Benzimidazole resistance-associated mutations improve the in silico dimerization of hookworm tubulin: an additional resistance mechanism?

Abstract

Background Benzimidazole resistance is conferred by mutations in the β-tubulin genes of helminths which result in diminished drug binding with the expressed protein. The impact of these resistance-associated mutations on tubulin dimerization has not been fully explored in soil-transmitted helminths. Hence, this research aims to determine the consequences of these mutations on the in silico dimerization of hookworm α- and β-tubulins using open-source bioinformatics tools. Methods The α- and β-tubulin amino acid sequences of Ancylostoma ceylanicum were used to predict the hookworm tubulin heterodimer fold via AlphaFold 3. Modeled complexes underwent several protein structure quality assurance checks. The complex’s binding free energies, overall binding affinity, dissociation constant, and interacting amino acids were determined. The dimer’s structural flexibility and motion were simulated through molecular dynamics. Results Benzimidazole resistance-associated amino acid substitutions in the β-tubulin isotype 1 protein of hookworms conferred alterations in tubulin dimerization. The E198K, E198V, and F200Y mutations provided the strongest and most stable binding between the α and β subunits, surpassing that of the wild-type. The opposite was observed in complexes with the Q134H and F200L mutations. The molecular dynamics simulations showed that both wild-type and mutated tubulin dimers shared similar dynamic behavior, except for slight alterations in those that have the F200L and E198K mutations. Conclusion Beyond hindering benzimidazole binding to the β-tubulin subunit, resistance-associated mutations enhance the hookworm's capacity to resist treatment through strengthening and stabilizing tubulin dimer interactions. Meanwhile, other mutations diminish the dimer’s interaction which could be to the detriment of the hookworm. Our results provide novel insights into helminth tubulin dimerization that are useful for research on new anthelmintics targeting tubulin dimerization.