Table 1.
Activity of gold standard drugs after 3-day treatment against intra- and extracellular L. donovani amastigotes.
Figure 1.
Linear correlation of the number of L. donovani amastigotes and the reduction of Alamar blue.
L. donovani amastigotes were isolated from the spleen of an infected Rag-2 −/− mouse. Up to 106 (A) or 105 (B) parasites were suspended in 200 µl/well in supplemented RPMI, Alamar blue was added and the plate incubated at 26°C. At the indicated time points, the fluorescence per well was determined and plotted over the initial number of parasites/well. The correlations between Alamar blue conversion and the initial parasite numbers are highly significant: P<0.0001 as shown by linear regression analysis (R224h– 0.9979, R248h – 0.9991, R272h – 0.9984).
Figure 2.
Release of parasites from infected macrophages in 96-well plates.
Bone marrow derived macrophages were infected overnight with L. donovani amastigotes. Cells were incubated for a further 3 days and then treated with DMEM containing the indicated concentration of saponin. Supplemented RPMI was added to the released amastigotes and plates were further incubated for 4 days at 26°C before addition of Alamar blue and subsequent measurement of fluorescence in a plate reader. The survival of freed amastigotes is dependent on the concentration of saponin used and the time of lysis. A 5-fold greater signal of reduced Alamar blue from freed amastigotes over uninfected macrophages was achieved with 2 mg·ml-1 saponin for 5 min; this concentration was used for all other experiments performed. All samples were run in triplicate; one representative experiment is shown. MΦ, uninfected macrophages; iMΦ, infected macrophages. *** denotes P<0.0001 as determined by a two-way ANOVA.
Figure 3.
Dose response curves for amphotericin B and miltefosine against intracellular L. donovani amastigotes.
Parasites were released by conditional lysis with saponin from bone marrow derived macrophages of BALB/c mice after 72 h incubation with anti-leishmanial standard drugs. Using this method, the measured activities of amphotericin B (EC50 29 nM) and miltefosine EC50 (1.45 µM) were reproducible and comparable to reported values against intracellular L. donovani (43–45). Representative plots for amphotericin B (n = 4) and miltefosine (n = 3) are shown. (R2AmphoB – 0.9905, R2Mil – 0.9963).
Figure 4.
Chemical structures of NMT inhibitors.
(A) Resynthesised T. brucei inhibitors identified by the Dundee Drug Discovery Unit as also inhibiting L. major NMT [31], [37]. Results summarized in Table 2. (B) Selective inhibitors identified by high throughput screening against L. donovani NMT [35]. Results summarised in Table 3.
Figure 5.
Cellular activity of compounds with good potency in the extracellular but varying potency in the intracellular model.
All three compounds tested showed cellular activity on the intracellular and extracellular L. donovani amastigotes. When tested against intracellular amastigotes, only DDD100887 generated a reproducible dose-response curve. It was not possible to generate dose-response curves for DDD85646 and DDD86211 as both compounds resulted in an reproducible “activity valley” at higher concentrations before resulting in complete killing of the parasites at the highest concentration tested (see text for details). One representative experiment is shown for each compound. The correlation coefficients for the extracellular amastigotes were R2>0.98, with almost all very close to or >0.99. The correlation coefficient for intracellular DDD100887 is R2 0.9212.
Table 2.
Activity values/properties of NMT inhibitors from DDD85646 series.*
Table 3.
Activity values/properties of NMT inhibitors identified in Pfizer high-throughput screen.1,2