Publications by authors named "Dilip K Tosh"

76 Publications

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

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

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

Design and in vivo activity of A adenosine receptor agonist prodrugs.

Purinergic Signal 2020 09 27;16(3):367-377. Epub 2020 Jul 27.

Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bldg. 8A, Rm. B1A-19, 9000 Rockville Pike, Bethesda, MD, 20892-0810, USA.

Prodrugs (MRS7422, MRS7476) of highly selective A adenosine receptor (AR) agonists Cl-IB-MECA and MRS5698, respectively, were synthesized by succinylation of the 2' and 3' hydroxyl groups, and the parent, active drug was shown to be readily liberated upon incubation with liver esterases. The prodrug MRS7476 had greatly increased aqueous solubility compared with parent MRS5698 and was fully efficacious and with a longer duration than MRS7422 in reversing mouse neuropathic pain (chronic constriction injury model, 3 μmol/kg, p.o.), a known AAR effect. MRS7476 (5 mg/kg, p.o., twice daily) was found to protect against non-alcoholic steatohepatitis (NASH) in the STAM mouse model, indicated by the NAFLD activity score. Hepatocyte ballooning, IL-10 production, and liver histology were significantly normalized in the MRS7476-treated mice, but not liver fibrosis (no change in ACTA2 levels) or inflammation. Hepatic expression of ADORA3 in human NAFLD patients was 1.9-fold lower compared to normal controls. Adora3 expression determined by qPCR in primary mouse liver was associated with the stellate cells, and its mouse full body AAR knockout worsened liver markers of inflammation and steatosis. Thus, we have introduced a reversible prodrug strategy that enables water solubility and in vivo activity of masked AAR agonists in models of two disease conditions.
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http://dx.doi.org/10.1007/s11302-020-09715-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524976PMC
September 2020

Chronic Morphine-Induced Changes in Signaling at the A Adenosine Receptor Contribute to Morphine-Induced Hyperalgesia, Tolerance, and Withdrawal.

J Pharmacol Exp Ther 2020 08 20;374(2):331-341. Epub 2020 May 20.

Department of Pharmacology and Physiology and Henry and Amelia Nasrallah Center for Neuroscience, Saint Louis University School of Medicine, St. Louis, Missouri (T.M.D., Z.C., D.S.); Department of Pharmacology, University of Arizona College of Medicine, Tucson, Arizona (T.M.L.-M., T.W.V.); Discipline of Physiology, Institute for Photonics and Advanced Sensing, ARC Centre of Excellence for Nanoscale BioPhotonics, University of Adelaide, Adelaide, Australia (V.S., M.R.H.); Department of Clinical and Experimental Medicine and Pharmacology, University of Messina, Messina, Italy (E.E., S.C.); Departments of Comparative Biology and Experimental Medicine and Physiology and Pharmacology, Hotchkiss Brain Institute, University of Calgary, Calgary, Canada (R.D., C.F., T.T.); Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (D.K.T., K.A.J.); and Department of Anesthesiology, University of California San Diego, La Jolla, California (G.J.B.)

Treating chronic pain by using opioids, such as morphine, is hampered by the development of opioid-induced hyperalgesia (OIH; increased pain sensitivity), antinociceptive tolerance, and withdrawal, which can contribute to dependence and abuse. In the central nervous system, the purine nucleoside adenosine has been implicated in beneficial and detrimental actions of morphine, but the extent of their interaction remains poorly understood. Here, we demonstrate that morphine-induced OIH and antinociceptive tolerance in rats is associated with a twofold increase in adenosine kinase (ADK) expression in the dorsal horn of the spinal cord. Blocking ADK activity in the spinal cord provided greater than 90% attenuation of OIH and antinociceptive tolerance through A adenosine receptor (AAR) signaling. Supplementing adenosine signaling with selective AAR agonists blocked OIH and antinociceptive tolerance in rodents of both sexes. Engagement of AAR in the spinal cord with an ADK inhibitor or AAR agonist was associated with reduced dorsal horn of the spinal cord expression of the NOD-like receptor pyrin domain-containing 3 (60%-75%), cleaved caspase 1 (40%-60%), interleukin (IL)-1 (76%-80%), and tumor necrosis factor (50%-60%). In contrast, the neuroinhibitory and anti-inflammatory cytokine IL-10 increased twofold. In mice, AAR agonists prevented the development of tolerance in a model of neuropathic pain and reduced naloxone-dependent withdrawal behaviors by greater than 50%. These findings suggest AAR-dependent adenosine signaling is compromised during sustained morphine to allow the development of morphine-induced adverse effects. These findings raise the intriguing possibility that AAR agonists may be useful adjunct to opioids to manage their unwanted effects. SIGNIFICANCE STATEMENT: The development of hyperalgesia and antinociceptive tolerance during prolonged opioid use are noteworthy opioid-induced adverse effects that reduce opioid efficacy for treating chronic pain and increase the risk of dependence and abuse. We report that in rodents, these adverse effects are due to reduced adenosine signaling at the AAR, resulting in NOD-like receptor pyrin domain-containing 3-interleukin-1β neuroinflammation in spinal cord. These effects are attenuated by AAR agonists, suggesting that AAR may be a target for therapeutic intervention with selective AAR agonist as opioid adjuncts.
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http://dx.doi.org/10.1124/jpet.120.000004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372916PMC
August 2020

Acute visceral pain relief mediated by A3AR agonists in rats: involvement of N-type voltage-gated calcium channels.

Pain 2020 09;161(9):2179-2190

Department of Neuroscience, Psychology, Drug Research and Child Health-Neurofarba-Division of Pharmacology and Toxicology, Florence, Italy.

Abstract: Pharmacological tools for chronic visceral pain management are still limited and inadequate. A3 adenosine receptor (A3AR) agonists are effective in different models of persistent pain. Recently, their activity has been related to the block of N-type voltage-gated Ca2+ channels (Cav2.2) in dorsal root ganglia (DRG) neurons. The present work aimed to evaluate the efficacy of A3AR agonists in reducing postinflammatory visceral hypersensitivity in both male and female rats. Colitis was induced by the intracolonic instillation of 2,4-dinitrobenzenesulfonic acid (DNBS; 30 mg in 0.25 mL 50% EtOH). Visceral hypersensitivity was assessed by measuring the visceromotor response and the abdominal withdrawal reflex to colorectal distension. The effects of A3AR agonists (MRS5980 and Cl-IB-MECA) were evaluated over time after DNBS injection and compared to that of the selective Cav2.2 blocker PD173212, and the clinically used drug linaclotide. A3AR agonists significantly reduced DNBS-evoked visceral pain both in the postinflammatory (14 and 21 days after DNBS injection) and persistence (28 and 35 days after DNBS) phases. Efficacy was comparable to effects induced by linaclotide. PD173212 fully reduced abdominal hypersensitivity to control values, highlighting the role of Cav2.2. The effects of MRS5980 and Cl-IB-MECA were completely abolished by the selective A3AR antagonist MRS1523. Furthermore, patch-clamp recordings showed that A3AR agonists inhibited Cav2.2 in dorsal root ganglia neurons isolated from either control or DNBS-treated rats. The effect on Ca2+ current was PD173212-sensitive and prevented by MRS1523. A3AR agonists are effective in relieving visceral hypersensitivity induced by DNBS, suggesting a potential therapeutic role against abdominal pain.
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http://dx.doi.org/10.1097/j.pain.0000000000001905DOI Listing
September 2020

Truncated (N)-Methanocarba Nucleosides as Partial Agonists at Mouse and Human A Adenosine Receptors: Affinity Enhancement by -(2-Phenylethyl) Substitution.

J Med Chem 2020 04 9;63(8):4334-4348. Epub 2020 Apr 9.

Department of Pharmacology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, United States.

Dopamine-derived -substituents, compared to -(2-phenylethyl), in truncated (N)-methanocarba (bicyclo[3.1.0]hexyl) adenosines favored high A adenosine receptor (AR) affinity/selectivity, e.g., C2-phenylethynyl analogue (MRS7591, = 10.9/17.8 nM, at human/mouse AAR). was a partial agonist in vitro (hAAR, cAMP inhibition, 31% ; mAAR, [S]GTP-γ-S binding, 16% ) and in vivo and also antagonized hAAR in vitro. Distal H-bonding substitutions of the -(2-phenylethyl) moiety particularly enhanced mAAR affinity by polar interactions with the extracellular loops, predicted using docking and molecular dynamics simulation with newly constructed mAAR and hAAR homology models. These hybrid models were based on an inactive antagonist-bound hAAR structure for the upper part of TM2 and an agonist-bound hAAR structure for the remaining TM portions. These species-independent AAR-selective nucleosides are low efficacy partial agonists and novel, nuanced modulators of the AAR, a drug target of growing interest.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443318PMC
April 2020

Assessment of biased agonism at the A adenosine receptor using β-arrestin and miniGα recruitment assays.

Biochem Pharmacol 2020 07 26;177:113934. Epub 2020 Mar 26.

Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Campus Heymans, Ottergemsesteenweg 460, B-9000 Ghent, Belgium. Electronic address:

The A adenosine receptor (AAR) is a G protein-coupled receptor that is involved in a wide variety of physiological and pathological processes, such as cancer. However, the use of compounds pharmacologically targeting this receptor remains limited in clinical practice, despite extensive efforts for compound synthesis. Moreover, the possible occurrence of biased agonism further complicates the interpretation of the functional characteristics of compounds. Hence the need for simple assays, which are comparable in terms of the used cell lines and read-out technique. We previously established a stable β-arrestin 2 (βarr2) bioassay, employing a simple, luminescent read-out via functional complementation of a split nanoluciferase enzyme. Here, we developed a complementary, new bioassay in which coupling of an engineered miniGα protein to activated AAR is monitored using a similar approach. Application of both bioassays for the concurrent determination of the potencies and efficacies of a set of 19 N-substituted adenosine analogues not only allowed for the characterization of structure-activity relationships, but also for the quantification of biased agonism. Although a broad distribution in potency and efficacy values was obtained within the test panel, no significant bias was observed toward either the βarr2 or miniGα pathway.
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http://dx.doi.org/10.1016/j.bcp.2020.113934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382964PMC
July 2020

Sexually dimorphic therapeutic response in bortezomib-induced neuropathic pain reveals altered pain physiology in female rodents.

Pain 2020 01;161(1):177-184

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

Chemotherapy-induced neuropathic pain (CINP) in both sexes compromises many current chemotherapeutics and lacks an FDA-approved therapy. We recently identified the sphingosine-1-phosphate receptor subtype 1 (S1PR1) and A3 adenosine receptor subtype (A3AR) as novel targets for therapeutic intervention. Our work in male rodents using paclitaxel, oxaliplatin, and bortezomib showed robust inhibition of CINP with either S1PR1 antagonists or A3AR agonists. The S1PR1 functional antagonist FTY720 (Gilenya) is FDA-approved for treating multiple sclerosis, and selective A3AR agonists are in advanced clinical trials for cancer and inflammatory disorders, underscoring the need for their expedited trials in patients with CINP as chemotherapy adjuncts. Our findings reveal that S1PR1 antagonists and A3AR agonists mitigate paclitaxel and oxaliplatin CINP in female and male rodents, but failed to block or reverse bortezomib-induced neuropathic pain (BINP) in females. Although numerous mechanisms likely underlie these differences, we focused on receptor levels. We found that BINP in male rats, but not in female rats, was associated with increased expression of A3AR in the spinal cord dorsal horn, whereas S1PR1 levels were similar in both sexes. Thus, alternative mechanisms beyond receptor expression may account for sex differences in response to S1PR1 antagonists. Morphine and duloxetine, both clinical analgesics, reversed BINP in female mice, demonstrating that the lack of response is specific to S1PR1 and A3AR agents. Our findings suggest that A3AR- and S1PR1-based therapies are not viable approaches in preventing and treating BINP in females and should inform future clinical trials of these drugs as adjuncts to chemotherapy.
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http://dx.doi.org/10.1097/j.pain.0000000000001697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923586PMC
January 2020

Evidence for the Interaction of A Adenosine Receptor Agonists at the Drug-Binding Site(s) of Human P-glycoprotein (ABCB1).

Mol Pharmacol 2019 08 24;96(2):180-192. Epub 2019 May 24.

Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute (B.A., S.R.D., M.M., S.V., S.V.A.), and Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases (D.K.T., K.A.J.), National Institutes of Health, Bethesda, Maryland

P-glycoprotein (P-gp) is a multidrug transporter that is expressed on the luminal surface of epithelial cells in the kidney, intestine, bile-canalicular membrane in the liver, blood-brain barrier, and adrenal gland. This transporter uses energy of ATP hydrolysis to efflux from cells a variety of structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs. In this regard, understanding the interaction with P-gp of drug entities in development is important and highly recommended in current US Food and Drug Administration guidelines. Here we tested the P-gp interaction of some A adenosine receptor agonists that are being developed for the treatment of chronic diseases, including rheumatoid arthritis, psoriasis, chronic pain, and hepatocellular carcinoma. Biochemical assays of the ATPase activity of P-gp and by photolabeling P-gp with its transport substrate [I]-iodoarylazidoprazosin led to the identification of rigidified (N)-methanocarba nucleosides (i.e., compound as a stimulator and compound as a partial inhibitor of P-gp ATPase activity). Compound significantly inhibited boron-dipyrromethene (BODIPY)-verapamil transport mediated by human P-gp (IC 2.4 ± 0.6 M); however, the BODIPY-conjugated derivative of (compound ) was not transported by P-gp. In silico docking of compounds and was performed using the recently solved atomic structure of paclitaxel (Taxol)-bound human P-gp. Molecular modeling studies revealed that both compounds and bind in the same region of the drug-binding pocket as Taxol. Thus, this study indicates that nucleoside derivatives can exhibit varied modulatory effects on P-gp activity, depending on structural functionalization. SIGNIFICANCE STATEMENT: Certain A adenosine receptor agonists are being developed for the treatment of chronic diseases. The goal of this study was to test the interaction of these agonists with the human multidrug resistance-linked transporter P-glycoprotein (P-gp). ATPase and photolabeling assays demonstrated that compounds with rigidified (N)-methanocarba nucleosides inhibit the activity of P-gp; however, a fluorescent derivative of one of the compounds was not transported by P-gp. Furthermore, molecular docking studies revealed that the binding site for these compounds overlaps with the site for paclitaxel in the drug-binding pocket. These results suggest that nucleoside derivatives, depending on structural functionalization, can modulate the function of P-gp.
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http://dx.doi.org/10.1124/mol.118.115295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6608608PMC
August 2019

Adenosine A3 receptor activation inhibits pronociceptive N-type Ca2+ currents and cell excitability in dorsal root ganglion neurons.

Pain 2019 05;160(5):1103-1118

Division of Pharmacology and Toxicology, Department of NEUROFARBA, University of Florence, Italy.

Recently, studies have focused on the antihyperalgesic activity of the A3 adenosine receptor (A3AR) in several chronic pain models, but the cellular and molecular basis of this effect is still unknown. Here, we investigated the expression and functional effects of A3AR on the excitability of small- to medium-sized, capsaicin-sensitive, dorsal root ganglion (DRG) neurons isolated from 3- to 4-week-old rats. Real-time quantitative polymerase chain reaction experiments and immunofluorescence analysis revealed A3AR expression in DRG neurons. Patch-clamp experiments demonstrated that 2 distinct A3AR agonists, Cl-IB-MECA and the highly selective MRS5980, inhibited Ca-activated K (KCa) currents evoked by a voltage-ramp protocol. This effect was dependent on a reduction in Ca influx via N-type voltage-dependent Ca channels, as Cl-IB-MECA-induced inhibition was sensitive to the N-type blocker PD173212 but not to the L-type blocker, lacidipine. The endogenous agonist adenosine also reduced N-type Ca currents, and its effect was inhibited by 56% in the presence of A3AR antagonist MRS1523, demonstrating that the majority of adenosine's effect is mediated by this receptor subtype. Current-clamp recordings demonstrated that neuronal firing of rat DRG neurons was also significantly reduced by A3AR activation in a MRS1523-sensitive but PD173212-insensitive manner. Intracellular Ca measurements confirmed the inhibitory role of A3AR on DRG neuronal firing. We conclude that pain-relieving effects observed on A3AR activation could be mediated through N-type Ca channel block and action potential inhibition as independent mechanisms in isolated rat DRG neurons. These findings support A3AR-based therapy as a viable approach to alleviate pain in different pathologies.
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http://dx.doi.org/10.1097/j.pain.0000000000001488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669900PMC
May 2019

Historical and Current Adenosine Receptor Agonists in Preclinical and Clinical Development.

Front Cell Neurosci 2019 28;13:124. Epub 2019 Mar 28.

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

Adenosine receptors (ARs) function in the body's response to conditions of pathology and stress associated with a functional imbalance, such as in the supply and demand of energy/oxygen/nutrients. Extracellular adenosine concentrations vary widely to raise or lower the basal activation of four subtypes of ARs. Endogenous adenosine can correct an energy imbalance during hypoxia and other stress, for example, by slowing the heart rate by AAR activation or increasing the blood supply to heart muscle by the AAR. Moreover, exogenous AR agonists, antagonists, or allosteric modulators can be applied for therapeutic benefit, and medicinal chemists working toward that goal have reported thousands of such agents. Thus, numerous clinical trials have ensued, using promising agents to modulate adenosinergic signaling, most of which have not succeeded. Currently, short-acting, parenteral agonists, adenosine and Regadenoson, are the only AR agonists approved for human use. However, new concepts and compounds are currently being developed and applied toward preclinical and clinical evaluation, and initial results are encouraging. This review focuses on key compounds as AR agonists and positive allosteric modulators (PAMs) for disease treatment or diagnosis. AR agonists for treating inflammation, pain, cancer, non-alcoholic steatohepatitis, angina, sickle cell disease, ischemic conditions and diabetes have been under development. Multiple clinical trials with two AAR agonists are ongoing.
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http://dx.doi.org/10.3389/fncel.2019.00124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6447611PMC
March 2019

Design and in Vivo Characterization of A Adenosine Receptor Agonists in the Native Ribose and Conformationally Constrained (N)-Methanocarba Series.

J Med Chem 2019 02 3;62(3):1502-1522. Epub 2019 Jan 3.

School of Pharmacy , Queen's University Belfast , 96 Lisburn Road , Belfast BT9 7BL , U.K.

(N)-Methanocarba ([3.1.0]bicyclohexyl) adenosines and corresponding ribosides were synthesized to identify novel A adenosine receptor (AAR) agonists for CNS or peripheral applications. Human and mouse AR binding was determined to assess the constrained ring system's AAR compatibility. N-Dicyclobutylmethyl ribose agonist (9, MRS7469, >2000-fold selective for AAR) and known truncated N-dicyclopropylmethyl methanocarba 7 (MRS5474) were drug-like. The pure diastereoisomer of known riboside 4 displayed high hAAR selectivity. Methanocarba modification reduced AAR selectivity of N-dicyclopropylmethyl and endo-norbornyladenosines but increased ribavirin selectivity. Most analogues tested (ip) were inactive or weak in inducing mouse hypothermia, despite mAAR full agonism and variable mAAR efficacy, but strong hypothermia by 9 depended on AAR, which reflects CNS activity (determined using AAR or AAR null mice). Conserved hAAR interactions were preserved in modeling of 9 and methanocarba equivalent 24 (∼400-fold AAR-selective). Thus, we identified, and characterized in vivo, ribose and methanocarba nucleosides, including with AAR-enhancing N-dicyclobutylmethyl-adenine and 1,2,4-triazole-3-carboxamide (40, MRS7451) nucleobases.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467784PMC
February 2019

Repurposing of a Nucleoside Scaffold from Adenosine Receptor Agonists to Opioid Receptor Antagonists.

ACS Omega 2018 Oct 4;3(10):12658-12678. Epub 2018 Oct 4.

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892, United States.

While screening off-target effects of rigid ()-methanocarba-adenosine 5'-methylamides as A adenosine receptor (AR) agonists, we discovered μM binding hits at the δ-opioid receptor (DOR) and translocator protein (TSPO). In an effort to increase OR and decrease AR affinity by structure activity analysis of this series, antagonist activity at κ-(K)OR appeared in 5'-esters (ethyl and propyl ), which retained TSPO interaction (μM). 7-Deaza modification of C2-(arylethynyl)-5'-esters but not 4'-truncation enhanced KOR affinity (MRS7299 and , ≈ 40 nM), revealed μ-OR and DOR binding, and reduced AR affinity. Molecular docking and dynamics simulations located a putative KOR binding mode consistent with the observed affinities, placing C7 in a hydrophobic region. 3-Deaza modification permitted TSPO but not OR binding, and 1-deaza was permissive to both; ribose-restored analogues were inactive at both. Thus, we have repurposed a known AR nucleoside scaffold for OR antagonism, with a detailed hypothesis for KOR recognition.
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http://dx.doi.org/10.1021/acsomega.8b01237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210068PMC
October 2018

Probing structure-activity relationship in β-arrestin2 recruitment of diversely substituted adenosine derivatives.

Biochem Pharmacol 2018 12 4;158:103-113. Epub 2018 Oct 4.

Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Campus Heymans, Ottergemsesteenweg 460, B-9000 Ghent, Belgium. Electronic address:

In the adenosine receptor (AR) subfamily of G protein-coupled receptors (GPCRs), biased agonism has been described for the human AAR, AAR and AAR. While diverse AAR agonists have been evaluated for receptor binding and G-mediated cAMP signalling, the β-arrestin2 (βarr2) pathway has been left largely unexplored. We screened nineteen diverse adenosine derivatives for βarr2 recruitment using a stable hAAR-NanoBit-βarr2 HEK293T cell line. Their activity profiles were compared with a cAMP accumulation assay in stable hAAR CHO cells. Structural features linked to βarr2 activation were further investigated by the evaluation of an additional ten AAR ligands. The AAR-selective reference agonist 2-Cl-IB-MECA, which is a full agonist in terms of cAMP inhibition, only showed partial agonist behaviour in βarr2 recruitment. Highly AAR-selective (N)-methanocarba 5'-uronamide adenosine derivatives displayed higher potency in both cAMP signalling and βarr2 recruitment than reference agonists NECA and 2-Cl-IB-MECA. Their AAR-preferred conformation tolerates C2-position substitutions, for increased βarr2 efficacy, better than the flexible scaffolds of ribose derivatives. The different amino functionalities in the adenosine scaffold of these derivatives each seem to be important for signalling as well. In conclusion, we have provided insights into ligand features that can help to guide the future therapeutic development of biased AAR ligands with respect to G-protein and βarr2 signalling.
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http://dx.doi.org/10.1016/j.bcp.2018.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6530469PMC
December 2018

Thermostabilization and purification of the human dopamine transporter (hDAT) in an inhibitor and allosteric ligand bound conformation.

PLoS One 2018 2;13(7):e0200085. Epub 2018 Jul 2.

Vollum Institute, Oregon Health & Science University, Portland, Oregon, United States of America.

The human dopamine transporter (hDAT) plays a major role in dopamine homeostasis and regulation of neurotransmission by clearing dopamine from the extracellular space using secondary active transport. Dopamine is an essential monoamine chemical messenger that regulates reward seeking behavior, motor control, hormonal release, and emotional response in humans. Psychostimulants such as cocaine primarily target the central binding site of hDAT and lock the transporter in an outward-facing conformation, thereby inhibiting dopamine reuptake. The inhibition of dopamine reuptake leads to accumulation of dopamine in the synapse causing heightened signaling. In addition, hDAT is implicated in various neurological disorders and disease-associated neurodegeneration. Despite its significance, the structural studies of hDAT have proven difficult. Instability of hDAT in detergent micelles has been a limiting factor in its successful biochemical, biophysical, and structural characterization. To overcome this hurdle, we identified ligands that stabilize hDAT in detergent micelles. We then screened ~200 single residue mutants of hDAT using a high-throughput scintillation proximity assay and identified a thermostable variant (I248Y). Here we report a robust strategy to overexpress and successfully purify a thermostable variant of hDAT in an inhibitor and allosteric ligand bound conformation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200085PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028122PMC
January 2019

Chemotherapy-induced pain is promoted by enhanced spinal adenosine kinase levels through astrocyte-dependent mechanisms.

Pain 2018 Jun;159(6):1025-1034

Departments of Pharmacology and Physiology and.

Development of chemotherapy-induced neuropathic pain (CINP) compromises the use of chemotherapy and greatly impacts thousands of lives. Unfortunately, there are no Food and Drug Administration-approved drugs to prevent or treat CINP. Neuropathological changes within CNS, including neuroinflammation and increased neuronal excitability, are driven by alterations in neuro-glia communication; but, the molecular signaling pathways remain largely unexplored. Adenosine is a potent neuroprotective purine nucleoside released to counteract the consequences of these neuropathological changes. Adenosine signaling at its adenosine receptors (ARs) is dictated by adenosine kinase (ADK) in astrocytes, which provides a cellular sink for the removal of extracellular adenosine. We now demonstrate that chemotherapy (oxaliplatin) in rodents caused ADK overexpression in reactive astrocytes and reduced adenosine signaling at the A3AR subtype (A3AR) within the spinal cord. Dysregulation of ADK and A3AR signaling was associated with increased proinflammatory and neuroexcitatory interleukin-1β expression and activation of nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome, but not putative oxaliplatin-associated GSK3β transcriptional regulation. Intrathecal administration of the highly selective A3AR agonist MRS5698 attenuated IL-1β production and increased the expression of potent anti-inflammatory and neuroprotective IL-10. The effects of MRS5698 were blocked by attenuating IL-10 signaling in rats with intrathecal neutralizing IL-10 antibody and in IL-10 knockout mice. These findings provide new molecular insights implicating astrocyte-based ADK-adenosine axis and nucleotide-binding oligomerization domain-like receptor protein 3 in the development of CINP and IL-10 in the mechanism of action of A3AR agonists. These findings strengthen the pharmacological rationale for clinical evaluation of A3AR agonists already in advanced clinical trials as anticancer agents as an adjunct to chemotherapy.
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http://dx.doi.org/10.1097/j.pain.0000000000001177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955834PMC
June 2018

A binding kinetics study of human adenosine A receptor agonists.

Biochem Pharmacol 2018 07 3;153:248-259. Epub 2018 Jan 3.

Division of Medicinal Chemistry, LACDR, Leiden University, The Netherlands. Electronic address:

The human adenosine A (hA) receptor has been suggested as a viable drug target in inflammatory diseases and in cancer. So far, a number of selective hA receptor agonists (e.g. IB-MECA and 2-Cl-IB-MECA) inducing anti-inflammatory or anticancer effects are under clinical investigation. Drug-target binding kinetics is increasingly recognized as another pharmacological parameter, next to affinity, for compound triage in the early phases of drug discovery. However, such a kinetics-driven analysis has not yet been performed for the hA receptor. In this study, we first validated a competition association assay for adenosine A receptor agonists to determine the target interaction kinetics. Affinities and Kinetic Rate Index (KRI) values of 11 ribofurano and 10 methanocarba nucleosides were determined in radioligand binding assays. Afterwards, 15 analogues were further selected (KRI <0.70 or KRI >1.35) for full kinetics characterization. The structure-kinetics relationships (SKR) were derived and longer residence times were associated with methanocarba and enlarged adenine N and C2 substitutions. In addition, from a k-k-K kinetic map we divided the agonists into three subgroups. A residence time "cliff" was observed, which might be relevant to (N)-methanocarba derivatives' rigid C2-arylalkynyl substitutions. Our findings provide substantial evidence that, next to affinity, additional knowledge of binding kinetics is useful for developing and selecting new hAR agonists in the early phase of the drug discovery process.
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http://dx.doi.org/10.1016/j.bcp.2017.12.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959751PMC
July 2018

Polypharmacology of conformationally locked methanocarba nucleosides.

Drug Discov Today 2017 12 3;22(12):1782-1791. Epub 2017 Aug 3.

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Bldg 8A, Rm B1A-19, Bethesda, MD 20892-0810, USA.

A single molecular scaffold can be adapted to interact with diverse targets, either separately or simultaneously. Nucleosides and nucleotides in which ribose is substituted with bicyclo[3.1.0]hexane are an example of a versatile drug-like scaffold for increasing selectivity at their classical targets: kinases, polymerases, adenosine and P2 receptors. Also, by applying structure-based functional group manipulations, rigidified adenosine derivatives can be repurposed to satisfy pharmacophoric requirements of various GPCRs, ion channels, enzymes and transporters, initially detected as off-target activities. Recent examples include 5HT serotonin receptor antagonists and novel dopamine transporter allosteric modulators. This directable target diversity establishes rigid nucleosides as privileged scaffolds.
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http://dx.doi.org/10.1016/j.drudis.2017.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705437PMC
December 2017

A Adenosine Receptors as Modulators of Inflammation: From Medicinal Chemistry to Therapy.

Med Res Rev 2018 07 6;38(4):1031-1072. Epub 2017 Jul 6.

Department of Medical Sciences, Pharmacology Section, University of Ferrara, Via Fossato di Mortara 17/19, 44121, Ferrara, Italy.

The A adenosine receptor (A AR) subtype is a novel, promising therapeutic target for inflammatory diseases, such as rheumatoid arthritis (RA) and psoriasis, as well as liver cancer. A AR is coupled to inhibition of adenylyl cyclase and regulation of mitogen-activated protein kinase (MAPK) pathways, leading to modulation of transcription. Furthermore, A AR affects functions of almost all immune cells and the proliferation of cancer cells. Numerous A AR agonists, partial agonists, antagonists, and allosteric modulators have been reported, and their structure-activity relationships (SARs) have been studied culminating in the development of potent and selective molecules with drug-like characteristics. The efficacy of nucleoside agonists may be suppressed to produce antagonists, by structural modification of the ribose moiety. Diverse classes of heterocycles have been discovered as selective A AR blockers, although with large species differences. Thus, as a result of intense basic research efforts, the outlook for development of A AR modulators for human therapeutics is encouraging. Two prototypical selective agonists, N6-(3-Iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA; CF101) and 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA; CF102), have progressed to advanced clinical trials. They were found safe and well tolerated in all preclinical and human clinical studies and showed promising results, particularly in psoriasis and RA, where the A AR is both a promising therapeutic target and a biologically predictive marker, suggesting a personalized medicine approach. Targeting the A AR may pave the way for safe and efficacious treatments for patient populations affected by inflammatory diseases, cancer, and other conditions.
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http://dx.doi.org/10.1002/med.21456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5756520PMC
July 2018

Scaffold Repurposing of Nucleosides (Adenosine Receptor Agonists): Enhanced Activity at the Human Dopamine and Norepinephrine Sodium Symporters.

J Med Chem 2017 04 20;60(7):3109-3123. Epub 2017 Mar 20.

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.

We have repurposed (N)-methanocarba adenosine derivatives (A adenosine receptor (AR) agonists) to enhance radioligand binding allosterically at the human dopamine (DA) transporter (DAT) and inhibit DA uptake. We extended the structure-activity relationship of this series with small N-alkyl substitution, 5'-esters, deaza modifications of adenine, and ribose restored in place of methanocarba. C2-(5-Halothien-2-yl)-ethynyl 5'-methyl 9 (MRS7292) and 5'-ethyl 10 (MRS7232) esters enhanced binding at DAT (EC ∼ 35 nM) and at the norepinephrine transporter (NET). 9 and 10 were selective for DAT compared to AAR in the mouse but not in humans. At DAT, the binding of two structurally dissimilar radioligands was enhanced; NET binding of only one radioligand was enhanced; SERT radioligand binding was minimally affected. 10 was more potent than cocaine at inhibiting DA uptake (IC = 107 nM). Ribose analogues were weaker in DAT interaction than the corresponding bicyclics. Thus, we enhanced the neurotransmitter transporter activity of rigid nucleosides while reducing AAR affinity.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501184PMC
April 2017

Structure-Based Scaffold Repurposing for G Protein-Coupled Receptors: Transformation of Adenosine Derivatives into 5HT/5HT Serotonin Receptor Antagonists.

J Med Chem 2016 12 9;59(24):11006-11026. Epub 2016 Dec 9.

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.

Adenosine derivatives developed to activate adenosine receptors (ARs) revealed micromolar activity at serotonin 5HT and 5HT receptors (5HTRs). We explored the structure-activity relationship at 5HTRs and modeled receptor interactions in order to optimize affinity and simultaneously reduce AR affinity. Depending on N substitution, small 5'-alkylamide modification maintained 5HTR affinity, which was enhanced upon ribose substitution with rigid bicyclo[3.1.0]hexane (North (N)-methanocarba), e.g., N-dicyclopropylmethyl 4'-CHOH derivative 14 (K 11 nM). 5'-Methylamide 23 was 170-fold selective as antagonist for 5HTR vs 5HTR. 5'-Methyl 25 and ethyl 26 esters potently antagonized 5HTRs with moderate selectivity in comparison to ARs; related 6-N,N-dimethylamino analogue 30 was 5HTR-selective. 5' position flexibility of substitution was indicated in 5HTR docking. Both 5'-ester and 5'-amide derivatives displayed in vivo t of 3-4 h. Thus, we used G protein-coupled receptor modeling to repurpose nucleoside scaffolds in favor of binding at nonpurine receptors as novel 5HTR antagonists, with potential for cardioprotection, liver protection, or central nervous system activity.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201133PMC
December 2016

Hypothermia in mouse is caused by adenosine A and A receptor agonists and AMP via three distinct mechanisms.

Neuropharmacology 2017 03 30;114:101-113. Epub 2016 Nov 30.

Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USA. Electronic address:

Small mammals have the ability to enter torpor, a hypothermic, hypometabolic state, allowing impressive energy conservation. Administration of adenosine or adenosine 5'-monophosphate (AMP) can trigger a hypothermic, torpor-like state. We investigated the mechanisms for hypothermia using telemetric monitoring of body temperature in wild type and receptor knock out (Adora1, Adora3) mice. Confirming prior data, stimulation of the A adenosine receptor (AR) induced hypothermia via peripheral mast cell degranulation, histamine release, and activation of central histamine H receptors. In contrast, AAR agonists and AMP both acted centrally to cause hypothermia. Commonly used, selective AAR agonists, including N-cyclopentyladenosine (CPA), N-cyclohexyladenosine (CHA), and MRS5474, caused hypothermia via both AAR and AAR when given intraperitoneally. Intracerebroventricular dosing, low peripheral doses of Cl-ENBA [(±)-5'-chloro-5'-deoxy-N-endo-norbornyladenosine], or using Adora3 mice allowed selective stimulation of AAR. AMP-stimulated hypothermia can occur independently of AAR, AAR, and mast cells. AAR and AAR agonists and AMP cause regulated hypothermia that was characterized by a drop in total energy expenditure, physical inactivity, and preference for cooler environmental temperatures, indicating a reduced body temperature set point. Neither AAR nor AAR was required for fasting-induced torpor. AAR and AAR agonists and AMP trigger regulated hypothermia via three distinct mechanisms.
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http://dx.doi.org/10.1016/j.neuropharm.2016.11.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5183552PMC
March 2017

Structure-Activity Analysis of Biased Agonism at the Human Adenosine A3 Receptor.

Mol Pharmacol 2016 07 2;90(1):12-22. Epub 2016 May 2.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, Victoria, Australia (J.-A.B., A.T.N.N., K.J.G., A.C., L.T.M); and Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland (S.P., D.K.T., K.A.J)

Biased agonism at G protein-coupled receptors (GPCRs) has significant implications for current drug discovery, but molecular determinants that govern ligand bias remain largely unknown. The adenosine A3 GPCR (A3AR) is a potential therapeutic target for various conditions, including cancer, inflammation, and ischemia, but for which biased agonism remains largely unexplored. We now report the generation of bias "fingerprints" for prototypical ribose containing A3AR agonists and rigidified (N)-methanocarba 5'-N-methyluronamide nucleoside derivatives with regard to their ability to mediate different signaling pathways. Relative to the reference prototypical agonist IB-MECA, (N)-methanocarba 5'-N-methyluronamide nucleoside derivatives with significant N(6) or C2 modifications, including elongated aryl-ethynyl groups, exhibited biased agonism. Significant positive correlation was observed between the C2 substituent length (in Å) and bias toward cell survival. Molecular modeling suggests that extended C2 substituents on (N)-methanocarba 5'-N-methyluronamide nucleosides promote a progressive outward shift of the A3AR transmembrane domain 2, which may contribute to the subset of A3AR conformations stabilized on biased agonist binding.
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http://dx.doi.org/10.1124/mol.116.103283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931866PMC
July 2016

Purine (N)-Methanocarba Nucleoside Derivatives Lacking an Exocyclic Amine as Selective A3 Adenosine Receptor Agonists.

J Med Chem 2016 Apr 18;59(7):3249-63. Epub 2016 Feb 18.

Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Building 8A, Room B1A-19, Bethesda, Maryland 20892-0810, United States.

Purine (N)-methanocarba-5'-N-alkyluronamidoriboside A3 adenosine receptor (A3AR) agonists lacking an exocyclic amine resulted from an unexpected reaction during a Sonogashira coupling and subsequent aminolysis. Because the initial C6-Me and C6-styryl derivatives had unexpectedly high A3AR affinity, other rigid nucleoside analogues lacking an exocyclic amine were prepared. Of these, the C6-Me-(2-phenylethynyl) and C2-(5-chlorothienylethynyl) analogues were particularly potent, with human A3AR Ki values of 6 and 42 nM, respectively. Additionally, the C2-(5-chlorothienyl)-6-H analogue was potent and selective at A3AR (MRS7220, Ki 60 nM) and also completely reversed mouse sciatic nerve mechanoallodynia (in vivo, 3 μmol/kg, po). The lack of a C6 H-bond donor while maintaining A3AR affinity and efficacy could be rationalized by homology modeling and docking of these hypermodified nucleosides. The modeling suggests that a suitable combination of stabilizing features can partially compensate for the lack of an exocyclic amine, an otherwise important contributor to recognition in the A3AR binding site.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4970510PMC
April 2016

Rigid Adenine Nucleoside Derivatives as Novel Modulators of the Human Sodium Symporters for Dopamine and Norepinephrine.

J Pharmacol Exp Ther 2016 Apr 26;357(1):24-35. Epub 2016 Jan 26.

VA Portland Health Care System, Research Service (R&D-22), and Departments of Psychiatry and Behavioral Neuroscience, Oregon Health and Science University, Portland, Oregon (A.J., A.J.E.); and Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (D.K.T., K.A.J.).

Thirty-two congeneric rigid adenine nucleoside derivatives containing a North (N)-methanocarba ribose substitution and a 2-arylethynyl group either enhanced (up to 760% of control) or inhibited [(125)I] methyl (1R,2S,3S)-3-(4-iodophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate (RTI-55) binding at the human dopamine (DA) transporter (DAT) and inhibited DA uptake. Several nucleosides also enhanced [(3)H]mazindol [(±)-5-(4-chlorophenyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol] binding to the DAT. The combination of binding enhancement and functional inhibition suggests possible allosteric interaction with the tropanes. The structure-activity relationship of this novel class of DAT ligands was explored: small N(6)-substition (methyl or ethyl) was favored, while the N1 of the adenine ring was essential. Effective terminal aryl groups include thien-2-yl (compounds 9 and 16), with EC50 values of 35.1 and 9.1 nM, respectively, in [(125)I]RTI-55 binding enhancement, and 3,4-difluorophenyl as in the most potent DA uptake inhibitor (compound 6) with an IC50 value of 92 nM (3-fold more potent than cocaine), but not nitrogen heterocycles. Several compounds inhibited or enhanced binding at the norepinephrine transporter (NET) and serotonin transporter (SERT) and inhibited function in the micromolar range; truncation at the 4'-position in compound 23 allowed for weak inhibition of the SERT. We have not yet eliminated adenosine receptor affinity from this class of DAT modulators, but we identified modifications that remove DAT inhibition as an off-target effect of potent adenosine receptor agonists. Thus, we have identified a new class of allosteric DAT ligands, rigidified adenosine derivatives, and explored their initial structural requirements. They display a very atypical pharmacological profile, i.e., either enhancement by increasing affinity or inhibition of radioligand binding at the DAT, and in some cases the NET and SERT, and inhibition of neurotransmitter uptake.
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http://dx.doi.org/10.1124/jpet.115.229666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4809312PMC
April 2016

Peripheral Adenosine A3 Receptor Activation Causes Regulated Hypothermia in Mice That Is Dependent on Central Histamine H1 Receptors.

J Pharmacol Exp Ther 2016 Feb 25;356(2):474-82. Epub 2015 Nov 25.

Diabetes, Endocrinology, and Obesity Branch (J.L.C., C.X., R.A.P., M.L.R.), Molecular Recognition Section, Laboratory of Bioorganic Chemistry (D.K.T., K.A.J.), and Mouse Metabolism Core (O.G.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; and Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Missouri (Z.C., D.S.)

Adenosine can induce hypothermia, as previously demonstrated for adenosine A1 receptor (A1AR) agonists. Here we use the potent, specific A3AR agonists MRS5698, MRS5841, and MRS5980 to show that adenosine also induces hypothermia via the A3AR. The hypothermic effect of A3AR agonists is independent of A1AR activation, as the effect was fully intact in mice lacking A1AR but abolished in mice lacking A3AR. A3AR agonist-induced hypothermia was attenuated by mast cell granule depletion, demonstrating that the A3AR hypothermia is mediated, at least in part, via mast cells. Central agonist dosing had no clear hypothermic effect, whereas peripheral dosing of a non-brain-penetrant agonist caused hypothermia, suggesting that peripheral A3AR-expressing cells drive the hypothermia. Mast cells release histamine, and blocking central histamine H1 (but not H2 or H4) receptors prevented the hypothermia. The hypothermia was preceded by hypometabolism and mice with hypothermia preferred a cooler environmental temperature, demonstrating that the hypothermic state is a coordinated physiologic response with a reduced body temperature set point. Importantly, hypothermia is not required for the analgesic effects of A3AR agonists, which occur with lower agonist doses. These results support a mechanistic model for hypothermia in which A3AR agonists act on peripheral mast cells, causing histamine release, which stimulates central histamine H1 receptors to induce hypothermia. This mechanism suggests that A3AR agonists will probably not be useful for clinical induction of hypothermia.
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http://dx.doi.org/10.1124/jpet.115.229872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4746492PMC
February 2016

Increased Signaling via Adenosine A1 Receptors, Sleep Deprivation, Imipramine, and Ketamine Inhibit Depressive-like Behavior via Induction of Homer1a.

Neuron 2015 Aug;87(3):549-62

Department of Psychiatry and Psychotherapy, University Medical Center Freiburg, 79104 Freiburg, Germany. Electronic address:

Major depressive disorder is among the most commonly diagnosed disabling mental diseases. Several non-pharmacological treatments of depression upregulate adenosine concentration and/or adenosine A1 receptors (A1R) in the brain. To test whether enhanced A1R signaling mediates antidepressant effects, we generated a transgenic mouse with enhanced doxycycline-regulated A1R expression, specifically in forebrain neurons. Upregulating A1R led to pronounced acute and chronic resilience toward depressive-like behavior in various tests. Conversely, A1R knockout mice displayed an increased depressive-like behavior and were resistant to the antidepressant effects of sleep deprivation (SD). Various antidepressant treatments increase homer1a expression in medial prefrontal cortex (mPFC). Specific siRNA knockdown of homer1a in mPFC enhanced depressive-like behavior and prevented the antidepressant effects of A1R upregulation, SD, imipramine, and ketamine treatment. In contrast, viral overexpression of homer1a in the mPFC had antidepressant effects. Thus, increased expression of homer1a is a final common pathway mediating the antidepressant effects of different antidepressant treatments.
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http://dx.doi.org/10.1016/j.neuron.2015.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4803038PMC
August 2015