Glycosylation switches spiramycin from an antibiotic to a hepatoprotective agent by targeting GSTP1.

Glycosylation commonly inactivates macrolide antibiotics, and the resulting glycosides are largely dismissed as inactive waste products. Whether such molecules retain hidden therapeutic potential remains underexplored. Here, we address this question using spiramycin I 2'-O-glucopyranoside as a proof-of-concept model. The glycoside was enzymatically synthesized via OleD and structurally confirmed by NMR and HR-ESI-MS. Activity evaluation showed that glycosylation at the 2'-OH position abolished antibacterial activity, adding a new example to glycosylation-mediated antibiotic inactivation. To explore whether this inactivated glycoside harbors alternative functions, we performed in silico target fishing and reverse docking. These approaches identified glutathione S-transferase P (GSTP1) as a high-confidence target, a prediction further supported by molecular dynamics simulations and MM/PBSA calculations. Guided by this hypothesis, we evaluated the glycoside in an acetaminophen (APAP)-induced HepG2 hepatocyte injury model and found that it significantly attenuated cell death, whereas the parent antibiotic exacerbated cytotoxicity. Moreover, glycosylation led to a markedly higher solubility. These findings demonstrate that antibacterially inert glycosides can retain valuable non-antibiotic activities, exemplified here by hepatoprotection via GSTP1 engagement. Beyond this specific case, our study establishes a paradigm for repurposing inactivated antibiotic glycosides as a previously overlooked source of bioactive molecules, advocating for broader re-evaluation of such "waste" metabolites in drug discovery.