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.

• Publikační typ
• časopisecké články MeSH
• preprinty MeSH

Protein arginine methyltransferases (PRMTs) are responsible for symmetric and asymmetric methylation of arginine residues of nuclear and cytoplasmic proteins. In the nucleus, PRMTs belong to important chromatin modifying enzymes of immense functional significance that affect gene expression, splicing and DNA repair. By time-lapse microscopy we have studied the sub-cellular localization and kinetics of PRMT1 after inhibition of PRMT1 and after irradiation. Both transiently expressed and endogenous PRMT1 accumulated in cytoplasmic bodies that were located in the proximity of the cell nucleus. The shape and number of these bodies were stable in untreated cells. However, when cell nuclei were microirradiated by UV-A, the mobility of PRMT1 cytoplasmic bodies increased, size was reduced, and disappeared within approximately 20 min. The same response occurred after γ-irradiation of the whole cell population, but with delayed kinetics. Treatment with PRMT1 inhibitors induced disintegration of these PRMT1 cytoplasmic bodies and prevented formation of 53BP1 nuclear bodies (NBs) that play a role during DNA damage repair. The formation of 53BP1 NBs was not influenced by PRMT1 overexpression. Taken together, we show that PRMT1 concentrates in cytoplasmic bodies, which respond to DNA injury in the cell nucleus, and to treatment with various PRMT1 inhibitors.

• MeSH
• 53BP1 MeSH
• chromozomální proteiny, nehistonové genetika   metabolismus   MeSH
• cytoplazma enzymologie   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• HeLa buňky MeSH
• intracelulární signální peptidy a proteiny genetika   metabolismus   MeSH
• lidé MeSH
• myši MeSH
• poškození DNA * MeSH
• proteinarginin-N-methyltransferasy antagonisté a inhibitory   genetika   metabolismus   MeSH
• represorové proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• ultrafialové záření * MeSH
• záření gama * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 53BP1 MeSH
• chromozomální proteiny, nehistonové MeSH
• DNA vazebné proteiny MeSH
• intracelulární signální peptidy a proteiny MeSH
• PRMT1 protein, human MeSH Prohlížeč
• Prmt1 protein, mouse MeSH Prohlížeč
• proteinarginin-N-methyltransferasy MeSH
• represorové proteiny MeSH
• TP53BP1 protein, human MeSH Prohlížeč
• Trp53bp1 protein, mouse MeSH Prohlížeč