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

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.

Chemicals derived from Panax ginseng are known to be pharmacologically active both in vivo and in vitro. This project will provide evidence for a direct interaction of ginsenosides with an ion channel expressed on immune cells and will explore the use of ginsenosides in modulating macrophage responses to intracellular bacteria.

Our first objective is to map a molecular binding site for ginsenosides on the P2X7 trimeric structure using a 3D homology model. Docking of the ginsenoside lead compound to the open P2X7 channel predicts an interaction with all three subunits at particular amino acid residues. These residues will be mutated to address whether the potentiating activity of ginsenosides is lost when the potential for hydrogen bonding is removed. A standard screening assay for P2X7 will be used measuring fluorescent dye uptake through the secondary permeability pathway. Patch clamp electrophysiology will also be employed to measure direct ion channel responses.

Secondly, the project will provide a detailed investigation into the structural requirements on the chemistry of the ginsenosides. A range of substitutions will be tested at key carbon atoms within the lead compound, ginsenoside CK, to determine the size and charge requirements for interaction with P2X7. In the third objective we will investigate whether enhancing apoptosis through P2X7 is the mechanism employed by macrophages to kill intracellular bacteria such as mycobacterium tuberculosis. The use of a P2X7-deficient J774 cell line through CRISPR-mediated gene editing will validate P2X7 as the receptor target for ginsenoside CK in macrophages. Killing of intracellular mycobacteria (BCG or tuberculosis) will be performed in human macrophages and we will address whether the ginsenoside CK can increase responses in macrophages carrying loss-of-function mutations of P2X7 thus rescuing a defective bacterial killing mechanism.

An alternative strategy for combatting antimicrobial resistance is to develop ways to enhance key physiological processes implemented by immune cells to kill pathogens. This project will contribute to the BBSRC responsive mode priority for microbial resistance by investigating whether the novel approach of enhancing P2X7-dependent apoptosis will result in increased microbial killing. We will enhance this process through plant-derived chemicals known to have immune system activity. This work may lead to the development of a new class of immunomodulators acting on P2X7.

Advances in scientific knowledge
This project will further academic knowledge on purinergic receptors and their important roles in immune cell signalling pathways which will foster future research projects and academic collaboration. High quality research publications in leading international journals and presentations at national/international conferences are critical for delivery of outcomes from the project. The work engages both national and international experts and the development and strengthening of such collaborations will increase the global impact of our findings.

Commercial benefits
The goal of this project is to provide mechanistic evidence for positive allosteric modulators enhancing bacterial killing via P2X7. Through our medicinal chemistry approach we will generate a library of synthetic ginsenosides, some of which may produce intellectual property and identify lead compound(s) for further development in the commercial biotech industry. We anticipate potential lead compounds will require proof-of-principle in vivo testing and this may attract future R&D investment from industrial or research council sources. Identification of potential industrial partners will be developed during this project.

Social impact
Many herbal products and supplements are widely available to the general public with little scientific evidence about their reported actions. This project will provide evidence regarding the pharmacological effects of purified chemicals from Panax ginseng (ginsenosides) and we will report our findings to the public in different formats (such as media reports, social media alerts, public lectures, school outreach programs, PI lab website). There is a real need to increase awareness of herbal products and plant derived chemicals and their effects in the body and we hope to contribute to community education on the value of natural products.

Training and development
The project will employ a postdoctoral researcher and contribute to their professional training and development in a variety of technical, academic and engagement skills. Opportunities for undergraduate students at UEA will be offered through short laboratory research projects. This would contribute to the training and development of future pharmacists and biologists with research techniques and general presentation, writing and data analysis skills.