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

<div><p>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 <i>Toxoplasma gondii</i> 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 <i>Toxoplasma</i> 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 <i>Toxoplasma</i> infection. These data therefore identify SB505124 as a novel small molecule inhibitor that acts by inhibiting two distinct targets, host HIF-1 and TgMAPK1.</p></div

Generation of SBR mutants.

<p>A. Relative plaque formation in HFFs was determined for each parasite strain in the presence of increasing concentrations of SB505124. B–D. Parasite replication was measured by infecting HFFs on glass coverslips in the presence or absence of 3 µM SB505124 and then fixing the cells 24 hours later. Parasites and nuclei were detected with anti-SAG1 antibody and DAPI, respectively. B. Representative images. C. For each replicate, 100 vacuoles were monitored for parasites per vacuole and nuclei per parasite. Vacuoles were designated as being irregular if they contained an irregular number of parasites/vacuole (non 2ⁿ). Shown are averaged percentages and standard deviations of 2 independent experiments with two replicates each. D. Averaged percentages and standard deviations of irregular vacuoles (show in C) by nuclei per parasite.</p

SB505124 reduces parasite growth in <i>Toxoplasma</i>-infected mice.

<p>RH-GFP infected mice were intraperitoneally injected daily with 10 mg/kg SB505124 or DMSO alone. After 5 days post-infection, mice were sacrificed and flow cytometric analysis was performed on peritoneal exudate cells (3–4 mice per treatment group per experiment, 2 independent experiments). A. FACS plots (upper) and histograms (lower) showing percentages of infected (GFP⁺) cells of two representative mice per treatment group. B. Mean percentages of infected cells between treatment groups with standard deviations. C. Relative MFI of infected (GFP⁺) cells with standard deviations. D. ELISA determination of serum IFNγ levels of mock- and drug-treated, intraperitoneally infected mice 5 days post-infection. Shown are average and standard deviations.</p

TgMAPK1 is an SBR gene.

<p>A. Venn diagram of whole genome sequencing data of codon-changing SNVs identified in each mutant. B. Amino acid positions of TgMAPK1^SBR mutations. C. TgMAPK1^SBR allelic replacement strategy. Primers 1 and 2 were used to amplify 944 bp fragments of genomic DNA containing the SBR allele and cloned into pCR2.1. Primers 3 and 4 were used to amplify 1055 bp fragments of genomic DNA to confirm allelic replacement by Sanger sequencing. D. RHΔku80 parasites were transfected with linearized TgMAPK1^WT or TgMAPK1^SBR replacement constructs and grown in 3 µM SB505124-treated HFFs. Shown are representative images depicting the ability of RHΔKu80:TgMAPK1^SBR1 to grow and form plaques after 5 days of growth in the presence of 3 µM SB505124.</p

SB505124 impacts HIF1 and TgMAPK1 through distinct pathways.

<p>A. HIF-1 luciferase reporter activity in mock- or SB505124-treated MEFs was measured after 18 h of infection with RHΔ or SBR1-3. Shown are averaged measurements and standard deviations from at least 3 independent experiments performed in triplicate. B. HIF-1αWT and HIF-1α-/- MEFs were grown in 24 well plates and infected with RHΔ or SBR1-3. The plates were grown for 66 h at 3% O₂ and then 5 µCi ³H-Uracil was added to each well to assess parasite growth. Shown are averaged data and standard deviations from 3 independent experiments performed in duplicate. C. HFFs grown on glass coverslips were infected with RH GFP at an MOI of 10 for 6 h in the presence or absence of 5 µM SB505124. Representative images are shown.</p

SB505124 directly targets TgMAPK1.

<p>A. Strategy for endogenously tagging TgMAPK1 with 3×HA tag. B. Immunoprecipitated TgMAPK1-HA was separated by SDS-PAGE for western blotting antibody and <i>in vitro</i> kinase assays. C. Equivalent volumes of RH WT or RH:TgMAPK1-HA lysate were added anti-HA sepharose beads, then washed and then processed for <i>in vitro</i> autokinase assays. The lysates were then separated by SDS-PAGE and visualized by autoradiography. Shown is a representative assay. D. Equivalent amounts of TgMAPK1-HA was immunoprecipitated from RH:TgMAPK1HA lysates using anti-HA sepharose beads and processed for <i>in vitro</i> kinase assays in the presence of increasing concentrations of SB505124. A representative assay with relative amounts of TgMAPK1 activity in each reaction is shown. E. Dose response curve showing averaged data and standard deviations from 3 experiments. F. Lysates (80 µg) were prepared from RHΔKu80ΔHPT (WT), RHΔKu80ΔHPT:TgMAPK1^WT-HA, and RHΔKu80ΔHPT:TgMAPK1<i>^ts</i>^-HA parasites grown at 34°C. Epitope-tagged TgMAPK1 was then detected using rat anti-HA antisera in the whole cell lysate and the flow through and immunoprecipitates following immunoprecipitation using rabbit anti-HA antibody conjugated beads. G. Immunoprecipitates of the indicated HA-tagged TgMAPK1 alleles were washed in kinase assay buffer and then incubated with γ³²P-ATP for 60′ at 34°C. Shown are triplicate samples prepared from the same lysates immunoprecipitated in F. The experiment was repeated 3 independent times and representative gels are shown.</p