Kinetic insights into cholera toxin B-biomimetic glycan interactions

Enterotoxins cause severe diseases worldwide, exacerbated by limited access to clean water, food, and medical care. Cholera is a diarrheal disease driven by cholera enterotoxin (CTX) and is secreted by Vibrio cholerae in the small intestinal (SI) epithelium. CTX consists of an enzymatic A subunit (CTA) and a pentameric B subunit (CTB), which mediates cellular uptake by binding to glycans. While CTB primarily recognizes GM1 glycosphingolipid or galactose ligands, GM1 is undetectable in the human SI, and fucose ligands are a key alternative binding target. Previously, we developed linear norbornenyl glycopolymers as biomimetic inhibitors that block CTB binding through interchain crosslinking, forming CTB-glycopolymer aggregates. To assess the polymers’ potential efficacy under flow conditions, we evaluated CTB-glycopolymer binding kinetics under dynamic conditions using surface plasmon resonance (SPR). Copolymer with a random display of galactose and fucose (pGal50Fuc50) showed nanomolar avidity with stable complex formation driven primarily by a slow dissociation rate in the multivalent phase (kd1). In contrast, an equimolar mixture of homopolymers (pGal100 + pFuc100) exhibited similar binding avidity and comparable inhibitory efficacy but did not display slow-dissociation. Moreover, random copolymer pGal25Fuc75 dissociated slowly even from low-CTB-density surfaces. These results highlight the superior stability of copolymers in forming CTB–copolymer aggregates under low CTB abundance and flow conditions. These findings underscore the importance of co-displaying galactose and fucose for complex formation and for developing clinically-useful therapies.