DRUG SYNTHESIS, SELECTION AND REPOSITIONING TO PREVENT SHIGA TOXIN-INDUCED RENAL INJURY IN POSTINFECTIOUS HAEMOLYTIC UREMIC SYNDROME

Shiga toxin 2 (Stx2) is an AB5 holotoxin and the major virulence factor of Shiga toxin–producing Escherichia coli (STEC), the causative agent of hemolytic uremic syndrome (HUS), a disease for which no specific treatment is currently available. The B5 subunit mediates cellular entry, while the A subunit exerts N-glycosidase activity on the ribosome, triggering ribotoxic stress and apoptosis. The catalytic deamination critically involves residues Arg170 and Glu167. The aim of this work is to identify small molecules capable of binding to Stx2 and preventing toxin-induced cellular damage. Cell viability assays were performed in Vero and HK-2 cells exposed to Stx2 in the presence or absence of candidate compounds, using a neutral red assay. In parallel, steered molecular dynamics simulations were conducted using AMBER to investigate potential binding mechanisms to the Stx2A1 subunit. Recombinant Stx2A1 was expressed in E. coli C43 from a pET28a plasmid, purified by nickel-affinity chromatography, and its folding assessed by circular dichroism spectroscopy. From a virtual screening of approximately 20,000 compounds, nine candidates were selected for experimental evaluation. One FDA-approved compound, B18, significantly increased cell viability in both cell lines compared to Stx2 alone (p < 0.0001, n = 8, 10⁻¹ μM). A second compound, FS1, synthesized and derivatized in-house to improve solubility, also showed a significant protective effect (p < 0.005, n = 8, 1 μM). Molecular dynamics simulations suggest that B18 binds within the Stx2 active site, near Arg170 and Glu167, interacting with Tyr77 and Tyr114 in a druggable pocket. Ongoing cell-free assays aim to further characterize the direct interaction between the compounds and the Stx2A1 subunit.