Bumped Kinase Inhibitors Inhibit both <i>Toxoplasma gondii</i> MAPKL1 and CDPK1

A subset of bumped kinase inhibitors (BKIs) has been optimized to inhibit the calcium dependent protein kinase 1 (<i>Tg</i>CDPK1; TGGT1_301440) of <i>Toxoplasma gondii</i> and pathogenic <i>Cryptosporidium</i> species. Extensive preclinical development of BKIs has identified BKI-1748 as a highly effective lead compound for toxoplasmosis. Phenotypic and therapeutic effects of BKIs have suggested that BKIs may have targets other than <i>Tg</i>CDPK1. The mechanism of BKI-1748 action was further investigated using a forward genetic screen of chemical mutagenesis, selection of BKI-1748 resistant clones, and whole genome sequence analysis. Resistant clones were found to have single nucleotide changes in the ATP binding site of the <i>T. gondii</i> mitogen-activated protein kinase-like 1 gene (<i>Tg</i>MAPKL1; TGGT1_312570). One of the mutations found in the ATP binding pocket, Leu162Gln, was introduced into a wild-type strain, resulting in 2 to 6-fold resistance to BKI-1748 and a set of structurally varied BKIs. A strain with a <i>Tg</i>CDPK1 gatekeeper substitution, Gly128Met, was also created and demonstrated a similar degree of resistance. The combination of <i>Tg</i>MAPKL1 and <i>Tg</i>CDPK1 substitutions together in a single strain resulted in a substantial increase in resistance to BKIs, up to 157-fold. The identification of <i>Tg</i>MAPKL1 in addition to <i>Tg</i>CDPK1 as a target of BKIs provides a greater understanding of the BKI mechanism of action that is important for further therapeutic development and suggests a high genetic barrier to meaningful drug resistance for this promising class of compounds.