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. 2005 Dec;49(12):4965-73.
doi: 10.1128/AAC.49.12.4965-4973.2005.

Potent antiviral activity of north-methanocarbathymidine against Kaposi's sarcoma-associated herpesvirus

Weimin Zhu  1 , Angela Burnette, Dorjbal Dorjsuren, Paula E Roberts, Mahmoud Huleihel, Robert H Shoemaker, Victor E Marquez, Riad Agbaria, Shizuko Sei
Affiliations

Potent antiviral activity of north-methanocarbathymidine against Kaposi's sarcoma-associated herpesvirus

Weimin Zhu et al. Antimicrob Agents Chemother. 2005 Dec.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) infection is a prerequisite for the development of Kaposi's sarcoma (KS). Blocking lytic KSHV replication may hinder KS tumorigenesis. Here, we report potent in vitro anti-KSHV activity of 2'-exo-methanocarbathymidine [North-methanocarbathymidine (N-MCT)], a thymidine analog with a pseudosugar ring locked in the northern conformation, which has previously been shown to block the replication of herpes simplex virus types 1 and 2. N-MCT inhibited KSHV virion production in lytically induced KSHV-infected BCBL-1 cells with a substantially lower 50% inhibitory concentration (IC50) than those of cidofovir (CDV) and ganciclovir (GCV) (IC50, mean +/- standard deviation: 0.08 +/- 0.03, 0.42 +/- 0.07, and 0.96 +/- 0.49 microM for N-MCT, CDV, and GCV, respectively). The reduction in KSHV virion production was accompanied by a corresponding decrease in KSHV DNA levels in the N-MCT-treated BCBL-1 cells, indicating that the compound blocked lytic KSHV DNA replication. A time- and dose-dependent accumulation of N-MCT-triphosphate (TP) was demonstrated in lytically induced BCBL-1 cells, while uninfected cells showed virtually no accumulation. The levels of N-MCT-TP were significantly decreased in the presence of 5'-ethynylthymidine, a potent inhibitor of herpesvirus thymidine kinase, resulting in the abrogation of anti-KSHV activity of N-MCT. N-MCT-TP more effectively blocked in vitro DNA synthesis by KSHV DNA polymerase with an IC50 of 6.24 +/- 0.08 microM (mean +/- standard deviation) compared to CDV-diphosphate (14.70 +/-2.47 microM) or GCV-TP (24.59 +/- 5.60 microM). Taken together, N-MCT is a highly potent and target-specific anti-KSHV agent which inhibits lytic KSHV DNA synthesis through its triphosphate metabolite produced in KSHV-infected cells expressing a virally encoded thymidine kinase.

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Figures

FIG. 1.
FIG. 1.
(A) Chemical structure of N-MCT. (B) The effects of N-MCT, cidofovir, and ganciclovir on KSHV DNA replication in PMA-stimulated BCBL-1 cells evaluated as KSHV virion-associated DNA copies in the culture supernatants and the amounts of KSHV DNA detected in LMW DNA. The mitochondrial DNA fragment amplified by a primer pair, MTC/F and MTC/R (see text), was included as an internal control for each sample. Shown as a reference is the level of KSHV DNA replication in unstimulated BCBL-1 (PMA −, farthest left lane). The data shown are representative of three independent experiments. (C) Cytotoxic effects of the three compounds examined in PMA-stimulated BCBL-1 cells at the concentrations tested up to 200 μM. The cell growth determined by the XTT method (58) was depicted as a % of the no-drug control (mean ± standard deviation of triplicate wells). The experiment shown is representative of three separate assays. Compounds: (▪) N-MCT; (▵) CDV; (○) GCV.
FIG. 2.
FIG. 2.
Intracellular phosphorylation profiles of N-MCT in KSHV-infected BCBL-1 cells (A) and uninfected CEM-SS cells (B) with (solid lines) and without (dotted lines) PMA stimulation. Shown are the levels of mono-, di-, and triphosphorylated N-MCT metabolites. The data shown are representative of two independent experiments. Compounds: (▪, □) N-MCT-MP; (▴, ▵) N-MCT-DP; (•, ○) N-MCT-TP in PMA-stimulated and unstimulated cells, respectively.
FIG. 3.
FIG. 3.
Intracellular phosphorylation profiles of N-MCT, CDV, and GCV in BCBL-1 cells with (+ PMA) and without PMA stimulation (no PMA). Shown are the levels of mono-, di-, and triphosphorylated metabolites of the test compounds analyzed at 24 h (top) and 72 h (bottom) postincubation. Of note, the phosphorylated metabolites of CDV were identified as CDV-monophosphate, CDV-DP (active metabolite), and a phosphate ester adduct of CDV (CDV-adduct) as previously described (28). The data shown are means ± standard deviations of two separate assays.
FIG. 4.
FIG. 4.
(A) Effects of a potent inhibitor of herpesvirus TK, 5′-ET (43), added at 10, 20, or 50 μM on the anti-KSHV activity of N-MCT (1 μM) and CDV (10 μM) in PMA-stimulated (PMA+) BCBL-1 cells. Shown are the levels of KSHV DNA evaluated by ORF65 PCR and mitochondrial DNA in the Hirt LMW DNA. (B) The effects of 5′-ET (50 μM) on anti-KSHV activity of N-MCT used at 1, 3, or 10 μM in PMA-stimulated BCBL-1. Shown are the amounts of virion-associated (supernatants) and cell-associated KSHV DNA (LMW) as determined by ORF65 PCR along with the levels of control mitochondrial DNA. (C) The levels of phosphorylated metabolites of N-MCT added at 1, 3, or 10 μM in the absence (top) or presence (bottom) of 50 μM 5′-ET in PMA-stimulated BCBL-1 cells.
FIG. 5.
FIG. 5.
Inhibitory activity of N-MCT-TP, CDV-DP, and GCV-TP on in vitro DNA synthesis, mediated by recombinant KSHV polymerase and polymerase processivity factor, depicted as % inhibition (mean ± standard deviation of triplicate wells). Shown in the inset are IC50 values (means ±standard deviations) from three independent experiments for each compound. Compounds: (▪) N-MCT-TP; (▵) CDV-DP; (•) GCV-TP.
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