Isofagomine Derivatives as TcdB Glucosyltransferase Inhibitors

Clostridioides difficile (C. difficile) is the leading cause of hospital-acquired life-threatening diarrhea. C. difficile toxins TcdA and TcdB contain a glucosyltransferase domain (GTD) that glucosylates and inactivates host GTPases, disrupting the actin cytoskeleton and compromising epithelial integrity. TcdB, the most potent virulence factor, drives disease progression and is a high-priority target forC. difficiletreatment and prevention. The iminosugar isofagomine has been shown to inhibit the GTD activity of TcdB by an uncompetitive inhibition mechanism, but requires the uridine 5'-diphosphate (UDP) reaction product. Compound classes synthesized here, ranging from isofagomine analogues to acyclic mimics, probe which modifications can tap into UDP-binding energy to enhance inhibition. Structure-activity relationship studies of isofagomine derivatives demonstrate remarkable specificity for isofagomine and limited advantage in accessing the UDP-binding site. Fluorescence and absorbance assays allowed facile inhibition assessment of TcdB's UDP-glucose hydrolysis. The molecules reported here guide scaffolds for future catalytic site inhibitors.