Inhibitors of eIF1A-ribosome interaction unveil uORF-dependent regulation of translation initiation and antitumor and antiviral effects.

During translation initiation, eIF1A binds the ribosome through its N- and C-terminal tails, but the functional importance of this temporal interaction in mammalian cells is lacking. Using a high-throughput drug screen targeting eIF1A-RPS10 interaction, we identified inhibitors (1Ais) for eIF1A, RPS10, or both. Applying 1Ais in biochemical assays along specific and global translation experiments, we confirmed known functions of eIF1A and uncovered new roles for both eIF1A and RPS10. Specifically, the eIF1A N-terminal tail (NTT) binding inhibitors revealed the requirement of eIF1A for translation re-initiation. Moreover, a cytosine at position +5 relative to the start codon AUG, located near eIF1A-NTT in the 48S structure, enhances sensitivity to 1Ais, suggesting that the initiating ribosome recognizes a broader AUG context than the conventional Kozak. Additionally, eIF1A-specific 1Ais predominately affect cancer-related pathways. In xenograft models of ovarian cancer, these 1Ais reduced tumor growth without apparent toxicity. Furthermore, inhibition of RPS10, but not eIF1A, modulates a context-dependent regulatory translation initiation at CUG codon of SARS-CoV-2 and impedes infection. Our study underscores 1Ais as effective means to study the role of eIF1A and RPS10 in translation and suggests their targeted inhibition as potential therapies for cancer and viral infections.