3,5-Substituted phenyl galactosides as leads in designing effective cholera toxin antagonists; synthesis and crystallographic studies

Abstract With the aim of developing high-affinity mono and multivalent antagonists of cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) we are using the galactose portion of the natural receptor ganglioside GM1 as an anchoring fragment in structure-based inhibitor design efforts. In order to establish a better structure–activity relationship for guiding these studies, we designed and prepared a small focused library of twenty 3,5-substituted phenylgalactosides based on two previous leads. The compounds were tested for their ability to block CTB 5 binding to immobilized ganglioside receptor and compared to the two previous leads. The crystal structures of the most promising compounds bound to either CTB 5 or LTB 5 were then determined in order to understand the basis for affinity differences. The most potent new compound yielded a six-fold improvement over our benchmark lead m -nitrophenyl-α- d -galactopyranoside (MNPG), and a two-fold improvement in IC 50 over a newer MNPG derivative. These results support the notion that the m -nitrophenyl moiety of MNPG and its derivatives is an important element to retain in future optimization efforts. Additionally, a consensus binding-pocket for the alkylmorpholine or piperazine moiety present in all of the designed antagonists was established as an important area of the GM1 binding site to target in future work.