Forward genetic screening reveals that SB505124 blocks Toxoplasma gondii growth by simultaneously inhibiting distinct host- and parasite-encoded kinases

Toxoplasma gondii is an obligate-intracellular parasite that causes significant morbidity in humans and other vertebrates. Like most intracellular organisms, Toxoplasma relies on host processes for growth. Hypoxia-inducible factor-1 (HIF-1) is a host cell transcription factor that is activated by and required for the Toxoplasma growth at physiological oxygen levels. Pharmacological inhibition of host Activin-Like Kinase (ALK) receptors 4, 5, and 7 by the small molecule SB505124 potently blocks the ability of Toxoplasma to activate HIF1 and replicate. Remarkably, SB505124 caused an even greater growth defect than loss of HIF-1. Therefore we hypothesized that SB505124 may inhibit an essential parasite gene in addition to blocking HIF-1 activation. To determine whether SB505124 also inhibited a parasite-encoded target, we used forward genetic screening to isolate parasites that could tolerate lethal doses of SB505124. We used whole-genome sequencing identify candidate SB505124-Resistance (SBR) mutations. We found that all of our SBR parasites had coding mutations in Toxoplasma MAP kinase, TgMAPK1. Allelic replacement of these coding mutations rescued growth in SB505124-treated parasites. Further biochemical analyses revealed that TgMAPK1 kinase activity is directly inhibited by SB505124. SB505124 independently impacts TgMAPK1 and ALK 4,5,7 signaling since drug resistant parasites could not activate HIF-1 in the presence of SB505124 or grow in HIF-1 deficient cells. Functionally, TgMAPK1 localized to mitotic structures and inhibition of TgMAPK1 disrupted the parasite's cellular counting mechanism. Thus, these data identify SB505124 as a novel small molecule inhibitor that acts by inhibiting two distinct targets, host HIF-1 and TgMAPK1.