Malaria is a severe disease that presents a significant threat to human health. As resistance to current drugs continues to increase, there is an urgent need for new antimalarial medications. Aminoacyl-tRNA synthetases (aaRSs) represent promising targets for drug development. In this study, we identified Plasmodium falciparum tyrosyl-tRNA synthetase (PfTyrRS) as a potential target for antimalarial drug development through a comparative analysis of the amino acid sequences and three-dimensional structures of human and plasmodium TyrRS, with particular emphasis on differences in key amino acids at the aminoacylation site. A total of 2141 bioactive compounds were screened using a high-throughput thermal shift assay (TSA). Okanin, known as an inhibitor of LPS-induced TLR4 expression, exhibited potent inhibitory activity against PfTyrRS, while showing limited inhibition of human TyrRS. Furthermore, bio-layer interferometry (BLI) confirmed the high affinity of okanin for PfTyrRS. Molecular dynamics (MD) simulations highlighted the stable conformation of okanin within PfTyrRS and its sustained binding to the enzyme. A molecular docking analysis revealed that okanin binds to both the tyrosine and partial ATP binding sites of the enzyme, preventing substrate binding. In addition, the compound inhibited the production of Plasmodium falciparum in the blood stage and had little cytotoxicity. Thus, okanin is a promising lead compound for the treatment of malaria caused by P. falciparum.

Influenza viruses are characterized by their high variability and pathogenicity, and effective therapeutic options remain limited. Given these challenges, targeting host cell proteins that facilitate viral replication presents a promising strategy for antiviral drug discovery. In the present study, we observed a significant upregulation of Glycyl-tRNA synthetase (GlyRS) within 24 h post-PR8 virus infection. The inhibition of GlyRS expression in A549 cells resulted in a marked reduction in infection rates across multiple influenza virus strains, while the overexpression of GlyRS led to an increase in viral infectivity during the early stages of infection. These findings suggest that GlyRS plays a critical role in the replication of influenza virus. Accordingly, we screened for potential inhibitors targeting GlyRS and identified Lycobetaine and Scutellarein using a multifaceted approach. Through a combination of molecular dynamics simulations, we further elucidated the mechanisms of action and potential binding sites of these compounds. Both inhibitors effectively suppressed the replication of influenza viruses, and their antiviral activity was confirmed to be mediated by GlyRS targeting. Therefore, GlyRS inhibitors, such as Lycobetaine and Scutellarein, represent promising candidates for combating influenza infections and provide novel insights into the treatment of influenza and aaRS-related diseases, opening new avenues for the development of aaRS-targeted therapeutics.