. 2021 Apr 6;35(1):108940.

doi: 10.1016/j.celrep.2021.108940. Epub 2021 Mar 18.

Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication

Gustavo Garcia Jr¹, Arun Sharma², Arunachalam Ramaiah³, Chandani Sen⁴, Arunima Purkayastha⁴, Donald B Kohn⁵, Mark S Parcells⁶, Sebastian Beck⁷, Heeyoung Kim⁸, Malina A Bakowski⁹, Melanie G Kirkpatrick⁹, Laura Riva⁹, Karen C Wolff⁹, Brandon Han¹⁰, Constance Yuen¹⁰, David Ulmert¹¹, Prabhat K Purbey¹², Philip Scumpia¹², Nathan Beutler¹³, Thomas F Rogers¹⁴, Arnab K Chatterjee⁹, Gülsah Gabriel⁷, Ralf Bartenschlager¹⁵, Brigitte Gomperts⁵, Clive N Svendsen¹⁶, Ulrich A K Betz¹⁷, Robert D Damoiseaux¹⁸, Vaithilingaraja Arumugaswami¹⁹

Affiliations

¹ Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
² Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA; Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
³ Department of Ecology and Evolutionary Biology, University of California, Irvine, Irvine, CA 92697, USA; Section of Cell and Developmental Biology, University of California, San Diego, San Diego, CA 92093, USA.
⁴ UCLA Children's Discovery and Innovation Institute, Mattel Children's Hospital UCLA, Department of Pediatrics, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA.
⁵ UCLA Children's Discovery and Innovation Institute, Mattel Children's Hospital UCLA, Department of Pediatrics, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, CA 90095, USA.
⁶ Department of Animal and Food Sciences, Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA.
⁷ Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany.
⁸ Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
⁹ Calibr, a division of Scripps Research Institute, 11119 North Torrey Pines Road, La Jolla, CA 92037, USA.
¹⁰ California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹¹ Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA 90095, USA.
¹² Department of Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹³ Department of Immunology and Microbiology, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
¹⁴ Department of Immunology and Microbiology, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA; UC San Diego Division of Infectious Diseases and Global Public Health, UC San Diego School of Medicine, La Jolla, CA 92093, USA.
¹⁵ Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; German Center for Infection Research, Heidelberg partner site, Heidelberg, Germany; Division Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany.
¹⁶ Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
¹⁷ Merck KGaA, Darmstadt, Germany. Electronic address: ulrich.betz@merckgroup.com.
¹⁸ Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA 90095, USA; California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: rdamoiseaux@mednet.ucla.edu.
¹⁹ Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, CA 90095, USA; California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: varumugaswami@mednet.ucla.edu.

PMID: 33784499
PMCID: PMC7969873
DOI: 10.1016/j.celrep.2021.108940

Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication

Gustavo Garcia Jr et al. Cell Rep. 2021.

. 2021 Apr 6;35(1):108940.

doi: 10.1016/j.celrep.2021.108940. Epub 2021 Mar 18.

Authors

Affiliations

¹ Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
² Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA; Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
³ Department of Ecology and Evolutionary Biology, University of California, Irvine, Irvine, CA 92697, USA; Section of Cell and Developmental Biology, University of California, San Diego, San Diego, CA 92093, USA.
⁴ UCLA Children's Discovery and Innovation Institute, Mattel Children's Hospital UCLA, Department of Pediatrics, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA.
⁵ UCLA Children's Discovery and Innovation Institute, Mattel Children's Hospital UCLA, Department of Pediatrics, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, CA 90095, USA.
⁶ Department of Animal and Food Sciences, Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA.
⁷ Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany.
⁸ Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
⁹ Calibr, a division of Scripps Research Institute, 11119 North Torrey Pines Road, La Jolla, CA 92037, USA.
¹⁰ California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹¹ Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA 90095, USA.
¹² Department of Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹³ Department of Immunology and Microbiology, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
¹⁴ Department of Immunology and Microbiology, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA; UC San Diego Division of Infectious Diseases and Global Public Health, UC San Diego School of Medicine, La Jolla, CA 92093, USA.
¹⁵ Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; German Center for Infection Research, Heidelberg partner site, Heidelberg, Germany; Division Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany.
¹⁶ Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
¹⁷ Merck KGaA, Darmstadt, Germany. Electronic address: ulrich.betz@merckgroup.com.
¹⁸ Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA 90095, USA; California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: rdamoiseaux@mednet.ucla.edu.
¹⁹ Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, CA 90095, USA; California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: varumugaswami@mednet.ucla.edu.

PMID: 33784499
PMCID: PMC7969873
DOI: 10.1016/j.celrep.2021.108940

Abstract

SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.

Keywords: ATR kinase; COVID-19; DNA-damage response pathway; SARS-CoV-2; berzosertib; high-throughput screen; mTOR-PI3K-AKT pathway; nucleoside analogs; protein kinase inhibitors.

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Conflict of interest statement

Declaration of interests U.A.K.B. is an employee of Merck KGaA, Darmstadt, Germany. Berzosertib compound is licensed by Merck KGaA, Darmstadt, Germany. The other authors declare no competing financial interests.

Figures

**Figure 1**
Drug-target kinase-connectivity network identifies key anti-viral protein kinase inhibitors (A) Workflow of drug screen is shown. (B) Connectivity map of drug hits from the primary screen is illustrated. The graphical representation shown is the confidence view in which stronger associations are represented by thicker lines, protein-protein interactions are shown in gray, chemical-protein interactions are in green, and interactions between chemicals are in red. Round shapes represent proteins, and oval shapes indicate hit compounds from the primary screen. The analysis indicated a protein-protein interaction enrichment score of 0.0026, which is statistically significant. (C) STRING analysis of host protein-protein network identified from the drug screen is shown. (D) Immunofluorescent images of SARS-CoV-2-infected cells (red) treated with the indicated drug compounds at various concentrations. AZD2014, torin2, and dactolisib were used at 500 nM. Scale bar, 100 μm. (E) Graphs show the percentage of inhibition of SARS-CoV-2 infectivity and cytotoxicity by the indicated compounds. Note: IC₅₀ of each compound is shown in the graph. Representative data from two independent experiments are presented.

**Figure 2**
Berzosertib inhibits SARS-CoV-2 replication in hiPSC-CMs (A) Graph shows beats per minute of SARS-CoV-2-infected hiPSC-CM cells treated with berzosertib (250 nM), dactolisib (250 nM), remdesivir (10 μM), and HQ (10 μM). (B) Graph shows viral titer (TCID₅₀/mL) of supernatant collected at the indicated time points after SARS-CoV-2 infection of drug-treated hiPSC-CMs. (C) Graph depicts quantification of SARS-CoV-2 and cleaved caspase-3-positive cells. (D) IFA images of hiPSC-CMs undergoing apoptosis after SARS-CoV-2 infection and drug treatment at 72 hpi. Scale bar, 25 μm. (E) hiPSC-CMs were stained with cardiac troponin T (cTnT) (green) to demonstrate that cells are protected from SARS-CoV-2-mediated cell injury (red) by berzosertib (250 nM). Scale bar, 25 μm. Statistical analysis of graphs (A and C) was conducted by multiple-comparison one-way analysis of variance (ANOVA) was conducted. ^∗∗p < 0.001, ^∗∗∗p < 0.0001. Representative data from three independent experiments are presented.

**Figure 3**
Berzosertib inhibits SARS-CoV-2, SARS-CoV-1, and MERS-CoV replication in human cells and is synergistic with remdesivir (A and B) Graphs show an eight-point dose-response curve of berzosertib (A) or remdesivir (B) in SARS-CoV-2-infected Calu-3 cells. Contrasted with cell viability of mock-infected cells. (C and D) Antiviral effect of berzosertib on SARS-CoV-1 (C) and MERS-CoV (D). (E) Graphs show antiviral activity measured with a SARS-CoV-2 immunostaining signal used for identification of infected A549-ACE2 cells. IC₅₀ values were calculated by non-linear regression sigmoidal dose-response analysis using the GraphPad Prism 7 software package. (F) Graph shows synergistic effect of berzosertib and remdesivir in infected A549-ACE2 cells. Dose-response curves obtained with mixtures of remdesivir and berzosertib, remdesivir alone (thick black line), and berzosertib alone (thick pink line) are shown. (G) Isobologram of drug combinations is depicted. (H) Combinatorial data were analyzed for inhibitory, additive, or synergistic effects (upper triangle, dotted line, and lower triangle, respectively) by using the Compusyn software package.

**Figure 4**
Berzosertib mode of antiviral activity in lung and kidney epithelial cells and effect on SARS-CoV-2-mediated inflammatory response (A) Graph shows eight-dose-response curve of berzosertib in SARS-CoV-2-infected human primary lung ALI culture. (B) Immunofluorescent images indicate dose-dependent reduction of SARS-CoV-2 replication in berzosertib-treated ALI culture (spike protein in red).Scale bar, 100 μm. (C) Western blot analysis shows time course of pCHK1 and virus replication kinetics in Vero E6 cells. Berzosertib treatment reduced CHK1 phosphorylation. In addition, it inhibited SARS-CoV-2 replication as early as 8 h after infection. By 24 h in untreated cells, SARS-CoV-2 signal intensity was oversaturated because of the high-level of viral proteins. Representative data from two independent experiments is shown. (D) SARS-CoV-2 genome replication kinetics in the presence of berzosertib treatment on Vero E6 cells. (E) Graph shows that berzosertib treatment reduces the expression of inflammatory IL-6 gene in SARS-CoV-2-infected Vero E6 cells. Statistical analysis of graphs (D and E) conducted with multiple-comparison two-way analysis of variance (ANOVA). ^∗p < 0.01, ^∗∗p < 0.001, ^∗∗∗p < 0.0001. Representative data from three independent experiments are presented.

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Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication

Affiliations

Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Miscellaneous