Anne Kelsen, Robyn S. Kent, Anne K. Snyde, Eddie Wehri, Stephen J. Bishop, Rachel V. Stadler, Cameron Powell, Bruno Martorelli di Genova, Pramod K. Rompikuntal, Martin J. Boulanger, David M. Warshaw, Nicholas J. Westwood, Julia Schaletzky, Gary E. Ward
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 determine whether the parasite’s motility and lytic cycle can be disrupted through pharmacological inhibition of TgMyoA, as an approach to altering disease progression in vivo. To this end, we first sought to identify inhibitors of TgMyoA by screening a collection of 50,000 structurally diverse small molecules for inhibitors of the recombinant 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 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, we present evidence that KNX-002 can slow disease progression in mice infected with wild-type parasites, but not parasites expressing the resistance-conferring TgMyoA T130A mutation. Taken together, these data demonstrate the specificity of KNX-002 for TgMyoA, both in vitro and in vivo, and validate TgMyoA as a druggable target in infections with T. gondii. 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.
Nearly one third of the world’s population is or has been infected with the apicomplexan parasite, Toxoplasma gondii. Although most infections are subclinical, acute toxoplasmosis can have severe consequences in neonates and immunocompromised individuals. Congenital infection can lead to spontaneous abortion or stillbirth, and even children born with subclinical infection frequently experience sequelae later in life including neurological damage and vision impairment . Among the immunocompromised, toxoplasmic encephalitis is a particularly significant risk in AIDS patients who are unaware of their HIV status and in the approximately 50% of HIV-infected individuals worldwide who do not have access to antiretroviral therapy . In individuals with AIDS, adverse effects of the currently available drugs to suppress toxoplasmosis cause the discontinuation of treatment in up to 40% of patients, highlighting the need for new, better-tolerated drugs to reduce toxoplasmosis-related morbidity and mortality.
Materials and method
Parasites were propagated by serial passage in HFFs. HFFs were grown to confluence in Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, Carlsbad, California, United States of America) containing 10% v/v heat-inactivated fetal bovine serum (FBS) (Life Technologies, Carlsbad, California, USA), 10 mM HEPES (pH 7), and 100 units/ml penicillin and 100 μg/ml streptomycin, as previously described. Prior to infection with T. gondii, the medium was changed to DMEM supplemented with 10 mM HEPES (pH 7), 100 units/ml penicillin and 100 μg/ml streptomycin, and 1% v/v FBS.
To identify inhibitors of TgMyoA motor activity, we used our previously described method for producing large amounts of functional motor, in which the TgMyoA heavy chain is co-expressed in insect cells with its 2 light chains, TgMLC1 and TgELC1, and a myosin co-chaperone protein. We developed a miniaturized coupled enzyme assay to measure actin-dependent ATPase activity of the purified recombinant motor and screened 50,000 compounds from the compound library at Cytokinetics Inc. for inhibitors of this ATPase activity. Hit follow-up and characterization included dose-response analysis using resupplied compound that was determined to be >95% pure by LC/MS analysis and control assays demonstrating the compound was inactive against an unrelated ATPase, hexokinase. The most potent hit was 1-(4-methoxyphenyl)-N-((3-(thiophen-2-yl)-1H-pyrazol-4-yl)methyl)cyclopropan-1-amine inset, subsequently named KNX-002. KNX-002 inhibits TgMyoA ATPase activity in a dose-dependent manner with an IC50 of 2.8 μM (95% confidence interval (CI) 2.4 to 3.2 μM;. In contrast, KNX-002 has little to no detectable effect on the actin-activated ATPase activity of bovine cardiac myosin, chicken gizzard smooth muscle myosin (SMM), rabbit fast skeletal muscle myosin, or bovine slow skeletal muscle myosin, up to the highest concentrations tested (40 μM; Figs 1 and S1). These data identify KNX-002 as a potentially specific inhibitor of T. gondii class XIVa myosin activity.
The intense interest in motility among those who study apicomplexan parasites reflects both the unique nature of the process and its importance in parasite biology and virulence. These parasites move without cilia, flagella, or a protruding leading edge that drives the substrate-dependent motility of most other eukaryotic cells. The class XIV myosins play a central role in motility, but they are unusual myosins in many respects, and precisely how these myosins and their interacting proteins function to drive motility remains controversial. The mechanisms underlying motility are therefore of fundamental cell biological interest. Since MyoA is essential for virulence, highly conserved in apicomplexan parasites, and different in several respects from the myosins found in humans, the MyoA motor complex also represents a potentially attractive target for drug development. We describe here the identification and characterization of a novel small molecule, KNX-002, which will be a useful chemical probe for future studies of the function of TgMyoA. Importantly, we also identified a mutation in TgMyoA that reduces the sensitivity of the motor to KNX-002. We used isogenic parasite lines expressing either the wild-type or mutant myosin to demonstrate that TgMyoA is a biologically relevant target of the compound in parasites and to show that pharmacological inhibition of TgMyoA can slow the progression of disease in mice infected with a lethal dose of T. gondii.
Citation: Kelsen A, Kent RS, Snyder AK, Wehri E, Bishop SJ, Stadler RV, et al. (2023) MyosinA is a druggable target in the widespread protozoan parasite Toxoplasma gondii. PLoS Biol 21(5): e3002110. https://doi.org/10.1371/journal.pbio.3002110
Academic Editor: Boris Striepen, University of Pennsylvania, UNITED STATES
Received: February 15, 2023; Accepted: April 5, 2023; Published: May 8, 2023
Copyright: © 2023 Kelsen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the National Institutes of Health (AI139201 and AI137767 to GEW, each including salary support; GM141743 to DMW, including salary support; F31AI145214 to RVS, including predoctoral fellowship stipend support; and T32AI055402 to GEW, including predoctoral fellowship stipend support for AKS). The work was also supported by the Canadian Institutes of Health Research (148596 to MJB), the Canada Research Chair program (to MJB, salary support) and the American Heart Association (20POST35220017 to RSK, including postdoctoral fellowship stipend support). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: CI, confidence interval; DSF, differential scanning fluorimetry; FBS, fetal bovine serum; HCM, hypertrophic cardiomyopathy; HFF, human foreskin fibroblast; RFU, relative fluorescence unit; RLU, relative luminescence unit; SAR, structure-activity relationship; SEC, size-exclusion chromatography; SMM, smooth muscle myosin