Mechanisms of Dinitroaniline Action, Selectivity and Resistance

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Microtubules (MTs) are essential components of all eukaryotic cells. Compounds that affect MT function are used to treat medical conditions including cancer, gout and helminth infection. Dinitroanilines (oryzalin, ethafluralin and trifluralin) disrupt MTs in plants and protozoa but are ineffective against vertebrate or fungal MTs. These compounds inhibit growth of diverse protozoan parasites (Trypanosoma spp., Leishmania spp., Entamoeba spp.,Plasmodium falcipamm, Cryptosporidium parvum and Toxoplasma gondii). Our previous studies used a combination of genetic analysis and computational biology to conclude that the dinitroanilines have a novel mechanism of action: they bind to a-tubulin and disrupt protofilament contacts. These conclusions evoke additional questions that we propose to address in this research. To begin with, our proposed dinitroaniline binding site is defined by amino acids that are not restricted to sensitive organisms and cannot explain why dinitroanilines only bind to plant or protozoan tubulin. We hypothesize that other residues that are restricted to plant/protozoan lineages influence the capacity of the binding site amino acids to interact with dinitroanilines. We will use directed changes to the Toxoplasma gondii and Saccharomyces cerevisiae a-tubulin genes to identify the basis of tubulin susceptibility and mechanisms of a-tubulin-based resistance. These data will be integrated with refined computational studies. Secondly, in previous work, we isolated a large number of dinitroaniline-resistant Toxoplasma lines which harbor point mutations to a-tubulin. We hypothesize that the a-tubulin mutations fall into two categories based on their underlying mechanism of action. Some of the mutations localize to regions of tubulin that are essential for dimer contacts within the MT lattice. We predict that these mutant tubulins continue to bind dinitroanilines but that the substitutions increase the dimer affinity within the MT lattice to compensate for the destabilizing effect of bound dinitroaniline. Other a-tubulin mutations localize to the region of our proposed binding site and we predict that these mutant a-tubulins have decreased affinity for dinitroanilines. We will measure dinitroaniline binding by isolated wild type and mutant Toxoplasma a-(3 tubulin dimers to assess whether the observed dinitroaniline affinities reflect our predicted resistance mechanisms. Lastly, all of the dinitroaniline resistant Toxoplasma lines that we have characterized to date have mutations to a-tubulin that confer resistance to 1-5 uM oryzalin. Parasites that have resistance to higher dinitroaniline concentrations (5 to >50 uM) harbor an additional non- tubulin mutation(s) in the mutant a-tubulin genetic background. We hypothesize that these other resistance alleles will have diverse activities including mutations to regulatory proteins that increase MT stability and mutations that decrease dinitroaniline concentration such as drug efflux pumps. We will use a "step-up" selection strategy to isolate these non- tubulin resistance alleles. In summary, we believe that further study of the dinitroanilines will elucidate new aspects of tubulin function and define novel ways to specifically disrupt parasite tubulins.