Discovery of Novel Adenosine Receptor Agonists That Exhibit Subtype Selectivity

A series of N6-bicyclic and N6-(2-hydroxy)cyclopentyl derivatives of adenosine were synthesized as novel A1R agonists and their A1R/A2R selectivity assessed using a simple yeast screening platform. We observed that the most selective, high potency ligands were achieved through N6-adamantyl substitution in combination with 5′-N-ethylcarboxamido or 5′-hydroxymethyl groups. In addition, we determined that 5′-(2-fluoro)thiophenyl derivatives all failed to generate a signaling response despite showing an interaction with the A1R. Some selected compounds were also tested on A1R and A3R in mammalian cells revealing that four of them are entirely A1R-selective agonists. By using in silico homology modeling and ligand docking, we provide insight into their mechanisms of recognition and activation of the A1R. We believe that given the broad tissue distribution, but contrasting signaling profiles, of adenosine receptor subtypes, these compounds might have therapeutic potential.

Adenosine 5'-Phosphosulfate Sulfotransferase and Adenosine 5'-Phosphosulfate Reductase Are Identical Enzymes

Adenosine 5′-phosphosulfate (APS) sulfotransferase and APS reductase have been described as key enzymes of assimilatory sulfate reduction of plants catalyzing the reduction of APS to bound and free sulfite, respectively. APS sulfotransferase was purified to homogeneity from Lemna minor and compared with APS reductase previously obtained by functional complementation of a mutant strain of Escherichia coli with an Arabidopsis thaliana cDNA library. APS sulfotransferase was a homodimer with a monomer M r of 43,000. Its amino acid sequence was 73% identical with APS reductase. APS sulfotransferase purified from Lemna as well as the recombinant enzyme were yellow proteins, indicating the presence of a cofactor. Like recombinant APS reductase, recombinant APS sulfotransferase used APS (K m = 6.5 μM) and not adenosine 3′-phosphate 5′-phosphosulfate as sulfonyl donor. TheV max of recombinant Lemna APS sulfotransferase (40 μmol min−1 mg protein−1) was about 10 times higher than the previously published V max of APS reductase. The product of APS sulfotransferase from APS and GSH was almost exclusively SO3 2−. Bound sulfite in the form ofS-sulfoglutathione was only appreciably formed when oxidized glutathione was added to the incubation mixture. Because SO3 2− was the first reaction product of APS sulfotransferase, this enzyme should be renamed APS reductase.

Plant Adenosine 5′-phosphosulfate Reductase Is a Novel Iron-Sulfur Protein

Adenosine 5′-phosphosulfate reductase (APR) catalyzes the two-electron reduction of adenosine 5′-phosphosulfate to sulfite and AMP, which represents the key step of sulfate assimilation in higher plants. Recombinant APRs from both Lemna minorand Arabidopsis thaliana were overexpressed inEscherichia coli and isolated as yellow-brown proteins. UV-visible spectra of these recombinant proteins indicated the presence of iron-sulfur centers, whereas flavin was absent. This result was confirmed by quantitative analysis of iron and acid-labile sulfide, suggesting a 4Fe-4S cluster as the cofactor. EPR spectroscopy of freshly purified enzyme showed, however, only a minor signal at g = 2.01. Therefore, Mössbauer spectra of 57Fe-enriched APR were obtained at 4.2 K in magnetic fields of up to 7 tesla, which were assigned to a diamagnetic 4Fe-4S2+ cluster. This cluster was unusual because only three of the iron sites exhibited the same Mössbauer parameters. The fourth iron site gave, because of the bistability of the fit, a significantly smaller isomer shift or larger quadrupole splitting than the other three sites. Thus, plant assimilatory APR represents a novel type of adenosine 5′-phosphosulfate reductase with a 4Fe-4S center as the sole cofactor, which is clearly different from the dissimilatory adenosine 5′-phosphosulfate reductases found in sulfate reducing bacteria.

Sulfate assimilation in higher plants. Characterization of a stable intermediate in the adenosine 5′-phosphosulfate reductase reaction

The enzyme catalysing the reduction of adenosine 5′-phosphosulfate (AdoPS) to sulfite in higher plants, AdoPS reductase, is considered to be the key enzyme of assimilatory sulfate reduction. In order to address its reaction mechanism, the APR2 isoform of this enzyme from Arabidopsis thaliana was overexpressed in Escherichia coli and purified to homogeneity. Incubation of the enzyme with [35S]AdoPS at 4 °C resulted in radioactive labelling of the protein. Analysis of APR2 tryptic peptides revealed 35SO2–3 bound to Cys248, the only Cys conserved between AdoPS and prokaryotic phosphoadenosine 5′-phosphosulfate reductases. Consistent with this result, radioactivity could be released from the protein by incubation with thiols, inorganic sulfide and sulfite. The intermediate remained stable, however, after incubation with sulfate, oxidized glutathione or AdoPS. Because truncated APR2, missing the thioredoxin-like C-terminal part, could be labelled even at 37 °C, and because this intermediate was more stable than the complete protein, we conclude that the thioredoxin-like domain was required to release the bound SO2–3 from the intermediate. Taken together, these results demonstrate for the first time the binding of 35SO2–3 from [35S]AdoPS to AdoPS reductase and its subsequent release, and thus contribute to our understanding of the molecular mechanism of AdoPS reduction in plants.

INCREASED EXPRESSION OF ONE OF TWO ADENOSINE DEAMINASE ALLELES IN A HUMAN CHORIOCARCINOMA CELL LINE FOLLOWING SELECTION WITH ADENINE NUCLEOSIDES

The human choriocarcinoma cell line JEG-3 is heterozygous at the adenosine deaminase (ADA) gene locus. Both allelic genes are under strong but incomplete repression causing a very low level expression of the gene locus. Because cytotoxic adenosine analogues such as 9-(beta)-D arabinofuranosyladenine (ara-A) and 9-(beta)-D xylofuranosyladenine (xyl-A) can be specifically detoxified by the action of ADA, these analogues were used to select for JEG-3 derived cells which had increased ADA expression. When JEG-3 cells were subjected to a multi-step, successively increasing dosage of either ara-A or xyl-A, resistant cells with increased ADA expression were generated. This increased ADA expression in the resistant cells was unstable, so that when the selective pressure was removed, cellular ADA expression would decrease. Subclone analysis of xyl-A resistant cells revealed that compared to parental JEG-3 cells, individual resistant cells had either elevated ADA levels or decreased adenosine kinase (ADK) levels or both. This altered ADA and ADK expression in the resistant cells were found to be independent events. Because of high endogenous tissue conversion factor (TCF) expression in the JEG-3 cells, the allelic nature of the increased ADA expression in most of the resistant cells could not be determined. However, several resistant subcloned cells were found to have lost TCF expression. These TCF('-) cells expressed only the ADA*2 allelic gene product. Cell fusion experiments demonstrated that the ADA*1 allelic gene was intact and functional in the A3-1A7 cell line. Chromosomal analysis of the A3-1A7 cells showed that they had no double-minutes or homogeneously staining chromosomal regions, although a pair of new chromosomes were found in these cells. Segregation analysis of the hybrid cells indicated that an ADA*2 allelic gene was probably located on this new chromosome. The analysis of the A3-1A7 cell line suggested that the expression of only ADA 2 in these cells was the result of possibly a cis-deregulation of the ADA gene locus or more probably an amplification of the ADA*2 allelic gene. Two effective positive selection systems for ADA('+) cells were also developed and tested. These selection systems should eventually lead to the isolation of the ADA gene.^

Adenosine mediated lung mast cell degranulation in the mouse

Adenosine has been implicated to play a role in inflammatory processes associated with asthma. Most notable is adenosine's ability to potentiate mediator release from mast cells. Mast cells are bone marrow derived inflammatory cells that can release mediators that have both immediate and chronic effects on airway constriction and inflammation. Most physiological roles of adenosine are mediated through adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B and A 3. The mechanisms by which adenosine can influence the release of mediators from lung tissue mast cells is not understood due to lack of in vivo models. Mice deficient in the enzyme adenosine deaminase (ADA) have been generated. ADA controls the levels of adenosine in tissues and cells, and consequently, adenosine accumulates in the lungs of ADA-deficient mice. ADA-deficient mice develop features seen in asthmatics, including lung eosinophilia and mucus hypersecretion. In addition, lung tissue mast cell degranulation was associated with elevated adenosine in ADA-deficient lungs and can be prevented by ADA enzyme therapy. We established primary murine lung mast cell cultures, and used real time RT-PCR and immunofluorescence to demonstrate that A 2A, A2B and A3 receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists and A3 receptor deficient (A3−/−) mast cells suggested that activation of A3 receptors could induce mast cell mediator release in vitro. Furthermore, this mediator release was associated with increases in intracellular Ca++ that appeared to be mediated through a Gi and PI3K pathway. In addition, nebulized A3 receptor agonist directly induced lung mast cell degranulation in wild type mice while having no effect in A3−/− mice. These results demonstrate that the A3 receptor plays an important role in adenosine mediated murine lung mast cell degranulation. Therefore, the A3 adenosine receptor and its signaling pathways may represent novel therapeutic targets for the treatment and prevention of asthma. ^

Gene regulation during placenta development: Murine adenosine deaminase as a model to study placenta specific expression

The formation of the placenta is one of the first and most important developmental events that occur in early mammalian embryogenesis. Even given this importance of the placenta, the academic community has largely ignored studying gene regulation during the development and maturation of the placenta. For this reason, an in-depth study of gene regulation in the trophoblast layer of the placenta using murine Adenosine Deaminase (Ada) as a model system has been undertaken. It has been determined that Ada is highly expressed in the placenta and is critical for embryo development. Dr. Kellems' laboratory has previously described a 1.8 kb fragment of the Ada 5 ′ flanking region that is capable of directing trophoblast specific expression in a transgenic model system. Preliminary studies have demonstrated several critical portions of this fragment that are necessary for the correct tissue specific expression in the placenta. My first specific aim was to elucidate the trans factor binding to one of these sequences, the FP3. Through electromobility shift assays (EMSA), the 30 bp FP3 was narrowed to a 5 bp sequence which computer databases predicted bound to Acute Myeloid Leukemia 1 (AML-1). This was confirmed by supershift analysis. The functional importance of this binding was demonstrated by a transgenic approach. A significant difference in expression of the reporter in the placenta was seen when the 5 bp sequence was mutated. This finding is a novel use for the AML-1 transcription factor which is the DNA binding portion of the heterodimer Core Binding Protein (CBP). The 5′ 240 bp region has also been demonstrated to contain functionally significant sequence. Through EMSA assays and computer predictions, the area has been narrowed to two pertinent regions that are predicted to contain GATA binding motifs. ^

Potential Role of Adenosine Signaling in Acetic Acid Activation of Murine Sensory Neurons

Chronic respiratory illnesses are a significant cause of morbidity and mortality, and acute changes in respiratory function often lead to hospitalization. Air pollution is known to exacerbate asthma, but the molecular mechanisms of this are poorly understood. The current studies were aimed at clarifying the roles of nerve subtypes and purinergic receptors in respiratory reflex responses following exposure to irritants. In C57Bl/6J female mice, inspired adenosine produced sensory irritation, shown to be mediated mostly by A-delta fibers. Secondly, the response to inhaled acetic acid was discovered to be dually influenced by C and A-delta fibers, as indicated by the observed effects of capsaicin pretreatment, which selectively destroys TRPV1-expressing fibers (mostly C fibers) and pretreatment with theophylline, a nonselective adenosine receptor antagonist. The responses to both adenosine and acetic acid were enhanced in the ovalbumin-allergic airway disease model, although the particular pathway altered is still unknown.

The contribution of A3 adenosine receptor signaling to pulmonary inflammation and mucus secretion in the mouse

Adenosine has been implicated in chronic lung diseases such as asthma and COPD. Most physiological actions of adenosine are mediated through G-protein coupled adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B, and A 3. However, the specific roles of the various adenosine receptors in processes central to asthma and COPD are not well understood in part due to the lack of adequate animal models that examine the effect of adenosine on the development of lung disease. In this study we have investigated the expression and function of the A3 adenosine receptor in pulmonary eosinophilia and mucus production/secretion in adenosine deaminase (ADA)-deficient mice in which adenosine levels are elevated. ADA-deficient mice develop features of asthma and COPD, including lung eosinophilia and mucus hyperplasia in association with elevated lung adenosine levels. The A3 receptor was found to be expressed in eosinophils and mucus producing cells in the airways of ADA-deficient. Disruption of A3 receptor signaling in ADA-deficient mice by genetic removal of the receptor or treatment with MRS 1523, a selective A3 adenosine receptor antagonist, prevented airway eosinophilia and mucus production. Although eosinophils were decreased in the airways of ADA-deficient mice with disrupted A3 receptor signaling, elevations in circulating and lung interstitial eosinophils persisted, suggesting signaling through the A3 receptor is needed for the migration of eosinophils into the airways. Further examination of the role of the A3 receptor in mucus biology demonstrated that the A3 receptor is neither required nor is overexpression of the receptor in clara cells sufficient for mucus production in naive mice. Transgenic overexpression of the A3 receptor did elucidate a role for the A3 receptor in the secretion of mucus into the airways of ovalbumin challenged mice. These findings identify an important role for the A3 adenosine receptor in regulating lung eosinophilia and mucus secretion in inflammatory lung diseases. Therefore, the A3 adenosine receptor may represent a novel therapeutic target for the treatment and prevention of asthma. ^

The role of adenosine in pulmonary angiogenesis

Angiogenesis is a feature of chronic lung diseases such as asthma and pulmonary fibrosis; however, the pathways controlling pathological angiogenesis during lung disease are not completely understood. Adenosine is a signaling nucleoside that accumulates as a result of tissue hypoxia and damage. Adenosine has been implicated in the exacerbation of chronic lung disease and in the regulation of angiogenesis; however, the relationship between these factors has not been investigated. The work presented in this dissertation utilized adenosine deaminase (ADA)-deficient mice to determine whether chronic elevations of adenosine in vivo result in pulmonary angiogenesis, and to identify factors that could potentially mediate this process. Results demonstrate that there is substantial angiogenesis in the tracheas of ADA-deficient mice in association with adenosine elevations. Replacement enzyme therapy with pegylated ADA resulted in a lowering of adenosine levels and reversal of tracheal angiogenesis, indicating that the increases in vessel number are dependent on adenosine elevations. Levels of the ELR+ angiogenic chemokine CXCL1 were found to be elevated in an adenosine-dependent manner in the lungs of ADA-deficient mice. Neutralization of CXCL1 and its putative receptor, CXCR2, in ADA-deficient lung lysates resulted in the inhibition of angiogenic activity suggesting that CXCL1 signaling through the CXCR2 receptor is responsible for mediating the observed increases in angiogenesis. Taken together, these findings suggest that adenosine plays an important role, via CXCL1, in the induction of pulmonary angiogenesis and may therefore represent an important therapeutic target for the treatment of pathological angiogenesis. ^

Adenosine induced pulmonary fibrosis and the contribution of the adenosine A2B receptor to the induction of osteopontin in a mouse model of pulmonary fibrosis

Pulmonary fibrosis is a devastating and lethal lung disease with no current cure. Research into cellular signaling pathways able to modulate aspects of pulmonary inflammation and fibrosis will aid in the development of effective therapies for its treatment. Our laboratory has generated a transgenic/knockout mouse with systemic elevations in adenosine due to the partial lack of its metabolic enzyme, adenosine deaminase (ADA). These mice spontaneously develop progressive lung inflammation and severe pulmonary fibrosis suggesting that aberrant adenosine signaling is influencing the development and/or progression of the disease in these animals. These mice also show marked increases in the pro-fibrotic mediator, osteopontin (OPN), which are reversed through ADA therapy that serves to lower lung adenosine levels and ameliorate aspects of the disease. OPN is known to be regulated by intracellular signaling pathways that can be accessed through adenosine receptors, particularly the low affinity A2BR receptor, suggesting that adenosine receptor signaling may be responsible for the induction of OPN in our model. In-vitro, adenosine and the broad spectrum adenosine receptor agonist, NECA, were able to induce a 2.5-fold increase in OPN transcripts in primary alveolar macrophages. This induction was blocked through antagonism of the A2BR receptor pharmacologically, and through the deletion of the receptor subtype in these cells genetically, supporting the hypothesis that the A2BR receptor was responsible for the induction of OPN in our model. These findings demonstrate for the first time that adenosine signaling is an important modulator of pulmonary fibrosis in ADA-deficient mice and that this is in part due to signaling through the A2BR receptor which leads to the induction of the pro-fibrotic molecule, otseopontin. ^

2-methylthio-N$\sp6$-dimethylallyl modification of adenosine 37 of tRNAs in {\it Escherichia coli\/} K-12

The hypermodified, hydrophobic 2-methylthio-N$\sp6$-(dimethylallyl)-adenosine (ms${2{\cdot}6}\atop1$A) residue occurs $3\sp\prime$ to the anticodon in tRNA species that read codons beginning with U. The first step (i$\sp6$A37 formation) of this modification is catalyzed by dimethylallyl diphosphate:tRNA dimethyallyltransferase (EC 2.5.1.8), which is the product of the miaA gene. Subsequent steps were proposed to be catalyzed by MiaB and MiaC enzymes to complete the ms${2{\cdot}6}\atop1$A37 modification. The study of functions of the ms${2{\cdot}6}\atop1$A37 is very important because this modified base is one of the best candidates for a role in global control in response to environmental stress. This dissertation describes the further delineation of functions of the ms${2{\cdot}6}\atop1$A37 modification in E. coli K-12 cells. This work provides significant information on functions of tRNA modifications in E. coli cells to adapt to stressful environmental conditions. Three hypotheses were tested in this work.^ The first hypothesis tested was that non-optimal translation processes cause increased spontaneous mutagenesis by the induction of SOS response in starving cells. To test this hypothesis, I measured spontaneous mutation rates of wild type cells and various mutant strains which are defective in tRNA modification, SOS response, or oxidative damage repair. I found that the miaA mutation acts as a mutator that increased Lac$\sp+$ reversion rates and Trp$\sp+$ reversion frequencies of the wild-type cells in starving conditions. However, the lexA3(Ind)(which abolishes the induction of SOS response) mutation abolished the mutator phenotype of the miaA mutant. The recA430 mutation, not other identified SOS genes, decreased the Lac$\sp+$ reversion to a less extent than that of the lexA3(Ind) mutation. These results suggest that RecA together with another unidentified SOS gene product are responsible for the process.^ The second hypothesis tested was that MiaA protein binds to full-length tRNA$\sp{\rm Phe}$ molecules in form of a protein dimer. To test this hypothesis, three versions of the MiaA protein and seven species of tRNA substrates were purified. Binding studies by gel mobility shift assays, filter binding assays and gel filtration shift assays support the hypothesis that MiaA protein binds to full-length tRNA$\sp{\rm Phe}$ as a protein dimer but as a monomer to the anticodon stem-and-loop. These results were further supported by using steady state enzyme kinetic studies.^ The third hypothesis tested in this work was that the miaB gene in E. coli exists and is clonable. The miaB::Tn10dCm insertion mutation of Salmonella typhimurium was transduced to E. coli K-12 cells by using P$\sb1$ and P$\sb{22}$ bacteriophages. The insertion was confirmed by HPLC analyses of nucleotide profiles of miaB mutants of E. coli. The insertion mutation was cloned and DNA sequences adjacent to the transposon were sequenced. These DNA sequences were 86% identical to the f474 gene at 14.97 min chromosome of E. coli. The f474 gene was then cloned by PCR from the wild-type chromosome of E. coli. The recombinant plasmid complemented the mutant phenotype of the miaB mutant of E. coli. These results support the hypothesis that the miaB gene of E. coli exists and is clonable. In summary, functions of the ms${2{\cdot}6}\atop1$A37 modification in E. coli cells are further delineated in this work in perspectives of adaptation to stressful environmental conditions and protein:tRNA interaction. (Abstract shortened by UMI.) ^

ATP, chlorophyll "a", and size structure of phytolpankton in seawater at stations in the Atlantic sector of the Southern Ocean

Chlorophyll "a" and adenozine triphosphate (ATP) concentrations together with size structure of microplankton were investigated in January-April 1989 in the Indian Ocean and in the Weddell Sea. ATP values varied from 11 to 92 ng/l, and chlorophyll "a" concentrations varied from 0.04 to 0.27 µg/l in the Indian Ocean, with prevailing nanoplankton and picoplankton fractions. Both ATP and chlorophyll "a" concentrations increased 2 times to the south of 40°S; in the Weddell Sea they exceeded 400 ng/l and 0.6 µg/l, respectively. Cells of nanophytoplankton and microphytoplankton (mainly diatoms) prevailed in size spectra. Spatial variabilities of the parameters were within one order of magnitude; their values decreased 3-4 times during 1 month. Size structure changed due to increased portion of nanoplankton and picolankton. ATP concentrations in the photic layer (0-200 m) varied from 31.96 mg/m**2 in February to 8.02 mg/m**2 in March to April. ATP concentrations were 61.5 and 98.8 mg/m**2 at depths of 4200 and 4700 m, respectively.