ORCID
0000-0003-3022-2213
Date of Award
2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Cellular, Molecular and Biomedical Sciences
First Advisor
Gary E. Ward
Second Advisor
David M. Warshaw
Abstract
The obligate intracellular parasites that make up the apicomplexan phylum disproportionately affect impoverished regions in tropical areas where they are common in immunocompromised individuals vulnerable to chronic parasitic disease. Among these pathogens is the causative agent of toxoplasmosis – Toxoplasma gondii – that infects up to one-third of the global population. The recommended treatments for T. gondii infections can lead to serious adverse effects. There is, therefore, a need for novel therapeutics. During active infections in the brain and other tissues, T. gondii uses a unique form of motility called “gliding motility” to invade and egress host cells, penetrate restrictive biological barriers, and disseminate throughout the host. Gliding motility does not use any motility appendages such as cilia or flagella nor actin protrusions at a leading edge. Instead, the parasitic motility machinery relies on force production by the unique class XIV myosin motor – TgMyoA. TgMyoA displaces actin filaments rearward in order to drive the parasite forward relative to the substrate. This TgMyoA motor is an attractive drug target for T. gondii infections because TgMyoA is conserved amongst apicomplexan parasites yet distinct from mammalian motors. As T. gondii parasites lacking the TgMyoA gene are also impaired in motility-related processes and avirulent in mice, we used small molecule inhibitors to both understand how the biophysical function of TgMyoA relates to parasite motility and determine the potential of TgMyoA as a drug target for future therapeutics. In this dissertation, we validate TgMyoA as a druggable target for T. gondii infections. With a team of collaborators, we discovered an inhibitor of TgMyoA (KNX-002), which is specific for T. gondii and has no effect on vertebrate myosins. KNX-002 inhibits recombinant TgMyoA activity and both motility and growth of T. gondii parasites. To verify TgMyoA as a target of KNX-002 in the parasite, we generated a mutation in TgMyoA (T130A) that was less sensitive to KNX-002 in our motility and growth assays. KNX-002 was also shown to alter disease progression in vivo by protecting mice against infection with WT parasites; however, the T130A KNX-002-resistant parasites did not receive this same protection. While this result demonstrated that TgMyoA is a druggable target in vivo, KNX-002 had only a modest protective effect and showed some liver toxicity. Thus, we performed an independent screen that identified a more potent inhibitor of TgMyoA – UCB-9721. UCB-9721 inhibited TgMyoA ATPase activity with nanomolar potency and was more potent in T. gondii growth and motility assays. With this more potent compound, we were able to further characterize the drug binding pocket and perform a directed structure-activity relationship (SAR) study to determine mechanisms of increased potency of this compound. In addition, we determined that UCB-9721 inhibits growth and motility of other apicomplexan parasites, which suggests that myosin A could also be a good drug target in these other parasites. Our findings provided both mechanistic and structural insight into a specific TgMyoA drug binding pocket and confirmed that targeting TgMyoA alters pathogenesis of a T. gondii infection.
Language
en
Number of Pages
293 p.
Recommended Citation
Snyder, Anne Kaditya, "Toxoplasma Gondii Myosin A Inhibitors Provide Insights Into Motor Function And Validate Myosin A As A Potential Drug Target" (2025). Graduate College Dissertations and Theses. 2078.
https://scholarworks.uvm.edu/graddis/2078