The class XIV myosin of Toxoplasma gondii, TgMyoA, is druggable in an animal model of infection

Toxoplasma gondii is a widespread apicomplexan parasite that can cause severe disease in its human hosts. The ability of T. gondii and other apicomplexan parasites to invade into, egress from, and move between cells of the hosts they infect is critical to parasite virulence and disease progression. An unusual and highly conserved parasite myosin motor (TgMyoA) plays a central role in T. gondii motility. The goal of this work was to test whether pharmacological inhibition of TgMyoA can alter disease progression in an animal model of infection. To this end, we sought to identify small molecule inhibitors of TgMyoA by screening a collection of 50,000 structurally diverse small molecules for inhibitors of the recombinant motors actin-activated ATPase activity. The top hit to emerge from the screen, KNX-002, inhibited TgMyoA with little to no effect on any of the vertebrate myosins tested. KNX-002 was also active against parasites, inhibiting parasite motility and growth in culture in a dose-dependent manner. We used chemical mutagenesis, selection in KNX-002, and targeted sequencing to identify a mutation in TgMyoA (T130A) that renders the recombinant motor less sensitive to compound. Compared to wild-type parasites, parasites expressing the T130A mutation showed reduced sensitivity to KNX-002 in motility and growth assays, confirming TgMyoA as a biologically relevant target of KNX-002. Finally, KNX-002 was shown to slow disease progression in mice infected with wild-type parasites, but not parasites expressing the resistance-conferring TgMyoA T130A mutation. These data demonstrate the specificity of KNX-002 for TgMyoA, both in vitro and in vivo, and validate TgMyoA as a druggable target for toxoplasmosis. Since TgMyoA is essential for virulence, conserved in apicomplexan parasites, and distinctly different from the myosins found in humans, pharmacological inhibition of MyoA offers a promising new approach to treating the devastating diseases caused by T. gondii and other apicomplexan parasites.


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Nearly one third of the world's population are or have been infected with the apicomplexan parasite, atom (in VEST1) confirmed the importance of this part of the molecule for biological activity (Figure 3 and differences were seen in either assay, suggesting that the effects of the T130A mutation on motor 229 function are not due to large structural changes in the protein caused by the amino acid substitution.

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Including KNX-002 in the in vitro motility assay with wild-type motor inhibits both the fraction of actin 231 filaments moving and the speed of those filaments that do move, each in a dose-dependent manner 232 ( Figure 6A, top and middle panels). Combining these two effects into a single "filament motility index" 233 (i.e., the fraction of filaments moving multiplied by the mean speed of the filaments that do move) 234 provides a better measure of the overall effect of the compound in the assay. KNX-002 induces a large 235 decrease in the filament motility index ( Figure 6A, bottom panel).

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In marked contrast to the wild-type motor, KNX-002 had little to no effect on the fraction of actin 237 filaments moved by the motor containing the T130A mutation ( Figure 6B, top panel). Furthermore, 238 although the basal filament sliding speed of the mutant motor was lower than that of wildtype, this 239 reduced sliding speed was also insensitive to compound ( Figure 6B, middle panel), as was the mutant 240 motor's overall filament motility index ( Figure 6B, bottom panel).

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Taken together, these data confirm that the T130A mutation renders TgMyoA less sensitive to the 242 inhibitory effects of KNX-002.

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Validation of the specificity of KNX-002 in parasites. The resistant parasite line generated by 244 mutagenesis and selection (clone R3, Figure 5B) is expected to contain as many as 70 different 245 mutations throughout its genome [40] in addition to the T130A mutation. To confirm that resistance to 246 KNX-002 in this parasite line was due to the identified mutation in TgMyoA, we recreated the single 247 T130A mutation in the wild-type background using CRISPR/Cas9 (Suppl. Figure 10). To quantitatively 248 evaluate effects of the mutation on the parasite's lytic cycle, we compared wild-type and T130A parasites 249 in a plaque assay, which encompasses the parasite's entire lytic cycle. The T130A mutation had no 250 significant effect on the number of plaques formed or plaque area (Suppl. Figure 11A, B), demonstrating 251 little to no effect of the mutation on the parasite's lytic cycle or growth in culture. Consistent with the 252 plaque assay results, the T130A mutation also had little to no effect on the fraction of the parasite 253 population moving in the 3D motility assay (compare the leftmost pair of panels in Figure 7A and two sets 254 of grey bars in Figure 7B) and only a minor effect on their mean trajectory speeds (compare the two sets 255 of grey bars, Figure 7C). Finally, the T130A mutation had no detectable effect on the virulence of the 256 parasite in a mouse model of infection (Suppl. Figure 11C). each condition. The wild-type parasite motility index is more severely impacted by KNX-002 treatment the T130A parasites moved farther than wildtype in the presence of 20 μM KNX-002 (compare the third 272 and sixth bars in Suppl. Figure 12). The decreased sensitivity of the T130A parasites to the effects of 273 KNX-002 is apparent in the maximum intensity projections from the 3D motility assay, which show more 274 T130A parasites than wildtype moving in the presence of 20 μM KNX-002, with longer average 275 displacements (compare right two sets of panels in Figure 7A).

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Together, these data show that KNX-002 can slow disease progression in mice infected with T. 297 gondii, and prove that the compound exerts its effects through inhibition of its intended target, TgMyoA.

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The intense interest in motility among those who study apicomplexan parasites reflects both the 302 unique nature of the process and its importance in parasite biology and virulence. These parasites move 303 without the cilia, flagella or protruding leading edge that drives the motility of most other eukaryotic cells.
characterization of a novel small molecule, KNX-002, which will be a useful chemical probe for future 311 studies of the function of TgMyoA. Importantly, we also identified a mutation in TgMyoA that reduces the 312 sensitivity of the motor to KNX-002. We used isogenic parasites lines expressing either the wild-type or 313 mutant myosin to unequivocally demonstrate that TgMyoA is a biologically relevant target of the     the filaments that move ( Figure 6A). Similar effects are seen in the 3D motility assay: compound approximately equally to the filament motility index in both the presence and absence of the compound.

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In wild-type parasites, the compound-induced changes to fraction moving are larger than to speed and    assays. This seems unlikely, since the mutant motor was indistinguishable from the wild-type motor by 390 SEC and DSF (Suppl. Figure 9). Second, the partially functional motor might be overexpressed in the 391 mutant parasites. This possibility was ruled out by quantitative western blotting (Suppl. Figure 14A).  Figure 14B, right panel). Furthermore, analysis of all differentially regulated transcripts 396 revealed that 57% of the genes up/down regulated more than log2-fold were annotated as hypothetical 397 proteins, and the remainder were largely associated with trafficking and metabolism (Suppl. Figure 14B,

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The mutagenesis protocol used to generate the T130A mutation kills 70% of the initial parasite     (Table 1), which contain the desired 528 mutation and using PfuUltra high fidelity taq polymerase in a thermocycling reaction to incorporate and 529 extend the primer product. The product was then digested by DpnI overnight at 37 °C and the resulting used to transform electro-competent DH5-alpha cells. Positive clones were screened, plasmid prepped 532 and the presence of the mutation verified by sequencing.

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In vitro motility. in vitro motility assays with chicken skeletal muscle actin were performed as previously

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All motility assays captured image stacks consisting of 41 z-slices, captured 1 um apart with a 16ms 592 exposure time. The data were analyzed using Imaris ×64 v. 9.2.0 (Bitplane AG, Zurich, Switzerland). The

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ImarisTrack module tracked the parasite nuclei using an estimated spot volume of 3.0 × 3.0 × 6.0 µm. A the growth assay (37.6μM; 95% C.I. 18.9-136.4 μM). Clonal lines were isolated by serial dilution in five 96-well plates per mutagenesis. Parasite sensitivity to KNX-002 was then determined by fluorescencemutations were compared to other strains to identify naturally occurring polymorphisms unlikely to be 614 responsible for resistance.

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Generation of TgMyoA T130A mutant using CRISPR/Cas9. All oligonucleotides were synthesized by 617 Sigma-Aldrich (The Woodlands, TX), and restriction enzymes were purchased from New England double stranded repair template was produced by annealing oligonucleotides P3 and P4 (Table 1). This 623 repair template contains the T130A mutation and two additional mutations that do not alter the amino 624 acid sequence: a mutation introducing a diagnostic BglII site and a mutation ablating the PAM sequence 625 (Suppl. Figure 10A). RH∆ku80∆hxgprt parasites were transfected with the ds repair template and

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To maximize cell lysis plates were mixed on an orbital shaker (700 -900rpm) for 5 minutes before 657 equilibration at room temperature for an additional 10 minutes. Quantitation was achieved by measuring 658 luminescence per well using a Biotek Synergy 2 plate reader.                      Figure 3). Green = less than 20% inhibition, yellow = 20-40% inhibition; orange = 45-70% inhibition; red = greater than 70% inhibition compared to vehicle (DMSO) controls. Growth assay data correspond to calculated IC50 and 95% confidence interval from a 5-6 day growth assay (Suppl. Figure 4). Red = IC50 less than 20 μM; orange = IC50 of 20-35 μM; yellow = IC50 of 35-50 μM; green = no detectable growth inhibition relative to vehicle (DMSO) controls. Toxicity assay entries summarize the results from 72-hour CellTox™ Green toxicity and CellTiter-Glo ® viability assays on both human foreskin fibroblasts and HepG2 cells. Thresholds for mild and moderate toxicity and loss of viability compared to vehicle (DMSO) controls are indicated by the colored bars on the right hand side of Suppl. Figure 5.  The corresponding parasite motility index (fraction of parasites moving x mean speed). Each data point in panels B-D represents a single biological replicate composed of three technical replicates. Three of the biological replicates were collected on the same three days (circles), and three of the biological replicates were collected on a different three days (squares). Bars show the mean of the biological replicates ± SEM. Sets of biological replicates using the same parasite line and collected on the same days were compared by Student's one-tailed paired t-tests (significance indicated above the graphs). Sets of biological replicates comparing different parasite lines were analyzed by Student's two-tailed unpaired t-tests (significance indicated below the graphs). ns = not significant (p > 0.05).