UC Irvine

Toxoplasma gondii is an apicomplexan pathogen and food-borne protozoan parasite that may cause life-threatening infections in humans. There is a demand for new treatments for Toxoplasma and related parasites that inhibit additional molecular targets with improved tolerability. New candidate drugs for inhibition of parasite replication may be identified by repurposing established medications for new applications, screening compound libraries for desired activity, or by rational design of ligands for drug binding pockets. Tubulin and microtubules represent a conserved broad-spectrum drug target shared by all eukaryotes, including protozoan pathogens. Perturbation of dynamic microtubule assembly and disassembly impairs mitotic spindle function and therefore replication. Small structural differences between amino acids that line the taxane, colchicine, and pironetin tubulin binding sites may be leveraged to identify selective ligands for protozoan rather than vertebrate tubulin. Prior studies indicate that protozoan-specific tubulin-targeting compounds disrupt T. gondii replication. This thesis work evaluates the specificity and activity of candidate compounds for inhibition of tubulin biogenesis (clemastine), microtubule assembly (parabulin) or microtubule disassembly (MMV676477) in T. gondii. Compounds were evaluated for parasite activity by plaque assays, vital imaging and quantification of tubulin and microtubules. To assess collateral toxicity to vertebrate cells, microtubule density and viability assays were performed in parallel. Selection for resistance was used to analyze compound binding sites, mechanisms of action and likelihood of resistance arising in clinical settings. Because haploid T. gondii tachyzoites express one primary α-tubulin isotype (α1) and two co-expressed β-tubulin isotypes (β1 and β2), we used CRISPR-Cas9 to knock-out β2-tubulin so resistance mutations would arise in single α- and β- loci. Although selection for oryzalin resistance in a wildtype background produces parasites with diverse single α-tubulin mutations, independently selected lines derived from the β2-knockout exclusively harbor the αL136F mutation. Although it is possible to confer oryzalin resistance by introducing other missense mutations into β2-knockout parasites, the αL136F is associated with greatest fitness in this context and therefore is the sole substitution that arises from bulk selection. My collective studies validate tubulin as a broad-spectrum antiparasitic drug target and illuminate the complexities that underlie tubulin regulation and proteostasis in apicomplexan parasites.