Indirubin derivatives are potent and selective anti-Trypanosoma cruzi agents

Figures & data

Table 1. Cytotoxicity against murine macrophages J774.1 and anti-Trypanosoma cruzi activity against trypomastigote and amastigote forms of indirubins.

Compound	EC50 Try. (µM)^a	CC50 (µM)^b	EC50 Ama. (µM)^c	SI^d
Compound 10	0.7 (± 0.2)	> 10	0.61 (± 0.14)	> 16
Compound 11	0.25 (± 0.05)	6.25	0.22 (± 0.11)	28
Compound 12	0.49 (± 0.2)	2.6	-	-
Compound 13	0.45 (± 0.16)	2.4	-	-
Compound 17	0.44 (± 0.14)	10	0.90 (± 0.24)	11
Compound 5	0.37 (± 0.16)	1.5	-	-
Compound 32	1.30 (± 0.46)	> 10	1.51 (± 0.45)	> 6.6
Benznidazole	11.4 (± 1.4)	> 100	13.4 (± 0.39)	> 7.5

CC₅₀ = cytotoxicity concentration 50 %. EC₅₀ = effective concentration 50 %. SI = Selective index. Values are means ± SD of three independent experiments performed in triplicate.

^aTrypanocidal effect on trypomastigotes forms determined 24 h post-treatment.

^bCell viability of murine macrophages J774.1 determined 72 h post-treatment.

^cTrypanocidal effect on amastigotes forms determined in infected macrophages treated for 6 h.

^dCalculated by dividing the CC₅₀/EC₅₀ Ama.

Figure 1. Transmission electron microscopy of T. cruzi trypomastigotes incubated with indirubins for 24 h. (a) Untreated cells, scale bar: 1 μm. (B-F) After incubation with indirubins, we observed: (b) loss of cytoplasm after incubation with the EC₅₀ concentration of the compound 10, scale bar: 0.5 μΜ, (c) dilatation of some Golgi cisternae after treatment with the EC₅₀ concentration of the compound 32, scale bar: 0.2 μm, (d) DNA degradation with pyknotic nuclei after incubation with the EC₅₀ concentration of the compound 11, scale bar: 0.5 μΜ, (e) DNA degradation with pyknotic nuclei after incubation with the EC₅₀ concentration of the compound 17, scale bar: 0.2 μΜ and (f) appearance of myelin figures after incubation with the EC₅₀ concentration of the compounds 10, 11, 17 and 32, scale bar: 0.5 μm. Black arrows indicate changes in the organelles.

Figure 2. Scanning electron microscopy of T. cruzi trypomastigotes incubated with indirubins for 24 h. Using SEM, we observed alterations in cell shape and loss of plasma membrane integrity after treatment with the EC₅₀ and 2 x EC₅₀ concentration of compound 11 (B and C respectively) and (A) untreated cells. White arrows indicate the alterations reported.

Figure 3. Flow cytometry analysis of trypomastigotes treated with indirubins 10, 11, 17 and 32 at the EC₅₀ concentration, upon propidium iodide (PI) and annexin-V staining. (A) Untreated trypomastigotes after 24 h of incubation; (B) trypomastigotes incubated with indirubins 10, 11, 17 and 32 for 6 and 24 h.

Table 2. Parasitemia and mortality in mice infected with Y Trypanosoma cruzi strain and treated daily with 20 mg/Kg of indirubins or 100 mg/Kg of benznidazole for 5 days.

Compounds	Dose (mg/Kg)	% Blood parasitemia reduction in mice at 12 dpi^a	Mortality^b
Compound 10	20	69.3%*	5/6
Compound 11	20	94.5%*	1/6*
Compound 17	20	85%*	1/6*
Compound 32	20	34.63*	4/6
Benznidazole	100	> 99*	0/6*
Vehicle		-	6/6

^aCalculated as ([average vehicle group − average treated group)/average vehicle group] × 100%).

^bMortality was monitored for 30 days after treatment. Bdz = benznidazole. *P < 0.05 compared to vehicle group.

Figure 4. Parasitemia and survival rate of BALB/c mice infected with T. cruzi and treated with 6-substituted indirubins. Female BALB/c mice were infected with 10⁴ Y strain trypomastigotes. Five days after infection, mice (n = 6) were treated orally with indirubins 10, 11 and 17 (20 mg/Kg) or benznidazole (100 mg/kg) daily, during five consecutive days. (A) Parasitemia was monitored by counting the number of trypomastigotes in fresh blood samples. Values represent the mean± SEM of 6 mice per group. (B) Survival was monitored by 30 days after infection. Results are from one experiment of two performed. ** P < 0.01; *** P < 0.001 compared to untreated-infected group (vehicle).

Figure 5. Parasitemia and survival rate of BALB/c mice infected with T. cruzi and treated with 7-substituted indirubin. Female BALB/c mice were infected with 10⁴ Y strain trypomastigotes. Five days after infection, mice (n = 6) were treated orally with indirubin 32 (20 mg/Kg) or benznidazole (100 mg/kg) daily, during five consecutive days. (A) Parasitemia was monitored by counting the number of trypomastigotes in fresh blood samples. Values represent the mean± SEM of 6 mice per group. (B) Survival was monitored by 30 days after infection. Results are from one experiment of two performed. ** P < 0.01; *** P < 0.001 compared to untreated-infected group (vehicle).