Fueling Open Innovation for Malaria Transmission-Blocking Drugs: Hundreds of Molecules Targeting Early Parasite Mosquito Stages

Abstract

Background: Despite recent successes at controlling malaria, progress has stalled with an estimated 219 million cases and 435,000 deaths in 2017 alone. Combined with emerging resistance to front line antimalarial therapies in Southeast Asia, there is an urgent need for new treatment options and novel approaches to halt the spread of malaria. Plasmodium, the parasite responsible for malaria propagates through mosquito transmission. This imposes an acute bottleneck on the parasite population and transmission-blocking interventions exploiting this vulnerability are recognized as vital for malaria elimination.

Methods: 13,533 small molecules with known activity against Plasmodium falciparum asexual parasites were screened for additional transmission-blocking activity in an ex vivo Plasmodium berghei ookinete development assay. Active molecules were then counterscreened in dose response against HepG2 cells to determine their activity/cytotoxicity window and selected non-toxic representative molecules were fully profiled in a range of transmission and mosquito infection assays. Furthermore, the entire dataset was compared to other published screens of the same molecules against P. falciparum gametocytes and female gametogenesis.

Results: 437 molecules inhibited P. berghei ookinete formation with an IC₅₀ < 10 μM. of which 273 showed >10-fold parasite selectivity compared to activity against HepG2 cells. Active molecules grouped into 49 chemical clusters of three or more molecules, with 25 doublets and 94 singletons. Six molecules representing six major chemical scaffolds confirmed their transmission-blocking activity against P. falciparum male and female gametocytes and inhibited P. berghei oocyst formation in the standard membrane feeding assay at 1 μM. When screening data in the P. berghei development ookinete assay was compared to published screens of the same library in assays against P. falciparum gametocytes and female gametogenesis, it was established that each assay identified distinct, but partially overlapping subsets of transmission-blocking molecules. However, selected molecules unique to each assay show transmission-blocking activity in mosquito transmission assays.

Conclusion: The P. berghei ookinete development assay is an excellent high throughput assay for efficiently identifying antimalarial molecules targeting early mosquito stage parasite development. Currently no high throughput transmission-blocking assay is capable of identifying all transmission-blocking molecules.

Keywords: drug; malaria; ookinete; screening; transmission.

FIGURE 1

Screening Progression Cascade. The Tres Cantos Antimalarial Set (TCAMS) was screened in the P. berghei ookinete development assay (Pb ODA) at 1 μM in duplicate identifying 550 compounds showing >50% inhibition. 437 compounds were reconfirmed in dose response to have an IC₅₀ < 10 μM. Active compounds were counterscreened against P. falciparum asexuals and HepG2 cells. Six potent and parasite-selective compounds representing major chemical scaffolds were selected and studied in detail.

FIGURE 2

Parasite selectivity and stage specificity. IC₅₀s of active compounds were determined in the P. berghei ookinete development assay (Pb ODA). (A) These were then compared to Tox₅₀ values against HepG2 cells to determine parasite selectivity. (B) In parallel, to determine parasite stage specificity, Pb ODA IC₅₀s were compared to corresponding activity in the P. falciparum asexual inhibition growth assay (Pf GIA).

FIGURE 3

Compound Clustering. Compounds active in the P. berghei ookinete development assay (Pb ODA) were clustered using the FragFP algorithm and visualized as a dendrogram. Terminal nodes represent individual compounds and linking nodes represent FragFP similarity toward the single central point in 0.1 unit increments. Colored regions highlight compounds possessing common major scaffolds. Graphs indicate the individual parasite selectivity (HepG2 IC₅₀/Pb ODA IC₅₀) of members of each scaffold.

FIGURE 4

Parasitological profiling. Six compounds representative of each of the major chemical scaffolds (pyridones, cyclic diaminopyrimidines, pyridyl derivatives, diaminopyrimidines, quinolones, tetrahydroisoquinolines and benzodiazoles) with potent activity against ookinetes and high parasite selectivity were selected for profiling in the P. berghei standard membrane feeding assay (Pb SMFA) and P. falciparum Dual Gamete Formation Assay (Pf DGFA).

FIGURE 5

Activity in other transmission-blocking assays. The six selected molecules were evaluated in the P. berghei standard membrane feeding assay (Pb SMFA). (A) Five molecules were tested at 1 μM to confirm transmission-blocking activity (n = 48–53 mosquitoes per treatment). Bars represent the mean of three biological repeats, error bars indicate the standard error of the mean (SEM) (B) The most potent compound in the Pb ODA, TCDMC-135461, was investigated over a range of concentrations in Pb SMFAs (n = 22–83 mosquitoes per condition). Bars represent the mean of three biological repeats, error bars indicate the standard error of the mean (SEM). (C) Additionally, the compounds were tested against male and female P. falciparum gametocytes at 1 μM in the Pf DGFA. Bars represent the mean of at least three biological repeats, error bars indicate the standard error of the mean (SEM).

FIGURE 6

Venn diagram comparing the TCAMS transmission-blocking across three transmission-blocking assay. Active compounds from the P. berghei ookinete development assay (Pb ODA) were compared with published activity against P. falciparum late stage gametocytes and P. falciparum female gametogenesis (Almela et al., 2015; Miguel-Blanco et al., 2017). To ensure parasite specificity, to be classed as active in a particular assay, parasite selectivity had to be greater than ten-fold compared to HepG2 cells (Supplementary Table S5). Where specific compounds have been reported to block transmission in mosquito feeds, this has been mapped onto the Venn diagram as mosquitoes. Their position on the diagram indicates their activity profile in the three assays and the color represents Pf SMFA (Black), Pb SMFA (White) or both (Black and White).