A high throughput screen identifies benzoquinoline compounds as inhibitors of Ebola virus replication

Abstract

Ebola virus (EBOV) is an enveloped negative-sense, single-stranded RNA virus of the filovirus family that causes severe disease in humans. Approved therapies for EBOV disease are lacking. EBOV RNA synthesis is carried out by a virus-encoded complex with RNA-dependent RNA polymerase activity that is required for viral propagation. This complex and its activities are therefore potential antiviral targets. To identify potential lead inhibitors of EBOV RNA synthesis, a library of small molecule compounds was screened against a previously established assay of EBOV RNA synthesis, the EBOV minigenome assay (MGA), in 384 well microplate format. The screen identified 56 hits that inhibited EBOV MGA activity by more than 70% while exhibiting less than 20% cell cytotoxicity. Inhibitory chemical scaffolds included angelicin derivatives, derivatives of the antiviral compound GSK983 and benzoquinolines. Structure-activity relationship (SAR) studies of the benzoquinoline scaffold produced ∼50 analogs and led to identification of an optimized compound, SW456, with a submicromolar IC₅₀ in the EBOV MGA and antiviral activity against infectious EBOV in cell culture. The compound was also active against a MGA for another deadly filovirus, Marburg virus. It also exhibited antiviral activity towards a negative-sense RNA virus from the rhabdovirus family, vesicular stomatitis virus, and a positive-sense RNA virus, Zika virus. Overall, these data demonstrate the potential of the EBOV MGA to identify anti-EBOV compounds and identifies the benzoquinoline series as a broad-spectrum antiviral lead.

Keywords: Antiviral; Ebola virus; Filovirus; Marburg virus; Vesicular stomatitis virus; Zika virus.

Figure 1.. A high throughput EBOV minigenome screen.

A. A workflow schematic of the EBOV MG high throughput screening assay. A representation of an optimized MG assay in 384 well format. HEK293T cells were transfected in bulk in a T75 flask. Twenty-four hours post-transfection cells were plated in a 384-well plate and allowed to rest for two hours after which the compound library was transferred via Biomek FX robotic liquid dispenser (final concentration 5 μM). Twenty-four hours following compound addition Renilla luciferase activity was measured. B. Results of a pilot assay testing the effects of 10 μM mycophenolic acid (MPA) and DMSO (1%) on the MG assay. Z’ was above 0.5 indicating the assay is suitable for HTS. No VP35, the VP35 expression plasmid was omitted from the transfection. C. The EBOV MG assay was performed with 3-fold serial dilution of SW539 starting from 50 μM in triplicate. Cell viability was determined in presence of SW539 using the CellTiter-Glo assay in parallel. Error bars represent the mean ± standard deviation of triplicates. D. A secondary assay was performed using a T7 promoter-firefly luciferase reporter plasmid and thymidine kinase promoter-Renilla luciferase reporter plasmid.

Figure 2.. Structure-activity relationship studies with the benzoquinoline series.

A. A series of analogs were generated for the benzoquinoline series and activity was determined in the EBOV MG assay. B. The MG assay activity and toxicity of SW456 was determined at 24h post compound addition. C. The effect of SW456 on the counter screen assay for which the readout is firefly and Renilla luciferase expression. D and E. The activity of SW539 (D) and SW456 (E) against the MARV MG assay.

Figure 3.. Antiviral activity of SW456 against EBOV.

The antiviral activity of SW456 was measured in Vero E6 cells in 24-well plate format. The cells were pretreated with increasing concentrations of compound for 1 h, after which they were infected with EBOV-GFP at an MOI of 2. Two days post-infection, (A) the expression of GFP in the cell monolayer was observed under a fluorescent microscope to observe the antiviral effects of the drugs and cell monolayer integrity, (B) the supernatant was collected and virus titers were determined by plaque assay (left axis, black bars). In parallel, cell toxicity of the compound was determined in uninfected Vero E6 cells using the Viral-Tox-Glo assay (right-axis, grey bars) at 48h post compound treatment. The graphs represent the mean and standard deviation of triplicates.

Figure 4.. Antiviral activity of SW456 against RNA viruses.

The antiviral activity of the compound was measured in Vero E6 cells. The cells were pretreated with 3-fold serial dilutions starting at 50 μM for 1h, and then infected with VSV-GFP at an MOI of 0.0001. (A) Virus activity was determined by quantifying GFP expression in 96 well plates at 21h post-infection. The y-axis represents percent mean fluorescence activity as determined by normalizing to the mock-treated VSV-GFP infected samples. (B) VSV-GFP viral titers were determined by plaque assay. (C) Vero E6 cells were treated with 4-fold serial dilutions of compound starting at 40 μM for 1h. The cells were infected with Zika virus at MOI 1. Zika virus protein production was quantified using antibody staining (pan-flavivirus antibody, 4G2) via a colorimetric assay at 48h post-infection by measuring absorbance at 650nm. The DMSO treated infected sample is set at 100% activity. Data represent the mean and standard deviation of triplicates.