Regulation of deoxyctidine kinase by deoxyctidine and deoxyctidine-5′ triphosphate in whole cells

Abstract

The antiviral activity and cytotoxicity of (E)-5-(2-bromovinyl)-2′-deoxycytidine (BrVdCyd) against herpes simplex virus type 1 (HSV-1), singly and in combination with deaminase inhibitors was determined using rabbit kidney (RK-13), HEP-2, BHK-21 and VERO cells. BrVdCyd was a potent inhibitor of HSV-1 replication with ED50 values of 0.30 to 1.20 μM depending on the cell line used. In the presence of tetrahydrouridine or tetrahydrodeoxyuridine (H4dUrd), potency of BrVdCyd increased approximately two fold (ED50: 0.54 μM) in HSV-infected VERO cells. The combination of BrVdCyd and H4dUrd was also effective in decreasing virus yield. Dihydrodeoxyuridine (H2dUrd) reversed the activity of BrVdCyd (ED50: 6 to 7 μM).The effect of (E)-5-(2-bromovinyl)-2′-deoxyuridine (BrVdUrd), BrVdCyd and BrVdCyd in combination with H4dUrd on deoxyribonucleoside triphosphate (dNTP) pools was assessed in VERO cells infected with a high multiplicity of infection (10 PFU/cell). Significant differences in dNTP poll sizes (pmol/106 cell) were observed with different treatments. BrVdUrd and BrVdCyd treatment resulted in marked expansion of the dTTP pool (> 1200 pmol) compared to HSV-infected VERO cells (303 pmol). Exposure to H4dUrd resulted in a 12-fold expansion of the dCTP pool (326 pmol) and barely detectable levels of dTTP (<1.0 pmol). BrVdCyd plus H4dUrd treatment resulted in a slight expansion of the dTTP pool (515 pmol). These results indicate: (i) H4dUrd inhibits de novo dCyd/dCMP deaminase pathway and (ii) exposure to BrVdCyd plus H4dUrd puts a strain on viral DNA synthesis to such an extent that even though dTTP is being formed from alternative pathways, its eventual utilization as a substrate is reduced and hence it builds up.