The emergence of P. falciparum resistant to current front-line artemisinin combination therapies underscores the urgent demand for new candidate molecules for a single exposure radical cure and prophylaxis drug. Developing a suitable candidate component that is both potent and effects a rapid rate of kill to replace artemisinins requires a new and innovative in vitro screening assays to support discovery. A standard Bioluminescence Relative Rate of Kill (BRRoK) assay to quickly triage rapid cytocidal antimalarial compounds in vitro has been developed. Recognizing limitations in the BRRoK assay necessitated a subsequent development of a modified-BRRoK assay. This mBRRoK assay explores a compound’s RoK and potency together in a fixed-concentration assay format more amenable to a high throughput screening of a large compound libraries.
Proof-of-principle for the mBRRoK assay was developed using the Medicine for Malaria Venture (MMV) Malaria Box compounds for which BRRoK data was available. A subsequent validation of mBRRoK was carried out using the MMV Pathogen Box open source discovery library. Potential new leads were identified, of a particular interest are novel PfeEF2 inhibitors (MMV634140 and MMV667494) that show a rapid initial relative rate of kill. These compounds are suggested for further optimization and characterization.
The mBRRoK assay was adapted, miniaturized and optimized for high throughput screening of 12,514 TCAMS library. The results demonstrated that this assay is simple, sensitive (81% true discovery rate), reliable and robust with Z´ value of 0.74-0.98 and S/B ratio of 160 to 475. Predicted fast-acting hits were selected and confirmed using the standard BRRoK assay in both the original Dd2luc reporter strain, but also in a new NF54luc (chloroquine-sensitive) strain. The results demonstrated the utility of mBRRoK assay not only for rapid screening of potent and fast-acting compound, but also to study drug-resistance profiles across different parasite strains.
The mBRRoK assay offers significant opportunities during early stage of antimalarial drug discovery and development to triage compound sets through understanding potency and initial rate of kill, but is also an assay system amenable to adaptations such as assays in artemisinin resistant reporter strains and the study of stage-specific action.