Morphological cell profiling of SARS-CoV-2 infection identifies drug repurposing candidates for COVID-19

Abstract

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10 to 15 y from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 US Food and Drug Administration (FDA)-approved compounds and clinical candidates, we identified 17 hits that inhibited SARS-CoV-2 infection and analyzed their antiviral activity across multiple cell lines, including lymph node carcinoma of the prostate (LNCaP) cells and a physiologically relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein found in secretory fluids including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.

Keywords: COVID-19; SARS-CoV-2; drug repurposing screening; lactoferrin.

Fig. 1.

Morphological profiling of SARS-CoV-2–infected Huh7 cells (MOI of 0.2 for 48 h). (A) Clockwise: Representative field with nuclei (cyan), neutral lipids (green), and SARS-CoV-2 N protein (magenta), N-protein image in the same area with “fire” false color LUT showing distinct morphologies of infected cells showing small/round cells with a hollow center, cells with protrusions, and large syncytia, CellMask image showing cell boundaries and syncytia formation. (B) UMAP embedding and phenotypic clustering of 3 million cells exhibiting viral stain show distinct morphologies, including small/bright cells (I), cells with protrusions (III), and syncytia (V). (C) Comparison of normalized cellular features in infected (brown) and uninfected (blue) cells showing differences in cytoplasmic organization, lipid content/distribution, and nuclear texture. All distributions were compared with the Mann–Whitney test and are statistically significant with P < 0.0001.

Fig. 2.

Screen to identify drugs with antiviral activity against SARS-CoV-2. (A) Schematic of the anti–SARS-CoV-2 drug repurposing screening. 1) Compounds are administered in qHTS to cells cultured on 384-well plates infected with SARS-CoV-2 and incubated for 48 h. Each plate contains 32 negative (infected) and 32 positive (noninfected) control wells. 2) Cells are fixed, stained, and imaged. Images are analyzed using Cell Profiler to identify nuclei, cell boundaries, neutral lipid content, and viral staining intensity. 3) Dose–response curves are fit to normalized percent infected cells per well. 4) Confirmation of antiviral activity in other cell lines, including a physiologically relevant iPSC-derived human alveolar epithelial cell (iAECs); (B) Dose–response curves of 17 compounds. Graphs represent median SEM of 10-point 1:2 dilution series of selected compounds for n = 3 biological replicates. IC₅₀ values were calculated based on normalization to the control and after fitting in GraphPad Prism.

Fig. 3.

Validation of candidate compounds in iAEC2 cells infected with SARS-CoV-2. (A) SARS-CoV-2–infected iAEC2 cells at MOI of 10, 48 h post infection (p.i.). Nuclei are in cyan, N protein in magenta, and acetylated tubulin in green. Representative image was acquired on a Yokogawa CQ1 high-content imager with a 60× lens and visualized with Fiji ImageJ. Cellular protrusions are indicated with white arrows. (B) Antiviral activity of bovine and human lactoferrin, remdesivir, and S1RA was assessed in iAEC2 cells infected with SARS-CoV-2 at MOI 10. Graphs represent median SEM of 10-point 1:2 dilution series of selected compounds for n = 3 biological replicates.

Fig. 4.

Time-of-addition study with the identified antiviral hits. (A) Experimental scheme where compounds are added 4 h prior (same treatment window as drug screening) or 1 h post infection (p.i.) with SARS-CoV-2 (MOI of 1). Huh7 cells are fixed, permeabilized, and subjected to RNAscope analysis 48 h p.i. (B) Representative image of SARS-CoV-2–infected and noninfected Huh7 cells acquired on the CX5 high-content platform at 10× and analyzed with Fiji ImageJ. Viral RNA is represented in red, and nuclei in cyan. (C) Time of drug-addition for selected antiviral hits (at 10× IC₅₀ dose) organized according to the compound class. Graphs represent the fold decrease of infection over the untreated condition. Infection was calculated on the viral RNA image after image segmentation with Cell Profiler. Graphs represent an average SEM of n = 3 biological replicates. Statistical significance determined using multiple Student’s t test with the Bonferroni–Dunn correction method, with α = 0.05. *P < 0.01.

Fig. 5.

Selective MEK inhibitors exacerbate SARS-CoV-2 infection. (A) Representative images of Huh7 cells infected with SARS-CoV-2 (MOI of 0.2) and treated with cobimetinib (250 nM), trametinib (250 nM), and binimetinib (250 nM) with nuclei in cyan and N protein in magenta. Viral infection was calculated on N-protein images after image segmentation with Cell Profiler. Bars represent n = 3 technical replicates and unpaired t tests with Welch’s correction were performed in GraphPad Prism. *P < 0.001. (B) Quantitation of percent infection with MEK inhibitors. (C) RNAscope of Huh7 infected with SARS-CoV-2 (MOI of 1) treated with cobimetinib (1,000 nM) and harvested at 24 and 48 h p.i. Graph represents average, SEM of n = 3 biological replicates. (D) Representative images of SARS-CoV-2–infected (MOI of 1) and cobimetinib (1,000 nM)-treated Huh7. Cells were harvested 48 h p.i., subjected to RNAscope to detect viral RNA (positive strand, in red) and counterstained with anti-S protein antibody (green) and Hoechst 33342 (nuclei in cyan). (E) SARS-CoV-2–infected (MOI of 1) Huh7 were treated with U-0126 (10 μM) and subjected to RNAscope 48 h p.i. Graph represents average SEM of n = 2 biological replicates, each with three technical replicates. Corresponding viability data for compound treatments is shown in SI Appendix, Fig. S3A.

Fig. 6.

Lactoferrin blocks SARS-CoV-2 at the attachment step. (A) Huh7 cells were infected with SARS-CoV-2 at MOI of 0.2 for 48 h and treated with increasing concentration of lactoferrin (6.25–6,250 nM). Cells were harvested and RNA was extracted. Viral genome copies were calculated by RT-qPCR with an absolute quantification method. (B) Huh7 were infected with SARS-CoV-2 (MOI of 1, 5, and 10; MOI of 0 indicates noninfected cells) and treated with 6,250 nM lactoferrin at 1 and 24 h p.i. Bars indicate the percentage of infected cells in different conditions. Data are an average of eight replicates. Statistical significance determined using multiple Student’s t test with the Bonferroni–Dunn method, with α = 0.05. Except for MOI of 0, all conditions (untreated vs. lactoferrin, 1 h or untreated vs. lactoferrin, 24 h) differ by P < 0.0001. (C) Percentage of SARS-CoV-2–infected Huh7 cells upon treatment with bovine apolactoferrin and hololactoferrin, native human lactoferrin, and transferrin at a concentration of 6,250 nM. (D) Binding assay. Huh7 cells were preincubated on ice with compounds: lactoferrin (1,250 and 6,250 nM) and remdesivir (10 nM) as negative controls for 1 h and then infected with SARS-CoV-2 (MOI = 10) for 1 h on ice. Cells were then washed thoroughly with PBS to remove unbound virus and viral RNA was quantified by RT-qPCR. Huh7 were cultured in NaClO₃ for 7 d, which strips heparan sulfate proteoglycans from the cell surface, and were subsequently used as a control for lactoferrin mode of action. (E) Synergy analysis of lactoferrin in combination with remdesivir. Cells were pretreated with combinations or single agents and infected with SARS-CoV-2 (MOI of 10) for 48 h. Data are shown after normalization to viral control (100%) and represent an average of n = 3 biological replicates with n = 2/3 technical replicates each. Unpaired t tests with Welch’s correction were performed in GraphPad Prism to determine significance. *P < 0.0001.

Fig. 7.

Lactoferrin indirect antiviral action is mediated by the up-regulation of cellular innate immune response. iAEC2 cells were treated with lactoferrin (6,250 nM) for 48 h, and then RNA was extracted and sequenced. (A) Volcano plot of genes down-regulated (blue) and up-regulated (red) upon treatment with lactoferrin. (B) Analysis of the top up-regulated and down-regulated pathways. (C) Heatmap of selected genes. In red are highlighted genes associated with inflammation and antiviral response.