P2X Inhibitor Research Articles

A shared mechanism for TNP-ATP recognition by members of the P2X receptor family

P2X receptors (P2X1–7) are non-selective cation channels involved in many physiological activities such as synaptic transmission, immunological modulation, and cardiovascular function. These receptors share a conserved mechanism to sense extracellular ATP. TNP-ATP is an ATP derivative acting as a nonselective competitive P2X antagonist. Understanding how it occupies the orthosteric site in the absence of agonism may help reveal the key allostery during P2X gating. However, TNP-ATP/P2X complexes (TNP-ATP/human P2X3 (hP2X3) and TNP-ATP/chicken P2X7 (ckP2X7)) with distinct conformations and different mechanisms of action have been proposed. Whether these represent species and subtype variations or experimental differences remains unclear. Here, we show that a common mechanism of TNP-ATP recognition exists for the P2X family members by combining enhanced conformation sampling, engineered disulfide bond analysis, and covalent occupancy. In this model, the polar triphosphate moiety of TNP-ATP interacts with the orthosteric site, while its TNP-moiety is deeply embedded in the head and dorsal fin (DF) interface, creating a restrictive allostery in these two domains that results in a partly enlarged yet ion-impermeable pore. Similar results were obtained from multiple P2X subtypes of different species, including ckP2X7, hP2X3, rat P2X2 (rP2X2), and human P2X1 (hP2X1). Thus, TNP-ATP uses a common mechanism for P2X recognition and modulation by restricting the movements of the head and DF domains which are essential for P2X activation. This knowledge is applicable to the development of new P2X inhibitors.

Inorganic polyphosphate regulates functions of thymocytes via activation of P2X purinoreceptors

Inorganic polyphosphate (polyP) is an ancient polymer, which was proven to be a signalling molecule in the mammalian brain, mediating the communication between astrocytes via activation of P2Y1 purinoreceptors and modulating the activity of neurons. There is very limited information regarding the ability of polyP to transmit the information as an agonist of purinoreceptors in other cells and tissues. Here, we show that application of polyP to the suspension of primary thymocytes increases the concentration of intracellular calcium. PolyP evoked calcium signal was dependent on the presence of P2X inhibitors but not P2Y1 inhibitor. PolyP dependent increase in intracellular calcium concentration caused mild mitochondrial depolarization, which was dependent on inhibitors of purinoreceptors, extracellular calcium and inhibitor of mitochondrial calcium uniporter but wasn't dependent on cyclosporin A. Application of polyP modulated cell volume regulation machinery of thymocytes in calcium dependent manner. Molecular docking experiments revealed that polyP can potentially bind to several types of P2X receptors with binding energy similar to ATP - natural agonist of P2X purinoreceptors. Further molecular dynamics simulations with P2X4 showed that binding of one molecule of polyP dramatically increases permeability of this receptor-channel for water molecules. Thus, in this research we for the first time showed that polyP can interact with P2X receptors in thymocytes and modulate physiological processes.

P2X3-selective mechanism of Gefapixant, a drug candidate for the treatment of refractory chronic cough

Gefapixant/AF-219, a selective inhibitor of the P2X3 receptor, is the first new drug other than dextromethorphan to be approved for the treatment of refractory chronic cough (RCC) in nearly 60 years. To date, seven P2X subtypes (P2X1-7) activated by extracellular ATP have been cloned, and subtype selectivity of P2X inhibitors is a prerequisite for reducing side effects. We previously identified the site and mechanism of action of Gefapixant/AF-219 on the P2X3 receptor, which occupies a pocket consisting of the left flipper (LF) and lower body (LB) domains. However, the mechanism by which AF-219 selectively acts on the P2X3 receptor is unknown. Here, we combined mutagenesis, chimera construction, molecular simulations, covalent occupation and chemical synthesis, and find that the negative allosteric site of AF-219 at P2X3 is also present in other P2X subtypes, at least for P2X1, P2X2 and P2X4. By constructing each chimera of AF-219 sensitive P2X3 and insensitive P2X2 subtypes, the insensitive P2X2 subtype was made to acquire the inhibitory properties of AF-219 and AF-353, an analog of AF-219 with higher affinity. Our results suggest that the selectivity of AF-219/AF-353 for P2X3 over the other P2X subtypes is determined by a combination of the accessibility of P2X3 binding site and the internal shape of this pocket, a finding that could provide new perspectives for drug design against P2X3-mediated diseases such as RCC, idiopathicpulmonaryfibrosis, hypertension and overactive bladder disorder.

Development of High-Throughput Fluorescent-Based Screens to Accelerate Discovery of P2X Inhibitors from Animal Venoms.

Animal venoms can play an important role in drug discovery, as they are a rich source of evolutionarily tuned compounds that target a variety of ion channels and receptors. To date, there are six FDA-approved drugs derived from animal venoms, with recent work using high-throughput platforms providing a variety of new therapeutic candidates. However, high-throughput methods for screening animal venoms against purinoceptors, one of the oldest signaling receptor families, have not been reported. Here, we describe a variety of quantitative fluorescent-based high-throughput screening (HTS) cell-based assays for screening animal venoms against ligand-gated P2X receptors. A diverse selection of 180 venoms from arachnids, centipedes, hymenopterans, and cone snails were screened, analyzed, and validated, both analytically and pharmacologically. Using this approach, we performed screens against human P2X3, P2X4, and P2X7 using three different fluorescent-based dyes on stable cell lines and isolated the active venom components. Our HTS assays are performed in 96-well format and allow simultaneous screening of multiple venoms on multiple targets, improving testing characteristics while minimizing costs, specimen material, and testing time. Moreover, utilizing our assays and applying them to the other natural product libraries, rather than venoms, might yield other novel natural products that modulate P2X activity.

LPS PROMOTES ASC‐SPECK FORMATION IN TREK‐1‐DEFICIENT MACROPHAGES

BACKGROUNDTREK‐1 is a 2‐pore potassium channel that we previously showed played a protective role against hyperoxia‐induced lung injury. To determine whether this protective effect was related to reduced inflammasome activation, we investigated LPS‐induced responses. Our preliminary studies with macrophages from TREK‐1−/− mice indicated a reduction in NLRP3 inflammasome priming and activation compared with wt mice, but we found that prolonged LPS exposure promoted increased IL‐1β release. We therefore investigatElied non‐canonical inflammasome pathways.METHODSWe stimulated alveolar macrophages (AMs) and bone marrow derived macrophages (BMDMs) using both a short‐term LPS+/−ATP model of inflammasome priming and activation and a prolonged‐LPS exposure. wt, TREK‐1−/−, and NLRP3−/− AMs and BMDMs were pre‐treated with a TREK‐1‐inhibitor (SPADIN), a TREK‐1‐activator, a Pannexin inhibitor, a P2X channel inhibitor, an ASC‐oligomerization inhibitor, or a JNK‐inhibitor. We measured IL‐1β, NLRP3, caspase‐1, ASC, TNFα as well as caspase−8, −7, and −11, and phosphorylated‐JNK using western blot, immunostaining, and ELISA. We used a membrane permeable potassium indicator to measure potassium efflux during ATP exposure. Using a caspase‐luciferace assay, we then measured caspase‐activity +/− MG132 (a proteasome inhibitor).RESULTSATP treatment of LPS‐primed TREK‐1‐inhibited and ‐deficient BMDMs exaggerated caspase‐7 and caspase‐11 activity compared to wt BMDMs, but both were dampened with co‐treatment with Pannexin and P2X inhibitors. Both TREK1 inhibition and deficiency caused elevated potassium efflux after short term LPS + ATP. With prolonged LPS exposure, both wt and TREK‐1−/− BMDMs contained more intracellular potassium than unstimulated wt BMDMs, with the LPS‐treated TREK‐1−/− BMDMs containing the highest concentration. Prolonged LPS exposure increased casp1‐p20, IL‐1β, and ASC‐speck formation in TREK‐1−/− BMDMs and AMs, compared with wt. Similarly, TREK‐1 inhibition with SPADIN increased ASC‐speck formation in both wt and NLRP3−/− BMDMs after prolonged LPS exposure. Additionally, MG132 completely blocked caspase activity in wt BMDMs treated with SPADIN after LPS treatment. JNK inhibition decreased ASC‐oligomerization in both wt and TREK‐1−/− BMDMs. However, prolonged LPS exposure led to increased caspase‐7 and JNK activity in TREK‐1−/− BMDMs compared to wt. Additionally, TREK‐1 inhibition increased caspase‐7 and caspase‐8 activity after LPS exposure in both wt and NLRP3−/− BMDMs.CONCLUSIONThese results suggest that the protective effect of TREK‐1 may be associated with regulation of apoptotic association ASC‐speck formation.Support or Funding InformationSupported by NIH grants HL123540, HL131526, and HL118118, and by the Le Bonheur Children's Hospital Research Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

VNUT and VMAT2 segregate within sympathetic varicosities and localize near preferred Cav2 isoforms in the rat tail artery.

ATP and norepinephrine (NE) are coreleased from peripheral sympathetic nerve terminals. Whether they are stored in the same vesicles has been debated for decades. Preferential dependence of NE or ATP release on Ca2+ influx through specific voltage-gated Ca2+ channel (Cav2) isoforms suggests that NE and ATP are stored in separate vesicle pools, but simultaneous imaging of NE and ATP containing vesicles within single varicosities has not been reported. We conducted an immunohistochemical study of vesicular monoamine transporter 2 (VMAT2/SLC18A2) and vesicular nucleotide translocase (VNUT/SLC17A9) as markers of vesicles containing NE and ATP in sympathetic nerves of the rat tail artery. A large fraction of varicosities exhibited neighboring, rather than overlapping, VNUT and VMAT2 fluorescent puncta. VMAT2, but not VNUT, colocalized with synaptotagmin 1. Cav2.1, Cav2.2, and Cav2.3 are expressed in nerves in the tunica adventitia. VMAT2 preferentially localized adjacent to Cav2.2 and Cav2.3 rather than Cav2.1. VNUT preferentially localized adjacent to Cav2.3 > Cav2.2 >> Cav2.1. With the use of wire myography, inhibition of field-stimulated vasoconstriction with the Cav2.3 blocker SNX-482 (0.25 µM) mimicked the effects of the P2X inhibitor suramin (100 µM) rather than the α-adrenergic inhibitor phentolamine (10 µM). Variable sensitivity to SNX-482 and suramin between animals closely correlated with Cav2.3 staining. We concluded that a majority of ATP and NE stores localize to separate vesicle pools that use different synaptotagmin isoforms and that localize near different Cav2 isoforms to mediate vesicle release. Cav2.3 appears to play a previously unrecognized role in mediating ATP release in the rat tail artery. NEW & NOTEWORTHY Immunofluorescence imaging of vesicular nucleotide translocase and vesicular monoamine transporter 2 in rat tail arteries revealed that ATP and norepinephrine, classical cotransmitters, localize to well-segregated vesicle pools. Furthermore, vesicular nucleotide translocase and vesicular monoamine transporter 2 exhibit preferential localization with specific Cav2 isoforms. These novel observations address long-standing debates regarding the mechanism(s) of sympathetic neurotransmitter corelease.

P2X receptors regulate adenosine diphosphate release from hepatic cells.

Extracellular nucleotides act as paracrine regulators of cellular signaling and metabolic pathways. Adenosine polyphosphate (adenosine triphosphate (ATP) and adenosine diphosphate (ADP)) release and metabolism by human hepatic carcinoma cells was therefore evaluated. Hepatic cells maintain static nanomolar concentrations of extracellular ATP and ADP levels until stress or nutrient deprivation stimulates a rapid burst of nucleotide release. Reducing the levels of media serum or glucose has no effect on ATP levels, but stimulates ADP release by up to 10-fold. Extracellular ADP is then metabolized or degraded and media ADP levels fall to basal levels within 2-4h. Nucleotide release from hepatic cells is stimulated by the Ca(2+) ionophore, ionomycin, and by the P2 receptor agonist, 2'3'-O-(4-benzoyl-benzoyl)-adenosine 5'-triphosphate (BzATP). Ionomycin (10μM) has a minimal effect on ATP release, but doubles media ADP levels at 5min. In contrast, BzATP (10-100μM) increases both ATP and ADP levels by over 100-fold at 5min. Ion channel purinergic receptor P2X7 and P2X4 gene silencing with small interference RNA (siRNA) and treatment with the P2X inhibitor, A438079 (100μM), decrease ADP release from hepatic cells, but have no effect on ATP. P2X inhibitors and siRNA have no effect on BzATP-stimulated nucleotide release. ADP release from human hepatic carcinoma cells is therefore regulated by P2X receptors and intracellular Ca(2+) levels. Extracellular ADP levels increase as a consequence of a cellular stress response resulting from serum or glucose deprivation.

Involvement of the P2X7 Purinergic Receptor and c-Jun N-Terminal and Extracellular Signal-Regulated Kinases in Cyclooxygenase-2 and Prostaglandin E2 Induction by LL-37

Periodontal disease is caused by microorganisms and host-derived inflammation involving increased cyclooxygenase-2 (COX-2) expression and prostaglandin E₂ (PGE₂) production. We previously demonstrated that human β-defensin-3 induces COX-2 and PGE₂ in human gingival fibroblasts (HGFs). We, therefore, aimed to examine the inducible effects of LL-37, the only cathelicidin expressed in humans, on COX-2 expression and PGE₂ synthesis in HGFs and to elucidate the relevant signaling pathways. The COX-2 expression was upregulated by LL-37 in dose- and time-dependent manners. Accordingly, the synthesis of PGE₂ in cell-free culture supernatants was raised by LL-37 (p < 0.01) and blocked by NS-398, a specific COX-2 inhibitor (p < 0.01). P2X inhibitors and a neutralizing antibody against P2X₇ purinergic receptor significantly abrogated COX-2 induction and PGE₂ production by LL-37 (p < 0.01). LL-37 upregulated COX-2 expression and PGE₂ synthesis via activation of extracellular signal-regulated kinase (ERK) and p46 c-Jun N-terminal kinase (JNK), while interleukin-1β did so via nuclear factor-ĸB and all three mitogen-activated protein kinases. In summary, LL-37 can control arachidonic acid metabolism by induction of COX-2 expression and PGE₂ synthesis via the P2X₇ receptor, ERK, and p46 JNK. The pro-inflammatory effects of LL-37 may be essential for initiating oral mucosal inflammation in periodontal disease.

P2X purinergic receptor ligands: recently patented compounds

Importance of the field: P2X channels are ionotropic purinergic receptors that are currently under scrutiny as attractive targets for novel therapeutics in areas including chronic inflammation, pain and depression. Their wide expression in the CNS, recent advances in the biochemical and pharmacological properties as well as increasing numbers of patents published in this research domain demand a review in this field.Areas covered in this review: The patent literature covering novel drug-like antagonists for each P2X receptor subtype (P2X1R to P2X7R) up to December 2009 is described in this review article together with their recent highlights in pharmacology.What the reader will gain: Readers will gain an up-to-date overview of patents covering drug-like antagonists for seven P2X receptor subtypes within the last 4 years.Take home message: P2X7R antagonists and other P2X inhibitors will probably be on the market for combating rheumatoid arthritis and other diseases. Some P2X7R antagonists are already in Phase I and II clinical trials.

Stimulation of P2X receptors enhances lipooligosaccharide-mediated apoptosis of endothelial cells

Exposure of endothelial cells to lipid A-containing molecules, such as lipopolysaccharide (LPS) or lipooligosaccharide (LOS), causes the release of purinergic compounds [e.g., adenosine 5'-triphosphate (ATP)] and can lead to apoptosis. The P2X family of purinergic receptors (e.g., P2X(7)) has been reported to modulate LPS signaling events and to participate in apoptosis. We investigated the role that P2X receptors play in the apoptosis that follows exposure of bovine endothelial cells to Haemophilus somnus LOS. Addition of P2X inhibitors, such as periodate-oxidized ATP (oATP) or pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium, significantly reduced LOS-induced apoptosis. Incubation of endothelial cells with apyrase, which degrades ATP, diminished LOS-induced apoptosis of endothelial cells. Concomitant addition of P2X agonists [e.g., 2',3'-(4-benzoyl)-benzoyl ATP or ATP] to LOS-treated endothelial cells significantly enhanced caspase-3 activation. The P2X antagonist oATP significantly blocked caspase-8 but not caspase-9 activation in LOS-treated endothelial cells. Together, these data indicate that stimulation of P2X receptors enhances LOS-induced apoptosis of endothelial cells, possibly as a result of endogenous release of ATP, which results in caspase-8 activation.