Identification of inhibitors of Plasmodium falciparum gametocyte development

Abstract

Background: Plasmodium falciparum gametocytes, specifically mature stages, are the only stage in man transmissible to the mosquito vector responsible for malaria transmission. Anti-malarial drugs capable of killing these forms are considered essential for the eradication of malaria. The comprehensive profiling of in vitro activity of anti-malarial compounds against both early (I-III) and late (IV-V) stage P. falciparum gametocytes, along with the high throughput screening (HTS) outcomes from the MMV malaria box are described.

Method: Two anti-gametocyte HTS assays based on confocal fluorescence microscopy, utilizing both a gametocyte specific protein (pfs16-Luc-GFP) and a viability marker (MitoTracker Red CM-H2XRos) (MTR), were used for the measurement of anti-gametocytocidal activity. This combination provided a direct observation of gametocyte number per assay well, whilst defining the viability of each gametocyte imaged.

Results: IC50 values were obtained for 36 current anti-malarial compounds for activities against asexual, early and late stage gametocytes. The MMV malaria box was screened and actives progressed for IC50 evaluation. Seven % of the "drug-like" and 21% of the "probe-like" compounds from the MMV malaria box demonstrated equivalent activity against both asexual and late stage gametocytes.

Conclusions: The assays described were shown to selectively identify compounds with gametocytocidal activity and have been demonstrated suitable for HTS with the capability of screening in the order of 20,000 compounds per screening campaign, two to three times per seven-day week.

Figure 1

Script analysis and performance of late stage gametocyte assay. A. Top panel no compound treatment. Bottom panel 5 μM puromycin, 72 hour treated late stage gametocytes. MTR channel mask, GFP channel Mask, analysed Gametocyte Mask and MTR-GFP overlay of Opera acquired images. B. Linearity graph- gametocyte number per well against script identified viable gametocytes per well. C. Opera acquired image of gametocytes after 72 hours incubation and MTR staining. A X60 water objective was used to capture detailed images. Classical stage V gametocytes are observed.

Figure 2

Compound activity distribution against early (I-III) and late (IV-V) stage gametocytes. Normal distribution of the activity of MMV malaria box compounds against the early (A) and late (B) stage gametocytes was not observed as the compound set has an asexual bias.

Figure 3

In plate control data for early (I-III) and late (IV-V) stage gametocyte assays. In-plate control data obtained during screening of the MMV malaria box against A. early (I-III) and B. late (IV-V) stage gametocytes is shown. Black squares represent no inhibition (vehicle control: 0.4% DMSO) and purple triangles indicate 100% inhibition (5 μM puromycin).

Figure 4

Early and late stage gametocytocidal activity of MMV Malaria Box “drug-like” compounds aligned with 3D7 asexual activity. A. Activity of compounds classified as "drug-like". B. MMV019918 IC₅₀ evaluation. C. MMV007907 IC₅₀ evaluation. Orange triangles are indicative of the early (I-III) stage gametocyte activity whereas the blue squares represent the late (IV-V) stage gametocyte activity.

Figure 5

Early and late stage gametocytocidal activity of MMV Malaria Box “probe-like” compounds aligned with 3D7 asexual activity. The activities of compounds from the MMV malaria box for the early (I-III) (orange) and late (IV-V) (blue) gametocyte stages are aligned with the activities determined for 3D7 asexual parasites (grey). All three assays were performed using image based assays and analysis.

Figure 6

Images with masked viable elongated gametocytes treated with five concentrations of artesunate. Representative images of the impact of increasing concentrations of artesunate on late stage (IV-V) gametocytes. Graphic illustration of the activity profile of artesunate against the late stage (IV-V) gametocytes activity.