Comparison of the substrate specificities of human thymidine kinase 1 and 2 and deoxycytidine kinase toward antiviral and cytostatic nucleoside analogs

Eriksson, Staffan; Kierdaszuk, Borys; Munch‐Petersen, Birgitte; Öberg, Bo; Johansson, Nils Gunnar

doi:10.1016/s0006-291x(05)80224-4

Cited by 214 publications

(137 citation statements)

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“…5-Halogenated 2'-deoxyuridine compounds are substrates for both TK1 and TK2. [23] To avoid the phosphorylation of these compounds to their monophosphate derivatives by cellular TKs, other modifications on the sugar moiety would be necessary. One possibility would be modification at the 5'-position: in this case, the compounds would no longer be substrates for TKs, with the drawback that they might become inhibitors of TKs.…”

Section: Discussionmentioning

confidence: 99%

Comparative Study of Purine and Pyrimidine Nucleoside Analogues Acting on the Thymidylate Kinases of Mycobacterium tuberculosis and of Humans

et al. 2003

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] Muriel Delepierre, [d] and He ¬le ¡ne Munier-Lehmann* [a] Thymidine monophosphate kinase (TMPK) from Mycobacterium tuberculosis (TMPKmt) is an attractive target for the design of specific inhibitors. This fact is the result of its key role in the thymidine pathway and of unique structural features in the active site observed by X-ray crystallography, especially in comparison to its human counterpart (TMPKh). Different 5-modified thymidine derivatives, as well as purine and pyrimidine analogues or C-nucleosides were tested on TMPKmt and TMPKh, and the results were rationalized by docking studies. 5-Halogenated 2'-deoxyuridines are the best inhibitors of TMPKmt found and present the highest selectivity indexes in favor of TMPKmt.

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Section: Discussionmentioning

confidence: 99%

Comparative Study of Purine and Pyrimidine Nucleoside Analogues Acting on the Thymidylate Kinases of Mycobacterium tuberculosis and of Humans

et al. 2003

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“…Phosphate transfer assay was performed as described by Eriksson et al [39] to determine the enzyme kinetics. Purified TK proteins were mixed with 1.6 µM [γ -32 P]ATP (6000 Ci/mmol), 50 mM Tris/HCl (pH 7.6), 0.5 mM MgCl 2 , 100 mM KCl, 0.5 mg/ml BSA and 0.2-100 µM nucleoside in a final volume of 50 µl.…”

Section: Phosphate Transfer Assay To Determine the Enzyme Kineticsmentioning

confidence: 99%

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

Chen

Chang

et al. 2004

Biochemical Journal

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Thymidine kinase (TK), encoded by EBV (Epstein-Barr virus), is an attractive target for antiviral therapy and provides a novel approach to the treatment of EBV-associated malignancies. Despite the extensive use of nucleoside analogues for the treatment of viral infections and cancer, the structure-function relationship of EBV TK has been addressed rarely. In the absence of any structural information, we sought to identify and elucidate the functional roles of amino acids in the nucleoside-binding site using site-directed mutagenesis. Through alignment with other human herpesviral TK protein sequences, we predicted that certain conserved regions comprise the nucleoside-binding site of EBV TK and, through site-directed mutagenesis, showed significant changes in activity and binding affinity for thymidine of site 3 (-DRH-) and 4 (-VFP-) mutants. For site 3, only mutants D392E (Asp 392 → Glu) and R393H retain activity, indicating that a negative charge is important for Asp 392 and a positive charge is required for Arg 393 . The increased binding affinities of these two mutants for 3 -deoxy-2 ,3 -didehydrothymidine suggest that the two residues are also important for substrate selection. Interestingly, the changed metal-ion usage pattern of D392E reveals that Asp 392 plays multiple roles in this region. His 394 cannot be compensated by other amino acids, also indicating a crucial role. In site 4, the F402Y mutant retains full activity; however, F402S retains only 60 % relative activity. Strikingly, when Phe 402 is substituted with serine residue, the original preferred pyrimidine substrates, such as 3 -azido-3 -deoxythymidine, iododeoxyuridine and β-L-5-iododioxolane uracil (L-form substrate), have decreased competitiveness with thymidine, suggesting that Phe 402 plays a crucial role in substrate specificity and that the aromatic ring is important for function.

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“…It is known, however, that cladribine is phosphorylated to its 5'-monophosphate by human deoxycytidine kinase and is a better substrate for this enzyme than the natural substrate, i.e. deoxycytidine [4].Correspondence to A. Bzowska, Department of Biophysics, University of Warsaw, Zwirki i Wigury 93, PL-02-089, Warsaw, Poland Fan: +48 22 22 02 48. Abbreviations.…”

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confidence: 99%

2‐Chloro‐2′ ‐deoxyadenosine (Cladribine) and its Analogues are Good Substrates and Potent Selective Inhibitors of Escherichia coli Purine‐Nucleoside Phosphorylase

Bzowska¹,

Kazimierczuk²

1995

European Journal of Biochemistry

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-EJB 95 1049/4 2-Chloro-2'-deoxyadenosine (CldAdo), a nucleoside that has proven useful in the treatment of several chronic lymphoid malignancies, and its analogue, 2-bromo-2'-deoxyadenosine, are both effective inhibitors of the bacterial (Escherichia coli) purine-nucleoside phosphorylase (PNP), with K, values of 4.5 pM and 6.3 pM, respectively. The examination of a series of base-modified analogues of CldAdo has shown that several other compounds have similar inhibitor properties, and has indicated that 6-benzyloxy-2-chloro-9-(2'-deoxy-~-~-ribofuranosyl)purine is the most potent inhibitor with a K, value of 0.5 pM, competitive with respect to inosine (Ino).CldAdo itself and its base-modified analogues, discounting those substituted at C(8), are also substrates for the E. coli PNP and undergo rapid glycosidic bond cleavage. CldAdo is degraded with substrate efficiency, i.e. V,,,,,IK,, similar to that observed for Ino (1 30 %I), although the individual kinetic constants, K,,, and V,,,, are both approximately an order of magnitude lower than for Ino.All compounds tested are totally inactive as substrates and inhibitors for mammalian (calf spleen) PNP and therefore constitute a new class of potent selective, although cleavable, inhibitors of bacterial phosphorylases.8-Bromo-2-chloro-2'-deoxyadenosine and 8-thio-2-chloro-2'-deoxyadenosine are the only base-modified CldAdo derivatives showing inhibitory activity against MOLT-3 (acute T-cell leukemia) and U-937 (histiocytic lymphoma) cells and, as shown in this study, are resistant to degradation by E. coli PNP. The above-mentioned results suggest that both analogues could be effective as oral cytotoxic agents that are noncleavable by enteric bacteria.Keywords: purine-nucleoside phosphorylase ; substratehnhibitor properties ; Escherichia coli; cladribine; 2-chloro-2'-deoxyadenosine.2-Chloro-2'-deoxyadenosine (CldAdo, cladribine, leustatin) is an adenosine-deaminase-resistant nucleoside that has been used for treatment of hairy cell leukemia and other hematological malignancies [I, 21. The therapeutical potential of cladribine has recently been extended to autoimmunoaggressive diseases such as multiple sclerosis 131.The metabolic pathways of this drug have not been clarified up to now. It is known, however, that cladribine is phosphorylated to its 5'-monophosphate by human deoxycytidine kinase and is a better substrate for this enzyme than the natural substrate, i.e. deoxycytidine [4].Correspondence to A. Bzowska, Department of Biophysics, University of Warsaw, Zwirki i Wigury 93, PL-02-089, Warsaw, Poland Fan: +48 22 22 02 48. Abbreviations. PNP, purine-nucleoside phosphorylase; CldAdo (cladribine, leustatin), 2-chloro-2'-deoxyadenosine; BrdAdo, 2-bromo-2'-deoxyadenosine; Nh-methyl-CldAdo, 2-chloro-9-(2'-deoxy-~-~-ribofuranosyl)-6-methylaminopurine (with similar connotations for other Nh-substituted analogues: Wbenzyl-, N"-hydroxyethyl-, W-cyclohexyl-); 8-bromo-CldAdo, 8-bromo-2-chloro-2'-deoxyadenosine (with similar connotations for other 8-substituted analogues : 8-...

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Comparison of the substrate specificities of human thymidine kinase 1 and 2 and deoxycytidine kinase toward antiviral and cytostatic nucleoside analogs

Cited by 214 publications

References 15 publications

Comparative Study of Purine and Pyrimidine Nucleoside Analogues Acting on the Thymidylate Kinases of Mycobacterium tuberculosis and of Humans

Comparative Study of Purine and Pyrimidine Nucleoside Analogues Acting on the Thymidylate Kinases of Mycobacterium tuberculosis and of Humans

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

2‐Chloro‐2′ ‐deoxyadenosine (Cladribine) and its Analogues are Good Substrates and Potent Selective Inhibitors of Escherichia coli Purine‐Nucleoside Phosphorylase

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