Identification of two Anti-Stx2 compounds: Stx2A expression and purification for drug binding studies

Shiga toxin–producing Escherichia coli (STEC) is a major foodborne pathogen associated with hemorrhagic colitis and hemolytic uremic syndrome (HUS), a disease that is endemic in Argentina. Among Shiga toxins, Stx2 is the most clinically relevant and there is currently no specific treatment available for HUS. In this work, we aimed to identify small molecules with anti-Stx2 activity using a structure-based drug design (SBDD) approach. A library of 20,105 compounds was screened through molecular docking and molecular dynamics (MD) simulations. Binding free energies were calculated and refined using multiple methods (MD, PBSA, and Jarzynski), leading to the selection of two candidate compounds: B18, an FDA-approved drug, and FS1, a synthesized and derivatized molecule with improved solubility. In vitro assays showed that both compounds significantly increased the viability of VERO cells exposed to Stx2. FS1 at 1 μM increased cell viability by 42% (p < 0.005, n = 8), while B18 at 10⁻¹ μM increased viability by 56% (p < 0.0001, n = 8), compared to Stx2 treatment alone (IC50 = 1 ng/mL). MD simulations suggest that B18 acts as a competitive inhibitor, binding near the catalytically relevant residues Arg170 and Glu167 of the Stx2 A subunit. To further investigate the Stx2–drug interaction mechanism, the Stx2A1 subunit was expressed using a pET28+ plasmid. Multiple expression conditions, plasmids, and E. coli strains were evaluated to optimize protein yield while maintaining host viability. The activity of purified Stx2A1 is currently being assessed using a cell-free luciferase inhibition assay, and circular dichroism experiments are underway to evaluate protein folding and potential interactions with the selected compounds.