Plasmodium falciparum parasites cause the most deadly form of malaria, a disease that kills approximately 1,000 children every day. Over the past two decades, significant advancements in the treatment and prevention of malaria have reduced the number of infections and associated deaths. This decrease in fatalities is largely attributed to a fast-acting, highly effective group of drugs known as artemisinins. However, early ring stage parasite tolerance to artesunate monotherapy has been documented in Southeast Asia manifesting as a delayed parasite clearance time, ultimately resulting in resistance to partner drugs. The ability of Pf early ring stage parasites to withstand a single high dose of the artemisinin derivatives has reinvigorated the impetus to identify alternative fast ring stage active compounds, with alternative mechanisms of action.
Traditionally, compound speed of action and parasite stage of effect is performed on a small number of compounds that pass a biological screening cascade, and an array of chemical filters, due in part to the time and complexity of performing such studies. Herein we present the adaptation of a highly validated automated high-throughput high-content imaging assay [1] for determining both speed and parasite stage of action. This approach classifies active anti-plasmodial compounds into fast, moderate, and slow acting (second generation), and provides an indication of the parasite stage of arrest based on a ring, trophozoite, or schizont classification.
We have applied this approach to current anti-malarial drugs and drug candidates in addition to compounds from the Medicines for Malaria Venture (MMV) Pathogen, Pandemic, and Global Priority Box open access compound collections. We also demonstrate the potential of this approach for screening large compound libraries, identifying fast ring stage active chemotypes from a two-dose testing regimen.
The ability to classify the speed of action for thousands of compounds simultaneously will expedite drug discovery, rapidly providing greater numbers of compounds with preferred activity profiles i.e., fast ring stage, enabling prioritisation for further downstream biological and chemical profiling.