High-Throughput Assay and Discovery of Small Molecules that Interrupt Malaria Transmission

Abstract

Preventing transmission is an important element of malaria control. However, most of the current available methods to assay for malaria transmission blocking are relatively low throughput and cannot be applied to large chemical libraries. We have developed a high-throughput and cost-effective assay, the Saponin-lysis Sexual Stage Assay (SaLSSA), for identifying small molecules with transmission-blocking capacity. SaLSSA analysis of 13,983 unique compounds uncovered that >90% of well-characterized antimalarials, including endoperoxides and 4-aminoquinolines, as well as compounds active against asexual blood stages, lost most of their killing activity when parasites developed into metabolically quiescent stage V gametocytes. On the other hand, we identified compounds with consistent low nanomolar transmission-blocking activity, some of which showed cross-reactivity against asexual blood and liver stages. The data clearly emphasize substantial physiological differences between sexual and asexual parasites and provide a tool and starting points for the discovery and development of transmission-blocking drugs.

Keywords: Plasmodium; chemotherapy; gametocytes; malaria; transmission.

Graphical abstract

Figure 1

Induction and Development of Pure, Stage-Specific Gametocytes (A) Gametocyte production protocol. To create stages I–IV, all steps were performed, but to create stage V only, steps indicated by red boxes were omitted. (B) Simplified schematic flow chart for the SaLSSA gametocyte assay. (C) Giemsa smears of gametocytes of stage I-V and gametocyte counts and percentages for each stage based on their morphology (numbers pooled from multiple cycles). (D) Z prime values for the different gametocyte stages and assay protocols. ¹384-well TSSA, stages I-V, ²384-well-SaLSSA, stages I-V, ³1,536-well SaLSSA, stage V. (E) Displayed is the number of viable stage V gametocytes in a dilution series over time (0.5 hr until 6 hr). These data were acquired using stage V gametocytes and 1,536-well SaLSSA (R² > 0.99).

Figure 2

Screening of the MMV Malaria Box in Comparison to GNF179 (A) Structures of MMV665941 and GNF179. (B) In vitro activity of MMV665941 and GNF179 against stage V gametocytes in dose response (1,536-well format, SaLSSA in duplicate). (C) Mean oocyst counts per midgut with gametocytes incubated with DMSO, GNF179 (5× EC₅₀), and MMV665941 (5× EC₅₀). Experiment was performed in duplicates. (D) Comparison of all 42 EC₅₀ values below 12.5μM of the MMV malaria box compounds when screened against gametocytes stages I, III, and V. The average EC₅₀ of compounds tested against stage V gametocytes is significantly higher compared to stage I gametocytes (p < 0.05, ANOVA test, Prism 6).

Figure 3

Starting Points for Transmission-Blocking Drugs All compounds in this study were clustered by their substructure similarity. All compounds with the same substructure (Tanimoto average compound similarity ≥ 0.85) were assigned to different scaffold families (indicated by different colors). Non-active compounds are shown as circle nodes and gametocyte-active compounds as diamonds. LogP is log probability of enrichment in the stage V gametocyte active set relative to rate expected by chance for each scaffold family. EC₅₀ values are for stage V gametocytes SaLSSA 1,536-well format.

Figure 4

Druggable Organelles and Processes in Gametocytes Infected red blood cell (red) with mature gametocyte (blue) and parasitophorous vacuole (white) as well as schematic parasite organelles known as P. falciparum drug targets. The upper (left) section displays compounds with similar activity against stage V gametocytes (SaLSSA) and asexual blood stage parasites as well as their presumed mode of action. The lower (right) section lists compounds with not active against stage V gametocytes compared to asexual blood stage parasites. Ery: erythrocyte; Gam: gametocyte; PPM: parasite plasma membrane; PV: parasitophorous vacuole; PVM: parasitophorous vacuole membrane.