Publications by authors named "Kenneth A Jacobson"

479 Publications

Synthesis and evaluation of adenosine derivatives as A, A, A and A adenosine receptor ligands containing boron clusters as phenyl isosteres and selective A agonists.

Eur J Med Chem 2021 Jun 11;223:113607. Epub 2021 Jun 11.

Laboratory of Medicinal Chemistry, Institute of Medical Biology PAS, Lodowa 106, 92-232, Łódź, Poland. Electronic address:

A series of adenosine and 2'-deoxyadenosine pairs modified with a 1,12-dicarba-closo-dodecaborane cluster or alternatively with a phenyl group at the same position was synthesized, and their affinity was determined at A, A, A and A adenosine receptors (ARs). While AR affinity differences were noted, a general tendency to preferentially bind A AR over other ARs was observed for most tested ligands. In particular, 5'-ethylcarbamoyl-N-(3-phenylpropyl)adenosine (18), N-(3-phenylpropyl)-2-chloroadenosine (24) and N-(3-phenylpropyl)adenosine (40) showed nanomolar A affinity (K 4.5, 6.4 and 7.5 nM, respectively). Among the boron cluster-containing compounds, the highest A affinity (K 206 nM) was for adenosine derivative 41 modified at C2. In the matched molecular pairs, analogs bearing boron clusters were found to show lower binding affinity for adenosine receptors than the corresponding phenyl analogs. Nevertheless, interestingly, several boron cluster modified adenosine ligands showed significantly higher A receptor selectivity than the corresponding phenyl analogs: 7vs. 8, 15vs. 16, 17vs. 18.
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http://dx.doi.org/10.1016/j.ejmech.2021.113607DOI Listing
June 2021

Ligand design by targeting a binding site water.

Chem Sci 2020 Nov 19;12(3):960-968. Epub 2020 Nov 19.

Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University SE-75124 Uppsala Sweden

Solvent reorganization is a major driving force of protein-ligand association, but the contribution of binding site waters to ligand affinity is poorly understood. We investigated how altered interactions with a water network can influence ligand binding to a receptor. A series of ligands of the A adenosine receptor, which either interacted with or displaced an ordered binding site water, were studied experimentally and by molecular dynamics simulations. An analog of the endogenous ligand that was unable to hydrogen bond to the ordered water lost affinity and this activity cliff was captured by molecular dynamics simulations. Two compounds designed to displace the ordered water from the binding site were then synthesized and evaluated experimentally, leading to the discovery of an A agonist with nanomolar activity. Calculation of the thermodynamic profiles resulting from introducing substituents that interacted with or displaced the ordered water showed that the gain of binding affinity was enthalpy driven. Detailed analysis of the energetics and binding site hydration networks revealed that the enthalpy change was governed by contributions that are commonly neglected in structure-based drug optimization. In particular, simulations suggested that displacement of water from a binding site to the bulk solvent can lead to large energy contributions. Our findings provide insights into the molecular driving forces of protein-ligand binding and strategies for rational drug design.
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http://dx.doi.org/10.1039/d0sc04938gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8179138PMC
November 2020

Optical control of adenosine A receptor function in psoriasis.

Pharmacol Res 2021 Jun 19;170:105731. Epub 2021 Jun 19.

Pharmacology Unit, Department of Pathology and Experimental Therapeutics, Faculty of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona, L'Hospitalet de Llobregat, Spain; Neuropharmacology and Pain Group, Neuroscience Program, Institut d'Investigació Biomèdica de Bellvitge, IDIBELL, L'Hospitalet de Llobregat, Spain. Electronic address:

Psoriasis is a chronic and relapsing inflammatory skin disease lacking a cure that affects approximately 2% of the population. Defective keratinocyte proliferation and differentiation, and aberrant immune responses are major factors in its pathogenesis. Available treatments for moderate to severe psoriasis are directed to immune system causing systemic immunosuppression over time, and thus concomitant serious side effects (i.e. infections and cancer) may appear. In recent years, the G protein-coupled A receptor (AR) for adenosine has been suggested as a novel and very promising therapeutic target for psoriasis. Accordingly, selective, and high affinity AR agonists are known to induce robust anti-inflammatory effects in animal models of autoimmune inflammatory diseases. Here, we demonstrated the efficacy of a selective AR agonist, namely MRS5698, in preventing the psoriatic-like phenotype in the IL-23 mouse model of psoriasis. Subsequently, we photocaged this molecule with a coumarin moiety to yield the first photosensitive AR agonist, MRS7344, which in photopharmacological experiments prevented the psoriatic-like phenotype in the IL-23 animal model. Thus, we have demonstrated the feasibility of using a non-invasive, site-specific, light-directed approach to psoriasis treatment.
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http://dx.doi.org/10.1016/j.phrs.2021.105731DOI Listing
June 2021

Spinal A adenosine receptor activation acutely restores morphine antinociception in opioid tolerant male rats.

J Neurosci Res 2021 Jun 1. Epub 2021 Jun 1.

Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, AB, Canada.

Opioids are potent analgesics, but their pain-relieving effects diminish with repeated use. The reduction in analgesic potency is a hallmark of opioid analgesic tolerance, which hampers opioid pain therapy. In the central nervous system, opioid analgesia is critically modulated by adenosine, a purine nucleoside implicated in the beneficial and detrimental actions of opioid medications. Here, we focus on the A adenosine receptor (A AR) in opioid analgesic tolerance. Intrathecal administration of the A AR agonist MRS5698 with daily systemic morphine in male rats attenuated the reduction in morphine antinociception over 7 days. In rats with established morphine tolerance, intrathecal MRS5698 partially restored the antinociceptive effects of morphine. However, when MRS5698 was discontinued, these animals displayed a reduced antinociceptive response to morphine. Our results suggest that MRS5698 acutely and transiently potentiates morphine antinociception in tolerant rats. By contrast, in morphine-naïve rats MRS5698 treatment did not impact thermal nociceptive threshold or affect antinociceptive response to a single injection of morphine. Furthermore, we found that morphine-induced adenosine release in cerebrospinal fluid was blunted in tolerant animals, but total spinal A AR expression was not affected. Collectively, our findings indicate that spinal A AR activation acutely potentiates morphine antinociception in the opioid tolerant state.
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http://dx.doi.org/10.1002/jnr.24869DOI Listing
June 2021

Structure-activity relationships of pyrimidine nucleotides containing a 5'-α,β-methylene diphosphonate at the P2Y receptor.

Bioorg Med Chem Lett 2021 Aug 26;45:128137. Epub 2021 May 26.

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

The G-coupled P2Y receptor (P2YR) is a component of the purinergic signaling system and functions in inflammatory, cardiovascular and metabolic processes. UDP, the native P2YR agonist and P2YR partial agonist, is subject to hydrolysis by ectonucleotidases. Therefore, we have synthesized UDP/CDP analogues containing a stabilizing α,β-methylene bridge as P2YR agonists and identified compatible affinity-enhancing pyrimidine modifications. A distal binding region on the receptor was explored with 4-benzyloxyimino cytidine 5'-diphosphate analogues and their potency determined in a calcium mobilization assay. A 4-trifluoromethyl-benzyloxyimino substituent in 25 provided the highest human P2YR potency (MRS4554, 0.57 µM), and a 5-fluoro substitution of the cytosine ring in 28 similarly enhanced potency, with >175- and 39-fold selectivity over human P2YR, respectively. However, 3-alkyl (31-33, 37, 38), β-d-arabinofuranose (39) and 6-aza (40) substitution prevented P2YR activation. Thus, we have identified new α,β-methylene bridged N-extended CDP analogues as P2YR agonists that are highly selective over the P2YR.
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http://dx.doi.org/10.1016/j.bmcl.2021.128137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8276771PMC
August 2021

Adipocyte P2Y14 receptors play a key role in regulating whole-body glucose and lipid homeostasis.

JCI Insight 2021 May 24;6(10). Epub 2021 May 24.

Molecular Recognition Section and.

Obesity is the major driver of the worldwide epidemic in type 2 diabetes (T2D). In the obese state, chronically elevated plasma free fatty acid levels contribute to peripheral insulin resistance, which can ultimately lead to the development of T2D. For this reason, drugs that are able to regulate lipolytic processes in adipocytes are predicted to have considerable therapeutic potential. Gi-coupled P2Y14 receptor (P2Y14R; endogenous agonist, UDP-glucose) is abundantly expressed in both mouse and human adipocytes. Because activated Gi-type G proteins exert an antilipolytic effect, we explored the potential physiological relevance of adipocyte P2Y14Rs in regulating lipid and glucose homeostasis. Metabolic studies indicate that the lack of adipocyte P2Y14R enhanced lipolysis only in the fasting state, decreased body weight, and improved glucose tolerance and insulin sensitivity. Mechanistic studies suggested that adipocyte P2Y14R inhibits lipolysis by reducing lipolytic enzyme activity, including ATGL and HSL. In agreement with these findings, agonist treatment of control mice with a P2Y14R agonist decreased lipolysis, an effect that was sensitive to inhibition by a P2Y14R antagonist. In conclusion, we demonstrate that adipose P2Y14Rs were critical regulators of whole-body glucose and lipid homeostasis, suggesting that P2Y14R antagonists might be beneficial for the therapy of obesity and T2D.
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http://dx.doi.org/10.1172/jci.insight.146577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262345PMC
May 2021

Adenosine Metabotropic Receptors in Chronic Pain Management.

Front Pharmacol 2021 16;12:651038. Epub 2021 Apr 16.

Department of Pharmacology and Physiology, Saint Louis University School of Medicine, St. Louis, MO, United States.

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http://dx.doi.org/10.3389/fphar.2021.651038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085424PMC
April 2021

Design and Synthesis of 2,6-Disubstituted-4'-Selenoadenosine-5'-,-Dimethyluronamide Derivatives as Human A Adenosine Receptor Antagonists.

Pharmaceuticals (Basel) 2021 Apr 14;14(4). Epub 2021 Apr 14.

Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 08826, Korea.

A new series of 4'-selenoadenosine-5'--dimethyluronamide derivatives as highly potent and selective human A adenosine receptor (hAAR) antagonists, is described. The highly selective AAR agonists, 4'-selenoadenosine-5'--methyluronamides were successfully converted into selective antagonists by adding a second -methyl group to the 5'-uronamide position. All the synthesized compounds showed medium to high binding affinity at the hAAR. Among the synthesized compounds, 2-H--3-iodobenzylamine derivative exhibited the highest binding affinity at hAAR. ( = 22.7 nM). The 2-H analogues generally showed better binding affinity than the 2-Cl analogues. The cAMP functional assay with 2-Cl--3-iodobenzylamine derivative demonstrated hAAR antagonist activity. A molecular modelling study suggests an important role of the hydrogen of 5'-uronamide as an essential hydrogen bonding donor for hAAR activation.
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http://dx.doi.org/10.3390/ph14040363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071163PMC
April 2021

Adenosine Kinase Expression Determines DNA Methylation in Cancer Cell Lines.

ACS Pharmacol Transl Sci 2021 Apr 16;4(2):680-686. Epub 2021 Feb 16.

Department of Neurosurgery, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey 08854, United States.

DNA methylation has a major role in cancer, and its inhibitors are used therapeutically. DNA methylation depends on methyl group flux through the transmethylation pathway, which forms adenosine. We hypothesized that an adenosine kinase isoform with nuclear expression (ADK-L) determines global DNA methylation in cancer cells. We quantified ADK-L expression (Western Blot) and global DNA methylation as percent 5-methyldeoxycytidine (5mdC, LC-MS/MS) in three cancer lines (HeLa, HepG2, and U373). ADK-L expression and global DNA methylation correlated positively with the highest levels in HeLa cells compared to U373 and HepG2 cells. To determine whether ADK increases global DNA methylation and to validate its potential therapeutics, we treated HeLa cells with potent ADK inhibitors MRS4203 and MRS4380 (IC 88 and 140 nM, respectively). Both nucleosides, but not a structurally related poor ADK inhibitor, significantly reduced global DNA methylation in HeLa cells in a concentration-dependent manner. Thus, ADK-L is a potential target for the therapeutic manipulation of DNA methylation levels in cancer.
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http://dx.doi.org/10.1021/acsptsci.1c00008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033756PMC
April 2021

Structure activity relationship of 3-nitro-2-(trifluoromethyl)-2H-chromene derivatives as P2Y receptor antagonists.

Bioorg Med Chem Lett 2021 06 6;41:128008. Epub 2021 Apr 6.

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

Various 6-alkynyl analogues of a known 3-nitro-2-(trifluoromethyl)-2H-chromene antagonist 3 of the G-coupled P2Y receptor (P2YR) were synthesized using a Sonogashira reaction to replace a 6-iodo group. The analogues were tested in a functional assay consisting of inhibition of calcium mobilization in P2YR-expressing astrocytoma cells elicited by native P2YR agonist UDP. 6-Ethynyl and 6-cyano groups were installed, and the alkynes were extended through both alkyl and aryl spacers. The most potent antagonists, with IC of ~1 µM, were found to be trialkylsilyl-ethynyl 7 and 8 (3-5 fold greater affinity than reference 3), t-butyl prop-2-yn-1-ylcarbamate 14 and p-carboxyphenyl-ethynyl 16 derivatives, and 3 and 8 displayed surmountable antagonism of UDP-induced production of inositol phosphates. Other chain-extended terminal carboxylate derivatives were less potent than the corresponding methyl ester derivatives. Thus, the 6 position in this chromene series is suitable for derivatization with flexibility of substitution, even with sterically extended chains, without losing P2YR affinity. However, a 3-carboxylic acid or 3-ester substitution did not serve as a nitro bioisostere, as the affinity was eliminated. These compounds provide additional ligand tools for the underexplored P2YR, which is a target for inflammatory, neurodegenerative and metabolic diseases.
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http://dx.doi.org/10.1016/j.bmcl.2021.128008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8240625PMC
June 2021

Adenosine A Receptors Are Upregulated in Peripheral Blood Mononuclear Cells from Atrial Fibrillation Patients.

Int J Mol Sci 2021 Mar 27;22(7). Epub 2021 Mar 27.

Pharmacology Unit, Department of Pathology and Experimental Therapeutics, Faculty of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona, 08907 L'Hospitalet de Llobregat, Spain.

Atrial fibrillation (AF) is the most common form of cardiac arrhythmia seen in clinical practice. While some clinical parameters may predict the transition from paroxysmal to persistent AF, the molecular mechanisms behind the AF perpetuation are poorly understood. Thus, oxidative stress, calcium overload and inflammation, among others, are believed to be involved in AF-induced atrial remodelling. Interestingly, adenosine and its receptors have also been related to AF development and perpetuation. Here, we investigated the expression of adenosine A2A receptor (A2AR) both in right atrium biopsies and peripheral blood mononuclear cells (PBMCs) from non-dilated sinus rhythm (ndSR), dilated sinus rhythm (dSR) and AF patients. In addition, plasma adenosine content and adenosine deaminase (ADA) activity in these subjects were also determined. Our results revealed increased A2AR expression in the right atrium from AF patients, as previously described. Interestingly, increased levels of adenosine content and reduced ADA activity in plasma from AF patients were detected. An increase was observed when A2AR expression was assessed in PBMCs from AF subjects. Importantly, a positive correlation (P=0.001) between A2AR expression in the right atrium and PBMCs was observed. Overall, these results highlight the importance of the A2AR in AF and suggest that the evaluation of this receptor in PBMCs may be potentially be useful in monitoring disease severity and the efficacy of pharmacological treatments in AF patients.
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http://dx.doi.org/10.3390/ijms22073467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036820PMC
March 2021

UDP-glucose and P2Y14 receptor amplify allergen-induced airway eosinophilia.

J Clin Invest 2021 Apr;131(7)

Immunity, Inflammation and Disease Laboratory.

Airway eosinophilia is a hallmark of allergic asthma and is associated with mucus production, airway hyperresponsiveness, and shortness of breath. Although glucocorticoids are widely used to treat asthma, their prolonged use is associated with several side effects. Furthermore, many individuals with eosinophilic asthma are resistant to glucocorticoid treatment, and they have an unmet need for novel therapies. Here, we show that UDP-glucose (UDP-G), a nucleotide sugar, is selectively released into the airways of allergen-sensitized mice upon their subsequent challenge with that same allergen. Mice lacking P2Y14R, the receptor for UDP-G, had decreased airway eosinophilia and airway hyperresponsiveness compared with wild-type mice in a protease-mediated model of asthma. P2Y14R was dispensable for allergic sensitization and for the production of type 2 cytokines in the lung after challenge. However, UDP-G increased chemokinesis in eosinophils and enhanced their response to the eosinophil chemoattractant, CCL24. In turn, eosinophils triggered the release of UDP-G into the airway, thereby amplifying eosinophilic recruitment. This positive feedback loop was sensitive to therapeutic intervention, as a small molecule antagonist of P2Y14R inhibited airway eosinophilia. These findings thus reveal a pathway that can be therapeutically targeted to treat asthma exacerbations and glucocorticoid-resistant forms of this disease.
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http://dx.doi.org/10.1172/JCI140709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011887PMC
April 2021

Structure-Activity Relationship of Heterocyclic P2Y Receptor Antagonists: Removal of the Zwitterionic Character with Piperidine Bioisosteres.

J Med Chem 2021 04 31;64(8):5099-5122. Epub 2021 Mar 31.

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States.

A known zwitterionic, heterocyclic P2YR antagonist was substituted with diverse groups on the central phenyl and terminal piperidine moieties, following a computational selection process. The most potent analogues contained an uncharged piperidine bioisostere, prescreened in silico, while an aza-scan (central phenyl ring) reduced P2YR affinity. Piperidine amide , 3-aminopropynyl , and 5-(hydroxymethyl)isoxazol-3-yl) congeners in the triazole series maintained moderate receptor affinity. Adaption of 5-(hydroxymethyl)isoxazol-3-yl gave the most potent naphthalene-containing (; MRS4654; IC, 15 nM) and less active phenylamide-containing () scaffolds. Thus, a zwitterion was nonessential for receptor binding, and molecular docking and dynamics probed the hydroxymethylisoxazole interaction with extracellular loops. Also, amidomethyl ester prodrugs were explored to reversibly block the conserved carboxylate group to provide neutral analogues, which were cleavable by liver esterase, and in vivo efficacy demonstrated. We have, in stages, converted zwitterionic antagonists into neutral molecules designed to produce potent P2YR antagonists for in vivo application.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00164DOI Listing
April 2021

Biological Evaluation of 5'-(-Ethylcarboxamido)adenosine Analogues as Grp94-Selective Inhibitors.

ACS Med Chem Lett 2021 Mar 1;12(3):373-379. Epub 2021 Mar 1.

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States.

The heat shock protein 90 kDa (Hsp90) family of chaperones is highly sought-after for the treatment of cancer and neurodegenerative diseases. Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum localized isoform that is responsible for the maturation of proteins involved in cell adhesion and the immune response, including Toll-like receptors, immunoglobulins, and integrins. Consequently, Grp94 has been implicated in many different diseases including cancer metastasis, glaucoma, and viral infection. 5'-(-Ethylcarboxamido)adenosine (NECA) was identified from a high-throughput screen as one of the first molecules to exhibit isoform selectivity toward Grp94, with the ethyl group projecting into a unique pocket within the ATP binding site of Grp94. This pocket has since been exploited by several groups to develop Grp94 selective inhibitors. Despite success in the development of other classes of inhibitors, relatively little work has been done to further develop inhibitors with the NECA scaffold. Unfortunately, NECA is also a potent adenosine receptor agonist, which is likely to confound any biological activity. Therefore, structure-activity relationship studies were performed on the NECA scaffold leading to the discovery of several molecules that displayed similar selectivity and affinity as the parent compound.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957913PMC
March 2021

Adenosine A3 agonists reverse neuropathic pain via T cell-mediated production of IL-10.

J Clin Invest 2021 Apr;131(7)

Department of Pharmacology and Physiology, Saint Louis University School of Medicine, St. Louis, Missouri, USA.

The A3 adenosine receptor (A3AR) has emerged as a therapeutic target with A3AR agonists to tackle the global challenge of neuropathic pain, and investigation into its mode of action is essential for ongoing clinical development. Immune cell A3ARs, and their activation during pathology, modulate cytokine release. Thus, the use of immune cells as a cellular substrate for the pharmacological action of A3AR agonists is enticing, but unknown. The present study discovered that Rag-KO mice lacking T and B cells, as compared with WT mice, are insensitive to the anti-allodynic effects of A3AR agonists. Similar findings were observed in interleukin-10 and interleukin-10 receptor knockout mice. Adoptive transfer of CD4+ T cells from WT mice infiltrated the dorsal root ganglion (DRG) and restored A3AR agonist-mediated anti-allodynia in Rag-KO mice. CD4+ T cells from Adora3-KO or Il10-KO mice did not. Transfer of CD4+ T cells from WT mice, but not Il10-KO mice, into Il10-KO mice or Adora3-KO mice fully reinstated the anti-allodynic effects of A3AR activation. Notably, A3AR agonism reduced DRG neuron excitability when cocultured with CD4+ T cells in an IL-10-dependent manner. A3AR action on CD4+ T cells infiltrated in the DRG decreased phosphorylation of GluN2B-containing N-methyl-D-aspartate receptors at Tyr1472, a modification associated with regulating neuronal hypersensitivity. Our findings establish that activation of A3AR on CD4+ T cells to release IL-10 is required and sufficient evidence for the use of A3AR agonists as therapeutics.
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http://dx.doi.org/10.1172/JCI139299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011899PMC
April 2021

Editorial: Geoffrey Burnstock - An Accidental Pharmacologist.

Biochem Pharmacol 2021 May 19;187:114421. Epub 2021 Jan 19.

Ohio State University, Columbus, OH, USA. Electronic address:

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http://dx.doi.org/10.1016/j.bcp.2021.114421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8236242PMC
May 2021

Novel cyanothiouracil and cyanothiocytosine derivatives as concentration-dependent selective inhibitors of U87MG glioblastomas: Adenosine receptor binding and potent PDE4 inhibition.

Eur J Med Chem 2021 Feb 31;212:113125. Epub 2020 Dec 31.

Istanbul Medipol University, School of Pharmacy, Department of Pharmaceutical Chemistry, Istanbul, Turkey.

Thiouracil and thiocytosine are important heterocyclic pharmacophores having pharmacological diversity. Antitumor and antiviral activity is commonly associated with thiouracil and thiocytosine derivatives, which are well known fragments for adenosine receptor affinity with many associated pharmacological properties. In this respect, 33 novel compounds have been synthesized in two groups: 24 thiouracil derivatives (4a-x) and 9 thiocytosine derivatives (5a-i). Antitumor activity of all the compounds was determined in the U87 MG glioblastoma cell line. Compound 5e showed an anti-proliferative IC of 1.56 μM, which is slightly higher activity than cisplatin (1.67 μM). The 11 most active compounds showed no signficant binding to adenosine A, A or A receptors at 1 μM. Brain tumors express high amounts of phosphodiesterases. Compounds were tested for PDE4 inhibition, and 5e and 5f showed the best potency (5e: 3.42 μM; 5f: 0.97 μM). Remakably, those compounds were also the most active against U87MG. However, the compounds lacked a cytotoxic effect on the HEK293 healthy cell line, which encourages further investigation.
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http://dx.doi.org/10.1016/j.ejmech.2020.113125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880896PMC
February 2021

Purinergic signaling in diabetes and metabolism.

Biochem Pharmacol 2021 May 25;187:114393. Epub 2020 Dec 25.

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892, USA. Electronic address:

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 (A, A, A and A), and several of the P2 subtypes (e.g. P2Y 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.
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http://dx.doi.org/10.1016/j.bcp.2020.114393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8096664PMC
May 2021

Activation of neuronal adenosine A1 receptors causes hypothermia through central and peripheral mechanisms.

PLoS One 2020 16;15(12):e0243986. Epub 2020 Dec 16.

Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland, United States of America.

Extracellular adenosine, a danger signal, can cause hypothermia. We generated mice lacking neuronal adenosine A1 receptors (A1AR, encoded by the Adora1 gene) to examine the contribution of these receptors to hypothermia. Intracerebroventricular injection of the selective A1AR agonist (Cl-ENBA, 5'-chloro-5'-deoxy-N6-endo-norbornyladenosine) produced hypothermia, which was reduced in mice with deletion of A1AR in neurons. A non-brain penetrant A1AR agonist [SPA, N6-(p-sulfophenyl) adenosine] also caused hypothermia, in wild type but not mice lacking neuronal A1AR, suggesting that peripheral neuronal A1AR can also cause hypothermia. Mice expressing Cre recombinase from the Adora1 locus were generated to investigate the role of specific cell populations in body temperature regulation. Chemogenetic activation of Adora1-Cre-expressing cells in the preoptic area did not change body temperature. In contrast, activation of Adora1-Cre-expressing dorsomedial hypothalamus cells increased core body temperature, concordant with agonism at the endogenous inhibitory A1AR causing hypothermia. These results suggest that A1AR agonism causes hypothermia via two distinct mechanisms: brain neuronal A1AR and A1AR on neurons outside the blood-brain barrier. The variety of mechanisms that adenosine can use to induce hypothermia underscores the importance of hypothermia in the mouse response to major metabolic stress or injury.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0243986PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7743955PMC
February 2021

Discovery and Structure-Activity Relationships of Novel Template, Truncated 1'-Homologated Adenosine Derivatives as Pure Dual PPARγ/δ Modulators.

J Med Chem 2020 12 16;63(24):16012-16027. Epub 2020 Dec 16.

Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 08826, Korea.

Following our report that A adenosine receptor (AR) antagonist exhibited a polypharmacological profile as a dual modulator of peroxisome proliferator-activated receptor (PPAR)γ/δ, we discovered a new template, 1'-homologated adenosine analogues , as dual PPARγ/δ modulators without AR binding. Removal of binding affinity to AAR was achieved by 1'-homologation, and PPARγ/δ dual modulation was derived from the structural similarity between the target nucleosides and PPAR modulator drug, rosiglitazone. All the final nucleosides were devoid of AR-binding affinity and exhibited high binding affinities to PPARγ/δ but lacked PPARα binding. 2-Cl derivatives exhibited dual receptor-binding affinity to PPARγ/δ, which was absent for the corresponding 2-H derivatives. 2-Propynyl substitution prevented PPARδ-binding affinity but preserved PPARγ affinity, indicating that the C2 position defines a pharmacophore for selective PPARγ ligand designs. PPARγ/δ dual modulators functioning as both PPARγ partial agonists and PPARδ antagonists promoted adiponectin production, suggesting their therapeutic potential against hypoadiponectinemia-associated cancer and metabolic diseases.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8201645PMC
December 2020

Geoffrey Burnstock - An accidental pharmacologist.

Biochem Pharmacol 2021 May 23;187:114300. Epub 2020 Oct 23.

Department of Biological Chemistry and Pharmacology, College of Medicine, Ohio State University, Columbus, OH, United States. Electronic address:

Geoffrey Burnstock, the founder of the field of purinergic signaling research passed away in Melbourne, Australia on June 3rd, 2020, at the age of 91. With his death, the world of biomedical research lost one of its most passionate, creative and unconventional thought leaders. He was an inspiration to the many researchers he interacted with for more than 50 years and a frequent irritation to those in the administrative establishment. Geoff never considered himself a pharmacologist having being trained as a zoologist and becoming an autonomic neurophysiologist based on his evolving interests in systems and disease-related research. By the end of his life he had: published some 1550 papers; been cited more than 125,000 times; had an h-index of 156 and had supervised over 100 Ph.D. students. His indelible legacy, based on a holistic, data-based, multidisciplinary, unconventional "outside the box" approach to research was reflected in two of the seminal findings in late 20th century biomedical research: the purinergic neurotransmitter hypothesis and the concept of co-neurotransmission, both of which were initially received by his peers with considerable skepticism that at times verged on disdain. Nonetheless, while raising hackles and threatening the status quo, Geoff persevered and prevailed, becoming a mentor for several generations of biomedical researchers. In this review we provide a joint perspective on Geoff Burnstock's legacy in research.
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http://dx.doi.org/10.1016/j.bcp.2020.114300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062576PMC
May 2021

Lack of adipocyte purinergic P2Y receptor greatly improves whole body glucose homeostasis.

Proc Natl Acad Sci U S A 2020 12 16;117(48):30763-30774. Epub 2020 Nov 16.

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892;

Uridine diphosphate (UDP)-activated purinergic receptor P2Y (P2YR) plays a crucial role in controlling energy balance through central mechanisms. However, P2YR's roles in peripheral tissues regulating energy and glucose homeostasis remain unexplored. Here, we report the surprising finding that adipocyte-specific deletion of P2YR protects mice from diet-induced obesity, improving glucose tolerance and insulin sensitivity with reduced systemic inflammation. These changes were associated with reduced JNK signaling and enhanced expression and activity of PPARα affecting downstream PGC1α levels leading to beiging of white fat. In contrast, P2YR deletion in skeletal muscle reduced glucose uptake, resulting in impaired glucose homeostasis. Interestingly, whole body P2YR knockout mice showed metabolic improvements similar to those observed with mice lacking P2YR only in adipocytes. Our findings provide compelling evidence that P2YR antagonists may prove useful for the treatment of obesity and type 2 diabetes.
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http://dx.doi.org/10.1073/pnas.2006578117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720204PMC
December 2020

Identification of a New Heterocyclic Scaffold for Inhibitors of the Polo-Box Domain of Polo-like Kinase 1.

J Med Chem 2020 11 11;63(22):14087-14117. Epub 2020 Nov 11.

Chemistry Section, Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States.

As a mitotic-specific target widely deregulated in various human cancers, polo-like kinase 1 (Plk1) has been extensively explored for anticancer activity and drug discovery. Although multiple catalytic domain inhibitors were tested in preclinical and clinical studies, their efficacies are limited by dose-limiting cytotoxicity, mainly from off-target cross reactivity. The C-terminal noncatalytic polo-box domain (PBD) of Plk1 has emerged as an attractive target for generating new protein-protein interaction inhibitors. Here, we identified a 1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-]quinazolin-5(1)-one scaffold that efficiently inhibits Plk1 PBD but not its related Plk2 and Plk3 PBDs. Structure-activity relationship studies led to multiple inhibitors having ≥10-fold higher inhibitory activity than the previously characterized Plk1 PBD-specific phosphopeptide, PLHSpT ( ∼ 450 nM). In addition, -methyl prodrugs effectively inhibited mitotic progression and cell proliferation and their metabolic stability was determined. These data describe a novel class of small-molecule inhibitors that offer a promising avenue for future drug discovery against Plk1-addicted cancers.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7769008PMC
November 2020

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

Biochem Pharmacol 2021 May 29;187:114311. Epub 2020 Oct 29.

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.

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 P2Y, P2Y, P2Y or P2X2 receptor antagonists. A P2X4 antagonist NC-2600 is in a clinical trial, and A 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.
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http://dx.doi.org/10.1016/j.bcp.2020.114311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8081756PMC
May 2021

Nucleotide P2Y receptor agonists are in vitro and in vivo prodrugs of A/A adenosine receptor agonists: implications for roles of P2Y and A/A receptors in physiology and pathology.

Purinergic Signal 2020 12 31;16(4):543-559. Epub 2020 Oct 31.

University of Texas Health at San Antonio, San Antonio, TX, USA.

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y receptor (P2YR) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A and AARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2YR activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.
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http://dx.doi.org/10.1007/s11302-020-09732-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855190PMC
December 2020

Update of P2X receptor properties and their pharmacology: IUPHAR Review 30.

Br J Pharmacol 2021 02 21;178(3):489-514. Epub 2020 Dec 21.

Department of Medical Sciences, Section of Experimental Medicine, University of Ferrara, Ferrara, Italy.

The known seven mammalian receptor subunits (P2X1-7) form cationic channels gated by ATP. Three subunits compose a receptor channel. Each subunit is a polypeptide consisting of two transmembrane regions (TM1 and TM2), intracellular N- and C-termini, and a bulky extracellular loop. Crystallization allowed the identification of the 3D structure and gating cycle of P2X receptors. The agonist-binding pocket is located at the intersection of two neighbouring subunits. In addition to the mammalian P2X receptors, their primitive ligand-gated counterparts with little structural similarity have also been cloned. Selective agonists for P2X receptor subtypes are not available, but medicinal chemistry supplied a range of subtype-selective antagonists, as well as positive and negative allosteric modulators. Knockout mice and selective antagonists helped to identify pathological functions due to defective P2X receptors, such as male infertility (P2X1), hearing loss (P2X2), pain/cough (P2X3), neuropathic pain (P2X4), inflammatory bone loss (P2X5), and faulty immune reactions (P2X7).
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http://dx.doi.org/10.1111/bph.15299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8199792PMC
February 2021

Direct Comparison of (N)-Methanocarba and Ribose-Containing 2-Arylalkynyladenosine Derivatives as A Receptor Agonists.

ACS Med Chem Lett 2020 Oct 14;11(10):1935-1941. Epub 2020 Feb 14.

Laboratory of Bioorganic Chemistry, Mouse Metabolism Core, and Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892, United States.

A side-by-side pharmacological comparison of ribose and (N)-methanocarba (bicyclo[3.1.0]hexane) nucleosides as AAR agonists indicated that the bicyclic pseudoribose ring constraint provided higher affinity/selectivity at human and mouse AAR. The mean affinity enhancement for 5 pairs of 5'-methylamides was 11-fold at hAAR and 42-fold at mAAR. Novel C2-(5-fluorothien-2-ylethynyl) substitution enhanced affinity in the methanocarba but not ribose series, with highly hAAR-selective (MRS7334) displaying K 280 pM and favorable pharmacokinetics and off-target activity profile. Molecular dynamics comparison of and its corresponding riboside suggested a qualitative entropic advantage of in hAAR binding. The 5-F substitution tended to increase hAAR affinity (cf. 5-Cl) for methanocarba but not ribose derivatives. A representative methanocarba agonist was shown to interact potently exclusively with AAR, among 240 GPCRs and 466 kinases. Thus, despite added synthetic difficulty, the (N)-methanocarba modification has distinct advantages for AAR agonists, which have translational potential for chronic disease treatment.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549272PMC
October 2020

Structure activity relationship of novel antiviral nucleosides against Enterovirus A71.

Bioorg Med Chem Lett 2020 12 5;30(23):127599. Epub 2020 Oct 5.

Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, 9000 Rockville Pike, Bethesda, MA 20892, USA. Electronic address:

Various (North)-methanocarba adenosine derivatives, containing rigid bicyclo[3.1.0]hexane ribose substitution, were screened for activity against representative viruses, and inhibition was observed after treatment of Enterovirus A71 with a 2-chloro-N-1-cyclopropyl-2-methylpropan-1-yl derivative (17). µM activity was also seen when testing 17 against other enteroviruses in the Picornaviridae family. Based on this hit, structural congeners of 17, containing other N-alkyl groups and 5' modifications, were synthesized and tested. The structure activity relationship is relatively narrow, with most modifications of the adenine or the methanocarba ring reducing or abolishing the inhibitory potency. 4'-Truncated 31 (MRS5474), 4'-fluoromethyl 48 (MRS7704) and 4'-chloromethyl 49 nucleosides displayed EC ~3-4 µM, and 31 and 48 achieved SI ≥10. However, methanocarba analogues of ribavirin and N-benzyladenosine, shown previously to have anti-EV-A71 activity, were inactive. Thus, we identified methanocarba nucleosides as a new scaffold for enterovirus inhibition with a narrow structure activity relationship and no similarity to previously published anti-enteroviral nucleosides.
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http://dx.doi.org/10.1016/j.bmcl.2020.127599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534897PMC
December 2020

Purinergic Signaling: Impact of GPCR Structures on Rational Drug Design.

ChemMedChem 2020 Nov 18;15(21):1958-1973. Epub 2020 Sep 18.

Laboratory of Bioorganic Chemistry, National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

The purinergic signaling system includes membrane-bound receptors for extracellular purines and pyrimidines, and enzymes/transporters that regulate receptor activation by endogenous agonists. Receptors include: adenosine (A , A , A and A ) and P2Y (P2Y , P2Y , P2Y , P2Y , P2Y , P2Y , P2Y , and P2Y ) receptors (all GPCRs), as well as P2X receptors (ion channels). Receptor activation, especially accompanying physiological stress or damage, creates a temporal sequence of signaling to counteract this stress and either mobilize (P2Rs) or suppress (ARs) immune responses. Thus, modulation of this large signaling family has broad potential for treating chronic diseases. Experimentally determined structures represent each of the three receptor families. We focus on selective purinergic agonists (A , A ), antagonists (A , P2Y ), and allosteric modulators (P2Y , A ). Examples of applying structure-based design, including the rational modification of known ligands, are presented for antithrombotic P2Y R antagonists and anti-inflammatory P2Y R antagonists and A AR agonists. A AR agonists are a potential, nonaddictive treatment for chronic neuropathic pain.
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http://dx.doi.org/10.1002/cmdc.202000465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8276773PMC
November 2020

Tribute to Prof. Geoffrey Burnstock: transition of purinergicsignaling to drug discovery.

Purinergic Signal 2021 03 14;17(1):3-8. Epub 2020 Aug 14.

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Rm. B1A-19, Bldg. 8A, 9000 Rockville Pike, Bethesda, MD, 20892, USA.

Geoffrey Burnstock made a chance observation early in his research career that did not fit the conventional scientific dogma-non-noradrenergic, non-cholinergic (NANC) nerves. Instead of rejecting these as an artifact, he followed their logical course to characterize the actions of extracellular ATP on nerves and muscles, eventually founding a large branch of pharmacology around purinergic signaling. The solid proof that validated his concept and dismissed many detractors was the cloning of seven ionotropic P2X receptors and eight metabotropic P2Y receptors, which are expressed in some combination in every tissue and organ. Given the broad importance of this signaling system in biology, medicinal chemists, inspired by Burnstock, began creating synthetic agonists and antagonists for these purinergic receptors. Various ligands have advanced to clinical trials, for disorders of the immune, nervous, cardiovascular, and other systems, and a few are already approved. Thus, medically important approaches have been derived from Burnstock's original pharmacological concepts and his constant guiding of the course of the field. The therapeutic potential of modulators of purinergic signaling is vast.
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http://dx.doi.org/10.1007/s11302-020-09717-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955018PMC
March 2021
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