A small bioactive glycoside inhibits epsilon toxin and protects host cell death.

Clostridium perfringens Epsilon toxin is categorized as third most lethal bioterrorism agent by CDC, with no therapeutic counter measures available for humans. Here we have developed a high affinity inhibitory compound by synthesizing and evaluating the structure activity relationship (SAR) of a library of diverse Glycosides 1-12. SAR of glycosides-Etx heptamer revealed exceptionally strong H-bond interactions of Glycoside-4 with a druggable pocket in the oligomerization and beta-hairpin region of Etx. Analysis of its structure suggested that Glycoside-4 might self-aggregate to form a robust micelle like supra-molecular complex due to its linear side chain architecture, which was authenticated by fluorescence spectroscopy. Further, this micelle hinders the Etx monomer-monomer interaction required for oligomerization, validated by both surface plasmon resonance (SPR) and immunoblotting. This phenomenon in-turn leads to blockage of pore formation. Downstream evaluation revealed that Glycoside-4 effectively blocked cell death of Etx treated cultured primary cells and maintained cellular homeostasis; via disrupting oligomerization, blocking pore formation, restoring calcium homeostasis, stabilizing mitochondrial membrane and impairing HMGB1 translocation from nucleus-to-cytoplasm. Furthermore, a single dosage of Glycoside-4 protected the Etx-challenged mice and restored normal function to multiple organs. This work for the first-time reports a potent, nontoxic glycoside with strong ability to occlude toxin lethality representing it as bio-arm therapeutics against Etx-based biological threat.pmid: 31492678status: publishe