GSK983: a novel compound with broad-spectrum antiviral activity

Abstract

GSK983, a novel tetrahydrocarbazole, inhibits the replication of a variety of unrelated viruses in vitro with EC(50) values of 5-20 nM. Both replication of the adenovirus Ad-5 and the polyoma virus SV-40, and episomal maintenance of human papillomaviruses (HPV) and Epstein-Barr virus (EBV) are susceptible to GSK983. The compound does not inhibit all viruses; herpes simplex virus (HSV-1), human immunodeficiency virus (HIV), and lytic replication of EBV were not susceptible at concentrations below 1 microM. GSK983 does inhibit the growth of cell lines immortalized by HTLV-1, EBV, HPV, SV40 and Ad-5, with EC(50) values in the range of 10-40 nM. Depending on the cell line, the compound induces either apoptosis or cytostasis at concentrations over 20 nM. GSK983 also inhibits cell lines immortalized by non-viral mechanisms, but has little effect on primary cells. The CC(50) values for keratinocytes, fibroblasts, lymphocytes, endothelial, and bone marrow progenitor cells are all above 10 microM. The pattern of inhibition, which includes diverse viruses as well as growth of immortalized cells of varied origins, suggests the target is a host cell protein, rather than a viral protein. Preliminary mechanism studies indicate that GSK983 acts by inducing a subset of interferon-stimulated genes.

Fig. 1

Structure of GSK983.

Fig. 2

Inhibition of adenovirus-5 replication and growth of HFF cells by GSK983. Cells were infected at an MOI of 3, for 2 h. Virus was removed and fresh medium containing the indicated concentrations of compound was added to each well. Adenovirus-5 DNA measurements were made by qPCR from cells 72 h after addition of compound. Measurement of cell growth in presence of compound was determined in parallel by MTS. (♦) Adenovirus-5 DNA; (▵) uninfected HFF cells by MTS.

Fig. 3

Inhibition of SV40 virus replication and growth of Vero cells by GSK983. Cells were infected at an MOI of 0.1, for 2 h. Virus was removed and fresh medium containing the indicated concentrations of compound was added to each well. SV40 DNA measurements were made by qPCR from cells 72 h after addition of compound. Measurement of cell growth in presence of compound was determined in parallel by MTS. (□) SV40 DNA; (♢) uninfected Vero cells, MTS; (♦) ratio, SV40 DNA/Vero MTS, plotted as % of ratio in the untreated control.

Fig. 4

Effects of GSK983 on growth of cell lines immortalized by episomal EBV or integrated HTLV-1 and on the growth of corresponding uninfected primary cells. Cells were plated in 96-well plates with medium containing the indicated concentrations of compound. Measurement of cell growth was determined by CTG after 72 h of incubation with the compound. Cell line labels are followed by the viruses they contain in parentheses. (♢) IM9 (EBV); (♦) MT-4 (HTLV-1); (□) activated primary B cells; (△) activated primary CD4 T cells; (■) activated PBMCs.

Fig. 5

Effects of GSK983 on HPV DNA in W12 cells and cell growth in W12 and human keratinocyte cells (HKC). Cells were plated in 96-well plates with medium containing the indicated concentrations of compound for 4 days. Measurement of W12 cell growth was determined by MTS followed by HPV16 DNA determination from the same cells by hybrid capture. Measurement of HKC cell growth in presence of compound was determined in parallel by MTS. (♢) W12 HPV DNA; (□) growth of W12 by MTS; (♦) W12, ratio HPV DNA/MTS, plotted as % of ratio in the untreated control; (△) growth of HKC by MTS.

Fig. 6

Inhibition by GSK983 of growth of cell lines bearing integrated adenovirus, SV40, EBV and HPV genomes. Cells were plated in 96-well plates with medium containing the indicated concentrations of compound. Measurement of cell growth was determined by CTG or MTS after 72 h of incubation with the compound. Cell line labels are followed by the viruses they contain in parentheses. (♦) HEK293 (Ad5); (♢) WI38 VA13 (SV40); (△) Namalwa (EBV); (▴) W12-20861 (HPV16); (□) CaSki (HPV16); (○) HeLa (HPV18).

Fig. 7

Time course of growth of (A) W12, (B) U937 and (C) IM9 cells in the presence of GSK983. The indicated concentrations of GSK983 were added to cells growing in 96-well plates at time zero. Rate of MTS reduction, to determine cell growth, was determined for 3 days starting at day zero. Growth of cells in untreated controls is indicated by solid squares in each case (GSK983, 0 nM).

Fig. 8

Apoptosis induced in cell lines after exposure to GSK983 for 20 h (IM9 and MT4 cells) or 40 h (W12 and U937). Cells were treated with GSK983 at the concentrations indicated for the time specified above. Apoptosis was estimated by measuring histone-associated DNA fragments in the cytoplasm by ELISA. Ctrl, no compound added; anti-Fas, positive control.

Fig. 9

Induction of luciferase under control of the IFIT1 promoter by GSK983 and α-interferon in HEK293 cells. HEK293-IFIT1 cells were grown in 96-well plates in the presence of GSK983 or α-IFN. Luciferase activity was measured at the time points indicated. (△) GSK983 10 nM; (▴) GSK983 30 nM; (□) α-IFN 10 units; (■) α-IFN 30 units; (♢) no addition.