N4-hydroxycytidine, the active compound of Molnupiravir, promotes SARS-CoV-2 mutagenesis and escape from a neutralizing nanobody

Arne Zibat; Xiaoxiao Zhang; Antje Dickmanns; Kim M Stegmann; Adrian W Dobbelstein; Halima Alachram; Rebecca Soliwoda; Gabriela Salinas; Uwe Groß; Dirk Görlich; Maik Kschischo; Bernd Wollnik; Matthias Dobbelstein

doi:10.1016/j.isci.2023.107786

N4-hydroxycytidine, the active compound of Molnupiravir, promotes SARS-CoV-2 mutagenesis and escape from a neutralizing nanobody

iScience. 2023 Aug 30;26(10):107786. doi: 10.1016/j.isci.2023.107786. eCollection 2023 Oct 20.

Authors

Arne Zibat¹, Xiaoxiao Zhang^{2

3}, Antje Dickmanns⁴, Kim M Stegmann⁴, Adrian W Dobbelstein⁵, Halima Alachram¹, Rebecca Soliwoda⁴, Gabriela Salinas⁶, Uwe Groß⁷, Dirk Görlich⁸, Maik Kschischo², Bernd Wollnik^{1

9}, Matthias Dobbelstein⁴

Affiliations

¹ Department of Human Genetics, University Medical Center Göttingen, 37073 Göttingen, Germany.
² Department of Mathematics and Technology, University of Applied Sciences Koblenz, 53424 Remagen, Germany.
³ Department of Informatics, Technical University of Munich, 81675 Munich, Germany.
⁴ Department of Molecular Oncology, Göttingen Center of Molecular Biosciences (GZMB), University Medical Center Göttingen, 37077 Göttingen, Germany.
⁵ Max Planck Institute for Biology, 72076 Tübingen, Germany.
⁶ NGS Integrative Genomics Core Unit, Department of Human Genetics, University Medical Center Göttingen, 37077 Göttingen, Germany.
⁷ Department of Medical Microbiology and Virology, University Medical Center Göttingen, 37075 Göttingen, Germany.
⁸ Max Planck Institute for Multidisciplinary Sciences, 37077 Göttingen, Germany.
⁹ Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, 37075 Göttingen, Germany.

Abstract

N4-hydroxycytidine (NHC), the active compound of the drug Molnupiravir, is incorporated into SARS-CoV-2 RNA, causing false base pairing. The desired result is an "error catastrophe," but this bears the risk of mutated virus progeny. To address this experimentally, we propagated the initial SARS-CoV-2 strain in the presence of NHC. Deep sequencing revealed numerous NHC-induced mutations and host-cell-adapted virus variants. The presence of the neutralizing nanobody Re5D06 selected for immune escape mutations, in particular p.E484K and p.F490S, which are key mutations of the Beta/Gamma and Omicron-XBB strains, respectively. With NHC treatment, nanobody resistance occurred two passages earlier than without. Thus, within the limitations of this purely in vitro study, we conclude that the combined action of Molnupiravir and a spike-neutralizing antagonist leads to the rapid emergence of escape mutants. We propose caution use and supervision when using Molnupiravir, especially when patients are still at risk of spreading virus.

Keywords: Immunology; Pharmaceutical science; Structural biology; Virology.