Repurposing the Medicines for Malaria Venture’s COVID Box to Discover Potent Inhibitors of Toxoplasma Gondii, and in Vivo Efficacy Evaluation of Almitrine Bismesylate (MMV1804175) in Chronically Infected Mice
Bruna Ramos dos Santos, Amanda Bruno da Silva Bellini Ramos, Renata Priscila Barros de Menezes, Marcus Tullius Scotti, Fábio Antônio Colombo, Marcos José Marques, Juliana Quero Reimão
Abstract
Toxoplasmosis, caused by the obligate intracellular parasite Toxoplasma gondii, affects about one-third of the world’s population and can cause severe congenital, neurological and ocular issues. Current treatment options are limited, and there are no human vaccines available to prevent transmission. Drug repurposing has been effective in identifying anti-T. gondii drugs. In this study, the screening of the COVID Box, a compilation of 160 compounds provided by the "Medicines for Malaria Venture" organization, was conducted to explore its potential for repurposing drugs to combat toxoplasmosis. The objective of the present work was to evaluate the compounds’ ability to inhibit T. gondii tachyzoite growth, assess their cytotoxicity against human cells, examine their absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties, and investigate the potential of one candidate drug through an experimental chronic model of toxoplasmosis.
Introduction
Toxoplasmosis is a disease that occurs in diverse ecosystems and has significant impacts on the health of humans, domestic animals, and wildlife. Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular parasite and one of the most well-adapted parasites in the world, capable of persisting for long periods in its hosts across different geographic regions [1]. Approximately one-third of the world’s population is affected by toxoplasmosis, a disease that can cause severe congenital neurological and ocular issues, especially in immunocompromised individuals [2]. The existing treatment regimen comprises only a limited number of medications, which can frequently lead to hypersensitivity and toxicity. Moreover, there is a notable absence of effective treatment options for chronic toxoplasmosis, highlighting the need for further research and development in this area [3].
Materials and methods
Drugs and chemicals
Pyrimethamine (PYR), dimethyl sulfoxide (DMSO), chlorophenol red-β-D-galactopyranosidase (CPRG), phosphate buffer saline (PBS) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Corporation. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), dithiothreitol (DTT), N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) and sodium dodecyl sulfate (SDS) were purchased from Thermo Fisher Scientific. The COVID Box was kindly donated by the Medicines for Malaria Venture (MMV) foundation. Almitrine bismesylate was purchased from CMS Científica do Brasil. Other analytical reagents were purchased from Sigma-Aldrich, unless otherwise stated.
Results
In vitro initial screening
The MMV COVID Box collection of 160 compounds was screened against T. gondii tachyzoites using a β-galactosidase assay and against HFF using the MTT assay to identify the most selective compounds. To do this, we applied a threshold that required compounds to induce at least 80% of inhibition of T. gondii at 1 uM and less than 50% of inhibition of HFF at the same concentration. This allowed us to identify compounds with submicromolar anti-T. gondii activity and low cytotoxicity against the host cell. As shown in Fig 1, we identified 29 compounds that inhibited >80% of T. gondii survival and <50% of HFF survival at 1 μM and selected them for further studies. S1 Fig shows the structures of selected compounds 1 to 29.
Discussion
The COVID Box was developed by the MMV in collaboration with scientists from industry and academia, to rapidly respond to the urgent need to combat COVID-19. As previously described, the COVID Box not only supports research against SARS-CoV-2, but also against other human threats [29, 30]. However, this is the first study to evaluate the COVID Box against a human protozoan parasite. The current study utilizing the COVID Box identified 29 compounds with submicromolar anti-Toxoplasma activity and high selectivity indexes. Out of these compounds, eight had previously been identified as anti-T. gondii agents and served as positive controls, such as itraconazole and tetracycline. Therefore, the present study contributes to the discovery of 21 new anti-T. gondii drug candidates through screening the COVID Box.
Conclusions
The screening of the COVID Box resulted in the identification of new anti-T. gondii agents, making this the first study to report their activity against this important human parasite. Based on the threshold applied (>80% of inhibition of T. gondii survival and <50% of inhibition of HFF survival at 1 μM), 29 compounds were found, all of them being at least 10-fold more lethal to the parasite than to the host cell. Based on their highly selective anti-T. gondii activity and desirable ADME properties, we have identified six compounds that we consider as promising candidates for further preclinical studies on chronic toxoplasmosis. Among them, we selected almitrine for further in vivo evaluation and found that almitrine bismesylate was effective in the treatment of mice chronically infected with T. gondii, resulting in parasite burden reduction in the brain in a dose-dependent manner. The identification of this novel drug candidate holds great relevance, given the lack of effective treatment options available for chronic toxoplasmosis. The results presented here provide further evidence of the potential of the MMV collections as a valuable source of drugs to be repositioned for infectious diseases.
Acknowledgments
We would like to thank the Medicines for Malaria Venture foundation (MMV; Switzerland) for having provided the COVID Box.
Citation: dos Santos BR, Ramos ABdSB, de Menezes RPB, Scotti MT, Colombo FA, Marques MJ, et al. (2023) Repurposing the Medicines for Malaria Venture’s COVID Box to discover potent inhibitors of Toxoplasma gondii, and in vivo efficacy evaluation of almitrine bismesylate (MMV1804175) in chronically infected mice. PLoS ONE 18(7): e0288335. https://doi.org/10.1371/journal.pone.0288335
Editor: Peter Mbugua Njogu, University of Nairobi, KENYA
Received: May 11, 2023; Accepted: June 24, 2023; Published: July 7, 2023
Copyright: © 2023 dos Santos 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 manuscript and its Supporting Information files.
Funding: JQR acknowledges support from the São Paulo Research Foundation (FAPESP https://fapesp.br/) (grant numbers 2018/18954-4 and 2020/03399-5). ABSBR acknowledges support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES https://www.gov.br/capes/pt-br). 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.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0288335#abstract0
