MyosinA is a druggable target in the widespread protozoan parasite Toxoplasma gondii
Anne Kelsen, Robyn S. Kent, Anne K. Snyder, 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
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 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.
Introduction
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 [1,2]. Among the immunocompromised, toxoplasmic encephalitis is a particularly significant risk in AIDS patients who are unaware of their HIV status [3–5] and in the approximately 50% of HIV-infected individuals worldwide who do not have access to antiretroviral therapy [6,7]. In individuals with AIDS, adverse effects of the currently available drugs to suppress toxoplasmosis [8] cause the discontinuation of treatment in up to 40% of patients [4,9–11], highlighting the need for new, better-tolerated drugs to reduce toxoplasmosis-related morbidity and mortality.
Material and methods
Cell and parasite culture
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 [69]. 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.
Results
High-throughput screening identifies a novel inhibitor of TgMyoA
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 [33]. 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.
Discussion
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 [17,23], and precisely how these myosins and their interacting proteins function to drive motility remains controversial [27,42–47]. 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.
Acknowledgments
We thank Cytokinetics, for their willingness to provide staff time and reagents to this project, Samantha Previs for technical support, Dr. Shane Nelson for help with the duty ratio analysis, and Dr. Roxanna del Rio-Guerra in the UVM Larner College of Medicine Harry Hood Basset Flow Cytometry and Cell Sorting Facility (RRID: SSR_022147) for advice and assistance with cell sorting. Genomic-scale data sets and ancillary information were obtained from the Toxoplasma Genome Database (ToxoDB.org).
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.
https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002110
