key: cord-0942513-xiqum6iz
authors: Chiu, Winston; Verschueren, Lore; Van den Eynde, Christel; Buyck, Christophe; De Meyer, Sandra; Jochmans, Dirk; Bojkova, Denisa; Ciesek, Sandra; Cinatl, Jindrich; De Jonghe, Steven; Leyssen, Pieter; Neyts, Johan; Van Loock, Marnix; Van Damme, Ellen
title: Development and optimization of a high‐throughput screening assay for in vitro anti‐SARS‐CoV‐2 activity: Evaluation of 5676 Phase 1 Passed Structures
date: 2022-03-23
journal: J Med Virol
DOI: 10.1002/jmv.27683
sha: f047fdf8f5ac29571ef070b3470e063f1e73e2ca
doc_id: 942513
cord_uid: xiqum6iz

Although vaccines are currently used to control the coronavirus disease 2019 (COVID‐19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS‐CoV‐2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS‐CoV‐2 and clinically successful against COVID‐19. As part of an immediate response to the COVID‐19 pandemic, a high‐throughput, high content imaging–based SARS‐CoV‐2 infection assay was developed in VeroE6 African green monkey kidney epithelial cells expressing a stable enhanced green fluorescent protein (VeroE6‐eGFP cells) and was used to screen a library of 5676 compounds that passed Phase 1 clinical trials. Eight drugs (nelfinavir, RG‐12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) were identified as inhibitors of in vitro anti–SARS‐CoV‐2 activity in VeroE6‐eGFP and/or Caco‐2 cell lines. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID‐19 treatment.

reported in Wuhan, China. 4 Common presenting symptoms of COVID-19 caused by Alpha variants include cough, fever, loss of taste or smell, and fatigue. 5 However, the more recent Delta variant seems to present slightly different symptoms, such as headache, runny nose, throat ache, fever, and coughing. 5 In cases that progress to severe disease, patients commonly experience dyspnea and hypoxemia followed by respiratory failure. 6 COVID-19 and its etiologic agent, SARS coronavirus 2 (SARS-CoV-2), have spread globally since the initial outbreak, infecting >400 million people and leading to ≥6 million deaths.

Even before the World Health Organization declared COVID-19 a public health emergency of international concern on January 30, 2020, repurposing of existing drugs and drug candidates was explored to accelerate the traditional research and development timelines to provide a rapid response to this unmet medical need. 7 Drug repurposing had been applied previously for SARS-CoV-1, MERS-CoV, and other viruses. 7, 8 Early in the pandemic, the development of cell-based systems was critical for the rapid evaluation of drugs with antiviral activity against SARS-CoV-2. Similar to SARS-CoV-1, Vero cells and Caco-2 cells were found to be susceptible to infection with SARS-CoV-2. 9,10 Therefore, a previously published high-throughput screening (HTS) assay for SARS-CoV-1 11 using authentic infection of VeroE6 African green monkey kidney epithelial cells expressing a stable enhanced green fluorescent protein (VeroE6-eGFP) was adapted, further developed, and miniaturized for screening of antiviral drugs against SARS-CoV-2, isolated from a Belgian patient. The resulting assay is an HTS 384-well cell-based SARS-CoV-2 infection assay with a high content imaging (HCI) readout of fluorescence that provides a measure for cytopathic effect (CPE). In parallel, a cellular toxicity assay was developed using ATPlite™; any toxicity of the compound to the cell line is then evaluated by luminescence.

To identify potential candidates for rapid clinical development, 5676 chemical structures that had passed Phase 1 clinical studies with applications in a variety of therapeutic fields, including oncology, neuroscience, and infectious diseases, were screened. The compounds were evaluated in a seven-point dose-response curve, starting at 20 µM, for their ability to inhibit SARS-CoV-2-induced CPE without causing general cellular toxicity. Selected hits were further evaluated in a second cellular model using Caco-2 cells that are susceptible to SARS-CoV-2 infection. 12, 13 2 | MATERIALS AND METHODS

Janssen Pharmaceutica maintains a regularly updated database of compounds that have been approved or tested in a successfully completed clinical Phase 1 study. One of the many uses of this Phase One Passed Structures (POPS) database is to serve as a starting point to identify high-priority compounds for possible repurposing. The goal of the POPS database is to be highly enriched in druggable, welldocumented, and diverse compounds from all disease areas, including oncology, neuroscience, and infectious diseases. The database is updated on a regular basis, both from a (virtual) annotation standpoint and with a physically available set of compounds that can be screened. Due to previous acquisition and synthesis efforts and active purchasing in the first months of 2020, approximately 5500 compounds that had sufficient availability and passed quality control for purity were identified and plated.

Plates were freshly prepared to ensure high-quality assays and were submitted for screening. CELLSTAR ® 384-well plates (Greiner Bio- 

VeroE6-eGFP cells were cloned and validated in-house as previously described. 11 

Assay quality performance (inter-and intraplate data comparison) and optimization (cell density, viral input, incubation time, Z′, signal/ noise) were performed on 384-well plates spotted with reference compounds. Raw data were normalized using the following formula:

where z i is the normalized value of the dataset, x i is the raw value of the dataset, min(x) is the minimum raw value of the dataset, and max(x) is the maximum raw value of the dataset. The Z′ value, which encompasses the dynamic range of the assay and well-to-well variability, was calculated for each plate as a measure of assay quality. 15, 16 All compounds were screened in a dose-response (seven dilutions) starting from 20 µM with half dilution steps and tested in triplicate on separate plates.

At the start of the pandemic, an immediate public health response was needed but tools to screen for antiviral agents were not available at that time. Due to the high sequence homology between SARS-CoV-1 and SARS-CoV-2, the primary focus was on VeroE6 cells, which were previously shown to be readily susceptible to infection with SARS-CoV-2. 9,10 Indeed, SARS-CoV-2 infected the cells and caused pronounced CPE, especially once the virus had been adapted to cell culture (three to five passages on VeroE6 cells). Next, a SARS-CoV-1 antiviral assay in VeroE6-eGFP cells 11 was adapted to be used for screening with SARS-CoV-2 ( Figure 1 ). In this system, SARS-CoV-2 infection causes cellular CPE and loss of fluorescent signal. Compound-mediated antiviral activity reduces CPE, leading to increased fluorescence compared with controls. To monitor CPE and inhibition thereof by antiviral molecules, the assay was developed in parallel tracks whereby readout was performed either by fluorescence (using a GFP PR) or HCI at low resolution to visualize and quantify CPE ( Figure 1 ).

In uninfected (CC) conditions, the mean normalized signal for the PR readout was 79.8 ± 10.9 (% CV = 13.7); for the HCI readout, the mean normalized signal was 94.8 ± 2.41 (% CV = 2.55). Under infected (virus control [VC]) conditions, a high % CV was calculated for both readouts with a large variation between the data points; because the mean value was low (PR < 5 and HCI < 1), the % CV was highly sensitive to small changes. In this type of biological assay, unavoidable "eGFP cell debris," found in residual organic waste after virus-induced cell death, contributes to variation in eGFP. For HTS quality assessment, the RZ′ and signal to noise ratio (S/N) were calculated for each method (PR readout: mean RZ′ the RZ′ and S/N were calculated for each experiment to determine F I G U R E 2 Signal-to-noise (A) and Z′ (B) comparison between PR (red) and HCI (blue) assays. The signal-to-noise ratio (raw data CC/ raw data VC) for the PR assay ranged from 1.84 to 6.22 with Z′ values ranging from -0.26 to 0.72. The HCI assay yielded much higher values for both parameters; the signal-to-noise ratio ranged from 17.95 to 312.3 while Z′ values ranged from 0.57 to 0.95. CC, uninfected conditions; HCI, high content imaging; PR, plate reader; VC, infected conditions. HTS quality. The assay performance of both the PR and HCI antiviral assays is shown in Table 1 . 

Slightly cytotoxic compounds may result in false negatives in this assay (i.e., activity caused by interference with cell proliferation).

Therefore, all compounds were evaluated using dose-response curves, in which bell-shaped curves indicate upcoming cytotoxicity.

In addition, every compound was tested in a parallel toxicity assay.

The drug repurposing screening hit rate was approximately 4.0%. All compounds were screened at the highest concentration available in 100% DMSO stock, which was either 100, 50, or 25 µM final concentration. Of the 5676 compounds screened, 228 compounds were found to be active (EC 50 value <20 µM in either PR or HCI readout) ( Figure 3 and Table S1 ).

All compounds were redissolved from neat and were tested again in seven-point dilution with ½ dilution steps dose-response (starting at the highest possible concentration: 100, 50, or 25 µM, final concentration) in triplicate for confirmation. In the confirmation run, 52 compounds remained active (maximum percent inhibition ≥30%). A thorough evaluation of these hits was made based on the quality of the curve, the chemistry, potential/known mode of action, literature, and toxicity to select only those with potential clinical relevance.

Finally, eight compounds with antiviral activity against SARS-CoV-2 in the VeroE6-eGFP cells were selected for further investigation: chloroquine, doxorubicin, hydroxychloroquine, itraconazole, nelfinavir, remdesivir, RG-12915, and sematilide (Table 3 ).

To exclude cell line-specific modes of antiviral activity, six compounds were further evaluated using a Caco-2 infection assay and a viral strain isolated from a different source (SARS-CoV-2/FFM1). Hydroxychloroquine and chloroquine were excluded from this evaluation because they had previously been found inactive in SARS-CoV-2-infected Caco-2 cells. 26 This system has been used previously for SARS-CoV-1 11 and was recently deployed for another drug repurposing screen. 26 All compounds were evaluated with a dose-response curve to obtain an EC 50 F I G U R E 3 Schematic overview of the screening campaign used on the Janssen POPS library containing 5676 compounds. The highthroughput screen was performed on 384-well plates with the described PR or HCI VeroE6-eGFP assay. After the initial screening, 228 compounds showed activity against SARS-CoV-2 with EC 50 values <20 µM in either assay. These compounds were retested in triplicate in the same assay for confirmation, which yielded 52 active compounds. Finally, the eight most promising compounds were selected for further profiling, and the top six candidates were selected for additional testing using the Caco-2 assay. EC 50 , concentration of the compound that inhibited 50% of the infection; HCI, high content imaging; POPS, Phase One Passed Structures; PR, plate reader; VeroE6-eGFP, VeroE6 African green monkey kidney epithelial cells expressing a stable enhanced green fluorescent protein.

signal upon cell death. 11 The HTS screening assay developed in this study was robust (Table 1) , automated, and represents a scalable and efficient system for identifying antiviral compounds. Although some previously developed assays have used HCI, 10, 28, 29 few have compared HCI and PR readout, as described in this study. In developing HTS assays, factors such as data storage, data acquisition speed, and logistics should be considered, especially when speed is of utmost importance, such as during a pandemic. In addition to the advantages of speed and storage afforded by PR, all parameters for HTS quality (RZ′, S/N), intraand interplate variation were overall substantially better using HCI compared with PR readout (Tables 1 and 2, Figure 2 ). This made HCI the preferred readout for screening.

A library of 5676 compounds that passed Phase 1 was screened.

The primary hit rate was 4.0%, which is relatively high among experimental repurposing screening studies that usually report hit rates of less than 2%. [31] [32] [33] This rather high hit rate is due to the low threshold that was set for primary hit selection and because toxicity was not taken into account for primary hit selection. In total, 52 hits were confirmed. After elimination of toxic compounds, screening artifacts (e.g., vitamin B2 and orantinib cause a false positive signal due to fluorescence of the compound), and unwanted modes of action such as influence on lysosomal function or phospholipidosis, 34 eight compounds remained ( Table 3) . Five of these compounds also exhibited antiviral activity in Caco-2 cells, a validated model for T A B L E 3 Observed antiviral activity (EC 50 ) against SARS-CoV-2 and cytotoxicity (CC 50 ) of the eight compounds identified using the VeroE6-eGFP cell assay a Pharmacological parameters of compounds (such as pharmacokinetics, pharmacodynamics, tissue distribution, and tolerability), as well as cytotoxicity/cytostatic assays, should be carefully considered when evaluating the feasibility of clinical applications. 40 For example, chloroquine and its analog hydroxychloroquine inhibit SARS-CoV-2 entry and replication in VeroE6 cells, 44 yet failed in clinical trials. 37, 45 Nonetheless, screening this repurposing library contributed to our understanding of fundamental features of SARS-CoV-2 and the rapid design/deployment needed for high-throughput assays, which may be instrumental during future outbreaks.

The findings of this study were comparable with other large screening studies that identified potential anti-SARS-CoV-2 agents and compounds, although in vivo efficacy, toxicity, and pharmacokinetic investigation of the selected hits in this report did not support novel applications against SARS-CoV-2. 10 sponsor, Janssen Pharmaceutica NV, contributed to the study design, data analysis and interpretation, the writing of the report, and the decision to submit the manuscript for publication.

The authors declare the following financial interests/personal relationships that may be considered as potential competing Ellen Van Damme contributed to study conceptualization, data curation and data analysis, methodology, project administration, and had supervisory duties.

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Data are available upon reasonable request.

Winston Chiu http://orcid.org/0000-0001-5236-623X

Marnix Van Loock http://orcid.org/0000-0003-4151-4588

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