Antimalarial and Plasmodium falciparum serpentine receptor 12 targeting effect of FDA approved purinergic receptor antagonist.

Intraerythrocytic stages of Plasmodium falciparum responsible for all clinical manifestations of malaria are regulated by array of signalling cascades that represent attractive targets for antimalarial therapy. G-protein coupled receptors (GPCRs) are druggable targets in the treatment of various pathological conditions, however, there is limited understanding about the role of GPCRs in malaria pathogenesis. In Plasmodium, serpentine receptors (PfSR1, PfSR10, PfSR12 and PfSR25) with GPCR-like membrane topology have been reported with the finite knowledge about their potential as antimalarial targets. We analyzed the localization of these receptors in malaria parasite by immunofluorescence assays. All four receptors were expressed in blood stages with PfSR12 expressing more in late intraerythrocytic stages. Further, we evaluated the druggability of PfSR12 using FDA-approved P2Y purinergic receptor antagonist, Prasugrel and its active metabolite R138727, which is proposed to be specific towards PfSR12. Interestingly, biophysical analysis indicated strong binding between PfSR12 and R138727 as compared to the prodrug Prasugrel. This binding interaction was further confirmed by thermal shift assay. Treatment of parasite with Prasugrel and R138727 resulted in growth inhibition of P. falciparum indicating an important role of purinergic signalling and PfSR12 in parasite survival. Next, progression studies indicated the inhibitory effect of Prasugrel begins in late erythrocyte stages corroborating with PfSR12 expression at these stages. Furthermore, Prasugrel also blocked in vivo growth of malaria parasite in a mouse experimental model. This study indicates the presence of P2Y type of purinergic signalling in growth and development of malaria parasite and suggests PfSR12, putative purinergic receptor druggability through Prasugrel.Communicated by Ramaswamy H. Sarma.

Purinergic receptor ligands: the cytokine storm attenuators, potential therapeutic agents for the treatment of COVID-19.

The coronavirus disease-19 (COVID-19), at first, was reported in Wuhan, China, and then rapidly became pandemic throughout the world. Cytokine storm syndrome (CSS) in COVID-19 patients is associated with high levels of cytokines and chemokines that cause multiple organ failure, systemic inflammation, and hemodynamic instabilities. Acute respiratory distress syndrome (ARDS), a common complication of COVID-19, is a consequence of cytokine storm. In this regard, several drugs have been being investigated to suppress this inflammatory condition. Purinergic signaling receptors comprising of P1 adenosine and P2 purinoceptors play a critical role in inflammation. Therefore, activation or inhibition of some subtypes of these kinds of receptors is most likely to be beneficial to attenuate cytokine storm. This article summarizes suggested therapeutic drugs with potential anti-inflammatory effects through purinergic receptors.

Synthesis, characterization and biological evaluation of indomethacin derived thioureas as purinergic (P2Y1, P2Y2, P2Y4, and P2Y6) receptor antagonists.

G-protein-coupled receptors for extracellular nucleotides are known as P2Y receptors and are made up of eight members that are encoded by distinct genes and can be classified into two classes based on their affinity for specific G-proteins. P2Y receptor modulators have been studied extensively, but only a few small-molecule P2Y receptor antagonists have been discovered so far and approved by drug agencies. Derivatives of indole carboxamide have been identified as P2Y12 and P2X7 antagonist, as a result, we developed and tested a series of indole derivatives4a-lhaving thiourea moiety as P2Y receptor antagonist by using a fluorescence-based assay to measure the inhibition of intracellular calcium release in 1321N1 astrocytoma cells that had been stably transfected with the P2Y1, P2Y2, P2Y4 and P2Y6 receptors. Most of the compounds exhibited moderate to excellent inhibition activity against P2Y1 receptor subtype. The series most potent compound, 4h exhibited an IC50 value of 0.36 ± 0.01 µM selectivity against other subtypes of P2Y receptor. To investigate the ligand-receptor interactions, the molecular docking studies were carried out. Compound 4h is the most potent P2Y1 receptor antagonist due to interaction with an important amino acid residue Pro105, in addition to Ile108, Phe119, and Leu102.

Roles of Cytokines in the Temporal Changes of Microglial Membrane Currents and Neuronal Excitability and Synaptic Efficacy in ATP-Induced Cortical Injury Model.

Cytokines are important neuroinflammatory modulators in neurodegenerative brain disorders including traumatic brain injury (TBI) and stroke. However, their temporal effects on the physiological properties of microglia and neurons during the recovery period have been unclear. Here, using an ATP-induced cortical injury model, we characterized selective effects of ATP injection compared to needle-control. In the damaged region, the fluorescent intensity of CX3CR1-GFP (+) cells, as well as the cell density, was increased and the maturation of newborn BrdU (+) cells continued until 28 day-post-injection (dpi) of ATP. The excitability and synaptic E/I balance of neurons and the inward and outward membrane currents of microglia were increased at 3 dpi, when expressions of tumor necrosis factor (TNF)-α/interleukin (IL)-1ß and IL-10/IL-4 were also enhanced. These changes of both cells at 3 dpi were mostly decayed at 7 dpi and were suppressed by any of IL-10, IL-4, suramin (P2 receptor inhibitor) and 4-AP (K+ channel blocker). Acute ATP application alone induced only small effects from both naïve neurons and microglial cells in brain slice. However, TNF-α alone effectively increased the excitability of naïve neurons, which was blocked by suramin or 4-AP. TNF-α and IL-1ß increased and decreased membrane currents of naïve microglia, respectively. Our results suggest that ATP and TNF-α dominantly induce the physiological activities of 3 dpi neurons and microglia, and IL-10 effectively suppresses such changes of both activated cells in K+ channel- and P2 receptor-dependent manner, while IL-4 suppresses neurons preferentially.

Capillaries communicate with the arteriolar microvascular network by a pannexin/purinergic-dependent pathway in hamster skeletal muscle.

We sought to determine if a pannexin/purinergic-dependent intravascular communication pathway exists in skeletal muscle microvasculature that facilitates capillary communication with upstream arterioles that control their perfusion. Using the hamster cremaster muscle and intravital microscopy, we locally stimulated capillaries and observed the vasodilatory response in the associated upstream 4A arteriole. We stimulated capillaries with vasodilators relevant to muscle contraction: 10-6 M S-nitroso-N-acetyl-dl-penicillamine (SNAP; nitric oxide donor), 10-6 M adenosine, 10 mM potassium chloride, 10-5 M pinacidil, as well as a known initiator of gap-junction-dependent intravascular communication, acetylcholine (10-5 M), in the absence and the presence of the purinergic membrane receptor blocker suramin (10-5 M), pannexin blocker mefloquine (2 × 10-5 M), or probenecid (5 × 10-6 M) and gap-junction inhibitor halothane (0.07%) applied in the transmission pathway, between the capillary stimulation site and the upstream 4A observation site. Potassium chloride, SNAP, and adenosine-induced upstream vasodilations were significantly inhibited by suramin, mefloquine, and probenecid but not halothane, indicating the involvement of a pannexin/purinergic-dependent signaling pathway. Conversely, SNAP-induced upstream vasodilation was only inhibited by halothane indicating that communication was facilitated by gap junctions. Both pinacidil and acetylcholine were inhibited by suramin but only acetylcholine was inhibited by halothane. These data demonstrate the presence of a pannexin/purinergic-dependent communication pathway between capillaries and upstream arterioles controlling their perfusion. This pathway adds to the gap-junction-dependent pathway that exists at this vascular level as well. Given that vasodilators relevant to muscle contraction can use both of these pathways, our data implicate the involvement of both pathways in the coordination of skeletal muscle blood flow.NEW & NOTEWORTHY Blood flow control during increased metabolic demand in skeletal muscle is not fully understood. Capillaries have been implicated in controlling blood flow to active skeletal muscle, but how capillaries communicate to the arteriolar vascular network is not clear. Our study uncovers a novel pathway through which capillaries can communicate to upstream arterioles to cause vasodilation and therefore control perfusion. This work implicates a new vascular communication pathway in blood flow control in skeletal muscle.

Purinergic Receptor Blockade with Suramin Increases Survival of Postnatal Neural Progenitor Cells In Vitro.

Neural progenitor cells (NPCs) are self-renewing and multipotent cells that persist in the postnatal and adult brain in the subventricular zone and the hippocampus. NPCs can be expanded in vitro to be used in cell therapy. However, expansion is limited, since the survival and proliferation of adult NPCs decrease with serial passages. Many signaling pathways control NPC survival and renewal. Among these, purinergic receptor activation exerts differential effects on the biology of adult NPCs depending on the cellular context. In this study, we sought to analyze the effect of a general blockade of purinergic receptors with suramin on the proliferation and survival of NPCs isolated from the subventricular zone of postnatal rats, which are cultured as neurospheres. Treatment of neurospheres with suramin induced a significant increase in neurosphere diameter and in NPC number attributed to a decrease in apoptosis. Proliferation and multipotency were not affected. Suramin also induced an increase in the gap junction protein connexin43 and in vascular endothelial growth factor, which might be involved in the anti-apoptotic effect. Our results offer a valuable tool for increasing NPC survival before implantation in the lesioned brain and open the possibility of using this drug as adjunctive therapy to NPC transplantation.

Nanobodies as probes to investigate purinergic signaling.

Nanobodies (VHHs) are the single variable immunoglobulin domains of heavy chain antibodies (hcAbs) that naturally occur in alpacas and other camelids. The two variable domains of conventional antibodies typically interact via a hydrophobic interface. In contrast, the corresponding surface area of nanobodies is hydrophilic, rendering these single immunoglobulin domains highly soluble, robust to harsh environments, and exceptionally easy to format into bispecific reagents. In homage to Geoffrey Burnstock, the pioneer of purinergic signaling, we provide a brief history of nanobody-mediated modulation of purinergic signaling, using our nanobodies targeting P2X7 and the NAD+-metabolizing ecto-enzymes CD38 and ARTC2.2 as examples.

Purinergic signalling in host innate immune defence against intracellular pathogens.

Purinergic signalling is an evolutionarily conserved signalling pathway mediated by extracellular nucleotides and nucleosides. Tri- and diphosphonucleotides released from host cells during intracellular pathogen infections activate plasma membrane purinergic type 2 receptors (P2 receptors) that stimulate microbicidal mechanisms in host innate immune cells. P2X ion channels and P2Y G protein-coupled receptors are involved in activating host innate immune defence mechanisms, phagocytosis, phagolysosomal fusion, production of reactive species, acidification of parasitophorous vacuoles, inflammasome activation, and the release of cytokines, chemokines, and other inflammatory mediators. In this review, as part of a special issue in tribute to Geoffrey Burnstock, we discuss advances in understanding the importance of P2 receptors in the host antimicrobial innate mechanisms against intracellular pathogen infections.

Purinergic receptors in airway hydration.

Airway epithelial purinergic receptors control key components of the mucociliary clearance (MCC), the dominant component of pulmonary host defense. In healthy airways, the periciliary liquid (PCL) is optimally hydrated, thus acting as an efficient lubricant layer over which the mucus layer moves by ciliary force. When the hydration of the airway surface decreases, the mucus becomes hyperconcentrated, the PCL collapses, and the "thickened" mucus layer adheres to cell surfaces, causing plaque/plug formation. Mucus accumulation is a major contributing factor to the progression of chronic obstructive lung diseases such as cystic fibrosis (CF) and chronic bronchitis (CB). Mucus hydration is regulated by finely tuned mechanisms of luminal Cl- secretion and Na+ absorption with concomitant osmotically driven water flow. These activities are regulated by airway surface liquid (ASL) concentrations of adenosine and ATP, acting on airway epithelial A2B and P2Y2 receptors, respectively. The goal of this article is to provide an overview of our understanding of the role of purinergic receptors in the regulation of airway epithelial ion/fluid transport and the mechanisms of nucleotide release and metabolic activities that contribute to airway surface hydration in healthy and chronically obstructed airways.

Purinergic signalling in the kidney: In physiology and disease.

Historically, the control of renal vascular and tubular function has, for the most part, concentrated on neural and endocrine regulation. However, in addition to these extrinsic factors, it is now appreciated that several complex humoral control systems exist within the kidney that can act in an autocrine and/or paracrine fashion. These paracrine systems complement neuroendocrine regulation by dynamically fine-tuning renal vascular and tubular function to buffer rapid changes in nephron perfusion and flow rate of tubular fluid. One of the most pervasive is the extracellular nucleotide/P2 receptor system, which is central to many of the intrinsic regulatory feedback loops within the kidney such as renal haemodynamic autoregulation and tubuloglomerular feedback (TGF). Although physiological actions of extracellular adenine nucleotides were reported almost 100 years ago, the conceptual framework for purinergic regulation of renal function owes much to the work of Geoffrey Burnstock. In this review, we reflect on our >20-year collaboration with Professor Burnstock and highlight the research that is still unlocking the potential of the renal purinergic system to understand and treat kidney disease.

Purinergic signaling in diabetes and metabolism.

Purinergic signaling, a concept originally formulated by the late Geoffrey Burnstock (1929-2020), was found to modulate pathways in every physiological system. In metabolic disorders there is a role for both adenosine receptors and P2 (nucleotide) receptors, of which there are two classes, i.e. P2Y metabotropic and P2X ionotropic receptors. The individual roles of the 19 receptors encompassed by this family have been dissected - and in many cases the effects associated with specific cell types, including adipocytes, skeletal muscle, liver cells and immune cells. It is suggested that ligands selective for each of the four adenosine receptors (A1, A2A, A2B and A3), and several of the P2 subtypes (e.g. P2Y6 or P2X7 antagonists) might have therapeutic potential for treating diabetes and obesity. This is a developing story with some conflicting conclusions relevant to drug discovery, which we summarize here.

From astrocytes to satellite glial cells and back: A 25 year-long journey through the purinergic modulation of glial functions in pain and more.

Fundamental progresses have been made in pain research with a comprehensive understanding of the neuronal pathways which convey painful sensations from the periphery and viscera to the central nervous system and of the descending modulating pathways. Nevertheless, many patients still suffer from various painful conditions, which are often associated to other primary pathologies, and get no or poor relief from available painkillers. Thus, the interest of many researchers has concentrated on new and promising cellular targets and biochemical pathways. This is the case of glia cells, both in the peripheral and in the central nervous system, and of purinergic receptors. Starting from many intuitions and hypotheses raised by Prof. Geoffrey Burnstock, data have accumulated which clearly highlight the fundamental role exerted by several nucleotide and nucleoside receptors in the modulation of glial cell reaction to pain triggers and of their cross-talk with sensory neurons which significantly contributes to the transition from acute to chronic pain. The purinergic system has therefore become an appealing pharmacological target in pain research, also based on the quite unexpected discovery that purines are involved in ancient analgesic techniques such as acupuncture. A more in-depth understanding of the complex and intricated purine-orchestrated scenario in pain conditions will hopefully lead to the identification and clinical development of new and effective analgesics.

The Therapeutic Potential of Purinergic Receptors in Alzheimer's Disease and Promising Therapeutic Modulators.

Recent studies have proven that the purinergic signaling pathway plays a key role in neurotransmission and neuromodulation, and is involved in various neurodegenerative diseases and psychiatric disorders. With the characterization of the subtypes of receptors in purinergic signaling, i.e. the P1 (adenosine), P2X (ion channel) and P2Y (G protein-coupled), more attention has been paid to the pathophysiology and therapeutic potential of purinergic signaling in the central nervous system disorders. Alzheimer's disease (AD) is a progressive and deadly neurodegenerative disease that is characterized by memory loss, cognitive impairment and dementia. However, as drug development aimed to prevent or control AD has series of failures in recent years, more researchers have focused on the neuroprotection-related mechanisms such as purinergic signaling in AD patients to find a potential cure. This article reviews the recent discoveries of purinergic signaling in AD, and summarizes the potential agents as modulators for the receptors of purinergic signaling in AD-related research and treatments. Thus, our paper provides an insight into purinergic signaling in the development of anti- AD therapies.

Human P2X7 receptors - Properties of single ATP-gated ion channels.

Patch clamp investigations of single ion channels give insight into the function of these proteins on the molecular level. Utilizing this technique, we performed detailed investigations of the human P2X7 receptor, which is a ligand gated ion channel opened by binding of ATP, like the other P2X receptor subtypes. P2X7 receptors become activated under pathological conditions of ATP release like hypoxia or cell destruction. They are involved in inflammatory and nociceptive reactions of the organism to these pathological events. Knowledge about the function of the P2X7 receptor might lead to a deeper insight into the signaling within these pathophysiological processes and to reveal targets of anti-inflammatory and anti-nociceptive therapies. We found that hP2X7 receptors become activated by ATP within a few milliseconds and are permeable only to cations. Their ion channel conductance remains constant across minutes of activation, which argues against dilation of the ion channel pore. Substitution of Na+ or Cl- ions not only influences the ion channel current amplitude but also the channel gating. Polar residues of the second transmembrane domains of the three protein subunits are important for ion conduction, with S342 constituting the ion selectivity filter and the gate of the channel. The specific long C-terminal domains are important for hP2X7 receptor ion channel function, as their loss strongly decreases ion channel currents.

Purinergic Receptors Crosstalk with CCR5 to Amplify Ca2+ Signaling.

Many G protein-coupled receptors (GPCRs) signal through more than one subtype of heterotrimeric G proteins. For example, the C-C chemokine receptor type 5 (CCR5), which serves as a co-receptor to facilitate cellular entry of human immunodeficiency virus 1 (HIV-1), normally signals through the heterotrimeric G protein, Gi. However, CCR5 also exhibits G protein signaling bias and certain chemokine analogs can cause a switch to Gq pathways to induce Ca2+ signaling. We want to understand how much of the Ca2+ signaling from Gi-coupled receptors is due to G protein promiscuity and how much is due to transactivation and crosstalk with other receptors. We propose a possible mechanism underlying the apparent switching between different G protein signaling pathways. We show that chemokine-mediated Ca2+ flux in HEK293T cells expressing CCR5 can be primed and enhanced by ATP pretreatment. In addition, agonist-dependent lysosomal exocytosis results in the release of ATP to the extracellular milieu, which amplifies cellular signaling networks. ATP is quickly degraded via ADP and AMP to adenosine. ATP, ADP and adenosine activate different cell surface purinergic receptors. Endogenous Gq-coupled purinergic P2Y receptors amplify Ca2+ signaling and allow for Gi- and Gq-coupled receptor signaling pathways to converge. Associated secretory release of GPCR ligands, such as chemokines, opioids, and monoamines, should also lead to concomitant release of ATP with a synergistic effect on Ca2+ signaling. Our results suggest that crosstalk between ATP-activated purinergic receptors and other Gi-coupled GPCRs is an important cooperative mechanism to amplify the intracellular Ca2+ signaling response.

History of ectonucleotidases and their role in purinergic signaling.

Ectonucleotidases are key for purinergic signaling. They control the duration of activity of purinergic receptor agonists. At the same time, they produce hydrolysis products as additional ligands of purinergic receptors. Due to the considerable diversity of enzymes, purinergic receptor ligands and purinergic receptors, deciphering the impact of extracellular purinergic receptor control has become a challenge. The first group of enzymes described were the alkaline phosphatases - at the time not as nucleotide-metabolizing but as nonspecific phosphatases. Enzymes now referred to as nucleoside triphosphate diphosphohydrolases and ecto-5'-nucleotidase were the first and only nucleotide-specific ectonucleotidases identified. And they were the first group of enzymes related to purinergic signaling. Additional research brought to light a surprising number of ectoenzymes with broad substrate specificity, which can also hydrolyze nucleotides. This short overview traces the development of the field and briefly highlights important results and benefits for therapies of human diseases achieved within nearly a century of investigations.

Adenosine kinase: A key regulator of purinergic physiology.

Adenosine (ADO) is an essential biomolecule for life that provides critical regulation of energy utilization and homeostasis. Adenosine kinase (ADK) is an evolutionary ancient ribokinase derived from bacterial sugar kinases that is widely expressed in all forms of life, tissues and organ systems that tightly regulates intracellular and extracellular ADO concentrations. The facile ability of ADK to alter ADO availability provides a "site and event" specificity to the endogenous protective effects of ADO in situations of cellular stress. In addition to modulating the ability of ADO to activate its cognate receptors (P1 receptors), nuclear ADK isoform activity has been linked to epigenetic mechanisms based on transmethylation pathways. Previous drug discovery research has targeted ADK inhibition as a therapeutic approach to manage epilepsy, pain, and inflammation. These efforts generated multiple classes of highly potent and selective inhibitors. However, clinical development of early ADK inhibitors was stopped due to apparent mechanistic toxicity and the lack of suitable translational markers. New insights regarding the potential role of the nuclear ADK isoform (ADK-Long) in the epigenetic modulation of maladaptive DNA methylation offers the possibility of identifying novel ADK-isoform selective inhibitors and new interventional strategies that are independent of ADO receptor activation.

Inflammation and thrombosis in COVID-19 pathophysiology: proteinase-activated and purinergic receptors as drivers and candidate therapeutic targets.

Evolving information has identified disease mechanisms and dysregulation of host biology that might be targeted therapeutically in coronavirus disease 2019 (COVID-19). Thrombosis and coagulopathy, associated with pulmonary injury and inflammation, are emerging clinical features of COVID-19. We present a framework for mechanisms of thrombosis in COVID-19 that initially derive from interaction of SARS-CoV-2 with ACE2, resulting in dysregulation of angiotensin signaling and subsequent inflammation and tissue injury. These responses result in increased signaling by thrombin (proteinase-activated) and purinergic receptors, which promote platelet activation and exert pathological effects on other cell types (e.g., endothelial cells, epithelial cells, and fibroblasts), further enhancing inflammation and injury. Inhibitors of thrombin and purinergic receptors may, thus, have therapeutic effects by blunting platelet-mediated thromboinflammation and dysfunction in other cell types. Such inhibitors include agents (e.g., anti-platelet drugs) approved for other indications, and that could be repurposed to treat, and potentially improve the outcome of, COVID-19 patients. COVID-19, caused by the SARS-CoV-2 virus, drives dysregulation of angiotensin signaling, which, in turn, increases thrombin-mediated and purinergic-mediated activation of platelets and increase in inflammation. This thromboinflammation impacts the lungs and can also have systemic effects. Inhibitors of receptors that drive platelet activation or inhibitors of the coagulation cascade provide opportunities to treat COVID-19 thromboinflammation.

Potential Therapeutic Role of Purinergic Receptors in Cardiovascular Disease Mediated by SARS-CoV-2.

Novel coronavirus disease 2019 (COVID-19) causes pulmonary and cardiovascular disorders and has become a worldwide emergency. Myocardial injury can be caused by direct or indirect damage, particularly mediated by a cytokine storm, a disordered immune response that can cause myocarditis, abnormal coagulation, arrhythmia, acute coronary syndrome, and myocardial infarction. The present review focuses on the mechanisms of this viral infection, cardiac biomarkers, consequences, and the possible therapeutic role of purinergic and adenosinergic signalling systems. In particular, we focus on the interaction of the extracellular nucleotide adenosine triphosphate (ATP) with its receptors P2X1, P2X4, P2X7, P2Y1, and P2Y2 and of adenosine (Ado) with A2A and A3 receptors, as well as their roles in host immune responses. We suggest that receptors of purinergic signalling could be ideal candidates for pharmacological targeting to protect against myocardial injury caused by a cytokine storm in COVID-19, in order to reduce systemic inflammatory damage to cells and tissues, preventing the progression of the disease by modulating the immune response and improving patient quality of life.