Validating a Firefly Luciferase-Based High-Throughput Screening Assay for Antimalarial Drug Discovery

Abstract

The emergence and spread of multidrug-resistant Plasmodium falciparum and recent detection of potential artemisinin-resistant strains in Southeast Asia highlight the importance of developing novel antimalarial therapies. Using a previously generated stable transgenic P. falciparum line with high-level firefly luciferase expression, we report the adaptation, miniaturization, optimization, and validation of a highthroughput screening assay in 384-well plates. Assay conditions, including the percentage of parasitemia and hematocrit, were optimized. Parameters of assay robustness, including Z 0 -value, coefficient variation (CV), and signal-to-background (S/B) ratio, were determined. The LOPAC 1280 small-compound library was used to validate this assay. Our results demonstrated that this assay is robust and reliable, with an average Z 0 -value of >0.7 and CV of <10%. Moreover, this assay showed a very low background, with the S/B ratio up to 71. Further, identified hits were selected and confirmed using a SYBR Green I-based confirmatory assay. It is evident that this assay is suitable for large-scale screening of chemical libraries for antimalarial drug discovery.

Luminescent multiplex viability assay for Trypanosoma brucei gambiense

“…Although nucleofection has been described to be very efficient for transient and for stable transfection of African trypanosomes [29,49,50], our study confirms that transfection with pHD309 RLuc is also successful by electroporation [40]. Due to the presence of hygromycin phosphotransferase as resistance selection marker for stable genomic integration of Renilla luciferase, cross resistance against trypanotoxic hygromycin analogues may pose a problem, as has been described for pyrimethamine resistance of a transgenic firefly luciferase Plasmodium strain [34]. To select the most RLuc transfected trypanosome population, two RLuc activity assays were used.…”

Evaluation of bioluminescence-based assays of anti-malarial drug activity

“…Here luc was flanked by the Pfhsp86 5 ′ and Pbdhfr ts 3 ′ sequences and integrated as a short concatamer into chromosome 7. The assay provided 50% inhibition concentration (IC 50 ) results comparable to those determined using 3 H-hypoxanthine or MSF, with a subsequent report demonstrating the feasibility of scaling the use of this transgenic parasite into a 384-multiwell format [16]. Screening the Library of Pharmacologically Active Compounds (LOPAC 1280 ) demonstrated the robustness of this assay format with Z’ scores >0.7 and S/N ratio of 71.…”

“…Correlating RLU against parasitaemia for each HCT used (Figure 1) shows a strong linear relationship (all R 2 > 0.97) irrespective of the HCT. Increasing the parasite number through fold-increases in HCT does initially (1 to 2% HCT) result in similar fold-increases in RLU; however, the fold-increase in RLU between 2% and 4% HCT is markedly reduced due to the quenching effect of the additional haemoglobin released at these higher HCT [16,22]. Starting conditions of 2% HCT and 2% trophozoite-stage parasitaemia were selected for all future experiments.…”

Identification of MMV Malaria Box Inhibitors of Plasmodium falciparum Early-Stage Gametocytes Using a Luciferase-Based High-Throughput Assay

“…Sexual differentiation was triggered by applying a combination of stress factors known to stimulate gametocytogenesis in vitro, namely, a drop in hct, a high parasitemia, and the consequent hemolysis (31) due to schizont rupture, as well as nutritional stress (32,33). Luciferase-based approaches have previously been shown to be useful for the development of high-throughput assays for compound screening against the asexual stages of P. falciparum (34)(35)(36). Our HTS assay for early gametocytogenesis was developed using the P. falciparum transgenic line NF54…”