Functionalized Congeners of 2H-Chromene P2Y6 Receptor Antagonists.

The P2Y6 receptor (P2Y6R), a Gq-coupled receptor, is a potential drug discovery target for various inflammatory and degenerative conditions. Antagonists have been shown to attenuate colitis, acute lung injury, etc. In the search for competitive antagonists, we have investigated the SAR of 3-nitro-2-(trifluoromethyl)-2H-chromene derivatives, although high affinity is lacking. We now reveal that long-chain amino-functionalized congeners display greatly enhanced affinity in the antagonism of UDP-induced Ca2+ mobilization in human (h) P2Y6R-transfected 1321N1 astrocytoma cells. A 6-(Boc-amino-n-heptylethynyl) analogue 30 (MRS4940) had an IC50 of 162 nM, which was a 123-fold greater affinity than the corresponding unprotected primary alkylamine, 107-fold greater than the corresponding pivaloyl derivative 30, and 132-fold selective compared to the P2Y14R. However, similar Boc-amino chains attached at the 8-position produced weak µM affinity. Thus, the P2Y6R affinity depended on the chain length, attachment point, and terminal functionality. Off-target activities, at 45 sites, were tested for acylamino derivatives 20, 24, 26, 30, 31, and 37, which showed multiple interactions, particularly at the biogenic amine receptors. The more potent analogues may be suitable for evaluation in inflammation and cancer models, which will be performed in future studies.

Discovery of a Series of 4-Amide-thiophene-2-carboxyl Derivatives as Highly Potent P2Y14 Receptor Antagonists for Inflammatory Bowel Disease Treatment.

The P2Y14 receptor has been proven to be a potential target for IBD. Herein, we designed and synthesized a series of 4-amide-thiophene-2-carboxyl derivatives as novel potent P2Y14 receptor antagonists based on the scaffold hopping strategy. The optimized compound 39 (5-((5-fluoropyridin-2-yl)oxy)-4-(4-methylbenzamido)thiophene-2-carboxylic acid) exhibited subnanomolar antagonistic activity (IC50: 0.40 nM). Moreover, compound 39 demonstrated notably improved solubility, liver microsomal stability, and oral bioavailability. Fluorescent ligand binding assay confirmed that 39 has the binding ability to the P2Y14 receptor, and molecular dynamics (MD) simulations revealed the formation of a unique intramolecular hydrogen bond (IMHB) in the binding conformation. In the experimental colitis mouse model, compound 39 showed a remarkable anti-IBD effect even at low doses. Compound 39, with a potent anti-IBD effect and favorable druggability, can be a promising candidate for further research. In addition, this work lays a strong foundation for the development of P2Y14 receptor antagonists and the therapeutic strategy for IBD.

Discovery of N-Substituted Acetamide Derivatives as Promising P2Y14R Antagonists Using Molecular Hybridization Based on Crystallographic Overlay.

P2Y14 receptor (P2Y14R) is activated by uridine 5'-diphosphate-glucose, which is involved in many human inflammatory diseases. Based on the molecular docking analysis of currently reported P2Y14R antagonists and the crystallographic overlap study between the reported P2Y14R antagonist compounds 6 and 9, a series of N-substituted-acetamide derivatives were designed, synthesized, and identified as novel and potent P2Y14R antagonists. The most potent antagonist, compound I-17 (N-(1H-benzo[d]imidazol-6-yl)-2-(4-bromophenoxy)acetamide, IC50 = 0.6 nM) without zwitterionic character, showed strong binding ability to P2Y14R, high selectivity, moderate oral bioactivity, and improved pharmacokinetic profiles. In vitro and in vivo evaluation demonstrated that compound I-17 had satisfactory inhibitory activity on the inflammatory response of monosodium urate (MSU)-induced acute gouty arthritis. I-17 decreased inflammatory factor release and cell pyroptosis through the NOD-like receptor family pyrin domain-containing 3 (NLRP3)/gasdermin D (GSDMD) signaling pathway. Thus, compound I-17, with potent P2Y14R antagonistic activity, in vitro and in vivo efficacy, and favorable bioavailability (F = 75%), could be a promising lead compound for acute gouty arthritis.

Bridged Piperidine Analogues of a High Affinity Naphthalene-Based P2Y14R Antagonist.

High affinity phenyl-piperidine P2Y14R antagonist 1 (PPTN) was modified with piperidine bridging moieties to probe receptor affinity and hydrophobicity. Various 2-azanorbornane, nortropane, isonortropane, isoquinuclidine, and ring-opened cyclopentylamino derivatives preserved human P2Y14R affinity (fluorescence binding assay), and their pharmacophoric overlay was compared. Enantiomeric 2-azabicyclo[2.2.1]hept-5-en-3-one precursors assured stereochemically unambiguous, diverse products. Pure (S,S,S) 2-azanorbornane enantiomer 15 (MRS4738) displayed higher affinity than 1 (3-fold higher affinity than enantiomer 16) and in vivo antihyperallodynic and antiasthmatic activity. Its double prodrug 143 (MRS4815) dramatically reduced lung inflammation in a mouse asthma model. Related lactams 21-24 and dicarboxylate 42 displayed intermediate affinity and enhanced aqueous solubility. Isoquinuclidine 34 (IC50 15.6 nM) and isonortropanol 30 (IC50 21.3 nM) had lower lipophilicity than 1. In general, rigidified piperidine derivatives did not lower lipophilicity dramatically, except those rings with multiple polar groups. P2Y14R molecular modeling based on a P2Y12R structure showed stable and persistent key interactions for compound 15.

Discovery and computational studies of 2-phenyl-benzoxazole acetamide derivatives as promising P2Y14R antagonists with anti-gout potential.

The P2Y14 nucleotide receptor, a subtype of P2Y receptors, is implicated in many human inflammatory diseases. Based on the identification of favorable residues of two screening hits in the almost symmetrical P2Y14 binding domain, we describe the structural optimization of previously identified virtual screening hits 6 and 7 that result in the development of P2Y14R antagonists with a novel 2-phenyl-benzoxazole acetamide chemical scaffold. Notably, compound 52 showed potent P2Y14R antagonistic activity (IC50 = 2 nM), and a stronger inhibitory effect on MSU-induced inflammatory in vitro, better than a previously described P2Y14R antagonist PPTN. In vivo evaluation demonstrated that compound 52 also had satisfactory inhibitory activity on the inflammatory response of gout flares in mice. Moreover, P2Y14R antagonist 52 decreased paw swelling and inflammatory cell infiltration through cAMP/NLRP3/GSDMD signaling pathways in MSU-induced acute gouty arthritis mice. The discussions on the binding mechanism that employ MM/GBSA free energy calculations/decompositions also provide some useful clues for further structural designing of compound 52. Taken together, 2-phenyl-benzoxazole acetamide derivative 52 with potent P2Y14R antagonistic activity and in vivo potency could be a promising strategy for gout therapy and deserves further optimization.

ATP induced calcium signaling activity in perivascular cells differ at different vascular branch levels in the porcine retina.

BACKGROUND: The purine adenosine triphosphate (ATP) plays a significant role in retinal blood flow regulation and recent evidence suggests that the vasoactive effect of the compound differs in vessels at different branching level. However, the cellular basis for the regulation of retinal blood flow mediated by ATP has only been scarcely studied. METHODS: Perfused porcine hemiretinas (n = 60) were loaded with the calcium-sensitive fluorophore Oregon Green ex vivo. Spontaneous oscillations in fluorescence were studied in perivascular cells at five different vascular branching levels ranging from the main arteriole to the capillaries, before and after the addition of intra- and extravascular ATP alone or in the presence of a P2-purinergic receptor antagonist. RESULTS: Intravascular ATP induced an overall significant (p < 0.01) constriction of (mean ± SD) -9.79 ± 13.40% and extravascular ATP an overall significant (p < 0.01) dilatation of (mean ± SD) 19.62 ± 13.47%. Spontaneous oscillations of fluorescence in perivascular cells were significantly more intense around third order arterioles than around vessels at both lower and higher branching levels (p < 0.05 for all comparisons). ATP increased intracellular fluorescence in perivascular cells of first and second order arterioles after extravascular application, and the increase correlated with the accompanying vasodilatation (p < 0.03). Blocking of P2-receptors reduced oscillating fluorescence in pre-capillary arterioles secondary to intravascular ATP (p = 0.03). CONCLUSIONS: Spontaneous oscillations of calcium-sensitive fluorescence in perivascular retinal cells differ at different vascular branching levels. Extravascular ATP increases fluorescence in cells around the larger retinal arterioles exposed to the retinal surface. Future studies should investigate calcium signaling activity in perivascular retinal cells during interventions that simulate retinal pathology such as hypoxia.

Expression of purinergic receptors on microglia in the animal model of choroidal neovascularisation.

To investigate the effect of P2 receptor on microglia and its inhibitor PPADS on choroidal neovascularization. Forty CX3CR1GFP/+ mice were randomly divided into 8 groups. In addition to the normal group, the rest of groups were receiving laser treatment. The retina and choroid from the second, third, fourth and fifth group of mice were taken in the 1, 4, 7, 14 days after laser treatment. The mice in the sixth and seventh group received intravitreal injection of 2 µl PPADS or PBS respectively immediately after laser treatment. The mice in the eighth group received topical application of PPADS once per day of three days. The mice in sixth, seventh and eighth group received AF and FFA examination on the fourth day after laser treatment. Immunofluorescence histochemical staining and real-time quantitative PCR were used to evaluate P2 expression and its effect on choroidal neovascularization. After laser treatment, activated microglia can express P2 receptors (P2X4, P2X7, P2Y2 and P2Y12). The expression of P2 increased on the first day after laser damage, peaked on the fourth day (tP2X4 = 6.05, tP2X7 = 2.95, tP2Y2 = 3.67, tP2Y12 = 5.98, all P < 0.01), and then decreased. After PPADS inhibition, compared with the PBS injection group, the mRNA of P2X4, P2X7, P2Y2 and P2Y12 were decreased significantly in the PPADS injection group (tP2X4 = 5.54, tP2X7 = 9.82, tP2Y2 = 3.86, tP2Y12 = 7.91, all P < 0.01) and the PPADS topical application group (tP2X4 = 3.24, tP2X7 = 5.89, tP2Y2 = 6.75, tP2Y12 = 4.97, all P < 0.01). Compared with the PBS injection group, not only the activity of microglia cells but also the leakage of CNV decreased significantly (P < 0.01) in the PPADS injection group and the PPADS topical application group. But between two PPADS groups, the leakage of CNV had no difference (P = 0.864). After laser induced CNV, activated microglia can express P2 receptors. The P2 receptor inhibitor, PPADS, can significantly affect the function of microglia and inhibit the formation of choroidal neovascularization.

Extracellular ATP and Adenosine in Cancer Pathogenesis and Treatment.

ATP hydrolysis and downstream signaling pathways in the extracellular space have a major impact upon tumor progression and metastasis. The complexity and interdependence of various cell types in the extracellular space have been increasingly appreciated in recent years. With increased awareness of the importance of this signaling pathway in the pathogenic development and progression of malignancies, there has been attention to therapeutic strategies targeting extracellular adenosine metabolism and signaling. This review summarizes the molecular and physiologic roles of extracellular ATP and adenosine in normal and disease states, and potential therapeutic applications.

Design, synthesis and evaluation of 3-amide-5-aryl benzoic acid derivatives as novel P2Y14R antagonists with potential high efficiency against acute gouty arthritis.

P2Y14 nucleotide receptor plays important roles in series of physiological and pathologic events especially associated with immune and inflammation. Based on the 3-amide benzoic acid scaffold reported by our group previously, a series of 5-aryl-3-amide benzoic acid derivatives were designed as novel P2Y14 antagonists with improved pharmacokinetic properties. Among which compound 11m showed most potent P2Y14 antagonizing activity with an IC50 value of 2.18 nM, furnishing greatly improved water solubility and bioavailability compared with PPTN. In MSU-induced acute gouty arthritis model in mice, 11m exerted promising in vivo efficacy in alleviating mice paw swelling and inflammatory infiltration. Mechanistically, compound 11m notably blocked pyroptosis of macrophages through inhibiting NLRP3 inflammasome activation. This work may contribute to the identification of potential therapeutic agents to intervene in acute gouty arthritis.

Enhanced sympathetic neurotransduction in the superior mesenteric artery in a rat model of heart failure: role of noradrenaline and ATP.

Heart failure (HF) is associated with neurohumoral activation, which in turn leads to an increased peripheral resistance. In mesenteric vasculature, perivascular innervation plays relevant role maintaining vascular tonus and resistance. Therefore, we aimed to determine the possible alterations in superior mesenteric artery (SMA) perivascular innervation function in HF rats. HF was induced by coronary artery occlusion in male Wistar rats, and sham-operated (SO) rats were used as controls. After 12 wk, a greater vasoconstrictor response to electrical field stimulation (EFS) was observed in endothelium-intact and endothelium-denuded SMA of HF rats. Alpha-adrenoceptor antagonist phentolamine diminished this response in a higher magnitude in HF than in SO animals. However, the noradrenaline (NA) reuptake inhibitor desipramine increased EFS-induced vasoconstriction more in segments from HF rats. Besides, EFS-induced NA release was greater in HF animals, due to a higher tyrosine hydroxylase expression and activity. P2 purinoceptor antagonist suramin reduced EFS-induced vasoconstriction only in segments from SO rats, and adenosine 5'-triphosphate (ATP) release was lower in HF than in SO. Moreover, nitric oxide (NO) synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) enhanced EFS-induced vasoconstriction in a similar extent in both groups. HF was not associated with changes in EFS-induced NO release or the vasodilator response to NO donor sodium nitroprusside. In conclusion, HF postmyocardial infarction enhanced noradrenergic function and diminished purinergic cotransmission in SMA and did not change nitrergic innervation. The net effect was an increased sympathetic participation on the EFS-induced vasoconstriction that could help to understand the neurotransduction involved on the control of vascular tonus in HF.NEW & NOTEWORTHY This study reinforces the pivotal role of noradrenergic innervation in the regulation of mesenteric vascular tone in a rat model of heart failure. Moreover, our results highlight the counteracting role of ATP and NA reuptake, and help to understand the signaling pathways involved on the control of vascular tonus and resistance in heart failure postmyocardial infarction.

Neonatal Seizures and Purinergic Signalling.

Neonatal seizures are one of the most common comorbidities of neonatal encephalopathy, with seizures aggravating acute injury and clinical outcomes. Current treatment can control early life seizures; however, a high level of pharmacoresistance remains among infants, with increasing evidence suggesting current anti-seizure medication potentiating brain damage. This emphasises the need to develop safer therapeutic strategies with a different mechanism of action. The purinergic system, characterised by the use of adenosine triphosphate and its metabolites as signalling molecules, consists of the membrane-bound P1 and P2 purinoreceptors and proteins to modulate extracellular purine nucleotides and nucleoside levels. Targeting this system is proving successful at treating many disorders and diseases of the central nervous system, including epilepsy. Mounting evidence demonstrates that drugs targeting the purinergic system provide both convulsive and anticonvulsive effects. With components of the purinergic signalling system being widely expressed during brain development, emerging evidence suggests that purinergic signalling contributes to neonatal seizures. In this review, we first provide an overview on neonatal seizure pathology and purinergic signalling during brain development. We then describe in detail recent evidence demonstrating a role for purinergic signalling during neonatal seizures and discuss possible purine-based avenues for seizure suppression in neonates.

P2 Receptors Influence hMSCs Differentiation towards Endothelial Cell and Smooth Muscle Cell Lineages.

BACKGROUND: Human mesenchymal stem cells (hMSCs) have shown their multipotential including differentiating towards endothelial and smooth muscle cell lineages, which triggers a new interest for using hMSCs as a putative source for cardiovascular regenerative medicine. Our recent publication has shown for the first time that purinergic 2 receptors are key players during hMSC differentiation towards adipocytes and osteoblasts. Purinergic 2 receptors play an important role in cardiovascular function when they bind to extracellular nucleotides. In this study, the possible functional role of purinergic 2 receptors during MSC endothelial and smooth muscle differentiation was investigated. METHODS AND RESULTS: Human MSCs were isolated from liposuction materials. Then, endothelial and smooth muscle-like cells were differentiated and characterized by specific markers via Reverse Transcriptase-PCR (RT-PCR), Western blot and immunochemical stainings. Interestingly, some purinergic 2 receptor subtypes were found to be differently regulated during these specific lineage commitments: P2Y4 and P2Y14 were involved in the early stage commitment while P2Y1 was the key player in controlling MSC differentiation towards either endothelial or smooth muscle cells. The administration of natural and artificial purinergic 2 receptor agonists and antagonists had a direct influence on these differentiations. Moreover, a feedback loop via exogenous extracellular nucleotides on these particular differentiations was shown by apyrase digest. CONCLUSIONS: Purinergic 2 receptors play a crucial role during the differentiation towards endothelial and smooth muscle cell lineages. Some highly selective and potent artificial purinergic 2 ligands can control hMSC differentiation, which might improve the use of adult stem cells in cardiovascular tissue engineering in the future.

Plant natural products as source of new P2 receptors ligands.

In recent years, interest in the research of P2 receptor (P2R)-mediated responses has grown significantly due to the recognition of the involvement of these receptors in various physiological and pathological processes. Despite all the progress made in the functional characterization of P2Rs, purinergic signaling research is still limited by the lack of selective or efficient ligands for different receptor subtypes. In this sense, several molecules have been tested towards these receptors as agonists or antagonists. Historically, natural products have always been sources of new bioactive substances for diverse purposes. However, compared to synthetic molecules, the number of natural products assessed for P2R ligands is still low. In this review, we present examples of studies that demonstrated plant natural products acting directly on P2R and modulating their functionality. In some cases, we highlight that the pharmacological activity previously described for the original organism could be correlated to an agonist or antagonist activity of a specific natural product on these receptors. These examples reinforce the need for more studies to investigate the pharmacological potential of new or known natural compounds targeting P2 receptors.

Exploration of Alternative Scaffolds for P2Y14 Receptor Antagonists Containing a Biaryl Core.

Various heteroaryl and bicyclo-aliphatic analogues of zwitterionic biaryl P2Y14 receptor (P2Y14R) antagonists were synthesized, and affinity was measured in P2Y14R-expressing Chinese hamster ovary cells by flow cytometry. Given this series' low water solubility, various polyethylene glycol derivatives of the distally binding piperidin-4-yl moiety of moderate affinity were synthesized. Rotation of previously identified 1,2,3-triazole attached to the central m-benzoic acid core (25) provided moderate affinity but not indole and benzimidazole substitution of the aryl-triazole. The corresponding P2Y14R region is predicted by homology modeling as a deep, sterically limited hydrophobic pocket, with the outward pointing piperidine moiety being the most flexible. Bicyclic-substituted piperidine ring derivatives of naphthalene antagonist 1, e.g., quinuclidine 17 (MRS4608, IC50 ≈ 20 nM at hP2Y14R/mP2Y14R), or of triazole 2, preserved affinity. Potent antagonists 1, 7a, 17, and 23 (10 mg/kg) protected in an ovalbumin/Aspergillus mouse asthma model, and PEG conjugate 12 reduced chronic pain. Thus, we expanded P2Y14R antagonist structure-activity relationship, introducing diverse physical-chemical properties.

The role of P2Y6R in cardiovascular diseases and recent development of P2Y6R antagonists.

As a member of the P2Y receptor family with a typical 7-transmembrane structure, P2Y6 purinergic receptor (P2Y6R) belongs to the G-protein-coupled nucleotide receptor activating the phospholipase-C signaling pathway. P2Y6R is widely involved in a range of human diseases, including atherosclerosis and other cardiovascular diseases, gradually attracting attention owing to its inappropriate or excessive activation. In addition, it was reported that P2Y6R might regulate inflammatory responses by governing the maturation and secretion of proinflammatory cytokines. Hence, several P2Y6R antagonists have been subjected to evaluation as new therapeutic strategies in recent years. This review was aimed at summarizing the role of P2Y6R in the pathogenesis of cardiovascular diseases, with an insight into the recent progress on discovery of P2Y6R antagonists.

Agonists and Antagonists for Purinergic Receptors.

Membrane receptors that are activated by the purine nucleoside adenosine (adenosine receptors) or by purine or pyrimidine nucleotides (P2Y and P2X receptors) transduce extracellular signals to the cytosol. They play important roles in physiology and disease. The G protein-coupled adenosine receptors comprise four subtypes: A1, A2A, A2B, and A3. The G-protein-coupled P2Y receptors are subdivided into eight subtypes: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14, while the P2X receptors represent ATP-gated homomeric or heteromeric ion channels consisting of three subunits; the most important subunits are P2X1, P2X2, P2X3, P2X4, and P2X7. This chapter provides guidance for selecting suitable tool compounds for studying these large and important purine receptor families.

P2 Receptors in Cardiac Myocyte Pathophysiology and Mechanotransduction.

ATP is a major energy source in the mammalian cells, but it is an extracellular chemical messenger acting on P2 purinergic receptors. A line of evidence has shown that ATP is released from many different types of cells including neurons, endothelial cells, and muscle cells. In this review, we described the distribution of P2 receptor subtypes in the cardiac cells and their physiological and pathological roles in the heart. So far, the effects of external application of ATP or its analogues, and those of UTP on cardiac contractility and rhythm have been reported. In addition, specific genetic alterations and pharmacological agonists and antagonists have been adopted to discover specific roles of P2 receptor subtypes including P2X4-, P2X7-, P2Y2- and P2Y6-receptors in cardiac cells under physiological and pathological conditions. Accumulated data suggest that P2X4 receptors may play a beneficial role in cardiac muscle function, and that P2Y2- and P2Y6-receptors can induce cardiac fibrosis. Recent evidence further demonstrates P2Y1 receptor and P2X4 receptor as important mechanical signaling molecules to alter membrane potential and Ca2+ signaling in atrial myocytes and their uneven expression profile between right and left atrium.

Effects of ticagrelor, empagliflozin and tamoxifen against experimentally-induced vascular reactivity defects in rats in vivo and in vitro.

BACKGROUND: In the current investigation, the effects of the P2Y12 blocker ticagrelor, the sodium/glucose transporter-2-inhibitor empagliflozin, and the selective estrogen receptor modulator tamoxifen were examined against rheumatoid arthritis (RA)/diabetes mellitus (DM)-co-morbidity-induced defects in vascular reactivity. METHODS: After model setting, rats were allocated into a normal control, an RA/DM-co-morbidity, and three treatment groups receiving ticagrelor, empagliflozin and tamoxifen. Aorta tissue was isolated for enzyme-linked immunosorbent assay (ELISA) and western blot estimation of the pro-inflammatory molecules angiotensin-II (Ang-II) and endothelin-1 (ET-1), the adhesion molecules P-selectin and vascular cell adhesion molecule-1 (VCAM-1), the energy preserving molecule adenosine-5'-monophosphate-activated protein kinase (AMPK), and the anti-inflammatory molecule vasoactive intestinal peptide (VIP). Estimations of endothelial nitric oxide synthase (eNOS) and matrix metalloproteinase (MMP)-2 were performed immunohistochemically, together with histopathological examination using hematoxylin and eosin and Masson trichrome staining. An in vitro study on rat aortic strips was conducted to assess aorta vasorelaxation. RESULTS: Ticagrelor, empagliflozin and tamoxifen significantly increased aorta tissue AMPK and eNOS and decreased Ang-II, ET-1, P-selectin, VCAM-1 and VIP levels compared with RA/DM-co-morbidity, coupled with improved acetylcholine vasorelaxation in vitro. CONCLUSION: Ticagrelor, empagliflozin and tamoxifen may correct vascular reactivity defects, where modulation of vascular AMPK, eNOS, Ang-II, ET-1, P-selectin, VCAM-1 and MMP-2 underline their protective effects.