Current Medicinal Chemistry - Volume 22, Issue 7, 2015

The publication of the first crystal structures of the zebrafish P2X4 receptor in 2009 was a pivotal moment; for the first time, researchers were able to interpret their experimental data in a structural context. Several research groups immediately set about using the data to make molecular models of the betterunderstood mammalian P2X receptors, in order to design and interpret the results of new, more focused structure- function experiments. In 2012, the publication of the crystal structure of zebrafish P2X4 in the ATPbound state gave further insights into the mechanism of ligand binding and its coupling to ion channel activation, initiating a new cycle of modelling, experimentation and interpretation. The purpose of this review is to describe our current understanding of the 3D-structure of P2X receptors, by highlighting the strengths and limitations of the zebrafish P2X4 crystal structures, discussing how the molecular models derived from them have been made, and what they have been used for, and explaining why crystal structures of mammalian P2X receptors are still needed to uncover the molecular mechanisms of differential agonist/antagonist potency, allosteric modulation, pore dilatation and desensitisation.

P2X receptors constitute a seven-member family (P2X1-7) of extracellular ATP-gated cation channels of widespread expression. Because P2X receptors have been implicated in neurological, inflammatory and cardiovascular diseases, they constitute promising drug targets. Since the first P2X cDNA sequences became available in 1994, numerous site-directed mutagenesis studies have been conducted to disclose key sites of P2X receptor function and oligomerization. The publication of the 3-A crystal structures of the zebrafish P2X4 (zfP2X4) receptor in the homotrimeric apo-closed and ATP-bound open states in 2009 and 2012, respectively, has ushered a new era by allowing for the interpretation of the wealth of molecular data in terms of specific three-dimensional models and by paving the way for designing more-decisive experiments. Thanks to these structures, the last five years have provided invaluable insight into our understanding of the structure and function of the P2X receptor class of ligandgated ion channels. In this review, we provide an overview of mutagenesis studies of the pre- and post-crystal structure eras that identified amino acid residues of key importance for ligand binding, channel gating, ion flow, formation of the pore and the channel gate, and desensitization. In addition, the sites that are involved in the trimerization of P2X receptors are reviewed based on mutagenesis studies and interface contacts that were predicted by the zfP2X4 crystal structures.

Potent actions of ATP in the central nervous system (CNS) were reported in the late 1940’s, but cloning and characterisation of receptors for purines and pyrimidines did not take place until the early 1990’s, which identified seven P2X ion channel receptor subtypes, three of which form the cation channel as homomultimers or heteromultimers. P2X receptor subtypes are widely expressed in the CNS and their distribution is described in different regions. They function in synaptic cotransmission and neuromodulation, as well as in trophic signalling. ATP released from nerves and astroglial cells are predominantly involved in neuron-glial interactions. Purinergic signalling is involved in normal behaviour, including learning and memory, sleep and arousal, locomotor and feeding activities and cognition. P2X receptors participate in CNS pathophysiology, including injury, inflammation, Alzheimer’s and Parkinson’s diseases, multiple sclerosis and amyotrophic lateral sclerosis, depression and anxiety. P2X4 and P2X7 receptor antagonists are effective via microglia against neuropathic pain, while P2X3 receptor antagonists also reduce neuropathic pain, but via a differernt mechanism.

Pain represents a very large social and clinical problem since the current treatment provides insufficient pain relief. Plasticity of pain receptors together with sensitisation of sensory neurons, and the role of soluble mediators released from non-neuronal cells render difficult to understand the spatial and temporal scale of pain development, neuronal responses and disease progression. In pathological conditions, ATP is one of the most powerful mediators that activates P2X receptors that behave as sensitive ATP-detectors, such as neuronal P2X3 receptor subtypes and P2X4 and P2X7 receptors expressed on non-neuronal cells. Dissecting the molecular mechanisms occurring in sensory neurons and in accessory cells allows to design appropriate tissue- and cell- targeted approaches to treat chronic pain.

This review considers the expression and roles of P2X receptors in the cardiovascular system in health and disease and their potential as therapeutic targets. P2X receptors are ligand gated ion channels which are activated by the endogenous ligand ATP. They are formed from the assembly of three P2X subunit proteins from the complement of seven (P2X1-7), which can associate to form homomeric or heteromeric P2X receptors. The P2X1 receptor is widely expressed in the cardiovascular system, being located in the heart, in the smooth muscle of the majority of blood vessels and in platelets. P2X1 receptors expressed in blood vessels can be activated by ATP coreleased with noradrenaline as a sympathetic neurotransmitter, leading to smooth muscle depolarisation and contraction. There is evidence that the purinergic component of sympathetic neurotransmission is increased in hypertension, identifying P2X1 receptors as a possible therapeutic target in this disorder. P2X3 and P2X2/3 receptors are expressed on cardiac sympathetic neurones and may, through positive feedback of neuronal ATP at this prejunctional site, amplify sympathetic neurotransmission. Activation of P2X receptors expressed in the heart increases cardiac myocyte contractility, and an important role of the P2X4 receptor in this has been identified. Deletion of P2X4 receptors in the heart depresses contractile performance in models of heart failure, while overexpression of P2X4 receptors has been shown to be cardioprotective, thus P2X4 receptors may be therapeutic targets in the treatment of heart disease. P2X receptors have been identified on endothelial cells. Although immunoreactivity for all P2X1-7 receptor proteins has been shown on the endothelium, relatively little is known about their function, with the exception of the endothelial P2X4 receptor, which has been shown to mediate endothelium-dependent vasodilatation to ATP released during shear stress. The potential of P2X receptors as therapeutic targets in the treatment of cardiovascular disease is discussed.

Until recently, P2X receptors have not received much attention in the context of immunology and inflammation. While this is justified to a certain extent for P2X1, P2X2, P2X3, P2X5 and P2X6, which still await identification of a convincing role in the pathophysiology of immune cells, it is clearly not any more the case for P2X4 and even more so for P2X7, a molecule that has achieved the status of an essential, nonredundant, immunomodulatory receptor. In this review I will highlight the most important inflammatory responses participated by P2X receptors.

Tumor microenvironment composition strongly conditions cancer growth and progression, acting not only at cancer itself but also modifying its interactions with immune, endothelial and nervous cells. Extracellular ATP and its receptors recently gained increasing attention in the oncological field. ATP accumulates in cancer milieu through spontaneous release, tumor necrosis or chemotherapy exerting a trophic activity on cancer cells, modulating the cross talk among tumor, and surrounding tissues. Accordingly, ATP gated P2X receptors emerged as central players in tumor development, invasion, progression and related symptoms. Indeed, P2X receptors are expressed and are functional not only on tumor cells but also in immune-infiltrate and nearby neurons. In this review, we summarize recent findings on P2X receptors role in tumor cell differentiation, bioenergetics, angiogenesis, metastasis and associated pain, giving an outline of the potential anti-neoplastic activity of receptor agonists and antagonists.

Diabetes is a metabolic disorder characterized by elevation of glucose levels in the blood that develops in humans as a result of genetic predisposition and environmental factors. Unbalanced glycemic control has been associated with the development of progressive and debilitating complications that dramatically affect the quality of life and life expectancy of people with diabetes. The purinergic system represents a widely diffused signaling pathway in mammalian cells of different tissues where it plays critical roles in both physiological and pathological conditions. Herein we review the increasing evidence supporting that the purinergic system plays an important role in the multiple facets of diabetes, including its physiopathology and complications. We also discuss the potential relevance of the purinergic pathway for diagnosis and treatment of diabetes.

Bone is a highly dynamic organ, being constantly modeled and remodeled in order to adapt to the changing need throughout life. Bone turnover involves the coordinated actions of bone formation and bone degradation. Over the past decade great effort has been put into the examination of how P2X receptors regulate bone metabolism and especially for the P2X7 receptor an impressive amount of evidence has now documented its expression in osteoblasts, osteoclasts, and osteocytes as well as important functional roles in proliferation, differentiation, and function of the cells of bone. Key evidence has come from studies on murine knockout models and from pharmacologic studies on cells and animals. More recently, the role of P2X receptors in human bone diseases has been documented. Loss-of-functions polymorphisms in the P2X7 receptorare associated with bone loss and increased fracture risk. Very recently a report from a genetic study in multiple myeloma demonstrated that decreased P2X7 receptor function was associated with increased risk of developing multiple myeloma. In contrast, the risk of developing myeloma bone disease and subsequent vertebral fractures was increased in subjects carrying P2X7 receptor gain-of-function alleles as compared to subjects only carrying loss-of-function or normal functioning alleles. It is evident that P2X receptors are important in regulating bone turnover and maintaining bone mass, and thereby holding great potential as novel drug targets for treatment of bone diseases. However, further research is needed before we fully understand the roles and effects of P2X receptors in bone.

In this work, we have highlighted data reported in the literature trying to draw a complete picture of the structures and biological activity of agonists and orthosteric antagonists of P2X receptors. Actually, only few P2X receptor agonists have been found and most of them are derived from modification of the natural ligand ATP and they are P2X receptor subtype unselective. In particular, BzATP (9) is one of the most potent P2X receptor agonists with EC50 value in the nanomolar range at some subtypes. Differently from agonists, P2X receptor antagonists belong to different chemical classes such as high molecular weight aryl polysulfonate molecules like suramin and its simplified derivatives and anthraquinone compounds. All these molecules proved to be non selective at P2X receptors, and they are endowed with micromolar activity and not favourable pharmacokinetic properties due to the presence of several charged groups. Also modification of the natural ligand ATP led to the discovery of P2X receptor antagonists like TNP-ATP (29), which, although not selective, showed high potency at P2X1, P2X3 (IC50 of 0.006 µM and 0.001 µM, respectively), and heteromeric P2X2/3 receptors. Also the dinucleotide inosine polyphosphate Ip5I (33) was found to be a potent and selective antagonist at P2X1 vs P2X3 receptors with IC50 = 0.003 µM. A significant improvement has been gained from the interest of pharmaceutical companies that in the last years discovered, through the use of high-throughput screening, potent and selective antagonists endowed with novel structures, some of which are currently in clinical trials for several therapeutic applications.

P2X receptors are trimeric ligand-gated ion channels whose potential as novel drug targets for a number of diseases has been recognized. They are mainly involved in inflammatory processes, including neuroinflammation, and pain sensation. The orthosteric binding site is lined by basic amino acid residues that bind the negatively charged agonist ATP. Therefore it is not easy to develop orthosteric ligands that possess drug-like properties for such a highly polar binding site. However, ligand-gated ion channels offer multiple additional binding sites for allosteric ligands, positive or negative allosteric modulators enhancing or blocking receptor function. So far, the P2X3 (and P2X2/3), as well as the P2X7 receptor subtype have been the main focus of drug development efforts. A number of potent and selective allosteric antagonists have been developed to block these receptors. We start to see the development of novel allosteric ligands also for the other P2X receptor subtypes, P2X1, P2X2 and especially P2X4. The times when only poor, non-selective, non-drug-like tools for studying P2X receptor function were available have been overcome. The first clinical studies with allosteric P2X3 and P2X7 antagonists suggest that P2X therapeutics may soon become a reality.