Biochemical and structural characterization of (South)-methanocarbathymidine that specifically inhibits growth of herpes simplex virus type 1 thymidine kinase-transduced osteosarcoma cells.
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.
Schelling
Pierre
P
Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH), Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Claus
Michael T
MT
Johner
Regula
R
Marquez
Victor E
VE
Schulz
Georg E
GE
Scapozza
Leonardo
L
D000998:Antiviral Agents / Q000494:pharmacology; D001665:Binding Sites; D002455:Cell Division; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D015774:Ganciclovir / Q000494:pharmacology; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D012516:Osteosarcoma / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D013936:Thymidine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D013937:Thymidine Kinase / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D000998:Antiviral Agents; D013937:Thymidine Kinase; C092717:thymidine kinase 1; C469995:(north)-methanocarbathymidine; D015774:Ganciclovir; D013936:Thymidine