Altiratinib blocks and development by selectively targeting a spliceosome kinase.

The Apicomplexa comprise a large phylum of single-celled, obligate intracellular protozoa that include , , and spp., which infect humans and animals and cause severe parasitic diseases. Available therapeutics against these diseases are limited by suboptimal efficacy and frequent side effects, as well as the emergence and spread of resistance. We use a drug repurposing strategy and identify altiratinib, a compound originally developed to treat glioblastoma, as a promising drug candidate with broad spectrum activity against apicomplexans. Altiratinib is parasiticidal and blocks the development of intracellular zoites in the nanomolar range and with a high selectivity index when used against . We have identified PRP4K of as the primary target of altiratinib using genetic target deconvolution, which highlighted key residues within the kinase catalytic site that conferred drug resistance when mutated. We have further elucidated the molecular basis of the inhibitory mechanism and species selectivity of altiratinib for PRP4K and for its counterpart, CLK3. Our data identified structural features critical for binding of the other CLK3 inhibitor, TCMDC-135051. Consistent with the splicing control activity of this kinase family, we have shown that altiratinib can cause global disruption of splicing, primarily through intron retention in both and . Thus, our data establish parasitic PRP4K/CLK3 as a potential pan-apicomplexan target whose repertoire of inhibitors can be expanded by the addition of altiratinib.