title: Development of a Novel High-Density [3H]Hypoxanthine Scintillation Proximity Assay To Assess Plasmodium falciparum Growth.
abstract: The discovery and development of new antimalarial drugs are becoming imperative because of the spread of resistance to current clinical treatments. The lack of robustly validated antimalarial targets and the difficulties with the building in of whole-cell activity in screening hits are hampering target-based approaches. However, phenotypic screens of structurally diverse molecule libraries are offering new opportunities for the identification of novel antimalarials. Several methodologies can be used to determine the whole-cell in vitro potencies of antimalarial hits. The [(3)H]hypoxanthine incorporation assay is considered the "gold standard" assay for measurement of the activity of antimalarial compounds against intraerythrocytic forms of Plasmodium falciparum However, the method has important limitations, as the assay is not amenable for high-throughput screening since it remains associated with the 96-well plate format. We have overcome this drawback by adapting the [(3)H]hypoxanthine incorporation method to a 384-well high-density format by coupling a homogeneous scintillation proximity assay (SPA) and thus eliminating the limiting filtration step. This SPA has been validated using a diverse set of 1,000 molecules, including both a representative set from the Tres Cantos Antimalarial Set (TCAMS) of compounds and molecules inactive against whole cells. The results were compared with those from the P. falciparum lactate dehydrogenase whole-cell assay, another method that is well established as a surrogate for parasite growth and is amenable for high-throughput screening. The results obtained demonstrate that the SPA-based [(3)H]hypoxanthine incorporation assay is a suitable design that is adaptable to high-throughput antimalarial drug screening and that maintains the features, robustness, and reliability of the standard filtration hypoxanthine incorporation method.
authors.affiliation: Diseases of the Developing World (DDW), GlaxoSmithKline, Tres Cantos, Spain cristina.d.de-cozar@gsk.com.
authors.forename: Cristina
authors.initials: C
authors.lastname: de Cózar
authors.affiliation: Platform Technology and Science, Centro de Investigación Básica, GlaxoSmithKline, Tres Cantos, Spain.
authors.forename: Iván
authors.initials: I
authors.lastname: Caballero
authors.affiliation: Platform Technology and Science, Centro de Investigación Básica, GlaxoSmithKline, Tres Cantos, Spain.
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Colmenarejo
authors.affiliation: Diseases of the Developing World (DDW), GlaxoSmithKline, Tres Cantos, Spain.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Sanz
authors.affiliation: Platform Technology and Science, Centro de Investigación Básica, GlaxoSmithKline, Tres Cantos, Spain.
authors.forename: Emilio
authors.initials: E
authors.lastname: Álvarez-Ruiz
authors.affiliation: Diseases of the Developing World (DDW), GlaxoSmithKline, Tres Cantos, Spain.
authors.forename: Francisco-Javier
authors.initials: FJ
authors.lastname: Gamo
authors.affiliation: Platform Technology and Science, Centro de Investigación Básica, GlaxoSmithKline, Tres Cantos, Spain.
authors.forename: Concepción
authors.initials: C
authors.lastname: Cid
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D019271:Hypoxanthine / Q000032:analysis / Q000378:metabolism; D020128:Inhibitory Concentration 50; D010963:Plasmodium falciparum / Q000187:drug effects / Q000254:growth & development*; D015203:Reproducibility of Results; D014316:Tritium
chemical_list: D000962:Antimalarials; D014316:Tritium; D019271:Hypoxanthine