Synergistic interference with SARS-CoV-2 replication by molnupiravir-derived N4-hydroxycytidine and inhibitors of CTP synthetase in cell culture

Kim M Stegmann; Antje Dickmanns; Hannah L Fuchs; David Scheibner; Björn-Patrick Mohl; Florian R Moeselaken; Wencke Reineking; Theresa Störk; Asisa Volz; Philip A Beer; Andrew E Parker; Veronika Pilchova; Christian Meyer Zu Natrup; Maren von Köckritz-Blickwede; Wolfgang Baumgärtner; Anne Balkema-Buschmann; Matthias Dobbelstein

doi:10.1016/j.virol.2025.110598

Synergistic interference with SARS-CoV-2 replication by molnupiravir-derived N⁴-hydroxycytidine and inhibitors of CTP synthetase in cell culture

Virology. 2025 Jun 6:610:110598. doi: 10.1016/j.virol.2025.110598. Online ahead of print.

Authors

Kim M Stegmann¹, Antje Dickmanns¹, Hannah L Fuchs¹, David Scheibner², Björn-Patrick Mohl², Florian R Moeselaken³, Wencke Reineking³, Theresa Störk³, Asisa Volz⁴, Philip A Beer⁵, Andrew E Parker⁵, Veronika Pilchova⁶, Christian Meyer Zu Natrup⁴, Maren von Köckritz-Blickwede⁶, Wolfgang Baumgärtner³, Anne Balkema-Buschmann², Matthias Dobbelstein⁷

Affiliations

¹ Institute of Molecular Oncology, Göttingen Center of Molecular Biosciences (GZMB), University Medical Center Göttingen, Justus von Liebig Weg 11, 37077, Göttingen, Germany.
² Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Suedufer 10, Insel Riems, 17493, Greifswald, Germany.
³ Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, 30559, Hannover, Germany.
⁴ Research Center for Emerging Infections and Zoonosis (RIZ), University of Veterinary Medicine Hannover, Bünteweg 17, 30559, Hannover, Germany; Institute of Virology, University of Veterinary Medicine Hannover, Bünteweg 17, 30559, Hannover, Germany.
⁵ Step Pharma, Technoparc du Pays-de-Gex, Saint-Genis-Pouilly, France.
⁶ Research Center for Emerging Infections and Zoonosis (RIZ), University of Veterinary Medicine Hannover, Bünteweg 17, 30559, Hannover, Germany; Institute of Biochemistry, University of Veterinary Medicine Hannover, Bünteweg 17, 30559, Hannover, Germany.
⁷ Institute of Molecular Oncology, Göttingen Center of Molecular Biosciences (GZMB), University Medical Center Göttingen, Justus von Liebig Weg 11, 37077, Göttingen, Germany; Max Planck Institute for Multidisciplinary Sciences, Am Fassberg 11, 37077, Göttingen, Germany. Electronic address: mdobbel@uni-goettingen.de.

PMID: 40561865
DOI: 10.1016/j.virol.2025.110598

Abstract

N⁴-hydroxycytidine (NHC), the active metabolite of molnupiravir, is incorporated into nascent RNA of SARS-CoV-2 and interferes with subsequent virus replication. We have previously described synergy between NHC and inhibitors of dehydroorotate dehydrogenase (DHODH), an enzyme required for pyrimidine synthesis. Upon DHODH inhibition, the lack of endogenous pyrimidines conceivably enhances NHC incorporation. However, the question remains whether preventing the synthesis of just one pyrimidine base, cytidine, might as well augment the antiviral efficacy of NHC. We tested this by inhibiting CTP synthetases (CTPSs), the cellular enzymes that directly catalyze the synthesis of a cytidine nucleotide. We observed that inhibitors of CTP synthetase (CTPSis), namely cyclopentenyl cytosine (CPEC) as well as STP938 and STP720, display a strong synergy with NHC for diminishing SARS-CoV-2 replication in cell culture, as shown earlier for DHODH inhibitors. NHC and CTPSis in combination prevented the cytopathic effect of SARS-CoV-2 and strongly reduced the release of viral RNA and infectious particles, as well as the synthesis of viral proteins. This combination was also active against an Omicron variant of SARS-CoV-2. Addition of cytidine, but not uridine, rescued virus growth under these conditions. Surprisingly, this synergy was not confirmed in the SARS-CoV-2 animal model in Syrian hamsters. While treatment with the CTPS1 inhibitor STP938 alone strongly diminished virus propagation and COVID pathology, addition of molnupiravir did not augment this effect and even counteracted the benefits of STP938 in vivo. We propose that, if further developed, CTPS inhibitors might represent candidates for antiviral therapy.

Keywords: COVID-19; CTP synthase; CTP synthetase (CTPS); Calu-3cells; Coronavirus; Cyclopentenyl cytosine (CPEC); Cytidine; EIDD-1931; EIDD-2801; Emorivir; Lagevrio; MK-4482; Molnupiravir; Monuvir; N(4)-hydroxycytidine (NHC); Nucleocapsid; Pyrimidine synthesis; RNA replication; SARS-CoV-2; STP720; STP938; Spike; Syrian gold hamster; Uridine; Vero E6 cells.