High-throughput drug screening using the Ebola virus transcription- and replication-competent virus-like particle system

Abstract

The massive epidemic of Ebola virus disease (EVD) in West Africa, followed in recent months by two outbreaks in the Democratic Republic of the Congo, underline the importance of this severe disease. Because Ebola virus (EBOV) must be manipulated under biosafety level 4 (BSL4) containment, the discovery and development of virus-specific therapies have been hampered. Recently, a transient transfection-based transcription- and replication competent virus-like particle (trVLP) system was described, enabling modeling of the entire EBOV life cycle under BSL2 conditions. Using this system, we optimized the condition for bulk co-transfection of multiple plasmids, developed a luciferase reporter-based assay in 384-well microtiter plates, and performed a high-throughput screening (HTS) campaign of an 8,354-compound collection consisting of U.S. Food & Drug Administration (FDA) -approved drugs, bioactives, kinase inhibitors, and natural products in duplicates. The HTS achieved a good signal-to-background ratio with a low percent coefficient of variation resulting in Z' = 0.7, and data points were reproducible with R = 0.89, indicative of a robust assay. After applying stringent hit selection criteria of ≥70% EBOV trVLP inhibition and ≥70% cell viability, 381 hits were selected targeting early, entry, and replication steps and 49 hits targeting late, maturation, and secretion steps in the viral life cycle. Of the total 430 hits, 220 were confirmed by dose-response analysis in the primary HTS assay. They were subsequently triaged by time-of-addition assays, then clustered and ranked according to their chemical structures, biological functions, therapeutic index, and maximum inhibition. Several novel drugs have been identified to very efficiently inhibit EBOV. Interestingly, most showed pharmacological activity in treatments for central nervous system-related diseases. We developed and screened an HTS assay using the novel EBOV trVLP system. Newly identified inhibitors are useful tools to study the poorly understood EBOV life cycle. In addition, they also provide opportunities to either repurpose FDA-approved drugs or develop novel viral interventions to combat EVD.

Antiviral activity of sertindole, raloxifene and ibutamoren against transcription and replication-competent Ebola virus-like particles

“…The majority of these compounds have been used to treat psychotic disorders or allergic diseases by binding to G protein-coupled receptors (GPCRs). This finding was partially consistent with previous reports of GPCR modulators as anti-EBOV compounds (19)(20)(21). Particularly, sertindole (EC50, 0.7 M; SI, ＞ 45.8), raloxifene hydrochloride (raloxifene; EC50, 0.5 M; SI, 47.1) and ibutamoren mesylate (ibutamoren; EC50, ＜ 0.4 M; SI, ＞ 81.1) showed the most prominent antiviral activity among the 15 major hit compounds.…”

“…Based on their target analysis, we found that GPCRs were closely linked to the EBOV life cycle. Using the same trVLP system, N. Lee et al found GPCR modulators as antiviral hits, suggesting experimental reliability (19). However, in contrast to the previous study, from a technical perspective, we adopted the dual luciferase assay to measure the antiviral activity and cytotoxicity simultaneously from the same sample, while the cytotoxicity was determined in an image-based manner via Hoechst staining.…”

Ebolavirus Entry: From Molecular Characterization to Drug Discovery

“…Recently, Lee and coworkers reported that the new antimalarial drug ferroquine inhibits EBOV entry, by affecting the pH dependent viral fusion step [77].…”

“…Iminodyn 17 is an inhibitor of the GTPase activity of dynamins, a class of proteins involved in the scission of newly formed membrane vesicles, which can block EBOV entry. Nobiletin and ML9 were reported to affect the trafficking of viral particles by targeting the PI3K-Akt pathway and the miosyn light chain kinase activity, respectively [77].…”

“…The use of cysteine cathepsin inhibitors as anti-EBOV agents have also been proposed [108]. Accordingly, a recent study showed that the inhibitors of cathepsin-L, N-acetyl-L-leucyl-L-leucyl-L methional and calpeptin, block infection of a pseudotyped virus [77].…”