Krzysztof Urbanowicz, Mikolaj Opielka, Kim M Stegmann, Antje Dickmanns, Matthias Dobbelstein, Godefridus J Peters, Ryszard T Smoleński
{"title":"利用液相色谱-质谱法直接测定 SARS-CoV-2 感染宿主细胞核酸中 N4-羟基胞嘧啶的掺入量。","authors":"Krzysztof Urbanowicz, Mikolaj Opielka, Kim M Stegmann, Antje Dickmanns, Matthias Dobbelstein, Godefridus J Peters, Ryszard T Smoleński","doi":"10.1080/15257770.2024.2346550","DOIUrl":null,"url":null,"abstract":"<p><p>Molnupiravir, an orally administered prodrug of β-d-N4-hydroxycytidine (NHC), is incorporated into newly synthesized RNA by viral RNA-dependent RNA polymerase (RdRp). It is used for treatment of SARS-CoV-2 infections. Incorporation of NHC triphosphate into viral RNA inhibits replication of the virus, at least in part by introducing deleterious mutations. However, there is limited information on NHC incorporation into host RNA and reports on the risk of mutagenicity that molnupiravir/NHC pose to the host are conflicting. We used two liquid chromatography-mass spectrometry (LC-MS) methods to evaluate the incorporation of NHC into RNA and DNA of host Vero E6 cells in a SARS-CoV-2 infection model. To test this, host and viral RNA were degraded to their ribonucleosides, while host DNA was degraded to deoxyribonucleosides. Subsequently, nucleic acid constituents were analyzed by LC-MS, which offers specific, direct, and quantitative determination of incorporation. Our findings revealed concentration dependent NHC incorporation into host cell RNA in both infected and uninfected cell cultures, reaching a maximum of 1 in 7,093 bases. Analysis of host DNA revealed no presence of deoxy-N4-hydroxycytidine down to a detection limit of 1 in 133,000 bases. Our findings therefore suggest minimal to no NHC incorporation into host DNA, indicating a low probability of significant host cell mutagenicity associated with its use.</p>","PeriodicalId":19343,"journal":{"name":"Nucleosides, Nucleotides & Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":1.1000,"publicationDate":"2024-05-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Evaluation of N4-hydroxycytidine incorporation into nucleic acids of SARS-CoV-2-infected host cells by direct measurement with liquid chromatography-mass spectrometry.\",\"authors\":\"Krzysztof Urbanowicz, Mikolaj Opielka, Kim M Stegmann, Antje Dickmanns, Matthias Dobbelstein, Godefridus J Peters, Ryszard T Smoleński\",\"doi\":\"10.1080/15257770.2024.2346550\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Molnupiravir, an orally administered prodrug of β-d-N4-hydroxycytidine (NHC), is incorporated into newly synthesized RNA by viral RNA-dependent RNA polymerase (RdRp). It is used for treatment of SARS-CoV-2 infections. Incorporation of NHC triphosphate into viral RNA inhibits replication of the virus, at least in part by introducing deleterious mutations. However, there is limited information on NHC incorporation into host RNA and reports on the risk of mutagenicity that molnupiravir/NHC pose to the host are conflicting. We used two liquid chromatography-mass spectrometry (LC-MS) methods to evaluate the incorporation of NHC into RNA and DNA of host Vero E6 cells in a SARS-CoV-2 infection model. To test this, host and viral RNA were degraded to their ribonucleosides, while host DNA was degraded to deoxyribonucleosides. Subsequently, nucleic acid constituents were analyzed by LC-MS, which offers specific, direct, and quantitative determination of incorporation. Our findings revealed concentration dependent NHC incorporation into host cell RNA in both infected and uninfected cell cultures, reaching a maximum of 1 in 7,093 bases. Analysis of host DNA revealed no presence of deoxy-N4-hydroxycytidine down to a detection limit of 1 in 133,000 bases. Our findings therefore suggest minimal to no NHC incorporation into host DNA, indicating a low probability of significant host cell mutagenicity associated with its use.</p>\",\"PeriodicalId\":19343,\"journal\":{\"name\":\"Nucleosides, Nucleotides & Nucleic Acids\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.1000,\"publicationDate\":\"2024-05-13\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Nucleosides, Nucleotides & Nucleic Acids\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1080/15257770.2024.2346550\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Nucleosides, Nucleotides & Nucleic Acids","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1080/15257770.2024.2346550","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Evaluation of N4-hydroxycytidine incorporation into nucleic acids of SARS-CoV-2-infected host cells by direct measurement with liquid chromatography-mass spectrometry.
Molnupiravir, an orally administered prodrug of β-d-N4-hydroxycytidine (NHC), is incorporated into newly synthesized RNA by viral RNA-dependent RNA polymerase (RdRp). It is used for treatment of SARS-CoV-2 infections. Incorporation of NHC triphosphate into viral RNA inhibits replication of the virus, at least in part by introducing deleterious mutations. However, there is limited information on NHC incorporation into host RNA and reports on the risk of mutagenicity that molnupiravir/NHC pose to the host are conflicting. We used two liquid chromatography-mass spectrometry (LC-MS) methods to evaluate the incorporation of NHC into RNA and DNA of host Vero E6 cells in a SARS-CoV-2 infection model. To test this, host and viral RNA were degraded to their ribonucleosides, while host DNA was degraded to deoxyribonucleosides. Subsequently, nucleic acid constituents were analyzed by LC-MS, which offers specific, direct, and quantitative determination of incorporation. Our findings revealed concentration dependent NHC incorporation into host cell RNA in both infected and uninfected cell cultures, reaching a maximum of 1 in 7,093 bases. Analysis of host DNA revealed no presence of deoxy-N4-hydroxycytidine down to a detection limit of 1 in 133,000 bases. Our findings therefore suggest minimal to no NHC incorporation into host DNA, indicating a low probability of significant host cell mutagenicity associated with its use.
期刊介绍:
Nucleosides, Nucleotides & Nucleic Acids publishes research articles, short notices, and concise, critical reviews of related topics that focus on the chemistry and biology of nucleosides, nucleotides, and nucleic acids.
Complete with experimental details, this all-inclusive journal emphasizes the synthesis, biological activities, new and improved synthetic methods, and significant observations related to new compounds.