Killing quickly – validation of an improved Rate-of-Kill assay to support antimalarial discovery.

Discussion

The emergence of Plasmodium falciparum resistant to current front-line antimalarials provides an impetus to develop and deliver new and innovative in vitro screening assays in the support of antimalarial drug discovery. New antimalarials are needed that are both potent as well as capable of affecting a high rate of initial cytocidal kill. Using a novel Bioluminescence Relative Rate-of-Kill (BRRoK) assay, an open source library of antimalarial drug development targets (Malaria Box) was previously screened for compounds with high RoK. Recognising some limitations with the BRRoK assay, subsequent development work utilised a modified BRRoK assay to explore RoK and potency together in a format much more amenable to a high throughput approach. This modified assay was applied to 13,000 antimalarial development targets from the Tres Cantos Antimalarial Set (TCAMS library) provided by GlaxoSmithKline. However, hits predicted from the modified assay need to be confirmed both in terms of their potency and initial cytocidal action to validate the screening approach.

The aim of this project was to validate the potency and RoK for hits identified in the modified BRRoK assay of the TCAMS library. The specific aims of this project were to (i) review the EC₅₀ data of 24 TCAMS compounds on 3D7 parasite strain, (ii) use the EC₅₀ data to determine their initial RoK, (iii) validate the predictions of RoK made from the modified BRRoK assay of TCAMS by repeating the RoK assessment using the standard BRRoK assay, (iv) determine the RoK in two genetic backgrounds of P. falciparum with distinct antimalarial drug resistant phenotypes.

Twenty-four TCAMS compounds were screened in two different parasite strains; Dd2^Luc and NF54. There were seven compounds predicted to be rapidly cytocidal, 12 compounds predicted to be intermediate, and five compounds predicted to be slow. Standardised BRRoK assays confirmed their relative RoK; providing data that validates the new high throughput approach when compared against a range of standard antimalarial benchmark compounds. Several compounds, however, did show some differences in RoK between the Dd2^Luc and NF54 strains used. Interestingly, examination of the structure of these compounds reveals a quinolone moiety, potentially affecting these different RoK in what are chloroquine resistant and sensitive parasite strains, respectively. 

This data supports validation of an improved in vitro assay that screens compounds in terms of both their antiplasmodial potency but also their initial cytocidal activity – both critical aspects of the Target Product Profile for new antimalarial drugs. The BRRoK assay can now be readily scaled to screen massive (>100,000– multi-million) compound libraries to support the discovery of new antimalarial drugs.