Biochemical and structural characterization of (South)-methanocarbathymidine that specifically inhibits growth of herpes simplex virus type 1 thymidine kinase-transduced osteosarcoma cells

J Biol Chem. 2004 Jul 30;279(31):32832-8. doi: 10.1074/jbc.M313343200. Epub 2004 May 25.


Two analogs of the natural nucleoside dT featuring a pseudosugar with fixed conformation in place of the deoxyribosyl residue (carbathymidine analogs) were biochemically and structurally characterized for their acceptance by both human cytosolic thymidine kinase isoenzyme 1 (hTK1) and herpes simplex virus type 1 thymidine kinase (HSV1 TK) and subsequently tested in cell proliferation assays. 3'-exo-Methanocarbathymidine ((South)-methanocarbathymidine (S)-MCT), which is a substrate for HSV1 TK, specifically inhibited growth of HSV1 TK-transduced human osteosarcoma cells with an IC(50) value in the range of 15 microM without significant toxicity toward both hTK1-negative (TK(-)) and non-transduced cells. 2'-exo-Methanocarbathymidine ((North)-methanocarbathymidine (N)-MCT), which is a weak substrate for hTK1 and a substantial one for HSV1 TK, induced a specific growth inhibition in HSV1 TK-transfected cells comparable to that of (S)-MCT and ganciclovir. A growth inhibition activity was also observed with (N)-MCT and ganciclovir in non-transduced cells in a cell line-dependent manner, whereas TK(-) cells were not affected. The presented 1.95-A crystal structure of the complex (S)-MCT.HSV1 TK explains both the more favorable binding affinity and catalytic turnover of (S)-MCT for HSV1 TK over the North analog. Additionally the plasticity of the active site of the enzyme is addressed by comparing the binding of (North)- and (South)-carbathymidine analogs. The presented study of these two potent candidate prodrugs for HSV1 TK gene-directed enzyme prodrug therapy suggests that (S)-MCT may be even safer to use than its North counterpart (N)-MCT.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antiviral Agents / pharmacology
  • Binding Sites
  • Cell Division
  • Cell Line, Tumor
  • Crystallography, X-Ray
  • Dose-Response Relationship, Drug
  • Ganciclovir / pharmacology
  • Herpesvirus 1, Human / enzymology*
  • Humans
  • Inhibitory Concentration 50
  • Kinetics
  • Models, Chemical
  • Models, Molecular
  • Osteosarcoma / metabolism*
  • Phosphorylation
  • Protein Binding
  • Protein Conformation
  • Thymidine / analogs & derivatives*
  • Thymidine / chemistry*
  • Thymidine / metabolism
  • Thymidine Kinase / metabolism*
  • Time Factors
  • Transfection


  • Antiviral Agents
  • Thymidine Kinase
  • thymidine kinase 1
  • (north)-methanocarbathymidine
  • Ganciclovir
  • Thymidine