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N4-Hydroxycytidine and Inhibitors of Dihydroorotate Dehydrogenase Synergistically Suppress SARS-CoV-2 Replication

55 Pages Posted: 25 Jan 2022 Publication Status: Published

See all articles by Kim M. Stegmann

Kim M. Stegmann

affiliation not provided to SSRN

Antje Dickmanns

affiliation not provided to SSRN

Natalie Heinen

affiliation not provided to SSRN

Claudia Blaurock

affiliation not provided to SSRN

Tim Karrasch

affiliation not provided to SSRN

Angele Breithaupt

World Health Organization (WHO) - Friedrich-Loeffler-Institut; Federal Research Institute for Animal Health - Friedrich-Loeffler-Institut

Robert Klopfleisch

affiliation not provided to SSRN

Nadja Uhlig

affiliation not provided to SSRN

Valentina Eberlein

affiliation not provided to SSRN

Leila Issmail

affiliation not provided to SSRN

Simon T. Herrmann

affiliation not provided to SSRN

Amelie Schreieck

affiliation not provided to SSRN

Evelyn Peelen

affiliation not provided to SSRN

Hella Kohlhof

affiliation not provided to SSRN

Alexander Riek

affiliation not provided to SSRN

John R. Speakman

University of Aberdeen

Uwe Groß

University of Goettingen (Göttingen) - Institute of Medical Microbiology

Dirk Görlich

affiliation not provided to SSRN

Daniel Vitt

affiliation not provided to SSRN

Thorsten Müller

affiliation not provided to SSRN

Thomas Grunwald

affiliation not provided to SSRN

Stephanie Pfaender

Ruhr-University Bochum - Department of Molecular and Medical Virology

Anne Balkema-Buschmann

affiliation not provided to SSRN

Matthias Dobbelstein

Georg August Universität - Institute of Molecular Oncology

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Abstract

SARS-CoV-2 incorporates the nucleoside analogue N4-hydroxycytidine (NHC) into its RNA through the RNA-dependent RNA polymerase (RdRp). NHC is the active metabolite of the prodrug Molnupiravir (MK-4482), which has recently been approved in the United Kingdom (UK) for the treatment of COVID-19. Likewise, inhibitors of dihydroorotate dehydrogenase (DHODH), by reducing the cellular synthesis of pyrimidines, also reduce virus yield upon infection. Here we show that NHC and DHODH inhibitors strongly synergize in the inhibition of SARS-CoV-2 replication. While single drug treatment of cultured cells only moderately limited virus progeny, the combination of both compounds reduced virus propagation by at least two orders of magnitude. This effect was also observed on the virus variants Alpha, Beta and Delta. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC into nascent viral RNA, thus precluding the correct synthesis of the viral genome and inhibiting the production of replicationcompetent virus particles. This concept is supported by the rescue of virus replication upon addition of pyrimidine nucleosides to the media. Moreover, we observed that the combination of DHODH inhibitors and Molnupiravir displayed increased antiviral efficiency not only in stem cell-derived organoids of human lung, but also in Syrian Gold hamsters and in K18-hACE2 mice. Combining Molnupiravir with DHODH inhibitors may thus improve available therapy options for COVID-19.

Funding:This work was funded by the COVID-19 Forschungsnetzwerk Niedersachsen (COFONI) to MD, by the Federal Ministry of Education and Research Germany (Bundesministerium für Bildung und Forschung; BMBF; OrganSARS, 01KI2058) to SP and TM, and by a grant of the Max Planck Foundation to DG.

Declaration of Interests: AS, HK, EP and DV are employees of Immunic AG and own shares and/or stock-options of the parent company of Immunic AG, Immunic Inc. Some of the Immunic AG employees also hold patents for the Immunic compounds described in this manuscript (WO2012/001148, WO03006425). KMS, AD and MD are employees of University Medical Center Göttingen, which has signed a License Agreement with Immunic AG covering the combination of DHODH inhibitors and nucleoside analogues to treat viral infections, including COVID-19 (inventors: MD, KMS, AD). The other authors declare no conflict of interest.

Ethics Approval Statement: Hamster experiments were carried out according to the German Regulations for Animal Welfare after obtaining the necessary approval from the authorized ethics committee of the State Office of Agriculture, Food Safety and Fishery in Mecklenburg – Western Pomerania (LALLF M-V) under permission number 7221.3-1-049/20 and approval of the commissioner for animal welfare at the Friedrich Loeffler Institute (FLI), representing the Institutional Animal Care and Use Committee (IACUC). Mouse experiments were carried out according to the German Regulations for Animal Welfare after obtaining the necessary approval from the authorized ethics committee of the State Saxony under the permission number 25-5121/515/7 (TVV 06/21).

Keywords: Coronavirus, SARS-COV-2, COVID-19, N4-hydroxycytidine (NHC), EIDD1931, EIDD-2801, Molnupiravir, MK-4482, Lagevrio, Emorivir, Monuvir, dihydroorotate dehydrogenase (DHODH), pyrimidine synthesis, Teriflunomide, IMU-838, Vidofludimus, BAY2402234, Brequinar, ASLAN003, Vero E6 cells, Calu-3 cells, RNA replication, spike, Nucleocapsid, ACE2, Syrian Gold hamster, K18-hACE2 transgenic mice

Suggested Citation

Stegmann, Kim M. and Dickmanns, Antje and Heinen, Natalie and Blaurock, Claudia and Karrasch, Tim and Breithaupt, Angele and Klopfleisch, Robert and Uhlig, Nadja and Eberlein, Valentina and Issmail, Leila and Herrmann, Simon T. and Schreieck, Amelie and Peelen, Evelyn and Kohlhof, Hella and Riek, Alexander and Speakman, John R. and Gross, Uwe and Görlich, Dirk and Vitt, Daniel and Müller, Thorsten and Grunwald, Thomas and Pfaender, Stephanie and Balkema-Buschmann, Anne and Dobbelstein, Matthias, N4-Hydroxycytidine and Inhibitors of Dihydroorotate Dehydrogenase Synergistically Suppress SARS-CoV-2 Replication. Available at SSRN: https://ssrn.com/abstract=4017904 or http://dx.doi.org/10.2139/ssrn.4017904
This version of the paper has not been formally peer reviewed.

Kim M. Stegmann

affiliation not provided to SSRN ( email )

No Address Available

Antje Dickmanns

affiliation not provided to SSRN ( email )

No Address Available

Natalie Heinen

affiliation not provided to SSRN ( email )

No Address Available

Claudia Blaurock

affiliation not provided to SSRN ( email )

No Address Available

Tim Karrasch

affiliation not provided to SSRN ( email )

No Address Available

Angele Breithaupt

World Health Organization (WHO) - Friedrich-Loeffler-Institut ( email )

Federal Research Institute for Animal Health - Friedrich-Loeffler-Institut ( email )

Greifswald-Insel Riems
Germany

Robert Klopfleisch

affiliation not provided to SSRN ( email )

No Address Available

Nadja Uhlig

affiliation not provided to SSRN ( email )

No Address Available

Valentina Eberlein

affiliation not provided to SSRN ( email )

No Address Available

Leila Issmail

affiliation not provided to SSRN ( email )

No Address Available

Simon T. Herrmann

affiliation not provided to SSRN ( email )

No Address Available

Amelie Schreieck

affiliation not provided to SSRN ( email )

No Address Available

Evelyn Peelen

affiliation not provided to SSRN ( email )

No Address Available

Hella Kohlhof

affiliation not provided to SSRN ( email )

No Address Available

Alexander Riek

affiliation not provided to SSRN ( email )

No Address Available

John R. Speakman

University of Aberdeen ( email )

Dunbar Street
Aberdeen, AB24 3QY
United Kingdom

Uwe Gross

University of Goettingen (Göttingen) - Institute of Medical Microbiology ( email )

Göttingen
Germany

Dirk Görlich

affiliation not provided to SSRN ( email )

No Address Available

Daniel Vitt

affiliation not provided to SSRN ( email )

No Address Available

Thorsten Müller

affiliation not provided to SSRN ( email )

No Address Available

Thomas Grunwald

affiliation not provided to SSRN ( email )

No Address Available

Stephanie Pfaender

Ruhr-University Bochum - Department of Molecular and Medical Virology ( email )

Germany

Anne Balkema-Buschmann

affiliation not provided to SSRN ( email )

No Address Available

Matthias Dobbelstein (Contact Author)

Georg August Universität - Institute of Molecular Oncology ( email )

Göttingen
Germany

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