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 10-15 years 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 FDA-approved compounds and clinical candidates, we identified 17 dose-responsive compounds with in vitro antiviral efficacy in human liver Huh7 cells and confirmed antiviral efficacy in human colon carcinoma Caco-2, human prostate adenocarcinoma LNCaP, and in a physiologic 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 classically 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.

Figure 1.

Morphological profiling of SARS-CoV-2 infected Huh7 cells (MOI of 0.2 for 48 hrs). 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 showing distinct morphologies present, 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.

Figure 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-hours. Each plate contains 32 negative (infected) and 32 positive (non-infected) 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 physiological 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.

Figure 3.

A) SARS-CoV-2 infected iAEC2 cells at MOI of 10, 48 hrs 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 60X lens and visualized with Fiji ImageJ. 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. Representative Images of nuclei (cyan), acetylated tubulin (green), and N protein (magenta) at compound IC₅₀ dose are also represented.

Figure 4.

Time of drug-addition of the identified antiviral hits. A) Experimental scheme where compounds are added 4 hrs prior (same treatment window as drug screening) or 1 hr postinfection (p.i.) with SARS-CoV-2 (MOI of 1). Huh7 cells are fixed, permeabilized and subjected to RNAscope analysis 48 hrs p.i. B) Representative image of SARS-CoV-2 infected and non-infected Huh7 cells acquired on the CX5 high-content platform at 10X and analyzed with Fiji ImageJ. Viral RNA is represented in red, nuclei in cyan. C) Time of drug-addition for selected antiviral hits (at 10x 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 alpha = 0.05. *p<0.01.

Figure 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) RNAscope of Huh7 infected with SARS-CoV-2 (MOI of 1) treated with cobimetinib (1000 nM) and harvested at 24 hrs and 48 hrs p.i. Graph represents average, SEM of N=3 biological replicates. D) Representative images of SARS-CoV-2-infected (MOI of 1) and cobimetinib (1000 nM)-treated Huh7. Cells were harvested 48 hrs 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 hrs p.i. Graph represents average SEM of N=2 biological replicates, each with three technical replicates.

Figure 6.

Lactoferrin blocks SARS-CoV-2 replication at different stages of the viral cycle. A) Huh7 cells were infected with SARS-CoV-2 at MOI of 0.2 for 48 hrs and treated with increasing concentration of lactoferrin (5.75 nM – 5750 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 non-infected cells) and treated with 2.3 μM of lactoferrin at 1 and 24 hrs p.i. Bars indicate the percentage of infected cells in different conditions. Data is an average of eight replicates. Statistical significance determined using multiple student’s t-test with the Bonferroni-Dunn method, with alpha = 0.05. Except for MOI of 0, all conditions (Untreated vs Lactoferrin, 1 hr or Untreated vs Lactoferrin, 24 hr) differ by p<0.0001. C) Binding assay. Huh7 cells were pre-incubated on ice with compounds: lactoferrin (100 μg/mL and 500 μg/mL) and remdesivir (10 nM), as a negative control, for one hour and then infected with SARS-CoV-2 (MOI of 10) for 1h on ice. Cells were then washed thoroughly with PBS to remove unbound virus and viral RNA was quantified by RT-qPCR. Huh7 cultured in NaClO₃ for 7 days, which strips heparan sulfate proteoglycans from the cell surface, were used as a control for lactoferrin mode of action. Data is 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. D) mRNA levels of cellular IFNβ, MX1, ISG15 and IFITM3 were calculated with ΔΔCt in SARS-CoV-2 infected and lactoferrin (5750 nM)-treated cells over uninfected Huh7. Data are average SD of N=2 biological replicates with n=3 technical replicates each. Statistical significance was determined using multiple student’s t-test with the Bonferroni-Dunn method with alpha = 0.05. *p<0.001. E) Percentage of SARSCoV-2 infected Huh7 cells upon treatment with bovine apolactoferrin and hololactoferrin, native human lactoferrin, and transferrin at a concentration of 2.3 μM. F) Dose response of lactoferrin (0 to 2.3 μM) in combination with remdesivir (0 to 30 nM). Cells were pre-treated with combination or single therapy and infected with SARS-CoV-2 (MOI of 0.2) for 48 hrs.