Forward Genetic Screening Identifies a Small Molecule That Blocks Toxoplasma gondii Growth by Inhibiting Both Host- and Parasite-Encoded Kinases

The simultaneous targeting of host and pathogen processes represents an untapped approach for the treatment of intracellular infections. Hypoxia-inducible factor-1 (HIF-1) is a host cell transcription factor that is activated by and required for the growth of the intracellular protozoan parasite Toxoplasma gondii at physiological oxygen levels. Parasite activation of HIF-1 is blocked by inhibiting the family of closely related Activin-Like Kinase (ALK) host cell receptors ALK4, ALK5, and ALK7, which was determined in part by use of an ALK4,5,7 inhibitor named SB505124. Besides inhibiting HIF-1 activation, SB505124 also potently blocks parasite replication under normoxic conditions. To determine whether SB505124 inhibition of parasite growth was exclusively due to inhibition of ALK4,5,7 or because the drug inhibited a second kinase, SB505124-resistant parasites were isolated by chemical mutagenesis. Whole-genome sequencing of these mutants revealed mutations in the Toxoplasma MAP kinase, TgMAPK1. Allelic replacement of mutant TgMAPK1 alleles into wild-type parasites was sufficient to confer SB505124 resistance. SB505124 independently impacts TgMAPK1 and ALK4,5,7 signaling since drug resistant parasites could not activate HIF-1 in the presence of SB505124 or grow in HIF-1 deficient cells. In addition, TgMAPK1 kinase activity is inhibited by SB505124. Finally, mice treated with SB505124 had significantly lower tissue burdens following Toxoplasma infection. These data therefore identify SB505124 as a novel small molecule inhibitor that acts by inhibiting two distinct targets, host HIF-1 and TgMAPK1.
Author Summary Understanding how a compound blocks growth of an intracellular pathogen is important not only for developing these compounds into drugs that can be prescribed to patients, but also because these data will likely provide novel insight into the biology of these pathogens. Forward genetic screens are one established approach towards defining these mechanisms. But performing these screens with intracellular parasites has been limited not only because of technical limitations but also because the compounds may have off-target effects in either the host or parasite. Here, we report the first compound that kills a pathogen by simultaneously inhibiting distinct host- and parasite-encoded targets. Because developing drug resistance simultaneously to two targets is less likely, this work may highlight a new approach to antimicrobial drug discovery.