Enhancing microbial killing through the novel action of Panax ginseng derivatives on P2X7

通过人参衍生物对 P2X7 的新作用增强微生物杀灭作用

• 批准号: BB/N018427/1
• 负责人: Leanne Stokes
• 金额: $ 51.12万
• 依托单位: University of East Anglia
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN018427%2F1
• 关键词: Enhancing; microbial; killing; through; novel

The immune system is critical for survival, fighting against microbial infections and providing continuous surveillance for abnormal cells in the body. In increasing numbers microbes are developing resistance to therapies designed to kill them directly therefore alternative strategies are required. Understanding how this complex system of immune cells co-ordinate their activities and attack a multitude of pathogens is vital for developing such strategies. One particular alternative to new anti-microbials is to enhance existing physiological mechanisms employed by immune cells to eliminate pathogens. P2X7 is an ion channel found on immune cells such as macrophages. Activation of this channel switches on a number of signalling pathways including a way to kill intracellular pathogens such as mycobacteria tuberculosis (TB), Chlamydia sp. or Toxoplasma gondii. In humans and domestic dogs the P2X7 gene is known to carry inherited mutations in the form of single nucleotide polymorphisms (SNPs) and these can dramatically affect the functioning of this ion channel. There is evidence that macrophages carrying mutations in P2X7 have defective killing of intracellular microbial pathogens. With the rising number of emerging resistant strains of mycobacteria and the global prevalence of other intracellular infections, we propose that understanding the activation of the P2X7 ion channel and developing strategies for modulating anti-microbial signalling pathways in macrophages is of great importance. We have recently discovered a family of natural products derived from the popular traditional Chinese herb Panax ginseng which act as positive allosteric modulators of the P2X7 ion channel. Ginseng is a herb taken by millions of people for a variety of claimed health benefits although much research is still required to understand how the ginsenoside chemicals exert their actions in the body. Our approach will develop synthetic ginsenoside analogues and focus on the major intestinal metabolite of ginsenosides, CK, as modulators of the P2X7 ion channel to enhance microbial killing in macrophages. We have published evidence for a direct interaction between ginsenoside CK and the P2X7 ion channel and we will use a combination of molecular modelling and chemical docking approaches together with site-directed mutagenesis of P2X7 to define the molecular binding site for ginsenosides. We will employ a medicinal chemistry approach to investigate the structure activity relationship for this modulator activity on P2X7. Combined with the mutagenesis approach this will provide detailed knowledge on the first known site for positive modulators on human P2X7. We will use patch clamp and fluorescent imaging assays to probe P2X7 channel activity in heterologous expression systems and in primary macrophages. Furthermore, we will test the hypothesis that enhancing P2X7 activity using ginsenosides will increase microbial killing in macrophages. We have preliminary evidence that ginsenosides can enhance P2X7-dependent cell death in macrophages and will investigate whether this is the major cellular mechanism for microbial killing. We will determine whether ginsenosides can increase killing of intracellular pathogens such as mycobacterium tuberculosis in human macrophages carrying loss-of-function SNPs in the P2X7 gene.

免疫系统对生存至关重要，它可以对抗微生物感染，并为体内的异常细胞提供持续的监测。越来越多的微生物对旨在直接杀死它们的疗法产生了抗药性，因此需要替代策略。了解这个复杂的免疫细胞系统如何协调它们的活动并攻击多种病原体对于开发此类策略至关重要。新的抗微生物药物的一个特别的替代方案是增强免疫细胞消除病原体的现有生理机制。P2 X7是在免疫细胞如巨噬细胞上发现的离子通道。该通道的激活开启了许多信号通路，包括杀死细胞内病原体如结核分枝杆菌（TB）、衣原体或弓形虫的途径。在人类和家犬中，已知P2 X7基因携带单核苷酸多态性（SNP）形式的遗传突变，这些突变可以显著影响该离子通道的功能。有证据表明，携带P2 X7突变的巨噬细胞对细胞内微生物病原体的杀伤有缺陷。随着分枝杆菌耐药菌株数量的不断增加和其他细胞内感染的全球流行，我们建议了解P2 X7离子通道的激活和开发用于调节巨噬细胞中抗微生物信号通路的策略是非常重要的。我们最近发现了一个家族的天然产物，来自流行的传统中药人参，作为积极的别构调节剂的P2 X7离子通道。人参是一种被数百万人服用的草药，具有各种声称的健康益处，尽管仍需要进行大量研究以了解这些化学物质如何在体内发挥作用。我们的方法将开发合成的β-内酰胺类似物，并专注于β-内酰胺类的主要肠道代谢产物CK，作为P2 X7离子通道的调节剂，以增强巨噬细胞中的微生物杀灭作用。我们已经发表了证据证明，直接之间的相互作用的P2 X7和P2 X7离子通道，我们将使用分子建模和化学对接的方法与定点诱变的P2 X7一起定义的分子结合位点的糖苷。我们将采用药物化学方法来研究这种调节剂对P2 X7活性的构效关系。结合诱变方法，这将提供关于人P2 X7上正调节剂的第一个已知位点的详细知识。我们将使用膜片钳和荧光成像分析来探测异源表达系统和原代巨噬细胞中的P2 X7通道活性。此外，我们将测试使用黄芪皂苷增强P2 X7活性将增加巨噬细胞中的微生物杀伤的假设。我们有初步证据表明，黄芪皂苷可以增强巨噬细胞中P2 X7依赖性细胞死亡，并将研究这是否是微生物杀灭的主要细胞机制。我们将确定在携带P2 X7基因功能缺失SNP的人巨噬细胞中，黄芪皂苷是否可以增加对细胞内病原体（如结核分枝杆菌）的杀伤。

项目成果

Oral Communication: Harnessing herbs to potentiate P2X7: elucidating the binding site of ginsenoside CK on P2X7

口头交流：利用草药增强 P2X7：阐明人参皂苷 CK 在 P2X7 上的结合位点

• 发表时间: 2018
• 期刊: PURINERGIC SIGNALLING
• 影响因子: 3.5
• 作者: Bidula S.

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

• DOI: 10.1124/molpharm.120.000129
• 发表时间: 2021-03
• 期刊: Molecular pharmacology
• 影响因子: 3.6
• 作者: Piyasirananda W;Beekman A;Ganesan A;Bidula S;Stokes L
• 通讯作者: Stokes L

Modulating P2X7-which way do we go?

调制P2X7——我们该走哪条路？

• 发表时间: 2018
• 期刊: PURINERGIC SIGNALLING
• 影响因子: 3.5
• 作者: Stokes Leanne

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

使用天然单核细胞和巨噬细胞的全细胞膜片钳记录 P2X 受体。

• DOI: 10.1007/978-1-4939-9717-6_20
• 发表时间: 2020
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Stokes L