Structure and Conformation of 1-β-D-Ribo Furanosyl Pyridin-2-one-5-Carboxamide: An Anti-Inflammatory Agent

The pyrimidine nucleoside, 1-β-D-ribofuranosyl pyridine-2-one-5-carboxamide, is an anti inflammatory agent used in the treatment of adjuvant-induced arthritis. It is the 2-one isomer of 1-β-D-ribofuranosyl pyridine-4-one 5-carboxamide, an unusual nucleoside isolated from the urine of patients with chronic myelogenic leukemia and an important cancer marker. Crystals of 1-β-D-ribofuranosyl pyridine-2-one-5-carboxamide are monoclinic, space group C2, with the cell dimensions a = 31.7920(13), b = 4.6872 (3), c = 16.1838(11), β = 93.071(3)°, V = 2408.2(2) ų, D_calc = 1.496 mg/m³ and Z = 8 (two molecules in the asymmetric unit). The structure was obtained by the application of direct methods to diffractometric data and refined to a final R value of 0.050 for 1669 reflections with I ≥ 3σ. The nucleoside exhibits an anti conformation across the glycosidic bond (χ_CN = ?15.5°, ?18.9°), a C3 ′- endo C2 ′ -exo [³ ₂T] ribose pucker and g⁺ across the C(4 ′)-C(5 ′) exocyclic bond. The amino group of the carboxamide group is distal from the 2-one and lacks the intramolecular hydrogen bonding found in the related 2-one molecule. Nuclear magnetic resonance studies shows also an anti conformation across the glycosidic bond but the solution conformation of the furanose ring is not the same as that found in the solid state.     

Cellulose-bound Peptide Arrays: Preparation and Applications

Spatial organization of metabolic enzymes may represent a general cellular mechanism to regulate metabolic flux. One recent example of this type of cellular phenomenon is the purinosome, a newly discovered multi-enzyme metabolic assembly that includes all of the enzymes within the de novo purine biosynthetic pathway. Our understanding of the components and regulation of purinosomes has significantly grown in recent years. This paper reviews the purine de novo biosynthesis pathway and its regulation, and presents the evidence supporting the purinosome assembly and disassembly processes under the control of G-protein-coupled receptor (GPCR) signaling. This paper also discusses the implications of purinosome and GPCR regulation in drug discovery.     

Crystal structure of the invertebrate bifunctional purine biosynthesis enzyme PAICS at 2.8 Å resolution

Two important steps of the de novo purine biosynthesis pathway are catalyzed by the 5‐aminoimidazole ribonucleotide carboxylase and the 4‐(N‐succinylcarboxamide)‐5‐aminoimidazole ribonucleotide synthetase enzymes. In most eukaryotic organisms, these two activities are present in the bifunctional enzyme complex known as PAICS. We have determined the 2.8‐Å resolution crystal structure of the 350‐kDa invertebrate PAICS from insect cells (Trichoplusia ni) using single‐wavelength anomalous dispersion methods. Comparison of insect PAICS to human and prokaryotic homologs provides insights into substrate binding and reveals a highly conserved enzymatic framework across divergent species. Proteins 2013; 81:1473–1478. © 2013 Wiley Periodicals, Inc.     

Biochemical analyses of ppGpp effect on adenylosuccinate synthetases,key enzymes in purine biosynthesis in rice

The ppGpp-signaling system functions in plant chloroplasts. In bacteria, a negative effect of ppGpp on adenylosuccinate synthetase (AdSS) has been suggested. Our biochemical analysis also revealed rice AdSS homologs are apparently sensitive to ppGpp. However, further investigation clarified that this phenomenon is cancelled by the high substrate affinity to the enzymes, leading to a limited effect of ppGpp on adenylosuccinate synthesis.     

A specific inhibitor of lactate dehydrogenase overcame the resistance toward gemcitabine in hypoxic mesothelioma cells,and modulated the expression of the human equilibrative transporter-1

ABSTRACT

Malignant pleural mesothelioma (MPM) is a very hypoxic malignancy, and hypoxia has been associated with resistance towards gemcitabine. The muscle-isoform of lactate dehydrogenase (LDH-A) constitutes a major checkpoint for the switch to anaerobic glycolysis. Therefore we investigated the combination of a new LDH-A inhibitor (NHI-1) with gemcitabine in MPM cell lines. Under hypoxia (O₂ tension of 1%) the cell growth inhibitory effects of gemcitabine, were reduced, as demonstrated by a 5- to 10-fold increase in IC₅₀s. However, the simultaneous addition of NHI-1 was synergistic (combination index < 1). Flow cytometry demonstrated that hypoxia caused a G1 arrest, whereas the combination of NHI-1 significantly increased gemcitabine-induced cell death. Finally, the mRNA expression levels of the human equilibrative transporter-1 (hENT1) were significantly down-regulated under hypoxia, but treatment with NHI-1 was associated with a recovery of hENT1 expression. In conclusion, our data show that hypoxia increased MPM resistance to gemcitabine. However, cell death induction and modulation of the key transporter in gemcitabine uptake may contribute to the synergistic interaction of gemcitabine with the LDH-A inhibitor NHI-1 and support further studies for the rational development of this combination.     

Synthesis and Anti-HCV Activity of 4-Methoxy-7H-Pyrrolo[2,3-d] Pyrimidine Carbocyclic Nucleosides

The present study includes the exploration of new possible nucleoside mimetics based on 4-methoxy-7H-pyrrolo[2,3-d]pyrimidine carbocyclic nucleosides (4a–g), which were synthesized by 10–15 synthetic steps and characterized adequately. We report the anti-HCV activities and cytotoxicities of 4a–g. Compound 4a was analyzed by single crystal X-ray diffraction which showed some puckering in the cyclopentene ring with a 2′-endo conformation and anti-base disposition (χ = ?125.7°).     

Mitochondrial purine and pyrimidine metabolism and beyond

ABSTRACT

Carefully balanced deoxynucleoside triphosphate (dNTP) pools are essential for both nuclear and mitochondrial genome replication and repair. Two synthetic pathways operate in cells to produce dNTPs, e.g., the de novo and the salvage pathways. The key regulatory enzymes for de novo synthesis are ribonucleotide reductase (RNR) and thymidylate synthase (TS), and this process is considered to be cytosolic. The salvage pathway operates both in the cytosol (TK1 and dCK) and the mitochondria (TK2 and dGK). Mitochondrial dNTP pools are separated from the cytosolic ones owing to the double membrane structure of the mitochondria, and are formed by the salvage enzymes TK2 and dGK together with NMPKs and NDPK in postmitotic tissues, while in proliferating cells the mitochondrial dNTPs are mainly imported from the cytosol produced by the cytosolic pathways. Imbalanced mitochondrial dNTP pools lead to mtDNA depletion and/or deletions resulting in serious mitochondrial diseases. The mtDNA depletion syndrome is caused by deficiencies not only in enzymes in dNTP synthesis (TK2, dGK, p53R2, and TP) and mtDNA replication (mtDNA polymerase and twinkle helicase), but also in enzymes in other metabolic pathways such as SUCLA2 and SUCLG1, ABAT and MPV17. Basic questions are why defects in these enzymes affect dNTP synthesis and how important is mitochondrial nucleotide synthesis in the whole cell/organism perspective? This review will focus on recent studies on purine and pyrimidine metabolism, which have revealed several important links that connect mitochondrial nucleotide metabolism with amino acids, glucose, and fatty acid metabolism.     

Synthesis and Bioactivity of Novel Trisubstituted Triazole Nucleosides

A series of novel trisubstituted 1,2,3-triazole purine nucleosides were efficiently synthesized via Huisgen 1,3-dipolar cycloaddition in good yields. Bioactivity against cytomegalovirus (CMV) and varicella-zoster virus (VZV) in human embryonic lung cell cultures was evaluated and all compounds show low antiviral activity.     

Three new double-headed nucleotides with additional nucleobases connected to C-5 of pyrimidines; synthesis,duplex and triplex studies

In the search for double-coding DNA-systems, three new pyrimidine nucleosides, each coded with an additional nucleobase anchored to the major groove face, are synthesized. Two of these building blocks carry a thymine at the 5-position of 2′-deoxyuridine through a methylene linker and a triazolomethylene linker, respectively. The third building block carries an adenine at the 6-position of pyrrolo-2′-deoxycytidine through a methylene linker. These double-headed nucleosides are introduced into oligonucleotides and their effects on the thermal stabilities of duplexes are studied. All studied double-headed nucleotide monomers reduce the thermal stability of the modified duplexes, which is partially compensated by using consecutive incorporations of the modified monomers or by flanking the new double-headed analogs with members of our former series containing propyne linkers. Also their potential in triplex-forming oligonucleotides is studied for two of the new double-headed nucleotides as well as the series of analogs with propyne linkers. The most stable triplexes are obtained with single incorporations of additional pyrimidine nucleobases connected via the propyne linker.