Auranofin Inhibits Ebola Virus Replication by Targeting NP-NP and NP-VP35 Interactions

Abstract

Ebola virus (EBOV) is a zoonotic pathogen that causes hemorrhagic fever with a high mortality rate in humans and non-human primates. The continuous outbreaks of filoviruses in Sub Saharan Africa highlight the urgency for development of effective antiviral. Current approved therapeutics to treat EBOV infection are Inmazed (REGN-EB3) and Ebanga monoclonal antibodies that target the viral glycoprotein (GP). Nevertheless, non-antiviral drugs are yet to be approved for targeting EBOV transcription and replication which are essential steps of the viral cycle. EBOV transcription requires viral L, NP, VP30 and VP35 proteins. Phosphorylation of VP35 on Thr-210 regulates EBOV transcription activity and is required for VP35 interaction with NP. The host protein phosphatase-1 (PP1) facilitates EBOV transcription and replication processes by switching the polymerase complex from transcription to replication activity upon the phosphorylation status of VP30. To that end, this thesis focused on (i) identifying small molecular inhibitors of EBOV replication, and (ii) elucidating their mechanisms of action for potential therapeutic. A recent high throughout screen conducted in Mathieu Bollen’s laboratory at KU Leuven identified several gold-containing compounds that affected the binding of PP1 to sds22 regulatory subunit. These compounds included auranofin, an FDA approved drug for rheumatoid arthritis. Auranofin and a gold-containing chloro(triethylphosphosphine) gold (CTPgold) compounds were further tested for inhibition of EBOV minigenome (transcription and replication) in HEK293T and Vero E-6 cells. Auranofin inhibited EBOV transcription with IC50 = 0.3 μM and also potently inhibited EBOV replication at sub-micromolar concentrations IC50< 0.3 μM. In contrast, CTP-gold inhibited EBOV transcription with IC50 > 100 μM. Analysis of cellular toxicity showed that auranofin was less toxic (IC50= 91 μM). Comparing it to CTP-gold (IC50= 91 μM) in MTT assay. We further analyzed the effect of auranofin on the expression level v of the host’s cellular genes in HEK293T cells using quantitative mass spectrometry. Auranofin treatment increased expression level of sds22 and decreased levels of PP1 regulatory subunits α, β/ δ and γ. It also induced expression level of proteins involved in the regulation of cell proliferation, survival, and differentiation and predicted to impact negatively viral infection. Next, we tested the effect of NP E619K on NP: VP35 interaction in the presence of PP1 using co-immunoprecipitation assay. Strong binding of VP35 to NP WT was observed than VP35 binding to NP E619K. In contrast, PP1α bound to NP E619K more efficiently compared to NP WT. We further tested the effect of auranofin and chloro-gold on NP: VP35 and NP: NP interactions using Split NanoBiT and co-immunoprecipitation assays to delineate their mechanism of action. In Split NanoBiT assay, auranofin potently inhibited NP: VP35 and NP: NP interactions with IC50 < 0.3 μM. Whereas the co-immunoprecipitation result showed increasing in NP: VP35 binding with higher concentrations of auranofin treatment. Overall, the findings presented here indicated that PP1-targeting is a viable approach for EBOV inhibition, although the mechanisms appear to differ based on Co-IP and crosslinker assays.