Pentachloropseudilin disrupts multiple steps of the Toxoplasma gondii lytic cycle, consistent with predicted multi-myosin engagement

The pharmacological targeting of apicomplexan myosins has emerged as a validated antiparasitic strategy, with selective inhibitors of MyoA demonstrating activity against parasite motility and invasion. However, apicomplexan parasites express multiple unconventional myosins with essential functions throughout their life cycles that remain unexplored pharmacologically. Here, we investigated pentachloropseudilin (PCIP), a reversible allosteric inhibitor characterized against class-I myosins, although canonical class-I myosins are absent from Apicomplexa. Structural modeling predicted PCIP- potentially compatible allosteric pockets in several parasite myosins, including MyoA, MyoF and MyoJ, suggesting interference with multiple myosin-dependent processes. To specifically resolve PCIP-induced phenotypes across the tachyzoite lytic cycle, we combined complementary high-content and high-resolution approaches, including real-time live-cell imaging, quantitative assays and ultrastructure expansion microscopy. This identified distinct steps at which parasite progression was impaired. PCIP rapidly and reversibly inhibited gliding motility and host-cell invasion in a dose- dependent manner within the submicromolar range. A pronounced effect was observed during intracellular development, where PCIP inhibited proliferation and arrested parasites near the onset of daughter-cell budding. Live imaging and expansion microscopy revealed failure to assemble daughter inner membrane complex scaffolds and subpellicular microtubule arrays, and to replicate apicoplast, consistent with disruption of MyoF-dependent processes. This arrest was not simply cytostatic, as even short PCIP exposure prevented a substantial proportion of intracellular tachyzoites from resuming replication following drug removal. At lower concentrations permitting daughter-cell formation, progeny displayed defective final maturation and frequently lost plasma-membrane integrity within an intact parasitophorous vacuole, preventing egress consistent with interference with MyoJ-dependent processes. Together, these findings reveal multiple points of PCIP-induced failure throughout the T. gondii lytic cycle and associate distinct phenotypes with MyoA-, MyoF- and MyoJ-dependent functions. They establish PCIP as a chemical probe for dissecting apicomplexan myosin-dependent processes and support multi-myosin interference as a promising antiparasitic strategy.