Human ribosome interactions reframe neomycin toxicity

Aminoglycosides like neomycin are widely used but clinically limited due to toxicity, traditionally attributed to mitoribosome inhibition. Here, we demonstrate that this assumption requires re-evaluation by comparing neomycin′s interactions with human ribosomes in vitro and in cells. While cryo-EM and biochemical assays reveal strong in vitro binding to both mitoribosomes and cytosolic ribosomes, especially at conserved regions such as the helix 44 (h44) decoding center despite sequence divergence, and H69. Cellular analyses show minimal impact on global translation, reduced occupancy on cytosolic ribosomes, and a complete absence of neomycin on mitoribosomes. These discrepancies suggest limited mitochondrial permeability rather than direct mitoribosome inhibition underlies neomycin′s toxicity. Our findings redefine the mechanistic basis of aminoglycosides side effects and call for a reassessment of their cellular targets.