guanosine-triphosphate and pyrimidine

The Filamenting temperature-sensitive mutant Z (FtsZ), an essential GTPase in bacterial cell division, is highly conserved among Gram-positive and Gram-negative bacteria and thus considered an attractive target to treat antibiotic-resistant bacterial infections. In this study, a new class of FtsZ inhibitors bearing the pyrimidine-quinuclidine scaffold was identified from structure-based virtual screening of natural product libraries. Iterative rounds of in silico studies and biological evaluation established the preliminary structure-activity relationships of the new compounds. Potent FtsZ inhibitors with low micromolar IC₅₀ and antibacterial activity against S. aureus and E. coli were found. These findings support the use of virtual screening and structure-based design for the rational development of new antibacterial agents with innovative mechanisms of action.

Topics: Animals; Anti-Bacterial Agents; Binding Sites; Cattle; Drug Design; Drug Evaluation, Preclinical; Enzyme Inhibitors; Escherichia coli; GTP Phosphohydrolases; Guanosine Triphosphate; Humans; Molecular Docking Simulation; Protein Conformation; Protein Multimerization; Protein Structure, Quaternary; Pyrimidines; Quinuclidines; Sequence Homology, Amino Acid; Staphylococcus aureus; Structure-Activity Relationship; Tubulin

The pyrH-encoded uridine 5'-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg²(+) as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents.

Topics: Adenosine Triphosphate; Allosteric Regulation; Amino Acid Sequence; Cloning, Molecular; Escherichia coli; Escherichia coli Proteins; Genes, Suppressor; Guanosine Triphosphate; Kinetics; Ligands; Molecular Sequence Data; Molecular Weight; Mycobacterium tuberculosis; Polymerase Chain Reaction; Pyrimidines; Sequence Alignment; Sequence Analysis, DNA; Spectrometry, Mass, Electrospray Ionization; Transferases; Uridine Triphosphate

The control of de novo pyrimidine biosynthesis in the industrially important patent strain "Pseudomonas alkanolytica" ATCC 21034 was investigated. Uracil supplementation of succinate-grown "P. alkanolytica" cells produced the greatest depression of the de novo pyrimidine biosynthetic pathway enzyme activities. After the pyrimidine limitation of a "P. alkanolytica" orotate phosphoribosyltransferase mutant strain grown on succinate, the pyrimidine biosynthetic pathway enzyme activities were derepressed. The pyrimidine biosynthetic pathway enzyme aspartate transcarbamoylase in "P. alkanolytica" was inhibited by pyrophosphate, cytidine 5'-triphosphate (CTP), uridine 5'-triphosphate (UTP), and guanosine 5'-triphosphate (GTP).

Topics: Aspartate Carbamoyltransferase; Cytidine Triphosphate; Diphosphates; Enzyme Repression; Gene Expression Regulation, Bacterial; Genes, Bacterial; Guanosine Triphosphate; Mutation; Orotate Phosphoribosyltransferase; Pseudomonas; Pyrimidines; Succinic Acid; Uracil; Uridine Triphosphate

Mycophenolate mofetil (MMF) is an effective immunosuppressant developed for use in organ transplantation. It specifically targets lymphocyte purine biosynthesis. However, side effects do occur. Understanding how the active metabolite of MMF, mycophenolic acid (MPA) affects the normally integrated interaction between intracellular purine and pyrimidine pathways might aid the development of improved therapeutic regimes.. We used a primary human T-lymphocyte model to study how preincubation with MPA (0.1-50 microM) affected normal ribonucleotide pool responses to phytohemagglutinin using radiolabeled precursors.. MPA not only restricted the mitogen-induced expansion of GTP pools, but actually induced a severe drop in both GTP (10% of unstimulated cells) and GDP-sugar pools, with a concomitant fall in ATP (up to 50%). These effects were concentration dependent. By contrast, uridine pools expanded whereas CTP pools remained at resting levels. These changes were confirmed by the altered incorporation of [14C]-bicarbonate and [14C]-glycine into nucleotides. Restriction of [14C]-hypoxanthine incorporation and reduction of [14C]-uridine uptake comparable to that of unstimulated cells indicated that MPA also inhibited both salvage routes of nucleotide synthesis.. MPA affects pyrimidine as well as purine responses to mitogens in T-lymphocytes, but not in an integrated way. The molecular mechanisms underlying these disproportionate changes can best be explained by MPA-related inhibition of amidophosphoribosyltransferase, catalysing the first step in purine biosynthesis. This would increase phosphoribosylpyrophosphate availability, thereby stimulating UTP biosynthesis. Such imbalances, coupled with ATP-depletion, could underlie reported side effects and might be overcome by appropriate combination therapies.

Topics: Adenosine Triphosphate; Bicarbonates; Cells, Cultured; Enzyme Inhibitors; Glycine; Guanosine Triphosphate; Humans; Hypoxanthine; Mitogens; Mycophenolic Acid; Osmolar Concentration; Phytohemagglutinins; Pilot Projects; Purine Nucleotides; Purines; Pyrimidines; Ribonucleotides; T-Lymphocytes; Uridine

Cancer chemopreventive effects of polyphenols from green tea (GTP) in mouse models of photocarcinogenesis are established. The present study is extended from mouse model to human system in vivo to determine the effect of topical application of GTP to human individuals against UV light-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) in the skin. UVB-induced CPDs were detected by immunohistochemical technique using monoclonal antibodies to thymine dimers. With the gradual increase in UVB dose, both erythema response and CPD formation in the skin was increased. GTP treatment inhibited both UVB-induced erythema response as well as CPD formation. Topical treatment with GTP (approximately 1 mg/cm2 of skin area) 20 min before human buttock skin (sun-protected site) exposure to UVB inhibited CPD formation in epidermis by 81, 70, 60, and 60% at 0.5, 1.0, 2.0, and 4.0 minimal erythema dose of UV exposure, respectively. Treatment of human skin with varying doses of GTP (1-4 mg/2.5 cm2 of skin area) before a single dose of UVB exposure (4.0 minimal erythema dose) decreased dose dependently the formation of UVB-induced CPDs in both epidermis and dermis. The inhibition of UVB-induced CPDs by GTP treatment may be, at least in part, responsible for the inhibition of photocarcinogenesis. Our data suggest that GTP may be used as a novel chemopreventive candidate and possible strategy to reduce UV-induced skin cancer risk in the human population.

Topics: Adult; Biopsy; Dimerization; DNA; DNA Damage; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Female; Flavonoids; Guanosine Triphosphate; Humans; Male; Microscopy, Fluorescence; Middle Aged; Phenols; Polymers; Polyphenols; Pyrimidines; Skin; Tea; Ultraviolet Rays