Structure of SARS-CoV-2 MTase nsp14 with the inhibitor STM957 reveals inhibition mechanism that is shared with a poxviral MTase VP39
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PROVIDER: S-EPMC11345338 | biostudies-literature | 2024 Jul
REPOSITORIES: biostudies-literature
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Project description:Upon SARS-CoV-2 infection, patients with severe forms of COVID-19 often suffer from a dysregulated immune response and hyperinflammation. Aberrant expression of cytokines and chemokines is associated with strong activation of the immunoregulatory transcription factor NF-κB, which can be directly induced by the SARS-CoV-2 protein NSP14. Here, we use NSP14 mutants and generated cells with host factor knockouts (KOs) in the NF-κB signaling pathways to characterize the molecular mechanism of NSP14-induced NF-κB activation. We demonstrate that full-length NSP14 requires methyltransferase (MTase) activity to drive NF-κB induction. NSP14 WT, but not an MTase-defective mutant, is poorly expressed and inherent post-translational instability is mediated by proteasomal degradation. Binding of SARS-CoV-2 NSP10 or addition of the co-factor S-adenosylmethionine (SAM) stabilizes NSP14 and augments its potential to activate NF-κB. Using CRISPR/Cas9-engineered KO cells, we demonstrate that NSP14 stimulation of canonical NF-κB activation relies on NF-κB factor p65/RELA downstream of the NEMO/IKK complex, while c-Rel or non-canonical RelB are not required to induce NF-κB transcriptional activity. However, NSP14 overexpression is unable to induce canonical IκB kinase β (IKKβ)/NF-κB signaling and in co-immunoprecipitation assays we do not detect stable associations between NSP14 and NEMO or p65, suggesting that NSP14 activates NF-κB indirectly through its methyltransferase activity. Taken together, our data provide a framework how NSP14 can augment basal NF-κB activation, which may enhance cytokine expression in SARS-CoV-2 infected cells.
| S-EPMC10776897 | biostudies-literature
Project description:Monkeypox is a disease with pandemic potential. It is caused by the monkeypox virus (MPXV), a double-stranded DNA virus from the Poxviridae family, that replicates in the cytoplasm and must encode for its own RNA processing machinery including the capping machinery. Here, we present crystal structures of its 2'-O-RNA methyltransferase (MTase) VP39 in complex with the pan-MTase inhibitor sinefungin and a series of inhibitors that were discovered based on it. A comparison of this 2'-O-RNA MTase with enzymes from unrelated single-stranded RNA viruses (SARS-CoV-2 and Zika) reveals a conserved sinefungin binding mode, implicating that a single inhibitor could be used against unrelated viral families. Indeed, several of our inhibitors such as TO507 also inhibit the coronaviral nsp14 MTase.
| S-EPMC10116469 | biostudies-literature
Project description:The emergence of SARS-CoV-2 variants and drug-resistant mutants underscores the urgent need for novel antiviral therapeutics. SARS-CoV-2 NSP14, an N7-guanosine methyltransferase, plays a critical role in viral RNA capping, enabling viral replication and immune evasion. While NSP14 has emerged as a promising drug target, its role in host-virus crosstalk and the cellular consequences of NSP14 inhibition remain poorly understood. Here, we present the identification and characterization of C10, a highly potent and selective first-in-class non-nucleoside inhibitor of the NSP14 S-adenosylmethionine (SAM)-binding pocket. C10 demonstrates robust antiviral activity against SARS-CoV-2, including its variants, with EC50 values ranging from 64.03 to 301.9 nM, comparable to the FDA-approved drug remdesivir in our cell-based assays. C10 also exhibits broad-spectrum activity against other betacoronaviruses and directly suppresses SARS-CoV-2 genomic replication. C10 specifically reversed NSP14-mediated alterations in host transcriptome and restored host cell cycle progression disrupted by NSP14. The antiviral efficacy of C10 was further validated in a transgenic mouse model of SARS-CoV-2 infection. Our findings highlight C10 as a promising candidate for the development of effective treatments against SARS-CoV-2 and its emerging variants. This study also uncovers a novel mechanism of NSP14 in SARS-CoV-2 pathogenesis and its therapeutic potential, providing insights that may extend to other viral capping methyltransferases.
2025-09-06 | GSE304814 | GEO
Project description:The emergence of SARS-CoV-2 variants and drug-resistant mutants necessitates additional antivirals. SARS-CoV-2 NSP14 N7-guanosine methyltransferase is responsible for viral RNA capping, facilitating replication and evading immune detection. NSP14 has emerged as a promising drug target, but the role of NSP14 in host-virus crosstalk and the cellular effects of NSP14 inhibition are poorly understood. Here, we performed structure-based virtual screen to identify non-nucleoside inhibitors targeting NSP14 SAM-binding pocket. Hit to Lead optimization resulted in the development of C10 that potently inhibited SARS-CoV-2 and variants with the EC50 values from 64.03 to 301.9 nM, comparable to FDA-approved drug remdesivir in our cell-based model. C10 is a selective inhibitor of β-coronavirus NSP14 and directly suppresses SARS-CoV-2 replication, as demonstrated by a SARS-CoV-2 replicon system. C10 specifically reversed NSP14-mediated host transcriptome alterations and, phenotypically, restored host cell cycle progression disrupted by NSP14. The antiviral efficacy of C10 was further validated in a transgenic mouse model of SARS-CoV-2 infection. Our findings indicate C10 holds promise for developing effective treatments against SARS-CoV-2 and emerging variants, as well as uncover a novel pathogenic role of NSP14 beyond its function in viral RNA capping, which may be also adaptable to other viral capping methyltransferase.
2025-09-06 | GSE290079 | GEO
Project description:The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has led to significant global morbidity and mortality. A crucial viral protein, the non-structural protein 14 (nsp14), catalyzes the methylation of viral RNA and plays a critical role in viral genome replication and transcription. Due to the low mutation rate in the nsp region among various SARS-CoV-2 variants, nsp14 has emerged as a promising therapeutic target. However, discovering potential inhibitors remains a challenge. In this work, we introduce a computational pipeline for the rapid and efficient identification of potential nsp14 inhibitors by leveraging virtual screening and the NCI open compound collection, which contains 250,000 freely available molecules for researchers worldwide. The introduced pipeline provides a cost-effective and efficient approach for early-stage drug discovery by allowing researchers to evaluate promising molecules without incurring synthesis expenses. Our pipeline successfully identified seven promising candidates after experimentally validating only 40 compounds. Notably, we discovered NSC620333, a compound that exhibits a strong binding affinity to nsp14 with a dissociation constant of 427 ± 84 nM. In addition, we gained new insights into the structure and function of this protein through molecular dynamics simulations. We identified new conformational states of the protein and determined that residues Phe367, Tyr368, and Gln354 within the binding pocket serve as stabilizing residues for novel ligand interactions. We also found that metal coordination complexes are crucial for the overall function of the binding pocket. Lastly, we present the solved crystal structure of the nsp14-MTase complexed with SS148 (PDB:8BWU), a potent inhibitor of methyltransferase activity at the nanomolar level (IC50 value of 70 ± 6 nM). Our computational pipeline accurately predicted the binding pose of SS148, demonstrating its effectiveness and potential in accelerating drug discovery efforts against SARS-CoV-2 and other emerging viruses.
| S-EPMC10592886 | biostudies-literature
Project description:The interaction of exoribonuclease (ExoN) nonstructural protein (NSP14) with NSP10 co-factors is crucial for high-fidelity proofreading activity of coronavirus replication and transcription. Proofreading function is critical for maintaining the large genomes to ensure replication proficiency; therefore, while maintaining the viral replication fitness, quick resistance has been reported to the nucleotide analogue (NA) drugs. Therefore, targeting the NSP14 and NSP10 interacting interface with small molecules or peptides could be a better strategy to obstruct replication processes of coronaviruses (CoVs). A comparative study on the binding mechanism of NSP10 with the NSP14 ExoN domain of SARS-CoV-2, SARS-CoV, MERS-CoV, and four SARS-CoV-2 NSP14mutant complexes has been carried out. Protein-protein interaction (PPI) dynamics, per-residue binding free energy (BFE) analyses, and the identification of interface hotspot residues have been studied using molecular dynamics simulations and various computational tools. The BFE of the SARS-CoV NSP14-NSP10 complex was higher when compared to novel SARS-CoV-2 and MERS. However, SARS-CoV-2 NSP14mutant systems display a higher BFE as compared to the wild type (WT) but lower than SARS-CoV and MERS. Despite the high BFE, the SARS-CoV NSP14-NSP10 complex appears to be structurally more flexible in many regions especially the catalytic site, which is not seen in SARS-CoV-2 and its mutant or MERS complexes. The significantly high residue energy contribution of key interface residues and hotspots reveals that the high binding energy between NSP14 and NSP10 may enhance the functional activity of the proofreading complex, as the NSP10-NSP14 interaction is essential in maintaining the stability of the ExoN domain for the replicative fitness of CoVs. The factors discussed for SARS-CoV-2 complexes may be responsible for NSP14 ExoN having a high replication proficiency, significantly leading to the evolution of new variants of SARS-CoV-2. The NSP14 residues V66, T69, D126, and I201and eight residues of NSP10 (L16, F19, V21, V42, M44, H80, K93, and F96) are identified as common hotspots. Overall, the interface area, hotspot locations, bonded/nonbonded contacts, and energies between NSP14 and NSP10 may pave a way in designing potential inhibitors to disrupt NSP14-NSP10 interactions of CoVs especially SARS-CoV-2.
| S-EPMC9397572 | biostudies-literature
Project description:The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3727 unique approved drugs and clinical compounds against SARS2 PLpro, identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent selfprocessing of nsp3 in cells, and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.
| S-SCDT-EMBOJ-2020-106275 | biostudies-other
Project description:SARS-CoV-2 NSP14 inhibitor exhibits potent antiviral activity and reverses NSP14-driven host modulation
| PRJNA1302786 | ENA
Project description:The search for new drugs against COVID-19 and its causative agent, SARS-CoV-2, is one of the major trends in the current medicinal chemistry. Targeting capping machinery could be one of the therapeutic concepts based on a unique mechanism of action. Viral RNA cap synthesis involves two methylation steps, the first of which is mediated by the nsp14 protein. Here, we rationally designed and synthesized a series of compounds capable of binding to both the S-adenosyl-l-methionine and the RNA-binding site of SARS-CoV-2 nsp14 N7-methyltransferase. These hybrid molecules showed excellent potency, high selectivity toward various human methyltransferases, nontoxicity, and high cell permeability. Despite the outstanding activity against the enzyme, our compounds showed poor antiviral performance in vitro. This suggests that the activity of this viral methyltransferase has no significant effect on virus transcription and replication at the cellular level. Therefore, our compounds represent unique tools to further explore the role of the SARS-CoV-2 nsp14 methyltransferase in the viral life cycle and the pathogenesis of COVID-19.
| S-EPMC10398685 | biostudies-literature
Project description:SARS-CoV-2 NSP14 Inhibitor Confers Potent Antiviral Efficacy and Reverses NSP14-triggered Host Cell Modulation
| PRJNA1226168 | ENA