Abstract
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 pathogenesis. An unusual and highly conserved myosin motor plays a central role in apicomplexan motility, and T. gondii depleted of this myosin (TgMyoA) are completely avirulent in animal models of infection. To identify small molecule inhibitors of TgMyoA, we used a chaperone-mediated strategy to express and purify large amounts of functional TgMyoA motor, and then screened a collection of 50,000 structurally diverse small molecules for inhibitors of the motor’s 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 inhibited parasite motility and growth in culture in a dose-dependent manner. We used a mutagenesis, selection, and targeted sequencing strategy to identify a mutation in TgMyoA (T130A) that renders the motor less sensitive to compound. Parasites expressing the T130A mutation showed reduced sensitivity to KNX-002 in both motility and growth assays, confirming TgMyoA as a biologically relevant target of KNX-002 in T. gondii. KNX-002 is the first specific inhibitor of an apicomplexan Class XIVa motor and a powerful new chemical probe for studying the function of TgMyoA. Furthermore, the availability of isogenic parasite lines that are either sensitive or partially resistant to KNX-002 will enable any effects observed upon compound treatment to be definitively ascribed to inhibition of TgMyoA. Since TgMyoA is essential for virulence, conserved in apicomplexan parasites, and distinctly different from the myosins found in humans, the MyoA inhibitor KNX-002 represents a promising new scaffold for the development of drugs to treat the devastating diseases caused by T. gondii and other apicomplexan parasites.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵♱ Communicating author: Gary.Ward{at}uvm.edu