Advanced piperazine-containing inhibitors target microbial β-glucuronidases linked to gut toxicity

Leveraging medicinal chemistry and structural biology to optimize microbial GUS inhibitors to UNC10206581, which displays potent L1 and FMN GUS activity – the structural subclasses linked to GI toxicity via small molecule glucuronide reactivation. The gut microbiome plays critical roles in human homeostasis, disease progression, and pharmacological efficacy through diverse metabolic pathways. Gut bacterial β-glucuronidase (GUS) enzymes reverse host phase 2 metabolism, in turn releasing active hormones and drugs that can be reabsorbed into systemic circulation to affect homeostasis and promote toxic side effects. The FMN-binding and loop 1 gut microbial GUS proteins have been shown to drive drug and toxin reactivation. Here we report the structure–activity relationships of two selective piperazine-containing bacterial GUS inhibitors. We explore the potency and mechanism of action of novel compounds using purified GUS enzymes and co-crystal structures. Our results establish the importance of the piperazine nitrogen placement and nucleophilicity as well as the presence of a cyclohexyl moiety appended to the aromatic core. Using these insights, we synthesized an improved microbial GUS inhibitor, UNC10206581, that potently inhibits both the FMN-binding and loop 1 GUS enzymes in the human gut microbiome, does not inhibit bovine GUS, and is non-toxic within a relevant dosing range. Kinetic analyses demonstrate that UNC10206581 undergoes a slow-binding and substrate-dependent mechanism of inhibition similar to that of the parent scaffolds. Finally, we show that UNC10206581 displays potent activity within the physiologically relevant systems of microbial cultures and human fecal protein lysates examined by metagenomic and metaproteomic methods. Together, these results highlight the discovery of more effective bacterial GUS inhibitors for the alleviation of microbe-mediated homeostatic dysregulation and drug toxicities and potential therapeutic development.