key: cord-0812791-sj6ts2vo
authors: Holwerda, Melle; V’kovski, Philip; Wider, Manon; Thiel, Volker; Dijkman, Ronald
title: Identification of five antiviral compounds from the Pandemic Response Box targeting SARS-CoV-2
date: 2020-05-17
journal: bioRxiv
DOI: 10.1101/2020.05.17.100404
sha: 63d42ad759fbfc7b3069dcff6979e5e34ee3e95b
doc_id: 812791
cord_uid: sj6ts2vo

With currently over 4 million confirmed cases worldwide, including more than 300’000 deaths, the current Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has a major impact on the economy and health care system. Currently, a limited amount of prophylactic or therapeutic intervention options are available against SARS-CoV-2. In this study, we screened 400 compounds from the antimicrobial ‘Pandemic Response Box’ library for inhibiting properties against SARS-CoV-2. We identified sixteen compounds that potently inhibited SARS-CoV-2 replication, of which five compounds displayed equal or even higher antiviral activity compared to Remdesivir. These results show that five compounds should be further investigated for their mode of action, safety and efficacy against SARS-CoV-2. Highlights 400 compounds from the pandemic response box were tested for antiviral activity against SARS-CoV-2. 5 compounds had an equal or higher antiviral efficacy towards SARS-CoV-2, compared to the nucleoside analogue Remdesivir.

developed the pandemic response box (PRB), a compound library containing 400 compounds with antibacterial, antifungal and antiviral properties. This compound library contains drugs that are already cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 in compound-well. Cell viability and cytotoxicity were assessed in parallel, in identically treated, uninfected plates.

Data representation.

Graphs were generated using GraphPad Prism software version 8.4.2 and the final figures were 127 assembled in Adobe Illustrator CS6. Brightness and contrast of microscopy picture were minimally 128 adjusted and processed identically to their corresponding control using FIJI. Images were assembled 129 using the FigureJ plugin in FIJI [16] .

To identify potential compounds that can be used as intervention option against SARS-CoV-2 concentration of 1 µM. Based on the documented inhibition of coronavirus replication, Remdesivir and 136 K22 were included as a positive control [8, 12] . Vero-E6 cells were pretreated for 2 hours and 137 subsequently infected with SARS-CoV-2 (MOI of 0.01) for 48 hours in drug-containing medium. Cell 138 survival was arbitrarily scored, from 0 to 2, upon visual inspection and evaluation of SARS-CoV-2-139 induced CPE (Supp. Fig. 1a ). This screen resulted in a total of five compounds that completely 

(three), antibacterial (six) and antiviral (seven) compounds (Table 1) , which, similarly to their vehicle 145 control (DMSO), Remdesivir and K22, did not influence cell cytotoxicity and cell viability (Supp. Fig.   146 1b, c). These results provide evidence for the relevance of a conservative and rapid screening of 147 libraries containing compounds that target viral replication.

Antiviral efficacy against SARS-CoV-2.

To further confirm and evaluate the extent of antiviral activity of the previously highlighted sixteen 151 compounds, cells were pretreated with the selected compounds at dosages ranging from 4 µM to 152 0.063 µM and infected with SARS-CoV-2 (MOI of 0.01). After 24 hours of infection, cells were fixed 153 and processed for immunofluorescence analysis using the anti-SARS-CoV nucleocapsid protein 154 antibody and DAPI. The efficacy of the selected compounds to inhibit SARS-CoV-2, as well as their 155 individual effects of cell viability and cytotoxicity, were compared to Remdesivir. The IC50 values for 156 each compound were inferred by calculating the ratio of the total intensity of the nucleocapsid protein remaining eleven compounds showed little or no inhibition when compared to the reference 164 compound, and therefore no reliable IC50 could be calculated (Supp. Fig 2) .

In parallel to the efficacy, we determined the half-maximum cytotoxicity concentration (CC50) of 

and PDNJ0803 (SI = 24.68) had comparable SI to that of Remdesivir (SI = 27.14) ( Table 2) .

Combined these results demonstrate that Retro-2.1 is the most potent antiviral candidate that should 175 be further evaluated on its mode of action, efficacy and safety in pre-clinical models such as human

In this study we demonstrate that a conservative in vitro screening approach of 400 179 compounds from the PRB resulted in the identification of five compounds with potent antiviral activity that Chloroquine, NN-DNJ, PDNJ0803, and URMC-099-C, all have a comparable characteristics to combination lopinavir, ritonavir, and interferon beta against MERS-CoV. Nat. Commun. 2020,

A novel coronavirus outbreak of global health 10

Treatment of chronic hepadnavirus infection in a woodchuck 468 animal model with an inhibitor of protein folding and trafficking

Discovery, synthesis, and characterization of 472 an orally bioavailable, brain penetrant inhibitor of mixed lineage kinase 3

lineage kinase 3 inhibitor URMC-099 is neuroprotective and anti-inflammatory in models of 477 human immunodeficiency virus-associated neurocognitive disorders

Knibbe-Hollinger, 64

inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in 496 mice

Broad-spectrum antiviral GS-5734 inhibits both 499 epidemic and zoonotic coronaviruses