The free-living amoeba Naegleria fowleri, which typically dwells within warm, freshwater environments, can opportunistically cause primary amoebic meningoencephalitis (PAM), a disease with a mortality rate of >97%. The lack of positive treatment outcomes for PAM has prompted the discovery and development of more effective therapeutics, yet most studies utilize only one or two clinical isolates. The inability to assess possible heterogenic responses to drugs among isolates from various geographical regions hinders progress in the discovery of more effective drugs. Here, we conducted drug efficacy and growth rate determinations for 11 different clinical isolates by applying a previously developed CellTiter-Glo 2.0 screening technique and flow cytometry. We found significant differences in the susceptibilities of these isolates to 7 of 8 drugs tested, all of which make up the cocktail that is recommended to physicians by the U.S. Centers for Disease Control and Prevention. We also discovered significant variances in growth rates among isolates, which draws attention to the differences among the amoeba isolates collected from different patients. Our results demonstrate the need for additional clinical isolates of various genotypes in drug assays and highlight the necessity for more targeted therapeutics with universal efficacy across N. fowleri isolates. Our data establish a needed baseline for drug susceptibility among clinical isolates and provide a segue for future combination therapy studies as well as research related to phenotypic or genetic differences that could shed light on mechanisms of action or predispositions to specific drugs.
IMPORTANCENaegleria fowleri, also known as the brain-eating amoeba, is ubiquitous in warm freshwater and is an opportunistic pathogen that causes primary amoebic meningoencephalitis. Although few cases are described each year, the disease has a case fatality rate of >97%. In most laboratory studies of this organism, only one or two well-adapted lab strains are used; therefore, there is a lack of data to discern if there are major differences in potency of currently used drugs for multiple strains and genotypes of the amoeba. In this study, we found significant differences in the susceptibilities of 11 N. fowleri isolates to 7 of the 8 drugs currently used to treat the disease. The data from this study provide a baseline of drug susceptibility among clinical isolates and suggest that new drugs should be tested on a larger number of isolates in the future.
Diseases caused by pathogenic free-living amoebae include primary amoebic meningoencephalitis (Naegleria fowleri), granulomatous amoebic encephalitis (Acanthamoeba spp.), Acanthamoeba keratitis, and Balamuthia amoebic encephalitis (Balamuthia mandrillaris). Each of these are difficult to treat and have high morbidity and mortality rates due to lack of effective therapeutics. Since repurposing drugs is an ideal strategy for orphan diseases, we conducted a high throughput phenotypic screen of 12,000 compounds from the Calibr ReFRAME library. We discovered a total of 58 potent inhibitors (IC50 <1 μM) against N. fowleri (n = 19), A. castellanii (n = 12), and B. mandrillaris (n = 27) plus an additional 90 micromolar inhibitors. Of these, 113 inhibitors have never been reported to have activity against Naegleria, Acanthamoeba or Balamuthia. Rapid onset of action is important for new anti-amoeba drugs and we identified 19 compounds that inhibit N. fowleri in vitro within 24 hours (halofuginone, NVP-HSP990, fumagillin, bardoxolone, belaronib, and BPH-942, solithromycin, nitracrine, quisinostat, pabinostat, pracinostat, dacinostat, fimepinostat, sanguinarium, radicicol, acriflavine, REP3132, BC-3205 and PF-4287881). These compounds inhibit N. fowleri in vitro faster than any of the drugs currently used for chemotherapy. The results of these studies demonstrate the utility of phenotypic screens for discovery of new drugs for pathogenic free-living amoebae, including Acanthamoeba for the first time. Given that many of the repurposed drugs have known mechanisms of action, these compounds can be used to validate new targets for structure-based drug design.
GRA Endowment helps researchers save lives through drug discovery
The Amoeba Summit in Orlando last year is where the importance of her work on drug discovery for deadly amoebae really hit home for Cassiopeia Russell.
It was there she learned the story of an 11-year-old boy who had gone on a family vacation to Costa Rica and was having a great time going down a waterslide at a hot spring there. Days later, he started complaining of a terrible headache. Then he started vomiting. Within a week, he was dead. The cause was a microscopic organism, Naegleria fowleri, living in the warm waters of the spring.
Russell, a doctoral student, had been working in the lab of Dennis Kyle, director of the UGA Center for Tropical and Emerging Global Diseases, for about a year when she was able to attend the conference thanks to a Georgia Research Alliance (GRA) endowment and learn how the research she does in the lab is making a real impact on people’s lives. The GRA was founded with the goal of expanding Georgia universities’ ability to conduct high-level research with the potential of bringing new and innovative products to market. Kyle is the GRA Eminent Scholar in Antiparasitic Drug Discovery, and his endowment enables him to run a 16-person staff of student researchers, postdocs, and research scientists.
“I asked myself at the beginning if I really wanted to work on a parasite this rare,” Russell says. “But if you look at the statistics, this parasite infects mainly young children. Hundreds have died.” She thinks about that every day in the lab.
More commonly referred to as the brain-eating amoeba, Naegleria fowleri is a surprisingly common amoeba found in warm water lakes, ponds, and rivers. When water is forced up the nose—like when diving into a body of water or repeatedly riding a waterslide—the parasite travels to the brain, where it attacks the organ’s cells. Though infections are rare, the brain-eating amoeba kills almost everyone it infects.
One reason Naegleria fowleri is so deadly is because symptoms of the infection resemble those of viral meningitis, a much more common and more treatable disease. “That misdiagnosis and waiting to see if the patient gets better after beginning treatment is wasting valuable time,” says Russell. She’s committed to finding faster, more effective ways to diagnose the condition so patients can get the right medications in time to stop the disease’s progression.
The Georgia Research Alliance really helped me set up this whole operation when I got here. Without the GRA, there’s no way that I could have had this team going for three years.” — Dennis Kyle, GRA Eminent Scholar in Antiparasitic Drug Discovery
As a member of Kyle’s lab, Russell also tests drug compounds to see which ones can kill an amoeba without destroying the human cells it infects. Current drugs used to treat the infection aren’t very effective and are highly toxic.
Despite being almost 99% fatal, not many federal dollars go toward research on brain-eating and other kinds of amoebae. That’s where the Georgia Research Alliance comes in.
“The Georgia Research Alliance really helped me set up this whole operation when I got here,” says Kyle. “Without the GRA, there’s no way that I could have had this team going for three years. This is something that we have concerns about in Georgia. Every summer, we hear of Naegleria fowleri cases on the news. But we don’t have many people worldwide working on it and very few doing the drug discovery needed to come up with a new drug that could save lives. And that’s really our goal.”
The other main area of research in the Kyle lab is malaria and how the parasite becomes resistant to the drugs commonly used to treat it. Additionally, a less commonly studied strain of malaria can go dormant inside its host, effectively hiding in the liver until flaring up weeks, months, or even years later. Kyle and his team were able to develop a model that simulates that dormant phase to test a variety of drugs to find a way to kill the parasite.
But in order to use that model, researchers in the lab must collect parasites from the field. The endowment helped the lab send assistant research scientist Steven Maher to Asia 25 times over the past four years to work with partners in Cambodia and Thailand.
“International travel has definitely changed my life,” Maher says. “Right now, we’re supporting people in Asia and their families, and I think having that human connection is really important. I think all too often we do research and we forget about ‘how is what I’m doing helping real people?’ I think a lot of researchers would benefit from that type of experience.”
The Georgia Research Alliance, private donors, and the UGA Athletic Association are committed to providing researchers like Russell and Maher with opportunities to advance their work on deadly infectious diseases threatening nations around the world.
Emma Troth, a Ph.D. trainee in Dennis Kyle‘s laboratory, is entering her fourth year at UGA. She is originally from Eureka, Illinois, and attended Bradley University where she majored in Biology with a minor in Ethics. While at UGA, Emma has served as president of the CTEGD Graduate Student Association (2019-2020) and is currently the CTEGD Graduate Student Association representative.
How did you get interested in neglected tropical diseases?
As an undergraduate, I participated in a Research Experience for Undergraduates program at the University of Notre Dame. At Notre Dame, I worked on a project characterizing malaria vectors in the Solomon Islands and Indonesia. My summer at Notre Dame sparked my interest in neglected tropical diseases.
Why did you choose UGA?
I chose UGA because of the diversity of research. Coming into graduate school, I knew I was interested in infectious diseases but did not have my heart set on a particular organism to work on. UGA works on the biggest selection of infectious organisms. With the Integrated Life Sciences Program, I had the opportunity to experience multiple labs working on different organisms, regardless of department, to help me identify where I would like to complete my doctoral degree.
What is your research focus? Why are you interested in this topic?
My project focuses on drug discovery for Naegleria fowleri, the brain-eating amoebae. My main project focuses on structure-based drug design to develop novel drug targets against N. fowleri. Additionally, I am working to develop phenotypic drug screening assays to complement our high-throughput drug discovery. I am fascinated by N. fowleri because it is such a mysterious, deadly organism. Though infections may not be as common as other parasitic diseases, nearly all cases of primary amebic meningoencephalitis (PAM) are fatal. This amoeba is grossly understudied; very few labs in the world research this organism. It is both a privilege and a challenge to be able to work on this neglected parasite.
Have you done any fieldwork or is there a collaborator/field site that you would like to visit in order to enhance your training?
I hope to complete an internship with the Task Force for Global Health. This internship will ideally include fieldwork with one of their neglected tropical disease programs.
What are your future professional plans?
Going forward, I would like to continue my career in neglected tropical diseases with an emphasis on global health. I am particularly interested in a career involving field research. Ultimately, I hope to work for a government agency like the Centers for Disease Control and Prevention (CDC) or a non-profit organization focused on neglected tropical diseases.
What is your favorite thing about Athens?
My favorite thing about Athens is the food! There is such a variety of local restaurants and new restaurants are continually opening. Coming from a small undergraduate institution, I really enjoy the atmosphere of a large university in a small college town. Athens is a very easy city to feel “at home”.
Any advice for a student interested in this field?
Never be afraid to reach out for help, wherever you need it! Coming into graduate school can be intimidating and at times, isolating. There are so many people eager to help you on your graduate school journey and ultimately want to see you succeed. Particularly within the CTEGD, I have always been met with kind and willing responses. All it takes is for you to take the step and reach out!
Support trainees like Emma by giving today to the Center for Tropical & Emerging Global Diseases.
Pathogenic free-living amoebae, Balamuthia mandrillaris, Naegleria fowleri, and several Acanthamoeba species are the etiological agents of severe brain diseases, with case mortality rates > 90%. A number of constraints including misdiagnosis and partially effective treatments lead to these high fatality rates. The unmet medical need is for rapidly acting, highly potent new drugs to reduce these alarming mortality rates. Herein, we report the discovery of new drugs as potential anti-amoebic agents. We used the CellTiter-Glo 2.0 high-throughput screening methods to screen the Medicines for Malaria Ventures (MMV) Pandemic Response Box in a search for new active chemical scaffolds. Initially, we screened the library as a single-point assay at 10 and 1 µM. From these data, we reconfirmed hits by conducting quantitative dose–response assays and identified 12 hits against B. mandrillaris, 29 against N. fowleri, and 14 against A. castellanii ranging from nanomolar to low micromolar potency. We further describe 11 novel molecules with activity against B. mandrillaris, 22 against N. fowleri, and 9 against A. castellanii. These structures serve as a starting point for medicinal chemistry studies and demonstrate the utility of phenotypic screening for drug discovery to treat diseases caused by free-living amoebae.
Balamuthia mandrillaris, is an under reported pathogenic free-living amoeba that causes Balamuthia amoebic encephalitis (BAE) and cutaneous skin infections. Although cutaneous infections are not typically lethal, BAE with or without cutaneous involvement usually is fatal. This is due to lack of drugs that are efficacious and that can cross the blood-brain barrier. We aimed to discover new leads for drug discovery by screening the open source MMV Malaria and MMV Pathogen boxes (800 compounds total). From an initial single point screen at 1 and 10 μM, we identified 54 hits that significantly inhibited the growth of B. mandrillaris in vitro. Hits were re-confirmed in quantitative dose response assays and 23 compounds (42.6 %) were confirmed with activity greater than miltefosine, the current standard of care.
Christopher A. Rice, Luis Fernando Lares-Jiménez, Fernando Lares-Villa, Dennis E. Kyle. Antimicrob Agents Chemother. 2020 Feb 18. pii: AAC.02233-19. doi: 10.1128/AAC.02233-19.