Novel pharmacological modulators of P2X7 receptor

Dhuna, K 2019, Novel pharmacological modulators of P2X7 receptor, Doctor of Philosophy (PhD), Health and Biomedical Sciences, RMIT University.

Document type: Thesis
Collection: Theses

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Title Novel pharmacological modulators of P2X7 receptor
Author(s) Dhuna, K
Year 2019
Abstract Inflammation is a biological response invoked by the body when challenged by harmful stimuli such as pathogens, injury or irritants. Inflammatory responses can be classified as being acute or chronic. In cases where the acute inflammatory response is unable to eliminate the infection or resolve the damage, the characteristics of the immune response are altered. The neutrophil response is supplanted by monocyte-derived macrophages and if there is infection, T cells from the adaptive immune system are employed as well. If the infection persists, a state of chronic inflammation ensues. This is accompanied by continued production of pro-inflammatory cytokines. The chronic inflammatory response may cause tissue destruction due to the production of reactive oxygen species (ROS) and hydrolytic enzymes. The over-production of pro-inflammatory cytokines contributes to the pathophysiology of many diseases such as heart disorders, cancer, obesity, stroke, chronic respiratory diseases and diabetes. According to the World Health Organisation (WHO), the chronic inflammatory diseases are the major cause of deaths in the world. According to the Australian Institute of Health and Welfare, 50% of Australians were reported having 1 of 8 chronic inflammatory diseases is high. The economic impact of chronic inflammatory disease is high. For example, the direct healthcare costs incurred by patients of chronic obstructive pulmonary disorders in the year 2010 were calculated to be approximately $50 million. There is a growing need to identify molecular targets (enzymes and receptors) which are key regulators of inflammatory pathways and to develop approaches to modulate them in order to attenuate inflammatory damage. Previous studies have indicated significant involvement of adenosine triphosphate in initiating and regulating inflammatory pathways.

Extracellular ATP (eATP) is involved in activating inflammatory responses and is  considered as an acute `danger signal¿ or DAMP (Danger Associated Molecular Pattern). eATP can bind to and activate purinergic receptors. Purinergic receptors, also known as purinoceptors, are a family of plasma membrane complexes that are found in almost all mammalian tissues and are activated by purines. P2X7, a trimeric purinergic ion channel is commonly expressed on immune cells such as microglia, monocytes, macrophages and dendritic cells. P2X7 is characterised by the formation of a large non-selective membrane pore following prolonged exposure to its ligand ATP, which allows the passage of small organic molecules (up to 900 Da in size) as well as high calcium flux into the cells. P2X7 regulates the maturation and secretion of inflammatory cytokines such as IL-1ß and IL-18. Alteration in function and expression of P2X7 has been implicated in the development and progression of many disease types such as neurodegenerative disorders, cancer, neuropathic and inflammatory pain and pulmonary fibrosis. Inhibition of receptor or deletion of the P2X7 gene has shown a down-regulation of inflammatory pathways. Thus, the P2X7 receptor is a potential drug target for treating inflammation-based disorders. Identification and characterisation of small molecular weight modulators for this receptor are important for establishing effective treatments for inflammation-related disorders. Several pharmaceutical companies (GSK, AstraZeneca, and Evotech) have been developing novel antagonists for the past decade and some of them have entered clinical trials for treatment of rheumatoid arthritis.

The project described in this thesis explores existing compounds that have been demonstrated to have pharmacological actions on P2X7-dependent signalling pathways and characterises the effects of two unrelated groups of compounds found to have pharmacological action on P2X7, namely, tetracycline antibiotics and ginsenosides derived from the Chinese herb ginseng.
Minocycline is a broad-spectrum antibiotic, which has been reported to have anti-inflammatory properties independent of its anti-microbial properties. Minocycline is an endogenous microglial inhibitor. In this study, it was observed that by inhibiting P2X7 responses, minocycline inhibited the influx of Ca2+ ions, and hence decreased pro-inflammatory cytokines secretion, ROS generation and subsequent cell death. These results provided evidence that the anti-inflammatory action of minocycline is due, in part, to blocking the P2X7 receptor.

Panax ginseng is a Chinese herb which has been used traditionally for treatment of anaemia, diabetes, inflammation of the stomach lining (gastritis), fever, chronic obstructive pulmonary disease (COPD), and asthma, all of which are diseases where resolution of inflammation or immune responses are compromised. The main bioactive components among these are ginseng saponins, also known as ginseng glycosides or ginsenosides. Previous work in our laboratory has indicated that specific ginsenosides potentiate P2X7 responses. In this study, selected ginsenosides (CK, Rd, Rb1 and Rh2) were shown to act as positive allosteric regulators of P2X7 receptors in HEK-293 cells overexpressing P2X7.  Potentiation of channel activity by these ginsenosides was found to translate to physiological responses mediated by P2X7 in both J774 macrophages and primary rat macrophages. These results indicate that ginsenosides may re-sensitize the immune system in individuals with sub-optimal immune responses by potentiating P2X7 responses in macrophages.

Overall, this thesis describes experiments which comprehensively characterise two unrelated classes of compounds; semi-synthetic tetracycline antibiotics and Panax ginseng derived ginsenosides, as effective pharmacological modulators of P2X7. Due to the involvement of P2X7 in numerous diseases, there is a mounting need to characterise physiological modulators of P2X7, which can act as tools to unravel the exact mechanism by which this receptor regulates inflammatory pathways. The information gathered with the help of these physiological tools may assist in designing safe, specific and selective drugs to target the P2X7 receptor and thereby develop novel treatments for inflammatory disorders.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Health and Biomedical Sciences
Subjects Immunology not elsewhere classified
Basic Pharmacology
Keyword(s) P2X7
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Created: Wed, 24 Jul 2019, 09:40:29 EST by Adam Rivett
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