Purine Nucleoside Monophosphates Research Articles

Development of Prolinol Containing Inhibitors of Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase: Rational Structure-Based Drug Design.

Inhibition of hypoxanthine-guanine-xanthine phosphoribosyltransferase activity decreases the pool of 6-oxo and 6-amino purine nucleoside monophosphates required for DNA and RNA synthesis, resulting in a reduction in cell growth. Therefore, inhibitors of this enzyme have potential to control infections, caused by Plasmodium falciparum and Plasmodium vivax, Trypanosoma brucei, Mycobacterium tuberculosis, and Helicobacter pylori. Five compounds synthesized here that contain a purine base covalently linked by a prolinol group to one or two phosphonate groups have Ki values ranging from 3 nM to >10 μM, depending on the structure of the inhibitor and the biological origin of the enzyme. X-ray crystal structures show that, on binding, these prolinol-containing inhibitors stimulated the movement of active site loops in the enzyme. Against TBr in cell culture, a prodrug exhibited an EC50 of 10 μM. Thus, these compounds are excellent candidates for further development as drug leads against infectious diseases as well as being potential anticancer agents.

Comprehensive Analysis of Prognostic Value and Immune Infiltration of the NT5DC Family in Hepatocellular Carcinoma.

Background Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, and its incidence is obviously increasing. The NT5DC family has been shown to be involved in the progression of many tumors. However, the biological function of NT5DC family members in HCC is still not well understood. Methods Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN, Kaplan–Meier plotter, cBioPortal, GeneMANIA, Metascape, and TIMER were applied to assess the biological function of NT5DC family members in HCC. Results Most of the NT5DC family members were highly expressed in HCC. High expression of NT5C2, NT5DC2, and NT5DC3 was closely associated with higher tumor stage and poor overall survival (OS). In addition, high NT5DC2 and NT5DC3 expression also predicted poor disease-free survival (DFS). Enrichment analysis revealed that the NT5DC family in HCC mainly involved the IMP metabolic process, purine ribonucleoside monophosphate metabolic process, and purine nucleoside monophosphate metabolic process. The expression of NT5DC family members was closely related to the infiltration of some immune cells, such as B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. Conclusion Our findings provided new insights into the biological function and prognostic value of NT5DC family members in HCC.

N,N-Bis(glycityl)amines as anti-cancer drugs

A series of N,N-bis(glycityl)amines with promising anti-cancer activity were prepared via the reductive amination of pentoses and hexoses, and subsequently screened for their ability to selectively inhibit the growth of cancerous versus non-cancerous cells. For the first time, we show that this class of compounds possesses anti-proliferative activity, and, while the selective killing of brain cancer (LN18) cells versus matched (SVG-P12) cells was modest, several of the amines, including d-arabinitylamine 1a and d-fucitylamine 1g, exhibited low micromolar IC50 values for HL60 cells. Moreover, these two amines showed good selectivity towards HL60 cells when compared to non-cancerous HEK-293 cells. The compounds also showed low micromolar inhibition of the leukaemic cell line, THP-1. The modes of action of amines 1a and 1g were then determined using yeast chemical genetics, whereby it was established that both compounds affect similar but distinct sets of biochemical pathways. Notably purine nucleoside monophosphate biosynthesis was identified as an enriched mechanism. The rapid synthesis of the amines and their unique mode of action thus make them attractive targets for further development as anti-cancer drugs.

Aza-acyclic Nucleoside Phosphonates Containing a Second Phosphonate Group As Inhibitors of the Human, Plasmodium falciparum and vivax 6-Oxopurine Phosphoribosyltransferases and Their Prodrugs As Antimalarial Agents

Hypoxanthine-guanine-[xanthine] phosphoribosyltransferase (HG[X]PRT) is considered an important target for antimalarial chemotherapy as it is the only pathway for the synthesis of the purine nucleoside monophosphates required for DNA/RNA production. Thus, inhibition of this enzyme should result in cessation of replication. The aza-acyclic nucleoside phosphonates (aza-ANPs) are good inhibitors of Plasmodium falciparum HGXPRT (PfHGXPRT), with Ki values as low as 0.08 and 0.01 μM for Plasmodium vivax HGPRT (PvHGPRT). Prodrugs of these aza-ANPs exhibit antimalarial activity against Pf lines with IC50 values (0.8-6.0 μM) and have low cytotoxicity against human cells. Crystal structures of six of these compounds in complex with human HGPRT have been determined. These suggest that the different affinities of these aza-ANPs could be due to the flexibility of the loops surrounding the active site as well as the flexibility of the inhibitors, allowing them to adapt to fit into three binding pockets of the enzyme(s).

An <i>H</i>-Phosphonate Approach for the Preparation of Purine-Nucleoside Monophosphates

Two purine-nucleoside monophosphates have been prepared from the corresponding nucleoside 5′-H-phosphonate precursors via sequential silylation, oxidation, and hydrolysis reactions in a one-pot manner. Compared to the reaction performed in the presence of pyridine, the hydrolysis of iodophosphate in the absence of pyridine generated nucleoside 5′-monophosphates as the major product. The experimental results indicated that the reaction between the formed nucleoside 5′-monophosphate with the residual iodophosphate intermediate was relatively slow, making the self-condensed dinucleoside diphosphate a minor product in this reaction.

Synthesis of 9-phosphonoalkyl and 9-phosphonoalkoxyalkyl purines: Evaluation of their ability to act as inhibitors of Plasmodium falciparum, Plasmodium vivax and human hypoxanthine–guanine–(xanthine) phosphoribosyltransferases

The purine salvage enzyme, hypoxanthine–guanine–(xanthine) phosphoribosyltransferase [HG(X)PRT], catalyses the synthesis of the purine nucleoside monophosphates, IMP, GMP or XMP essential for DNA/RNA production. In protozoan parasites, such as Plasmodium, this is the only route available for their synthesis as they lack the de novo pathway which is present in human cells. Acyclic nucleoside phosphonates (ANPs), analogs of the purine nucleoside monophosphates, have been found to inhibit Plasmodium falciparum (Pf) HGXPRT and Plasmodium vivax (Pv) HGPRT with Ki values as low as 100nM. They arrest parasitemia in cell based assays with IC50 values of the order of 1–10μM. ANPs with phosphonoalkyl and phosphonoalkoxyalkyl moieties linking the purine base and phosphonate group were designed and synthesised to evaluate the influence of this linker on the potency and/or selectivity of the ANPs for the human and malarial enzymes. This data shows that variability in the linker, as well as the positioning of the oxygen in this linker, influences binding. The human enzyme binds the ANPs with Ki values of 0.5μM when the number of atoms in the linker was 5 or 6 atoms. However, the parasite enzymes have little affinity for such long chains unless oxygen is included in the three-position. In comparison, all three enzymes have little affinity for ANPs where the number of atoms linking the base and the phosphonate group is of the order of 2–3 atoms. The chemical nature of the purine base also effects the Ki values. This data shows that both the linker and the purine base play an important role in the binding of the ANPs to these three enzymes.

Adenosine Deaminase-like Protein 1 (ADAL1): Characterization and Substrate Specificity in the Hydrolysis of N6- or O6-Substituted Purine or 2-Aminopurine Nucleoside Monophosphates

Human N(6)-methyl-AMP/dAMP aminohydrolase has been shown to be involved in metabolism of pharmacologically important N(6)-substituted purine nucleosides and 5'-monophosphate prodrugs thereof. This enzyme was cloned and expressed in E. coli, and mass spectroscopic analysis followed by amino acid sequence analyses indicated that the protein was adenosine deaminase-like protein isoform 1 (ADAL1). An extensive structure-activity relationship study showed that ADAL1 was able to catalyze removal of different alkyl groups not only from N(6)-substituted purine or 2-aminopurine nucleoside monophosphates but also from O(6)-substituted compounds. The ADAL1 activity was susceptible to modifications in the phosphate moiety but not to changes in the sugar moiety. Overall, our data indicated that ADAL1 specifically acts at the 6-position of purine and 2-aminopurine nucleoside monophosphates. Our results may help designing of new therapeutic nucleoside/nucleotide prodrugs with desired metabolic profiles. Furthermore, amino acid sequence analysis in conjunction with crystallographic data and metal analysis suggested that ADAL1 contains a catalytic zinc ion. Finally, a potential physiological role of ADAL1 is discussed.

Adenosine Deaminase-like Protein 1 (ADAL1) Catalyzes Removal of Different Alkyl Groups from N6- or O6-substituted Purine or 2-Aminopurine Nucleoside Monophosphates

Adenosine Deaminase-like Protein 1 (ADAL1) Catalyzes Removal of Different Alkyl Groups from N6- or O6-substituted Purine or 2-Aminopurine Nucleoside Monophosphates

ITPase-deficient mice show growth retardation and die before weaning

Inosine triphosphate pyrophosphatase (ITPase), the enzyme that hydrolyzes ITP and other deaminated purine nucleoside triphosphates to the corresponding purine nucleoside monophosphate and pyrophosphate, is encoded by the Itpa gene. In this study, we established Itpa knockout (KO) mice and used them to show that ITPase is required for the normal organization of sarcomeres in the heart. Itpa(-/-) mice died about 2 weeks after birth with features of growth retardation and cardiac myofiber disarray, similar to the phenotype of the cardiac alpha-actin KO mouse. Inosine nucleotides were found to accumulate in both the nucleotide pool and RNA of Itpa(-/-) mice. These data suggest that the role of ITPase in mice is to exclude ITP from the ATP pool, and the main target substrate of this enzyme is rITP. Our data also suggest that cardiomyopathy, which is mainly caused by mutations in sarcomeric protein-encoding genes, is also caused by a defect in maintaining the quality of the ATP pool, which is an essential requirement for sarcomere function.

Mutational, inhibitory and microcalorimetric analyses of Plasmodium falciparum TMP kinase. Implications for drug discovery

Plasmodium falciparum thymidylate kinase (PfTMK) can tolerate a range of substrates, which distinguishes it from other thymidylate kinases. The enzyme not only phosphorylates TMP and dUMP but can also tolerate bulkier purines, namely, dGMP, GMP, and dIMP. In order to probe the flexibility of PfTMK in accommodating ligands of various sizes, we developed 6 mutant enzymes and subjected these to thermodynamic, inhibitory and catalytic evaluation. Kinase activity was markedly affected by introducing a larger lysine residue instead of A111. The lack of the hydroxyl group after inducing mutation of Y107F affected enzyme activity, and had a more severe impact on dGMP kinase activity. PfTMK can be inhibited by both purine and pyrimidine nucleosides, raising the possibility of developing highly selective drugs. Thermodynamic analysis revealed that enthalpic forces govern both purine and pyrimidine nucleoside monophosphate binding, and the binding affinity of both substrates was highly comparable. The heat produced due to dGMP binding is lower than that attributable to TMP. This indicates that additional interactions occur with TMP, which may be lost with larger dGMP. Targeting PfTMK not only affects thymidine nucleotide synthesis but may also affect purine nucleotides, and thus the enzyme represents an attractive antimicrobial target.

The Crystal Structure of Free Human Hypoxanthine-guanine Phosphoribosyltransferase Reveals Extensive Conformational Plasticity Throughout the Catalytic Cycle

Human hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyses the synthesis of the purine nucleoside monophosphates, IMP and GMP, by the addition of a 6-oxopurine base, either hypoxanthine or guanine, to the 1-beta-position of 5-phospho-alpha-d-ribosyl-1-pyrophosphate (PRib-PP). The mechanism is sequential, with PRib-PP binding to the free enzyme prior to the base. After the covalent reaction, pyrophosphate is released followed by the nucleoside monophosphate. A number of snapshots of the structure of this enzyme along the reaction pathway have been captured. These include the structure in the presence of the inactive purine base analogue, 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and PRib-PP.Mg2+, and in complex with IMP or GMP. The third structure is that of the immucillinHP.Mg(2+).PP(i) complex, a transition-state analogue. Here, the first crystal structure of free human HGPRT is reported to 1.9A resolution, showing that significant conformational changes have to occur for the substrate(s) to bind and for catalysis to proceed. Included in these changes are relative movement of subunits within the tetramer, rotation and extension of an active-site alpha-helix (D137-D153), reorientation of key active-site residues K68, D137 and K165, and the rearrangement of three active-site loops (100-128, 165-173 and 186-196). Toxoplasma gondii HGXPRT is the only other 6-oxopurine phosphoribosyltransferase structure solved in the absence of ligands. Comparison of this structure with human HGPRT reveals significant differences in the two active sites, including the structure of the flexible loop containing K68 (human) or K79 (T.gondii).

Expression and characterization of recombinant bovine cytosolic 5'-nucleotidase IMP-GMP specific.

Cytosolic 5′-nucleotidase specific for IMP-GMP and their respective deoxyderivatives (5′N) is an ubiquitous enzyme able to catalyse the hydrolysis of purine nucleoside monophosphates or the transfer of the phosphate to an acceptor nucleoside. The enzyme has been purified from several sources and its kinetic parameters and molecular characteristics have been studied.1,2 All the cytosolic 5′-nucleotidases described, although sometimes differ remarkably in molecular mass, exhibit very similar properties, such as the activation by ATP and the inhibition by orthophosphate (Pi). A reaction mechanism proceeding via the formation of a covalent enzyme-phosphate intermediate was proposed by Worku and Newby.3 The validity of this mechanism was recently demonstrated by the isolation of an enzyme-phosphate covalent complex. Even though there is indirect evidence for an involvement of histidine and cysteine residues in catalysis, nothing is known about the nature of the amino acid acceptor of the phosphate.4 KeywordsRecombinant ProteinXanthine OxidasePurine NucleosideImmobilise Metal Affinity ChromatographyPurine Nucleoside PhosphorylaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Complexation of Nickel(II) with Guanosine 5'-Monophosphate and Inosine 5'-Monophosphate: A Potentiometric and Calorimetric Study.

The constants (K(s)) and enthalpies (DeltaH(s)) for stacking interactions between purine nucleoside monophosphates were determined by calorimetry; the values thus obtained were guanosine as follows: K(s) = 2.1 +/- 0.3 M(-)(1) and DeltaH(s) = -41.8 +/- 0.8 kJ/mol for adenosine 5'-monophosphate (5'AMP); K(s) = 1.5 +/- 0.3 M(-1) and DeltaH(s) = -42.0 +/- 1.5 kJ/mol for guanosine 5'-monophosphate (5'GMP); and K(s) = 1.0 +/- 0.2 M(-1) and DeltaH(s) = -42.3 +/- 1.1 kJ/mol for inosine 5'-monophosphate (5'IMP). The interaction of nickel(II) with purine nucleoside monophosphates was studied using potentiometric and calorimetric methods, with 0.1 M tetramethylammonium bromide as the background electrolyte, at 25 degrees C. The presence in solution of the complexes [Ni(5'GMP)(2)](2)(-) and [Ni(5'IMP)(2)](2)(-) was observed. The thermodynamic parameters obtained were log K(ML) = 3.04 +/- 0.02, log K(ML2) = 2.33 +/- 0.02, DeltaH(ML) = -18.4 +/- 0.9 kJ/mol and DeltaH(ML2) = -9.0 +/- 1.9 kJ/mol for 5'GMP; and log K(ML) = 2.91 +/- 0.01, log K(ML2) = 1.92 +/- 0.01, DeltaH(ML) = -16.2 +/- 0.9 kJ/mol and DeltaH(ML2) = -0.1 +/- 2.3 kJ/mol for 5'IMP. The relationships between complex enthalpies and the degree of macrochelation, as well as the stacking interaction between purine bases in the complexes are discussed in relation to previously reported calorimetric data.

Nucleic-base recognition. Intramolecular stack formation in mixed ligand complexes containing a purine-nucleoside monophosphate (NMP)

Nucleic-base recognition. Intramolecular stack formation in mixed ligand complexes containing a purine-nucleoside monophosphate (NMP)

Characterization of adenine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum

Because of their inability to synthesize purines de novo, malaria parasites rely on purine phosphoribosyltransferases (PRTases) to convert purine bases salvaged from the host cell (the erythrocyte) into the corresponding purine nucleoside monophosphates. Our studies with late trophozoites of the human malaria parasite, Plasmodium falciparum, showed that virtually all of the purine PRTase activity is accounted for by two distinct enzymes. One enzyme utilizes hypoxanthine, guanine and xanthine (Queen, S.A., Vander Jagt, D. and Reyes, P. (1988) Mol. Biochem. Parasitol. 30, 123–134). The second enzyme utilizes only adenine and is the subject of this paper. This latter enzyme exhibits a biphasic pH-activity profile and is moderately to weakly inhibited by several divalent metal ions. Several of the properties of the P. falciparum enzyme were found to differ significantly from those of human erythrocyte adenine PRTase. (1) The molecular weight (18 000) of the parasite enzyme is smaller than that of the host cell enzyme. (2) The parasite enzyme, unlike the erythrocyte enzyme, is not significantly inhibited by sulfuhydryl reagents. (3) 6-Mercaptopurine and 2,6-diaminopurine proved to be competitive inhibitors of the parasite enzyme ( K i 0.70 and 1.0 mM, respectively); on the other hand, the human enzyme is not inhibited by these agents. (4) The K m for adenine (0.80 μM) and 5-phosphoribosyl-1-pyrophosphate (0.70 μM) displayed by the parasite enzyme are significantly smaller than the corresponding K m values shown by the erythrocyte enzyme. These distinctions between the parasite and host enzymes point to the possibility that adenine PRTase of P. falciparum may represent a potential target for chemotherapeutic attack.

OH-induced Free Radicals in Purine Nucleoside Monophosphates: E.S.R. and Spin-trapping

Free radicals produced by the reactions of OH radicals with six purine nucleoside monophosphates (3'-AMP, 5'-AMP, 5'-dAMP, 3'-GMP, 5'-GMP and 5'-dGMP) were investigated by a method combining e.s.r. spin-trapping and high-performance liquid chromatography (HPLC). The N2O-saturated aqueous solutions of purine nucleoside monophosphates, containing 2-methyl-2-nitrosopropane as a spin-trap, were X-irradiated and the resulting spin-adducts were separated by reverse-phase HPLC in the ion suppression mode. The separated spin-adducts were characterized by e.s.r. spectrometry and UV spectrophotometry. Consequently, the radicals due to H-abstraction at the C4' position of the sugar moiety were identified arising from 5'-dAMP and 5'-dGMP. In all cases, e.s.r. spectra consisting of a secondary doublet were observed and assigned to the radical due to H-abstraction at the C5' position of the sugar moiety.

Measurement of cytoplasmic free Ca2+ and cyclic purine nucleoside monophosphates in rat hepatocytes during receptor-mediated endocytosis of transferrin and asialoglycoproteins

Measurement of cytoplasmic free Ca2+ and cyclic purine nucleoside monophosphates in rat hepatocytes during receptor-mediated endocytosis of transferrin and asialoglycoproteins

Biochemical mechanisms of the antileishmanial activity of sodium stibogluconate

Pentavalent antimonial agents such as sodium stibogluconate (Pentostam; Burroughs Wellcome Co., London, United Kingdom) are the drugs of choice for the treatment of leishmaniasis, but their biochemical mechanisms of action are virtually unknown. The viability of Leishmania mexicana (WR 227) promastigotes and amastigotes was decreased 40 to 61% by a 4-h exposure to 500 micrograms of Sb (in the form of stibogluconate) per ml. Such exposure also resulted in a 51 to 65% decrease in incorporation of label into DNA, RNA, and protein; a 56 to 65% decrease in incorporation of label into purine nucleoside triphosphate; and a 34 to 60% increase in incorporation of label into purine nucleoside monophosphate and diphosphate. It is postulated that the apparent decrease in ATP and GTP synthesis from ADP and GDP contributes to decreased macromolecular synthesis and to decreased Leishmania viability. Further experiments suggested that inhibition of glycolysis and the citric acid cycle may partially explain the inability to phosphorylate ADP.

Evidence on the existence of a purine ligand induced conformational change in the active site of bovine pancreatic ribonuclease A studied by proton nuclear magnetic resonance spectroscopy.

The titration curves of the C-2 histidine protons of RNase A and of derivative II--a covalent derivative obtained by reaction of the enzyme with the halogenated nucleotide 9-beta-D-ribofuranosyl-6-chloropurine 5'-phosphate--in the presence of a number of purine nucleosides, nucleoside monophosphates, and nucleoside diphosphates were studied by means of proton nuclear magnetic resonance at 270 MHz. The examination of the perturbations found on the chemical shifts and pKs of the C-2 protons of His-12, -48, and -119 are consistent with the following conclusions: (1) The interaction of adenosine in the primary purine binding site of the enzyme (B2R2) induces a conformational change in the active center of the enzyme [for the nomenclature of the RNase A binding subsites, see Parés et al. [Parés, X., Llorens, R., Arús, C., & Cuchillo, C. M. (1980) Eur. J. Biochem. 105, 571-579]]. (2) The phosphate moiety of the ligands, independently of its position, probably acts as a general carrier of the nucleotide to the active center, while the substituents of the base are the generators of the specificity of the binding and control the binding equilibrium between subsites B2R2 and B1R1. (3) There is no overlapping between the binding sites occupied by the labeling nucleotide in derivative II (B3R3p2) and the primary binding site for purine mononucleotides (B2R2p1).

Inhibition by Cyclic Guanosine 3':5'-Monophosphate of the Soluble DNA Polymerase Activity, and of Partially Purified DNA Polymerase A (DNA Polymerase I) from the Yeast Saccharomyces cerevisiae

DNA polymerase activity from extracts of growing yeast cells is inhibited by cGMP. Experiments with partially purified yeast DNA polymerases show, that cGMP inhibits DNA polymerase A (DNA polymerase I from Chang), which is the main component of the soluble DNA polymerase activity in yeast extracts, by competing for the enzyme with the primer-template DNA. Since the enzyme is not only inhibited by 3', 5'-cGMP, but also by 3',5'-cAMP, the 3'--:5'-phosphodiester seems to be crucial for the competition between cGMP and primer. This would be inconsistent with the concept of a 3'-OH primer binding site in the enzyme. The existence of such a site in the yeast DNA polymerase A is indicated from studies with various purine nucleoside monophosphates. When various DNA polymerases are compared, inhibition by cGMP seems to be restricted to those enzymes, which are involved in DNA replication, DNA polymerases with an associated nuclease activity are not inhibited, DNA polymerase B from yeast is even activated by cGMP. Though some relations between the cGMP effect and the presumed function of the enzymes in the living cell are apparent, the biological meaning of the observations in general remains open.