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.

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.

Effects of single-dose antipurinergic therapy on behavioral and molecular alterations in the valproic acid-induced animal model of autism.

Autism spectrum disorder (ASD) is characterized by deficits in communication and social interaction, restricted interests, and stereotyped behavior. Environmental factors, such as prenatal exposure to valproic acid (VPA), may contribute to the increased risk of ASD. Since disturbed functioning of the purinergic signaling system has been associated with the onset of ASD and used as a potential therapeutic target for ASD in both clinical and preclinical studies, we analyzed the effects of suramin, a non-selective purinergic antagonist, on behavioral, molecular and immunological in an animal model of autism induced by prenatal exposure to VPA. Treatment with suramin (20 mg/kg, intraperitoneal) restored sociability in the three-chamber apparatus and decreased anxiety measured by elevated plus maze apparatus, but had no impact on decreased reciprocal social interactions or higher nociceptive threshold in VPA rats. Suramin treatment did not affect VPA-induced upregulation of P2X4 and P2Y2 receptor expression in the hippocampus, and P2X4 receptor expression in the medial prefrontal cortex, but normalized an increased level of interleukin 6 (IL-6). Our results suggest an important role of purinergic signaling modulation in behavioral, molecular, and immunological aberrations described in VPA model, and indicate that the purinergic signaling system might be a potential target for pharmacotherapy in preclinical studies of ASD.

Antipurinergic therapy for autism-An in-depth review.

Are the symptoms of autism caused by a treatable metabolic syndrome that traces to the abnormal persistence of a normal, alternative functional state of mitochondria? A small clinical trial published in 2017 suggests this is possible. Based on a new unifying theory of pathogenesis for autism called the cell danger response (CDR) hypothesis, this study of 10 boys, ages 5-14years, showed that all 5 boys who received antipurinergic therapy (APT) with a single intravenous dose of suramin experienced improvements in all the core symptoms of autism that lasted for 5-8weeks. Language, social interaction, restricted interests, and repetitive movements all improved. Two children who were non-verbal spoke their first sentences. None of these improvements were observed in the placebo group. Larger and longer studies are needed to confirm this promising discovery. This review introduces the concept of M2 (anti-inflammatory) and M1 (pro-inflammatory) mitochondria that are polarized along a functional continuum according to cell stress. The pathophysiology of the CDR, the complementary functions of M1 and M2 mitochondria, relevant gene-environment interactions, and the metabolic underpinnings of behavior are discussed as foundation stones for understanding the improvements in ASD behaviors produced by antipurinergic therapy in this small clinical trial.

Reversal of autism-like behaviors and metabolism in adult mice with single-dose antipurinergic therapy.

Autism spectrum disorders (ASDs) now affect 1-2% of the children born in the United States. Hundreds of genetic, metabolic and environmental factors are known to increase the risk of ASD. Similar factors are known to influence the risk of schizophrenia and bipolar disorder; however, a unifying mechanistic explanation has remained elusive. Here we used the maternal immune activation (MIA) mouse model of neurodevelopmental and neuropsychiatric disorders to study the effects of a single dose of the antipurinergic drug suramin on the behavior and metabolism of adult animals. We found that disturbances in social behavior, novelty preference and metabolism are not permanent but are treatable with antipurinergic therapy (APT) in this model of ASD and schizophrenia. A single dose of suramin (20 mg kg(-1) intraperitoneally (i.p.)) given to 6-month-old adults restored normal social behavior, novelty preference and metabolism. Comprehensive metabolomic analysis identified purine metabolism as the key regulatory pathway. Correction of purine metabolism normalized 17 of 18 metabolic pathways that were disturbed in the MIA model. Two days after treatment, the suramin concentration in the plasma and brainstem was 7.64 µM pmol µl(-1) (±0.50) and 5.15 pmol mg(-1) (±0.49), respectively. These data show good uptake of suramin into the central nervous system at the level of the brainstem. Most of the improvements associated with APT were lost after 5 weeks of drug washout, consistent with the 1-week plasma half-life of suramin in mice. Our results show that purine metabolism is a master regulator of behavior and metabolism in the MIA model, and that single-dose APT with suramin acutely reverses these abnormalities, even in adults.

Identification of 2-oxo-N-(phenylmethyl)-4-imidazolidinecarboxamide antagonists of the P2X(7) receptor.

A backup molecule to compound 2 was sought by targeting the most likely metabolically vulnerable site in this molecule. Compound 18 was subsequently identified as a potent P2X(7) antagonist with very low in vivo clearance and high oral bioavailability in all species examined. Some evidence to support the role of P2X(7) in the etiology of pain is also presented.

Absorption, distribution, metabolism, and excretion of ticagrelor in healthy subjects.

Ticagrelor [(1S,2S,3R,5S)-3-[7-[[(1R,2S)-2-(3,4-difluorophenyl) cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol)] is a reversibly binding oral P2Y(12) receptor antagonist in development for the prevention of thrombotic events in patients with acute coronary syndromes. The pharmacokinetics, metabolism, and excretion of ticagrelor were investigated over 168 h in six healthy male subjects receiving a single oral suspension dose of 200 mg of [(14)C]ticagrelor. Ticagrelor was rapidly absorbed with a maximum plasma concentration at 1.5 h. The major active metabolite, AR-C124910XX, is formed by O-deethylation. Exposure to AR-C124910XX was 29% of peak and 40% of overall exposure to ticagrelor. In most subjects, radioactivity was undetectable in plasma after 20 h and whole blood after 12 h (half-life values of 6.3 and 4.6 h, respectively). The ratio of radioactivity in plasma to whole blood was 1.69, suggesting that ticagrelor and its metabolites are largely restricted to the plasma space. Mean radioactivity recovery was 26.5% in urine and 57.8% in feces. Major circulating components in the plasma and feces were identified as ticagrelor and AR-C124910XX, whereas in urine the major components were metabolite M5 (AR-C133913XX) and its glucuronide conjugate M4. Levels of unchanged ticagrelor and AR-C124910XX were <0.05% in the urine, indicating that renal clearance of ticagrelor and AR-C124910XX is of minor importance. Interindividual variability was small in both urine and fecal extracts with only small quantitative differences. All 10 of the metabolites were fully or partially characterized and a full biotransformation pathway was proposed for ticagrelor, in which oxidative loss of the hydroxyethyl side chain from ticagrelor forms AR-C124910XX and a second oxidative pathway leads to N-dealkylation of ticagrelor, forming AR-C133913XX.