title: Imaging-based high-throughput screening assay to identify new molecules with transmission-blocking potential against Plasmodium falciparum female gamete formation.
abstract: In response to a call for the global eradication of malaria, drug discovery has recently been extended to identify compounds that prevent the onward transmission of the parasite, which is mediated by Plasmodium falciparum stage V gametocytes. Lately, metabolic activity has been used in vitro as a surrogate for gametocyte viability; however, as gametocytes remain relatively quiescent at this stage, their ability to undergo onward development (gamete formation) may be a better measure of their functional viability. During gamete formation, female gametocytes undergo profound morphological changes and express translationally repressed mRNA. By assessing female gamete cell surface expression of one such repressed protein, Pfs25, as the readout for female gametocyte functional viability, we developed an imaging-based high-throughput screening (HTS) assay to identify transmission-blocking compounds. This assay, designated the P. falciparum female gametocyte activation assay (FGAA), was scaled up to a high-throughput format (Z' factor, 0.7 ± 0.1) and subsequently validated using a selection of 50 known antimalarials from diverse chemical families. Only a few of these agents showed submicromolar 50% inhibitory concentrations in the assay: thiostrepton, methylene blue, and some endoperoxides. To determine the best conditions for HTS, a robustness test was performed with a selection of the GlaxoSmithKline Tres Cantos Antimalarial Set (TCAMS) and the final screening conditions for this library were determined to be a 2 μM concentration and 48 h of incubation with gametocytes. The P. falciparum FGAA has been proven to be a robust HTS assay faithful to Plasmodium transmission-stage cell biology, and it is an innovative useful tool for antimalarial drug discovery which aims to identify new molecules with transmission-blocking potential.
authors: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors: Celia
authors: C
authors: Miguel-Blanco
authors: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors: Joël
authors: J
authors: Lelièvre
authors: Department of Life Sciences, Imperial College, London, United Kingdom.
authors: Michael J
authors: MJ
authors: Delves
authors: Screening and Compound Profiling, Centro de Investigación Básica, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors: Ana I
authors: AI
authors: Bardera
authors: Screening and Compound Profiling, Centro de Investigación Básica, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors: Jesús L
authors: JL
authors: Presa
authors: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors: María José
authors: MJ
authors: López-Barragán
authors: Department of Life Sciences, Imperial College, London, United Kingdom.
authors: Andrea
authors: A
authors: Ruecker
authors: Department of Life Sciences, Imperial College, London, United Kingdom.
authors: Sara
authors: S
authors: Marques
authors: Department of Life Sciences, Imperial College, London, United Kingdom.
authors: Robert E
authors: RE
authors: Sinden
authors: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Madrid, Spain esperanza.a.herreros@gsk.com.
authors: Esperanza
authors: E
authors: Herreros
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D005260:Female; D057166:High-Throughput Screening Assays; D020128:Inhibitory Concentration 50; D008751:Methylene Blue / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D013883:Thiostrepton / Q000494:pharmacology
chemical_list: D000962:Antimalarials; D012333:RNA, Messenger; D013883:Thiostrepton; D008751:Methylene Blue