guanosine-monophosphate and acivicin

Topics: Adenosine Monophosphate; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Deoxyribonucleotides; Gene Expression Regulation; Gluconeogenesis; Guanosine Monophosphate; Humans; Inosine Monophosphate; Isoxazoles; Kidney Neoplasms; Liver Neoplasms; Liver Regeneration; Models, Biological; Neoplasms; Purines; Pyrimidines; Ribonucleotides

The discovery that genetic mutations in several cellular pathways can increase lifespan has lent support to the notion that pharmacological inhibition of aging pathways can be used to extend lifespan and to slow the onset of age-related diseases. However, so far, only few compounds with such activities have been described. Here, we have conducted a chemical genetic screen for compounds that cause the extension of chronological lifespan of Schizosaccharomyces pombe. We have characterized eight natural products with such activities, which has allowed us to uncover so far unknown anti-aging pathways in S. pombe. The ionophores monensin and nigericin extended lifespan by affecting vacuolar acidification, and this effect depended on the presence of the vacuolar ATPase (V-ATPase) subunits Vma1 and Vma3. Furthermore, prostaglandin J₂ displayed anti-aging properties due to the inhibition of mitochondrial fission, and its effect on longevity required the mitochondrial fission protein Dnm1 as well as the G-protein-coupled glucose receptor Git3. Also, two compounds that inhibit guanosine monophosphate (GMP) synthesis, mycophenolic acid (MPA) and acivicin, caused lifespan extension, indicating that an imbalance in guanine nucleotide levels impinges upon longevity. We furthermore have identified diindolylmethane (DIM), tschimganine, and the compound mixture mangosteen as inhibiting aging. Taken together, these results reveal unanticipated anti-aging activities for several phytochemicals and open up opportunities for the development of novel anti-aging therapies.

Topics: Dynamins; Garcinia mangostana; Guanosine Monophosphate; Hydroxybenzoates; Indoles; Isoxazoles; Mitochondrial Dynamics; Monensin; Mycophenolic Acid; Nigericin; Reactive Oxygen Species; Receptors, G-Protein-Coupled; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Time Factors; Vacuolar Proton-Translocating ATPases; Vacuoles

GMP synthetase (EC 6.3.5.2) is an amidotransferase that catalyzes the amination of xanthosine 5'-monophosphate to form GMP in the presence of glutamine and ATP. Glutamine hydrolysis produces the necessary amino group while ATP hydrolysis drives the reaction. Ammonia can also serve as an amino group donor. GMP synthetase contains two functional domains, which are well coordinated. The "glutamine amide transfer" or glutaminase domain is responsible for glutamine hydrolysis. The synthetase domain is responsible for ATP hydrolysis and GMP formation. Inorganic pyrophosphate inhibits the synthetase and uncouples the two domain functions by allowing glutamine hydrolysis to take place in the absence of ATP hydrolysis or GMP formation. Acivicin, a glutamine analog, selectively abolishes the glutaminase activity. It inhibits the synthetase activity only when glutamine is the amino donor. When ammonia is used in place of glutamine, acivicin has no effect on the synthetase activity. Acivicin inhibits GMP synthetase irreversibly by covalent modification. Enzyme inactivation is greatly facilitated by the presence of substrates. Acivicin labels GMP synthetase at a single site, and a tryptic peptide containing the modified residue was isolated. Mass spectrometry and Edman sequence analysis show that Cys104 is the site of modification. This residue is conserved among GMP synthetases and is located within a predicted glutamine amide transfer domain. These data suggest that Cys104 is an essential residue involved in the hydrolysis of glutamine to produce an amino group and is not needed for the hydrolysis of ATP or amination of xanthosine 5'-monophosphate to produce GMP.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Binding Sites; Carbon-Nitrogen Ligases; Cysteine; Enzyme Inhibitors; Glutamine; Guanosine Monophosphate; Humans; Hydrolysis; In Vitro Techniques; Isoxazoles; Kinetics; Ligases; Molecular Sequence Data; Peptide Fragments

The flux activities of de novo and salvage purine synthesis were compared in rat hepatoma 3924A cells in various growth phases. The initial rate assays of [14C]adenine, [14C]hypoxanthine, and [14C]guanine incorporation yielded Michaelis-Menten kinetics with Kms of 5, 7, and 7 microM, respectively. After replating plateau phase cells in lag and log phases the activity of purine de novo pathway increased 4.5- to 8-fold with a preferential rise in guanylate synthesis, whereas purine salvage activities increased only 1.6- to 2.1-fold. However, for the syntheses of IMP, AMP, and GMP, the activities of purine salvage pathways were 2- to 7-fold, 5- to 28-fold, and 2- to 32-fold higher than those of the de novo purine pathway. Treatment of cells with acivicin, an inhibitor of the activity of amidophosphoribosyltransferase, phosphoribosylformylglycinamidine synthase, and GMP synthase, inhibited the flux activities of de novo purine, adenylate, and guanylate syntheses to 37, 73, and 3% of the controls and decreased the concentration of GTP to 42%; the concentration of ATP did not change and that of 5-phosphoribosyl 1-pyrophosphate increased 3.1-fold. Under these conditions the activities of salvage synthesis from hypoxanthine and guanine were enhanced 2.5-fold. Treatment of hepatoma cells with IMP dehydrogenase inhibitors, tiazofurin, ribavirin, and 4-carbamoylimidazolium 5-olate, to block de novo guanylate synthesis accelerated the flux activity of guanine salvage pathway. The higher capacity of purine salvage pathway than that of the de novo one and the further rise of the activity in response to the drugs targeted against the de novo pathway highlight the important role salvage synthesis might play in circumventing the impact of antimetabolites of de novo purine synthesis in cancer chemotherapy.

Topics: Animals; Antimetabolites, Antineoplastic; Guanosine Monophosphate; IMP Dehydrogenase; Inosine Monophosphate; Isoxazoles; Kinetics; Liver Neoplasms, Experimental; Phosphoribosyl Pyrophosphate; Purines; Rats