Quindoline-derivatives display potent G-quadruplex-mediated antiviral activity against herpes simplex virus 1

Abstract

G-quadruplexes (G4s) are non-canonical nucleic acid structures that regulate key biological processes, from transcription to genome replication both in humans and viruses. The herpes simplex virus-1 (HSV-1) genome is prone to form G4s that, along with proteins, regulate its viral cycle. General G4 ligands have been shown to hamper the viral cycle, pointing to viral G4s as original antiviral targets. Because cellular G4s are also normally present in infected cells, the quest for improved anti-HSV-1 G4 ligands is still open. Here, we evaluated a series of new quindoline-derivatives which showed high binding to and stabilization of the viral G4s. They displayed nanomolar-range anti-HSV-1 activity paralleled by negligible cytotoxicity in human cells, thus proving remarkable selectivity. The best-in-class compound inhibited the viral life cycle at the early times post infection up to the step of viral genome replication. In infected human cells, it reduced expression of ICP4, the main viral transcription factor, by stabilizing the G4s embedded in ICP4 promoter. Quindoline-derivatives thus emerge as a new class of G4 ligands with potent dual anti HSV-1 activity.

Keywords: Antiviral activity; G-quadruplex; HSV-1; ICP4; Quindoline derivatives.

Graphical abstract

Scheme 1

Synthesis of quindoline analogs.

Fig. 1

Antiviral activity of Quindoline and derivatives in HSV-1-infected U-2 OS cells. Infected cells were treated with increasing concentrations of each compound and newly produced infective viral particles were quantified by PRA. Antiviral activity is expressed as plaque forming units per milliliter (PFU/ml), calculated over the viral titre of vehicle treated cells. Cell viability was measured by MTT assay on uninfected cells treated with the same compound concentration used in the antiviral assay. Viral titre reduction (bars) and cell viability (line) at each tested concentration are reported. Mean values of two independent experiments, with three replicates per condition, and SD are reported.

Fig. 2

Thermal unfolding on the HSV-1 G4 sequences folded in 2.5 mM KCl in the absence and presence of GSA-0932. (A) un3 G4 alone and (B) with GSA-0932; (C) un2 G4 alone and (D) with GSA-0932; (E) gp054a G4 alone and (F) with GSA-0932. Spectra were recorded over a temperature range of 20–90 °C. Oligonucleotide folding was tested in two independent assays, one replicate per condition. Representative spectra per each oligonucleotide are shown. Arrows indicate the direction of changes in molar ellipticity.

Fig. 3

Taqpolymerase stop assay on HSV-1 G4s. (A) Un2, gp054a, un3 templates were amplified by Taq polymerase in the absence (lanes 5,9,13) and presence of K⁺, combined with increasing amounts (2–8 μM) of GSA-0932 (lanes 7–8, 11–12, 15–16) or the same amount of DMSO as that in the ligand (lanes 6,10,14). Un3 was analyzed at 50 mM of K⁺, while the other sequences were investigated at 0.5 mM of K⁺. A template (non-G4 ctrl) unable to fold into G4 was also used as control (lanes 1–4). P stands for unreacted labeled primer, FL stands for full-length product. G4-specific Taq polymerase stop sites are highlighted by vertical bars. (B) Full-length and G4 stop bands intensity quantification relative to the Taq polymerase stop assay shown in panel (A).

Fig. 4

GSA-0932 hinders viral genome replication and strongly affects ICP4 expression levels in infected cells. (A) Time-of-addition of GSA-0932 in U-2 OS cells infected with HSV-1 (MOI 1). GSA-0932 (12.4 μM, 75-fold its IC₅₀) was added at different times (0, 2, 4, 6, 8, 10 h) post infection (Red line). Acyclovir was used as a reference drug (45 μM, 75-fold its IC₅₀, green line). Vehicle-treated cells were used as control (black line,CTR). (B) U-2 OS cells were infected with HSV-1 (MOI 1) and treated with increasing concentrations of GSA-0932 or ACV (0–10 μM). Total RNA was extracted from infected cells at 6 hpi and mRNA expression levels of ICP4 were measured. ACV was used as the reference antiviral drug for HSV-1. (C) Western Blot analysis of ICP4 expression upon GSA-0932 treatment of HSV-1 infected U-2 OS cells (6 hpi, 0–10 μM). The alpha-tubulin protein was used as housekeeping control gene (D) Western Blot quantification of ICP4 levels normalized to the housekeeping protein. Data are mean ± s.e.m. of n = 2 biological replicates. Each condition was tested in duplicate per replicate. S.e.m. stands for standard error of the mean.

Fig. 5

Colocalization between GSA-0932 and the recombinant VP16-GFP virus. U-2 OS cells were seeded on coverslips, infected with the recombinant HSV-1, and treated for 2 h with the tested compound. The 8 hpi time point is shown. (A) GSA-0932 (6 μM) signal in blue; (B) VP16-GFP HSV-1 signal in green; (C) merge of the two signals. Arrows indicate colocalization areas. White lines are scale bars (50 μm).