Microbial β-glucuronidases (GUSs) cause severe gut toxicities that limit the efficacy of
cancer drugs and other
therapeutics. Selective inhibitors of bacterial GUS have been shown to alleviate these side effects. Using structural and chemical biology, mass spectrometry, and cell-based assays, we establish that
piperazine-containing GUS inhibitors intercept the glycosyl-
enzyme catalytic intermediate of these retaining glycosyl
hydrolases. We demonstrate that
piperazine-based compounds are substrate-dependent GUS inhibitors that bind to the GUS-GlcA catalytic intermediate as a
piperazine-linked
glucuronide (GlcA,
glucuronic acid). We confirm the GUS-dependent formation of inhibitor-
glucuronide conjugates by LC-MS and show that methylated
piperazine analogs display significantly reduced potencies. We further demonstrate that a range of approved
piperazine- and
piperidine-containing drugs from many classes, including those for the treatment of depression,
infection, and
cancer, function by the same mechanism, and we confirm through gene editing that these compounds selectively inhibit GUS in living bacterial cells. Together, these data reveal a unique mechanism of GUS inhibition and show that a range of
therapeutics may impact GUS activities in the human gut.