Gametocyte-specific and all-blood-stage transmission-blocking chemotypes discovered from high throughput screening on Plasmodium falciparum gametocytes

Abstract

Blocking Plasmodium falciparum human-to-mosquito transmission is essential for malaria elimination, nonetheless drugs killing the pathogenic asexual stages are generally inactive on the parasite transmissible stages, the gametocytes. Due to technical and biological limitations in high throughput screening of non-proliferative stages, the search for gametocyte-killing molecules so far tested one tenth the number of compounds screened on asexual stages. Here we overcome these limitations and rapidly screened around 120,000 compounds, using not purified, bioluminescent mature gametocytes. Orthogonal gametocyte assays, selectivity assays on human cells and asexual parasites, followed by compound clustering, brought to the identification of 84 hits, half of which are gametocyte selective and half with comparable activity against sexual and asexual parasites. We validated seven chemotypes, three of which are, to the best of our knowledge, novel. These molecules are able to inhibit male gametocyte exflagellation and block parasite transmission through the Anopheles mosquito vector in a standard membrane feeding assay. This work shows that interrogating a wide and diverse chemical space, with a streamlined gametocyte HTS and hit validation funnel, holds promise for the identification of dual stage and gametocyte-selective compounds to be developed into new generation of transmission blocking drugs for malaria elimination.

Isoliensinine from Cissampelos pariera rhizomes exhibits potential gametocytocidal and anti-malarial activities against Plasmodium falciparum clinical isolates

“…The analysis revealed an enrichment of; Pf3D7_0904900 (Cu 2+ ATPase), Pf3D7_1352100 (Mdr6), Pf3D7_1303500 (Nhe-1), Pf3D7_1235200 (Vp2), and Pf3D7_0830500 highlighting an active interaction with transmembrane solute transporters of both monovalent and divalent cations, as well as amino acids trafficking. Previous studies have demonstrated that solute carrier inhibitors are potent gametocytocidal [ 22 , 30 , 41 ]. As high expression of membrane enriched proteins was reported in late-stage gametocytes [ 88 ], and going by the fact that these aforementioned compounds target membrane ion transporters, our data, therefore, support the argument that late-stage gametocytes are likely permeable to ionic homeostasis disruptors.…”

Generation of aPlasmodium falciparumreporter line for studies of parasite biology throughout the life cycle

“…In an attempt to find a neutral locus for transgene insertion into P. falciparum genome, we initially selected the cg6 locus, which has been shown to be dispensable for asexual and gametocyte parasite development in vitro and has been previously used as a docking site (34,35). However, we found out that disruption of cg6 caused a significant reduction in oocyst loads (data not shown).…”

A versatile Plasmodium falciparum reporter line expressing NanoLuc enables highly sensitive multi-stage drug assays

“…Luminescence from expressed luciferase has been used as a readout for drug assays against P. falciparum at multiple life cycle stages 17 , 26 – 31 . Although HTS for gametocytocidal drugs, which utilises luciferase-expressing P. falciparum , was recently achieved by a relatively robust and simple assay method, the reporter line has not been optimised for assays targeting asexual blood- and liver-stage parasites because luciferase expression is confined to the gametocyte stage 32 .…”