High-throughput phenotypic screen identifies a new family of potent anti-amoebic compounds
Conall Sauvey, Ittipat Meewan, Gretchen Ehrenkaufer, Jonathan Blevitt, Paul Jackson, Ruben Abagyan
Abstract
Entamoeba histolytica is a disease-causing parasitic amoeba which affects an estimated 50 million people worldwide, particularly in socioeconomically vulnerable populations experiencing water sanitation issues. Infection with E. histolytica is referred to as amoebiasis, and can cause symptoms such as colitis, dysentery, and even death in extreme cases. Drugs exist that are capable of killing this parasite, but they are hampered by downsides such as significant adverse effects at therapeutic concentrations, issues with patient compliance, the need for additional drugs to kill the transmissible cyst stage, and potential development of resistance. Past screens of small and medium sized chemical libraries have yielded anti-amoebic candidates, thus rendering high-throughput screening a promising direction for new drug discovery in this area. In this study, we screened a curated 81,664 compound library from Janssen pharmaceuticals against E. histolytica trophozoites in vitro, and from it identified a highly potent new inhibitor compound.
Introduction
Entamoeba histolytica is a parasitic protozoan amoeba that infects the human intestinal tract and causes intestinal disease with symptoms include stomach cramping, abdominal pain, and diarrheal disease amoebiasis, also known as amoebic colitis [1, 2]. In some cases, the localized diseases were disseminated to more severe diseases such as liver abscess, pneumonia, purulent pericarditis, and cerebral amoebiasis [3, 4]. It is estimated to infect around 50 million people globally at any given time, resulting in approximately 50,000 to 70,000 deaths annually, and as such represents a significant problem from a global health perspective [5–7]. It exists in a two-stage life cycle consisting of an environmental-resistant, infective cyst stage, and a mobile, invasive trophozoite stage. It is transmitted in a characteristic fecal-oral route, where cysts can be ingested from contaminated water or food. Once ingested, cysts pass through to the host intestinal tract where they release trophozoites. Trophozoites feed and multiply, and under certain conditions invade and infect the surrounding host tissues. They can further re-form into cysts which are passed in the host’s feces, and potentially onwards to other hosts [6]. Due to this mode of infection, amoebiasis is most widespread in places where fecal contamination of water or food is likely, such as those where water sanitation is insufficient or non-existent.
Material and methods
Compound library and chemical properties prediction
The chemical compound library screened against E. histolytica trophozoites was obtained in collaboration with Janssen Pharmaceuticals. The library, referred to as the “Jump-stARter” library, contains a diverse collection of 81,664 drug-like small molecules intended for maximum potential efficacy in collaboration drug-discovery projects with external research groups [23–26, 28]. The Jump-stARter library was originally selected from millions of proprietary compounds by Janssen Pharmaceuticals medicinal chemists using “drug-likeness,” structural diversity, and favorable physical properties as criteria [27, 28]. For this study the library was spotted into black, clear-bottom 1536-well plates (Greiner) at 50nL per well in the Janssen compound logistics facility in Beerse, Belgium, and shipped directly to the University of California—San Diego. Top compounds in this study were predicted the additional key pharmacokinetic property including solubility, lipophilicity and ICM ToxScore by the method implemented in ICM-Pro v3.9 [29]. ICM ToxScore of a compound was calculated based on the present of substructure or functional group that were identified as toxic [30, 31].
Entamoeba cell culture
E. histolytica strain HM-1:IMSS trophozoites were maintained in 50 ml culture flasks (Greiner Bio-One) containing TYI-S-33 media, 10% (v/v) heat-inactivated adult bovine serum (Sigma), 1% (v/v) MEM Vitamin Solution (Gibco), supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) (Omega Scientific) [20]. E. invadens strain IP-1 were cultured in LYI-S-2 at 25°C [32, 33].
Results
High-throughput screen of the Jump-stARter library against E. histolytica trophozoites in vitro
In order to identify new inhibitors of the human parasite E. histolytica we established a collaboration with scientists from Janssen Pharmaceuticals, inc., and from them, obtained a copy of their Jump-stARter chemical library. This library consists of 81,664 small molecules selected by Janssen medicinal chemists for their chemical diversity and optimal physical properties for drug discovery and development efforts [23, 24, 27, 28]. We screened the Jump-stARter library against E. histolytica trophozoites in vitro using a semi-automated, high-throughput methodology. Previous screens using this organism have been accomplished using 96-well or 384-well plate formats, but due to the large size of the Jump-stARter library, we developed and utilized a novel 1536-well-plate-based methodology. All compounds in the library were tested at 25 μM, and the viability of the parasite cells after incubation for 48 hours was measured with the luciferase-based CellTiter-glo assay. 297 compounds achieved greater than 70% inhibition values and were thus designated as ‘hits’ and selected for further investigation (Fig 1).
Discussion
Amoebiasis caused by infection with E. histolytica remains a significant public health issue in many places throughout the world, despite the longstanding availability of the drug metronidazole as a treatment [3, 4]. Multiple factors may contribute to this, including infrastructural problems in affected areas such as difficulties with water and food sanitation [4]. However, another important factor may be the shortcomings of metronidazole itself. Strong side effects and metronidazole’s inability to kill transmissible cysts complicate the course of treatment and have been suggested to contribute to patient non-compliance and resulting increased disease spread [13, 14]. As such, research into new and better anti-amoebic drugs is ongoing. For much of the history of this research, efforts have focused on inhibiting or disrupting specific cellular processes or targets within the parasite such as redox metabolism or kinase proteins [19].
Acknowledgments
The authors would like to thank the following for their contributions to this work:
Monica Mendes Kangussu Marcolino for her assistance with Entamoeba invadens culture and screening.
Kirti Khandwal Chahal for providing human HEK293 cells.
Jean Bernatchez and Danielle Skinner for their assistance with screening equipment training.
Jim McKerrow for establishing and maintaining the high-throughput screening core and equipment as head of the Center for Discovery and Innovation in Parasitic Diseases at the Skaggs School of Pharmacy and Pharmaceutical Sciences at the University of California–San Diego.
Citation: Sauvey C, Meewan I, Ehrenkaufer G, Blevitt J, Jackson P, Abagyan R (2023) High-throughput phenotypic screen identifies a new family of potent anti-amoebic compounds. PLoS ONE 18(5): e0280232. https://doi.org/10.1371/journal.pone.0280232
Editor: Jesús Valdés, Centro de Investigación y de Estudios Avanzados del I.P.N., MEXICO
Received: January 18, 2022; Accepted: December 23, 2022; Published: May 9, 2023
Copyright: © 2023 Sauvey 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: CS received funding from Janssen Pharmaceuticals (https://www.janssen.com) (Janssen Pharmaceuticals / UCSD Biomedical Sciences Program Graduate Student Collaboration 2019) GE received funding from the National Institute of Allergy and Infectious Diseases (https://www.niaid.nih.gov/) (Grant no. R21AI146651) RA received funding from the United States National Institute of Health National Institute of General Medical Sciences (https://www.nigms.nih.gov/) (grant no. R35GM131881) 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.0280232#abstract0
