Identification of small molecule estrogen‐related receptor α–specific antagonists and homology modeling to predict the molecular determinants as the basis for selectivity over ERRβ and ERRγ

The estrogen‐related receptor α (ERRα) was one of the first orphan receptors identified through a search for genes encoding proteins related to the steroid nuclear receptor, Estrogen Receptor α (ERα). The physiological role of ERRα has not yet been established nor has a natural ligand been elucidated. Importantly, research indicates that ERRα may be a novel drug target to treat breast cancer and/or metabolic disorders. A homogeneous time‐resolved fluorescence (HTRF) assay has been developed to screen for ERRα‐specific antagonists. This assay uses the ERR ligand binding domain and the coactivator interaction domain of Proliferator‐activated Receptor γ Coactivator‐1α (PGC‐1α) to examine the ability of compounds to antagonize the constitutive interaction between ERRα and the coactivator. A dissociation‐enhanced lanthanide fluorescence immunoassay (DELFIA) was also created to counter screen compounds identified in the HTRF screen. Here we report the discovery of high‐affinity ERRα subtype selective antagonists. Additionally, a homology model of ERRα in an antagonist conformation has been developed and after subsequent docking studies, we offer a model showing the molecular determinants that suggest why our novel tri‐cyclic antagonist, N‐[(2Z)‐3‐(4,5‐dihydro‐1,3‐thiazol‐2‐yl)‐1,3‐thiazolidin‐2‐yl idene]‐5 H dibenzo[a,d][7]annulen‐5‐amine, binds to ERRα with high affinity but does not bind to either ERRβ or ERRγ. Drug Dev Res 69:203–218, 2008. © 2008 Wiley‐Liss, Inc.     

Discovery of naphthyl‐N‐acylhydrazone p38α MAPK inhibitors with in vivo anti‐inflammatory and anti‐TNF‐α activity

Protein kinases constitute attractive therapeutic targets for development of new prototypes to treat different chronic diseases. Several available drugs, like tinibs, are tyrosine kinase inhibitors; meanwhile, inhibitors of serine/threonine kinases, such as mitogen‐activated protein kinase (MAPK), are still trying to overcome some problems in one of the steps of clinical development to become drugs. So, here we reported the synthesis, the in vitro kinase inhibitory profile, docking studies, and the evaluation of anti‐inflammatory profile of new naphthyl‐N‐acylhydrazone derivatives using animal models. Although all tested compounds ( 3a–d ) have been characterized as p38α MAPK inhibitors and have showed in vivo anti‐inflammatory action, LASSBio‐1824 ( 3b ) presented the best performance as p38α MAPK inhibitor, with IC₅₀ = 4.45 μm , and also demonstrated to be the most promising anti‐inflammatory prototype, with good in vivo anti‐TNF‐α profile after oral administration.     

Molecular regulation of skeletal muscle mass

1. The maintenance of skeletal muscle mass is determined by a fine balance between protein synthesis and protein degradation. Skeletal mass is increased when there is a net gain in protein synthesis, which can occur following progressive exercise training. In contrast, skeletal muscle mass is lost when degradation occurs more rapidly than synthesis and is observed in numerous conditions, including neuromuscular disease, chronic disease, ageing, as well as following limb immobilization or prolonged bed rest due to injury or trauma. 2. Understanding the molecular pathways that regulate skeletal muscle protein synthesis and degradation is vital for identifying potential therapeutic targets that can attenuate muscle atrophy during disease and disuse. 3. The regulation of skeletal mass is complex and involves the precise coordination of several intracellular signalling pathways. The present review focuses on the role and regulation of pathways involving Akt, atrogin‐1 and muscle ring finger‐1 (MuRF1; atrogenes), peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) and striated activator of Rho signalling (STARS), with exercise and disease.     

Up‐regulation of Gadd45α after exposure to metal nanoparticles: The role of hypoxia inducible factor 1α

The increased development and use of nanoparticles in various fields may lead to increased exposure, directly affecting human health. Our current knowledge of the health effects of metal nanoparticles such as cobalt and titanium dioxide (Nano‐Co and Nano‐TiO₂) is limited but suggests that some metal nanoparticles may cause genotoxic effects including cell cycle arrest, DNA damage, and apoptosis. The growth arrest and DNA damage‐inducible 45α protein (Gadd45α) has been characterized as one of the key players in the cellular responses to a variety of DNA damaging agents. The aim of this study was to investigate the alteration of Gadd45α expression in mouse embryo fibroblasts (PW) exposed to metal nanoparticles and the possible mechanisms. Non‐toxic doses of Nano‐Co and Nano‐TiO₂ were selected to treat cells. Our results showed that Nano‐Co caused a dose‐ and time‐dependent increase in Gadd45α expression, but Nano‐TiO₂ did not. To investigate the potential pathways involved in Nano‐Co‐induced Gadd45α up‐regulation, we measured the expression of hypoxia inducible factor 1α (HIF‐1α) in PW cells exposed to Nano‐Co and Nano‐TiO₂. Our results showed that exposure to Nano‐Co caused HIF‐1α accumulation in the nucleus. In addition, hypoxia inducible factor 1α knock‐out cells [HIF‐1α (?/?)] and its wild‐type cells [HIF‐1α (+/+)] were used. Our results demonstrated that Nano‐Co caused a dose‐ and time‐dependent increase in Gadd45α expression in wild‐type HIF‐1α (+/+) cells, but only a slight increase in HIF‐1α (?/?) cells. Pre‐treatment of PW cells with heat shock protein 90 inhibitor, 17‐(Allylamino)?17‐demethoxygeldanamycin (17‐AAG), prior to exposure to Nano‐Co significantly abolished Nano‐Co‐induced Gadd45α expression. These results suggest that HIF‐1α accumulation may be partially involved in the increased Gadd45α expression in cells exposed to Nano‐Co. These findings may have important implications for understanding the potential health effects of metal nanoparticle exposure. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 490–499, 2015.     

Novel role for β‐adrenergic signalling in skeletal muscle growth,development and regeneration

1. In adult mammals, skeletal muscle mass is maintained through a precise balance of protein synthesis and protein degradation, whereas during development cellular (not protein) turnover predominates. When protein balance is shifted towards synthesis, skeletal muscle hypertrophy ensues. In contrast, increased protein degradation leads to skeletal muscle atrophy. Insulin‐like growth factor (IGF)‐I is among the best documented of the growth factors and regulates skeletal muscle mass by increasing protein synthesis and decreasing protein degradation. However, an IGF‐I‐independent growth pathway has been identified that involves the activation of β‐adrenoceptors and subsequent skeletal muscle growth, development and hypertrophy. 2. Although the importance of β‐adrenergic signalling in the heart has been well documented and continues to receive significant attention, it is only more recently that we have started to appreciate the importance of this signalling pathway in skeletal muscle structure and function. Studies have identified an important role for β‐adrenoceptors in myogenesis and work from our laboratory has identified a novel role for β‐adrenoceptors in regulating skeletal muscle regeneration after myotoxic injury. In addition, new data suggest that β‐adrenoceptors are markedly upregulated during differentiation of C2C12 cells. 3. It is now clear that β‐adrenoceptors play an important role in regulating skeletal muscle structure and function. Importantly, a clearer understanding of the pathways regulating skeletal muscle mass may lead to the identification of novel therapeutic targets for the treatment of muscle wasting disorders, including sarcopenia, cancer cachexia and the muscular dystrophies.     

Synthesis,p38α MAP kinase inhibition,anti‐inflammatory activity,and molecular docking studies of 1,2,4‐triazole‐based benzothiazole‐2‐amines

Recent studies have demonstrated that inhibition of p38α MAP kinase could effectively inhibit pro‐inflammatory cytokines including TNF‐α and interleukins. Thus, inhibition of this enzyme can prove greatly beneficial in the therapy of chronic inflammatory diseases. A new series of N‐[3‐(substituted‐4H‐1,2,4‐triazol‐4‐yl)]‐benzo[d]thiazol‐2‐amines ( 4a–n ) were synthesized and subjected to in vitro evaluation for anti‐inflammatory activity (BSA anti‐denaturation assay) and p38α MAPK inhibition. Among the compounds selected for in vivo screening of anti‐inflammatory activity ( 4b , 4c , 4f , 4g , 4j , 4m , and 4n ), compound 4f was found to be the most active with an in vivo anti‐inflammatory efficacy of 85.31% when compared to diclofenac sodium (83.68%). It was also found to have a low ulcerogenic risk and a protective effect on lipid peroxidation. The p38α MAP kinase inhibition of this compound (IC₅₀ = 0.036 ± 0.12 μM) was also found to be superior to the standard SB203580 (IC₅₀ = 0.043 ± 0.27 μM). Furthermore, the in silico binding mode of the compound on docking against p38α MAP kinase exemplified stronger interactions than those of SB203580.

     

α1‐ and α2‐Adrenoceptor hyporesponsiveness in isolated bisected vas deferens of bile duct‐ligated rats

1 It has been suggested that cholestasis accompanied with changes in autonomic balance and hyporesponsiveness in muscarinic and adrenergic receptors of some organs, e.g. cardiovascular system. Increased plasma levels of epinephrine and norepinephrine has been shown during cholestasis suggesting augmented activity of sympathetic nervous system. In this study we evaluate both α₁ and α₂ responsiveness in isolated rat vas deferens, as a tissue with rich adrenergic innervations. 2 Epididymal and prostatic halves of vas deferens responsiveness have been studied to phenylephrine and clonidine respectively in three groups of un‐operated, sham‐operated (sham), and bile duct‐ligated (BDL) rats. 3 Our results indicate that in vas deferens of BDL animals, the concentration‐response curve of both phenylephrine and clonidine shifted to rightward compared to control group, while the position of concentration‐response curve of sham group did not change significantly (P > 0.05). EC₅₀ of phenylephrine and IC₅₀ of clonidine were increased showing a decreased responsiveness of tissue to phenylephrine (P < 0.05) and clonidine (P < 0.001) in BDL rats. 4 In this study, both subtype of α‐adrenoceptors (α₁ and α₂) has been studied in cholestatic rat vas deference. Our results showed that cholestasis induce hyporesponsiveness to phenylephrine and clonidine. These results are consistent with previous reports, suggesting the hyporesponsiveness of α₁‐adrenoceptors in pulmonary artery and papillary muscle and mesenteric beds. Our conclusion is that the cholestasis induces hyporesponsiveness to phenylephrine and clonidine in epididymal (α₁‐adrenoceptors) and prostatic (α₂‐adrenoceptors) halves of rat vas deferens respectively. Although the logical explanation to this hyporesponsiveness is the down regulation but it has been suggested that it is not because of down regulation.     

Nephropreventing effect of hypoxia‐inducible factor 1α in a rat model of ischaemic/reperfusion acute kidney injury

Acute kidney injury (AKI) occurs in 5% of hospitalized patients and in 50% of sepsis patients with acute renal dysfunction. However, there have been no safe and effective therapeutic strategies. The hypoxia condition is closely related to renal injury and function under AKI. As hypoxia‐inducible factor 1α (HIF‐1α) is critical for the cellular response to hypoxia, we investigated the protective effect of HIF‐1α in a rat AKI model. We found that HIF‐1α injection improved the survival of rat with AKI, and the level of creatinine and blood urea nitrogen (BUN) was also increased. Our data showed that HIF‐1α treatment significantly alleviated ischaemic/reperfusion injury to kidney tubules and nephrocytes. We also found the downstream factors, such as EPOR, VEGF, and PHD3, were also upregulated by HIF‐1α. Finally, it was observed that HIF‐1α treatment also increased the percentage of adult resident progenitor cells (ARPC) in vitro and in vivo. In conclusion, HIF‐1α plays a protective role in the ischaemic AKI model through stimulating the proliferation of ARPC, and our study provided a potential therapeutic strategy for AKI.     

Drug resistance and cancer stem cells: the shared but distinct roles of hypoxia‐inducible factors HIF1α and HIF2α

Chemotherapy resistance is a major contributor to poor treatment responses and tumour relapse, the development of which has been strongly linked to the action of cancer stem cells (CSCs). Mounting evidence suggests that CSCs are reliant on low oxygen conditions and hypoxia‐inducible factors 1α and 2α (HIF1α and HIF2α) to maintain their stem cell features. Research in the last decade has begun to clarify the functional differences between the two HIFα subtypes (HIFαs). Here, we review and discuss these differences in relation to CSC‐associated drug resistance. Both HIFαs contribute to CSC survival but play different roles –HIF1α being more responsible for survival functions and HIF2α for stemness traits such as self‐renewal – and are sensitive to different degrees of hypoxia. Failure to account for physiologically relevant oxygen concentrations in many studies may influence the current understanding of the roles of HIFαs. We also discuss how hypoxia and HIFαs contribute to CSC drug resistance via promotion of ABC drug transporters Breast cancer resistance protein (BCRP), MDR1, and MRP1 and through maintenance of quiescence. Additionally, we explore the PI3K/AKT cell survival pathway that may support refractory cancer by promoting CSCs and activating both HIF1α and HIF2α. Accordingly, HIF1α and HIF2α inhibition, potentially via PI3K/AKT inhibitors, could reduce chemotherapy resistance and prevent cancer relapse.     

Specific α7 nicotinic acetylcholine receptor agonist ameliorates isoproterenol‐induced cardiac remodelling in mice through TGF‐β1/Smad3 pathway

It is well‐accepted that inflammation plays an important role in the development of cardiac remodelling and that therapeutic approaches targeting inflammation can inhibit cardiac remodelling. Although a large amount of evidence indicates that activation of α7 nicotinic acetylcholine receptor (α7nAChR) causes an anti‐inflammatory effect, the role of α7nAChR in cardiac remodelling and the underlying mechanism have not been established. To investigate the effect of the specific α7nAChR agonist, PNU282987, on cardiac remodelling induced by isoproterenol (ISO 60 mg/kg per day) in mice, the cardiomyocyte cross‐sectional area (CSA) and collagen volume fraction were evaluated by hematoxylin and eosin (HE) and Masson staining, respectively. Cardiac function and ventricular wall thickness were measured by echocardiography. The protein expressions of collagen I, matrix metalloproteinase 9 (MMP‐9), transforming growth factor β1 (TGF‐β1), and Smad3 were analyzed by Western blot. ISO‐induced cardiac hypertrophy, characterized by an increase in the heart weight/body weight ratio, CSA and ventricular wall thickness. Moreover, cardiac fibrosis indices, such as collagen volume fraction, MMP‐9 and collagen I protein expression, were also increased by ISO. PNU282987 not only attenuated cardiac hypertrophy but also decreased the cardiac fibrosis induced by ISO. Furthermore, PNU282987 suppressed TGF‐β1 protein expression and the phosphorylation of Smad3 induced by ISO. In conclusion, PNU282987 ameliorated the cardiac remodelling induced by ISO, which may be related to the TGF‐β1/Smad3 pathway. These data imply that the α7nAChR may represent a novel therapeutic target for cardiac remodelling in many cardiovascular diseases.     

Estrogen and ERα enhanced β‐catenin degradation and suppressed its downstream target genes to block the metastatic function of HA22T hepatocellular carcinoma cells via modulating GSK‐3β and β‐TrCP expression

In our previous experiments, we found β‐catenin was highly expressed in the tumor area with high invasive ability and poor prognosis. In this study, we have examined the mechanism by which ERα regulates β‐catenin expression as well as the metastasis ability of hepatocellular cancer HA22T cells. To identify whether the anticancer effect of estrogen and ERα is mediated through suppression of β‐catenin expression, we co‐transfected pCMV‐β‐catenin and ERα into HA22T cells, and determined the cell motility by wound healing, invasion, and migration assays. Results showed that estrogen and/or ERα inhibited β‐catenin gene expression and repressed HA22T cell motility demonstrated that similar data was observed in cells expressing the ERα stable clone. Moreover, we examined the protein‐protein interaction between ERα and β‐catenin by immunostain, co‐immunoprecipitation, and Western blotting. E2 enhanced the binding of ERα with β‐catenin and then triggered β‐catenin to bind with E3 ligase (βTrCP) to promote β‐catenin degradation. Finally by employing systematic ChIP studies, we showed ERα can interact directly with the β‐catenin promoter region following E2 treatment. All our results reveal that estrogen and ERα blocked metastatic function of HA22T cells by modulating GSK3β and βTrCP expression and further enhanced β‐catenin degradation and suppressed its downstream target genes. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 519–529, 2017.     

Protective effects of β‐sheet breaker α/β‐hybrid peptide against amyloid β‐induced neuronal apoptosis in vitro

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid‐β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid‐β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β‐sheet breaker α/β‐hybrid peptide (BSBHp) and the underlying mechanisms against Aβ₄₀‐induced neurotoxicity in human neuroblastoma SH‐SY5Y cells. Cells were pretreated with the peptide Aβ₄₀ to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca²⁺, and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ₄₀‐induced toxicity by retaining cell viability, suppressing generation of ROS, Ca²⁺ levels, and effectively protects neuronal apoptosis by suppressing pro‐apoptotic protein Bax and up‐regulating antiapoptotic protein Bcl‐2. These results suggest that α/β‐hybrid peptide has neuroprotective effects against Aβ₄₀‐induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.     

Andrographolide inhibits hypoxia‐induced hypoxia‐inducible factor 1α and endothelin 1 expression through the heme oxygenase 1/CO/cGMP/MKP‐5 pathways in EA.hy926 cells

Andrographolide is a potent anti‐inflammatory agent found in Andrographis paniculata. Endothelin 1 (ET‐1) is an endothelium‐derived vasoconstrictor with pro‐inflammatory properties secreted in response to hypoxia. Mitogen‐activated protein kinase phosphatase 5 (MKP‐5) is a dual‐specificity phosphatase that dephosphorylates threonine and tyrosine residues of MAPKs. We showed previously that hypoxia‐induced HIF‐1α expression and ET‐1 secretion are dependent on p38 MAPK in EA.hy926 cells. Here, we investigate what role MKP‐5 plays in andrographolide's inhibition of hypoxia‐induced expression of HIF‐1α and ET‐1. Hypoxic conditions were created using the hypoxia‐mimetic agent CoCl₂. Andrographolide enhanced HO‐1 and MKP‐5 expression and cellular cGMP content in addition to inhibiting hypoxia‐induced ROS generation. Concomitantly, the HO‐1 byproduct CO and the cGMP analogue 8‐bromoguanosine 3′,5′‐cyclic monophosphate (8‐Br‐cGMP) increased MKP‐5 expression, and pretreatment with CO and 8‐Br‐cGMP inhibited hypoxia‐induced HIF‐1α and ET‐1 expression. Transfection of HO‐1 siRNA or pretreatment with the HO‐1 inhibitor ZnPP‐9 or 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one, a specific inhibitor of soluble guanylate cyclase, reduced andrographolide‐induced MKP‐5 expression. Moreover, silencing MKP‐5 or treatment with the phosphatase inhibitor vanadate abrogated andrographolide's suppressing hypoxia‐induced p38 MAPK activation and HIF‐1α expression. The inhibition of hypoxia‐induced HIF‐1α and ET‐1 expression by andrographolide is likely associated with HO‐1/CO/cGMP/MKP‐5 pathways, which is involved in inhibiting hypoxia‐induced p38 MAPK activation.     

Comparative assessment of HIF‐1α and Akt responses in human lung and skin cells exposed to benzo[α]pyrene: Effect of conditioned medium from pre‐exposed primary fibroblasts

Exposure to atmospheric pollutants has been accused for many adverse health effects. Benzo[α]pyrene (Β[α]Ρ) in particular, the most extensively studied member of pollutants, is implicated in both cancer initiation and promotion. In the present study, we compared the effects of noncytotoxic doses of Β[α]Ρ, between human skin and lung epithelial cells A431 and A549, respectively, focusing on Akt kinase and HIF‐1α, as it is well known that these proteins are upregulated in various human cancers promoting survival, angiogenesis and metastasis of tumor cells. Also, taking into consideration that fibroblasts are involved in cancer progression, we tested the possible modulation of epithelial cell response by paracrine factors secreted by Β[α]Ρ‐treated fibroblasts. Low doses of Β[α]Ρ were found to enhance epithelial cell proliferation and upregulate both Akt kinase and HIF‐1α, with A549 cells exhibiting a more sustained profile of upregulation. It is to notice that, the response of HIF‐1α was remarkably early, acting as a sensitive marker in response to airborne pollutants. Also, HIF‐1α was induced by Β[α]Ρ in both lung and skin fibroblasts indicating that this effect may be conserved throughout different cell types and tissues. Interestingly however, the response of both proteins was differentially modified upon treatment with conditioned medium from Β[α]Ρ‐exposed fibroblasts. This is particularly evident in A459 cells and confirms the critical role of intercellular and paracrine factors in the modulation of the final response to an extracellular signal. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1103–1112, 2016.     

Synthesis of 1,4‐Anthracene‐9,10‐dione Derivatives and Their Regulation of Nitric Oxide,IL‐1β and TNF‐α in Activated RAW264.7 Cells

Mitoxantrone is an anthracenedione antineoplastic and immunosuppressive agent approved for multiple sclerosis treatment. Novel mono‐ and disubstituted anthraquinone derivatives, analogues of mitoxantrone, were synthesized through the addition of lipophilic amino alcohols and evaluated for their effect on IL‐1β, TNF‐α and nitric oxide production by LPS/IFN‐γ‐stimulated RAW264.7 cells. The disubstituted 1,4‐anthracene‐9,10‐dione 10 showed significant inhibition of nitric oxide, TNF‐α and IL‐1β production at the concentration of 5 μg/mL, with a much lower cytotoxicity than mitoxantrone. The monosubstituted 3 , 4 , 11, 12 and 13 also displayed a moderate to good inhibitory capacity on IL‐1β production. However, the methylated compounds 11, 12 and 13 failed to inhibit the TNF‐α production, and compound 13 was the only one to decrease the production of nitric oxide. None of these derivatives was toxic at the tested concentrations. Compounds 10 and 13 had better inhibitory capacity of the inflammatory mediators analyzed, with reliable viability of the cells.