N4-Hydroxycytidine and Inhibitors of Dihydroorotate Dehydrogenase Synergistically Suppress SARS-CoV-2 Replication : [Preprint]

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).