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

免疫系统对生存、对抗微生物感染和持续监测体内异常细胞至关重要。越来越多的微生物对旨在直接杀死它们的疗法产生抗药性，因此需要替代策略。了解这种复杂的免疫细胞系统是如何协调它们的活动并攻击多种病原体的，对于开发这样的策略至关重要。新型抗微生物药物的一个特殊替代方案是增强免疫细胞用来消除病原体的现有生理机制。P2X7是一种存在于巨噬细胞等免疫细胞上的离子通道。这一通道的激活开启了许多信号通路，包括一种杀死细胞内病原体的方法，如结核分枝杆菌(TB)、衣原体(Chlamydia Sp)。或者弓形虫。在人类和家犬中，已知P2X7基因携带单核苷酸多态(SNPs)形式的遗传突变，这些突变可以极大地影响这种离子通道的功能。有证据表明，携带P2X7突变的巨噬细胞对细胞内病原体的杀灭存在缺陷。随着新出现的分枝杆菌耐药菌株数量的增加和其他细胞内感染的全球流行，我们认为了解P2X7离子通道的激活和开发策略来调节巨噬细胞中的抗微生物信号通路具有重要意义。我们最近发现了一系列从流行的中草药人参中提取的天然产物，它们是P2X7离子通道的正性变构调节剂。人参是一种被数百万人服用的草药，声称对健康有多种益处，尽管仍需进行大量研究，以了解人参皂苷化学物质如何在人体内发挥作用。我们的方法将开发合成人参皂苷类似物，并将重点放在人参皂苷的主要肠道代谢物CK，作为P2X7离子通道的调节剂，以增强巨噬细胞中的微生物杀伤。我们已经发表了人参皂苷CK与P2X7离子通道之间直接相互作用的证据，我们将结合分子模拟和化学对接方法以及P2X7的定点突变来确定人参皂苷的分子结合部位。我们将使用药物化学的方法来研究该调节剂在P2X7上的活性的构效关系。结合突变方法，这将为人类P2X7上第一个已知的正调节剂位点提供详细的知识。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P2X7通道活性。此外，我们将验证使用人参皂苷增强P2X7活性将增加巨噬细胞中微生物杀伤力的假设。我们有初步证据表明，人参皂苷可以促进巨噬细胞中依赖于P2X7的细胞死亡，并将探讨这是否是微生物死亡的主要细胞机制。我们将确定人参皂苷是否可以增加携带P2X7基因功能缺失SNPs的人巨噬细胞对细胞内病原体的杀伤力，如结核分枝杆菌。

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.