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J. Biol. Chem., Vol. 279, Issue 31, 32832-32838, July 30, 2004

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Biochemical and Structural Characterization of (South)-Methanocarbathymidine That Specifically Inhibits Growth of Herpes Simplex Virus Type 1 Thymidine Kinase-transduced Osteosarcoma Cells^*

Pierre Schelling¶, Michael T. Claus||, Regula Johner**, Victor E. Marquez, Georg E. Schulz||, and Leonardo Scapozza

From the Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH), Winterthurerstrasse 190, CH-8057 Zurich, Switzerland, the ^||Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Albertstrasse 21, D-79104 Freiburg im Breisgau, Germany, and the Laboratory of Medicinal Chemistry, Center for Cancer Research, NCI, National Institutes of Health, Frederick, Maryland 21702-1201

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 IC₅₀ value in the range of 15 µM 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-Å 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.

Received for publication, December 5, 2003 , and in revised form, May 25, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^¶ Supported by the Stipendienfonds der Basler Chemischen Industrie. Present address: Laboratory of Developmental Immunology, Massachusetts General Hospital and Harvard Medical School, 55 Fruit St. GRJ1402, Boston, MA 02114.

^** Supported by European Community Grant QLK3-CT-2001-01265.

To whom correspondence should be addressed. Tel.: 41-1-635-6036; Fax: 41-1-635-6884; E-mail: Leonardo.Scapozza{at}pharma.ethz.ch.

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