title: Exploring the Potent Inhibition of CTP Synthase by Gemcitabine-5'-Triphosphate.
abstract: CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a target for the development of antiviral, anticancer, antiprotozoal, and immunosuppressive agents. Exposure of cell lines to the antineoplastic cytidine analogue gemcitabine causes depletion of intracellular CTP levels, but the direct inhibition of CTPS by its metabolite gemcitabine-5'-triphosphate (dF-dCTP) has not been demonstrated. We show that dF-dCTP is a potent competitive inhibitor of Escherichia coli CTPS with respect to UTP [Ki=(3.0±0.1) μm], and that its binding affinity exceeds that of CTP ≈75-fold. Site-directed mutagenesis studies indicated that Glu149 is an important binding determinant for both CTP and dF-dCTP. Comparison of the binding affinities of the 5'-triphosphates of 2'-fluoro-2'-deoxycytidine and 2'-fluoro-2'-deoxyarabinocytidine revealed that the 2'-F-arabino group contributes markedly to the strong binding of dF-dCTP. Geminal 2'-F substitution on UTP (dF-dUTP) did not result in an increase in binding affinity with CTPS. Remarkably, CTPS catalyzed the conversion of dF-dUTP into dF-dCTP, thus suggesting that dF-dCTP might be regenerated in vivo from its catabolite dF-dUTP.
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: McCluskey
authors.affiliation: Faculty of Pharmacy, The British University in Egypt, 11837, Cairo, Egypt.
authors.forename: Samy
authors.initials: S
authors.lastname: Mohamady
authors.affiliation: Department of Chemistry, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada.
authors.forename: Scott D
authors.initials: SD
authors.lastname: Taylor
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada.|Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada.
authors.forename: Stephen L
authors.initials: SL
authors.lastname: Bearne
mesh_terms: D019731:Carbon-Nitrogen Ligases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003565:Cytidine Diphosphate / Q000031:analogs & derivatives; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D015394:Molecular Structure; D013329:Structure-Activity Relationship
chemical_list: D004791:Enzyme Inhibitors; C523676:gemcitabine-5'-diphosphate; D003565:Cytidine Diphosphate; D003570:Cytidine Triphosphate; C533693:gemcitabine triphosphate; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase
keywords: antitumor agents; fluorinated nucleotides; gemcitabine; inhibitors; molecular recognition