Bumped Kinase Inhibitors (BKI) inhibit Toxoplasma gondii MAPKL-1 and CDPK1

Bumped Kinase Inhibitors (BKIs) based on a 5‑aminopyrazole-4-carboxamide (AC) core are a series of compounds that can be optimized to inhibit the calcium-dependent protein kinase 1 (CDPK1) of Toxoplasma gondii, Cryptosporidium species, and other apicomplexan pathogens. Extensive preclinical development of BKIs has identified AC-BKI-1748 as a promising lead compound for toxoplasmosis that is highly effective and non-toxic in animal models. Phenotypic and therapeutic effects of BKIs and previous experiments have suggested other potential inhibitory mechanisms of action in addition to TgCDPK1. The mechanism of BKI-1748 was therefore investigated using a forward genetic screen of chemical mutagenesis, selection of BKI-1748 resistant clones, and whole genome sequencing. Three clones were isolated with unique single nucleotide changes in the ATP binding site of T. gondii mitogen-activated protein kinase-like 1 (MAPKL1) gene in BKI-1748 resistant clones. One of these mutations, Leu162Gln, was introduced into a wild-type strain, resulting in 2- to 9-fold resistance to BKI-1748 and other BKIs including 1-NM-PP1. Because these compounds also were potent inhibitors of the isolated TgCDPK1 enzyme, we introduced a Gly128Met substitution into the TgCDPK1 gatekeeper position and found 2- to 8-fold resistance across BKIs. To further investigate the significance of these targets, we created a clone with both the above substitutions, which demonstrated a 14-fold to 153-fold resistance to BKIs. The identification of TgMAPKL-1 and TgCDPK1 as dual targets of multiple BKIs provides a greater understanding of the BKI mechanism of action and suggests a high genetic barrier to meaningful drug resistance for this promising class of compounds.