Identifying the Target of an Antiparasitic Compound in Toxoplasma Using Thermal Proteome Profiling

Abstract

Apicomplexan parasites include the causative agents of malaria and toxoplasmosis. Cell-based screens in Toxoplasma previously identified a chemical modulator of calcium signaling (ENH1) that blocked parasite egress from host cells and exhibited potent antiparasitic activity. To identify the targets of ENH1, we adapted thermal proteome profiling to Toxoplasma, which revealed calcium-dependent protein kinase 1 (CDPK1) as a target. We confirmed the inhibition of CDPK1 by ENH1 in vitro and in parasites by comparing alleles sensitive or resistant to ENH1. CDPK1 inhibition explained the block in egress; however, the effects of ENH1 on calcium homeostasis and parasite viability were CDPK1-independent, implicating additional targets. Thermal proteome profiling of lysates from parasites expressing the resistant allele of CDPK1 identified additional candidates associated with the mitochondria and the parasite pellicle-compartments that potentially function in calcium release and homeostasis. Our findings illustrate the promise of thermal profiling to identify druggable targets that modulate calcium signaling in apicomplexan parasites.

Figure 1.. Thermal profiling identifies putative targets of ENH1.

(a) Structure of ENH1. (b) Thermal profiling strategy. Parasites (gray) treated with ENH1 or vehicle (DMSO) were heated, and soluble protein was extracted following lysis. Peptide abundance at each temperature was quantified by mass spectrometry to generate protein stability curves. (c) Proteins ranked by p-value, based on the responsiveness of their thermal profiles to ENH1. (d) Representative stability curves of proteins unaffected (TUB1: TGGT1_316400B), stabilized (CDPK1: TGGT1_301440), or destabilized (FHA domain-containing protein: TGGT1_267600) by ENH1. The p-value (p) corresponds to an F-statistic based nonparametric model comparing the shapes of protein stability curves corrected for multiple hypothesis testing; the degrees of freedom (dof) required for the curves is indicated. Circles and triangles denote replicates.

Figure 2.. A chemical-genetic strategy confirms CDPK1 as a target of ENH1.

(a) Addition of ENH1 to wild-type CDPK1^G and CDPK1^M parasites followed by Western blot detection of protein thermal stability. (b) TUB1 thermal stability in the presence of ENH1. (c) Kinase assays of recombinantly expressed and purified CDPK1^G or CDPK1^M in the presence of ENH1 or the known gatekeeper-specific competitive inhibitor 3-MB.

Figure 3.. ENH1 inhibits parasite growth through CDPK1-dependent and -independent pathways.

(a) Egress assays of wild-type CDPK1^G and CDPK1^M parasites treated with ENH1 followed by the egress agonist zaprinast. (b) Plaque assays of CDPK1^G and CDPK1^M parasites treated with ENH1. (c) Selected frames of time-lapse microscopy of CDPK1^G and CDPK1^M parasites expressing the genetically encoded calcium indicator GCaMP6f with ENH1. Scale bar is 10 μm. Time is indicated as minutes:seconds. (d) Kymographs showing the median fluorescence intensities relative to the initial intensity of 99 parasite vacuoles (rows) 10 minutes following treatment with ENH1.

Figure 4.. Thermal profiling in CDPK1^M parasite lysates reveals other potential targets of ENH1.

(a) Thermal profiling strategy in CDPK1^M parasite lysates with 2 mM calcium. Detergent-solubilized parasite lysates were combined with ENH1 or vehicle, heated, and processed analogously to the intact-cell profiling experiment. (b) A side-by-side comparison of the CDPK1 thermal stability profiles obtained from the in-cell (CDPK1^G) and lysate (CDPK1^M) experiment. Circles and triangles denote replicates. (c) A comparison of thermal stability shifts measured in the in-cell (CDPK1^G) and lysate (CDPK1^M) experiment. Proteins exhibiting significant shifts in both experiments are highlighted. (d) A table of proteins exhibiting significant ENH1-dependent changes in thermal stabilization. The direction of alteration to the thermal profile in each experiment is indicated by + (stabilization) or – (destabilization).