The thymidylate synthesis cycle and anticancer drugs

Douglas, Kenneth T.

doi:10.1002/med.2610070405

Cited by 53 publications

(28 citation statements)

References 124 publications

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“…Enzyme-DNA interactions mediate processes that allow for destabilization of the DNA helix and extrahelical stabilization of the target base. This shift in DNA conformation is utilized in a variety of biological processes, including DNA methylation and DNA repair (23)(24)(25)(26), by providing access to buried functionalities on DNA bases that would be inaccessible in a B-form DNA helix. Current proposals on the mechanisms by which enzymes facilitate base flipping include enzymatic manipulations to either 5Ј-or 3Ј-phosphates (8,27), "pinchpull-push" mechanisms (28), and passive mechanisms involving stabilization of transiently flipped nucleotides in B-form DNA (29).…”

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confidence: 99%

The Coupling of Tight DNA Binding and Base Flipping

Estabrook

Lipson

Hopkins

et al. 2004

Journal of Biological Chemistry

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The molecular basis of enzymatic catalysis is of broad interest, with implications for biocatalyst design and drug development. The abundance of detailed three-dimensional structures and investigational methods provides newly addressable aspects of enzymatic function. We are interested in the importance of protein motion, and particularly correlated motions, to catalysis. The underlying premise is that protein-solvent interactions are converted into peptide motions, resulting in the transient stabilization of active site elements with preferred reactivities (1, 2).Recent studies (1) have provided highly suggestive evidence for this concept. Molecular dynamics investigations of dihydrofolate reductase demonstrate that strong coupled motions in the reactive complex disappear in the product complexes, indicating that these motions may be linked to catalysis. Mutants that alter the kinetics of particular catalytic steps are concentrated within segments of the protein structure shown to participate in highly correlated motions (1). Solid state NMR and solution NMR relaxation studies have measured substrate and protein dynamics that are matched to the turnover time of the respective enzymes (3). Studies of hydrogen and electron tunneling during enzyme catalysis provide further evidence for the importance of protein dynamics to catalytic events at the active site (4, 5).Molecular dynamic simulations of catechol O-methyltransferase and M.HhaI 1 DNA methyltransferase provided initial evidence for correlated motions within the active sites of these enzymes (6, 7). We sought to test the importance to catalysis of motions made by specific distal residues (His 127 -Thr 132 ) in facilitating active site chemistries by altering the position and orientation of critical residues such as Val 121 . Alanine scan point mutagenesis and kinetic characterization of individual steps in the catalytic cycle were used to probe the effects of such mutations and provide insights into the roles of correlated motions. M.HhaI provides an excellent structurally and functionally tractable enzyme to study various aspects of catalysis, including base flipping and the importance of motions to catalysis. M.HhaI, from Haemophilus haemolyticus, is an AdoMetdependent C 5 -cytosine methyltransferase that methylates the central cytosine (C) in the recognition sequence 5Ј-GCGC-3Ј after stabilizing the target base in an extrahelical position. Many M.HhaI crystal structures provide structural insights into the mechanisms of DNA methylation and base flipping (8). Functional analysis of the WT M.HhaI has been extensive (9 -12), including K D DNA determination for a variety of DNA substrates (13,14). Many structural components of the M.HhaI mechanism have been examined by mutagenesis including Gln 237 , which positions itself into the DNA helix and interacts with the lone guanine (15), and Cys 81 , which forms a covalent bond to the target cytosine (16). Other mutational studies have examined protein-phosphate interactions (17) and conserved residues within the...

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confidence: 99%

The Coupling of Tight DNA Binding and Base Flipping

Estabrook

Lipson

Hopkins

et al. 2004

Journal of Biological Chemistry

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“…This step represents the sole de novo source of 2'-deoxythymidine-5'-monophosphate and hence inhibition of thymidylate synthase, in the absence of salvage, leads to "thymineless death". Thus inhibition of thymidylate synthase is an attractive goal for the development of antitumor agents [3,4].…”

Section: Introductionmentioning

confidence: 99%

Synthesis of 2-amino-4-oxo-5-substitutedbenzylthiopyrrolo[2,3-d]-pyrimidines as potential inhibitors of thymidylate synthase

Gangjee

Jain

Phan

et al. 2005

Journal of Heterocyclic Chemistry

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A series of ten novel 2-amino-4-oxo-5-[(substitutedbenzyl)thio]pyrrolo [2,3-d]pyrimidines 2-11 were synthesized as potential inhibitors of thymidylate synthase and as antitumor agents. The analogues contain various electron withdrawing and electron donating substituents on the benzylsulfanyl ring of the side chains and were synthesized from the key intermediate 2-amino-4-oxo-6-methylpyrrolo[2,3-d]pyrimidine, 14. Appropriately substituted benzyl mercaptans were appended to the 5-position of 14 via an oxidative addition reaction using iodine, ethanol and water. The compounds were evaluated against human, Escherichia coli and Toxoplasma gondii thymidylate synthase and against human, Escherichia coli and Toxoplasma gondii dihydrofolate reductase. The most potent inhibitor, (6) which has a 4'-methoxy substituent on the side chain, has an IC 50 =25 µM against human thymidylate synthase. Contrary to analogues of general structure 1, electron donating or electron withdrawing substituents on the side chain of 2-11 had little or no influence on the human thymidylate synthase inhibitory activity.

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“…Proliferating cells (including cancer cells) have higher levels of TS than nonproliferating cells (Derenzini et al, 2002). TS has been an important drug target for cancer treatment for more than 50 years (Douglas, 1987;Costi, 1998). Fluorinated pyrimidines, including 5-fluorouracil (5FU) and its metabolite 5-fluorodeoxyuridine (5FUdR), inhibit TS by mimicking dUMP and irreversibly binding to the enzyme (Heidelberger et al, 1957;Berg et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Combining Small Interfering RNAs Targeting Thymidylate Synthase and Thymidine Kinase 1 or 2 Sensitizes Human Tumor Cells to 5-Fluorodeoxyuridine and Pemetrexed

Cresce¹,

Figueredo²,

Ferguson³

et al. 2011

J Pharmacol Exp Ther

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Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis and repair. Drugs targeting TS protein are a mainstay in cancer treatment, but off-target effects and toxicity limit their use. Cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2) contribute to an alternative dTMP-producing pathway, by salvaging thymidine from the tumor milieu, and may modulate resistance to TStargeting drugs. Combined down-regulation of these enzymes is an attractive strategy to enhance cancer therapy. We have shown previously that antisense-targeting TS enhanced tumor cell sensitivity to TS-targeting drugs in vitro and in vivo. Because both TS and TKs contribute to increased cellular dTMP, we hypothesized that TKs mediate resistance to the capacity of TS small interfering RNA (siRNA) to sensitize tumor cells to TS-targeting anticancer drugs. We assessed the effects of targeting TK1 or TK2 with siRNA alone and in combination with siRNA targeting TS and/or TS-protein targeting drugs on tumor cell proliferation. Down-regulation of TK with siRNA enhanced the capacity of TS siRNA to sensitize tumor cells to traditional TS protein-targeting drugs [5-fluorodeoxyuridine (5FUdR) and pemetrexed]. The sensitization was greater than that observed in response to any siRNA used alone and was specific to drugs targeting TS. Up-regulation of TK1 in response to combined 5FUdR and TS siRNA suggests that TK knockdown may be therapeutically useful in combination with these agents. TKs may be useful targets for cancer therapy when combined with molecules targeting TS mRNA and TS protein.

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The thymidylate synthesis cycle and anticancer drugs

Cited by 53 publications

References 124 publications

The Coupling of Tight DNA Binding and Base Flipping

The Coupling of Tight DNA Binding and Base Flipping

Synthesis of 2-amino-4-oxo-5-substitutedbenzylthiopyrrolo[2,3-d]-pyrimidines as potential inhibitors of thymidylate synthase

Combining Small Interfering RNAs Targeting Thymidylate Synthase and Thymidine Kinase 1 or 2 Sensitizes Human Tumor Cells to 5-Fluorodeoxyuridine and Pemetrexed

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