In Silico Discovery and Validation of Amide Based Small Molecule Targeting the Enzymatic Site of Shiga Toxin

Shiga
toxin (Stx), a category B biothreat agent, is a ribosome
inactivating protein and toxic to human and animals. Here, we designed
and synthesized small molecules that block the active site of the
Stx A subunit. On the basis of binding energy, 20 molecules were selected
for synthesis and evaluation. These molecules were primarily screened
using fluorescence-based thermal shift assay and in vitro in Vero cells. Among 32 molecules (including 12 reported), six molecules
offered protection with IC50 of 2.60–23.90 μM.
4-Nitro-N-[2-(2-phenylsulfanyl­ethylamino)­ethyl]­benzamide
hydrochloride is the most potent inhibitor with IC50 at
7.96 μM and selectivity index of 22.23 and is better than any
known small molecule inhibitor of Stx. Preincubation with Stx offered
full protection against Shiga toxin in mice. Surface plasmon resonance
assay further confirmed that these molecules bind specifically to
Stx A subunit. Further optimization is continued to identify a potential
candidate which will be in vivo effective.