Characterisation of benzimidazole binding with recombinant tubulin from Giardia duodenalis, Encephalitozoon intestinalis, and Cryptosporidium parvum

MacDonald, L.M., Armson, A., Thompson, R.C.A. and Reynoldson, J.A. (2004) Characterisation of benzimidazole binding with recombinant tubulin from Giardia duodenalis, Encephalitozoon intestinalis, and Cryptosporidium parvum. Molecular and Biochemical Parasitology, 138 (1). pp. 89-96.

Abstract

The binding kinetics of several benzimidazole compounds were determined with recombinant tubulin from benzimidazole-sensitive and -insensitive organisms. This study utilised the naturally occurring high efficacy of the benzimidazoles for the parasitic protozoa Giardia duodenalis and Encephalitozoon intestinalis, and low efficacy with Cryptosporidium parvum. Direct kinetic analysis of the benzimidazole-β-tubulin interaction was performed using a fluorescence-based quenching method to determine the apparent association (k on) and dissociation (k off) rate constants from which the affinity constant (K a) was calculated. The binding kinetics were determined with recombinant α- and β-tubulin from the parasitic protozoa with several benzimidazole R 2-carbamate analogues. The affinity constant for the binding of several benzimidazoles with β-tubulin from benzimidazole-sensitive protozoa was found to be significantly greater than binding to β-tubulin from benzimidazole-insensitive protozoa. Additionally, the high affinity of several benzimidazole derivatives (albendazole, fenbendazole, mebendazole) for monomeric β-tubulin and heterodimeric αβ-tubulin from benzimidazole-sensitive protozoa was also clearly demonstrated. The affinity constants determined with β-tubulin from G. duodenalis and E. intestinalis also supported the observed in vitro efficacy of these compounds. The binding characteristics of the benzimidazoles with the highest in vitro efficacy (albendazole, fenbendazole, mebendazole) was reflected in their high association and slow dissociation rates with the β-tubulin monomer or dimer from benzimidazole-sensitive protozoa compared with insensitive ones. Benzimidazole-bound αβ-tubulin heterodimers also had a significantly lower rate of microtubule assembly compared with benzimidazole-free αβ-heterodimers. The incorporation of benzimidazole-bound αβ-heterodimers into assembling microtubules was shown to arrest polymerisation in vitro although the addition of benzimidazole compounds to assembled microtubules did not result in depolymerisation. These findings indicate that a benzimidazole-β-tubulin cap may be formed at the growing end of the microtubule and this cap prevents elongation of the microtubule.