Screening herbal and natural product libraries to aid discovery of novel allosteric modulators of human P2X7.

 P2X7 is an emerging therapeutic target for several disorders and diseases due to its role in inflammatory signalling. This study aimed to exploit the unique chemical libraries of plants used in traditional medicinal practices to discover novel allosteric modulators from natural sources. We identified several compounds from the NCI Natural Product library as P2X7 antagonists including confertifolin and digallic acid (IC values 3.

Anti-Inflammatory Activities of Yataprasen Thai Traditional Formulary and Its Active Compounds, Beta-Amyrin and Stigmasterol, in RAW264.7 and THP-1 Cells.

Yataprasen (YTPS) remedy formulary, a national Thai traditional medicine formulary, comprises 13 herbal plants. It has been extensively prescribed to relieve osteoarthritis and musculoskeletal pain in the Thai traditional medicine healthcare system. The aim of this study was to investigate the antioxidant and anti-inflammatory properties of the bioactive compounds (β-amyrin and stigmasterol) of YTPS remedy formulary ethanolic extract, along with its composition.

Methods for studying P2X4 receptor ion channels in immune cells.

The P2X4 receptor is a trimeric ligand-gated ion channel activated by adenosine 5'-triphosphate (ATP). P2X4 is present in immune cells with emerging roles in inflammation and immunity, and related disorders. This review aims to provide an overview of the methods commonly used to study P2X4 in immune cells, focusing on those methods used to assess P2RX4 gene expression, the presence of the P2X4 protein, and P2X4 ion channel activity in these cells from humans, dogs, mice and rats.

Ginsenosides enhance P2X7-dependent cytokine secretion from LPS-primed rodent macrophages.

The activation of P2X7 is a well-known stimulus for the NLRP3-caspase 1 inflammasome and subsequent rapid IL-1β secretion from monocytes and macrophages. Here we show that positive allosteric modulators of P2X7, ginsenosides, can enhance the release of three important cytokines, IL-1β, IL-6 and TNF-α from LPS-primed rodent macrophages using the J774 mouse macrophage cell line and primary rat peritoneal macrophages. We compared the immediate P2X7 responses in un-primed and LPS-primed macrophages and found no difference in calcium response amplitude or kinetics.

P2X receptors: Insights from the study of the domestic dog.

Fifty years ago, the late Geoffrey Burnstock described the concept of purinergic nerves and transmission bringing into existence the broader concepts of purinergic signaling including P2X receptors. These receptors are trimeric ligand-gated cation channels activated by extracellular adenosine 5'-triphosphate (ATP). P2X receptors have important roles in health and disease and continue to gain interest as potential therapeutic targets in inflammatory, neurological, cardiovascular and many other disorders including cancer.

P2X receptor antagonists and their potential as therapeutics: a patent review (2010-2021).

Introduction: Purinergic receptors play a critical role in neurotransmission, and modulation of complex physiological functions and thus have implications in numerous disease states. The past decade has seen substantial progress in the design of novel chemical compounds that act on the P2X class of receptors and warrants an updated review of this field.

Areas Covered: This review provides a summary of the patent literature describing the discovery and clinical uses of P2X receptor antagonists published between 2010 and September 2021.

Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors.

P2X7 is an important ligand-gated ion channel expressed in multiple immune cell populations. This study aimed to investigate the chemical requirements of triterpenoid glycosides within a new binding pocket to characterize the structure-activity relationship. A set of glycosides were screened for positive modulator activity at human P2X7 using a YO-PRO-1 dye uptake assay in HEK-293 cells stably expressing the wild-type human P2X7 variant (HEK-hP2X7 cells).

Revisiting the Idea That Amyloid-β Peptide Acts as an Agonist for P2X7.

The P2X7 receptor (P2X7) is a cell surface ligand-gated ion channel, activated by its physiological nucleotide agonist ATP and a synthetic analog (BzATP). However, it has also been suggested that there may be structurally unrelated, non-nucleotide agonists such as the amyloidogenic β peptide. Here we aimed to reassess the effect of amyloid β peptides in various cell models, namely HEK293 overexpressing human P2X7, the microglial BV-2 cell line, and BV-2 cells lacking P2X7.

To Inhibit or Enhance? Is There a Benefit to Positive Allosteric Modulation of P2X Receptors?

The family of ligand-gated ion channels known as P2X receptors were discovered several decades ago. Since the cloning of the seven P2X receptors (P2X1-P2X7), a huge research effort has elucidated their roles in regulating a range of physiological and pathophysiological processes. Transgenic animals have been influential in understanding which P2X receptors could be new therapeutic targets for disease.

Pharmacological and genetic characterisation of the canine P2X4 receptor.

Background And Purpose: P2X4 receptors are emerging therapeutic targets for treating chronic pain and cardiovascular disease. Dogs are well-recognised natural models of human disease, but information regarding P2X4 receptors in dogs is lacking. To aid the development and validation of P2X4 receptor ligands, we have characterised and compared canine and human P2X4 receptors.

A P2RX7 single nucleotide polymorphism haplotype promotes exon 7 and 8 skipping and disrupts receptor function.

P2X7 is an ATP-gated membrane ion channel that is expressed by multiple cell types. Brief exposure to ATP induces the opening of a nonselective cation channel; while repeated or prolonged exposure induces formation of a transmembrane pore. This process may be partially regulated by alternative splicing of full-length P2RX7A pre-mRNA, producing isoforms that delete or retain functional domains.

Bombesin receptor-targeted liposomes for enhanced delivery to lung cancer cells.

Gastrin-releasing peptide is a member of the bombesin family of peptides. Its cognate receptor, gastrin releasing peptide receptor (GRPR), is widely expressed in cancers of the lung, pancreas and ovaries. Gastrin releasing peptide (GRP) is an autocrine growth factor in small cell lung cancer, which has very poor patient outcomes.

Positive allosteric modulation of P2X7 promotes apoptotic cell death over lytic cell death responses in macrophages.

P2X7 is an ATP-gated ion channel that is highly expressed by leukocytes, such as macrophages. Here, P2X7 has been demonstrated to be involved in the regulation of various cell death pathways; including apoptosis, pyroptosis, necrosis, and autophagy. However, cell death induction via P2X7 is complex and is reliant upon the nature of the stimulus, the duration of the stimulus, and the cell type investigated.

Recording P2X Receptors Using Whole-Cell Patch Clamp from Native Monocytes and Macrophages.

Investigating ion channels in their native cell type is important when striving to understand their regulation and function, but this comes with added complexities due to the plethora of channels and receptors present. Details of recording ATP-gated ion channels in macrophages are presented together with information on how to prepare the primary cells for electrophysiological analysis.

Development of High-Throughput Fluorescent-Based Screens to Accelerate Discovery of P2X Inhibitors from Animal Venoms.

Animal venoms can play an important role in drug discovery, as they are a rich source of evolutionarily tuned compounds that target a variety of ion channels and receptors. To date, there are six FDA-approved drugs derived from animal venoms, with recent work using high-throughput platforms providing a variety of new therapeutic candidates. However, high-throughput methods for screening animal venoms against purinoceptors, one of the oldest signaling receptor families, have not been reported.

Mapping a novel positive allosteric modulator binding site in the central vestibule region of human P2X7.

P2X7 receptors are important in the regulation of inflammatory responses and immune responses to intracellular pathogens such as Mycobacterium tuberculosis and Toxoplasma gondii. Enhancement of P2X7 receptor responses may be useful in pathogen clearance particularly in individuals with defective microbial killing mechanisms. Ginsenosides from Panax ginseng have been discovered to act as positive allosteric modulators of P2X7.

Ginsenosides Act As Positive Modulators of P2X4 Receptors.

We investigated the selectivity of protopanaxadiol ginsenosides from acting as positive allosteric modulators on P2X receptors. ATP-induced responses were measured in stable cell lines overexpressing human P2X4 using a YOPRO-1 dye uptake assay, intracellular calcium measurements, and whole-cell patch-clamp recordings. Ginsenosides CK and Rd were demonstrated to enhance ATP responses at P2X4 by ∼twofold, similar to potentiation by the known positive modulator ivermectin.

Correlation between thyroidal and peripheral blood total T cells, CD8 T cells, and CD8 T- regulatory cells and T-cell reactivity to calsequestrin and collagen XIII in patients with Graves' ophthalmopathy.

Unlabelled: Purpose/aim of the study: Graves' ophthalmopathy (GO) is closely related to the thyroid autoimmune disorder Graves' disease. Previous studies have suggested roles for thyroidal CD8 T cells and autoimmunity against calsequestrin-1 (CASQ)-1 in the link between thyroidal and orbital autoimmune reactions in GO. A role for autoimmunity against CollXIII has also been suggested.

Probenecid directly impairs activation of the canine P2X7 receptor.

The current study aimed to determine if probenecid could directly impair the canine P2X7 receptor, a ligand-gated cation channel activated by extracellular adenosine 5'-triphosphate (ATP). Patch clamp measurements demonstrated that probenecid impairs ATP-induced inward currents in HEK-293 cells expressing canine P2X7. Flow cytometric measurements of ethidium uptake into HEK-293 cells expressing canine P2X7 showed that probenecid impairs ATP-induced pore formation in a concentration-dependent manner, with a half maximal inhibitory concentration of 158 µM.

Rac1 plays a role in CXCL12 but not CCL3-induced chemotaxis and Rac1 GEF inhibitor NSC23766 has off target effects on CXCR4.

Cell migration towards a chemotactic stimulus relies on the re-arrangement of the cytoskeleton, which is triggered by activation of small G proteins RhoA, Rac1 and Cdc42, and leads to formation of lamellopodia and actin polymerisation amongst other effects. Here we show that Rac1 is important for CXCR4 induced chemotaxis but not for CCR1/CCR5 induced chemotaxis. For CXCL12-induced migration via CXCR4, breast cancer MCF-7 cells are reliant on Rac1, similarly to THP-1 monocytes and Jurkat T-cells.

Pharmacological Evaluation of Novel Bioisosteres of an Adamantanyl Benzamide P2X Receptor Antagonist.

Adamantanyl benzamide 1 was identified as a potent P2XR antagonist but failed to progress further due to poor metabolic stability. We describe the synthesis and SAR of a series of bioisosteres of benzamide 1 to explore improvements in the pharmacological properties of this lead. Initial efforts investigated a series of heteroaromatic bioisosteres, which demonstrated improved physicochemical properties but reduced P2XR antagonism.

P2X4 Receptor Function in the Nervous System and Current Breakthroughs in Pharmacology.

Adenosine 5'-triphosphate is a well-known extracellular signaling molecule and neurotransmitter known to activate purinergic P2X receptors. Information has been elucidated about the structure and gating of P2X channels following the determination of the crystal structure of P2X4 (zebrafish), however, there is still much to discover regarding the role of this receptor in the central nervous system (CNS). In this review we provide an overview of what is known about P2X4 expression in the CNS and discuss evidence for pathophysiological roles in neuroinflammation and neuropathic pain.

Are you okay to drive? Commuting behavior and blood alcohol concentrations among restaurant diners.

Objective: Drink driving is widely recognized as a major road safety problem. In Australia, health promotion messages encourage monitoring the number of standard drinks consumed prior to driving. This pilot research aimed to investigate commuting behavior and blood alcohol concentration (BAC) of diners, including intended drivers, at Sunshine Coast restaurants.

Store-Operated Ca Entry (SOCE) and Purinergic Receptor-Mediated Ca Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation.

Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells.