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

Abstract

The design of new antimalarial combinations to treat Plasmodium falciparum infections requires drugs that, in addition to resolving disease symptoms caused by asexual blood stage parasites, can also interrupt transmission to the mosquito vector. Gametocytes, which are essential for transmission, develop as sexual blood stage parasites in the human host over 8 to 12 days and are the most accessible developmental stage for transmission-blocking drugs. Considerable effort is currently being devoted to identifying compounds active against mature gametocytes. However, investigations on the drug sensitivity of developing gametocytes, as well as screening methods for identifying inhibitors of early gametocytogenesis, remain scarce. We have developed a luciferase-based high-throughput screening (HTS) assay using tightly synchronous stage I to III gametocytes from a recombinant P. falciparum line expressing green fluorescent protein (GFP)-luciferase. The assay has been used to evaluate the earlystage gametocytocidal activity of the MMV Malaria Box, a collection of 400 compounds with known antimalarial (asexual blood stage) activity. Screening this collection against early-stage (I to III) gametocytes yielded 64 gametocytocidal compounds with 50% inhibitory concentrations (IC 50 s) below 2.5 M. This assay is reproducible and suitable for the screening of large compound libraries, with an average percent coefficient of variance (%CV) of <5%, an average signal-to-noise ratio (S:N) of >30, and a Z= of ϳ0.8. Our findings highlight the need for screening efforts directed specifically against early gametocytogenesis and indicate the importance of experimental verification of early-stage gametocytocidal activity in the development of new antimalarial candidates for combination therapy. P lasmodium falciparum malaria remains a primary global cause of death and disability from infectious disease, particularly in infants and pregnant women (1). Malaria treatment currently relies on artemisinin-based combination therapy (ACT); however, emerging resistance to artemisinins in the field (2, 3) underscores the need to progress new antimalarial candidates through the drug development pipeline. Recent in vitro high-throughput screening (HTS) campaigns against P. falciparum asexual blood stages, the forms responsible for the clinical manifestations of the disease, have identified a wealth of active chemical classes, representing a promising starting point for the discovery of new therapeutic agents (4-7). The current malaria elimination strategy has also highlighted the need for all new antimalarial combination therapies to include components capable of interrupting the transmission of sexual-stage gametocytes to Anopheles mosquitoes (8, 9). Methods to enable prioritization of inhibitors of the asexual parasite stages based on their additional transmission-blocking activity are therefore urgently required.P. falciparum gametocytes develop through five morphologically distinct stages in the human blood over 8 to 12 days (10) and thereafter ...

In vitro inhibition of Plasmodium falciparum early and late stage gametocyte viability by extracts from eight traditionally used South African plant species

“…While it is highly active against the asexual parasites (Clarkson et al, 2004) there has been no report of its potency on P. falciparum NF54 gametocyte stages. However, A. annua (Asteraceae), that produces a sesquiterpene lactone, artemisinin, has gametocytocidal properties in vitro Lucantoni et al, 2013). The genus Artemisia is well known for the production of sesquiterpene lactones (Al-khathlan et al, 1992) and A. afra is no exception (Al-khathlan et al, 1992;Jakupovic et al, 1988) but there is no evidence for the presence of artemisinin in the latter specie (Van der Kooy et al, 2008;Liu et al, 2009).…”

“…that are structurally similar to those known to be gametocytocidal either in vitro, in vivo or ex vivo Adjalley et al, 2011;D'Alessandro et al, 2013;Duffy and Avery, 2013;Jones et al, 1994;Lucantoni et al, 2013Lucantoni et al, , 2010Saenz et al, 2013;Sun et al, 2013;Yerbanga et al, 2014). Using these two parallel selection strategies, the ethnobotanical set was narrowed down to a hundred plant species from which eight were collected and screened in the current study.…”

“…Dried crude acetone extracts were dissolved in DMSO as stock solutions and diluted in culture medium prior to their use in assays (highest concentration of DMSO to which parasites were exposed to was 0.4% (v/v) which is non-toxic to gametocytes (D'Alessandro et al, 2015(D'Alessandro et al, , 2013Duffy and Avery, 2013;Lucantoni et al, 2013). These extracts were transferred into 96 well sterile plates and seeded with an equal volume (100 µl/well) of gametocyte suspension (0.8-3% gametocytaemia) to achieve a final haematocrit of 1% et al, 2013).…”

“…falciparum gametocytes (Abay, 2013;Butcher, 1997;Lucantoni et al, 2013;Mackerras and Ercole, 1949;Price et al, 1996;White et al, 2014), they are not effective at clinically relevant concentrations on the late stage, transmittable gametocytes. There is therefore an urgent need to find new safe and efficacious transmission blocking drugs that can target late stage gametocytes.…”

Discovering New Transmission-Blocking Antimalarial Compounds: Challenges and Opportunities

“…The Medicines for Malaria Venture (MMV) Malaria Box of 400 compounds (selectively active against asexual parasites [22]) has been screened worldwide for activity against stage IV/V gametocytes [20,[23][24][25][26][27][28][29][30][31]. Primary hit rates (>80% inhibition at 5 μM) of 4.5-24% [20,23,27,29] were achieved when screened against stage IV/V gametocytes; this increased to 33% against stage I-III gametocytes [23,26].…”