-of-Concept Study for a Combination therapy and Potential Mechanisms

Background: Whether and how any anti-inflammatory agents and their combination with conventional pulmonary vasodilators protect against pulmonary arterial hypertension (PAH) is unknown. Hypothesis: The disruption of C-C motif chemokine receptor (CCR) -2, a receptor for CCL-2, ameliorates PAH in any models with the reversal of the associated pro-inflammatory state and vascular cell phenotypes and synergizes with any conventional pulmonary vasodilators. Methods and Results: By using Ccr2 (-/-) rats generated by CRISPR/ Cas9 , we investigated PAH in Ccr2 (+/+) or Ccr2 (-/-) rats treated with monocrotaline (MCT), SU5416/hypoxia (SuHx), and chronic hypoxia (CH). Ccr2 (-/-) decreased the right ventricular systolic pressure, right ventricular hypertrophy, and mortality rate, and reversed increased expression of inflammatory cytokines/chemokines (IL-6, TNF-α, CCL2, IL-1β, TGF-β) in MCT-treated rats, but not in SuHx or CH models. Consistently, Ccr2 (-/-) decreased indices of pulmonary vascular diseases (PVDs) and perivascular macrophage infiltration, as well as reversed impaired BMPR2 signaling, increased endothelial apoptosis, impaired NO signaling (eNOS, peNOS/eNOS) and decreased phosphodiesterase (PDE)-5 expression in lungs in MCT-treated rats. Gene expression of the receptor for prostaglandin I 2 and endothelin was not altered in MCT-treated rats by Ccr2 (-/-). In cultured pulmonary arterial smooth muscle cells (PASMCs), Ccr2 (-/-) suppressed CCL2-induced hyperproliferation and dedifferentiation as well as reversed CCL2-induced decrease in PDE5 expression. Consistently, the whole-genome RNA sequencing analysis identified differentially expressed genes in CCL2-stimulated Ccr2 (-/-) PASMCs, which are related to cellular differentiation and contraction. Based on studies in rats and cultured PASMCs, we investigated whether a PDE5 inhibitor, tadalafil, synergizes with Ccr2 (-/-). Tadalafil administration ameliorated PH and PVDs in MCT-treated Ccr2 (-/-) rats but not in Ccr2 (+/+) rats, and further improved survival in Ccr2 (-/-) rats. Conclusions: PDE5 inhibition synergized with Ccr2 disruption in protecting against PAH in MCT-treated rats, which was mechanistically related to the restoration of proinflammatory states and associated vascular cell phenotypes. These findings confer a fundamental basis supporting that CCR2 may be a therapeutic target in intractable PAH patients with a certain CCR2-related inflammatory phenotype and refractory to conventional pulmonary vasodilators.

SGC stimulator (BAY 41-8543) combined with PDE9 inhibitor (BAY 73-6691) reduces renal fibrosis in 5/6 nephrectomized rats.

Renal fibrosis is closely related to the prognosis of chronic kidney disease (CKD). The increase in cGMP reduces renal fibrosis. Soluble guanylate cyclase (sGC) and phosphodiesterase (PDE) are key enzymes that maintain cGMP levels. BAY 41-8543 (1mg/kg/day) and/or BAY 73-6691 (1mg/kg/day) were used to treat 5/6 nephrectomized rats for 13 weeks. 5/6 Nephrectomy caused an increase in cystatin C, proteinuria and glomerulosclerosis and renal interstitial fibrosis. Neither sGC stimulation nor PDE9 inhibition alone improved kidney function and morphology, whereas BAY 41-8543 in combination with BAY 73-6691 attenuated renal interstitial fibrosis. This beneficial effect could not be explained by alterations in blood pressure and the renal immune system. BAY 41-8543 in combination with BAY 73-6691 had no effect on renal macrophage, CD4 + T-cell and CD8 + T-cell in the late-stage of 5/6 nephrectomy. RNA sequencing revealed BAY 41-8543 in combination with BAY 73-6691 down-regulated the expression of fibrosis-related genes such as Collagen Type I Alpha 1, Collagen Type III Alpha 1 Chain and Collagen Type XIV Alpha 1 Chain. sGC stimulator combined with PDE9 inhibitor attenuated renal fibrosis in 5/6 nephrectomized rats by down-regulating fibrosis-related gene expression. This novel approach of using low-dose combination therapies to minimize side effects while maintaining therapeutic efficacy offers a promising strategy for the treatment of CKD.

Closed-perfusion transretinal ERG setup for preclinical drug and nanostructure testing.

The isolated mammalian retina may serve as a sensitive biosensor for preclinical drug testing, including eye drugs and a broader range of pharmaceuticals. To facilitate testing with minimal amounts of drug molecules or nanostructures, we developed a closed-perfusion transretinal electroretinography (tERG) setup. The major challenge with small amounts of circulating perfusate was maintaining retinal viability and stability during long experiments. We conducted ex vivo tERG using WT C57BL/6J and Gnat1-/- mice to assess rod- and cone-mediated light signals. The dark-adapted retina was stimulated with full-field light flashes while perfused at 5-6 ml/min. The minimum perfusate needed in our closed-circulation was around 50 ml. Penicillin-Streptomycin (Pen-Strep) was indispensable for long recordings. Rod responses remained stable for at least 42 hours, the longest recording we conducted, with the retina still responsive, and rod and cone bipolar cell responses for up to 12 hours. IBMX (3-isobutyl-1-methylxanthine), a non-specific phosphodiesterase (PDE) inhibitor with reversible effects, validated our setup. We used our setup to test the zwitterionic polymer poly(sulfobetaine methacrylate) (PSMBA), serving as a promising material for thermoresponsive nanostructures, and the corresponding monomer SBMA for possible harmful effects on mouse rod and bipolar cell functioning. Our closed-perfusion tERG setup enables long experiments with small amounts of perfusate. PSMBA or SBMA had no effect on rod and bipolar cell responses. This method is applicable for assessing drug functionality, as well as conducting preliminary biocompatibility and toxicity testing using small amounts of molecules or nanostructures that could impact neuronal or synaptic function.

Cyclic di-GMP incorporates the transcriptional factor FleQ03 in Pseudomonas syringae MB03 to elicit biofilm-dependent resistance in response to Caenorhabditis elegans predation

Bacteria usually form biofilms as a defense mechanism against predation by bacterivorous nematodes. In this context, the second messenger c-di-GMP from the wild-type Pseudomonas syringae MB03 actuates the transcriptional factor FleQ03 to elicit biofilm-dependent nematicidal activity against Caenorhabditis elegans N2. P. syringae MB03 cells exhibited nematicidal activity and c-di-GMP content in P. syringae MB03 cells was increased after feeding to nematodes. Expression of a diguanylate cyclase (DGC) gene in P. syringae MB03 resulted in an increased c-di-GMP content, biofilm yield and nematicidal activity, whereas converse effects were obtained when expressing a phosphodiesterase (PDE) gene. Molecular docking and isothermal titration calorimetry assays verified the affinity activity between c-di-GMP and the FleQ03 protein. The disruption of the fleQ03 gene in P. syringae MB03, while increasing c-di-GMP content, significantly diminished both biofilm formation and nematicidal activity. Interestingly, P. syringae MB03 formed a full-body biofilm around the worms against predation, probably extending from the tail to the head, whereas it was not observed in the fleQ03 gene disrupted cells. Thus, we hypothesized that c-di-GMP incorporated FleQ03 to reinforce bacterial biofilm and biofilm-dependent pathogenicity in response to C. elegans predation, providing insights into a possible means of resisting bacterivorous nematodes by bacteria in natural ecosystems.

Real-time imaging of cGMP signaling shows pronounced differences between glomerular endothelial cells and podocytes.

Recent clinical trials of drugs enhancing cyclic guanosine monophosphate (cGMP) signaling for cardiovascular diseases have renewed interest in cGMP biology within the kidney. However, the role of cGMP signaling in glomerular endothelial cells (GECs) and podocytes remains largely unexplored. Using acute kidney slices from mice expressing the FRET-based cGMP biosensor cGi500 in endothelial cells or podocytes enabled real-time visualization of cGMP. Stimulation with atrial natriuretic peptide (ANP) or SNAP (NO donor) and various phosphodiesterase (PDE) inhibitors elevated intracellular cGMP in both cell types. GECs showed a transient cGMP response upon particulate or soluble guanylyl cyclase activation, while the cGMP response in podocytes reached a plateau following ANP administration. Co-stimulation (ANP + SNAP) led to an additive response in GECs. The administration of PDE inhibitors revealed a broader basal PDE activity in GECs dominated by PDE2a. In podocytes, basal PDE activity was mainly restricted to PDE3 and PDE5 activity. Our data demonstrate the existence of both guanylyl cyclase pathways in GECs and podocytes with cell-specific differences in cGMP synthesis and degradation, potentially suggesting new therapeutic options for kidney diseases.

Effects of the restrictive cardiomyopathy-associated cardiac troponin I K178E mutation on cAMP signalling at the myofilament

Abstract Background Catecholaminergic stimulation of cardiac β-adrenergic receptors (β-ARs) and subsequent cAMP signalling control cardiac inotropy and lusitropy. cAMP signalling is known to be compartmentalised to optimise sympathetic control by producing heterogeneous cAMP signals and protein kinase A (PKA) activation at distinct subcellular sites. This signalling may be disrupted in diseases such as restrictive cardiomyopathy (RCM) characterised by diastolic dysfunction, which is associated with certain mutations including the K178E missense mutation in cardiac troponin I (TPNI). The impaired ventricular relaxation associated with this mutation is thought to arise from myofilament calcium hypersensitivity, which can be modulated by PKA-dependent TPNI phosphorylation. However, a detailed understanding of the pathological effects mediated by the K178E-TPNI mutation is lacking. Purpose This study aims to determine whether the K178E-TPNI mutation alters local cAMP/PKA signalling at TPNI, potentially affecting myofilament calcium sensitivity and cardiac myocyte relaxation. Methods Real-time imaging of cAMP was carried out using the Fluorescence Resonance Energy Transfer (FRET)-based sensor CUTie (cAMP Universal Tag for imaging experiments) [1] fused to wild type (WT)- or K178E-TPNI. The sensors were expressed by transfection in neonatal rat ventricular myocytes (NRVMs) and FRET-changes were recorded at the myofilament on application of the β-AR stimulant isoproterenol (ISO). We further assessed the involvement of cAMP hydrolysing phosphodiesterases (PDEs) in the regulation of cAMP levels selectively at TPNI. Results The peak FRET-change and FRET-change after 5mins on ISO application were significantly higher in NRVMs expressing the mutant compared to the WT sensor, as was the rate of cAMP accumulation. A strong trend showed that the rise in FRET following PDE4 inhibition was larger in NRVMs expressing the WT compared to the mutant sensor, suggesting the involvement of this isoform in degrading cAMP locally at TPNI and reduced PDE4 activity in the presence of the K178E mutation. Co-immunoprecipitation analysis in HEK293 cells demonstrated interaction between WT-TPNI and a PDE4D isoform, and a weaker interaction of this isoform with the K178E-TPNI mutant. Conclusions This study shows that the K178E-TPNI mutation attenuates the TPNI/PDE4D interaction and alters the magnitude and kinetics of the local cAMP response at the myofilament. This mechanistic insight may aid the development of therapeutics targeting the TPNI/PDE4D interaction to treat RCM-associated diastolic dysfunction.The TPNI-CUTie SensorFRET Response Trace

The CNP analogue vosoritide reduces arrhythmia in diabetic cardiomyopathy via cGMP-dependent PDE2 stimulation on cellular and organ level

Abstract Background Detrimental arrhythmia occurs frequently in patients with diabetic cardiomyopathy (DiabCM), but safe and effective therapies are limited. Diabetes-associated neuropathological alterations increase the sympathetic tone, leading to hyperactivation of β-adrenoceptor-mediated cAMP signalling in cardiomyocytes (CM). Increased activity of deleterious cAMP-dependent kinases causes pro-arrhythmic dysregulation of intracellular Ca2+ homeostasis. Phosphodiesterases (PDE) can reduce cAMP levels. Exceptionally, the cAMP-hydrolysing activity of PDE2 can be cGMP-dependently increased by C-type natriuretic peptide (CNP). Thus, the CNP analogue vosoritide (approved for the treatment of achondroplasia) might mediate this cGMP/cAMP crosstalk, reducing arrhythmia. Purpose Here, we aim to study whether the cGMP-dependent activation of PDE2 by vosoritide (VO) is sufficient to reduce arrhythmia and arrhythmogenic triggers in DiabCM. Methods Diabetes was induced by 5 consecutive injections of streptozotocin (50 mg/kg) in control (WT) mice and mice with a CM-specific PDE2 knockout (KO). All experiments were performed 5 weeks later. Cardiac function was characterised by echocardiography. Primary ventricular CM were isolated to assess protein expression and phosphorylation levels by Western blot (WB) and cAMP levels by ELISA. Patch-clamp and Ca2+ imaging experiments were used to investigate pro-arrhythmic triggers. Arrhythmic events were quantified in ex vivo perfused hearts after ischaemia-reperfusion (I/R) injury. Results Compared to healthy controls, diabetic animals showed impaired systolic function with a ~9 % reduction in ejection fraction. The significant increase in E/E’ ratio by ~34 % indicated a diastolic dysfunction. Interestingly, the cardiac function was further worsened in diabetic PDE2 KO. WB analysis revealed upregulation of the CNP receptor NPR-B and PDE2 in diabetic CM and altered regulation of proteins involved in cAMP-dependent Ca2+ cycling such as protein kinase A, Ca2+/calmodulin-dependent kinase II and phospholamban. Interestingly, VO significantly reduced isoprenaline (Iso)-induced cAMP levels in diabetic CM. The effect of VO was prevented by specific PDE2 inhibition with BAY 60-7550 (BAY). Furthermore, VO was able to reduce the Iso-induced increase in L-type Ca2+ current, whereas BAY or genetic PDE2 deletion counteracted this effect. Accordingly, VO significantly decreased the Iso-triggered Ca2+ spark frequency by ~40 %. Concomitant PDE2 inhibition or PDE2 KO prevented this anti-arrhythmic effect. Finally, ECG analysis revealed that VO clearly reduced arrhythmia development after I/R in ex vivo perfused diabetic hearts via PDE2 activation. Conclusion Our data indicate that vosoritide-induced PDE2 stimulation may attenuate abnormal cardiac Ca2+ cycling and thereby reduce arrhythmia in diabetic hearts. Thus, vosoritide may be repurposed as a novel anti-arrhythmic drug in DiabCM.

A phase 2, randomized, double-blind, placebo-controlled study to assess the tolerability and pharmacodynamic effects of CRD-740, a PDE9 inhibitor, in participants with chronic heart failure

Abstract Background Natriuretic peptide (NP) receptor activation has a beneficial role in the treatment of heart failure (HF). The enzyme phosphodiesterase (PDE) 9 breaks down cGMP, the second messenger stimulated by natriuretic peptides. PDE9 inhibition may increase intracellular cGMP signaling and, potentially, have beneficial effects in HF. We examined the effects of the selective PDE9 inhibitor CRD-740 on the NP signaling pathway in HF. Purpose The objectives of this early phase 2 trial were to assess the tolerability of CRD-740 and its effects on plasma and urinary cGMP (markers of potential clinical effiacy) in patients with HFrEF, including those receving background treatment with sacubitril/valsartan. Methods Key inclusion criteria were patients with a history of HFrEF of > 6 mos, NYHA II or III, EF < 40% and NT-proBNP ≥600 pg/ml at screening (≥1000 pg/mL if in atrial fibrillation), on treatment with stable guideline-directed therapy for a minimum of 4 weeks. Patients were randomized 2:1 double-blind to CRD-740 (10mg twice daily for 2 weeks, then 25mg twice daily for 10 weeks) or placebo. Tolerability and safety were assessed. The primary pharmacodynamic endpoint was the between-group change from baseline in plasma cGMP at Week 4. Exploratory endpoints included urine cGMP levels, KCCQ and biomarkers. Results 60 patients were randomized to CRD-740 (n=40) or placebo (n=20). Baseline characteristics included age 67+13 yrs, 85% men, EF 28+7%, BMI 30+10, with 73% of patients taking sacubitril/valsartan, and 47% on SGLT2 inhibitors. The primary endpoint was positive, with placebo-corrected change in plasma cGMP significantly increased at all time points at week 4 in those treated with CRD-740 (see Table). cGMP levels were also increased on day 1 (Table) and week 2. A significant increase in urinary cGMP also was observed with CRD-740 vs placebo (Figure) in 6-hour collections at day 1 (p=0.012) and week 2 (p=0.014). In this small study, not powered for exploratory endpoints, directionally favorable placebo-corrected, baseline-adjusted changes also were seen in the KCCQ Clinical (+7.1 [95%CI, -1.6, +15.7], p=0.11) and Overall Summary Scores (+7.4 [-0.8, +15.5], p=0.075) for patients on CRD-740. No significant changes in NT-proBNP were observed between treatment groups. Adverse events (AEs) were observed in 62% and 50% of CRD-740 and placebo-treated patients, with discontinuation in 5% and 10% respectively, with no between-group difference in change in BP. There were no hypotension AEs, and no treatment-related serious adverse events. Conclusions In this early phase 2 trial, PDE9 inhibition with CRD-740 was well-tolerated and resulted in substantial elevations of plasma and urinary cGMP on top of standard care, including sacubitril/valsartan. These results support the potential of PDE9 inhibition to further activate the beneficial effects of the NP receptor-cGMP pathway incrementally to that achieved by existing HF treatments.Changes in Plasma cGMP over Time (ng/mL)Changes in Urinary cGMP over Time

Phosphodiesterase inhibition and Gucy2C activation enhance tyrosine hydroxylase Ser40 phosphorylation and improve 6-hydroxydopamine-induced motor deficits

BackgroundParkinson’s disease is characterized by a progressive loss of dopaminergic neurons in the nigrostriatal pathway, leading to dopamine deficiency and motor impairments. Current treatments, such as L-DOPA, provide symptomatic relief but result in off-target effects and diminished efficacy over time. This study explores an alternative approach by investigating the activation of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Specifically, we explore the effects of phosphodiesterase (PDE) inhibition and guanylate cyclase-C (GUCY2C) activation on tyrosine hydroxylase Ser40 phosphorylation and their impact on motor behavior in a 6-hydroxydopamine (6-OHDA) Parkinson's disease model.ResultsOur findings demonstrate that increasing cyclic nucleotide levels through PDE inhibition and GUCY2C activation significantly enhances tyrosine hydroxylase Ser40 phosphorylation. In a Pitx3-deficient mouse model, which mimics the loss of dopaminergic neurons seen in Parkinson’s disease, Ser40 phosphorylation remained manipulable despite reduced tyrosine hydroxylase protein levels. Moreover, we observed no evidence of tyrosine hydroxylase degradation due to Ser40 phosphorylation, challenging previous reports. Furthermore, both PDE inhibition and GUCY2C activation resulted in improved motor behavior in the 6-OHDA Parkinson’s disease mouse model, highlighting the potential therapeutic benefits of these approaches.ConclusionsThis study underscores the therapeutic potential of enhancing tyrosine hydroxylase Ser40 phosphorylation to improve motor function in Parkinson’s disease. Both PDE inhibition and GUCY2C activation represent promising non-invasive strategies to modulate endogenous dopamine biosynthesis and address motor deficits. These findings suggest that targeting cyclic nucleotide pathways could lead to novel therapeutic approaches, either as standalone treatments or in combination with existing therapies like L-DOPA, aiming to provide more durable symptom relief and potentially mitigate neurodegeneration in Parkinson's disease.

Dual Inhibition of Phosphodiesterase 3 and 4 Enzymes by Ensifentrine Protects against MRSA-Induced Lung Endothelial and Epithelial Dysfunction.

Acute Respiratory Distress Syndrome (ARDS) is a severe lung condition with a high mortality rate for which there are no effective therapeutics. The failure of the alveolar-capillary barrier, composed of lung endothelial (EC) and alveolar epithelial (AEC) cells, is a critical factor leading to excessive inflammation and edema characteristic of acute lung injury (ALI) pathophysiology. Phosphodiesterases (PDE) are enzymes well-recognized for their roles in regulating endothelial permeability and inflammation. Although PDE inhibitors are used as therapeutics for inflammatory diseases like COPD (chronic obstructive pulmonary disease), their efficacy in treating ARDS has not yet been established. In this study, we investigated the effects of ensifentrine, an FDA-approved novel dual PDE 3/4 inhibitor, on lung endothelial and epithelial dysfunction caused by methicillin-resistant S. aureus (MRSA), a pathogen involved in bacterial ARDS. Human primary lung endothelial cells and alveolar epithelial cell lines (A549 and immortalized AEC) were treated with heat-killed MRSA, and their responses were assessed in the presence or absence of ensifentrine. Ensifentrine given either pre- or post-exposure attenuated MRSA-induced increased lung endothelial permeability. VE-cadherin junctions, which serve to stabilize the EC barrier, were disrupted by MRSA; however, ensifentrine effectively prevented this disruption. Pre-treatment with ensifentrine protected against MRSA-induced EC pro-inflammatory signaling by inhibiting the expression of VCAM-1, ICAM-1, and by reducing the IL-6 and IL-8 release. In AEC, MRSA caused the upregulation of ICAM-1, the activation of NF-kB, and the production of IL-8, all of which were inhibited by ensifentrine. These results indicate that the dual inhibition of phosphodiesterases 3 and 4 by ensifentrine is barrier protective and attenuates MRSA-induced inflammation in both lung endothelial and epithelial cells. The PDE3/4 inhibitor ensifentrine may represent a promising novel strategy for the treatment of MRSA-induced ARDS.

Nerandomilast Improves Bleomycin-Induced Systemic Sclerosis-Associated Interstitial Lung Disease in Mice by Regulating the TGF-β1 Pathway.

Systemic sclerosis (SSc) is a rare connective tissue disease with a heterogeneous clinical course. Interstitial lung disease (ILD) is a common complication of SSc and a major contributor to SSc-related deaths. Besides nintedanib and tocilizumab, there are currently no clinically approved drugs for SSc-ILD, highlighting the urgent need for new treatment strategies. Previous studies have shown that cyclic adenosine monophosphate (cAMP) plays a crucial role in the pathogenesis of SSc and lung fibrosis. Phosphodiesterases (PDEs) are enzymes that specifically hydrolyze cAMP, making PDE inhibitors promising candidates for SSc-ILD treatment. Nerandomilast, a preferential phosphodiesterase 4B (PDE4B) inhibitor currently undergoing phase III clinical trials for idiopathic pulmonary fibrosis and progressive fibrosing interstitial lung diseases (PF-ILD), has good preference for PDE4B but lacks studies for SSc-ILD. Our research demonstrates that nerandomilast effectively inhibits skin and lung fibrosis in a bleomycin-induced mouse model of SSc-ILD. For lung fibrosis, we found that nerandomilast could improve bleomycin-induced SSc-ILD through inhibiting PDE4B and the TGF-β1-Smads/non-Smads signaling pathways, which provides a theoretical basis for potential therapeutic drug development for SSc-ILD.

Functional analysis of cyclic diguanylate-modulating proteins in Vibrio fischeri.

As bacterial symbionts transition from a motile free-living state to a sessile biofilm state, they must coordinate behavior changes suitable to each lifestyle. Cyclic diguanylate (c-di-GMP) is an intracellular signaling molecule that can regulate this transition, and it is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. Generally, c-di-GMP inhibits motility and promotes biofilm formation. While c-di-GMP and the enzymes that contribute to its metabolism have been well studied in pathogens, considerably less focus has been placed on c-di-GMP regulation in beneficial symbionts. Vibrio fischeri is the sole beneficial symbiont of the Hawaiian bobtail squid (Euprymna scolopes) light organ, and the bacterium requires both motility and biofilm formation to efficiently colonize. c-di-GMP regulates swimming motility and cellulose exopolysaccharide production in V. fischeri. The genome encodes 50 DGCs and PDEs, and while a few of these proteins have been characterized, the majority have not undergone comprehensive characterization. In this study, we use protein overexpression to systematically characterize the functional potential of all 50 V. fischeri proteins. All 28 predicted DGCs and 10 of the 14 predicted PDEs displayed at least one phenotype consistent with their predicted function, and a majority of each displayed multiple phenotypes. Finally, active site mutant analysis of proteins with the potential for both DGC and PDE activities revealed potential activities for these proteins. This work presents a systems-level functional analysis of a family of signaling proteins in a tractable animal symbiont and will inform future efforts to characterize the roles of individual proteins during lifestyle transitions.IMPORTANCECyclic diguanylate (c-di-GMP) is a critical second messenger that mediates bacterial behaviors, and Vibrio fischeri colonization of its Hawaiian bobtail squid host presents a tractable model in which to interrogate the role of c-di-GMP during animal colonization. This work provides systems-level characterization of the 50 proteins predicted to modulate c-di-GMP levels. By combining multiple assays, we generated a rich understanding of which proteins have the capacity to influence c-di-GMP levels and behaviors. Our functional approach yielded insights into how proteins with domains to both synthesize and degrade c-di-GMP may impact bacterial behaviors. Finally, we integrated published data to provide a broader picture of each of the 50 proteins analyzed. This study will inform future work to define specific pathways by which c-di-GMP regulates symbiotic behaviors and transitions.

Development of a Candidate 11C-Labeled Selective Phosphodiesterase 1 Radioligand for Positron Emission Tomography.

Phosphodiesterases (PDEs) constitute a superfamily of phosphohydrolytic enzymes that regulate intracellular second messenger signaling by hydrolyzing cyclic adenosine monophosphate and cyclic guanosine monophosphate. Among the 11 subfamilies of PDEs, phosphodiesterase 1 (PDE1) stands out due to its broad implications in central and peripheral pathologies. There are three subtypes of PDE1: PDE1A, PDE1B, and PDE1C. While PDE1A and PDE1C are distributed in both the brain and peripheral organs, PDE1B is predominantly expressed in the brain, rendering it an attractive drug target for neurological and psychological disorders. Despite continuous efforts dedicated to the development of novel PDE1 inhibitors, a suitable PDE1 radioligand for human use is currently lacking. In this study, we present the identification and preclinical evaluation of [11C]PF-04822163, a selective radioligand candidate for imaging PDE1 with positron emission tomography. PF-04822163 exhibits excellent potency toward PDE1 and demonstrates great target selectivity over other PDEs. Then, PF-04822163 was labeled with carbon-11 (half-life, 20 min) in favorable radiochemical yields (25 ± 10%, decay-corrected) and high molar activities (106-194 GBq/μmol). Further, in vitro and in vivo evaluations in rodents suggested that [11C]PF-04822163 displayed good brain penetration and a rapid washout. Despite these promising performance characteristics of [11C]PF-04822163, only marginal specific binding was observed in vivo. Further optimization of the scaffold is warranted to obtain favorable pharmacological and ADME properties.

Phosphodiesterase 5 and its inhibitors with ischemic heart disease: a Mendelian randomization analysis and a real-world study.

Accumulating studies reported that several phosphodiesterases (PDEs) inhibitors might have cardiovascular benefits. This study aimed to explore the relationship between genetically-predicted PDEs and ischemia heart disease via drug target Mendelian randomization (MR) approach, and then examine the effect of inhibitors of identified target on the outcomes by using real-world data. In the two-sample MR study, the expression of genes encoding PDEs was used to proxy the level of PDEs and available expression quantitative trait loci (eQTLs) for each target gene were identified as the genetic instruments. The outcomes included coronary heart disease (CHD) and myocardial infarction (MI). In the real-world study, a retrospective cohort was conducted to compare the incidence of outcomes between PDE5 inhibitors and alprostadil use by linking Swedish nationwide registers. MR analyses identified two types of PDEs, PDE5 and PDE8, genetically-predicted expression in blood of the encoded genes was significantly associated with the risk of CHD (ORPDE5A=1.22,95% CI=1.06-1.40; ORPDE8A=1.26,95% CI=1.07-1.49) and MI (ORPDE5A=1.27,95% CI=1.09-1.48; ORPDE8A=1.24,95% CI=1.04-1.48). Notably, the highest expression of PDE5A was observed in artery aorta, which was also positively related to CHD (OR=1.17,95% CI=1.05-1.32) and MI (OR=1.15,95% CI=1.02-1.30). Real-world study provided supportive evidence that as compared to alprostadil use, PDE5 inhibitors use significantly reduced the incidence of CHD (adjusted HR=0.70,95% CI=0.66-0.73) and MI (adjusted HR=0.79,95% CI=0.73-0.84). This study provided observational and genetic evidence about the protective role of PDE5 inhibition against ischemic heart disease, indicating the potential of these drugs to be repurposed for ischemia heart disease prevention and treatment.

Phosphodiesterase Inhibitors in Fetal Growth Restriction: Do Not Forget to Consider Fetal Sex and Subcellular Compartmentation.

Fetal growth restriction (FGR) is a common complication of pregnancy, associated with major perinatal mortality and morbidity, and with an increased risk to develop cardiometabolic diseases later in life. There is currently no effective approach to prevent or treat FGR, despite numerous animal and human studies assessing substances likely to improve fetal growth. Phosphodiesterase (PDE) inhibitors appeared as promising drugs to improve FGR management. However, to date, studies have led to somewhat disappointing or controversial results. In this Opinion article, we would like to draw attention to the need to consider the biological sex and the relative reactivity of human umbilical vein and arteries when developing therapeutic interventions to improve human umbilical circulation using PDE inhibitors. Indeed, we suspect that fetal sex, vessel type and the presence of FGR may influence subcellular compartmentation, which could jeopardize beneficial effects of PDE inhibitors.

Effects of the phosphodiesterase 2 inhibitor BI 474121 on central nervous system cyclic guanosine monophosphate concentrations: Translational studies.

Phosphodiesterase 2 (PDE2) regulates intracellular cyclic adenosine monophosphate and guanosine monophosphate (cAMP/cGMP) levels, which contribute to processes crucial for learning and memory. BI 474121, a potent and selective PDE2 inhibitor, is in development for treating cognitive impairment associated with schizophrenia. The effects of BI 474121 on cGMP concentrations were first assessed in rat cerebrospinal fluid (CSF) to demonstrate central nervous system (CNS) and functional target engagement. Next, a Phase I study in healthy participants assessed the pharmacokinetics of BI 474121 in CSF vs. plasma, the pharmacodynamics of BI 474121 by measuring cGMP concentrations in the CSF, and the safety of BI 474121. In rats, BI 474121 was associated with a dose-dependent increase (71% at the highest dose tested [3.0mg kg-1]) in cGMP levels in the CSF relative to vehicle (P < 0.001). In healthy participants, the maximum-measured concentration CSF-to-plasma ratio for BI 474121 exposure was similar following single oral doses of BI 474121 2.5, 10, 20 and 40 mg (dose-adjusted geometric mean: 8.96% overall). BI 474121 2.5-40 mg administration in healthy participants also increased cGMP levels in CSF (maximum exposure-related change from baseline ratio, BI 474121: 1.44-2.20 vs. placebo: 1.26). The most common treatment-emergent adverse event (AE) was mild-to-moderate post-lumbar puncture syndrome, which resolved with standard treatment. No AEs of special interest were observed. BI 474121 crosses the blood-brain barrier to inhibit PDE2, supporting cGMP as a translational marker to monitor CNS target engagement. These findings promote further clinical development of BI 474121. gov number (NCT04672954).

Identification of potent and orally efficacious phosphodiesterase inhibitors in Cryptosporidium parvum-infected immunocompromised male mice

Cryptosporidium parvum and C. hominis are parasites that cause life-threatening diarrhea in children and immunocompromised people. There is only one approved treatment that is modestly effective for children and ineffective for AIDS patients. Here, screening 278 compounds from the Merck KGaA, Darmstadt, Germany collection and accelerated follow-up enabled by prior investigation of the compounds identifies a series of pyrazolopyrimidine human phosphodiesterase (PDE)-V (hPDE-V) inhibitors with potent anticryptosporidial activity and efficacy following oral administration in C. parvum-infected male mice. The lead compounds affect parasite host cell egress, inhibit both C. parvum and C. hominis, work rapidly, and have minimal off-target effects in a safety screening panel. Interestingly, the hPDE-V inhibitors sildenafil and the 4-aminoquinoline compound 7a do not affect Cryptosporidium. C. parvum expresses one PDE (CpPDE1) continuously during asexual growth, the inhibited life stage. According to homology modeling and docking, the lead compounds interact with CpPDE1. Bulkier amino acids (Val900 and His884) in the CpPDE1 active site replace alanines in hPDE-V and block sildenafil binding. Supporting this, sildenafil kills a CRISPR-engineered Cryptosporidium CpPDE1 V900A mutant. The CpPDE1 mutation also alters parasite susceptibility to pyrazolopyrimidines. CpPDE1 is therefore a validated pyrazolopyrimidine molecular target to exploit for target-based optimization for improved anticryptosporidial development.

Phosphodiesterases: Evolving Concepts and Implications for Human Therapeutics.

Phosphodiesterases (PDEs) are a superfamily of enzymes that hydrolyze cyclic nucleotides. While the 11 PDE subfamilies share common features, key differences confer signaling specificity. The differences include substrate selectivity, enzymatic activity regulation, tissue expression, and subcellular localization. Selective inhibitors of each subfamily have elucidated the protean role of PDEs on normal cell function. PDEs are also linked to diseases, some of which affect the immune, cardiac, and vascular systems. Selective PDE inhibitors are clinically used to treat these specific disorders. Ongoing preclinical studies and clinical trials are likely to lead to the approval of additional PDE-targeting drugs for therapy in human disease. In this review, we discuss the structure and function of PDEs and examine current and evolving therapeutic uses of PDE inhibitors, highlighting their mechanisms and innovative applications that could further leverage this crucial family of enzymes in clinical settings.