R430: A potent inhibitor of DNA and RNA viruses

Abstract

Acyclovir (ACV) is an effective antiviral agent for treating lytic Herpes Simplex virus, type 1 (HSV-1) infections, and it has dramatically reduced the mortality rate of herpes simplex encephalitis. However, HSV-1 resistance to ACV and its derivatives is being increasingly documented, particularly among immunocompromised individuals. The burgeoning drug resistance compels the search for a new generation of more efficacious anti-herpetic drugs. We have previously shown that trans-dihydrolycoricidine (R430), a lycorane-type alkaloid derivative, effectively inhibits HSV-1 infections in cultured cells. We now report that R430 also inhibits ACV-resistant HSV-1 strains, accompanied by global inhibition of viral gene transcription and enrichment of H3K27me3 methylation on viral gene promoters. Furthermore, we demonstrate that R430 prevents HSV-1 reactivation from latency in an ex vivo rodent model. Finally, among a panel of DNA viruses and RNA viruses, R430 inhibited Zika virus with high therapeutic index. Its therapeutic index is comparable to standard antiviral drugs, though it has greater toxicity in non-neuronal cells than in neuronal cells. Synthesis of additional derivatives could enable more efficacious antivirals and the identification of active pharmacophores.

Figure 1

R430 is effective against acyclovir-resistant strains of HSV-1. Vero cells were infected with ACV-resistant HSV-1 strains Tk- and PAAv, or HSV-1 KOS strain, and incubated with acyclovir or R430 at concentrations varying from 0.1–50 µM. At 48 hours post-infection cells were fixed and stained for ICP4 protein, counterstained with Hoechst 33342, and imaged with Nikon AS1 microscope. Cells were counted and IC50 was determined using the drc package for R, based on the proportion of positive-staining cells. CC50 for R430 was determined using drc package based on total number of remaining cells. No CC50 is reported for ACV due to negligible toxicity.

Figure 2

Effects of R430 on HSV-1 transcript levels. hiPSC-neurons were incubated with HSV-1 (MOI 0.3) and R430 (10 µM) or vehicle in triplicate. Total RNA was extracted, quantified and sequenced following ribosome depletion. Sequences were trimmed and mapped to GRCH38 using sequence and annotation provided by Ensembl (release 82). The remaining unmapped reads were collected and mapped to the Human herpesvirus 1 strain KOS genome (GenBank: JQ780693.1).

Figure 3

Enrichment of repressive mark H3K27me3 at HSV-1 genes in infected hiPSC-neurons treated with R430 (10 µM) or ACV (50 µM) for 24 hours. ChIPs using anti-histone H#K27me3 antibody were subjected to real-time PCR using primers specific for the HSV-1 target genes indicated, and the results were graphed as % Input. The data represent an average of three independent experiments. Error bars represent standard deviations.

Figure 4

Potency and toxicity of R430 and conventional antivirals on selected RNA and DNA viruses. A panel of antiviral drugs were incubated with cell cultures and R430, conventional antivirals or vehicle as described, in triplicate. The 50% inhibitory concentration (EC50) values are shown (upper panel). Separately, the 50% cytotoxic concentrations (CC50) were estimated following incubation of cell lines with drugs or vehicle in the absence of virus. ZIKA: Zika virus; HCV: Hepatitis C virus, HSV-2: Herpes Simplex virus, type 2; HCMV: Human cytomegalovirus; MCMV: murine cytomegalovirus; HBV: Hepatitis B virus.

Figure 5

R430 suppresses EGFP expression in a transient plasmid transfection assay. Vero cells were transfected with pEGFP-N1. Two hours after the transfection cells were treated with R430 (10 µM) or ACV (50 µM). The corresponding EGFP expression under different conditions is depicted (a). The percentage of EGFP-positive cells was measured 24 hours after the transfection by flow cytometry (b).