Authors

M Famodimu¹; P Horrocks¹;  ¹ Keele University

Discussion

The bioluminescence relative rate of kill assay (BRRoK) offers a rapid, 6 hours, assay format to determine a compound immediate cytocidal effect (1). An important limitation in scaling the BRRoK assay is the requirement to measure IC_50, a process that takes longer than determining initial rate of kill. Using fixed concentrations of discovery compounds would greatly assist in scaling the assay format such that large compounds sets, e.g. TCAMS (2), can be tested.
We hypothesized that the loss of bioluminescence signal following fixed dose drug perturbation would combine the effect of both the IC₅₀ (multiples achieved with the fixed concentration employed, 10�M and 2�M) and immediate rate of kill. Exploring this effect using 10 antimalarial drugs and 100 compounds from the MMV Malaria Box for which IC₅₀ and immediate rate of kill are known, we were able to derive cut offs based on the specificity and sensitivity of the assay that enable potent and rapidly cytocidal drugs to be identified using this simpler assay format. Validation of this approach is demonstrated by screening all 400 compounds in the MMV Pathogen Box, with hits identified within nano-molar potency range and immediate rate of kill greater than that of chloroquine. We propose this revised BRRoK assay offers an important in vitro tool for lead discovery of antimalarial compounds.
(1) ULLAH, I., SHARMA, R., BIAGINI, G.A. and HORROCKS, P., 2016. A validated bioluminescence-based assay for the rapid determination of the initial rate of kill for discovery antimalarials. The Journal of antimicrobial chemotherapy,72: 717-726
(2) GAMO, F., SANZ, L.M., VIDAL, J., DE COZAR, C., ALVAREZ, E., LAVANDERA, J., VANDERWALL, D.E., GREEN, D.V.S., KUMAR, V., HASAN, S., BROWN, J.R., PEISHOFF, C.E., CARDON, L.R. and GARCIA-BUSTOS, J., 2010. Thousands of chemical starting points for antimalarial lead identification.