STRUCTURAL FOUNDATION FOR THE DESIGN OF RECEPTOR ANTAGONISTS TARGETING ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN

Background: Escherichia coli heat-labile enterotoxin (LT) is the causa tive agent of traveller's diarrhoea, and it is also responsible for th e deaths of hundreds of thousands of children per year in developing c ountries. LT is highly homologous in sequence, structure and function to cholera toxin (CT). Both toxins attack intestinal epithelial cells via specific binding to the branched pentasaccharide of ganglioside G( M1) at the cell surface. A receptor-binding antagonist which blocked t his interaction would potentially constitute a prophylactic drug confe rring protection both against the severe effects of cholera itself and against the milder but more common disease caused by LT. Results: Fou r derivatives of the simple sugar galactose, members of a larger serie s of receptor antagonists identified by computer modeling and competit ive binding studies, have been co-crystallized with either the full LT AB(5) holotoxin or the LT B pentamer. These crystal structures have p rovided detailed views of the toxin in complex with each of the four a ntagonists: melibionic acid at 2.8 A resolution, lactulose at 2.65 Ang strom resolution, metanitrophenylgalactoside (MNPG) at 2.2 Angstrom re solution and thiodigalactoside (TDG) at 1.7 Angstrom resolution. The b inding mode of each galactose derivative was observed 5-15 times, depe nding on the number of crystallographically independent toxin B pentam ers per asymmetric unit. There is a remarkable consistency, with one i mportant exception, in the location and hydrogen-bonding involvement o f well-ordered water molecules at the receptor-binding site. Conclusio ns: The bound conformations of these receptor antagonist compounds pre serve the toxin-galactose interactions previously observed for toxin-s ugar complexes, but gain additional favorable interactions. The highes t affinity compound, MNPG, is notable in that it displaces a water mol ecule that is observed to be well-ordered in all other previous and cu rrent crystal structures of toxin-sugar complexes. This could be a fav orable entropic factor contributing to the increased affinity. The hig hest affinity members of the present set of antagonists (MNPG and TDG) bury roughly half (400 Angstrom(2)) Of the binding-site surface cover ed by the full receptor G(M1) pentasaccharide, despite being considera bly smaller. This provides an encouraging basis for the creation of su bsequent generations of derived compounds that can compete effectively with the natural receptor.