In vitro activity of salinomycin and monensin derivatives against Trypanosoma brucei
© The Author(s). 2016
Received: 5 May 2016
Accepted: 14 July 2016
Published: 25 July 2016
African trypanosomes are the causative agents of sleeping sickness in humans and nagana disease in livestock animals. As the few drugs available for treatment of the diseases have limited efficacy and produce adverse reactions, new and better tolerated therapies are required. Polyether ionophores have been shown to display anti-cancer, anti-microbial and anti-parasitic activity. In this study, derivatives of the polyether ionophores, salinomycin and monensin were tested for their in vitro activity against bloodstream forms of Trypanosoma brucei and human HL-60 cells.
Most polyether ionophore derivatives were less trypanocidal than their corresponding parent compounds. However, two salinomycin derivatives (salinomycin n-butyl amide and salinomycin 2,2,2-trifluoroethyl ester) were identified that showed increased anti-trypanosomal activity with 50 % growth inhibition values in the mid nanomolar range and minimum inhibitory concentrations of below 1 μM similar to suramin, a drug used in the treatment of sleeping sickness. In contrast, human HL-60 cells were considerably less sensitive towards all polyether ionophore derivatives. The cytotoxic to trypanocidal activity ratio (selectivity) of the two promising compounds was greater than 250.
The data indicate that polyether ionophore derivatives are interesting lead compounds for rational anti-trypanosomal drug development.
African trypanosomiasis is an infectious parasitic disease of humans and animals of similar aetiology and epidemiology. The causative agents of the disease are flagellated protozoans of the genus Trypanosoma. The parasites are transmitted by the bite of infected tsetse flies (Glossina sp.) and live and multiply in the blood and tissue fluids of their mammalian host. The distribution of trypanosomiasis in Africa corresponds to the range of tsetse flies and comprises an area of 8 million km2 between 14°N and 20°S latitude . In this so-called tsetse belt, millions of people and cattle are at risk of contracting the disease [2, 3]. Throughout history, African trypanosomiasis has severely repressed the economic and cultural development of central Africa .
For treatment of African trypanosomiasis only a handful drugs are available. All the drugs are outdated, require parenteral administration, induce significant toxic side effects, have limited efficacy and are being increasingly subject to drug resistance [5–7]. Thus, there is an urgent need for the development of new, more effective and safer treatments for African trypanosomiasis.
In recent years, polyether ionophores have received attention as promising anti-cancer candidate drugs . However, compounds displaying anti-cancer activity often also exhibit trypanocidal activity . Recently it has been shown that the ionophore salinomycin inhibits the growth of bloodstream forms of T. brucei in vitro at sub-micromolar concentration . Although salinomycin was shown to be less toxic to human cells, its selectivity (cytotoxic/trypanocidal ratio) was in a moderate range (< 100) . Therefore, we were interested whether chemical modification of polyether ionophores, such as salinomycin and monensin, could lead to compounds with improved trypanocidal activity and better selectivity.
Bloodstream forms of T. brucei (clone 427-221a)  and human myeloid leukaemia HL-60 cells  were grown in Baltz medium  and RPMI medium , respectively. Both culture media were supplemented with 16.7 % (v/v) heat-inactivated foetal calf serum. All cultures were maintained in a humidified atmosphere containing 5 % CO2 at 37 °C.
Cells were seeded in 96-well plates in a final volume of 200 μl of their respective culture medium containing 10-fold serial dilutions of ionophore derivatives (10-4 to 10-10 M) and 1 % DMSO. Wells containing medium and 1 % DMSO served as controls. The initial cell densities were 1 × 104/ml for T. brucei and 1 × 105/ml for HL-60 cells. After 24 h incubation at 37 °C in a humidified atmosphere containing 5 % CO2, 20 μl of a 0.44 mM resazurin solution prepared in PBS was added and the cells were incubated for a further 48 h so that the total incubation time was 72 h. Thereafter, the plates were read on a microplate reader using a test wavelength of 570 nm and a reference wavelength of 630 nm. The 50 % growth inhibition (GI50) value, i.e. the concentration of a compound necessary to reduce the growth rate of cells by 50 % compared to the control was determined by linear interpolation according to the method described in . The minimum inhibitory concentration (MIC) values, i.e. the concentration of the drug at which all trypanosome and human cells were killed, was determined microscopically. Each compound was independently tested three times.
Measurement of changes in cell volume
Change in cell volume was determined by light scattering as previously described . In brief, bloodstream forms of T. brucei were seeded at a density of 5 × 107 cells/ml in 96-well plates in a final volume of 200 μl culture medium containing 100 μM of salinomycin or the salonimycin derivatives SAL-E7 or SAL-AM2 and 1 % DMSO. Absorbance of the cultures was measured at 490 nm every 15 min. A decrease in absorbance corresponded to an increase in cell volume. The experiment was repeated three times.
Results and discussion
GI50 and MIC values and ratios of salinomycin and monensin derivatives for T. brucei and HL-60 cells
0.18 ± 0.06
0.44 ± 0.21
3.08 ± 0.18
35.5 ± 2.4
3.25 ± 0.26
34.6 ± 1.9
3.10 ± 0.21
33.8 ± 3.5
3.12 ± 0.08
32.9 ± 2.5
3.21 ± 0.02
38.4 ± 4.2
3.01 ± 0.06
0.057 ± 0.029
16.4 ± 1.9
3.23 ± 0.21
38.9 ± 3.2
0.040 ± 0.007
14.5 ± 1.3
2.94 ± 0.20
7.92 ± 1.95
2.69 ± 0.51
24.5 ± 3.7
2.69 ± 0.20
39.2 ± 1.6
0.029 ± 0.002
1.48 ± 0.56
3.06 ± 0.06
34.1 ± 2.2
2.76 ± 0.10
25.3 ± 5.0
1.68 ± 0.56
20.3 ± 3.1
0.31 ± 0.06
23.3 ± 6.6
0.035 ± 0.002
For determination of the general cytotoxicity of salinomycin and monensin derivatives, HL-60 cells were used as reference because their sensitivity for approved trypanocides is well established [24, 25]. All derivatives were less cytotoxic towards HL-60 cells than their parent compounds with MIC values of 100 μM and GI50 values ranging between 7.9–39 μM (Table 1). Compound SAL-E6 did not affect HL-60 cells, even at 100 μM, indicating that salinomycin 2,4-dinitrobenzyl ester displays no cytotoxicity (Table 1). Overall, the observed cytotoxic activities of the salinomycin derivatives were in good agreement with previously reported findings [11–13].
With the exception of SAL-AM3 (benzyl amide), the selectivity indices (MIC and GI50 ratios) of salinomycin derivatives were found to be better than those of the parent compound salinomycin. Most derivatives had selectivity indices of around 10 (Table 1). Compounds SAL-E7 and SAL-AM2 had promising MIC and GI50 ratios of > 100 (Table 1). In contrast to salinomycin derivatives, the selectivity indices of most monensin derivatives were found to be inferior to the parent compound monensin (Table 1). Only MON-UR1 (alkyl urethane) had MIC and GI50 ratios similar to those of monensin (Table 1). By comparison, drugs used for treatment of African trypanosomiasis have much higher selectivity indices [24, 25]. For example, the reference drug suramin displayed no toxicity towards HL-60 cells with MIC and GI50 values greater than 100 μM. Accordingly, the MIC and GI50 ratios for suramin were > 1000 and > 2857, respectively (Table 1).
This study has shown that the polyether ionophores can be modified into derivatives with improved trypanocidal and reduced cytotoxic activity. Two salinomycin derivatives, SAL-E7 and Sal-AM2, were identified that, in this respect, were superior to the parent compound. In contrast to salinomycin, derivatization of monensin did not result in compounds with increased trypanocidal activity. One reason for this maybe that monensin itself is already quite trypanocidal (about 6–10 times more active than salinomycin) and, therefore, it might be difficult to improve its trypanocidal activity further by chemical modification.
Both, SAL-E7 and SAL-AM2, match the activity criteria for drug candidates for African trypanosomiasis (GI50 < 1 μM; selectivity > 100) . However, it should be noted that in this study a cancer cell line was used for determining selectivity and that, compared with non-malignant cells, cytotoxicity of both compounds are therefore likely to be overestimated as has been shown for the parent compound salinomycin. For example, the cytotoxicity of salinomyicn in human peripheral blood mononuclear and nasal mucosa cells was determined to be in the mid-micromolar range with 50 % effective concentrations of 30 and 11 μM, respectively [10, 27]. Before developing any salinomycin derivatives into trypanocides, animal experiments should be carried out to establish the in vivo activity of the compounds.
DMSO, dimethyl sulfoxide; GI50, 50 % growth inhibition; MIC, minimum inhibitory concentration; PBS, phosphate-buffered saline
The work received financial support from the Polish National Science Centre (NCN) [2011/03/D/ST5/05884]. MA wishes to thank the Polish National Science Centre (NCN) for the doctoral scholarship “ETIUDA” [2014/12/T/ST5/00710] and the Adam Mickiewicz University Foundation for a scholarship granted in 2015/2016.
Availability of data and materials
All data are disclosed as tables and figures in the main document.
DS and AH conceived and designed the study. MA and AH carried out the synthesis of compounds. DS performed the in vitro toxicity assays. DS and AH analysed the data and drafted the manuscript. All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethical approval and consent to participate
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