title: A Replicative In Vitro Assay for Drug Discovery against Leishmania donovani.
abstract: The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis, a disease potentially fatal if not treated. Current available treatments have major limitations, and new and safer drugs are urgently needed. In recent years, advances in high-throughput screening technologies have enabled the screening of millions of compounds to identify new antileishmanial agents. However, most of the compounds identified in vitro did not translate their activities when tested in in vivo models, highlighting the need to develop more predictive in vitro assays. In the present work, we describe the development of a robust replicative, high-content, in vitro intracellular L. donovani assay. Horse serum was included in the assay media to replace standard fetal bovine serum, to completely eliminate the extracellular parasites derived from the infection process. A novel phenotypic in vitro infection model has been developed, complemented with the identification of the proliferation of intracellular amastigotes measured by EdU incorporation. In vitro and in vivo results for miltefosine, amphotericin B, and the selected compound 1 have been included to validate the assay.
authors.affiliation: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors.forename: Diana
authors.initials: D
authors.lastname: Tegazzini
authors.affiliation: London School of Hygiene & Tropical Medicine, London, United Kingdom.
authors.forename: Rosario
authors.initials: R
authors.lastname: Díaz
authors.affiliation: London School of Hygiene & Tropical Medicine, London, United Kingdom.
authors.forename: Fernando
authors.initials: F
authors.lastname: Aguilar
authors.affiliation: Molecular Discovery Research, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors.forename: Imanol
authors.initials: I
authors.lastname: Peña
authors.affiliation: Molecular Discovery Research, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors.forename: Jesús L
authors.initials: JL
authors.lastname: Presa
authors.affiliation: London School of Hygiene & Tropical Medicine, London, United Kingdom.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Yardley
authors.affiliation: Molecular Discovery Research, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors.forename: Julio J
authors.initials: JJ
authors.lastname: Martin
authors.affiliation: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Madrid, Spain.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Coteron
authors.affiliation: London School of Hygiene & Tropical Medicine, London, United Kingdom.
authors.forename: Simon L
authors.initials: SL
authors.lastname: Croft
authors.affiliation: Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Madrid, Spain juan.2.cantizani-perez@gsk.com.
authors.forename: Juan
authors.initials: J
authors.lastname: Cantizani
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology*; D000818:Animals; D000981:Antiprotozoal Agents / Q000627:therapeutic use*; D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005260:Female; D006801:Humans; D007893:Leishmania donovani / Q000187:drug effects / Q000254:growth & development*; D007898:Leishmaniasis, Visceral / Q000188:drug therapy*; D008264:Macrophages / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D021261:Parasitic Sensitivity Tests; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D000981:Antiprotozoal Agents; D010767:Phosphorylcholine; C039128:miltefosine; D000666:Amphotericin B