Biochemical characterization of dinitroaniline-tubulin interactions

Dinitroanilines are small, synthetic molecules that selectively disrupt microtubules in plants and protozoa, and lack activity against vertebrate or fungal microtubules. This selective property is of interest for the development of anti-parasitic drugs to disrupt protozoan microtubules and block replication. Studies presented in this dissertation exploit expression of tubulin from Tetrahymena thermophila in order to characterize the biochemical properties of dinitroaniline interaction with sensitive tubulin and the altered features of tubulin bearing resistance mutations. Previous research has indicated that dinitroaniline analogs bearing meta amine substitutions have the greatest efficacy for inhibiting replication of the human parasite Toxoplasma gondii. Since T. gondii and other apicomplexans are obligate intracellular parasites which are not amenable to bulk culture, we used tubulin purified from the related alveolate T. thermophila to characterize dinitroaniline analog binding. These experiments indicate that the most effective compounds for inhibiting T. gondii growth show the highest affinity for Tetrahymena tubulin. In turn, these data indicate that the meta-substitution most likely influences dinitroaniline interaction with the tubulin binding site and not a non-specific property such as membrane permeability. Since Tetrahymena is a genetically tractable organism, it was exploited to express alpha-tubulin with Ile252Leu or Leu136Phe mutations (previously shown to confer dinitroaniline resistance in T. gondii) as the sole source of alpha-tubulin. The resulting T. thermophila lines are dinitroaniline resistant and serve as a source of purified tubulin for biochemical assays. Replacement of the wild-type alpha-tubulin gene with amino acid mutations Leu136Phe and Ile252Leu is sufficient to alter both dinitroaniline binding and assembly of tubulin in the presence of these compounds. Additionally, a line was generated that complements ATU1 null Tetrahymena with T. gondii alpha-tubulin to form functional dimers to next express true T. gondii alpha-beta dimers in Tetrahymena. To this end, inducible constructs were generated to express His6 tagged T. gondii beta-tubulin in the T. gondii alpha-tubulin-complemented Tetrahymena line. Completion of this line will permit future studies of interactions of tubulin binding drugs with functional T. gondii tubulin dimers.