Biology subjects

Troth, E. V.

Publications and source records attributed to Troth, E. V..

2 recordsLinked to original sources

bioRxiv2021

EdU incorporation to assess cell proliferation and drug susceptibility for the brain-eating amoeba, Naegleria fowleri

Naegleria fowleri is a pathogenic free-living amoeba that is commonly found in warm, freshwater and can cause a rapidly fulminant disease known as primary amoebic meningoencephalitis (PAM). New drugs are urgently needed to treat PAM, as the fatality rate is >97%. Until recently, few advances have been made in the discovery of new drugs for N. fowleri and one drawback is the lack of validated tools and methods to enhance drug discovery and diagnostics research. In this study we aimed to validate alternative methods to assess cell proliferation that are commonly used for other cell types and develop a novel drug screening assay to evaluate drug efficacy on N. fowleri replication. EdU (5-ethynyl-2'-deoxyuridine) is a pyrimidine analog of thymidine that can be used as a quantitative endpoint for cell proliferation. EdU incorporation is detected via a copper catalyzed click reaction with an Alexa Fluor linked azide. EdU incorporation in replicating N. fowleri was validated using fluorescence microscopy and quantitative methods for assessing EdU incorporation were developed by using an imaging flow cytometer. Currently used PAM therapeutics inhibited N. fowleri replication and EdU incorporation in vitro. EdA (5'ethynyl-2'-deoxyadenosine), an adenine analog, also was incorporated by N. fowleri, but was more cytotoxic than EdU. In summary, EdU incorporation could be used as a complimentary method for drug discovery for these neglected pathogens.

microbiology↗

bioRxiv2020

Discovery of anti-amoebic inhibitors from screening the MMV Pandemic Response Box on Balamuthia mandrillaris, Naegleria fowleri and Acanthamoeba castellanii

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 {micro}M. 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.

microbiology↗