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J Biol Chem
. 2025 Mar 4:108378.
doi: 10.1016/j.jbc.2025.108378. Online ahead of print. Covalent inhibition of the SARS-CoV-2 NiRAN domain via an active-site cysteine
Genaro Hernandez 1 , Adam Osinski 2 , Abir Majumdar 2 , Jennifer L Eitson 3 , Monika Antczak 4 , Krzysztof Pawłowski 2 , Hanspeter Niederstrasser 4 , Kelly A Servage 2 , Bruce Posner 4 , John W Schoggins 3 , Joseph M Ready 4 , Vincent S Tagliabracci 5
Affiliations
The kinase-like NiRAN domain of nsp12 in SARS-CoV-2 catalyzes the formation of the 5' RNA cap structure. This activity is required for viral replication, offering a new target for the development of antivirals. Here, we develop a high-throughput assay to screen for small molecule inhibitors targeting the SARS-CoV-2 NiRAN domain. We identified NCI-2, a compound with a reactive chloromethyl group that covalently binds to an active site cysteine (Cys53) in the NiRAN domain, inhibiting its activity. NCI-2 can enter cells, bind to, and inactivate ectopically expressed nsp12. A cryo-EM reconstruction of the SARS-CoV-2 replication-transcription complex (RTC) bound to NCI-2 offers a detailed structural blueprint for rational drug design. Although NCI-2 showed limited potency against SARS-CoV-2 replication in cells, our work lays the groundwork for developing more potent and selective inhibitors targeting the NiRAN domain. This approach presents a promising therapeutic strategy for effectively combating COVID-19 and potentially mitigating future coronavirus outbreaks.
Keywords: COVID-19; NiRAN; RNA capping; SARS-CoV-2; drug discovery; drug screening; enzyme inhibitor; enzyme structure; inhibitor; pseudokinase.
. 2025 Mar 4:108378.
doi: 10.1016/j.jbc.2025.108378. Online ahead of print. Covalent inhibition of the SARS-CoV-2 NiRAN domain via an active-site cysteine
Genaro Hernandez 1 , Adam Osinski 2 , Abir Majumdar 2 , Jennifer L Eitson 3 , Monika Antczak 4 , Krzysztof Pawłowski 2 , Hanspeter Niederstrasser 4 , Kelly A Servage 2 , Bruce Posner 4 , John W Schoggins 3 , Joseph M Ready 4 , Vincent S Tagliabracci 5
Affiliations
- PMID: 40049411
- DOI: 10.1016/j.jbc.2025.108378
The kinase-like NiRAN domain of nsp12 in SARS-CoV-2 catalyzes the formation of the 5' RNA cap structure. This activity is required for viral replication, offering a new target for the development of antivirals. Here, we develop a high-throughput assay to screen for small molecule inhibitors targeting the SARS-CoV-2 NiRAN domain. We identified NCI-2, a compound with a reactive chloromethyl group that covalently binds to an active site cysteine (Cys53) in the NiRAN domain, inhibiting its activity. NCI-2 can enter cells, bind to, and inactivate ectopically expressed nsp12. A cryo-EM reconstruction of the SARS-CoV-2 replication-transcription complex (RTC) bound to NCI-2 offers a detailed structural blueprint for rational drug design. Although NCI-2 showed limited potency against SARS-CoV-2 replication in cells, our work lays the groundwork for developing more potent and selective inhibitors targeting the NiRAN domain. This approach presents a promising therapeutic strategy for effectively combating COVID-19 and potentially mitigating future coronavirus outbreaks.
Keywords: COVID-19; NiRAN; RNA capping; SARS-CoV-2; drug discovery; drug screening; enzyme inhibitor; enzyme structure; inhibitor; pseudokinase.