Crystal structures of the thymidine kinase from herpes simplex virus type-I in complex with deoxythymidine and Ganciclovir

Brown, David G.; Visse, Robert; Sandhu, G.; Davies, Anna M.; Rizkallah, P.J.; Melitz, C.; Summers, William C.; Sanderson, Mark

doi:10.1038/nsb1095-876

Cited by 133 publications

(144 citation statements)

References 29 publications

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“…While the mutagenesis and selection studies were carried out before the solution of the crystal structure, the involvement of the region targeted for mutagenesis in binding substrate (thymidine and GCV) is supported by the determined structure. 20 We have used molecular modeling to help visualize the amino acid substitutions found in mutant 30 ( Figures 6 and 7). Four of the six substitutions appear not to play a major role in substrate binding or structure stability.…”

Section: Discussionmentioning

confidence: 99%

“…37 Coordinates for the TK structure with thymidine bound were obtained from the Protein Data Bank file 1KIN. 20 Coordinates with GCV bound to TK were generously provided by Mark Sanderson et al (King's College, London, UK). The six residues in each structure were replaced with mutated side chains such that each was roughly oriented in the same direction as in the unmutated structure.…”

Section: Molecular Modelingmentioning

confidence: 99%

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Enhancement of tumor ablation by a selected HSV-1 thymidine kinase mutant

Kokoris¹,

Sabo²,

Adman

et al. 1999

Gene Ther

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With the advent of gene therapy, herpes simplex virus type fold lower than one that impedes growth of wild-type TK-I (HSV-1) thymidine kinase (TK) has garnered much interexpressing tumors. Furthermore, in the presence of GCV est as a suicide gene for cancer ablation. As a means to a substantial bystander effect is observable when only 20% improve the overall efficacy of the prodrug-gene activation of the tumor cells express mutant 30 whereas no restriction approach, as well as to reduce ganciclovir-mediated toxin tumor growth is seen in tumors bearing the wild-type TK icity, a large library of mutant thymidine kinases was generunder the same conditions. The enhanced sensitization to ated and screened for the ability to enhance in vitro cell prodrugs conferred by mutant 30 is apparently due to a sensitivity to the prodrugs, ganciclovir (GCV) and acyclovir 35-fold increase in thymidine K m which results in reduced (ACV). Enzyme kinetics of one thymidine kinase mutant competition between prodrug and thymidine at the active from this library that contains six amino acid substitutions site. This provides mutant 30 a substantial kinetic advanat or near the active site reveals a distinct mechanism for tage despite very high K m s for both ganciclovir and acycloproviding enhanced prodrug-mediated killing in mamvir. Molecular modeling of the mutations within the active malian cells. In in vitro rat C6 cell prodrug sensitivity assays site suggests that a tyrosine substitution at alanine 168 the TK mutant (mutant 30) achieves nanomolar IC 50 values (A168) alters thymidine and prodrug interactions by causwith GCV and ACV, in contrast to IC 50 values of 30 M ing catalytically important residues to move. The use of and Ͼ100 M, respectively, for wild-type TK. In a mouse mutant 30 in place of the wild-type TK should provide a xenograft tumor model, growth of mutant 30 expressing more effective gene therapy of cancer. tumors is restricted by ganciclovir at a dose at least 10-

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Section: Discussionmentioning

confidence: 99%

Section: Molecular Modelingmentioning

confidence: 99%

Enhancement of tumor ablation by a selected HSV-1 thymidine kinase mutant

Kokoris¹,

Sabo²,

Adman

et al. 1999

Gene Ther

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“…As shown in Figure 1, there are five conserved regions in these TK protein sequences. Among them, sites 3 and 4 are highly conserved and have been suggested to be involved in nucleoside binding through studies of crystal structure and mutation analysis of HSV-1 TK [32][33][34][35][36]. Site 3, consisting of the motif -DRH-, comprises three hydrophilic residues.…”

Section: Alignment Of Five Human Herpesvirus Tksmentioning

confidence: 99%

“…Sites 1, 3 and 4 are supposed to be involved in substrate binding. HSV-1 TK has three substrate-binding sites, site 1 for ATP and sites 3 and 4 for thymidine binding, which have been confirmed by mutagenetic methods and structural analysis [31][32][33][34][35][36]. Thymidinebinding sites are also the regions that are responsible for binding to various nucleoside analogues.…”

Section: Introductionmentioning

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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“…After conversion of these analogues into the corresponding monophosphates, they are further phosphorylated to nucleoside diphosphates, by TK in the case of the thymine derivatives or by cellular kinases in the other cases. The crystal structure of HSV-1 TK [4] reveals the presence of a fold common with the nucleoside monophosphate kinase (NMPK) family, made up of a five-stranded parallel b sheet and other additional structural elements. This fold, part of the protein core, contains the active site.…”

Section: Introductionmentioning

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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Crystal structures of the thymidine kinase from herpes simplex virus type-I in complex with deoxythymidine and Ganciclovir

Cited by 133 publications

References 29 publications

Enhancement of tumor ablation by a selected HSV-1 thymidine kinase mutant

Enhancement of tumor ablation by a selected HSV-1 thymidine kinase mutant

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

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

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