Synthesis and biological evaluation of some phosphate triester derivatives of the anti-cancer drug araC.

A number of novel phosphate triester derivatives of the anti-cancer nucleoside analogue araC have been prepared by a rapid 2-step procedure, not necessitating prior sugar protection. Spectroscopic and lipophilicity data have been collected on these compounds. An in vitro assay indicated inhibition of thymidine incorporation by mammalian epithelial cells, by each of these compounds, in the range 3-300 microM. Moreover, the degree of inhibition showed a close correlation to chemical structure; in particular, there was a clear relationship between inhibition of thymidine incorporation and log(P). These results are consistent with cellular penetration by the intact phosphate triesters and intracellular action by an unspecified mechanism. Triethyl phosphate is inactive under the conditions of the test.     

The role of nucleoside triphosphate hydrolysis in transducing systems: p21ras and muscle.

A variety of systems use nucleoside triphosphate hydrolysis to control or provide energy for biological processes, mediated through protein-protein interactions. The nature of this coupling may vary, but often there is a degree of similarity. In this paper, two systems are compared: actomyosin in muscle and p21ras in a signal transduction pathway as yet undefined. The mechanism of the nucleotide triphosphate hydrolysis and the consequent changes in the protein-nucleotide complex have been investigated, to understand how the coupling to biological function is achieved. The basal nucleoside triphosphatase mechanisms are compared and the roles of proteins that activate the hydrolysis, actin and GAP, are discussed. The cleavage process was probed by stereochemical techniques to determine the basic mechanism, of either a phosphorylated enzyme intermediate or direct displacement of nucleoside diphosphate by water. Phosphate-water oxygen exchange probes were used to investigate nucleoside triphosphate and inorganic phosphate release steps. A new method of probing the kinetics of inorganic phosphate release directly has been developed. In muscle, this process seems likely to be related directly to force generation. In the GAP-ras system, measurement of phosphate release is allowing the mechanism of the GAP-p21ras interaction to be probed.     

Nucleoside phosphate analogues of biological interest, and their synthesis via aryl nucleoside H-phosphonates as intermediates.

This review presents a brief account of the chemistry and mechanistic aspects of aryl H-phosphonates, and selected applications of this class of compounds as intermediates in the synthesis of a wide range of biologically important analogues of nucleoside phosphates, and oligonucleotides, in which the phosphate moieties are replaced by other structurally related groups. The aryl nucleoside H-phosphonates, compounds of controlled reactivity, have proven to be more versatile and superior to various mixed anhydrides as synthetic intermediates, particularly for preparation of nucleotide analogues bearing P-N or P-S bonds in various configurational arrangements at the phosphate moiety.     

Synthesis and evaluation of some novel phosphate and phosphinate derivatives of araA. Studies on the mechanism of action of phosphate triesters.

A number of novel phosphinate and phosphate triester derivatives of the anti-viral nucleoside analogue araA have been prepared. Spectroscopic and analytical data have been collected on both the reagents and the nucleotides. An in vitro assay indicated inhibition of DNA synthesis by mammalian cells, by each of the nucleotide derivatives, in the range 3-30 microM. Inhibition was reduced, but not abolished, for the phosphinates relative to the phosphates. These results are consistent with a mode of action involving release of the free nucleoside araA and the nucleotide araAMP.     

Synthesis and enzymic activity of 8-acyl and 8-alkyl derivatives of guanosine 3, 5-cyclic phosphate.

Several new 8-alkyl and 8-acyl derivatives of quanosie 3',5'-cyclic phosphate (cGMP) and inosine 3',5'-cyclic phosphate (cGMP) were prepared by direct alkylation or acylation of the parent cyclic nucleotide via free radicals generated in situ. These compounds have been examined for their ability to stimulate a cGMP-dependent protein kinase, and several of the cGMP derivatives were as active in this regard as cGMP. These compounds proved to be quite ineffective when tested for their ability to activate an adenosine 3',5'-cyclic phosphate (cAMP) dependent protein kinase. In addition, these 8-substituted cGMP derivatives are not substrates for a phosphodiesterase preparation from rabbit kidney, but do show inhibition of the hydrolysis of cAMP by crude phosphodiesterase preparations from rabbit lung and beef heart.     

Synthesis, hydrolysis and anti-EBV activity of a series of 3'-modified cycloSal-BVDUMP pronucleotides

A series of cycloSal-BVDUMP phosphate triesters has been prepared. The prototype compound was 3-methyl-cycloSal-BVDUMP 2. Furthermore, a series of 3'-O-acyl-modified derivatives having carboxylic acids with different lipophilicity or a L-configurated alpha-amino acid (phenylalanine) was prepared. The hydrolysis properties in phosphate buffer PBS as well as in PBS containing pig liver esterase (PLE) will be described. Finally, the biological activity against EBV has been determined.     

Nucleotides as nucleophiles: reactions of nucleotides with phosphoimidazolide activated guanosine.

An earlier study of the reaction of phosphoimidazolide activated nucleosides (ImpN) in aqueous phosphate buffers indicated two modes of reaction of the phosphate monoanion and dianion. The first mode is catalysis of the hydrolysis of the P-N bond in ImpN's which leads to imidazole and nucleoside 5'-monophosphate. The second represents a nucleophilic substitution of the imidazole to yield the nucleoside 5'-diphosphate. This earlier study thus served as a model for the reaction of ImpN with nucleoside monophosphates (pN) because the latter can be regarded as phosphate derivatives. In the present study we investigated the reaction of guanosine 5'-phosphate-2-methylimidazolide, 2-MeImpG, in the presence of pN (N = guanosine, adenosine and uridine) in the range 6.9 less than or equal to pH less than or equal to 7.7. We observed that pN's do act as nucleophiles to form NppG, and as general base to enhance the hydrolysis of the P-N bond in 2-MeImpG, i.e. pN show the same behavior as inorganic phosphate. The kinetic analysis yields the following rate constants for the dianion pN2-: knpN = 0.17 +/- 0.02 M-1 h-1 for nucleophilic attack and khpN = 0.11 +/- 0.07 M-1 h-1 for general base catalysis of the hydrolysis. These rate constants which are independent of the nucleobase compare with kp.2 = 0.415 M-1 h-1 and khp2. = 0.217 M-1 h-1 for the reactions of HPO4(2-). In addition, this study shows that under conditions where pN presumably form stacks, the reaction mechanism remains unchanged although in quantitative terms stacked pN are somewhat less reactive. Attack by the 2'-OH and 3'-OH groups of the ribose moiety in amounts greater than or equal to 1% is not observed; this is attributed to the large difference in nucleophilicity in the neutral pH range between the phosphate group and the ribose hydroxyls. This nucleophilicity rank is not altered by stacking.     

Catalytic mechanism of α-phosphate attack in dUTPase is revealed by X-ray crystallographic snapshots of distinct intermediates, 31P-NMR spectroscopy and reaction path modelling

Enzymatic synthesis and hydrolysis of nucleoside phosphate compounds play a key role in various biological pathways, like signal transduction, DNA synthesis and metabolism. Although these processes have been studied extensively, numerous key issues regarding the chemical pathway and atomic movements remain open for many enzymatic reactions. Here, using the Mason–Pfizer monkey retrovirus dUTPase, we study the dUTPase-catalyzed hydrolysis of dUTP, an incorrect DNA building block, to elaborate the mechanistic details at high resolution. Combining mass spectrometry analysis of the dUTPase-catalyzed reaction carried out in and quantum mechanics/molecular mechanics (QM/MM) simulation, we show that the nucleophilic attack occurs at the α-phosphate site. Phosphorus-31 NMR spectroscopy (³¹P-NMR) analysis confirms the site of attack and shows the capability of dUTPase to cleave the dUTP analogue α,β-imido-dUTP, containing the imido linkage usually regarded to be non-hydrolyzable. We present numerous X-ray crystal structures of distinct dUTPase and nucleoside phosphate complexes, which report on the progress of the chemical reaction along the reaction coordinate. The presently used combination of diverse structural methods reveals details of the nucleophilic attack and identifies a novel enzyme–product complex structure.     

Effect of change in nucleoside structure on the activation and antiviral activity of phosphoramidate derivatives

Changing the nucleoside group of a series of phosphoramidate derivatives affects the enzyme mediated hydrolysis rate of the compounds. d4T and AZT-substituted analogs were activated by enzymes such as lipases, esterases, and proteases. On the other hand, 3dT-substituted derivatives were comparatively less prone to hydrolysis under similar experimental conditions. From the experimental results, we propose that the most preferable nucleoside group for enzyme activation is d4T rather than AZT or 3dT. Additionally, we also observed that depending on the enzymes used the chiral selectivity of the enzymes for the phosphorus center of these phosphoramidate derivatives differed, demonstrating the importance of the nucleoside structure for this class of compounds.     

The enzymatic synthesis of antiviral agents.

The majority of potential antiviral agents which are currently undergoing clinical trials are inhibitors of the replication of nucleic acids. The most common class of these inhibitors are nucleoside analogues and the elucidation of synthetic routes to these compounds has been of interest for many years as many are anticancer agents. One synthetic development has been the application of bio-transformations to nucleoside syntheses. This topic has been reviewed recently (Shirae et al., 1991) but this review is not widely available. In the present review, the application of biotechnology to the synthesis of antiviral agents including those which are not nucleoside analogues will be discussed. Enzymatic syntheses of nucleosides can be simpler and quicker than syntheses carried out by chemical methods. The most useful enzymes are those found in catabolic pathways. Nucleoside phosphorylases and N-deoxyribosyltransferases have both been widely used for nucleoside synthesis catalysing the transfer of sugar residues from a donor nucleoside to a heterocyclic base. Enzymatic methods have also been applied to the resolution of racemic mixtures and adenosine deaminase is a convenient catalyst for the hydrolysis of amino groups on purines and purine analogues. Regioselective deprotection of nucleoside esters has been achieved with lipases and these enzymes have also been applied to the synthesis of esters of sugar-like alkaloids. The latter have potential as inhibitors of the replication of HIV. Esterases have also been used in combined chemical and enzymatic syntheses of organophosphorus antiviral agents.     

Adrenal cortex mitochondrial enzyme with ATP-dependent protease and protein-dependent ATPase activities. Purification and properties

We have purified an ATP-dependent protease with protein-dependent ATPase activity from bovine adrenal cortex mitochondria to near homogeneity. The subunit molecular weight is 108,000 and the enzyme appears to be a hexamer with approximately identical subunits. Based on the experiments using various nucleoside triphosphates and their related compounds, it is concluded that hydrolysis of the high-energy bond in nucleoside triphosphates is not an absolute requirement for proteolysis. Nucleotide specificity of this enzyme varies, depending on the protein or peptide substrates used. When casein was the substrate, ATP and dATP were quite effective, but other nucleotides were not. When insulin and angiotensinogen were used as substrate, ATP, other nucleoside triphosphates, ADP, inorganic triphosphate, pyrophosphate, and phosphate were effective. One of the cleaving linkages hydrolyzed by this enzyme was revealed to be the Leu-Leu bond of angiotensinogen. However, the specificity appears to be broad in view of the hydrolysis pattern of glucagon.     

Lipophilicity and antiproliferative activity profiling of 2-benzylidencycloalkanones

High performance liquid chromatographic (HPLC) method has been developed to separate the members of a library including 24 benzylidenecycloalkanone-type structures and to characterize their lipophilicity. The experimental lipophilicity data (k) of the compounds have been compared with their calculated lipophilicity parameters (CLOGP). In general, good correlations between the measured and calculated lipophilicities have been found and these results were in good accordance with our previously data obtained in case of structurally related molecular libraries. In addition, cytotoxicity screening has been performed to determine the antiproliferative activity of these compounds. Some of the investigated compounds possessed noticeable inhibitory potential. Based on the correlation between the antiproliferative activity and experimentally determined lipophilicity of the molecules investigated, limited structural demands to obtain more potent compounds can be exhibited to support the synthetic design.     

Trypanosoma cruzi: inhibition of parasite growth and respiration by oxazolo(thiazolo)pyridine derivatives and its relationship to redox potential and lipophilicity

Chagas' disease constitutes a therapeutic challenge because presently available drugs have wide toxicity to the host and are generally ineffective in the chronic stages of the disease. A series of oxazolo(thiazolo)pyridene derivatives were studied on Trypanosoma cruzi epimastigote growth and oxygen consumption and their electrochemical (redox) potentials and lipophilicity. The derivatives produced different degrees of parasite growth and respiration inhibition on CL Brener, LQ, and Tulahuen strains of T. cruzi epimastigotes. Respiratory chain inhibition appears to be a determinant of the trypanosomicidal activity of these compounds, since a significant correlation between respiration and culture growth inhibition was found. A similar correlation was found, within the different structural subfamilies, between toxic effects and the ability of the compounds to be oxidized in aqueous media. The inhibition of respiration and of parasite growth in culture increases with the lipophilicity of the substituents on the oxazolopyridine nucleus. No difference in the action of these derivatives was found among the different parasite strains. It is concluded that these compounds may have a potential usefulness in the treatment of Chagas' disease.     

A novel human phosphotransferase highly specific for adenosine.

A novel nucleoside phosphotransferase, referred to as adenosine phosphotransferase (Ado Ptase), was partially purified 1230-fold from human placenta. This enzyme differed from other known nucleoside phosphotransferases in its substrate specificity. Using AMP as the phosphate donor, it readily phosphorylated Ado. Changes in the sugar moiety were tolerated. dAdo and ddAdo were phosphate acceptors and dAMP was a donor. No other nucleotide or nucleoside common in nature displayed appreciable activity as donor or acceptor substrate, respectively. In the absence of nucleoside, the enzyme catalyzed the hydrolysis of AMP, typical of other nucleoside phosphotransferases. However, in the presence of Ado, little, if any, hydrolysis occurred. Ado Ptase had an absolute requirement for a metal cation, with Mg2+ and, to a lesser extent, Mn2+ fulfilling this requisite. The apparent Km for Ado was 0.2 mM. However, the donor AMP displayed cooperativity in both transfer and hydrolytic reactions. This cooperativity was eliminated by nucleotides, 2,3-diphosphoglycerate, and inorganic phosphate. ADP and 2,3-diphosphoglycerate were especially potent. In the presence of these effectors, the apparent Km for AMP was 3.0 mM in the transfer reaction and 4.0 mM in the hydrolytic reaction. Kinetic data suggest that there are two nucleotide binding sites on Ado Ptase, one for the donor, the other for an effector. AMP appeared to bind to both sites. Although this novel enzyme might play a role in the anabolism of nucleoside analogues, the normal physiological role of this nucleoside phosphotransferase is not understood.     

Lipophilicity plays a major role in modulating the inhibition of monoamine oxidase B by 7-substituted coumarins

A series of coumarin derivatives (1-22), bearing at the 7-position ether, ketone, ester, carbamate, or amide functions of varying size and lipophilicity, were synthesized and investigated for their in vitro monoamine oxidase-A and -B (MAO-A and -B) inhibitory activities. Most of the compounds acted preferentially as MAO-B inhibitors, with IC(50) values in the micromolar to low-nanomolar range. A structure-activity-relationship (SAR) study highlighted lipophilicity as an important property modulating the MAO-B inhibition potency of 7-substituted coumarins, as shown by a linear correlation (n=20, r(2)=0.72) between pIC(50) and calculated log P values. The stability of ester-containing coumarin derivatives in rat plasma provided information on factors that either favor (lipophilicity) or decrease (steric hindrance) esterase-catalyzed hydrolysis. Two compounds (14 and 22) were selected to investigate how lipophilicity and enzymatic stability may affect in vivo MAO activities, as assayed ex vivo in rat. The most-potent and -selective MAO-B inhibitor 22 (=7-[(3,4-difluorobenzyl)oxy]-3,4-dimethyl-1-benzopyran-2(2H)-one) within the examined series significantly inhibited (>60%) ex vivo rat-liver and striatal MAO-B activities 1 h after intraperitoneal administration of high doses (100 and 300 mumol kg(-1)), revealing its ability to cross the blood-brain barrier. At the same doses, liver and striatum MAO-A was less inhibited in vivo, somehow reflecting MAO-B selectivity, as assessed in vitro. In contrast, the metabolically less stable derivative 14, bearing an isopropyl ester in the lateral chain, had a weak effect on hepatic MAO-B activity in vivo, and none on striatal MAO-B, but, surprisingly, displayed inhibitory effects on MAO-A in both peripheral and brain tissues.     

THIAMINE IN NEURAL MEMBRANES ENZYMIC HYDROLYSIS OF THIAMINE DIPHBSPHATE

Abstract— The membrane-associated diphosphatase from rat brain which catalyses the hydrolysis of thiamine diphosphate and nucleoside diphosphate is described. The parallel sub-cellular distribution of thiamine diphosphatase and nucleoside diphosphatase activity and the equal inhibition of both activities by adenosine methylenediphosphonate, a non-hydrolysable structural analogue of ADP, suggests that a single enzyme is involved. The divalent cation requirement and basic kinetic properties of this enzyme have been determined. This nucleoside diphosphatase is not activated by ATP.     

Pathway of phospholipase C activation initiated with platelet-derived growth factor is different from that initiated with vasopressin and bombesin

The mode of phospholipase C activation initiated with platelet-derived growth factor (PDGF) has been studied in comparison with that initiated with vasopressin and bombesin in a rat fibroblast line, WFB. Stimulation of WFB cells by PDGF, vasopressin, and bombesin elicites rapid hydrolysis of polyphosphoinositides and an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i). On stimulation by PDGF, there was a lag period of about 10 s before an increase in [Ca2+]i. No measurable lag period was observed in the [Ca2+]i response induced by vasopressin or bombesin. Pretreatment of WFB cells with phorbol 12-myristate 13-acetate profoundly inhibited inositol phosphate formation evoked by vasopressin and bombesin, but enhanced to some extent inositol phosphate formation stimulated by PDGF. In membranes prepared from WFB cells, GTP markedly augmented inositol polyphosphate formation induced by vasopressin and bombesin. It was not successful in showing the PDGF-stimulated formation of inositol phosphates in the membrane preparation. The effects of GTP, guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) on polyphosphoinositide hydrolysis stimulated by growth factors were studied in WFB cells made permeable to nucleotides by treatment with either saponin or Pseudomonas aeruginosa cytotoxin. PDGF, vasopressin, and bombesin elicited inositol phosphate production in the permeabilized WFB cells in the absence of added GTP. GDP beta S, a competitive inhibitor of GTP-binding proteins (G-proteins), markedly reduced the bombesin- and vasopressin-stimulated production of inositol phosphates. However, the PDGF-stimulated production of inositol phosphates was not affected by the addition of GDP beta S. GTP gamma S, an agonist of G-proteins, largely enhanced the vasopressin- and bombesin-stimulated hydrolysis of inositol lipids when added at 10-100 microM. In the presence of GTP gamma S, the PDGF-stimulated hydrolysis of inositol lipids was not enhanced, but was reduced: 100 microM GTP gamma S reduced the stimulated hydrolysis to about a half of the control level. Only GTP gamma S, and no other nucleoside triphosphates, was found to have these effects. Activation of G-proteins in WFB cells by fluoroaluminate resulted in the inhibition of inositol phosphate production elicited with not only PDGF, but also with vasopressin and bombesin. These results indicate that a G-protein couples vasopressin and bombesin receptors to the activation of phospholipase C. Moreover, these results suggest that coupling of the PDGF receptor to phospholipase C is not mediated through a G-protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Methods for the determination of intracellular levels of ribose phosphates

Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway or stem from the phosphorolytic cleavage of the N-glycosidic bond of ribonucleosides. The two major pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, can be readily interconverted by phosphopentomutase. Ribose-5-phosphate is also the direct precursor of 5-phosphoribosyl-1-pyrophosphate, which is used for both de novo and salvage synthesis of nucleotides. On the other hand, the phosphorolysis of deoxyribonucleosides is the major source of deoxyribose phosphates. While the destiny of the nucleobase stemming from nucleoside phosphorolysis has been extensively investigated, the fate of the sugar moiety has been somehow neglected. However, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. Nevertheless, many aspects of pentose phosphate metabolism, and the possible involvement of these compounds in a number of cellular processes still remain obscure. The comprehension of the role played by pentose phosphates may be greatly facilitated by the knowledge of their steady-state intracellular levels and of their changes in response to variations of intra- and extracellular signals.     

Further studies of urea-catalyzed phosphorylation reactions

Summary We have analyzed the products formed when mixtures of a nucleoside and ammonium dihydrogen phosphate are heated with an excess of urea. If there is more phosphate than nucleoside in the mixture, compounds containing pyrophosphate bonds are obtained. If uridine, as nucleoside, is in excess over phosphate, di- and oligonucleotides are formed.On leave from Department of Medical Biochemistry, University of Göteborg, Sweden.     

Recent advances in structure and function of cytosolic IMP-GMP specific 5′nucleotidase II (cN-II)

Cytosolic 5′ nucleotidase II (cN-II) catalyses both the hydrolysis of a number of nucleoside monophosphates (e.g., IMP + H₂O→ inosine + Pi), and the phosphate transfer from a nucleoside monophosphate donor to the 5′ position of a nucleoside acceptor (e.g., IMP + guanosine → inosine + GMP). The enzyme protein functions through the formation of a covalent phosphoenzyme intermediate, followed by the phosphate transfer either to water (phosphatase activity) or to a nucleoside (phosphotransferase activity). It has been proposed that cN-II regulates the intracellular concentration of IMP and GMP and the production of uric acid. The enzyme might also have a potential therapeutic importance, since it can phosphorylate some anti-tumoral and antiviral nucleoside analogues that are not substrates of known kinases. In this review we summarise our recent studies on the structure, regulation and function of cN-II. Via a site-directed mutagenesis approach, we have identified the amino acids involved in the catalytic mechanism and proposed a structural model of the active site. A series of in vitro studies suggests that cN-II might contribute to the regulation of 5-phosphoribosyl-1-pyrophosphate (PRPP) level, through the so-called oxypurine cycle, and in the production of intracellular adenosine, formed by ATP degradation.