Protaetia brevitarsis larvae extract protects against lipopolysaccharides-induced ferroptosis and inflammation by inhibiting acid sphingomyelinase.

BACKGROUND/OBJECTIVES: Inflammation and ferroptosis are implicated in various diseases and lipopolysaccharides (LPS) have been linked with these disorders. Recently, many edible insects, such as Gryllus bimaculatus, Protaetia brevitarsis larvae (PB) and Tenebrio molitor larvae, have been recommended as alternative foods because they contain lots of nutritional sources. In this study, we explored the potential of PB extract in preventing LPS-induced inflammation and ferroptosis in Hep3B cells. MATERIALS/METHODS: PB powder was extracted using 70% ethanol and applied to Hep3B cells. Co-treatment with LPS was conducted to induce ferroptosis and inflammation. The anti-inflammatory and anti-ferroptosis mechanisms of the PB extract were confirmed using Western blot, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction analysis. RESULTS: PB extract effectively prevented LPS-induced cell death and restored LPS-induced inflammatory cytokine production, NF-κB signaling, endoplasmic reticulum (ER) stress and ferroptosis. Interestingly, PB extract reduced LPS-induced ceramide increase and acid sphingomyelinase (ASMase) expression. The use of the ASMase inhibitor, desipramine, also demonstrated a reduction in these pathways, highlighting the pivotal role of ASMase in inflammation and ferroptosis. Treatment with each inhibitor revealed that ferroptosis causes ER stress and that NF-κB and MAP kinase pathways are involved in inflammation. CONCLUSION: PB emerges as a potential functional food with inhibitory effects on LPS-induced inflammation and ferroptosis, making it a promising candidate for nutritional interventions.

Sildenafil as Bridge Therapy for Inhaled Nitric Oxide in Preterm Neonates.

OBJECTIVE: Inhaled nitric oxide (iNO) is a mainstay of treatment for infants with persistent pulmonary -hypertension. However, abrupt discontinuation of inhaled nitric oxide can result in rebound pulmonary -hypertension. The objective of this analysis is to describe the use of sildenafil to facilitate the weaning from iNO in preterm neonates. METHODS: This retrospective chart review identified all infants who were receiving iNO and subsequently received sildenafil between 2017 and 2021. Neonates were included if they met the following criteria: gestational age at birth less than 34 weeks, receiving iNO, and started on sildenafil with the indication to facilitate weaning or discontinuation of iNO. Patients were excluded if they had major congenital anomalies, including congenital heart disease or congenital diaphragmatic hernia. RESULTS: We identified 7 neonates with a gestational age range of 22 5/7 weeks to 31 0/7 weeks and birth weight range of 545 to 910 g with previously failed attempts at iNO weaning. The most common starting dose for sildenafil was 0.125 mg/kg intravenously every 8 hours or 0.25 mg/kg enterally every 8 hours. Four infants were able to discontinue iNO within 48 hours of sildenafil initiation, 1 patient discontinued iNO within 5 days, and 1 patient within 10 days of sildenafil initiation. One patient experienced weaning failure from iNO despite initiation of sildenafil. No adverse events, such as hypotension or deaths, were reported in any of the 7 infants. CONCLUSIONS: Sildenafil facilitated weaning off iNO in most preterm neonates evaluated without adverse side effects.

Theophylline derivatives promote primordial follicle activation via cAMP-PI3K/Akt pathway and ameliorate fertility deficits in naturally aged mice.

In elderly women and patients with premature ovarian insufficiency (POI), activating their remaining dormant primordial follicles in vivo is challenging. In this study, we found that phosphodiesterase (PDE) subtypes were expressed mainly in primordial follicle oocytes. The specific PDE inhibitors and theophylline derivatives (aminophylline, dyphylline, and enprofylline) activated primordial follicles in neonatal mice by ovary culture and intraperitoneal injection. These inhibitors also increased the levels of ovarian cyclic adenosine monophosphate (cAMP) and oocyte phosphorylated protein kinase B (p-Akt). The blockade of gap junctions using carbenoxolone (CBX) increased the levels of ovarian cAMP and pre-granulosa cell phosphorylated mammalian target of rapamycin (p-mTOR), suggesting that oocyte PDEs hydrolyze cAMP from pre-granulosa cells through gap junctions to maintain primordial follicle dormancy. Importantly, oral aminophylline improved ovulated oocyte quantity and quality, and increased offspring numbers in naturally aged mice. In addition, theophylline derivatives also activated human primordial follicles and increased p-Akt levels. Thus, theophylline derivatives activate primordial follicles by accumulating cAMP levels and activating phosphatidylinositol 3-kinase (PI3K)/Akt pathway in oocytes, and oral aminophylline increased fertility in naturally aged female mice by improving ovulated oocyte quantity and quality. As oral medications, theophylline derivatives may be used to improve fertility in elderly women and patients with POI.

Measurement of PDE5 concentration in human serum: proof-of-concept and validation of methodology in control and prostate cancer patients.

PURPOSE: We aimed to investigate if the type 5 phosphodiesterase (PDE5), an enzyme with cardinal biological functions in sexual and cardiovascular health, can be detected and quantited in human serum. METHODS: Blood samples were collected from control male and female subjects. PDE5 levels were measured by a specific ELISA kit. ROC curves weighted for age and serum levels of PSA (male subjects), or age (female subjects) were used to identify the predictive ability in the detection of PCa. Sensitivity, specificity, PPV and NPV values were determined for cut-off value determined during ROC curve analysis. RESULTS: 41 control male subjects, 18 control female subjects, and 55 consecutive subjects, of which 25 were affected by benign prostatic hypertrophy (BPH) and 30 with histologically confirmed prostate cancer (PCa), were studied. PDE5 serum levels were detectable in all subjects (range: 5 to 65 ng/ml). Analysis by MANCOVA identified a significant difference in serum PDE5 between control subjects or hyperplasia patients and PCa patients. Marginal means of serum PDE5 concentrations showed a significant difference (p < 0.001). The ROC curve demonstrated that PDE5 serum levels can predict men with or without PCa, with 0.806 AUC value (p < 0.0001). Using a 12.705 ng/ml PDE5 serum cut-off yielded sensitivity, specificity, PPV, and NPV of 83.3%, 77.27%, 62.5%, and 91.1% in detecting men with histologically proven PCa, respectively. CONCLUSIONS: We demonstrated, for the first time, that PDE5 levels can be detected in human sera and that PCa patients have significantly higher PDE5 concentration compared to BPH patients or male and female controls. While serum PDE5 level measurement may open new research avenues, the clinical relevance of PDE5 levels in PCa patients deserves further investigation.

Effects of sildenafil on gas exchange, ventilatory, and sensory responses to exercise in subjects with mild-to-moderate COPD: A randomized cross-over trial.

Excess exercise ventilation (high ventilation (V̇E)/carbon dioxide output (V̇CO2)) contributes significantly to dyspnea and exercise intolerance since the earlier stages of chronic obstructive pulmonary disease (COPD). A selective pulmonary vasodilator (inhaled nitric oxide) has shown to increase exercise tolerance secondary to lower V̇E/V̇CO2 and dyspnea in this patient population. We aimed to assess whether a clinically more practical option - oral sildenafil - would be associated with similar beneficial effects. In a randomized, placebo-controlled study, twenty-four patients with mild-to-moderate COPD completed, on different days, two incremental cardiopulmonary exercise tests (CPET) one hour after sildenafil or placebo. Eleven healthy participants performed a CPET in a non-interventional visit for comparative purposes with patients when receiving placebo. Patients (FEV1= 69.4 ± 13.5 % predicted) showed higher ventilatory demands (V̇E/V̇CO2), worse pulmonary gas exchange, and higher dyspnea during exercise compared to controls (FEV1= 98.3 ±11.6 % predicted). Contrary to our expectations, however, sildenafil (50 mg; N= 15) did not change exertional V̇E/V̇CO2, dead space/tidal volume ratio, operating lung volumes, dyspnea, or exercise tolerance compared to placebo (P>0.05). Due to the lack of significant beneficial effects, nine additional patients were trialed with a higher dose (100 mg). Similarly, active intervention was not associated with positive physiological or sensory effects. In conclusion, acute oral sildenafil (50 or 100 mg) failed to improve gas exchange efficiency or excess exercise ventilation in patients with predominantly moderate COPD. The current study does not endorse a therapeutic role for sildenafil to mitigate exertional dyspnea in this specific patient subpopulation. Clinical trial registry: https://ensaiosclinicos.gov.br/rg/RBR-4qhkf4 Web of Science Researcher ID: O-7665-2019.

VP3.15 reduces acute cerebellum damage after germinal matrix-intraventricular hemorrhage of the preterm newborn.

Germinal matrix-intraventricular hemorrhage (GM-IVH) is one of the most common complications of the preterm newborn. The pathology of the GM-IVH is not completely understood and even regions distant from the lesion area are severely affected. It has been suggested that cerebellar diaschisis may underlie the neurodevelopmental problems that many of these kids show, including cerebral palsy, attention deficit disorders or hyperactivity. Additionally, GM-IVH has no successful treatment. VP3.15 is a dual action phosphodiesterase 7 (PDE7) and glycogen synthase kinase-3ß (GSK-3ß) inhibitor that limits neuroinflammation and neuronal loss. Therefore, it might also provide a relevant tool to reduce complications associated with GM-IVH. We have used a murine model of GM-IVH to analyze the short and long-term effects of VP3.15 in brain pathology and behavioral complications. In our hands, the induction of unilateral GM-IVH to P7 CD1 mice results in a short-term (P14) compromise of the cerebellar neuronal population and Purkinje cells arborization, an increase of microglia burden in the nuclei and an overall increase of punctuate cerebellar hemorrhages. Whereas brain alterations are no longer observed in the long term (P110), these animals present overt hyperactivity when analyzed in the adulthood, supporting the long-term behavioral impairment. Also, hyperactivity significantly correlates with ipsi and contralateral cerebellar sizes, neuronal densities and myelin basic protein levels. Importantly, treatment with VP3.15 significantly reduces neuronal loss, Purkinje cells simplification, the presence of cerebellar hemorrhages, as well as hyperactivity. Altogether, our data support the neuroprotective effects of VP3.15 in GM-IVH of the PT.

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.

Inhibition of phosphodiesterase 10A mitigates neuronal injury by modulating apoptotic pathways in cold-induced traumatic brain injury.

Brain injury develops from a complex series of pathophysiological phases, resulting in acute necrotic or delayed apoptotic cell death after traumatic brain injury (TBI). Inhibition of apoptotic cell death is critical for the treatment of acute neurodegenerative disorders, such as TBI. Here, we investigated the role of phosphodiesterase 10A (PDE10A) in the development of neuronal injury, particularly in apoptotic cell death. Using the PDE10A inhibitor TAK-063, we found that PDE10A inhibition is associated with decreased brain injury, brain swelling, and blood brain barrier disruption 48 h after cold-induced TBI. Furthermore, a particularly notable result was observed with 3 mg/kg TAK-063, which reduced disseminated neuronal injury. Protein abundance analysis revealed that PDE10A inhibition activates survival kinases AKT and ERK-1/-2, which were associated with the decreased activation of MMP-9 and PTEN. Additionally, iNOS and nNOS levels significantly reduced in the TAK-063 group, playing roles in inflammation and apoptosis. A planar surface immunoassay was performed for in-depth analyses of the apoptotic signaling pathways. We observed that inhibition of PDE10A resulted in the decreased expression of TNFRSF1A, TNFRSF10B, and TNFRSF6 receptors, particularly inducing apoptotic cell death. Moreover, these findings correlated with reduced levels of pro-apoptotic proteins, including PTEN, p27, Cytochrome-c, cleaved Caspase-3, Bad, and p53. Interestingly, TAK-063 treatment reduced levels of anti-apoptotic proteins or enzymes, including XIAP, Claspin, and HIF1α, without affecting Bcl-x, MCL-1, SMAC, HO-1, HO-2, HSP27, HSP60, and HSP70. The findings suggest that PDE10A regulates cellular signaling predominantly pro-apoptotic pathways, and inhibition of this protein is a promising approach for the treatment of acute brain injury.

Diminished nuclear-localized ß-adrenoceptor signalling activates YAP to promote kidney fibrosis in diabetic nephropathy.

BACKGROUND AND PURPOSE: Diabetic nephropathy (DN) is a leading cause of chronic kidney disease (CKD), which is characterized by mesangial matrix expansion that involves dysfunctional mesangial cells (MCs). However, the underlying mechanisms remain unclear. This study aims to delineate the spatiotemporal contribution of adrenergic signalling in diabetic kidney fibrosis to reveal potential therapeutic targets. EXPERIMENTAL APPROACH: A model of diabetic nephropathy was induced by in db/db mice. Gene expression in kidneys was profiled by RNA-seq analyses, western blot and immunostaining. Subcellular-localized fluorescence resonance energy transfer (FRET) biosensors determined adrenergic signalling microdomains in MCs. Effects of oral rolipram, a phosphodiesterase 4 (PDE4) inhibitor, on the model were measured. KEY RESULTS: Our model exhibited impaired kidney function with elevated expression of adrenergic and fibrotic genes, including Adrb1, PDEs, Acta2 and Tgfß. RNA-seq analysis revealed that MCs with dysregulated YAP pathway were crucial to the extracellular matrix secretion in kidneys from diabetic nephropathy patients. In cultured MCs, TGF-ß promoted profibrotic gene transcription, which was regulated by nuclear-localized ß-adrenoceptor signalling. Mechanistically, TGF-ß treatment diminished nuclear-specific cAMP signalling in MCs and reduced PKA-dependent phosphorylation of YAP, leading to its activation. In parallel, db/db mouse kidneys showed increased expressions of PDE4B and PDE4D. Treatment with oral rolipram alleviated kidney fibrosis in db/db mice. CONCLUSION AND IMPLICATIONS: Diabetic nephropathy impaired nuclear-localized ß1-adrenoceptor-cAMP signalling microdomain through upregulating PDE4 expression, promoting fibrosis in MCs via PKA dephosphorylation-dependent YAP activation. Our results suggest PDE4 inhibition as a promising strategy for alleviating kidney fibrosis in diabetic nephropathy.

Autosomal recessive hypophosphatemic rickets type 2 due to ENPP1 deficiency (ARHR2).

Autosomal recessive hypophosphatemic rickets type 2 (ARHR2; MIM #613312) is a very rare disorder caused by biallelic loss-of-function mutations in the ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) gene. ENPP1 deficiency encompasses a spectrum of phenotypes that includes, in addition to ARHR2, generalized arterial calcification of infancy (GACI), ossification of the posterior longitudinal ligament (OPLL), and pseudoxanthoma elasticum. ARHR2 can be found in GACI survivors, but it may also be the first manifestation of ENPP1 deficiency. Although the precise mechanisms are not fully elucidated, patients with GACI and ARHR2 have elevated serum FGF23 levels, leading to renal phosphate wasting and hypophosphatemia. As a result, the clinical and radiological phenotype of ARHR2 patients is very similar to that of patients affected with other forms of hypophosphatemic rickets, such as X-linked hypophosphatemia. Patients show signs of rickets (abnormal mineralization of growth plates in children) and osteomalacia (abnormal bone mineralization in children and adults) of varying severity. Clinical manifestations specific to ENPP1 loss-of-function mutations and common to GACI, such as ectopic calcifications (valvular, arterial, or periarticular), deafness, OPLL, and PXE, may also be found. Genetic confirmation of the disease is important so as to ensure that patients receive the appropriate treatment or have the opportunity to participate in clinical trials to evaluate the safety and efficacy of novel and promising recombinant enzyme therapies.

Safety, tolerability and pharmacokinetics of the phosphodiesterase 2 inhibitor BI 474121: An overview of phase I randomized trials in healthy volunteers.

BACKGROUND: Cognitive impairment associated with schizophrenia predicts poor functional outcomes, but currently no efficacious pharmacotherapies are available. AIMS: Four phase I trials examined the safety, tolerability and pharmacokinetics of the phosphodiesterase 2 inhibitor BI 474121, along with potential drug-drug interactions. METHODS: Trial 1 evaluated single rising doses (SRDs) of BI 474121 versus placebo in healthy males. The influence of drug formulation and food on drug bioavailability was also examined. Trial 2 evaluated SRD of BI 474121 versus placebo in healthy Japanese males. Trial 3 evaluated multiple rising doses of BI 474121 in healthy young (with/without midazolam) and elderly (without midazolam) participants versus placebo. Trial 4 investigated interactions between itraconazole and single-dose BI 474121 in healthy males. RESULTS/OUTCOMES: No deaths, serious adverse events (AEs), severe AEs or protocol-specified AEs of special interest were observed. BI 474121 absorbed rapidly during fasting, achieved maximum concentration of analyte in plasma and dose proportionality via tablet formulation, and decreased in a multiphasic manner. BI 474121 steady state occurred within 11 days of multiple oral administration. Multiple doses increased BI 474121 plasma concentrations, but did not alter the time course of plasma concentrations. Urinary excretion of unchanged BI 474121 was negligible. No clinically relevant inhibition or induction of CYP3A4 by BI 474121 was observed. Itraconazole co-administration produced higher exposures of BI 474121 versus BI 474121 alone. CONCLUSIONS/INTERPRETATION: BI 474121 demonstrated favourable safety and pharmacokinetic profiles in healthy Caucasian and Japanese individuals, supporting further clinical development.

Vacuum Erection Device Plus Once-Daily Tadalafil Improve Clinical Outcomes after Extracorporeal Shock Wave Therapy in Men Affected by Erectile Dysfunction Associated with Peyronie's Disease.

BACKGROUND: The purpose of this study is to examine the combination of the mechanical effects of penile therapy with vacuum erection devices (VEDs) plus PDE5i, which improve clinical outcomes after extracorporeal shockwave therapy (ESWT) in men affected by erectile dysfunction (ED) associated with Peyronie's disease (PD). METHODS: A total of 153 medical records of patients affected by PD in stable stage with ED and treated with ESWT were divided into two groups. Group A (GA) included 72 men treated with ESWT, mechanical stretching with VEDs and PDE5ì (Tadalafil 5 mg), and Group B (GB) included 81 men who received only ESWT plus Tadalafil 5 mg with the same protocol of GA. The patients in both groups were assessed at baseline and follow-up for erectile function, painful erections, penile plaque size, and penile curvature. The results were evaluated at baseline and 3, 6, and 12 months after the treatments. RESULTS: Three months after the treatment, GA patients had a reduction in penile curvature degree from a mean ± SD of 33.91 ± 8.34° at baseline to a mean ± SD of 19.46 ± 7.15° after 12 months, whereas pain in an erection or during intercourse was resolved completely in 88.9% of the patients. The mean ± SD IIEF-15 score of patients affected by severe/moderate ED further improved significantly in the GA group (p < 0.001) after 3, 6, and 12 months of treatment. There were no permanent adverse sequelae after treatments. CONCLUSIONS: The regular use of a VED plus Tadalafil in patients who had undergone ESWT significantly provided more benefit in patients with PD in terms of penile deformity, pain, and erectile function.

Cheminformatic Identification of Tyrosyl-DNA Phosphodiesterase 1 (Tdp1) Inhibitors: A Comparative Study of SMILES-Based Supervised Machine Learning Models.

BACKGROUND: Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs damages in DNA induced by abortive topoisomerase 1 activity; however, maintenance of genetic integrity may sustain cellular division of neoplastic cells. It follows that Tdp1-targeting chemical inhibitors could synergize well with existing chemotherapy drugs to deny cancer growth; therefore, identification of Tdp1 inhibitors may advance precision medicine in oncology. OBJECTIVE: Current computational research efforts focus primarily on molecular docking simulations, though datasets involving three-dimensional molecular structures are often hard to curate and computationally expensive to store and process. We propose the use of simplified molecular input line entry system (SMILES) chemical representations to train supervised machine learning (ML) models, aiming to predict potential Tdp1 inhibitors. METHODS: An open-sourced consensus dataset containing the inhibitory activity of numerous chemicals against Tdp1 was obtained from Kaggle. Various ML algorithms were trained, ranging from simple algorithms to ensemble methods and deep neural networks. For algorithms requiring numerical data, SMILES were converted to chemical descriptors using RDKit, an open-sourced Python cheminformatics library. RESULTS: Out of 13 optimized ML models with rigorously tuned hyperparameters, the random forest model gave the best results, yielding a receiver operating characteristics-area under curve of 0.7421, testing accuracy of 0.6815, sensitivity of 0.6444, specificity of 0.7156, precision of 0.6753, and F1 score of 0.6595. CONCLUSIONS: Ensemble methods, especially the bootstrap aggregation mechanism adopted by random forest, outperformed other ML algorithms in classifying Tdp1 inhibitors from non-inhibitors using SMILES. The discovery of Tdp1 inhibitors could unlock more treatment regimens for cancer patients, allowing for therapies tailored to the patient's condition.

Aerosol of Enoximone/Hydroxypropyl-ß-Cyclodextrin Inclusion Complex, Biopharmaceutical Evidence for ARDS Applicability.

BACKGROUND: Phosphodiesterase (PDE) inhibitors are gaining interest in the context of pulmonary pathologies. In particular, the PDE3 inhibitor enoximone (ENXM) has shown potential relative to the cure of asthma, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS). Despite its administration via inhalation being planned for use against COVID-19 related ARDS (C-ARDS), presently, no inhalable medicine containing ENXM is available. OBJECTIVES: This study aims to develop a new formulation suitable for pulmonary administration of ENXM. METHODS: A solution for nebulization, based on the complex between ENXM and Hydroxypropyl-ß-Cyclodextrin (HPßCD) (ENXM/HPßCD) is developed. The obtained solution is characterized in terms of aerodynamic distributions and biopharmaceutical features. RESULTS: The evaluation of the aerosol droplets indicates a good bronchi-lung distribution of the drug. Biological evaluations of the air-liquid interface (ALI) in an in vitro lung cell model demonstrates that ENXM/HPßCD is capable of a local direct effect, increasing intracellular cyclic adenosine monophosphate (cAMP) levels and protecting from oxidative stress. CONCLUSIONS: This study offers a promising advance in the optimization of enoximone delivery to the lungs.

Roflumilast-loaded nanostructured lipid carriers attenuate oxidative stress and neuroinflammation in Parkinson's disease model.

Parkinson's disease (PD) is a progressive neurodegenerative disorder with limited symptomatic treatment options. Targeting phosphodiesterase 4 (PDE4) has shown a promising result in several preclinical studies. In our study, we aim to repurpose US FDA-approved PDE4 inhibitor for PD. Through in-silico study, we identified roflumilast (ROF) as the potential candidate targeting PDE4B2. In Drosophila PD expressing the A30P mutant α-synuclein model, ROF exhibited anti-PD effects as indicated by negative geotaxis and antioxidant activities. Given the low brain distribution of ROF (<50%) at clinical doses, incorporation into nanostructured lipid carriers (NLCs) was carried out to enhanced blood-brain barrier permeability. In vitro release studies indicated sustained ROF release from NLCs (≈75%) over 24 h. Single-dose oral toxicity studies reported no mortality or toxicity signs. ROF-loaded NLCs significantly alleviated behavioural deficits, increased antioxidant parameters (p < 0.05), and reduced TNF-α and IL-6 levels (p < 0.5) in the striatum compared to pure ROF. ROF-loaded NLCs demonstrated potential anti-PD effects with high efficacy than pure ROF. Our study suggests that nanostructured lipid carriers (NLCs) can be a promising drug delivery system to overcome limitations associated with poor brain bioavailability of lipophilic drugs like ROF for PD treatment. Further investigation related to brain occupancy and underlying mechanisms of our formulation is warranted to confirm and strengthen our current findings.

Mutations in the N-domain of aryl hydrocarbon receptor interacting protein affect interactions with heat shock protein 90ß and phosphodiesterase 4A5.

The aryl hydrocarbon receptor interacting protein (AIP) is a cytoplasmic molecular co-chaperone and tumour suppressor that assists in protein stability and complex formation involving the aryl hydrocarbon receptor. Germline mutations in the AIP gene predispose to pituitary tumourigenesis with patients exhibiting an aggressive clinical phenotype. Full length AIP proteins harbouring N-domain mutations (R9Q, R16H, V49 M and K103R) were purified from E.coli utilizing a methodology that maintained structural integrity and monomeric stability. Mutations did not significantly affect the thermal stability of the protein and caused no overall disruptive effect in the protein structure. The mutations studied lowered the binding affinity of AIP towards two of its binding partners; heat shock protein 90ß and phosphodiesterase 4A5 (PDE4A5). The inhibition of phosphodiesterase activity by AIP was also greatly reduced by all mutants. While previously published data has mainly concentrated on the tetratricopeptide repeats of the C-domain of AIP, we present clear evidence that AIP N-domain mutations play a significant role in two protein:protein interactions with partner proteins. The complex interactome of AIP suggests that any observable change in one or more of its binding partners cannot be disregarded as it may have repercussions on other biochemical pathways.

Hua-Shi-Bai-Du decoction inactivates NLRP3 inflammasome through inhibiting PDE4B in macrophages and ameliorates mouse acute lung injury.

BACKGROUND: Hua-Shi-Bai-Du decoction (HSBD) exerts significant effects on the prevention and treatment of COVID-19 in China. The activation of the NLRP3 inflammasome of macrophages plays a vital role in COVID-19 pathology. However, no previous studies have focused on this pathological process to explore the effect of HSBD. PURPOSE: Our aim is to uncover the effect of HSBD on NLRP3 inflammasome activation and the underlying mechanisms. METHODS: The NLRP3-activated J774A.1 cells primed by LPS and activated by nigericin/ATP/MSU were used to evaluate NLRP3 activation in vitro. ASC oligomerization and speck formation were assessed by western blot and immunofluorescence imaging. Intracellular K+ levels were determined by the colorimetric assay. Mitochondrial ROS (mtROS) level was detected by the flow cytometry and the fluorescence spectrophotometry. The intracellular cAMP level was determined by chemiluminescence method and ELISA, while phosphodiesterase (PDE) activity was measured using the fluorescent substrate MANT-cAMP. siRNA was applied to knockdown PDE4B. Two in vivo mouse models, MSU-induced peritonitis and LPS-induced acute lung injury (ALI), were used to evaluate the effects of HSBD on IL-1ß and other inflammatory cytokines. Pathological changes in lung tissue were observed by histopathological examination. RESULTS: HSBD not only decreased supernatant IL-1ß, caspase-1 p20, and cleaved gasdermin D (GSDMD) in NLRP3-activated J774A.1 cells, but also reduced IL-1ß in the peritoneal lavage fluid of mice with MSU-induced peritonitis, demonstrating the suppressive effect on NLRP3 inflammasome activation. The mechanism study showed that HSBD blocked ASC oligomerization and speck formation without affecting K+ efflux or mtROS production. Furthermore, it prevented the decrease of intracellular cAMP by inhibiting PDE4B activity. And in the PDE4B-deficient cells, its suppressive effect on IL-1ß release was abolished. In LPS-induced ALI mice, oral administration of HSBD decreased several proinflammatory cytokines (IL-1ß, IL-6, TNF-α, and CXCL-1) and attenuated the pathological damage to the lung. CONCLUSION: HSBD suppresses the activation of NLRP3 inflammasome by inhibiting PDE4B activity to counteract the decrease of intracellular cAMP, thereby blocking ASC oligomerization in macrophages. Our findings may provide new insight into the clinical effets of HSBD for the treatment of COVID-19.

Discovery and optimization of 4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amine derivatives as novel phosphodiesterase 4 inhibitors.

Phosphodiesterases (PDEs) are important intracellular enzymes that hydrolyze the second messengers cAMP and/or cGMP. Now several studies have shown that PDE4 received particular attention due to which it represents the most prominent cAMP-metabolizing enzyme involved in many diseases. In this study, we performed prescreening of our internal compound library and discovered the compound (PTC-209) with moderate PDE4 inhibitory activity (IC50 of 4.78 ± 0.08 µM). And a series of 4-(imidazo[1,2-a]pyrimidin-3-yl)thiazol-2-amine derivatives as novel PDE4 inhibitors starting from PTC-209 were successfully designed and synthesized using a structure-based discovery strategy. L19, the most potent inhibitor, exhibited good inhibitory activity (IC50 of 0.48 ± 0.02 µM) and remarkable metabolic stability in rat liver microsomes. Our study presents an example of discovery novel PDE4 inhibitors, which would be helpful for design and optimization of novel inhibitors in future.

The Effect of Chronic Treatment with the Inhibitor of Phosphodiesterase 5 (PDE5), Sildenafil, in Combination with L-DOPA on Asymmetric Behavior and Monoamine Catabolism in the Striatum and Substantia Nigra of Unilaterally 6-OHDA-Lesioned Rats.

The use of phosphodiesterase inhibitors in the treatment of Parkinson's disease is currently widely discussed. The study aimed to investigate the impact of acute and chronic treatment with the phosphodiesterase 5 inhibitor, sildenafil, at low and moderate doses of 2 mg/kg and 6 mg/kg, and L-DOPA (12.5 mg/kg), alone or in combination, on asymmetric behavior and dopamine (DA) and serotonin metabolism in the striatum and substantia nigra of unilaterally 6-OHDA-lesioned rats. Acute administration of sildenafil at both tested doses jointly with L-DOPA significantly increased the number of contralateral rotations during a 2 h measurement compared to L-DOPA alone. The effect of a lower dose of sildenafil combined with L-DOPA was much greater in the second hour of measurement. However, the acute combined administration of a higher dose of sildenafil with L-DOPA resulted in an immediate and much stronger increase in the number of contralateral rotations compared to L-DOPA alone, already visible in the first hour of measurement. Interestingly, the chronic combined administration of 2 mg/kg of sildenafil and L-DOPA significantly reduced the number of contralateral rotations, especially during the first hour of measurement, compared to the long-term treatment with L-DOPA alone. Such an effect was not observed after the long-term combined treatment of a higher dose of sildenafil and L-DOPA compared to L-DOPA alone. The concentration of DA in the ipsilateral striatum and substantia nigra after the last combined chronic dose of sildenafil (2 or 6 mg/kg) and L-DOPA (12.5 mg/kg) was significantly higher than after L-DOPA alone. In spite of much stronger increases in the DA concentration in the ipsilateral striatum and substantia nigra, the number of contralateral rotations was reduced in the group of rats treated with the combination of 2 mg/kg sildenafil and L-DOPA compared to the group receiving L-DOPA alone. Moreover, the combined treatment with a low dose of sildenafil and L-DOPA had an opposite effect on DA catabolism, as assessed by DOPAC/DA and HVA/DA indexes, and these indexes were reduced in the ipsilateral striatum but increased in the contralateral striatum and substantia nigra compared to the treatment with L-DOPA alone. The results of the present study show that the addition of a low dose of a PDE5 inhibitor to the standard L-DOPA therapy differently modulates rotational behavior, the tissue DA concentration and its catabolism in the striatum and substantia nigra.