Abstract
The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets.
Highlights
The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment
To identify compounds that could inhibit replication of SARS-CoV-2 in human cells, we developed a high-content imaging (HCI) 384-well format assay using HeLa cells expressing the human SARS-CoV-2 receptor, the angiotensin-converting enzyme 2, or ACE2 (HeLa-ACE2)
The assay relies on immunofluorescent detection of SARS-CoV-2 proteins with plasma purified from patients exposed to the virus, which together with host-cell nuclear staining allows for quantification of the percent infected cells in each well (Fig. 1b)
Summary
The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. We report results from ReFRAME screens in two different cell-based SARS-CoV-2 infection assays and in a remdesivir potentiation format, and the profiling of the identified hits in secondary orthogonal assays. This screening cascade and subsequent hit prioritization identified and validated our most promising hit, MK-4482, as a potent inhibitor of SARS-CoV-2, in vitro findings which translated to an in vivo hamster model of SARS-CoV-2 infection. Other hits identified in these studies have the potential for repurposing following further evaluation in advanced models or may serve as tool compounds in elucidation of coronavirus replication pathways
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