Purine Compounds Research Articles

PH-Dependent mismatch discrimination of oligonucleotide duplexes containing 2′-deoxytubercidin and 2- or 7-substituted derivatives: protonated base pairs formed between 7-deazapurines and cytosine

Oligonucleotides incorporating 2′-deoxytubercidin (1a), its 2-amino derivative 2a and related 2-, or 7-substituted analogs (1d, 2b–d, 3 and 4) are synthesized. For this purpose, a series of novel phosphoramidites are prepared and employed in solid-phase synthesis. Hybridization experiments performed with 12mer duplexes indicate that 7-halogenated nucleosides enhance the duplex stability both in antiparallel and parallel DNA, whereas 2-fluorinated 7-deaza-2′-deoxyadenosine residues destabilize the duplex structure. The 7-deazaadenine nucleosides 1a, 1d and their 2-amino derivatives 2a–d form stable base pairs with dT but also with dC and dG. The mispairing with dC is pH-dependent. Ambiguous base pairing is observed at pH 7 or under acid conditions, whereas base discrimination occurs in alkaline medium (pH 8.0). This results from protonated base pairs formed between 1a or 2a and dC under neutral or acid condition, which are destroyed in alkaline medium. It is underlined by the increased basicity of the pyrrolo[2,3-d]pyrimidine nucleosides over that of the parent purine compounds (pKa values: 1a = 5.30; 2a = 5.71; dA = 3.50).

Effect of short‐term salt stress on the metabolic profiles of pyrimidine, purine and pyridine nucleotides in cultured cells of the mangrove tree, Bruguiera sexangula

To investigate the short‐term (3 h) effect of salt on the metabolism of purine, pyrimidine and pyridine nucleotides in mangrove (Bruguiera sexangula) cells, we examined the uptake and overall metabolism of radiolabelled intermediates involved in the de novo pathways and substrates of salvage pathways for nucleotide biosynthesis in the presence and absence of 100 mM NaCl. Uptake by the cells of substrates for the salvage pathways was much faster than uptake of intermediates of the de novo pathways. The activity of the de novo pyrimidine biosynthesis estimated by [2‐14C]orotate metabolism was not significantly affected by the salt. About 20–30% of [2‐14C]uridine, [2‐14C]uracil and more than 50% of [2‐14C]cytidine were salvaged for pyrimidine nucleotide biosynthesis. However, substantial quantities of these compounds were degraded to 14CO2 via β‐ureidopropionate (β‐UP), and degradation of β‐UP was increased by the salt. The activities of the de novo pathway, estimated by [2‐14C] 5‐aminoimidazole‐4‐carboxamide ribonucleoside, and the salvage pathways from [8‐14C]adenosine and [8‐14C]guanosine for the purine nucleotide biosynthesis were not influenced by the salt. Most [8‐14C]hypoxanthine was catabolised to 14CO2, and other purine compounds are also catabolised via xanthine. Purine catabolism was stimulated by the salt. [3H]Quinolinate, [carbonyl‐14C]nicotinamide and [carboxyl‐14C]nicotinic acid were utilised for the biosynthesis of pyridine nucleotides. The salvage pathways for pyridine nucleotides were significantly stimulated by the salt. Trigonelline was synthesised from all pyridine precursors that were examined; its synthesis was also stimulated by the salt. We discuss the physiological role of the salt‐stimulated reactions of nucleotide metabolism.

Modified Pyrimidines Specifically Bind the Purine Riboswitch

The purine riboswitch is a genetic regulatory element found in the 5'-untranslated regions of Gram-positive bacteria that regulates expression of the mRNA specifically in response to either guanine or adenine. We report that the adenine-responsive RNA element is also capable of specifically recognizing pyrimidine compounds bearing modifications at the 6- or 5,6-positions in a fashion similar to that of purine compounds. Using isothermal titration calorimetry and X-ray crystallography, the binding of these compounds is characterized.

A Three-Dimensional Quantitative Structure-Activity Relationship Study of the Inhibition of the ATPase Activity and the Strand Passing Catalytic Activity of Topoisomerase IIα by Substituted Purine Analogs

Based on the topoisomerase IIalpha catalytic inhibitory activity of a previous hit compound, NSC35866, we screened 40 substituted purines or purine-like compounds from the National Cancer Institute repository for their ability to inhibit the ATPase activity of human topoisomerase IIalpha. Several compounds, including NSC348400, NSC348401 and NSC348402, were inhibitory at submicromolar concentrations. Three-dimensional quantitative structure-activity relationship models using comparative molecular field and comparative molecular similarity indices analyses were constructed using 24 of these compounds. The ability of 10 selected compounds to inhibit the complete DNA strand passage reaction of topoisomerase IIalpha correlated well with their potency as ATPase inhibitors. None of the 40 compounds significantly increased levels of the topoisomerase IIalpha-DNA covalent complex, suggesting that they functioned as catalytic topoisomerase II inhibitors and not as topoisomerase II poisons. Although some of these compounds could antagonize the effect of etoposide on the level of topoisomerase IIalpha-DNA covalent complex formation in vitro, in contrast to NSC35866, they were not capable of antagonizing etoposide-induced cytotoxicity and DNA strand breaks in cells. Two independently selected human SCLC cell lines with reduced topoisomerase IIalpha expression displayed cross-resistance to NSC348400, NBSC348401, and NSC348402, whereas an MDR1 line was fully sensitive. These results suggest that topoisomerase IIalpha is a functional cellular target for most of these substituted purine compounds and that these compounds do not display MDR1 liability.

The purine metabolism of human erythrocytes

This review summarizes currently available information about a crucial part of erythrocyte metabolism, that is, purine nucleotide conversions and their relationships with other conversion pathways. We describe the cellular resynthesis, interconversion, and degradation of purine compounds, and also the regulatory mechanisms in the conversion pathways. We also mention purine metabolism disorders and their clinical consequences. The literature is fragmentary because studies have concentrated only on selected aspects of purine metabolism; hence the need for a synthetic approach.

Postnatal development of P2 receptors in the murine gastrointestinal tract

The actions of purine and pyrimidine compounds on isolated segments of the mouse intestine were investigated during postnatal development. The localization of P2Y 1, P2Y 2, P2Y 4, P2X 1, P2X 2 and P2X 3 receptors were examined immunohistochemically, and levels of expression of P2Y 1, P2X 1 and P2X 2 were studied by Western immunoblot. From day 12 onwards, the order of potency for relaxation of longitudinal muscle of all regions was 2-MeSADP ≥ α,β-meATP ≥ ATP = UTP = adenosine, suggesting P2Y 1 receptors. This was supported by the sensitivity of responses to 2-MeSADP to the selective antagonist MRS 2179 and P2Y 1 receptor immunoreactivity on longitudinal muscle and a subpopulation of myenteric neurons. A further α,β-meATP-sensitive P2Y receptor subtype was also indicated. ATP and UTP were equipotent suggesting a P2Y 2 and/or P2Y 4 receptor. Adenosine relaxed the longitudinal muscle in all regions via P1 receptors. The efficacy of all agonists to induce relaxation of raised tone preparations increased with age, being comparable to adult by day 20, the weaning age. During postnatal development the contractile response of the ileum and colon was via P2Y 1 receptors, while the relaxant response mediated by P2Y 1 receptors gradually appeared along the mouse gastrointestinal tract, being detectable in the stomach from day 3 and in the duodenum from day 6. In the ileum and colon relaxant responses to 2-MeSADP were not detected until days 8 and 12, respectively. 2-MeSADP induced contractions on basal tone preparations from day 3, but decreased significantly at day 12 and disappeared by day 20. At day 8, contractions of colonic longitudinal muscle to ATP showed no desensitisation suggesting the involvement of P2X 2 receptors. Immunoreactivity to P2X 2 receptors only was observed on the longitudinal muscle of the colon and ileum from day 1 and on a subpopulation of myenteric neurons from day 3. These data suggest that P2Y 1 receptors undergo postnatal developmental changes in the mouse gut, with a shift from contraction to relaxation. Such changes occur 1 week before weaning and may contribute to the changes that take place in the gut when the food composition changes from maternal milk to solid food.

Rigidity and Flexibility of Dipeptidyl Peptidase IV: Crystal Structures of and Docking Experiments with DPIV

Dipeptidyl peptidase IV (DPIV) is an alpha,beta-hydrolase-like serine exopeptidase, which removes dipeptides, preferentially with a C-terminal l-Pro residue, from the N terminus of longer peptide substrates. Previously, we determined the tetrameric 1.8A crystal structure of native porcine DPIV. Each monomer is composed of a beta-propeller and a catalytic domain, which together embrace an internal cavity housing the active centre. This cavity is connected to the bulk solvent by a "propeller opening" and a "side opening". Here, we analyse DPIV complexes with a t-butyl-Gly-Pro-Ile tripeptide, Pro-boroPro, a piperazine purine compound, and aminoethyl phenyl sulfonylfluoride. The latter two compounds bind to the active-site groove in a compact and a quite bulky manner, respectively, causing considerable shifts of the catalytic Ser630 side-chain and of the Tyr547 phenolic group, which forms the oxyanion hole. The tripeptide, mimicking a peptide substrate, is clamped to the active site through tight interactions via its N-terminal alpha-ammonium group, the P2 carbonyl group, the P1-l-Pro side-chain, the C-terminal carboxylate group, and the stable orthoacid ester amide formed between the scissile peptide carbonyl group and Ser630 O(gamma). This stable trapping of the tripeptide could be due to stabilization of the protonated His740 imidazolium cation by the adjacent negatively charged C-terminal carboxylate group, preventing proton transfer to the leaving group nitrogen atom. Docking experiments with the compact rigid 58 residue protein aprotinin, which had been shown to be processed by DPIV, indicate that the Arg1-Pro2 N terminus can access the DPIV active site only upon widening of its side openings, probably by separation of the first and the last propeller blades, and/or of the catalytic and the propeller domain.

AMP Deaminase Activation Contributes to Accelerated Adenine Nucleotide Pool Depletion during Periods of Energy Imbalance in Sickle Cell Erythrocytes.

Mature erythrocytes are unable to synthesize AMP from IMP due to the developmental loss of adenylosuccinate synthetase, which also effectively prevents the synthesis of adenine nucleotides from circulating hypoxanthine, the major salvageable purine compound. Consequently, AMP deaminase (AMPD) activity must be tightly regulated in order to avoid irreversible depletion of the erythrocyte adenine nucleotide pool during periods of energy imbalance. However, increased intracellular calcium promotes activation of erythrocyte AMPD (AMPD3) through a protein-protein interaction with calmodulin (Mahnke and Sabina, Biochemistry 44:5551, 2005). Therefore, we hypothesized that Sickle Cell Disease (SCD) erythrocytes would more rapidly accumulate IMP and deplete the adenine nucleotide pool during periods of energy imbalance. In order to test this hypothesis, acid-soluble pools of these metabolites were quantified by anion-exchange HPLC in SCD and control erythrocytes during autoincubation and glucose starvation, both at 37C. Data in the table show that SCD erythrocytes accumulate more IMP during a 6-hour autoincubation compared to control cells, and this is accompanied by a decrease in AMP.Effect of Autoincubation on Erythrocyte AMP and IMP PoolsGROUPHOURSAMPaIMPIMP/AMPControl (N=4)0.29±.08.15±.06.58±.286.29±.01.33±.11†1.15±.35†SCD (N=5)0.22±.04.08±.03.36±.106.14±.03†.74±.24†‡5.15±1.25†‡a, metabolite concentrations reported as μmol/g Hb; †, p<.05 when compared to 0 time in a paired student's t-test; ‡, p<.05 when compared to control in an unpaired student's t-testData in the figure show an accelerated depletion of ATP and increased accumulation of IMP in glucose-starved SCD erythrocytes compared to control cells (*, p<.05 when compared to control in an unpaired student's t-test). [Display omitted] These combined results demonstrate AMPD3 enzyme activation and accelerated adenine nucleotide pool depletion during periods of energy imbalance in SCD erythrocytes. This metabolic imbalance in SCD erythrocytes could contribute to 1) an increased rate of hemolysis, 2) cation channel dysregulation, 3) modulation of adhesive membrane components, and 4) diminished protein phosphorylation. Furthermore, strategies directed against this metabolic imbalance, e.g., diffusible AMPD inhibitors and adenine supplementation, may have a therapeutic benefit in SCD.

Purine Metabolites in Gout and Asymptomatic Hyperuricemia: Analysis by HPLC–Electrospray Tandem Mass Spectrometry

Hyperuricemia, a serum urate concentration >0.45 mmol/L (7.0 mg/dL) in men and 0.36 mmol/L (6.0 mg/dL) in women, is the biochemical hallmark of gout (1)(2)(3), but many individuals with life-long hyperuricemia do not develop gouty arthritis (4). Conversely, serum urate concentrations may be within reference values in some patients with acute gout, particularly during the early phases of the disorder. We hypothesized that gout patients may have a different profile of purine precursors than do asymptomatic people with hyperuricemia and that concentrations of these compounds may reflect disorders of purine metabolism. To our knowledge, purine metabolites have not been studied and compared in gout and asymptomatic hyperuricemia. With established methods for measuring purine compounds in blood and urine (5)(6)(7)(8)(9), retention time is not always adequate for identification of every peak because urine and blood usually contain many interfering compounds. Quantification by tandem mass spectrometry (MS/MS) (10)(11) may require an internal standard, which often is hard to obtain. In this study of 10 purine compounds in the blood of gout patients and hyperuricemia patients with no gout symptoms, we used HPLC for serum sample separation, MS/MS for peak identification, and ultraviolet (UV) detection for quantification. Purines were purchased from Fluka and Sigma. The HPLC-MS experiment was performed on an Agilent 1100 series liquid chromatograph with a mass spectrometric detector trap. Separation was carried out on a Supelcosil™ LC-18-DB column [25 cm × 4.6 mm (i.d.); 5-μm film thickness]. The column temperature was maintained at 25 °C. The mobile phases were as follows: 10 mmol/L ammonium acetate, adjusted to pH 6.5 with glacial acetic acid (eluant A), and methanol (eluant B). The elution gradient was as follows (flow rate, 1 mL/min): 0 to 10 …

Purine- and pyrimidine-induced responses and P2Y receptor characterization in the hamster proximal urethra.

1. Purine and pyrimidine compounds were investigated on hamster proximal urethral circular smooth muscle preparations. In situ hybridization studies were carried out to localize P2Y(1), P2Y(2), P2Y(4) and P2Y(6) mRNA. Protein expression was studied using Western blotting analysis with antibodies against P2Y(1) and P2Y(2) receptors. 2. The hamster urethra relaxed with an agonist potency order of: 2-MeSADP>beta,gamma-meATP=ATP=adenosine=ADP>2-MeSATP>alpha,beta-meATP>TTP>CTP=UTP>GTP=UDP. The high potency of 2-MeSADP is suggestive of an action via P2Y(1) receptors. Although the order is not characteristic for any known single P2Y receptor subtype, it may represent a combination of P2Y receptor subtypes. 4. The selective P2Y(1) receptor antagonist MRS2179 inhibited ATP-, 2-MeSADP-, 2-MeSATP-, beta,gamma-meATP-, and to a lesser degree alpha,beta-meATP-induced responses. 3. Adenosine, but not ATP, was inhibited by the adenosine receptor antagonist 8-phenyltheophylline, indicating that ATP was not acting via adenosine following enzymatic breakdown. 5. Western blotting analysis showed the expression of both P2Y(1) and P2Y(2) receptors, confirming the results obtained with in situ hybridization that showed the expression of both P2Y(1) and P2Y(2), but not P2Y(4) or P2Y(6) mRNA, in smooth muscle layers of the hamster proximal urethra. 6. It is proposed that the relaxant response of the urethra to ATP may be evoked through the activation of the combination of receptors for P2Y(1) and to a lesser extent P2Y(2) receptors, which may mediate a trophic effect in addition. A P2Y subtype responsive to alpha,beta-meATP and P1 receptors may contribute to urethral smooth muscle relaxation.

Selectivity of febuxostat, a novel non-purine inhibitor of xanthine oxidase/xanthine dehydrogenase

The purine analogue, allopurinol, has been in clinical use for more than 30 years as an inhibitor of xanthine oxidase (XO) in the treatment of hyperuricemia and gout. As consequences of structural similarities to purine compounds, however, allopurinol, its major active product, oxypurinol, and their respective metabolites inhibit other enzymes involved in purine and pyrimidine metabolism. Febuxostat (TEI-6720, TMX-67) is a potent, non-purine inhibitor of XO, currently under clinical evaluation for the treatment of hyperuricemia and gout. In this study, we investigated the effects of febuxostat on several enzymes in purine and pyrimidine metabolism and characterized the mechanism of febuxostat inhibition of XO activity. Febuxostat displayed potent mixed-type inhibition of the activity of purified bovine milk XO, with Ki and Ki′ values of 0.6 and 3.1 nM respectively, indicating inhibition of both the oxidized and reduced forms of XO. In contrast, at concentrations up to 100 μM, febuxostat had no significant effects on the activities of the following enzymes of purine and pyrimidine metabolism: guanine deaminase, hypoxanthine-guanine phosphoribosyltransferase, purine nucleoside phosphorylase, orotate phosphoribosyltransferase and orotidine-5′-monophosphate decarboxylase. These results demonstrate that febuxostat is a potent non-purine, selective inhibitor of XO, and could be useful for the treatment of hyperuricemia and gout.

Adenine nucleotides in snail muscles as one of biomarkers of fluoride toxicity

The aim of this work was to determine the extent of bioaccumulation of fluorides in tissues of Helix aspersa maxima. The toxicity of fluorides administered orally on the energy balance of the snail's foot was investigated based on measurements of concentrations of adenine nucleotides and their metabolism degradation products. Quantitation of fluoride levels was done in soft tissues (foot, hepatopancreas) and shells of mature snails. Qualitative and quantitative analysis of purine compounds was performed in slices of foot from mature snails. Fluoride concentrations in pulverized shells were measured using an ion-selective electrode. Gas chromatography was used to determine fluoride concentrations in soft tissues (hepatopancreas and foot). Purines were measured in foot muscle slices with high-performance liquid chromatography (HPLC). Fluoride levels in soft tissues of the snail cannot serve as an indicator for biomonitoring purposes as no significant accumulation was observed during exposure to maximum allowable concentrations of fluoride in drinking water. Contrary to this, levels of fluoride in the shell rose significantly with this concentration of fluoride in drinking water. The effect of fluorides on energy metabolism of foot muscle was evidenced by elevated AMP levels, increased adenine nucleotide pool and reduced conversion of ADP to ATP. Exposure to rising F(-) concentrations was accompanied by decreasing values of the adenylate energy charge AEC. Determination of AMP or AEC in foot muscle of exposed snails seems to be a useful indicator of fluoride effects on metabolic activity.

Ultra-Sensitive Anodic Stripping Voltammetry for the Determination of Xanthine at a Glassy Carbon Electrode

An electrochemical anodic stripping procedure for ultra-trace assay of xanthine in Cu2+ solution at a glassy carbon electrode (GCE) is described. Cyclic voltammetry was used to characterize the nature of the process taking place at the GCE. The anodic stripping response in the presence of Cu2+, at 150 mV (peak I) and 600 mV (peak II), is evaluated with respect to various experimental and instrumental conditions. Voltammetric studies show that the mechanism of the overall reaction is similar to that of the oxidation of purine derivatives at a pyrolytic graphite electrode. It is found that the copper metal deposited onto the GCE was oxidized to Cu+ at around −180 mV vs. Ag/AgCl and the generated Cu+ reacted with xanthine to accumulate on the GCE as an insoluble compound. The Cu+-xanthine compound accumulated on the GCE was redissolved by the oxidation of Cu+ to Cu2+ at ca. 150 mV, and the concentration of xanthine in the vicinity of the GCE increased. The results enabled us to use the measurement of the oxidation peak current as the basis of a simple, accurate and rapid method of determining xanthine within a concentration range of 19.9 to 166 nM for peak (I) and 0.24 to 17.2 µM for peak (II). Promising results were obtained for xanthine determination by using an external mixing step prior to stripping measurements, which yielded a detection limit of 0.138 µg L−1 (9.1×10−10 M) xanthine. The effect of some interferences (e.g. purine compounds, amino acids and some metal ions) was considered.

The Solid Surface Room Temperature Phosphorescence of Three Purine Compounds and Analytical Application

Solid surface room temperature phosphorescence (SS‐RTP) of 6‐mercaptopurine (6‐MP), azathiopurine (BAN), and 8‐azaguanine (8‐Azan) was investigated. Various factors affecting the SS‐RTP signal were discussed in detail. The corresponding analytical determination methods were established. The linear dynamic range (LDR) was 2.00 × 10−3 mmol/L–0.401 mmol/L for 6‐MP, 2.01 × 10−3 mmol/L–0.600 mmol/L for BAN, 1.09 × 10−3 mmol/L–0.218 mmol/L for 8‐Azan, respectively. The limit of detection (LOD) was 1.30 µmol/L for 6‐MP, 1.21 µmol/L for BAN, 7.35 × 10−2 µmol/L for 8‐Azan, respectively. Average recoveries in urine sample were 97.1%–99.9% for 6‐MP, 95.4%–101.7% for BAN, 96.2%–101.3% for 8‐Azan. The recovery of 6‐MP added to pharmaceutical preparation was 99.6%–100.5%.

Capillary electrophoresis microchip with a carbon nanotube-modified electrochemical detector.

Significant improvements in the performance of a capillary electrophoresis (CE) microchip with an electrochemical detector are observed using a carbon nanotube (CNT)-modified working electrode. The CNT-modified electrode allows CE amperometric detection at significantly lower operating potentials and yields substantially enhanced signal-to-noise characteristics. The electrocatalytic detection is coupled to resistance to surface fouling and hence enhanced stability. Such advantages are illustrated in connection with several classes of hydrazine, phenol, purine, and amino acid compounds. Substantial minimization of surface fouling effects has been demonstrated in connection with the monitoring of phenol and tyrosine. Factors affecting the performance of the new CNT detector were assessed and optimized. CNTs from different sources are evaluated, and the effect of an anodic pretreatment is explored. The broad and significant catalytic activity exhibited by CNT-based CE detectors indicates great promise for a wide range of bioanalytical and environmental applications.

Discovery of nonxanthine adenosine A2A receptor antagonists for the treatment of Parkinson's disease.

During a program to investigate the biochemical basis of side effects associated with the antimalarial drug mefloquine, the authors made the unexpected discovery that the (-)-(R,S)-enantiomer of the drug is a potent adenosine A2A receptor antagonist. Although the compound was ineffective in in vivo animal models of central adenosine receptor function, it provided a unique nonxanthine adenosine A2A receptor antagonist lead structure and encouraged the initiation of a medicinal chemistry program to develop novel adenosine A2A antagonists for the management of Parkinson's disease (PD). The authors have synthesized and screened more than 2,000 chemically diverse and novel adenosine A(2A antagonists. Early examples from two distinct chemical series are the thieno[3,2-dy]pyrimidine VER-6623 and the purine compounds VER-6947 and VER-7835, which have high affinity at adenosine A2A receptors (K(i) values 1.4, 1.1, and 1.7 nmol/L, respectively) and act as competitive antagonists. In particular, VER-6947 and VER-7835 demonstrate potent in vivo activity reversing the locomotor deficit caused by the D2 receptor antagonist haloperidol, with minimum effective doses comparable with that of KW6002 (0.3 to 1 mg/kg). In conclusion, the authors have discovered potent, selective, and in vivo active nonxanthine adenosine A2A antagonists that have considerable promise as a new therapy for PD.

Microchip capillary electrophoresis with a boron-doped diamond electrode for rapid separation and detection of purines

Microchip capillary electrophoresis (CE) coupled with a boron-doped diamond (BDD) electrode has been employed for the separation and detection of several purines and purine-containing compounds. The BDD end-channel amperometric detector offers favorable signal-to-noise (S/N) characteristics at the high detection potential (+1.3 V) essential for detecting purine-related compounds. Factors influencing the separation and detection processes were examined and optimized. Five purines (guanine, hypoxanthine, guanosine, xanthine, and uric acid) have been separated within 6 min at a separation voltage of 1000 V using a borate/phosphate run buffer (pH 8.2). Linear calibration plots are observed for micromolar concentrations of the purine compounds. Good stability and reproducibility (R.S.D.<5%) are obtained reflecting the minimal adsorption of purines at the BDD surface. Applicability for the detection of nucleosides, nucleotides, and oligonucleotides is illustrated. The new microchip protocol offers great promise for a wide range of bioanalytical applications involving assays of purines and purine-containing compounds.

5‐Aza‐7‐deazapurines: Synthesis and Properties of Nucleosides and Oligonucleotides

AbstractFor Abstract see ChemInform Abstract in Full Text.

Bone-targeted 2,6,9-trisubstituted purines: novel inhibitors of Src tyrosine kinase for the treatment of bone diseases.

Novel bone-targeted 2,6,9-trisubstituted purine template-based inhibitors of Src tyrosine kinase are described. Drug design studies of known purine compounds revealed that both positions-2 and -6 were suitable for incorporating bone-seeking moieties. A variety of bone-targeting groups with different affinity to hydroxyapatite were utilized in the study. Compound 3d was determined to be a potent Src inhibitor and was quite selective against a panel of other protein kinases.

Glutamine and the maintenance of meiotic arrest in mouse oocytes: influence of culture medium, glucose, and cumulus cells.

The selection of culture media and supplements therein has a tremendous impact on the regulation of oocyte maturation in vitro. In the present study, we have evaluated how altering the levels of glutamine in the presence or absence of glucose affects meiotic arrest in cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) when cultured in either the simple medium M16 or the more complex Eagle's minimum essential medium (MEM). We have also tested the effectiveness of follicle-stimulating hormone (FSH) in triggering germinal vesicle breakdown (GVB) and purine de novo synthesis in differing MEM culture conditions. When DO were cultured 17-18 hr in hypoxanthine (HX)- or dbcAMP-supplemented M16 medium, neither glucose nor glutamine had any effect on oocyte maturation, with dbcAMP the more effective inhibitor. In the absence of glutamine, cumulus cells promoted meiotic resumption, since significantly lower levels of meiotic arrest were maintained in CEO than in DO by either HX or dbcAMP, but addition of the amino acid dose-dependently decreased the maturation percentage in CEO below that observed in DO. In MEM, glutamine and glucose again had little effect on the maturation of DO, although the percentage of maturing DO in HX-supplemented medium was about 20% lower than that in M16 medium. In the absence of glucose, high levels of maturation were observed in CEO in glutamine-free medium that were dose-dependently lowered by the amino acid. However, when glucose was present, CEO were as effectively arrested as DO when glutamine was absent, with no further effect of the amino acid. This inhibitory action of glucose was dependent on the essential amino acids present in MEM. The effects of glutamine were not due to changes in metabolic coupling between the oocyte and cumulus cells. Measurement of purine de novo synthesis indicated that the maintenance of meiotic arrest as well as FSH induction of meiotic resumption were associated with increases in purine synthesis. We conclude that glucose and glutamine act cooperatively to promote the synthesis of new purine compounds within the somatic compartment and that the timing and duration of such synthesis determines whether meiotic resumption will be suppressed or promoted.