High-Throughput Luciferase-Based Assay for the Discovery of Therapeutics That Prevent Malaria

Abstract

In order to identify the most attractive starting points for drugs that can be used to prevent malaria, a diverse chemical space comprising tens of thousands to millions of small molecules may need to be examined. Achieving this throughput necessitates the development of efficient ultra-high-throughput screening methods. Here, we report the development and evaluation of a luciferase-based phenotypic screen of malaria exoerythrocytic-stage parasites optimized for a 1536-well format. This assay uses the exoerythrocytic-stage of the rodent malaria parasite, Plasmodium berghei, and a human hepatoma cell line. We use this assay to evaluate several biased and unbiased compound libraries, including two small sets of molecules (400 and 89 compounds, respectively) with known activity against malaria erythrocytic-stage parasites and a set of 9,886 Diversity-Oriented Synthesis (DOS)-derived compounds. Of the compounds screened we obtain hit rates of 12–13% and 0.6% in preselected and naïve libraries, respectively, and identify 52 compounds with exoerythrocytic-stage activity less than 1 μM, and having minimal host cell toxicity. Our data demonstrate the ability of this method to identify compounds known to have causal prophylactic activity in both human and animal models of malaria, as well as novel compounds, including some exclusively active against parasite exoerythrocytic stages.

The use of transgenic parasites in malaria vaccine research

“…Transgenic rodent and human malaria parasites that express fluorescent and luminescent reporter proteins have been used in screening assays to efficiently and rapidly measure inhibition of parasite growth at different points of the parasite life cycle [6,17,22,51]. These assays have been used to identify and characterize anti-Plasmodium drugs and small-molecule inhibitors, as well as vaccines targeting parasite development at different points of the life cycle.…”

“…For example, flow cytometricbased assays counting GFP (or mCherry)-positive parasiteinfected red blood cells [20,52,53] (Figure 1(a)) or quantification of luminescence signals to determine parasite numbers or parasite loads in blood, liver, or other organs [19,21] (Figure 1(b)). Infecting mice with defined numbers of luciferase expressing parasites and subsequent quantifying parasite loads (luminescence signal) in the liver by real-time imaging of live mice is frequently used to establish the in vivo effect of either inhibitors and vaccines on liver-stage development [6,17,22,23]. Bioluminescence imaging is simple to execute and can be used to monitor the course of an infection without sacrificing the animal [19] (Figure 1(b)).…”

“…These parasites have been exploited in screening assays to measure (inhibition of) parasite growth at different points of the parasite life cycle. Fluorescent and luminescent P. falciparum parasites have been used in vitro to examine the effect of drugs and other inhibitors on blood-stage growth and on gametocytes [6,[14][15][16][17]and fluorescent P. cynomolgi parasites have been generated to screen for compounds that target the hypnozoite stage in the liver [18]. Transgenic fluorescent and luminescent rodent parasites have been used in in vitro screening assays to test inhibitors that target parasite development in the blood and liver [6,[19][20][21][22].…”

“…In addition to measuring growth inhibition in vitro, transgenic rodent parasites have been used to examine the impact of drug or vaccine interventions in vivo, where inhibition of parasite development is measured as the reduction of reporter signal (mostly luminescent) in organs of the treated (compared to unimmunized/untreated) rodent host [6,17,19,22,23]. As the life cycle and basic biology of rodent and human Plasmodium parasites are very similar and since the vast majority of genes within their genomes are conserved [24], transgenic rodent parasites are frequently used to evaluate protective immunity against candidate Plasmodium antigens in vivo and are used to assess different vaccine delivery platforms and vaccination regiments.…”

Phenotypic Screens in Antimalarial Drug Discovery

“…Ultra high throughput campaigns involving million compound libraries have previously been conducted with asexual blood stage parasites [15-17]; however, the screening capacity against some of the other Plasmodium lifecycle stages is orders of magnitude lower than the asexual blood stage (ABS) screens (Figure 2) [18, 19]. Only recently has the antimalarial drug discovery community begun to develop assays that are compatible with screening for activity against other lifecycle stages at the same throughput as blood stage screens [20, 21]. Recent advances in high throughput technologies for exoerythrocytic stage screening will be discussed below.…”