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Apicomplexan parasites, such as Toxoplasma gondii, Plasmodium spp., Babesia spp., and Cryptosporidium spp., cause significant morbidity and mortality. Existing treatments are problematic due to toxicity and the emergence of drug-resistant parasites. Because protozoan tubulin can be selectively disrupted by small molecules to inhibit parasite growth, we assembled an in vitro testing cascade to fully delineate effects of candidate tubulin-targeting drugs on Toxoplasma gondii and vertebrate host cells. Using this analysis, we evaluated clemastine, an antihistamine that has been previously shown to inhibit Plasmodium growth by competitively binding to the CCT/TRiC tubulin chaperone as a proof-of-concept. We concurrently analyzed astemizole, a distinct antihistamine that blocks heme detoxification in Plasmodium. Both drugs have EC50 values of ~2 µM and do not demonstrate cytotoxicity or vertebrate microtubule disruption at this concentration. Parasite subpellicular microtubules are shortened by treatment with either clemastine or astemizole but not after treatment with pyrimethamine, indicating that this effect is not a general response to antiparasitic drugs. Immunoblot quantification indicates that the total α-tubulin concentration of 0.02 pg/tachyzoite does not change with clemastine treatment. In conclusion, the testing cascade allows profiling of small-molecule effects on both parasite and vertebrate cell viability and microtubule integrity.

Infectious diseases caused by apicomplexan parasites remain a global public health threat. The presence of multiple ligand‐binding sites in tubulin makes this protein an attractive target for anti‐parasite drug discovery. However, despite remarkable successes as anti‐cancer agents, the rational development of protozoan parasite‐specific tubulin drugs has been hindered by a lack of structural and biochemical information on protozoan tubulins. Here, we present atomic structures for a protozoan tubulin and microtubule and delineate the architectures of apicomplexan tubulin drug‐binding sites. Based on this information, we rationally designed the parasite‐specific tubulin inhibitor parabulin and show that it inhibits growth of parasites while displaying no effects on human cells. Our work presents for the first time the rational design of a species‐specific tubulin drug providing a framework to exploit structural differences between human and protozoa tubulin variants enabling the development of much‐needed, novel parasite inhibitors., In an effort to discover novel drug‐scaffolds targeting unique parasite proteins and pathways, specific inhibition of parasite tubulin was achieved using structure‐guided rational drug design.