Design of EBVTK substrates to eradicate EBV+ tumors

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DESCRIPTION (provided by applicant): The unique properties of aherpesvirus replicative enzymes have permitted development of antiviral drugs that are converted to cytotoxic compounds exclusively in virus-infected cells. Certain of these proteins form the basis for cytotoxic gene therapy (HSV-1TK and ganciclovir). Although the gherpesvirus EBV encodes similar genes, expression is silent during immortalizing/tumorigenic infection. However, EBV-transformed B-cells can be induced to express replicative proteins by various chemical/biological agents, suggesting TK induction in vivo is feasible and that drugs destroying EBVinfected tumors can be developed. A protocol to treat EBV-associated malignancies with Argbutyrate to induce EBV-TK and ganciclovir to effect TK-mediated killing was therefore developed. However, experiments to validate the protocol surprisingly revealed that ganciclovir was a poor substrate for EBVTK based on competition, phosphorylation and differential cytoxicity assays. HSV-1 TK is a polynucleoside kinase, whereas EBV TK phosphorylates thymidine. Our Phase-I goal is to synthesize and characterize thymidine (5-substituted uracil) analogs that preliminary studies suggest will effectively kill cells in an EBV-TK-dependent manner. Synthetic schemes are described. Nucleosides will be systematicalily analyzed for (1) recognition by EBV-TK but not cellular TK- 1, TK-2 or nucleoside kinases (2) effective incorporation into cellular DNA by host polymerases, producing irreversible inhibition of replication in EBV-TK+ cells. Successful candidates will enter Phase II. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE