Purine Riboside Research Articles

Plant nucleoside N-ribohydrolases: riboside binding and role in nitrogen storage mobilization.

Cells save their energy during nitrogen starvation by selective autophagy of ribosomes and degradation of RNA to ribonucleotides and nucleosides. Nucleosides are hydrolyzed by nucleoside N-ribohydrolases (nucleosidases, NRHs). Subclass I of NRHs preferentially hydrolyzes the purine ribosides while subclass II is more active towards uridine and xanthosine. Here, we performed a crystallographic and kinetic study to shed light on nucleoside preferences among plant NRHs followed by in vivo metabolomic and phenotyping analyses to reveal the consequences of enhanced nucleoside breakdown. We report the crystal structure of Zea mays NRH2b (subclass II) and NRH3 (subclass I) in complexes with the substrate analog forodesine. Purine and pyrimidine catabolism are inseparable because nucleobase binding in the active site of ZmNRH is mediated via a water network and is thus unspecific. Dexamethasone-inducible ZmNRH overexpressor lines of Arabidopsis thaliana, as well as double nrh knockout lines of moss Physcomitrium patents, reveal a fine control of adenosine in contrast to other ribosides. ZmNRH overexpressor lines display an accelerated early vegetative phase including faster root and rosette growth upon nitrogen starvation or osmotic stress. Moreover, the lines enter the bolting and flowering phase much earlier. We observe changes in the pathways related to nitrogen-containing compounds such as β-alanine and several polyamines, which allow plants to reprogram their metabolism to escape stress. Taken together, crop plant breeding targeting enhanced NRH-mediated nitrogen recycling could therefore be a strategy to enhance plant growth tolerance and productivity under adverse growth conditions.

Structure activity relationship, 6-modified purine riboside analogues to activate hSTING, stimulator of interferon genes

Twenty-nine nucleoside analogues have been synthesized and evaluated in a cell based assay for their ability to activate the human Stimulator of Interferon Genes (hSTING), a key protein of the innate immune defense. Some 6-O-alkyl nucleoside analogues activate hSTING without associated cytotoxicity. SAR and combination studies were performed to decipher possible activation mechanism. The described nucleoside hSTING activators represent first-in-class modulators of the innate immune defense; a highly relevant target for antiviral, antibacterial, anticancer or Alzheimer’s disease treatments and may present advantages over other types of hSTING activators.

C2-substituted aromatic cytokinin sugar conjugates delay the onset of senescence by maintaining the activity of the photosynthetic apparatus

Cytokinins are plant hormones with biological functions ranging from coordination of plant growth and development to the regulation of senescence. A series of 2-chloro-N6-(halogenobenzylamino)purine ribosides was prepared and tested for cytokinin activity in detached wheat leaf senescence, tobacco callus and Amaranthus bioassays. The synthetic compounds showed significant activity, especially in delaying senescence in detached wheat leaves. They were also tested in bacterial receptor bioassays using both monocot and dicot members of the cytokinin receptor family. Most of the derivatives did not trigger cytokinin signaling via the AHK3 and AHK4 receptors from Arabidopsis thaliana in the bacterial assay, but some of them specifically activated the ZmHK1 receptor from Zea mays and were also more active than the aromatic cytokinin BAP in an ARR5::GUS cytokinin bioassay using transgenic Arabidopsis plants. Whole transcript expression analysis was performed using an Arabidopsis model to gather information about the reprogramming of gene transcription when senescent leaves were treated with selected C2-substituted aromatic cytokinin ribosides. Genome-wide expression profiling revealed that the synthetic halogenated derivatives induced the expression of genes related to cytokinin signaling and metabolism. They also prompted both up- and down-regulation of a unique combination of genes coding for components of the photosystem II (PSII) reaction center, light-harvesting complex II (LHCII), and the oxygen-evolving complex, as well as several stress factors responsible for regulating photosynthesis and chlorophyll degradation. Chlorophyll content and fluorescence analyses demonstrated that treatment with the halogenated derivatives increased the efficiency of PSII photochemistry and the abundance of LHCII relative to DMSO- and BAP-treated controls. These findings demonstrate that it is possible to manipulate and fine-tune leaf longevity using synthetic aromatic cytokinin analogs.

Immobilized purine nucleoside phosphorylase from Aeromonas hydrophila as an on-line enzyme reactor for biocatalytic applications

We described the development of a biochromatographic system which uses a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) for the evaluation of the substrate specificity on nucleoside libraries. AhPNP has been covalently immobilized on a fused silica Open Tubular Capillary (OTC) via Schiff base chemistry. The resulting bioreactor has been characterized by the determination of kinetic constants (Km and Vmax) for a natural substrate (inosine) and then assayed versus all natural purine (deoxy)ribonucleosides and a small library of 6-substituted purine ribosides. Characterization of the bioreactor has been carried out through a bidimensional chromatographic system with the sample on-line transfer from the bioreactor to the analytical column for the separation and quantification of substrate and product. Comparison with the soluble enzyme showed that the AhPNP-based bioreactor is reliable as the same ranking order, with respect to the standard activity assay, was obtained. The stability of the IMER was also assessed and the system was found to be stable up to 60 reactions.

Structure and Function of Nucleoside Hydrolases from Physcomitrella patens and Maize Catalyzing the Hydrolysis of Purine, Pyrimidine, and Cytokinin Ribosides

We present a comprehensive characterization of the nucleoside N-ribohydrolase (NRH) family in two model plants, Physcomitrella patens (PpNRH) and maize (Zea mays; ZmNRH), using in vitro and in planta approaches. We identified two NRH subclasses in the plant kingdom; one preferentially targets the purine ribosides inosine and xanthosine, while the other is more active toward uridine and xanthosine. Both subclasses can hydrolyze plant hormones such as cytokinin ribosides. We also solved the crystal structures of two purine NRHs, PpNRH1 and ZmNRH3. Structural analyses, site-directed mutagenesis experiments, and phylogenetic studies were conducted to identify the residues responsible for the observed differences in substrate specificity between the NRH isoforms. The presence of a tyrosine at position 249 (PpNRH1 numbering) confers high hydrolase activity for purine ribosides, while an aspartate residue in this position confers high activity for uridine. Bud formation is delayed by knocking out single NRH genes in P. patens, and under conditions of nitrogen shortage, PpNRH1-deficient plants cannot salvage adenosine-bound nitrogen. All PpNRH knockout plants display elevated levels of certain purine and pyrimidine ribosides and cytokinins that reflect the substrate preferences of the knocked out enzymes. NRH enzymes thus have functions in cytokinin conversion and activation as well as in purine and pyrimidine metabolism.

ChemInform Abstract: Synthesis and Evaluation of the Substrate Activity of C‐6 Substituted Purine Ribosides with E. coli Purine Nucleoside Phosphorylase: Palladium Mediated Cross‐Coupling of Organozinc Halides with 6‐Chloropurine Nucleosides.

AbstractPd(IV)‐Mediated cross‐coupling reactions of ribofuranosylpurine (I) with organozinc halides (II) and subsequent deprotection afford the C‐6 substituted purine ribosides (III) in satisfactory to high yields.

ChemInform Abstract: A New Method for Synthesis of 2‐Alkoxyadenosine Analogues.

AbstractThe 2‐amino group in purine ribosides (I) and (V) is converted into the corresponding 2‐alkoxy group by employing a one‐pot de novo synthesis involving non‐aqueous diazotization‐dediazoniation reactions.

Synthesis and evaluation of the substrate activity of C-6 substituted purine ribosides with E. coli purine nucleoside phosphorylase: Palladium mediated cross-coupling of organozinc halides with 6-chloropurine nucleosides

A series of C-6 alkyl, cycloalkyl, and aryl-9-(β- d-ribofuranosyl)purines were synthesized and their substrate activities with Escherichia coli purine nucleoside phosphorylase ( E. coli PNP) were evaluated. (Ph 3P) 4Pd-mediated cross-coupling reactions of 6-chloro-9-(2,3,5-tri- O-acetyl-β- d-ribofuranosyl)-purine ( 6) with primary alkyl (Me, Et, n-Pr, n-Bu, isoBu) zinc halides followed by treatment with NH 3/MeOH gave the corresponding 6-alkyl-9-(β- d-ribofuranosyl)purine derivatives 7– 11, respectively, in good yields. Reactions of 6 with cycloalkyl(propyl, butyl, pentyl)zinc halides and aryl (phenyl, 2-thienyl)zinc halides gave under similar conditions the corresponding 6-cyclopropyl, cyclobutyl, cyclopentyl, phenyl, and thienyl -9-(β- d-ribofuranosyl)purine derivatives 12– 16, respectively in high yields. E. coli PNP showed a high tolerance to the steric and hydrophobic environment at the 6-position of the synthesized purine ribonucleosides. Significant cytotoxic activity was observed for 8, 12, 15, and 16. Evaluation of 12 and 16 against human tumor xenografts in mice did not demonstrate any selective antitumor activity. In addition, 6-methyl-9-(β- d-arabinofuranosyl)purine ( 18) was prepared and evaluated.

Use of a QM/MM-Based FEP Method to Evaluate the Anomalous Hydration Behavior of Simple Alkyl Amines and Amides: Application to the Design of FBPase Inhibitors for the Treatment of Type-2 Diabetes

Standard molecular mechanics (MM) force fields predict a nearly linear decrease in hydration free energy with each successive addition of a methyl group to ammonia or acetamide, whereas a nonadditive relationship is observed experimentally. In contrast, the non-additive hydration behavior is reproduced directly using a quantum mechanics (QM)/MM-based free-energy perturbation (FEP) method wherein the solute partial atomic charges are updated at every window. Decomposing the free energies into electrostatic and van der Waals contributions and comparing the results with the corresponding free energies obtained using a conventional FEP method and a QM/MM method wherein the charges are not updated suggests that inaccuracies in the electrostatic free energies are the primary reason for the inability of the conventional FEP method to predict the experimental findings. The QM/MM-based FEP method was subsequently used to evaluate inhibitors of the diabetes drug target fructose-1,6-bisphosphatase adenosine 5'-monophosphate and 6-methylamino purine riboside 5'-monophosphate. The predicted relative binding free energy was consistent with the experimental findings, whereas the relative binding free energy predicted using the conventional FEP method differed from the experimental finding by an amount consistent with the overestimated relative solvation free energies calculated for alkylamines. Accordingly, the QM/MM-based FEP method offers potential advantages over conventional FEP methods, including greater accuracy and reduced user input. Moreover, since drug candidates often contain either functionality that is inadequately treated by MM (e.g., simple alkylamines and alkylamides) or new molecular scaffolds that require time-consuming development of MM parameters, these advantages could enable future automation of FEP calculations as well as greatly increase the use and impact of FEP calculations in drug discovery.

A New Method for Synthesis of 2-Alkoxyadenosine Analogs

O 6 -Methylguanosine and 2-amino-6-chloropurine riboside derivative were treated with isoamylnitrite in the presence of an appropriate alcohol to give the corresponding 2-alkoxy-6-methoxy (or chloro) purine riboside derivatives.

Purine nucleoside phosphorylase activity and expression are upregulated in sites affected by periodontal disease

Purine nucleoside phosphorylase (PNP) is an enzyme that catalyzes the reversible phosphorolysis of purine nucleosides, playing a key role in the purine salvage pathway. Activated T cells seem to rely heavily on PNP to remain functionally active and are particularly sensitive to PNP deficiency. The role of PNP in periodontal tissues has not been characterized thus far. The aim of this study therefore was to assess the activity and expression of PNP in the gingival tissues of periodontitis patients. Ten patients consecutively admitted for treatment had their periodontal clinical variables recorded and their gingival crevicular fluid collected. After periodontal treatment the patients were seen once a month for plaque and bleeding control, and had their periodontal variables recorded and gingival crevicular fluid collected at 90 and 180 d. Purine nucleoside phosphorylase-specific activity was assessed using a spectrophotometer through the addition of the PNP substrate analog 2-amino-6mercapto-7-methyl purine riboside to the gingival crevicular fluid. In parallel, PNP expression was assessed by immunohistochemistry and real-time PCR in gingival biopsies and cell culture. Purine nucleoside phosphorylase activity was higher in the gingival crevicular fluid of periodontally diseased sites, which was positively correlated with improvements of the clinical variables. Treatment of periodontal disease induced a striking decrease of PNP activity in periodontally diseased sites. Expression of PNP was more pronounced in mononuclear cells and endothelial cells of the gingiva, and the mRNA levels were 5.7-fold higher in inflamed tissues compared with control samples. Purine nucleoside phosphorylase activity and expression are upregulated in periodontally diseased sites and can be detected in the gingival crevicular fluid.

Modulation of AMP Deaminase in Rat Hearts Subjected to Ischemia and Reperfusion by Purine Riboside

Changes in AMP deaminase (AMPD) activity influence heart function and progression of heart disease, but the underlying mechanism is unknown. We evaluated the effect of purine riboside (Purr) on the activity of AMPD in perfused rat hearts and in isolated rat cardiomyocytes. Brief perfusion of the pre-ischemic heart with 200 μ M Purr resulted in activation of AMPD, more pronounced degradation of the adenine nucleotides, and reduced recovery of the adenine nucleotide pool during reperfusion. Brief incubation of rat cardiomyocytes with 200 μ M Purr also activated AMPD, while prolonged exposure resulted in enzyme inhibition. We conclude that Purr activates AMPD, whereas metabolites of this compound may inhibit the enzyme.

Novel immunoaffinity purification approach for isolation of new cytokinin based anticancer drug and its identification in plant tissues

Cytokinins [1] are plant hormones that trigger a wide range of biological processes in plants. Among their effects the control of cell division is the crucial function. Ribosides are one of the forms in which cytokinins naturally occur. In addition to their actions in plants, it has been discovered that these substances are also very effective inhibitors of cancer cell proliferation and very potent inducers of apoptosis in several human leukemia cell lines with typical morphological and biochemical hallmarks [2,3,4]. We have been studying cytotoxic effect of several synthetic cytokinin ribosides against various leukaemia cell lines. Surprisingly, we have proved considerable cytotoxicity of 6-(2-hydroxy-3-methoxybenzylamino)purine riboside (20H3MeOBAPR) toward CEM and HL-60 leukaemia cell lines with IC50 values of 0.3µM and 0.15µM, respectively [5]. In order to study this interesting compound in more details, we prepared monoclonal antibodies against 20H3MeOBAPR and we developed effective batch immunoaffinity extraction technique for its purification from various biological materials [6]. Consequently, we performed a study on per oral bioavailability [6] and pharmacokinetic quantitative analyses of this compound and its proposed pro-drug form in murine blood samples by high performance liquid chromatography-mass spectrometry (HPLC/MS) measurement. Further, we undertook a general screening for new di- and trisubstituted cytokinin ribosides as endogenous cytokinins in various biological materials, namely in Agrobacterium tumefaciens and Arabidopsis thaliana tissue. By the use of high resolution measurement, on a hybrid quadrupole-time of flight (Q-TOF) mass analyzer with electrospray ionization technique connected to capillary liquid chromatography (capLC), we were able to identify two new disubstituted benzyladenosines in both investigated biological materials [5].

Isolation and biochemical characterization of δ-aminolevulinic acid dehydratase from Streptomyces yokosukanensis ATCC 25520

In this study, delta-aminolevulinic acid dehydratase from Streptomyces yokosukanensis ATCC 25520, producer of an unusual purine riboside antibiotic called nebularine, was purified and characterized. Purification procedures involved with ammonium sulphate precipitation and gel filtration techniques by use of Sephacryl S-200. After gel filtration a 90.76-fold purification was obtained. The maximum enzymic activity was observed in the supernatant after 100% precipitation. According to the data obtained from investigation, the enzyme was found to be a single polypeptide having molecular mass around 34.8 kDa. This was determined by SDS-PAGE. Its optimal temperature around 45 degrees C, and optimal pH was found to be 8.0. Some heavy metals, Pb2+, Zn2+, Fe3+, Co2+, Mn2+, Mg2+ inhibited its activity between 20-51%, Ni2+ increased its activity up to 15%.

Constrained NBMPR analogue synthesis, pharmacophore mapping and 3D-QSAR modeling of equilibrative nucleoside transporter 1 (ENT1) inhibitory activity

Conformationally constrained analogue synthesis was undertaken to aid in pharmacophore mapping and 3D-QSAR analysis of nitrobenzylmercaptopurine riboside (NBMPR) congeners as equilibriative nucleoside transporter 1 (ENT1) inhibitors. In our previous study [ J. Med. Chem. 2003, 46, 831–837], novel regioisomeric nitro-1,2,3,4-tetrahydroisoquinoline conformationally constrained analogues of NBMPR were synthesized and evaluated as ENT1 ligands. 7-NO 2-1,2,3,4-Tetrahydroisoquino-2-yl purine riboside was identified as the analogue with the nitro group in the best orientation at the NBMPR binding site of ENT1. In the present study, further conformational constraining was introduced by synthesizing 5′- O,8-cyclo derivatives. The flow cytometrically determined binding affinities indicated that the additional 5′- O,8-cyclo constraining was unfavorable for binding to the ENT1 transporter. The structure–activity relationship (SAR) acquired was applied to pharmacophore mapping using the PHASE program. The best pharmacophore hypothesis obtained embodied an anti-conformation with three hydrogen-bond acceptors, one hydrophobic center, and two aromatic rings involving the 3′-OH, 4′-oxygen, the NO 2 group, the benzyl phenyl and the imidazole and pyrimidine portions of the purine ring, respectively. A PHASE 3D-QSAR model derived with this pharmacophore yielded an r 2 of 0.916 for four ( 4) PLS components, and an excellent external test set predictive r 2 of 0.78 for 39 compounds. This pharmacophore was used for molecular alignment in a comparative molecular field analysis (CoMFA) 3D-QSAR study that also afforded a predictive model with external test set validation predictive r 2 of 0.73. Thus, although limited, this study suggests that the bioactive conformation for NBMPR at the ENT1 transporter could be anti. The study has also suggested an ENT1 inhibitory pharmacophore, and established a predictive CoMFA 3D-QSAR model that might be useful for novel ENT1 inhibitor discovery and optimization.

An efficient convergent synthesis of adenosine-5′- N-alkyluronamides

Herein we report an efficient synthesis of adenosine-5′- N-alkyluronamides in which an enzyme-mediated deacetylation reaction is a key to the selective modification of the 5′- N-position, prior to coupling the ribose and purine components via a microwave-assisted Vorbrüggen coupling. This approach provides access to highly functionalised adenosines with 2- and N 6-substitutents, which can be incorporated before or after the ribose-coupling step. In all cases the microwave-assisted Vorbrüggen coupling conditions afforded anomerically pure purine ribosides in good to excellent yields.

Substrate analogs induce an intermediate conformational change inToxoplasma gondiiadenosine kinase

Adenosine kinase (AK) is a key enzyme in purine metabolism in the ubiquitous intracellular parasite Toxoplasma gondii and is a potential chemotherapeutic target for the treatment of T. gondii infections. To better understand the structure-activity relationship of 6-substituted purine ribosides, the structures of the T. gondii AK-N6,N6-dimethyladenosine (DMA) complex, the AK-DMA-AMP-PCP complex, the AK-6-methyl mercaptopurine riboside (MMPR) complex and the AK-MMPR-AMP-PCP complex were determined to 1.35, 1.35, 1.75 and 1.75 A resolution, respectively. These structures reveal a conformation intermediate between open and closed, with a small lid-domain rotation of 12 degrees . Residues Gly143-X-X-Gly146 undergo torsional changes upon substrate binding, which together with a Gly68-Gly69 switch induces a hinge bending of the lid domain. The intermediate conformation suggests that ATP binding is independent of adenosine binding. Orienting the gamma-phosphate group of ATP into the optimal catalytic position may be the last step before the onset of chemical catalysis and may require the translocation of Arg136 following the complete closure of the lid domain. 6-Substituted purine-nucleoside analogs are accommodated in a hydrophobic cavity. Modification at the N6 or C6 position of the nucleoside would affect the interactions with the surrounding residues and the binding affinity.

Purine analog substitution of the HIV-1 polypurine tract primer defines regions controlling initiation of plus-strand DNA synthesis

Despite extensive study, the mechanism by which retroviral reverse transciptases (RTs) specifically utilize polypurine tract (PPT) RNA for initiation of plus-strand DNA synthesis remains unclear. Three sequence motifs within or adjacent to the purine-rich elements are highly conserved, namely, a rU:dA tract region immediately 5′ to the PPT, an rA:dT-rich sequence constituting the upstream portion of the PPT and a downstream rG:dC tract. Using an in vitro HIV-1 model system, we determined that the former two elements define the 5′ terminus of the (+)-strand primer, whereas the rG:dC tract serves as the primary determinant of initiation specificity. Subsequent analysis demonstrated that G→A or A→G substitution at PPT positions −2, −4 and +1 (relative to the scissile phosphate) substantially reduces (+)-strand priming. We explored this observation further using PPT substrates substituted with a variety of nucleoside analogs [inosine (I), purine riboside (PR), 2-aminopurine (2-AP), 2,6-diaminopurine (2,6-DAP), isoguanine (iG)], or one of the naturally occurring bases at these positions. Our results demonstrate that for PPT positions −2 or +1, substituting position 2 of the purine was an important determinant of cleavage specificity. In addition, cleavage specificity was greatly affected by substituting −4G with an analog containing a 6-NH2 moiety.

Cytokinins inhibit epiphyllous plantlet development on leaves of Bryophyllum (Kalanchoe) marnierianum

When leaves of Bryophyllum marnierianum are detached from the plant, plantlets develop from primordia located at their margins. Leaves excised with a piece of stem attached do not produce plantlets. Severing the major leaf veins overcomes the inhibitory effect of the attached stem, indicating that the control agent is transmitted through the vascular system. A possible mechanism is that an inhibitory substance, possibly a known plant hormone, transported from the stem to the leaf, suppresses plantlet development. A number of hormones were tested for their ability to inhibit plantlet primordium development in whole isolated leaves. Auxins had no effect, indicating that apical dominance is not involved. The cytokinins zeatin, kinetin, and benzylaminopurine (BAP) strongly inhibited plantlet development, suggesting that they may be the or a factor involved in maintenance of plantlet primordium dormancy when the leaf is attached to the plant. This hypothesis was strongly supported by the finding that treatment of leaves attached to stems with a cytokinin antagonist (purine riboside) released the primordia from inhibition. In contrast to whole leaves, plantlet primordium development on leaf explants incubated on Murashige Skoog medium containing 3% sucrose was strongly stimulated by cytokinins. A possible explanation of these observations is that in whole leaves the cytokinin signal is transduced into an inhibitory signal whereas in the isolated primordium cytokinin has a direct stimulatory effect. The inhibitory cytokinin pathway must be dominant as long as the leaf is attached to the plant. A model is proposed which could explain these findings. This study points to a novel role of cytokinins in the maintenance of foliar plantlet primordium dormancy.

INTERACTION OF AROMATIC CYTOKININS WITH HUMAN LIVER MICROSOMAL CYTOCHROMES P450

Aromatic cytokinins (ortho-topolin riboside, 6-benzylaminopurine riboside and 6-(2-hydroxy-3-methoxybenzyla mino)purine riboside) were tested for their possible interaction with human liver microsomal cytochromes P450 by absorption difference spectroscopy. All three compounds were shown to bind to the CYP enzymes producing a high to low spin shift of the heme iron yielding a Soret absorption band shift to approximately 425 nm. As this type of spectral change means that the substance is able to bind directly to the heme iron, the results obtained open the possibility of an interaction of these compounds with metabolism of other drugs or, in general, with other substrates of cytochromes P450.