Identify old drugs as selective bacterial β-GUS inhibitors by structural-based virtual screening and bio-evaluations.

Irinotecan (CPT-11) is a cytotoxic drug that has wide applicability and usage in cancer treatment. Despite its success, patients suffer dose-dependent diarrhea, limiting the drug's efficacy. No effective therapy is available for this unmet medical need. The bacterial β-glucuronidase (β-GUS) plays pivotal role in CPT-11-induced diarrhea (CID) via activating the non-toxic SN-38G to toxic SN-38 inside intestine. By using structural-based virtual screening, three old drugs (N-Desmethylclozapine, Aspartame, and Gemifloxacin) were firstly identified as selective bacterial β-GUS inhibitors. The IC50 values of the compounds in the enzyme-based and cell-based assays range from 0.0389 to 3.6040 and 0.0105 to 5.3730 μM, respectively. The compounds also showed good selectivity against mammalian β-GUS and no significant cytotoxicity in bacteria. Molecular docking and molecular dynamics simulations were performed to further investigate the binding modes of compounds with bacterial β-GUS. Binding free energy decomposition revealed that the compounds formed strong interactions with E413 in catalytic trail from primary monomer and F365' on the bacterial loop from the other monomer of bacterial β-GUS, explaining the selectivity against mammalian β-GUS. The old drugs identified here may be used as bacterial β-GUS inhibitors for CID or other bacterial β-GUS-related disorders.