Pathophysiological significance and modulation of the transient receptor potential canonical 3 ion channel.

Transient receptor potential canonical 3 (TRPC3) protein belongs to the TRP family of nonselective cation channels. Its activation occurs by signaling through a G protein-coupled receptor (GPCR) and a phospholipase C-dependent (PLC) pathway. Perturbations in the expression of TRPC3 are associated with a plethora of pathophysiological conditions responsible for disorders of the cardiovascular, immune, and central nervous systems. The recently solved cryo-EM structure of TRPC3 provides detailed inputs about the underlying mechanistic aspects of the channel, which in turn enables more efficient ways of designing small-molecule modulators. Pharmacologically targeting TRPC3 in animal models has demonstrated great efficacy in treating diseases including cancers, neurological disorders, and cardiovascular diseases. Despite extensive scientific evidence supporting some strong correlations between the expression and activity of TRPC3 and various pathophysiological conditions, therapeutic strategies based on its pharmacological modulations have not led to clinical trials. The development of small-molecule TRPC3 modulators with high safety, sufficient brain penetration, and acceptable drug-like profiles remains in progress. Determining the pathological mechanisms for TRPC3 involvement in human diseases and understanding the requirements for a drug-like TRPC3 modulator will be valuable in advancing small-molecule therapeutics to future clinical trials. In this review, we provide an overview of the origin and activation mechanism of TRPC3 channels, diseases associated with irregularities in their expression, and new development in small-molecule modulators as potential therapeutic interventions for treating TRPC3 channelopathies.

The inducible prostaglandin E synthase (mPGES-1) in neuroinflammatory disorders.

The cyclooxygenase (COX)/prostaglandin E2 (PGE2) signaling pathway has emerged as a critical target for anti-inflammatory therapeutic development in neurological diseases. However, medical use of COX inhibitors in the treatment of various neurological disorders has been limited due to well-documented cardiovascular and cerebrovascular complications. It has been widely proposed that modulation of downstream microsomal prostaglandin E synthase-1 (mPGES-1) enzyme may provide more specificity for inhibiting PGE2-elicited neuroinflammation. Heightened levels of mPGES-1 have been detected in a variety of brain diseases such as epilepsy, stroke, glioma, and neurodegenerative diseases. Subsequently, elevated levels of PGE2, the enzymatic product of mPGES-1, have been demonstrated to modulate a multitude of deleterious effects. In epilepsy, PGE2 participates in retrograde signaling to augment glutamate release at the synapse leading to neuronal death. The excitotoxic demise of neurons incites the activation of microglia, which can become overactive upon further stimulation by PGE2. A selective mPGES-1 inhibitor was able to reduce gliosis and the expression of proinflammatory cytokines in the hippocampus following status epilepticus. A similar mechanism has also been observed in stroke, where the overactivation of microglia by PGE2 upregulated the expression and secretion of proinflammatory cytokines. This intense activation of neuroinflammatory processes triggered the secondary injury commonly observed in stroke, and blockade of mPGES-1 reduced infarction size and edema, suppressed induction of proinflammatory cytokines, and improved post-stroke well-being and cognition. Furthermore, elevated levels of PGE2 have been shown to intensify the proliferation of glioma cells, mediate P-glycoprotein expression at the blood-brain barrier (BBB) and facilitate breakdown of the BBB. For these reasons, targeting mPGES-1, the central and inducible enzyme of the COX cascade, may provide a more specific therapeutic strategy for treating neuroinflammatory diseases.

Complementary roles of EP2 and EP4 receptors in malignant glioma.

BACKGROUND AND PURPOSE: Glioblastoma (GBM) is the most aggressive brain tumour in the central nervous system, but the current treatment is very limited and unsatisfactory. PGE2 -initiated cAMP signalling via EP2 and EP4 receptors is involved in the tumourigenesis of multiple cancer types. However, whether or how EP2 and EP4 receptors contribute to GBM growth largely remains elusive. EXPERIMENTAL APPROACH: We performed comprehensive data analysis of gene expression in human GBM samples and determined their expression correlations through multiple bioinformatics approaches. A time-resolved fluorescence energy transfer (TR-FRET) assay was utilized to characterize PGE2 -mediated cAMP signalling via EP2 and EP4 receptors in human glioblastoma cells. Using recently reported potent and selective small-molecule antagonists, we determined the effects of inhibition of EP2 and EP4 receptors on GBM growth in subcutaneous and intracranial tumour models. KEY RESULTS: The expression of both EP2 and EP4 receptors was upregulated and highly correlated with a variety of tumour-promoting cytokines, chemokines, and growth factors in human gliomas. Further, they were heterogeneously expressed in human GBM cells, where they compensated for each other to mediate PGE2 -initiated cAMP signalling and to promote colony formation, cell invasion and migration. Inhibition of EP2 and EP4 receptors revealed that these receptors might mediate GBM growth, angiogenesis, and immune evasion in a compensatory manner. CONCLUSION AND IMPLICATIONS: The compensatory roles of EP2 and EP4 receptors in GBM development and growth suggest that concurrently targeting these two PGE2 receptors might represent a more effective strategy than inhibiting either alone for GBM treatment.

Bacterial biosynthesis of flavonoids: Overview, current biotechnology applications, challenges, and prospects.

Flavonoids are secondary metabolites present in plant organs and tissues. These natural metabolites are the most prevalent and display a wide range of beneficial physiological effects, making them usually intriguing in several scientific fields. Due to their safety for use and protective attributes, including antioxidant, anti-inflammatory, anticancer, and antimicrobial functions, flavonoids are broadly utilized in foods, pharmaceuticals, and nutraceuticals. However, conventional methods for producing flavonoids, such as plant extraction and chemical synthesis, entailed dangerous substances, and laborious procedures, with low product yield. Recent studies have documented the ability of microorganisms, such as fungi and bacteria, to synthesize adequate amounts of flavonoids. Bacterial biosynthesis of flavonoids from plant biomass is a viable and environmentally friendly technique for producing flavonoids on a larger scale and has recently received much attention. Still, only a few bacteria species, particularly Escherichia coli, have been extensively studied. The most recent developments in bacterial biosynthesis of flavonoids are reviewed and discussed in this article, including their various applications as natural food biocontrol agents. In addition, the challenges currently faced in bacterial flavonoid biosynthesis and possible solutions, including the application of modern biotechnology approaches for developing bacterial strains that could successfully produce flavonoids on an industrial scale, were elucidated.

The discovery of cyclic γ-AApeptides as the promising ligands targeting EP2.

EP2 is a G protein-coupled receptor for prostaglandin E2 (PGE2) derived from cell membrane-released arachidonic acid upon various harmful and injurious stimuli. It is commomly upregulated in tumors and injured brain tissues, as its activation by PGE2 is widely believed to be involved in the pathophysiological mechanisms underlying these conditions via promoting pro-inflammatory reactions. Herein, we report the discovery of two novel macrocyclic peptidomimetics based on the screening of a cyclic γ-AApeptides combinatorial library. These two cyclic γ-AApeptides showed excellent binding affinity with the EP2 protein, and they may lead to the development of novel therapeutic agents and/or molecular probes to modulate the PGE2/EP2 signaling.

Prostaglandin E2 in neuroblastoma: Targeting synthesis or signaling?

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor arising from neural crest cells of the developing sympathetic nervous system. Despite marked advances in cancer treatment, the survival rate of high-risk NB remains unsatisfactory. As a key pro-inflammatory mediator regulating tumor microenvironment, prostaglandin E2 (PGE2) promotes NB proliferation, angiogenesis, and immune evasion via acting on four G protein-coupled receptors, particularly the EP2 subtype. Recent studies have been vigorously focused on developing and evaluating compounds targeting PGE2-regulated tumor inflammation in animal models of NB. In this review, we revisit these translational efforts and examine the feasibility of pharmacological inhibition of enzymes responsible for PGE2 biosynthesis or its signaling receptors as emerging therapeutic strategies for NB. We also explore the potential downstream oncogenic pathways upon the activation of PGE2 receptors, aiming to bridge the knowledge gap between tumorigenesis and the role of elevated PGE2/EP2 signaling, which is widely observed in high-risk NBs.

TRIM22 actives PI3K/Akt/mTOR pathway to promote psoriasis through enhancing cell proliferation and inflammation and inhibiting autophagy.

OBJECTIVE: To reveal the function and underlying mechanism of Tri-domain protein 22 (TRIM22) in psoriasis. METHODS: M5 cytokines were applied in HaCat cells to mimic psoriasis in vitro. The TRIM22-silencing viruses were established to knockdown TRIM22 in HaCat cells. Western blot and/or real-time PCR were used to detect the expression of TRIM22, KRT1, KRT6, p-P65, P65, LC3, Beclin 1, P62, p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR. ELISA kits were applied to assess levels of TNF-α, IL-1ß, IL-18, and HMGB1. RESULTS: TRIM22 expression levels were upregulated in M5-treated HaCat cells. M5 treatment enhanced cell proliferation and inflammation, and inhibited autophagy in HaCat cells which were effectively reversed by TRIM22 deficiency. Activation of PI3K/Akt/mTOR pathway is an essential promoter of cell proliferation and inflammation, and inhibitor of autophagy in psoriasis. TRIM22 deficiency blocked M5-induced activation of PI3K/Akt/mTOR pathway in HaCat cells. CONCLUSIONS: TRIM22 facilitates cell proliferation and inflammation, and suppresses autophagy in M5-treated HaCat cells through activating PI3K/Akt/mTOR pathway, and inhibition of TRIM22 can be a novel potential treatment for psoriasis.

Distinct Cell-specific Roles of NOX2 and MyD88 in Epileptogenesis.

It is well established that temporal lobe epilepsy (TLE) is often related to oxidative stress and neuroinflammation. Both processes subserve alterations observed in epileptogenesis and ultimately involve distinct classes of cells, including astrocytes, microglia, and specific neural subtypes. For this reason, molecules associated with oxidative stress response and neuroinflammation have been proposed as potential targets for therapeutic strategies. However, these molecules can participate in distinct intracellular pathways depending on the cell type. To illustrate this, we reviewed the potential role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) and myeloid differentiation primary response 88 (MyD88) in astrocytes, microglia, and neurons in epileptogenesis. Furthermore, we presented approaches to study genes in different cells, employing single-cell RNA-sequencing (scRNAseq) transcriptomic analyses, transgenic technologies and viral serotypes carrying vectors with specific promoters. We discussed the importance of identifying particular roles of molecules depending on the cell type, endowing more effective therapeutic strategies to treat TLE.

Ablation of Siglec-E augments brain inflammation and ischemic injury.

Sialic acid immunoglobulin-like lectin E (Siglec-E) is a subtype of pattern recognition receptors found on the surface of myeloid cells and functions as a key immunosuppressive checkpoint molecule. The engagement between Siglec-E and the ligand α2,8-linked disialyl glycans activates the immunoreceptor tyrosine-based inhibitory motif (ITIM) in its intracellular domain, mitigating the potential risk of autoimmunity amid innate immune attacks on parasites, bacteria, and carcinoma. Recent studies suggest that Siglec-E is also expressed in the CNS, particularly microglia, the brain-resident immune cells. However, the functions of Siglec-E in brain inflammation and injuries under many neurological conditions largely remain elusive. In this study, we first revealed an anti-inflammatory role for Siglec-E in lipopolysaccharide (LPS)-triggered microglial activation. We then found that Siglec-E was induced within the brain by systemic treatment with LPS in mice in a dose-dependent manner, while its ablation exacerbated hippocampal reactive microgliosis in LPS-treated animals. The genetic deficiency of Siglec-E also aggravated oxygen-glucose deprivation (OGD)-induced neuronal death in mouse primary cortical cultures containing both neurons and glial cells. Moreover, Siglec-E expression in ipsilateral brain tissues was substantially induced following middle cerebral artery occlusion (MCAO). Lastly, the neurological deficits and brain infarcts were augmented in Siglec-E knockout mice after moderate MCAO when compared to wild-type animals. Collectively, our findings suggest that the endogenous inducible Siglec-E plays crucial anti-inflammatory and neuroprotective roles following ischemic stroke, and thus might underlie an intrinsic mechanism of resolution of inflammation and self-repair in the brain.

Targeting EP2 receptor with multifaceted mechanisms for high-risk neuroblastoma.

Prostaglandin E2 (PGE2) promotes tumor cell proliferation, migration, and invasion, fostering an inflammation-enriched microenvironment that facilitates angiogenesis and immune evasion. However, the PGE2 receptor subtype (EP1-EP4) involved in neuroblastoma (NB) growth remains elusive. Herein, we show that the EP2 receptor highly correlates with NB aggressiveness and acts as a predominant Gαs-coupled receptor mediating PGE2-initiated cyclic AMP (cAMP) signaling in NB cells with high-risk factors, including 11q deletion and MYCN amplification. Knockout of EP2 in NB cells blocks the development of xenografts, and its conditional knockdown prevents established tumors from progressing. Pharmacological inhibition of EP2 by our recently developed antagonist TG6-129 suppresses the growth of NB xenografts in nude mice and syngeneic allografts in immunocompetent hosts, accompanied by anti-inflammatory, antiangiogenic, and apoptotic effects. This proof-of-concept study suggests that the PGE2/EP2 signaling pathway contributes to NB malignancy and that EP2 inhibition by our drug-like compounds provides a promising strategy to treat this deadly pediatric cancer.

Targeting NLRP3 signaling by a novel-designed sulfonylurea compound for inhibition of microglial inflammation.

The nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome plays an important role in microglia-mediated inflammation. Dysregulation of NLRP3 signaling results in microglial activation and triggers inflammatory responses contributing to the development of neurological disorders including ischemic stroke, schizophrenia, Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Inhibition of the NLRP3-linked inflammatory pathways reduces microglia-induced inflammation and is considered as a promising therapeutic approach for neuro-inflammatory diseases. In the present study, we report the development of AMS-17, a rationally-designed tertiary sulfonylurea compound for inhibition of inflammation in microglia. AMS-17 inhibited expression of the NLRP3, and its downstream components and cytokines such as caspase-1, tumor necrosis factor-α (TNF-α), IL-1ß and inducible nitric oxide synthase (iNOS). It also suppressed lipopolysaccharide (LPS)-induced N9 microglial cell phagocytosis in vitro and activation of the microglia in mouse brain in vivo. Together, these results provide promising evidences for the inhibitory effects of AMS-17 in inflammation. This proof-of-concept study provides a new chemical scaffold, designed with the aid of pharmacophore modeling, with NLRP3 inhibitory activity which can be further developed for the treatment of inflammation-associated neurological disorders.

Inducible Prostaglandin E Synthase as a Pharmacological Target for Ischemic Stroke.

As the inducible terminal enzyme for prostaglandin E2 (PGE2) synthesis, microsomal PGE synthase-1 (mPGES-1) contributes to neuroinflammation and secondary brain injury after cerebral ischemia via producing excessive PGE2. However, a proof of concept that mPGES-1 is a therapeutic target for ischemic stroke has not been established by a pharmacological strategy mainly due to the lack of drug-like mPGES-1 inhibitors that can be used in relevant rodent models. To this end, we recently developed a series of novel small-molecule compounds that can inhibit both human and rodent mPGES-1. In this study, blockade of mPGES-1 by our several novel compounds abolished the lipopolysaccharide (LPS)-induced PGE2 and pro-inflammatory cytokines interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNF-α) in mouse primary brain microglia. Inhibition of mPGES-1 also decreased PGE2 produced by neuronal cells under oxygen-glucose deprivation (OGD) stress. Among the five enzymes for PGE2 biosynthesis, mPGES-1 was the most induced one in cerebral ischemic lesions. Systemic treatment with our lead compound MPO-0063 (5 or 10 mg/kg, i.p.) in mice after transient middle cerebral artery occlusion (MCAO) improved post-stroke well-being, decreased infarction and edema, suppressed induction of brain cytokines (IL-1ß, IL-6, and TNF-α), alleviated locomotor dysfunction and anxiety-like behavior, and reduced the long-term cognitive impairments. The therapeutic effects of MPO-0063 in this proof-of-concept study provide the first pharmacological evidence that mPGES-1 represents a feasible target for delayed, adjunct treatment - along with reperfusion therapies - for acute brain ischemia.

Assessment of the in vitro toxicity of calixarenes and a metal-seamed calixarene: a chemical pathway for clinical application.

Calixarenes are known to form host-guest complexes and supramolecular nanoassemblies with well-defined architectures. However, the use of these materials in conjunction with drug moieties is still under explored. One reason is the insuffcient biocompatibility studies. Our present study represents a systematic in vitro investigation of the cytotoxicity associated with C-methylresorcin[4]arene, C-methylpyrogallol[4]arene, p-phosphonated calix[8]arene and a metal-seamed calixarene-copper(II) complex, using human HEK293 and rat C6G cell lines and two different cell viability assays (MTT and CellTiter-Glo) to avoid species-biased results. All compounds showed low to moderate toxicity. The trend in the CC50 values indicated that the suppression of the coordination ability and the presence of phosphonate groups decrease the overall cytotoxicity of the compounds. The results of this study not only establish calixarenes and their immediate families as potential drug carriers and drug modifiers, but also reveal a pathway for fine-tuning their toxicological behaviour by appropriate chemical modification.

Small-molecule inhibition of prostaglandin E receptor 2 impairs cyclooxygenase-associated malignant glioma growth.

BACKGROUND AND PURPOSE: An up-regulation of COX-2 in malignant gliomas causes excessive synthesis of PGE2 , which is thought to facilitate brain tumour growth and invasion. However, which downstream PGE2 receptor subtype (i.e., EP1 -EP4 ) directly contributes to COX activity-promoted glioma growth remains largely unknown. EXPERIMENTAL APPROACH: Using a publicly available database from The Cancer Genome Atlas research network, we compared the expression of PGE2 signalling-associated genes in human lower grade glioma and glioblastoma multiforme (GBM) samples. The Kaplan-Meier analysis was performed to determine the relationship between their expression and survival probability. A time-resolved FRET method was used to identify the EP subtype that mediates COX-2/PGE2 -initiated cAMP signalling in human GBM cells. Taking advantage of a recently identified novel selective bioavailable brain-permeable small-molecule antagonist, we studied the effect of pharmacological inhibition of the EP2 receptor on glioma cell growth in vitro and in vivo. KEY RESULTS: The EP2 receptor is a key Gαs -coupled receptor that mediates COX-2/PGE2 -initiated cAMP signalling pathways in human malignant glioma cells. Inhibition of EP2 receptors reduced COX-2 activity-driven GBM cell proliferation, invasion, and migration and caused cell cycle arrest at G0-G1 and apoptosis of GBM cells. Glioma cell growth in vivo was also substantially decreased by post-treatment with an EP2 antagonist in both subcutaneous and intracranial tumour models. CONCLUSION AND IMPLICATIONS: Taken together, our results suggest that PGE2 signalling via the EP2 receptor increases the malignant potential of human glioma cells and might represent a novel therapeutic target for GBM.

Discovery of 2-Piperidinyl Phenyl Benzamides and Trisubstituted Pyrimidines as Positive Allosteric Modulators of the Prostaglandin Receptor EP2.

Prostaglandin E2 (PGE2) via its Gαs-coupled EP2 receptor protects cerebral cortical neurons from excitotoxic and anoxic injury, though EP2 receptor activation can also cause secondary neurotoxicity in chronic inflammation. We performed a high-throughput screen of a library of 292 000 small molecules and identified several compounds that have a 2-piperidinyl phenyl benzamide or trisubstituted pyrimidine core as positive modulators for human EP2 receptor. The most active compounds increased the potency of PGE2 on EP2 receptor 4-5-fold at 20 µM without altering efficacy, indicative of an allosteric mechanism. These compounds did not augment the activity of the other Gαs-coupled PGE2 receptor subtype EP4 and showed neuroprotection against N-methyl-d-aspartate (NMDA)-induced excitotoxicity. These newly developed compounds represent second-generation allosteric potentiators for EP2 receptor and shed light on a promising neuroprotective strategy. They should prove valuable as molecular tools to achieve a better understanding of the dichotomous action of brain EP2 receptor activation.

Simultaneous determination of isomeric substituted anilines by imidization with benzaldehyde and gas chromatography-mass spectrometry.

The chromatographic separation of several isomeric anilines is a challenging issue. Herein, a simple method for the simultaneous determination of four groups of isomeric primary aromatic amines, including chloroanilines, methylanilines, methoxylanilines, and dimethylanilines, was presented. In this method, all of the 15 primary aromatic amines were easily transformed into the corresponding imine derivative by treatment with benzaldehyde under mild conditions. The formed isomeric imine derivatives were completely separated on a commercial capillary gas chromatography column. The effects of several derivatization parameters were investigated and optimized. Linearity in the optimized method ranged from 0.050 to 50 µg/mL with the squared correlation coefficients (R2 ) between 0.9981 and 0.9999. Reasonable reproducibility was obtained, with the intraday relative standard deviation (N = 5) ranging from 0.89 to 4.57% and interday relative standard deviation ranging from 2.26 to 7.69% at the concentration of 5.0 µg/mL. The developed method has been successfully applied to determine these isomeric aromatic amines in real samples.

Cyclooxygenase-2 contributes to oxidopamine-mediated neuronal inflammation and injury via the prostaglandin E2 receptor EP2 subtype.

Cyclooxygenase-2 (COX-2) triggers pro-inflammatory processes that can aggravate neuronal degeneration and functional impairments in many neurological conditions, mainly via producing prostaglandin E2 (PGE2) that activates four membrane receptors, EP1-EP4. However, which EP receptor is the culprit of COX-2/PGE2-mediated neuronal inflammation and degeneration remains largely unclear and presumably depends on the insult types and responding components. Herein, we demonstrated that COX-2 was induced and showed nuclear translocation in two neuronal cell lines - mouse Neuro-2a and human SH-SY5Y - after treatment with neurotoxin 6-hydroxydopamine (6-OHDA), leading to the biosynthesis of PGE2 and upregulation of pro-inflammatory cytokine interleukin-1ß. Inhibiting COX-2 or microsomal prostaglandin E synthase-1 suppressed the 6-OHDA-triggered PGE2 production in these cells. Treatment with PGE2 or EP2 selective agonist butaprost, but not EP4 agonist CAY10598, increased cAMP response in both cell lines. PGE2-initiated cAMP production in these cells was blocked by our recently developed novel selective EP2 antagonists - TG4-155 and TG6-10-1, but not by EP4 selective antagonist GW627368X. The 6-OHDA-promoted cytotoxicity was largely blocked by TG4-155, TG6-10-1 or COX-2 selective inhibitor celecoxib, but not by GW627368X. Our results suggest that PGE2 receptor EP2 is a key mediator of COX-2 activity-initiated cAMP signaling in Neuro-2a and SH-SY5Y cells following 6-OHDA treatment, and contributes to oxidopamine-mediated neurotoxicity.

Prostaglandin E2 Signaling: Alternative Target for Glioblastoma?

Elevated cyclooxygenase-2 (COX-2) and the associated inflammation within the brain contribute to glioblastoma development. However, medical use of COX inhibitors in glioblastoma treatment has been limited due to their well-documented vascular toxicity and inconsistent outcomes from recent human studies. Prostaglandin E2 (PGE2) has emerged as a principal mediator for COX-2 cascade-driven gliomagenesis. Are PGE2 terminal synthases and receptors feasible therapeutic targets for glioblastoma?