Medicinal chemistry of P2 and adenosine receptors: Common scaffolds adapted for multiple targets.

Prof. Geoffrey Burnstock originated the concept of purinergic signaling. He demonstrated the interactions and biological roles of ionotropic P2X and metabotropic P2Y receptors. This review paper traces the historical origins of many currently used antagonists and agonists for P2 receptors, as well as adenosine receptors, in early attempts to identify ligands for these receptors - prior to the use of chemical libraries for screening. Rather than presenting a general review of current purinergic ligands, we focus on common chemical scaffolds (privileged scaffolds) that can be adapted for multiple receptor targets. By carefully analyzing the structure activity relationships, one can direct the selectivity of these scaffolds toward different receptor subtypes. For example, the weak and non-selective P2 antagonist reactive blue 2 (RB-2) was derivatized using combinatorial synthetic approaches, leading to the identification of selective P2Y2, P2Y4, P2Y12 or P2X2 receptor antagonists. A P2X4 antagonist NC-2600 is in a clinical trial, and A3 adenosine agonists show promise, for chronic pain. P2X7 antagonists have been in clinical trials for depression (JNJ-54175446), inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, inflammatory pain and chronic obstructive pulmonary disease (COPD). P2X3 antagonists are in clinical trials for chronic cough, and an antagonist named after Burnstock, gefapixant, is expected to be the first P2X3 antagonist filed for approval. We are seeing that the vision of Prof. Burnstock to use purinergic signaling modulators, most recently at P2XRs, for treating disease is coming to fruition.

P2Y4, P2Y6 and P2Y11 receptors: From the early days of cloning to their function.

The family of P2Y nucleotide receptors is composed of eight members differentiated by their pharmacology and their coupling to specific G-proteins and transduction mechanisms. The laboratory studying these nucleotide receptors at IRIBHM institute (Free University of Brussels) has participated actively in their cloning. We used classical cloning by homology strategies relying on polymerase chain reactions with degenerate primers or on DNA libraries screening with P2Y receptors-related primers or probes, respectively. We identified and characterised four of the eight human P2Y receptors cloned so far: P2Y4, P2Y6, P2Y11 and P2Y13 receptors. These human receptors displayed specific features in terms of pharmacology such as affinity for pyrimidine nucleotides for P2Y4 and P2Y6 receptors and differential G-protein coupling. Their specific and restricted tissue distribution compared to ubiquitous P2Y1 and P2Y2 receptors led us to study their physiological role in chosen cell systems or using mice deficient for these P2Y subtypes. These studies revealed over the years that the P2Y11 receptor was able to confer tolerogenic and tumorigenic properties to human dendritic cells and that P2Y4 and P2Y6 receptors were involved in mouse heart post-natal development and cardioprotection. P2Y receptors and their identified target genes could constitute therapeutic targets to regulate cardiac hypertrophy and regeneration. The multiple roles of P2Y receptors identified in the ischemic heart and cardiac adipose tissue could have multiple innovative clinical applications and present a major interest in the field of cardiovascular diseases. P2Y receptors can induce cardioprotection by the regulation of cardiac inflammation and the modulation of the volume and composition of cardiac adipose tissue. These findings might lead to the pre-clinical validation of P2Y receptors as new targets for the treatment of myocardial ischemia.

Extracellular adenosine 5'-triphosphate in pulmonary disorders.

Adenosine 5'-triphosphate (ATP) is found in every cell of the human body where it plays a critical role in cellular energetics and metabolism. ATP is released from cells under physiologic and pathophysiologic condition; extracellular ATP is rapidly degraded to adenosine 5'-diphosphate (ADP) and adenosine by ecto-enzymes (mainly, CD39 and CD73). Before its degradation, ATP acts as an autocrine and paracrine agent exerting its effects on targeted cells by activating cell surface receptors named P2 Purinergic receptors. The latter are expressed by different cell types in the lungs, the activation of which is involved in multiple pulmonary disorders. This succinct review summarizes the role of ATP in inflammation processes associated with these disorders including bronchoconstriction, cough, mechanical ventilation-induced lung injury and idiopathic pulmonary fibrosis. All of these disorders still constitute unmet clinical needs. Therefore, the various ATP-signaling pathways in pulmonary inflammation constitute attractive targets for novel drug-candidates that would improve the management of patients with multiple pulmonary diseases.

Activation of basal forebrain purinergic P2 receptors promotes wakefulness in mice.

The functions of purinergic P2 receptors (P2Rs) for extracellular adenosine triphosphate (ATP) are poorly understood. Here, for the first time, we show that activation of P2Rs in an important arousal region, the basal forebrain (BF), promotes wakefulness, whereas inhibition of P2Rs promotes sleep. Infusion of a non-hydrolysable P2R agonist, ATP-γ-S, into mouse BF increased wakefulness following sleep deprivation. ATP-γ-S depolarized BF cholinergic and cortically-projecting GABAergic neurons in vitro, an effect blocked by antagonists of ionotropic P2Rs (P2XRs) or glutamate receptors. In vivo, ATP-γ-S infusion increased BF glutamate release. Thus, activation of BF P2XRs promotes glutamate release and excitation of wake-active neurons. Conversely, pharmacological antagonism of BF P2XRs decreased spontaneous wakefulness during the dark (active) period. Together with previous findings, our results suggest sleep-wake regulation by BF extracellular ATP involves a balance between excitatory, wakefulness-promoting effects mediated by direct activation of P2XRs and inhibitory, sleep-promoting effects mediated by degradation to adenosine.

Nucleotide P2Y13-stimulated phosphorylation of CREB is required for ADP-induced proliferation of late developing retinal glial progenitors in culture.

Nucleotides stimulate phosphorylation of CREB to induce cell proliferation and survival in diverse cell types. We report here that ADP induces the phosphorylation of CREB in a time- and concentration-dependent manner in chick embryo retinal progenitors in culture. ADP-induced increase in phospho-CREB is mediated by P2 receptors as it is blocked by PPADS but not by the adenosine antagonists DPCPX or ZM241385. Incubation of the cultures with the CREB inhibitor KG-501 prevents ADP-induced incorporation of [3H]-thymidine, indicating that CREB is involved in retinal cell proliferation. No effect of this compound is observed on the viability of retinal progenitors. While no significant increase in CREB phosphorylation is observed with the P2Y1 receptor agonist MRS2365, ADP-induced phosphorylation of CREB is blocked by the P2Y13 receptor selective antagonist MRS2211, but not by MRS2179 or PSB0739, two antagonists of the P2Y1 and P2Y12 receptors, respectively, suggesting that ADP-induced CREB phosphorylation is mediated by P2Y13 receptors. ADP-induced increase in phospho-CREB is attenuated by the PI3K inhibitor LY294002 and completely prevented by the MEK inhibitor U0126, suggesting that at least ERK is involved in ADP-induced CREB phosphorylation. A pharmacological profile similar to the activation and inhibition of CREB phosphorylation is observed in the phosphorylation of ERK, suggesting that P2Y13 receptors mediate ADP induced ERK/CREB pathway in the cultures. While no increase in [3H]-thymidine incorporation is observed with the P2Y1 receptor agonist MRS2365, both MRS2179 and MRS2211 prevent ADP-mediated increase in [3H]-thymidine incorporation, but not progenitor's survival, suggesting that both P2Y1 and P2Y13 receptor subtypes are involved in ADP-induced cell proliferation. P2Y1 receptor-mediated increase in [Ca2+]i is observed in glial cells only when cultures maintained for 9days are used. In glia from cultures cultivated for only 2days, no increase in [Ca2+]i is detected with MRS2365 and no inhibition of ADP-mediated calcium response is observed with MRS2179. In contrast, MRS2211 attenuates ADP-mediated increase in [Ca2+]i in glial cells from cultures at both stages, suggesting the presence of P2Y13 receptors coupled to calcium mobilization in proliferating retinal glial progenitors in culture.

P2Y13 receptors mediate presynaptic inhibition of acetylcholine release induced by adenine nucleotides at the mouse neuromuscular junction.

It is known that adenosine 5'-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12-13 receptors, 2-methylthioadenosine 5'-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12-13 antagonists AR-C69931MX or 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5'-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y13. This study provides new insights into the types of purinergic receptors that contribute to the fine-tuning of cholinergic transmission at mammalian neuromuscular junction.

ATP and the purine type 2 X7 receptor affect sleep.

Sleep is dependent upon prior brain activities, e.g., after prolonged wakefulness sleep rebound occurs. These effects are mediated, in part, by humoral sleep regulatory substances such as cytokines. However, the property of wakefulness activity that initiates production and release of such substances and thereby provides a signal for indexing prior waking activity is unknown. We propose that extracellular ATP, released during neuro- and gliotransmission and acting via purine type 2 (P2) receptors, is such a signal. ATP induces cytokine release from glia. Cytokines in turn affect sleep. We show here that a P2 receptor agonist, 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP), increased non-rapid eye movement sleep (NREMS) and electroencephalographic (EEG) delta power while two different P2 receptor antagonists, acting by different inhibitory mechanisms, reduced spontaneous NREMS in rats. Rat P2X7 receptor protein varied in the somatosensory cortex with time of day, and P2X7 mRNA was altered by interleukin-1 treatment, by sleep deprivation, and with time of day in the hypothalamus and somatosensory cortex. Mice lacking functional P2X7 receptors had attenuated NREMS and EEG delta power responses to sleep deprivation but not to interleukin-1 treatment compared with wild-type mice. Data are consistent with the hypothesis that extracellular ATP, released as a consequence of cell activity and acting via P2 receptors to release cytokines and other sleep regulatory substances, provides a mechanism by which the brain could monitor prior activity and translate it into sleep.