Lipid Trolling to Optimize A3 Adenosine Receptor-Positive Allosteric Modulators (PAMs).

A3 adenosine receptor (A3AR) positive allosteric modulators (PAMs) (2,4-disubstituted-1H-imidazo[4,5-c]quinolin-4-amines) allosterically increase the Emax of A3AR agonists, but not potency, due to concurrent orthosteric antagonism. Following mutagenesis/homology modeling of the proposed lipid-exposed allosteric binding site on the cytosolic side, we functionalized the scaffold, including heteroatom substitutions and exocyclic phenylamine extensions, to increase allosteric binding. Strategically appended linear alkyl-alkynyl chains with terminal amino/guanidino groups improved allosteric effects at both human and mouse A3ARs. The chain length, functionality, and attachment position were varied to modulate A3AR PAM activity. For example, 26 (MRS8247, p-alkyne-linked 8 methylenes) and homologues increased agonist Cl-IB-MECA's Emax and potency ([35S]GTPγS binding). The putative mechanism involves a flexible, terminally cationic chain penetrating the lipid environment for stable electrostatic anchoring to cytosolic phospholipid head groups, suggesting "lipid trolling", supported by molecular dynamic simulation of the active-state model. Thus, we have improved A3AR PAM activity through rational design based on an extrahelical, lipidic binding site.

Extrahelical Binding Site for a 1H-Imidazo[4,5-c]quinolin-4-amine A3 Adenosine Receptor Positive Allosteric Modulator on Helix 8 and Distal Portions of Transmembrane Domains 1 and 7.

This study describes the localization and computational prediction of a binding site for the A3 adenosine receptor (A3AR) positive allosteric modulator 2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-(3,4-dichlorophenyl)amine (LUF6000). The work reveals an extrahelical lipid-facing binding pocket disparate from the orthosteric binding site that encompasses transmembrane domain (TMD) 1, TMD7, and Helix (H) 8, which was predicted by molecular modeling and validated by mutagenesis. According to the model, the nearly planar 1H-imidazo[4,5-c]quinolinamine ring system lies parallel to the transmembrane segments, inserted into an aromatic cage formed by π-π stacking interactions with the side chains of Y2847.55 in TMD7 and Y2938.54 in H8 and by π-NH bonding between Y2847.55 and the exocyclic amine. The 2-cyclohexyl group is positioned "upward" within a small hydrophobic subpocket created by residues in TMDs 1 and 7, while the 3,4-dichlorophenyl group extends toward the lipid interface. An H-bond between the N-1 amine of the heterocycle and the carbonyl of G291.49 further stabilizes the interaction. Molecular dynamics simulations predicted two metastable intermediates, one resembling a pose determined by molecular docking and a second involving transient interactions with Y2938.54; in simulations, each of these intermediates converges into the final bound state. Structure-activity-relationships for replacement of either of the identified exocyclic or endocyclic amines with heteroatoms lacking H-bond donating ability were consistent with the hypothetical pose. Thus, we characterized an allosteric pocket for 1H-imidazo[4,5-c]quinolin-4-amines that is consistent with data generated by orthogonal methods, which will aid in the rational design of improved A3AR positive allosteric modulators. SIGNIFICANCE STATEMENT: Orthosteric A3AR agonists have advanced in clinical trials for inflammatory conditions, liver diseases, and cancer. Thus, the clinical appeal of selective receptor activation could extend to allosteric enhancers, which would induce site- and time-specific activation in the affected tissue. By identifying the allosteric site for known positive allosteric modulators, structure-based drug discovery modalities can be enabled to enhance the pharmacological properties of the 1H-imidazo[4,5-c]quinolin-4-amine class of A3AR positive allosteric modulators.

Evidence of Histone H2A.Z Deacetylation and Cardiomyocyte Dedifferentiation in Infarcted/Tip60-depleted Hearts.

Myocardial infarction (MI) in the human heart causes death of billions of cardiomyocytes (CMs), resulting in cardiac dysfunction that is incompatible with life or lifestyle. In order to re-muscularize injured myocardium, replacement CMs must be generated via renewed proliferation of surviving CMs. Approaches designed to induce proliferation of CMs after injury have been insufficient. Toward this end, we are targeting the Tip60 acetyltransferase, based on the rationale that its pleiotropic functions conspire to block the CM cell-cycle at several checkpoints. We previously reported that genetic depletion of Tip60 in a mouse model after MI reduces scarring, retains cardiac function, and activates the CM cell-cycle, although it is unclear whether this culminates in the generation of daughter CMs. For pre-existing CMs in the adult heart to resume proliferation, it is becoming widely accepted that they must first dedifferentiate, a process highlighted by loss of maturity, epithelial to mesenchymal transitioning (EMT), and reversion from fatty acid oxidation to glycolytic metabolism, accompanied by softening of the myocardial extracellular matrix. Findings in hematopoietic stem cells, and more recently in neural progenitor cells, have shown that Tip60 induces and maintains the differentiated state via site-specific acetylation of the histone variant H2A.Z. Here, we report that genetic depletion of Tip60 from naïve or infarcted hearts results in the near-complete absence of acetylated H2A.Z in CM nuclei, and that this is accordingly accompanied by altered gene expressions indicative of EMT induction, ECM softening, decreased fatty acid oxidation, and depressed expression of genes that regulate the TCA cycle. These findings, combined with our previous work, support the notion that because Tip60 has multiple targets that combinatorially maintain the differentiated state and inhibit proliferation, its transient therapeutic targeting to ameliorate the effects of cardiac injury should be considered.

First Potent Macrocyclic A3 Adenosine Receptor Agonists Reveal G-Protein and ß-Arrestin2 Signaling Preferences.

(N)-Methanocarba adenosine derivatives (A3 adenosine receptor (AR) agonists containing bicyclo[3.1.0]hexane replacing furanose) were chain-extended at N6 and C2 positions with terminal alkenes for ring closure. The resulting macrocycles of 17-20 atoms retained affinity, indicating a spatially proximal orientation of these receptor-bound chains, consistent with molecular modeling of 12. C2-Arylethynyl-linked macrocycle 19 was more A3AR-selective than 2-ether-linked macrocycle 12 (both 5'-methylamides, human (h) A3AR affinities (Ki): 22.1 and 25.8 nM, respectively), with lower mouse A3AR affinities. Functional hA3AR comparison of two sets of open/closed analogues in ß-arrestin2 and Gi/o protein assays showed certain signaling preferences divergent from reference agonist Cl-IB-MECA 1. The potencies of 1 at all three Gαi isoforms were slightly less than its hA3AR binding affinity (Ki: 1.4 nM), while the Gαi1 and Gαi2 potencies of macrocycle 12 were roughly an order of magnitude higher than its radioligand binding affinity. Gαi2-coupling was enhanced in macrocycle 12 (EC50 2.56 nM, ∼40% greater maximal efficacy than 1). Di-O-allyl precursor 18 cyclized to form 19, increasing the Gαi1 potency by 7.5-fold. The macrocycles 12 and 19 and their open precursors 11 and 18 potently stimulated ß-arrestin2 recruitment, with EC50 values (nM) of 5.17, 4.36, 1.30, and 4.35, respectively, and with nearly 50% greater efficacy compared to 1. This example of macrocyclization altering the coupling pathways of small-molecule (nonpeptide) GPCR agonists is the first for potent and selective macrocyclic AR agonists. These initial macrocyclic derivatives can serve as a guide for the future design of macrocyclic AR agonists displaying unanticipated pharmacology.

Pharmacological inhibition of the acetyltransferase Tip60 mitigates myocardial infarction injury.

Pharmacologic strategies that target factors with both pro-apoptotic and anti-proliferative functions in cardiomyocytes (CMs) may be useful for the treatment of ischemic heart disease. One such multifunctional candidate for drug targeting is the acetyltransferase Tip60, which is known to acetylate both histone and non-histone protein targets that have been shown in cancer cells to promote apoptosis and to initiate the DNA damage response, thereby limiting cellular expansion. Using a murine model, we recently published findings demonstrating that CM-specific disruption of the Kat5 gene encoding Tip60 markedly protects against the damaging effects of myocardial infarction (MI). In the experiments described here, in lieu of genetic targeting, we administered TH1834, an experimental drug designed to specifically inhibit the acetyltransferase domain of Tip60. We report that, similar to the effect of disrupting the Kat5 gene, daily systemic administration of TH1834 beginning 3 days after induction of MI and continuing for 2 weeks of a 4-week timeline resulted in improved systolic function, reduced apoptosis and scarring, and increased activation of the CM cell cycle, effects accompanied by reduced expression of genes that promote apoptosis and inhibit the cell cycle and reduced levels of CMs exhibiting phosphorylated Atm. These results support the possibility that drugs that inhibit the acetyltransferase activity of Tip60 may be useful agents for the treatment of ischemic heart disease.

Characterization of Dual-Acting A3 Adenosine Receptor Positive Allosteric Modulators That Preferentially Enhance Adenosine-Induced Gαi3 and GαoA Isoprotein Activation.

The A3 adenosine receptor (A3AR) is a promising therapeutic target for inflammatory diseases, cancer, and chronic neuropathic pain, with agonists already in advanced clinical trials. Here we report an in-depth comparison of the pharmacological properties and structure-activity relationships of existing and expanded compound libraries of 2-substituted 1H-imidazo[4,5-c]quinolin-4-amine and 4-amino-substituted quinoline derivatives that function as A3AR positive allosteric modulators (PAMs). We also show that our lead compound from each series enhances adenosine-induced A3AR signaling preferentially toward activation of Gαi3 and GαoA isoproteins, which are coexpressed with the A3AR in immune cells and spinal cord neurons. Finally, utilizing an extracellular/intracellular chimeric A3AR approach composed of sequences from a responding (human) and a nonresponding (mouse) species, we provide evidence in support of the idea that the imidazoquinolin-4-amine class of PAMs variably interacts dually with the orthosteric ligand binding site as well as with a separate allosteric site located within the inner/intracellular regions of the receptor. This study has advanced both structural and pharmacological understanding of these two classes of A3AR PAMs, which includes leads for future pharmaceutical development.

Conditional depletion of the acetyltransferase Tip60 protects against the damaging effects of myocardial infarction.

Injury from myocardial infarction (MI) and consequent post-MI remodeling is accompanied by massive loss of cardiomyocytes (CM), a cell type critical for contractile function that is for all practical purposes non-regenerable due to its profound state of proliferative senescence. Identification of factors that limit CM survival and/or constrain CM renewal provides potential therapeutic targets. Tip60, a pan-acetyltransferase encoded by the Kat5 gene, has been reported to activate apoptosis as well as multiple anti-proliferative pathways in non-cardiac cells; however, its role in CMs, wherein it is abundantly expressed, remains unknown. Here, using mice containing floxed Kat5 alleles and a tamoxifen-activated Myh6-MerCreMer recombinase transgene, we report that conditional depletion of Tip60 in CMs three days after MI induced by permanent coronary artery ligation greatly improves functional recovery for up to 28 days. This is accompanied by diminished scarring, activation of cell-cycle transit markers in CMs within the infarct border and remote zones, reduced expression of cell-cycle inhibitors pAtm and p27, and reduced apoptosis in the remote regions. These findings implicate Tip60 as a novel, multifactorial target for limiting the damaging effects of ischemic heart disease.

Evidence that the acetyltransferase Tip60 induces the DNA damage response and cell-cycle arrest in neonatal cardiomyocytes.

Tip60, a pan-acetyltransferase encoded by the Kat5 gene, is enriched in the myocardium; however, its function in the heart is unknown. In cancer cells, Tip60 acetylates Atm (Ataxia-telangiectasia mutated), enabling its auto-phosphorylation (pAtm), which activates the DNA damage response (DDR). It was recently reported that activation of pAtm at the time of birth induces the DDR in cardiomyocytes (CMs), resulting in proliferative senescence. We therefore hypothesized that Tip60 initiates this process, and that depletion of Tip60 accordingly diminishes the DDR while extending the duration of CM cell-cycle activation. To test this hypothesis, an experimental model was used wherein a Myh6-driven Cre-recombinase transgene was activated on postnatal day 0 (P0) to recombine floxed Kat5 alleles and induce Tip60 depletion in neonatal CMs, without causing pathogenesis. Depletion of Tip60 resulted in reduced numbers of pAtm-positive CMs during the neonatal period, which correlated with reduced numbers of pH2A.X-positive CMs and decreased expression of genes encoding markers of the DDR as well as inflammation. This was accompanied by decreased expression of the cell-cycle inhibitors Meis1 and p27, activation of the cell-cycle in CMs, reduced CM size, and increased numbers of mononuclear/diploid CMs. Increased expression of fetal markers suggested that Tip60 depletion promotes a fetal-like proliferative state. Finally, infarction of Tip60-depleted hearts at P7 revealed improved cardiac function at P39 accompanied by reduced fibrosis, increased CM cell-cycle activation, and reduced apoptosis in the remote zone. These findings indicate that, among its pleiotropic functions, Tip60 induces the DDR in CMs, contributing to proliferative senescence.

Myh6-driven Cre recombinase activates the DNA damage response and the cell cycle in the myocardium in the absence of loxP sites.

Regeneration of muscle in the damaged myocardium is a major objective of cardiovascular research, for which purpose many investigators utilize mice containing transgenes encoding Cre recombinase to recombine loxP-flanked target genes. An unfortunate side effect of the Cre-loxP model is the propensity of Cre recombinase to inflict off-target DNA damage, which has been documented in various eukaryotic cell types including cardiomyocytes (CMs). In the heart, reported effects of Cre recombinase include contractile dysfunction, fibrosis, cellular infiltration and induction of the DNA damage response (DDR). During experiments on adult mice containing a widely used Myh6-merCremer transgene, the protein product of which is activated by tamoxifen, we observed large, transient, off-target effects of merCremer, some of which have not previously been reported. On Day 3 after the first of three daily tamoxifen injections, immunofluorescent microscopy of heart sections revealed that the presence of merCremer protein in myonuclei was nearly uniform, thereafter diminishing to near extinction by Day 6; during this time, cardiac function was depressed as determined by echocardiography. On Day 5, peaks of apoptosis and expression of DDR-regulatory genes were observed, highlighted by >25-fold increased expression of Brca1 Concomitantly, the expression of genes encoding cyclin-A2, cyclin-B2 and cyclin-dependent kinase 1, which regulate the G2/S cell-cycle transition, were dramatically increased (>50- to 100-fold). Importantly, immunofluorescent staining revealed that this was accompanied by peaks in Ki67, 5'-bromodeoxyuridine and phosphohistone H3 labeling in non-CMs, as well as CMs. We further document that tamoxifen-induced activation of merCremer exacerbates cardiac dysfunction following myocardial infarction. These findings, when considered in the context of previous reports, indicate that the presence of merCremer in the nucleus induces DNA damage and unscheduled cell-cycle activation. Although these effects are transient, the inclusion of appropriate controls, coupled with an awareness of the defects caused by Cre recombinase, are required to avoid misinterpreting results when using Cre-loxP models for cardiac regeneration studies.This article has an associated First Person interview with the first author of the paper.

Llgl1 regulates zebrafish cardiac development by mediating Yap stability in cardiomyocytes.

The Hippo-Yap pathway regulates multiple cellular processes in response to mechanical and other stimuli. In Drosophila, the polarity protein Lethal (2) giant larvae [L(2)gl], negatively regulates Hippo-mediated transcriptional output. However, in vertebrates, little is known about its homolog Llgl1. Here, we define a novel role for vertebrate Llgl1 in regulating Yap stability in cardiomyocytes, which impacts heart development. In contrast to the role of Drosophila L(2)gl, Llgl1 depletion in cultured rat cardiomyocytes decreased Yap protein levels and blunted target gene transcription without affecting Yap transcript abundance. Llgl1 depletion in zebrafish resulted in larger and dysmorphic cardiomyocytes, pericardial effusion, impaired blood flow and aberrant valvulogenesis. Cardiomyocyte Yap protein levels were decreased in llgl1 morphants, whereas Notch, which is regulated by hemodynamic forces and participates in valvulogenesis, was more broadly activated. Consistent with the role of Llgl1 in regulating Yap stability, cardiomyocyte-specific overexpression of Yap in Llgl1-depleted embryos ameliorated pericardial effusion and restored blood flow velocity. Altogether, our data reveal that vertebrate Llgl1 is crucial for Yap stability in cardiomyocytes and its absence impairs cardiac development.

Ability of CP-532,903 to protect mouse hearts from ischemia/reperfusion injury is dependent on expression of A3 adenosine receptors in cardiomyoyctes.

A3 adenosine receptor (A3AR) agonists are effective at limiting injury caused by ischemia/reperfusion injury of the heart in experimental animal models. However, understanding of their mechanism of action, which is likely multifactorial, remains incomplete. In prior studies, it has been demonstrated that A3AR-mediated ischemic protection is blocked by glibenclamide and is absent in Kir6.2 gene ablated mice that lack the pore-forming subunit of the ATP-sensitive potassium (KATP) channel, suggesting one contributing mechanism may involve accelerated activation of KATP channels. However, presence of A3ARs in the myocardium has yet to be established. Utilizing a whole-cell recording technique, in this study we confirm functional expression of the A3AR in adult mouse ventricular cardiomyocytes, coupled to activation of ATP-dependent potassium (KATP) channels via Gi inhibitory proteins. We further show that ischemic protection provided by the selective A3AR agonist CP-532,903 in an isolated, buffer-perfused heart model is lost completely in Adora3LoxP/LoxP;Myh6-Cre mice, which is a newly developed model developed and comprehensively described herein whereby the A3AR gene (Adora3) is deleted exclusively in cardiomyocytes. Our findings, taken together with previously published work, are consistent with the hypothesis that A3AR agonists provide ischemic tolerance, at least in part, by facilitating opening of myocardial KATP channels.

IL-13 promotes in vivo neonatal cardiomyocyte cell cycle activity and heart regeneration.

There is great interest in identifying signaling mechanisms by which cardiomyocytes (CMs) can enter the cell cycle and promote endogenous cardiac repair. We have previously demonstrated that IL-13 stimulated cell cycle activity of neonatal CMs in vitro. However, the signaling events that occur downstream of IL-13 in CMs and the role of IL-13 in CM proliferation and regeneration in vivo have not been explored. Here, we tested the role of IL-13 in promoting neonatal CM cell cycle activity and heart regeneration in vivo and investigated the signaling pathway(s) downstream of IL-13 specifically in CMs. Compared with control, CMs from neonatal IL-13 knockout (IL-13-/-) mice showed decreased proliferative markers and coincident upregulation of the hypertrophic marker brain natriuretic peptide ( Nppb) and increased CM nuclear size. After apical resection in anesthetized newborn mice, heart regeneration was significantly impaired in IL-13-/- mice compared with wild-type mice. Administration of recombinant IL-13 reversed these phenotypes by increasing CM proliferation markers and decreasing Nppb expression. RNA sequencing on primary neonatal CMs treated with IL-13 revealed activation of gene networks regulated by ERK1/2 and Akt. Western blot confirmed strong phosphorylation of ERK1/2 and Akt in both neonatal and adult cultured CMs in response to IL-13. Our data demonstrated a role for endogenous IL-13 in neonatal CM cell cycle and heart regeneration. ERK1/2 and Akt signaling are important pathways known to promote CM proliferation and protect against apoptosis, respectively; thus, targeting IL-13 transmembrane receptor signaling or administering recombinant IL-13 may be therapeutic approaches for activating proregenerative and survival pathways in the heart. NEW & NOTEWORTHY Here, we demonstrate, for the first time, that IL-13 is involved in neonatal cardiomyocyte cell cycle activity and heart regeneration in vivo. Prior work has shown that IL-13 promotes cardiomyocyte cell cycle activity in vitro; however, the signaling pathways were unknown. We used RNA sequencing to identify the signaling pathways activated downstream of IL-13 in cardiomyocytes and found that ERK1/2 and Akt signaling was activated in response to IL-13.

Species differences and mechanism of action of A3 adenosine receptor allosteric modulators.

Activity of the A3 adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A3AR species homologs used in preclinical drug development. In guanosine 5'-[γ-[35S]thio]triphosphate ([35S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A3ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A3ARs but provided only weak activity at mouse A3ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A3AR agonist 2-chloro-N 6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide as well as adenosine > 2-fold, while slightly reducing potency in human and dog. Based on results from N 6-(4-amino-3-[125I]iodobenzyl)adenosine-5'-N-methylcarboxamide ([125I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A3AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [125I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A3AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.

Hypothermia in mouse is caused by adenosine A1 and A3 receptor agonists and AMP via three distinct mechanisms.

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 A3 adenosine receptor (AR) induced hypothermia via peripheral mast cell degranulation, histamine release, and activation of central histamine H1 receptors. In contrast, A1AR agonists and AMP both acted centrally to cause hypothermia. Commonly used, selective A1AR agonists, including N6-cyclopentyladenosine (CPA), N6-cyclohexyladenosine (CHA), and MRS5474, caused hypothermia via both A1AR and A3AR when given intraperitoneally. Intracerebroventricular dosing, low peripheral doses of Cl-ENBA [(±)-5'-chloro-5'-deoxy-N6-endo-norbornyladenosine], or using Adora3-/- mice allowed selective stimulation of A1AR. AMP-stimulated hypothermia can occur independently of A1AR, A3AR, and mast cells. A1AR and A3AR 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 A1AR nor A3AR was required for fasting-induced torpor. A1AR and A3AR agonists and AMP trigger regulated hypothermia via three distinct mechanisms.

Honokiol Decreases Lung Cancer Metastasis through Inhibition of the STAT3 Signaling Pathway.

Lung cancer is the leading cause of cancer death in the United States. Metastasis to lymph nodes and distal organs, especially brain, leads to severe complications and death. Preventing lung cancer development and metastases is an important strategy to reduce lung cancer mortality. Honokiol (HNK), a natural compound present in the extracts of magnolia bark, has a favorable bioavailability profile and recently has been shown to readily cross the blood-brain barrier. In the current study, we evaluated the antimetastatic effects of HNK in both the lymph node and brain mouse models of lung tumor metastasis. We tested the efficacy of HNK in preventing 18 H2030-BrM3 cell (brain-seeking human lung tumor cells) migration to lymph node or brain. In an orthotopic mouse model, HNK significantly decreased lung tumor growth compared with the vehicle control group. HNK also significantly reduced the incidence of lymph node metastasis and the weight of mediastinal lymph nodes. In a brain metastasis model, HNK inhibits metastasis of lung cancer cells to the brain to approximately one third of that observed in control mice. We analyzed HNK's mechanism of action, which indicated that its effect is mediated primarily by inhibiting the STAT3 pathway. HNK specifically inhibits STAT3 phosphorylation irrespective of the mutation status of EGFR, and knockdown of STAT3 abrogated both the antiproliferative and the antimetastatic effects of HNK. These observations suggest that HNK could provide novel chemopreventive or therapeutic options for preventing both lung tumor progression and lung cancer metastasis. Cancer Prev Res; 10(2); 133-41. ©2016 AACR.

Characterization of Dahl salt-sensitive rats with genetic disruption of the A2B adenosine receptor gene: implications for A2B adenosine receptor signaling during hypertension.

The A(2B) adenosine receptor (AR) has emerged as a unique member of the AR family with contrasting roles during acute and chronic disease states. We utilized zinc-finger nuclease technology to create A(2B)AR gene (Adora2b)-disrupted rats on the Dahl salt-sensitive (SS) genetic background. This strategy yielded a rat strain (SS-Adora2b mutant rats) with a 162-base pair in-frame deletion of Adora2b that included the start codon. Disruption of A(2B)AR function in SS-Adora2b mutant rats was confirmed by loss of agonist (BAY 60-6583 or NECA)-induced cAMP accumulation and loss of interleukin-6 release from isolated fibroblasts. In addition, BAY 60-6583 produced a dose-dependent increase in glucose mobilization that was absent in SS-Adora2b mutants. Upon initial characterization, SS-Adora2b mutant rats were found to exhibit increased body weight, a transient delay in glucose clearance, and reduced proinflammatory cytokine production following challenge with lipopolysaccharide (LPS). In addition, blood pressure was elevated to a greater extent (∼15-20 mmHg) in SS-Adora2b mutants as they aged from 7 to 21 weeks. In contrast, hypertension augmented by Ang II infusion was attenuated in SS-Adora2b mutant rats. Despite differences in blood pressure, indices of renal and cardiac injury were similar in SS-Adora2b mutants during Ang II-augmented hypertension. We have successfully created and validated a new animal model that will be valuable for investigating the biology of the A(2B)AR. Our data indicate varying roles for A(2B)AR signaling in regulating blood pressure in SS rats, playing both anti- and prohypertensive roles depending on the pathogenic mechanisms that contribute to blood pressure elevation.

Effect of human 15-lipoxygenase-1 metabolites on vascular function in mouse mesenteric arteries and hearts.

Lipoxygenases regulate vascular function by metabolizing arachidonic acid (AA) to dilator eicosanoids. Previously, we showed that endothelium-targeted adenoviral vector-mediated gene transfer of the human 15-lipoxygenase-1 (h15-LO-1) enhances arterial relaxation through the production of vasodilatory hydroxyepoxyeicosatrienoic acid (HEETA) and trihydroxyeicosatrienoic acid (THETA) metabolites. To further define this function, a transgenic (Tg) mouse line that overexpresses h15-LO-1 was studied. Western blot, immunohistochemistry and RT-PCR results confirmed expression of 15-LO-1 transgene in tissues, especially high quantity in coronary arterial wall, of Tg mice. Reverse-phase HPLC analysis of [(14)C]-AA metabolites in heart tissues revealed enhanced 15-HETE synthesis in Tg vs. WT mice. Among the 15-LO-1 metabolites, 15-HETE, erythro-13-H-14,15-EETA, and 11(R),12(S),15(S)-THETA relaxed the mouse mesenteric arteries to the greatest extent. The presence of h15-LO-1 increased acetylcholine- and AA-mediated relaxation in mesenteric arteries of Tg mice compared to WT mice. 15-LO-1 was most abundant in the heart; therefore, we used the Langendorff heart model to test the hypothesis that elevated 15-LO-1 levels would increase coronary flow following a short ischemia episode. Both peak flow and excess flow of reperfused hearts were significantly elevated in hearts from Tg compared to WT mice being 2.03 and 3.22 times greater, respectively. These results indicate that h15-LO-1-derived metabolites are highly vasoactive and may play a critical role in regulating coronary blood flow.

Adenosine A1 receptors heterodimerize with ß1- and ß2-adrenergic receptors creating novel receptor complexes with altered G protein coupling and signaling.

G protein coupled receptors play crucial roles in mediating cellular responses to external stimuli, and increasing evidence suggests that they function as multiple units comprising homo/heterodimers and hetero-oligomers. Adenosine and ß-adrenergic receptors are co-expressed in numerous tissues and mediate important cellular responses to the autocoid adenosine and sympathetic stimulation, respectively. The present study was undertaken to examine whether adenosine A1ARs heterodimerize with ß1- and/or ß2-adrenergic receptors (ß1R and ß2R), and whether such interactions lead to functional consequences. Co-immunoprecipitation and co-localization studies with differentially epitope-tagged A1, ß1, and ß2 receptors transiently co-expressed in HEK-293 cells indicate that A1AR forms constitutive heterodimers with both ß1R and ß2R. This heterodimerization significantly influenced orthosteric ligand binding affinity of both ß1R and ß2R without altering ligand binding properties of A1AR. Receptor-mediated ERK1/2 phosphorylation significantly increased in cells expressing A1AR/ß1R and A1AR/ß2R heteromers. ß-Receptor-mediated cAMP production was not altered in A1AR/ß1R expressing cells, but was significantly reduced in the A1AR/ß2R cells. The inhibitory effect of the A1AR on cAMP production was abrogated in both A1AR/ß1R and A1AR/ß2R expressing cells in response to the A1AR agonist CCPA. Co-immunoprecipitation studies conducted with human heart tissue lysates indicate that endogenous A1AR, ß1R, and ß2R also form heterodimers. Taken together, our data suggest that heterodimerization between A1 and ß receptors leads to altered receptor pharmacology, functional coupling, and intracellular signaling pathways. Unique and differential receptor cross-talk between these two important receptor families may offer the opportunity to fine-tune crucial signaling responses and development of more specific therapeutic interventions.

Structure-guided design of A(3) adenosine receptor-selective nucleosides: combination of 2-arylethynyl and bicyclo[3.1.0]hexane substitutions.

(N)-Methanocarba adenosine 5'-methyluronamides containing known A(3) AR (adenosine receptor)-enhancing modifications, i.e., 2-(arylethynyl)adenine and N(6)-methyl or N(6)-(3-substituted-benzyl), were nanomolar full agonists of human (h) A(3)AR and highly selective (K(i) ∼0.6 nM, N(6)-methyl 2-(halophenylethynyl) analogues 13 and 14). Combined 2-arylethynyl-N(6)-3-chlorobenzyl substitutions preserved A(3)AR affinity/selectivity in the (N)-methanocarba series (e.g., 3,4-difluoro full agonist MRS5698 31, K(i) 3 nM, human and mouse A(3)) better than that for ribosides. Polyaromatic 2-ethynyl N(6)-3-chlorobenzyl analogues, such as potent linearly extended 2-p-biphenylethynyl MRS5679 34 (K(i) hA(3) 3.1 nM; A(1), A(2A), inactive) and fluorescent 1-pyrene adduct MRS5704 35 (K(i) hA(3) 68.3 nM), were conformationally rigid; receptor docking identified a large, mainly hydrophobic binding region. The vicinity of receptor-bound C2 groups was probed by homology modeling based on recent X-ray structure of an agonist-bound A(2A)AR, with a predicted helical rearrangement requiring an agonist-specific outward displacement of TM2 resembling opsin. Thus, the X-ray structure of related A(2A)AR is useful in guiding the design of new A(3)AR agonists.

Polyamidoamine (PAMAM) dendrimer conjugate specifically activates the A3 adenosine receptor to improve post-ischemic/reperfusion function in isolated mouse hearts.

BACKGROUND: When stimulated by small molecular agonists, the A3 adenosine receptor (AR) mediates cardioprotective effects without inducing detrimental hemodynamic side effects. We have examined pharmacologically the protective properties of a multivalent dendrimeric conjugate of a nucleoside as a selective multivalent agonist for the mouse A3AR. RESULTS: A PAMAM dendrimer fully substituted by click chemistry on its peripheral groups with 64 moieties of a nucleoside agonist was shown to be potent and selective in binding to the mouse A3AR and effective in cardioprotection in an isolated mouse heart model of ischemia/reperfusion (I/R) injury. This conjugate MRS5246 and a structurally related model compound MRS5233 displayed binding Ki values of 0.04 and 3.94 nM, respectively, and were potent in in vitro functional assays to inhibit cAMP production. A methanocarba (bicyclo[3.1.0]hexane) ring system in place of ribose maintained a North conformation that is preferred at the A3AR. These analogues also contained a triazole linker along with 5'-N-methyl-carboxamido and 2-alkynyl substitution, previously shown to be associated with species-independent A3AR selectivity. Both MRS5233 and MRS5246 (1 and 10 nM) were effective at increasing functional recovery of isolated mouse hearts after 20 min ischemia followed by 45 min reperfusion. A statistically significant greater improvement in the left ventricular developed pressure (LVDP) by MRS5246 compared to MRS5233 occurred when the hearts were observed throughout reperfusion. Unliganded PAMAM dendrimer alone did not have any effect on functional recovery of isolated perfused mouse hearts. 10 nM MRS5246 did not improve functional recovery after I/R in hearts from A3AR gene "knock-out" (A3KO) mice compared to control, indicating the effects of MRS5246 were A3AR-specific. CONCLUSIONS: Covalent conjugation to a versatile drug carrier enhanced the functional potency and selectivity at the mouse A3AR and maintained the cardioprotective properties. Thus, this large molecular weight conjugate is not prevented from extravasation through the coronary microvasculature.