Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

Abstract

Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a readout for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in numerous cell culture models, independently of cytopathogenic effect formation. Compared to other models, the Caco-2 subline Caco-2-F03 displayed superior performance. It possesses a stable SARS-CoV-2 susceptibility phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1,796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of phosphoglycerate dehydrogenase (PHGDH), CDC like kinase 1 (CLK-1), and colony stimulating factor 1 receptor (CSF1R). The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the hexokinase II (HK2) inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2-F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false-positive hits.

Keywords: Drugs; Screening in health technology; Virology.

Graphical abstract

Figure 1

Susceptibility of Caco-2 cells to SARS-CoV-2 infection (A) Percentage of SARS-CoV-2-infected cells detected in Caco-2 cell lines from different sources infected with different SARS-CoV-2 isolates at a MOI 0.01 as determined by immunostaining for the viral spike (S protein) 48 h postinfection. (B) Cytopathogenic effect (CPE) formation in SARS-CoV-2 (MOI 0.01)-infected Caco-2 cell lines from different sources as determined 48 h postinfection. (C) Susceptibility of Caco-2-F03 cells to a broad range of SARS-CoV-2 isolates after different times of cultivation. Cells had been frozen at passage 14 and were resuscitated and cultivated for a further 30 passages. SARS-CoV-2 susceptibility was determined by immunostaining for S 48h after SARS-CoV-2 (MOI 0.01) infection 3 and 30 passages post-resuscitation. (D) ACE2 and TMPRSS2 levels in Caco-2-F03, Caco-2A, and single-cell derived clones from Caco-2A. (E) Susceptibility of Caco-2A clones to selected SARS-CoV-2 isolates as indicated by immunostaining for S and CPE formation in SARS-CoV-2 (MOI 0.01)-infected cells 48 h postinfection. (F) Correlation of S staining and CPE formation with cellular ACE2 levels. Correlations were determined using the Pearson correlation coefficient. Results are expressed as the mean ± standard deviation.

Figure 2

Caspase 3/7 activity for the quantification of the replication of SARS-CoV-2 and other coronaviruses (A) Caspase 3/7, caspase 8, and caspase 9 activity in Caco-2-F03 cells infected with a range of different SARS-CoV-2 isolates (MOI 0.01), as determined by Caspase-Glo assay (Promega) 48 h postinfection. Higher signal-to-basal (S/B) ratios and Z′ scores indicate higher assay robustness. (B–D) Caspase 3/7 activity as determined by Caspase-Glo assay, C) SARS-CoV-2 Spike (S) protein staining, and D) virus titers as indicated by genomic RNA copy numbers determined by qPCR in Caco-2-F03 cells infected with a wide range of uncharacterized SARS-CoV-2 isolates (MOI 0.01) 48 h postinfection. (E) Representative images indicating CPE formation in G614 (MOI 0.01)-infected Caco-2-F03 and Vero cells 48 h postinfection as indicated by phase contrast microscopy and immunofluoresce staining for the viral S protein in combination with DAPI-stained nuclei. (F) Quantification of cellular S protein levels in Caco-2-F03 cells infected with G614 (MOI 0.01) 48 h postinfection by immunostaining. (G) Only caspase 3/7 activity but not viability assays (MTT, CellTiter-Glo measuring ATP production) reflects G614 (MOI 0.01) replication 48h postinfection in Vero cells, which do not display a virus-induced CPE. G614 (MOI 0.01)-infected Caco-2-F03 cells served as a control that displays a CPE. p values were calculated by one-way ANOVA. (H) Caspase 3/7 activity in Caco-2-F03 cells infected with MERS-CoV, SARS-CoV, and HCoV-229E (MOI 0.01) as determined 48 h postinfection including S/B ratios and Z′ scores. Results are expressed as the mean ± standard deviation.

Figure 3

Caspase 3/7 activity for the determination of the antiviral activity of anti-SARS-CoV-2 agents and neutralization assays (A) Dose-response curves and concentrations that inhibit virus infection by 50% (IC50) of antiviral agents as determined by caspase 3/7 activity and immunostaining for the coronavirus S protein in G614 (MOI 0.01)-infected Caco-2-F03 cells 24 h postinfection. (B) Effects of the approved anti-SARS-CoV-2 drug remdesivir on cellular levels of the viral NP protein in G614 (MOI 1)-infected air liquid interface (ALI) cultures of primary human bronchial epithelial (HBE) cells 120 h postinfection. (C) Effects of remdesivir on caspase 3/7 activity and virus titers (genomic RNA copy numbers determined by PCR) in G614 (MOI 1)-infected ALI HBE cultures 120 h postinfection. (D) Effects of remdesivir on caspase 3/7 activity and virus titers in G614 (MOI 1)-infected primary human cardiomyocytes (CMS) 48 h postinfection. (E) Correlation of the neutralization capacity of sera derived from seven donors two weeks after their second dose of the mRNA-1273 vaccine determined by caspase 3/7 activity or cytopathogenic effect (CPE) scoring in D614-, Alpha-, and Delta-infected Caco-2-F03 cells 48 h postinfection. Correlations were determined using the Pearson correlation coefficient. (F) Determination of neutralization titers by caspase 3/7 activity or CPE scoring using sera derived from seven donors two weeks after their second dose of the mRNA-1273 vaccine in Caco-2-F03 cells infected with D614, Alpha, and Delta isolates 72 h postinfection. p values were calculated using paired t-test. P-values were determined by ANOVA. Results are expressed as the mean ± standard deviation.

Figure 4

Caspase 3/7 activity for the phenotypic resistance testing of SARS-CoV-2 strains (A) Drug dose-response curves and concentrations that reduce cellular levels of the SARS-CoV-2 S protein by 50% (IC50) in Caco-2-F03 and Calu-3 cells as determined by immunostaining 24 h (Caco-2-F03) or 48 h (Calu-3) postinfection with the SARS-CoV-2 strain FFM3 or its remdesivir-adapted substrain FFM3rREM at MOI 0.01. (B) Drug concentrations that reduce caspase 3/7 activity in FFM3 and FFM3rREM (MOI 0.01)-infected Caco-2-F03 cells 48 h postinfection. P-values were determined by Student’s t-test. (C) Sequence variants in FFM3rREM compared to FFM3. (D) The polymerase complex with nsp7 and nsp8 and a template-primer RNA (cyan and deep teal) and remdesivir (magenta) bound. Gly671Ser is shown in red (as serine). (E) The SARS-CoV-2 polymerase Gly671Ser sequence variant. Residue 671 is shown in red as serine, which would be able to form a hydrogen bond with Thr402 which would not be present as Gly671. All p values were calculated by two-way ANOVA. Results are expressed as the mean ± standard deviation.

Figure 5

Proof-of-concept screen for anti-SARS-CoV-2 drug candidates using the Caco-2-F03 cell line platform and caspase 3/7 activity as readout method (A) Overview of the proof-of-concept screen for anti-SARS-CoV-2 compounds using the Kinase inhibitor library L-1200 (Selleckchem, Germany) containing 1,796 compounds (Selleckchem, Germany) in Delta (MOI 0.01)-infected Caco-2-F03 cells using caspase 3/7 activity as readout 48 h postinfection. For the screen, every compound was tested at a concentration of 10 and 1 μM. 21 selected hits were then confirmed by determining drug-response curves. (B) Quality controls, Z’scores served as quality controls (left). Only plates with a Z'score ≥0.5 were further analyzed. Remdesivir (10 μM) was used as positive control on each plate and produced consistent results (right). (C) Number of hits at different inhibition cutoffs. (D) Visualization of the distribution of hits according to their targets. Targets for which inhibitors were selected for confirmation are indicated. (E) Heatmaps of the anti-SARS-CoV-2 activity of 21 hits by the determination of dose-response in Delta and Omicron (MOI 0.01)-infected Caco-2-F03 cells using immunostaining for the viral S protein as readout 24 h postinfection.

Figure 6

Investigation of the anti-SARS-CoV-2 effects of the PHGDH inhibitor NCT-503 alone or in in combination with 2-Deoxy-D-glucose (2DG) (A–E) Scheme of the testing of NCT-503 for anti-SARS-CoV-2 activity in air liquid interface (ALI) cultures of primary human bronchial epithelial (HBE) cells. Effect of NCT-503 on (B) caspase 3/7 activity, (C) virus titers (determined as genomic RNA copy numbers by qPCR), (D) transepithelial electrical resistance (TEER), and (E) LDH release in ALI HBE cultures infected with Delta (MOI 1) 120 h postinfection. (F) Anti-SARS-CoV-2 effects of NCT-503 in combination with 2-Deoxy-D-glucose (2DG). Illustration of how NCT-503 and 2DG can exert combined effects on a common metabolic pathway. (G) Representative fluorescence images indicating the number of Delta- and Omicron (MOI 0.01)-infected cells in NCT503 and/or 2DG-treated Caco-2-F03 cultures 24 h postinfection. (H) and I) Weighted combination indices (CIwt) determined by the method of Chou and Talalay indicating a strong synergism of NCT-503 and 2DG. Results are expressed as the mean ± standard deviation.