ATP Derivatives Research Articles

Bisphosphonates activate the 5-fluorouracil/uracil phosphoribosyltransferase activity present in Saccharomyces cerevisiae cell extracts : Implications for tumor treatments

Most of the effects described for bisphosphonates (pC(R1)(R2)p) are related, directly or indirectly with a pyrophosphate moiety. Bisphosphonates are (i) analogs of pyrophosphate in the synthesis of ATP derivatives (AppC(R1)(R2)p) catalyzed by ligases and (ii) inhibitors of enzymes of the mevalonate pathway with substrates containing a terminal pyrophosphate. Searching for the role of bisphosphonates on other reactions involving pyrophosphate, we explored their effect on a phosphoribosyltransferase activity, present in Saccharomyces cerevisiae cell extracts, using 5-fluorouracil or uracil as substrates. Unexpectedly, bisphosphonates increased the initial rate of synthesis of 5-FUMP (from 5-fluorouracil and phosphoribosylpyrophosphate): etidronate (2.8 ± 0.3 times); pamidronate (2.6 ± 0.4 times); alendronate (2.5 ± 0.6 times) and clodronate (2.0 ± 0.1 times). Similar values for the synthesis of UMP (from uracil and phosphoribosylpyrophosphate) were obtained in the presence of bisphosphonates. The values of the activation constants determined for alendronate and clodronate for the synthesis of UMP were 0.05 ± 0.02 mM and 0.32 ± 0.22 mM, respectively. These results raise the possibility that bisphosphonates enhance the effect of 5-fluorouracil (or other uracil prodrugs) in the treatment of bone tumors or bone tumor metastases.

Effects of extracellular ATP on the physiology ofStreptomyces coelicolorA3(2)

Because ATP is an extracellular effector in animal and plant systems and derivatives of ATP, such as S-adenosylmethionine and cAMP, can control antibiotic production and morphological differentiation in Streptomyces, we hypothesized that extracellular ATP (exATP) can also affect physiologies of Streptomyces. We found that the addition of 10 microM exATP to Streptomyces coelicolor A3(2) cultures resulted in enhanced actinorhodin and undecylprodigiosin production and morphological differentiation on solid medium. However, these phenotypes were reduced by the addition of a 10-fold higher concentration of exATP (100 microM). Intracellular ATP concentrations were also modulated in response to changes in exATP. ATP analogs, added at a 100-fold lower concentration, affected Streptomyces similarly to that seen for 10 microM exATP. The enhanced promoter activity of actII-orf4 indicated that 10 microM exATP affect the transcriptional level for actinorhodin production. Results from this study suggest that exATP is an effector for the physiology of S. coelicolor and careful manipulation of exATP may significantly enhance the high-yield production of antibiotics by S. coelicolor.

Functional Characterization of Escherichia coli MsbA: INTERACTION WITH NUCLEOTIDES AND SUBSTRATES

The Escherichia coli MsbA protein is a 65-kDa member of the ATP-binding cassette superfamily. It is thought to function as an ATP-dependent lipid translocase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. MsbA with high ATPase activity was isolated and found to be homodimeric in detergent solution. The protein ATPase activity was inhibited by vanadate and showed variable patterns of stimulation and inhibition by lipid A and other compounds. The intrinsic tryptophan fluorescence of the protein was characterized, and dynamic quenching using acrylamide showed that a conformational change took place on binding of lipid A. Fluorescence quenching was used to characterize the interactions of MsbA with nucleotides and various putative substrates, including lipids, lipid-like compounds, and drugs. MsbA had an apparent binding affinity for ATP of approximately 2 mm and also bound nonhydrolyzable ATP analogs and fluorescent ATP derivatives. The putative substrate lipid A interacted with the protein with an affinity of 6.4 microm. Drugs that are known to be substrates for ABC multidrug transporters also interacted with MsbA with affinities in the range 0.25-50 microm. This study represents the first use of fluorescence approaches to estimate MsbA binding affinities for nucleotides and putative transport substrates.

Theoretical Modeling of PrkCc, Serine–Threonine Protein Kinase Intracellular Domain, Complexed with ATP Derivatives

AbstractWith the advent of the Molecular Dynamics (MD) methods two complexes of PrkC catalytic domain with N6‐methoxy‐ATP and N6‐pyrrolidino‐ATP analogues were studied. Structures of the intracellular domain of the enzyme and the ATP derivatives were modeled theoretically. We report here the spatial arrangement of the PrkC kinase and its two complexes as well as their behavior during the MD simulations.

Inhibition of Ca2+signaling and glucagon secretion in mouse pancreatic α-cells by extracellular ATP and purinergic receptors

Glucagon secreted from pancreatic alpha-cells plays a critical role in glycemia, mainly by hepatic glucose mobilization. In diabetic patients, an impaired control of glucagon release can worsen glucose homeostasis. Despite its importance, the mechanisms that regulate its secretion are still poorly understood. Since alpha-cells are particularly sensitive to neural and paracrine factors, in this report we studied the role of purinergic receptors and extracellular ATP, which can be released from nerve terminals and beta-cell secretory granules. Using immunocytochemistry, we identified in alpha-cells the P2 receptor subtype P2Y1, as well as the P1 receptors A1 and A2A. In contrast, only P2Y1 and A1 receptors were localized in beta-cells. To analyze the role of purinergic receptors in alpha-cell function, we studied their participation in Ca2+ signaling. At low glucose concentrations, mouse alpha-cells exhibited the characteristic oscillatory Ca2+ signals that lead to secretion. Application of ATP (1-10 microM) abolished these oscillations or reduced their frequency in alpha-cells within intact islets and isolated in culture. ATPgammaS, a nonhydrolyzable ATP derivative, indicated that the ATP effect was mainly direct rather than through ATP-hydrolytic products. Additionally, adenosine (1-10 microM) was also found to reduce Ca2+ signals. ATP-mediated inhibition of Ca2+ signaling was accompanied by a decrease in glucagon release from intact islets in contrast to the adenosine effect. Using pharmacological agonists, we found that only P2Y1 and A2A were likely involved in the inhibitory effect on Ca2+ signaling. All these findings indicate that extracellular ATP and purinergic stimulation are effective regulators of the alpha-cell function.

Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases

T4 DNA ligase and the ubiquitin activating enzyme (E1), catalyze the synthesis of ATP β,γ-bisphosphonate derivatives. Concerning T4 DNA ligase: (i) etidronate (pC(OH)(CH 3)p) displaced the AMP moiety of the complex E-AMP in a concentration dependent manner; (ii) the K m values and the rate of synthesis k cat (s −1), determined for the following compounds were, respectively: etidronate, 0.73 ± 0.09 mM and (70 ± 10) × 10 −3 s −1; clodronate (pCCl 2p), 0.08 ± 0.01 mM and (4.1 ± 0.3) × 10 −3 s −1; methylenebisphosphonate (pCH 2p), 0.024 ± 0.001 mM and (0.6 ± 0.1) × 10 −3 s −1; tripolyphosphate (P 3) (in the synthesis of adenosine 5′-tetraphosphate, p 4A), 1.30 ± 0.30 mM and (6.2 ± 1.1) × 10 −3 s −1; (iii) in the presence of GTP and ATP, inhibition of the synthesis of Ap 4G was observed with clodronate but not with pamidronate (pC(OH)(CH 2–CH 2–NH 3)p). Concerning the ubiquitin activating enzyme (E1): methylenebisphosphonate was the only bisphosphonate, out of the ones tested, that served as substrate for the synthesis of an ATP derivative ( K m = 0.36 ± 0.09 mM and k cat = 0.15 ± 0.02 s −1). None of the above bisphosphonates were substrates of the reaction catalyzed by luciferase or by acyl-CoA synthetase. The ability of acetyl-CoA synthetase to use methylenebisphosphonate as substrate depended on the commercial source of the enzyme. In our view this report widens our knowledge of the enzymes able to metabolize bisphosphonates, a therapeutic tool widely used in the treatment of osteoporosis.

Studies on the Contribution of Phosphoryl Group to the Non-Covalent Interaction between ATP and α-Aminophosphonic Acids Derivatives by ESI-MS and Molecular Modeling

In this work, Diisopropyl [phenyl(pyridine-4-carboxamido)methyl]phosphonate was synthesized for investigating the interaction with ATP. The noncovalent complex was observed by the Electrospray ionization Mass Spectrometry (ESI-MS). Through Molecular modeling, it was found that the non-covalent complex was stabilized by two intermolecular hydrogen bonds, which has the binding energy of -8.999 kcals/mol. In order to investigate the function of phosphoryl group in the interaction, benzoyl phenylglycine isopropyl ester was also tested, but no interaction with ATP was observed by ESI-MS. These results implied that phosphoryl group was a very important functional group to provide effective interaction site between α-Aminophosphonic acids and ATP.

Nucleotide binding to human UMP‐CMP kinase using fluorescent derivatives − a screening based on affinity for the UMP‐CMP binding site

Methylanthraniloyl derivatives of ATP and CDP were used in vitro as fluorescent probes for the donor-binding and acceptor-binding sites of human UMP-CMP kinase, a nucleoside salvage pathway kinase. Like all NMP kinases, UMP-CMP kinase binds the phosphodonor, usually ATP, and the NMP at different binding sites. The reaction results from an in-line phosphotransfer from the donor to the acceptor. The probe for the donor site was displaced by the bisubstrate analogs of the Ap5X series (where X = U, dT, A, G), indicating the broad specificity of the acceptor site. Both CMP and dCMP were competitors for the acceptor site probe. To find antimetabolites for antivirus and anticancer therapies, we have developed a method of screening acyclic phosphonate analogs that is based on the affinity of the acceptor-binding site of the human UMP-CMP kinase. Several uracil vinylphosphonate derivatives had affinities for human UMP-CMP kinase similar to those of dUMP and dCMP and better than that of cidofovir, an acyclic nucleoside phosphonate with a broad spectrum of antiviral activities. The uracil derivatives were inhibitors rather than substrates of human UMP-CMP kinase. Also, the 5-halogen-substituted analogs inhibited the human TMP kinase less efficiently. The broad specificity of the enzyme acceptor-binding site is in agreement with a large substrate-binding pocket, as shown by the 2.1 A crystal structure.

The Non-mevalonate Pathway of Isoprenoid Precursor Biosynthesis

The recently discovered non-mevalonate biosynthetic route to isoprenoid precursors is an essential metabolic pathway in plants, apicomplexan parasites, and many species of bacteria. The pathway relies on eight enzymes exploiting different cofactors and metal ions. Structural and mechanistic data now exist for most components of the pathway though there remain some gaps in our knowledge. The individual enzymes represent new, validated targets for broad spectrum antimicrobial drug and herbicide development. Detailed knowledge of the pathway may also be exploited to genetically modify microorganisms and plants to produce compounds of agricultural and medical interest.

Nucleotide degradation in sardine (Sardina pilchardus) stored in different storage condition at 4°C

Degradation products of nucleotide in sardine stored up to 15 days in stored in boxes without ice, vacuum package (VP) and modified atmosphere package (MAP) at 4oC were investigated using a rapid HPLC method. Nucleotide degradation was more rapid in sardine stored in air than those stored in MAP and VP. The lowest value from production of ATP derivatives were observed in sardine held in MAP, followed by VP. ATP content was not detected under the three conditions. AMP and ADP levels were less than 0.2 μmol/g on day 0 in all cases. The shelf-life of fish stored in MAP was 12 days according to sensory assessment, at which the content of IMP, Hx and Ino were approximately 0.2, 0.7 and 0.6 μmoles/g, respectively. Although IMP and Ino showed fluctuations under all of storage conditions, Hx content increased linearly with storage time. Thus, it might be used as a freshness indicator for sardine stored under MAP and VP. The maximum levels of Hx compound were 0.79 μmoles/g for MAP and 0.75 μmoles/g for VP at day 15, respectively.

ATP as effector of inorganic pyrophosphatase of Escherichia coli. The role of residue Lys112 in binding effectors

It has been shown that PP(i), methylenediphosphonate, and ATP act as effectors of Escherichia coli inorganic pyrophosphatase (E-PPase), and that they compete for binding at the allosteric regulatory site. On the basis of chemical modification and computer modeling of a structure of the enzyme-ATP complex, a number of amino acid residues presumably involved in binding effectors has been revealed. Mutant variants Lys112Gln, Lys112Gln/Lys148Gln, and Lys112Gln/Lys115Ala of E-PPase have been obtained, as well as a modified variant of wild type E-PPase ((Ad)wt PPase) with a derivative of ATP chemically attached to the amino group of Lys146. Kinetic properties of these variants have been investigated and compared to the earlier described variants Lys115Ala, Arg43Gln, and Lys148Gln. Analysis of the data confirms the proposed location of an effector binding site in a cluster of positively charged amino acid residues including the side chains of Arg43, Lys146 (subunit A), Lys112, and Lys115 (subunit B). Lys112 is supposed to play a key role in forming contacts with the phosphate groups of the three studied effectors.

ATP as effector of inorganic pyrophosphatase of Escherichia coli. Identification of the binding site for ATP

The interaction of Escherichia coli inorganic pyrophosphatase (E-PPase) with effector ATP has been studied. The E-PPase has been chemically modified with the dialdehyde derivative of ATP. It has been established that in the experiment only one molecule of effector ATP is bound to each subunit of the hexameric enzyme. Tryptic digestion of the adenylated protein followed by isolation of a modified peptide by HPLC and its mass-spectrometric identification has showed that it is an amino group of Lys146 that undergoes modification. Molecular docking of ATP to E-PPase indicates that the binding site for effector ATP is located in a cluster of positively charged amino acid residues proposed earlier on the basis of site-directed mutagenesis to participate in binding of effector pyrophosphate. Molecular docking also reveals several other amino acid residues probably involved in the interaction with effectors.

Design of Dual‐Emission Chemosensors for Ratiometric Detection of ATP Derivatives

Nucleoside pyrophosphate (nucleoside PP) derivatives are widespread in living cells and play pivotal roles in various biological events. We report novel fluorescence chemosensors for nucleoside PPs that make use of coordination chemistry. The chemosensors, which contain two Zn(II)-dipicolylamine units, bind strongly to nucleoside PPs (K(app)>10(6) M(-1)) in aqueous solution and sense them by a dual-emission change. Detailed fluorescence and UV/Vis spectral studies revealed that the emission changes of the chemosensors upon binding to nucleoside PPs can be ascribed to the loss of coordination between Zn(II) and the acridine fluorophore. This is a unique sensing system based on the anion-induced rearrangement of the coordination. Furthermore, we demonstrated the utility of these chemosensors in real-time monitoring of two important biological processes involving nucleoside PP conversion: the apyrase-catalyzed hydrolysis of nucleoside PPs and the glycosyl transfer catalyzed by beta-1,4-galactosyltransferase.

Opposite diastereoselective activation of P2Y1 and P2Y11 nucleotide receptors by adenosine 5′‐O‐(α‐boranotriphosphate) analogues

We explored the stereoselective activation of the P2Y11 receptor, stably expressed and tagged with GFP, in 1321N1 cells, in comparison to its closest homologue, the P2Y1 receptor. The potency of several chiral ATP analogues was determined by measuring increases in intracellular calcium concentration ([Ca2+]i). In a series of ATP-alpha-B and ATP-alpha-S analogues, a non-bridging oxygen atom of Palpha was substituted by BH3 or sulphur, respectively, introducing a chiral center at Palpha. The pairs of diastereoisomers (A and B isomers) were each applied as purified compounds. The (B) isomers (ATP-alpha-B Sp isomers and ATP-alpha-S Rp isomers) of all derivatives tested were more potent at the P2Y11 receptor than the corresponding (A) isomers (ATP-alpha-B Rp isomers and ATP-alpha-S Sp isomers) and the parent compounds. This characteristic of the P2Y11 receptor is opposite to the behaviour of the same diastereoisomers at the P2Y1 receptor, at which the (A) isomers are more active. The distinctly opposite diastereoselective activity of ATP derivatives at the P2Y11 and the P2Y1 receptor will allow the deciphering of structural differences of the ligand recognition sites between these receptor subtypes and may aid in the development of subtype-selective agonists. Moreover, ATP-alpha-B diastereoisomers are not active at the P2Y2 receptor. Thus, they are compounds suitable for distinguishing the functional contribution of the two ATP-activated P2Y receptors, the P2Y2 and P2Y11 receptor, in physiological or pathophysiological responses of cells.

Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase

T4 RNA ligase catalyzes the synthesis of ATP β,γ-bisphosphonate analogues, using the following substrates with the relative velocity rates indicated between brackets: methylenebisphosphonate (pCH 2p) (100), clodronate (pCCl 2p) (52), and etidronate (pC(OH)(CH 3)p) (4). The presence of pyrophosphatase about doubled the rate of these syntheses. Pamidronate (pC(OH)(CH 2–CH 2–NH 2)p), and alendronate (pC(OH)(CH 2–CH 2–CH 2–NH 2)p) were not substrates of the reaction. Clodronate displaced the AMP moiety of the complex E-AMP in a concentration dependent manner. The K m values and the rate of synthesis ( k cat) determined for the bisphosphonates as substrates of the reaction were, respectively: methylenebisphosphonate, 0.26 ± 0.05 mM (0.28 ± 0.05 s −1); clodronate, 0.54 ± 0.14 mM (0.29 ± 0.05 s −1); and etidronate, 4.3 ± 0.5 mM (0.028 ± 0.013 s −1). In the presence of GTP, and ATP or AppCCl 2p the relative rate of synthesis of adenosine 5′,5‴-P 1,P 4-tetraphosphoguanosine (Ap 4G) was around 100% and 33%, respectively; the methylenebisphosphonate derivative of ATP (AppCH 2p) was a very poor substrate for the synthesis of Ap 4G. To our knowledge this report describes, for the first time, the synthesis of ATP β,γ-bisphosphonate analogues by an enzyme different to the classically considered aminoacyl-tRNA synthetases.

Intermolecular Cross-Linking of a Novel Rice Kinesin K16 Motor Domain with a Photoreactive ATP Derivative

A fluorescent photoreactive ATP derivative, 2'(3')-O-(4-benzoylbenzoyl)-1,N(6)-etheno-ATP (Bz(2)-epsilonATP), was synthesized and reacted with the rice kinesin K16 motor domain (K16MD). In the presence of ADP or ATP, UV irradiation of the K16MD solution containing Bz(2)-epsilonATP resulted in a new 100 kDa band, which was an intermolecular cross-linked product of motor domains. In contrast, no cross-linking was observed in the absence of nucleotides. For the motor domain of mouse brain kinesin and skeletal muscle myosin subfragment-1, no such intermolecular photo cross-linking by Bz(2)-epsilonATP was observed. Our results indicate that Bz(2)-epsilonATP acts unusually as a photoreactive crosslinker to detect conformational changes in K16MD induced by nucleotide binding resulting in the formation of dimers.

Dichotomous but stringent substrate selection by the dual-function Cdk7 complex revealed by chemical genetics

Cdk7 performs two essential but distinct functions as a CDK-activating kinase (CAK) required for cell-cycle progression and as the RNA polymerase II (Pol II) CTD kinase of general transcription factor IIH. To investigate the substrate specificity underlying this dual function, we created an analog-sensitive (AS) Cdk7 able to use bulky ATP derivatives. Cdk7-AS-cyclin H-Mat1 phosphorylates approximately 10-15 endogenous polypeptides in nuclear extracts. We identify seven of these as known and previously unknown Cdk7 substrates that define two classes: proteins such as Pol II and transcription elongation factor Spt5, recognized efficiently only by the fully activated Cdk7 complex, through sequences surrounding the site of phosphorylation; and CDKs, targeted equivalently by all active forms of Cdk7, dependent on substrate motifs remote from the phosphoacceptor residue. Thus, Cdk7 accomplishes dual functions in cell-cycle control and transcription not through promiscuity but through distinct, stringent modes of substrate recognition.

Optimizing Gas Mixtures for Modified Atmosphere Packaging of Fresh King Salmon (Oncorhynchus tshawytscha)

Researchers have evaluated the effect of modified atmosphere packaging (MAP) on the shelf life of fish products. However, there is little published basis for recommended gas mixtures. Most studies have examined the effects of empirical evidence of gas mixes on particular species, but they cannot be used to predict the effect of different gas mixes on shelf life. Further, while the fish:gas ratio has been shown to be a key determinant of bacterial growth under MAP, most studies did not control this parameter. The aim of our research was to optimize gas mixes and fish:gas ratios for the MAP storage of king salmon. Flesh portions from defined fillet positions were placed on drip pads and boxed, giving a standardized nominal volume of 27.4 cm3 and an exposed surface area of 14.4 cm2. Each sample was placed in a foil barrier bag containing an oxygen absorber. Controlled volumes (0-100 cm3) of CO2 or 100 cm3 of a 40:60 mixture of CO2:N2 were then hand-injected through a septa. Bags were stored in melting ice (0 ± 0.05°C) and analyzed after 0, 8, 22 and 90 days for headspace CO2 and O2, EH, fish freshness meter (Distell, West Lothian, Scotland) reading, drip loss, texture, raw and cooked sensory characteristics, microbial numbers, bacterial flora composition, total volatile base nitrogen (TVBN) and ATP derivatives. High CO2 levels reduced bacterial counts, but resulted in poor cooked sensory characteristics due to carbonated flavor. We calculated the threshold level of CO2 at which this could be detected by our trained panel. We also determined the solubility of CO2 in salmon flesh and calculated Henry's constant to resolve the relationship between gas mixes and the fish:gas ratio. We now have the basis of a simple mathematical model optimizing these to give maximum shelf life and sensory acceptability.

Evidence of a novel cell signaling role for extracellular adenosine triphosphates and diphosphates in Arabidopsis.

Extracellular ATP is a known receptor agonist in animals and was previously shown to alter plant growth, and so we investigated whether ATP derivatives could function outside plant cells as signaling agents. Signaling responses induced by exogenous nucleotides in animal cells typically include increases in free cytoplasmic calcium concentration ([Ca(2+)](cyt)). We have evaluated the ability of exogenously applied adenosine 5'-[gamma-thio]triphosphate (ATPgammaS), adenosine 5'-[beta-thio]diphosphate (ADPbetaS), and adenosine 5'-O-thiomonophosphate to alter [Ca(2+)](cyt) in intact apoaequorin transgenic Arabidopsis thaliana seedlings. ATPgammaS and ADPbetaS increase [Ca(2+)](cyt), and this increase is enhanced further when the nucleotides are added with the elicitor oligogalacturonic acid. Exogenous treatment with ATP also increases the level of transcripts encoding mitogen-activated protein kinases and proteins involved in ethylene biosynthesis and signal transduction. The increase in [Ca(2+)](cyt) induced by nucleotide derivatives can be ablated by Ca(2+)-channel blocking agents and by the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and the changes in gene expression can be partially blocked by these agents. These observations suggest that extracellular ATP can activate calcium-mediated cell-signaling pathways in plants, potentially playing a physiological role in transducing stress and wound responses.

Molecular recognition in purinergic receptors. 1. A comprehensive computational study of the h-P2Y1-receptor.

P2Y receptors (P2Y-Rs) are attractive pharmaceutical targets due to their involvement in the modulation of many tissues and organs. The lack of experimental structural data on P2Y-Rs impedes structure-based drug design. The need to elucidate the receptor's molecular recognition, together with the limitations of previous receptor models, triggered the construction of a new molecular model for the h-P2Y1-R. Therefore, a h-P2Y1-R model was constructed by homology modeling using the 2.6 A crystal structure of bovine rhodopsin as a template and subsequently refined by constrained molecular dynamics (MD) simulations in a fully hydrated lipid bilayer environment. ATP was docked into the receptor binding site, followed by binding site refinement using Monte Carlo and MD simulations. Analysis of the h-P2Y1-R-ATP complex suggests that the triphosphate moiety is tightly bound by a multitude of interactions possibly including a Mg2+ ion, the ribose ring is not involved in specific interactions, and the adenine ring is bound via N1, N7, and N6. The molecular recognition of the h-P2Y1-R was further probed by ATP derivatives modified on the adenine ring, and correlated with EC50 values for these derivatives. Analysis of receptor:ligand complexes and quantum mechanical studies on model compounds support the role of both steric and electronic effects in improving H-bonding (via N1 and N6) and pi-stacking interactions. The computed h-P2Y1-R model was validated with respect to our previous biochemical results. We believe that this new model of the h-P2Y1-R provides the means for understanding phenomena such as the ligand's potency and receptor subtype selectivity.