Fig 5.

The casein kinase 1 inhibitor D4476 causes death in L. donovani promastigotes. (A) Modulation of endogenous Leishmania kinase activities by staurosporine and D4476. L. donovani cell extracts were treated with vehicle alone (0.5% DMSO, control [co]) or treated with either 10 μM staurosporine (S) or D4476 (D) for 5 min before adding the peptide substrate and ATP. The percentage of phosphorylation of the substrate compared to that of untreated cell extracts (determined as described in Materials and Methods) is reported in the form of a heat map, with gray corresponding to phosphorylation and black to the absence or inhibition of phosphorylation. Abbreviations: cyclic AMP-dependent protein kinase (PKA), protein kinase B (PKB), protein kinase CΔ (PKC), casein kinases (CK1 and -2), cyclin-dependent kinases (CDK2 and -5), and AMP-activated protein kinase (AMPK). (B) Inhibition of CK1 activity by D4476 kills Leishmania through necrosis. Cell growth analysis (left). Cell growth was determined following measurement of optical density (OD) at 600 nm in the presence of 30 μM (●) and 60 μM (○) D4476. Right, FACS analysis. The three markers TMRE (tetramethylrhodamine ethyl), AV (annexin V), and PI (propidium iodide) were followed to define the mode of cell death induced in L. donovani in the presence of D4476 at 60 μM. The percentages of cells showing mitochondrial membrane depolarization (negative %) or hyperpolarization (positive %) (●), annexin V binding (○), and propidium iodide incorporation (▼) are represented. The experiment was carried out twice, and one representative result is shown.