title: A novel Leishmania major amastigote assay in 96-well format for rapid drug screening and its use for discovery and evaluation of a new class of leishmanicidal quinolinium salts.
abstract: In most laboratories, the screening for leishmanicidal compounds is carried out with Leishmania promastigotes or axenic amastigotes. However, the best approach to identify leishmanicidal compounds is the use of amastigotes residing in macrophages. Reporter gene-based assays are relatively new tools in the search for drugs against eucaryotic protozoa, permitting the development of faster, more automated assays. In this paper, we report on the establishment of a rapid screening assay in a 96-well format. A luciferase-transgenic (Luc-tg) Leishmania major strain was generated and used to infect bone marrow-derived macrophages (BMDM). Amastigote-infected BMDM were treated with different compound concentrations. Cells were lysed with a luciferin-containing buffer, and the resulting luminescence was measured to determine the half-maximal inhibitory concentration (IC50). To validate this new amastigote screening assay, a library of a new class of quinolinium salts was synthesized and tested for leishmanicidal activity. Some of the quinolinium salts showed very promising activities, with IC50s against intracellular amastigotes (IC50 < 1 μg/ml) and selectivity indices (SI > 20) that match the criteria of World Health Organization (WHO) for hits. Compound 21c (IC50 = 0.03 μg/ml; SI = 358) could become a new lead structure for the development of improved chemotherapeutic drugs against L. major. In summary, we describe the establishment of a new 96-well format assay with Luc-transgenic L. major for the rapid screening of compounds for leishmanicidal activity against intracellular amastigotes and its application to the identification of a new class of quinolinium salts with most promising leishmanicidal activity.
authors.affiliation: Institute of Organic Chemistry, University of Würzburg, Würzburg, Germany. bringman@chemie.uni-wuerzburg.de
authors.forename: Gerhard
authors.initials: G
authors.lastname: Bringmann
authors.forename: Katja
authors.initials: K
authors.lastname: Thomale
authors.forename: Sebastian
authors.initials: S
authors.lastname: Bischof
authors.forename: Christoph
authors.initials: C
authors.lastname: Schneider
authors.forename: Martina
authors.initials: M
authors.lastname: Schultheis
authors.forename: Tobias
authors.initials: T
authors.lastname: Schwarz
authors.forename: Heidrun
authors.initials: H
authors.lastname: Moll
authors.forename: Uta
authors.initials: U
authors.lastname: Schurigt
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D061245:Axenic Culture; D002478:Cells, Cultured; D018320:Leishmania major / Q000187:drug effects*; D016773:Leishmaniasis, Cutaneous / Q000188:drug therapy*; D017737:Macrophages, Peritoneal / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D021261:Parasitic Sensitivity Tests; D011806:Quinolinium Compounds / Q000494:pharmacology*
chemical_list: D000981:Antiprotozoal Agents; D011806:Quinolinium Compounds