Integrated mutagenesis, bio-informatic and fluorescence approaches to characterize the molecular basis of antagonist action at P2X7 receptors for ATP

Damage to cells leads to the release of the chemical ATP, this then acts as a danger signal that is sensed by P2X7 receptors. Binding of ATP to P2X7 receptors on the surface of immune cells triggers their stimulation and an inflammatory response that can contribute to a range of clinical conditions. Blocking P2X7 receptor stimulation can reduce the symptoms of pain, arthritis, Crohn's disease, high blood pressure, as well as kidney injury and damage to the heart following a heart attack. Drugs that block the action of ATP at P2X7 receptors (antagonists) therefore have considerable potential for treating disease and some of these are undergoing clinical trials. However we do not understand where on the receptor these drugs bind. In this study we aim to determine how four chemically distinct types of P2X7 receptor antagonist bind to the receptor.

We have constructed a 3D model of the P2X7 receptor based on the known structure of a related P2X receptor. We then used computer based simulations, with the chemical structure of the drugs and the receptor, to make predictions on where the antagonists could bind to the receptor. These suggested several different solutions for binding that were clustered into two regions on the receptor surface. We therefore propose to test these predictions by generating P2X7 receptors that have been modified by systematically replacing parts of the P2X7 receptor predicted to bind antagonists with the corresponding region from an antagonist insensitive P2X1 receptor. We can inject the cDNA for the mutated P2X receptors into unfertilized frog eggs that then synthesise the receptors, and make electrical recordings from the eggs to measure responses to added ATP and whether this is blocked by the antagonists. If the region that is replaced is important we would expect the mutant P2X7 receptor to have reduced sensitivity to the antagonist, if there is no change in sensitivity this would indicate the variant region that was swapped did not contribute to drug action. Therefore we can use this replacement strategy to identify regions of the receptor that are important for antagonist binding.

P2X7 receptors are made up from amino acid building blocks (there are 20 different types of these that vary in their chemical properties and thus interaction with drugs). In a second round of mutagenesis studies we will determine which amino acids within a region shown to affect antagonist action are involved directly in drug binding. To do this we will systematically mutate individual amino acids to a cysteine residue and characterize receptor properties in frog eggs. If a cysteine mutation reduces antagonist inhibition that will identify an important residue for drug action. The advantage of cysteine is that this amino acid has unique properties in that it can be chemically modified and fluorescently labelled. We can use this fluorescent labelling to measure directly how accessible the introduced cysteine residue is. To do this we will take oocytes expressing the cysteine mutant and treat them with the fluorescent label. We will then determine whether this level of "control" fluorescence is different from that where the receptors have also been treated with the antagonist. If a cysteine residue is part of the drug binding site we expect the antagonist to block accessibility to the cysteine and so reduce fluorescent labelling. These fluorescent methods will thus show which residues directly line the antagonist binding pocket. We will then undertake additional computer modelling and mutagenesis with other amino acid substitutions to determine the exact nature of the chemical interaction between the drug and the P2X7 receptor. This information will help in the improvement of P2X7 receptor drugs and be useful to develop drugs to block the related P2X4 receptor that is involved in pain but for which there are currently no effective blockers.

Activation of P2X7 receptors by ATP released from damaged cells plays a significant role in inflammatory conditions, and the receptor is an important therapeutic target. However we know little about the molecular basis of antagonist action at these receptors. Our pilot mutagenesis and bioinformatics studies have identified potential orthosteric and allosteric binding sites for four chemically distinct P2X7 receptor selective antagonists. We will use an iterative cycle of mutagenesis and in silico molecular docking to test and refine models for binding of each of the P2X7 receptor antagonists. We will initially generate chimeras replacing stretches of residues comprising the predicted antagonist binding site of the human P2X7 receptor with those from the P2X7 receptor antagonist insensitive P2X1 receptor. These will be expressed in Xenopus oocytes and their pharmacological properties characterized electrophysiologically to identify the regions of the receptor important for antagonist action. We will then generate individual cysteine point mutants of variant residues within these regions to identify which residues are important for antagonist action. These data will be used to further refine molecular models and enable us to make predictions about the nature of the interaction between the antagonist and the residues required for high affinity antagonist binding that can be tested with specific mutations. To further substantiate the binding site model(s) the accessibility of introduced cysteine residues will be probed using biochemical methods to determine whether the incorporation of cysteine reactive fluorescence compounds is sensitive to antagonists. In addition voltage clamp fluorometry of labelled P2X7 receptor cysteine mutants adjacent to the antagonist binding site will be used to directly measure the kinetics of drug action. These studies will increase our fundamental knowledge of drug action at this distinct family of ion channels.

The primary impact of our work will be directly to extend knowledge of the molecular basis of antagonist action at P2X receptors. The work has direct relevance to the Academic Community, Pharmaceutical Industry and ultimately the Healthcare sector as there is significant therapeutic interest in P2X receptor drugs. For example P2X7 receptors are involved in a number of disease states including pain, inflammation, multiple sclerosis, Alzheimer's and Parkinson's diseases, glomerulonephritis, hypertension, renal injury as well as damage following acute myocardial infarction. Our work will provide models of antagonist binding that can be used to develop potential novel and more selective antagonists. Development of new P2X receptor drugs through sharing our work/collaborating with the Pharmaceutical Industry could have economic impact on UK based Pharmaceutical companies. Our research therefore has the potential to provide a significant impact in the long term in meeting clinical need, improving therapies for a range of debilitating conditions and contributing to human health and wellbeing.

Our models may be used to predict/develop drugs for other P2X receptor subtypes. This would be of particular relevance in the case of the P2X4 receptor that has been shown to be involved in a range of pain phenotypes and has been suggested as a target for novel forms of pain management. For example, back pain costs the UK economy ~ £5-7 billion a year in sickness leave and treatment costs (British Pain Society/BBC). P2X4 receptor activation has been shown to be a significant contributor to spinal pain following injury and development of selective antagonists to block this receptor could have a major impact on treatment of this condition, improving quality of life but also economic productivity.

The work will be of direct interest to the academic community working on the structure-function, and properties, of the distinct P2X receptor family of ligand gated ion channels. The chimeras and mutants may reveal regions/residues that are determinants of the low agonist sensitivity and distinct properties of the P2X7 receptor that would be of interest to understanding the molecular basis of differences between the P2X receptor subtype properties. An understanding of the molecular interactions in the receptor will also be of relevance to those interested in other ion channels. It is likely that some P2X receptor antagonists bind in an orthosteric way at the ATP binding site. These findings will also be relevant to those interested in other ATP binding proteins, for example, ATP binding at ATP sensitive potassium channels, and ATP action at kinases.

The impact of our work therefore will be initially through the publication of our research findings that will be of interest to the academic community and pharmaceutical industry. In the medium term this may translate into the development of new P2X receptor drugs that have the long term potential to impact on health and wellbeing as well as have economic returns.

The postdoc will benefit from continued scientific training and will receive training in the interpretation of molecular modelling and bioinformatics data. In addition there will be continuing ongoing professional development of presentation skills (oral, written and numerical analysis). The project will also provide training opportunities for MSc students doing 3 months projects on P2X receptor modelling. This will give them direct practical experience as well as training in presentation skills and time management.