Development of an aggregation assay to screen FimH antagonists

α-d-Mannopyranosides are potent FimH antagonists, which inhibit the adhesion of Escherichia coli to highly mannosylated uroplakin Ia on the urothelium and therefore offer an efficient therapeutic opportunity for the treatment and prevention of urinary tract infection. For the evaluation of the therapeutic potential of FimH antagonists, their effect on the disaggregation of E. coli from Candida albicans and guinea pig erythrocytes (GPE) was studied. The mannose-specific binding of E. coli to yeast cells and erythrocytes is mediated by type 1 pili and can be monitored by aggregometry. Maximal aggregation of C. albicans or GPE to E. coli is reached after 600s. Then the FimH antagonist was added and disaggregation determined by light transmission over a period of 1400s. A FimH-deleted mutant of E. coli, which does not induce any aggregation, was used in a control experiment. The activities of FimH antagonists are expressed as IC50s, the half maximal inhibitory concentration of the disaggregation potential. n-Heptyl α-d-mannopyranoside (1) was used as a reference compound and exhibits an IC50 of 77.14μM , whereas methyl α-d-mannopyranoside (2) does not lead to any disaggregation at concentrations up to 800μM. o-Chloro-p-[N-(2-ethoxy-3,4-dioxocyclobut-1-enyl)amino]phenyl α-d-mannopyranoside (3) shows a 90-fold and 2-chloro-4-nitrophenyl α-d-mannopyranoside (4) a 6-fold increased affinity compared to 1. Finally, 4-nitrophenyl α-d-mannopyranoside (5) exhibits an activity similar to 1. As negative control, d-galactose (6) was used. The standardized aggregation assay generates concentration-dependent, reproducible data allowing the evaluation of FimH antagonists according to their potency to inhibit E. coli adherence and can therefore be employed to select candidates for experimental and clinical studies for treatment and prevention of urinary tract infections.