Inhibitors of dihydroorotate dehydrogenase cooperate with molnupiravir and N4-hydroxycytidine to suppress SARS-CoV-2 replication

Kim M. Stegmann; Antje Dickmanns; Natalie Heinen; Claudia Blaurock; Tim Karrasch; Angele Breithaupt; Robert Klopfleisch; Nadja Uhlig; Valentina Eberlein; Leila Issmail; Simon T. Herrmann; Amelie Schreieck; Evelyn Peelen; Hella Kohlhof; Balal Sadeghi; Alexander Riek; John R. Speakman; Uwe Groß; Dirk Görlich; Daniel Vitt; Thorsten Müller; Thomas Grunwald; Stephanie Pfaender; Anne Balkema-Buschmann; Matthias Dobbelstein

doi:10.1016/j.isci.2022.104293

Inhibitors of dihydroorotate dehydrogenase cooperate with molnupiravir and N4-hydroxycytidine to suppress SARS-CoV-2 replication

Kim M. Stegmann, Antje Dickmanns, Natalie Heinen, Claudia Blaurock, Tim Karrasch, Angele Breithaupt, Robert Klopfleisch, Nadja Uhlig, Valentina Eberlein, Leila Issmail, Simon T. Herrmann, Amelie Schreieck, Evelyn Peelen, Hella Kohlhof, Balal Sadeghi, Alexander Riek, John R. Speakman, Uwe Groß, Dirk Görlich, Daniel VittThorsten Müller, Thomas Grunwald, Stephanie Pfaender, Anne Balkema-Buschmann, Matthias Dobbelstein^*

^*Corresponding author for this work

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Abstract

The nucleoside analog N4-hydroxycytidine (NHC) is the active metabolite of the prodrug molnupiravir, which has been approved for the treatment of COVID-19. SARS-CoV-2 incorporates NHC into its RNA, resulting in defective virus genomes. Likewise, inhibitors of dihydroorotate dehydrogenase (DHODH) reduce virus yield upon infection, by suppressing the cellular synthesis of pyrimidines. Here, we show that NHC and DHODH inhibitors strongly synergize in the inhibition of SARS-CoV-2 replication in vitro. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC into nascent viral RNA. This concept is supported by the rescue of virus replication upon addition of pyrimidine nucleosides to the media. DHODH inhibitors increased the antiviral efficiency of molnupiravir not only in 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.

Original language	English
Article number	104293
Number of pages	27
Journal	iScience
Volume	25
Issue number	5
Early online date	6 May 2022
DOIs	https://doi.org/10.1016/j.isci.2022.104293
Publication status	Published - 20 May 2022

Bibliographical note

Funding Information:
We thank Thorsten Wolff, Daniel Bourquain, Jessica Schulz, and Christian Mache from the Robert-Koch Institute and Martin Beer from the Friedrich Loeffler Institute (FLI) for providing isolates of SARS-CoV-2 variants. We thank Anna Kraft and Gabriele Czerwinski (both FLI) for support in the preparation of samples for pathology, and Catherine Hambly (University of Aberdeen) for help with daily energy expenditure measurements. We would like to thank Cathrin Bierwirth (University Medical Center Göttingen), Isabell Schulz, Anne-Kathrin Donner, and Frank-Thorben Peters for excellent technician assistance and Jasmin Fertey and Alexandra Rockstroh for providing the virus stocks for the mice experiment (Fraunhofer Institute IZI Leipzig). We acknowledge support by the Open Access Publication Funds of the Göttingen University. KMS was a member of the Göttingen Graduate School GGNB during this work. 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 (WO2012/001,148, 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 analogs to treat viral infections, including COVID-19 (inventors: MD, KMS, and AD). The other authors declare no conflict of interest.

Data Availability Statement

All data reported in this paper will be shared by the Lead contact (Matthais Dobbelstein) upon request.

This paper does not report original code.

Any additional information required to reanalyze the data reported in this paper is available from the Lead contact upon request.

Keywords

Drugs
Virology

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Stegmann, K. M., Dickmanns, A., Heinen, N., Blaurock, C., Karrasch, T., Breithaupt, A., Klopfleisch, R., Uhlig, N., Eberlein, V., Issmail, L., Herrmann, S. T., Schreieck, A., Peelen, E., Kohlhof, H., Sadeghi, B., Riek, A., Speakman, J. R., Groß, U., Görlich, D., ... Dobbelstein, M. (2022). Inhibitors of dihydroorotate dehydrogenase cooperate with molnupiravir and N4-hydroxycytidine to suppress SARS-CoV-2 replication. iScience, 25(5), Article 104293. https://doi.org/10.1016/j.isci.2022.104293

Stegmann, KM, Dickmanns, A, Heinen, N, Blaurock, C, Karrasch, T, Breithaupt, A, Klopfleisch, R, Uhlig, N, Eberlein, V, Issmail, L, Herrmann, ST, Schreieck, A, Peelen, E, Kohlhof, H, Sadeghi, B, Riek, A, Speakman, JR, Groß, U, Görlich, D, Vitt, D, Müller, T, Grunwald, T, Pfaender, S, Balkema-Buschmann, A & Dobbelstein, M 2022, 'Inhibitors of dihydroorotate dehydrogenase cooperate with molnupiravir and N4-hydroxycytidine to suppress SARS-CoV-2 replication', iScience, vol. 25, no. 5, 104293. https://doi.org/10.1016/j.isci.2022.104293

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abstract = "The nucleoside analog N4-hydroxycytidine (NHC) is the active metabolite of the prodrug molnupiravir, which has been approved for the treatment of COVID-19. SARS-CoV-2 incorporates NHC into its RNA, resulting in defective virus genomes. Likewise, inhibitors of dihydroorotate dehydrogenase (DHODH) reduce virus yield upon infection, by suppressing the cellular synthesis of pyrimidines. Here, we show that NHC and DHODH inhibitors strongly synergize in the inhibition of SARS-CoV-2 replication in vitro. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC into nascent viral RNA. This concept is supported by the rescue of virus replication upon addition of pyrimidine nucleosides to the media. DHODH inhibitors increased the antiviral efficiency of molnupiravir not only in 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.",

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note = "Funding Information: We thank Thorsten Wolff, Daniel Bourquain, Jessica Schulz, and Christian Mache from the Robert-Koch Institute and Martin Beer from the Friedrich Loeffler Institute (FLI) for providing isolates of SARS-CoV-2 variants. We thank Anna Kraft and Gabriele Czerwinski (both FLI) for support in the preparation of samples for pathology, and Catherine Hambly (University of Aberdeen) for help with daily energy expenditure measurements. We would like to thank Cathrin Bierwirth (University Medical Center G{\"o}ttingen), Isabell Schulz, Anne-Kathrin Donner, and Frank-Thorben Peters for excellent technician assistance and Jasmin Fertey and Alexandra Rockstroh for providing the virus stocks for the mice experiment (Fraunhofer Institute IZI Leipzig). We acknowledge support by the Open Access Publication Funds of the G{\"o}ttingen University. KMS was a member of the G{\"o}ttingen Graduate School GGNB during this work. This work was funded by the COVID-19 Forschungsnetzwerk Niedersachsen (COFONI) to MD, by the Federal Ministry of Education and Research Germany ( Bundesministerium f{\"u}r 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 (WO2012/001,148, WO03006425). KMS, AD, and MD are employees of University Medical Center G{\"o}ttingen, which has signed a License Agreement with Immunic AG covering the combination of DHODH inhibitors and nucleoside analogs to treat viral infections, including COVID-19 (inventors: MD, KMS, and AD). The other authors declare no conflict of interest. ",

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AU - Blaurock, Claudia

AU - Karrasch, Tim

AU - Breithaupt, Angele

AU - Klopfleisch, Robert

AU - Uhlig, Nadja

AU - Eberlein, Valentina

AU - Issmail, Leila

AU - Herrmann, Simon T.

AU - Schreieck, Amelie

AU - Peelen, Evelyn

AU - Kohlhof, Hella

AU - Sadeghi, Balal

AU - Riek, Alexander

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AU - Groß, Uwe

AU - Görlich, Dirk

AU - Vitt, Daniel

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AU - Grunwald, Thomas

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N2 - The nucleoside analog N4-hydroxycytidine (NHC) is the active metabolite of the prodrug molnupiravir, which has been approved for the treatment of COVID-19. SARS-CoV-2 incorporates NHC into its RNA, resulting in defective virus genomes. Likewise, inhibitors of dihydroorotate dehydrogenase (DHODH) reduce virus yield upon infection, by suppressing the cellular synthesis of pyrimidines. Here, we show that NHC and DHODH inhibitors strongly synergize in the inhibition of SARS-CoV-2 replication in vitro. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC into nascent viral RNA. This concept is supported by the rescue of virus replication upon addition of pyrimidine nucleosides to the media. DHODH inhibitors increased the antiviral efficiency of molnupiravir not only in 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.

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Inhibitors of dihydroorotate dehydrogenase cooperate with molnupiravir and N4-hydroxycytidine to suppress SARS-CoV-2 replication

Abstract

Bibliographical note

Data Availability Statement

Keywords

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