Leishmania donovani Project

L. donovani screen

L. donovani amastigote hits

High Throughput Protocol

Leishmania donovani (strain MHOM/ET/67/HU3) promastigotes. Promastigote stages of L. donovani (5 x 10 5 cells/ml) were cultured in medium 199 (Sigma-Aldrich), supplemented with 10% heat-inactivated FBS (Omega Scientific, Tarzana, CA). 100 mL aliquots of the parasites were transferred into 96-well culture plates containing 1 micromolar (final working concentration) FDA library compounds and incubated at 27°C. Cultures treated with amphotericin B (10 micromolar) were incubated alongside as positive leishmanicidal controls. Parasite viability was determined at Day 7 of culture using the CellTiter-Glo™ luminescent cell viability assay (Promega, WI) according to the manufacturer's instructions. Compound activity was expressed as the percent reduction in parasite viability relative to that in DMSO-treated cultures.

Low Throughput Protocol

Leishmania amastigotes in macrophage screen.
1 x10 5, irradiated J774 murine macrophage cell line, were seeded onto 4-well chamber glass slides and incubated overnight at 37°C in RPMI+ 5%FBS and then infected with Leishmania donovani amastigotes at an MOI (multiplicity of infection) of 10.  After 3 hours, cells were washed twice with 800uL 37°C PBS. RPMI + 5% FBS containing either 1 or 10 µM of drug (diluted from 20mM stocks in DSMO) was added and cultures incubated for 48 hours. Leishmanicidal controls of 1 micromolar and 10 micromolar Amphotericin B were included. After 48 hours the slides were fixed and subjected to DiffQuick staining. Amastigotes per 100 macrophages was determined by counting at 200 x magnification.  Results were compared to DMSO controls in culture medium.