Inhibition of Leishmania mexicana Growth by the Tuberculosis Drug SQ109

Abstract

We report that the tuberculosis drug SQ109 [N-adamantan-2-yl-N′-((E)-3,7-dimethyl-octa-2,6-dienyl)-ethane-1,2-diamine] has potent activity against the intracellular amastigote form of Leishmania mexicana (50% inhibitory concentration [IC₅₀], ∼11 nM), with a good selectivity index (>500). It is also active against promastigotes (IC₅₀, ∼500 nM) and acts as a protonophore uncoupler, in addition to disrupting Ca²⁺ homeostasis by releasing organelle Ca²⁺ into the cytoplasm, and as such, it is an interesting new leishmaniasis drug hit candidate.

TEXT

There is a need for new drugs to treat the neglected tropical diseases, in particular, Chagas disease and leishmaniasis. In previous work (1–5), we discovered that the antiarrhythmia drugs amiodarone (Fig. 1, compound 1) and dronedarone (Fig. 1, compound 2) had activity against Trypanosoma cruzi as well as Leishmania mexicana, the causative agents of Chagas disease and one form of cutaneous leishmaniasis, respectively. In addition, amiodarone was found to have partial in vivo activity against T. cruzi in mice, which was considerably increased when added in combination with posaconazole (1), and in initial clinical work in humans, it has been used to treat parasitic infections (6, 7). The mechanism of action of compounds 1 and 2 is thought to involve uncoupling activity, with the release of Ca²⁺ from intracellular organelles (acidocalcisomes and mitochondria), as well as inhibition of oxidosqualene synthase and hence, ergosterol biosynthesis. Interestingly, another type of uncoupler, the nitrothiazole nitazoxanide (Fig. 1, compound 3), and its active metabolite, tizoxanide (Fig. 1, compound 4), also have activity against T. cruzi and L. mexicana (8), and compound 4 has been shown to act, at least in part, as an uncoupler, in Mycobacterium tuberculosis (9).

FIG 1.

Structures of compounds discussed in the text.

Since we and others recently reported (10–13) that another M. tuberculosis drug/drug lead (13–15), SQ109 (Fig. 1, compound 5) [N-adamantan-2-yl-N′-((E)-3,7-dimethyl-octa-2,6-dienyl)-ethane-1,2-diamine], also acted as an uncoupler in Mycobacterium smegmatis, we tested it against T. cruzi, finding 50% inhibitory concentrations (IC₅₀s) of ∼50 nM against trypomastigotes, ∼5 μM against epimastigotes, and ∼1 μM against amastigotes (13). The amastigote result was disappointing, being less effective than that we found with dronedarone (∼1 nM); however, since both amiodarone (Cordarone) and dronedarone (Multaq) come with “black box” warnings, we elected to test SQ109 against L. mexicana, since it seemed possible that it might have good activity against this parasitic protozoan, much in the same way as it does against M. tuberculosis (and M. smegmatis).

We show in Fig. 2A and B the effects of SQ109 on the viability of L. mexicana promastigotes and intracellular amastigotes (inside J774 macrophages) and on macrophage viability (Fig. 2C and D). As can be seen in Fig. 2A and B, SQ109 inhibits the viability of L. mexicana promastigotes in a dose-dependent manner, and as shown in Fig. 2B, the IC₅₀ (after 72 h of treatment) is 0.53 ± 0.06 μM (plus/minus indicates the standard error of the mean for at least three independent experiments). There is little effect on macrophage viability, since the IC₅₀ (after 72 h of treatment) is 5.8 ± 0.1 μM (Fig. 2C).

FIG 2.

Effects of SQ109 on L. mexicana and J774 macrophages. (A) Promastigote growth as a function of time and SQ109 concentration. (B) Effects of SQ109 on the viability of L. mexicana promastigotes. The IC₅₀ (after 72 h of treatment) is 0.53 ± 0.06 μM. Miltefosine is shown as a positive control. The asterisks represent statistically significant differences, determined using the Student t test, P ≤ 0.05 (*) and P ≤ 0.01 (**). (C) Effects of SQ109 on J774 macrophage viability; IC₅₀ (after 72 h of treatment) is 5.8 ± 0.1 μM. (D) Effects of SQ109 on macrophages infected with L. mexicana amastigotes. SQ109 potently inhibits amastigote proliferation, yielding an IC₅₀ (after 48 h of treatment) of 11 ± 0.9 nM. The protocols used in panels A and D are the same as those described in reference 5 and for panels B and C in reference 16 (the error bars represent standard deviation for least three independent experiments).

More significantly, in the intracellular assay (Fig. 2D), the 50% inhibitory concentration against amastigotes in infected macrophages (after 48 h of treatment) was 11 ± 0.9 nM, with no observable effect on uninfected macrophages. These results are clearly much more impressive than those we reported earlier with SQ109 in T. cruzi and represent a good selectivity index (calculated by IC₅₀ of J774 macrophages/IC₅₀ of L. mexicana amastigotes) of >500. The question then arises, what is the mechanism of action of SQ109 in L. mexicana?

As noted above, in earlier work on M. smegmatis, we and others (10–12) showed that a major mechanism of action of SQ109 (as well as amiodarone) was on the proton motive force (PMF), with SQ109 collapsing pH gradients, as determined by nuclear magnetic resonance spectroscopy, and electrochemical potentials (in both M. smegmatis and Escherichia coli membrane vesicles), as determined by fluorescence spectroscopy. Similar results were obtained with amiodarone and dronedarone in T. cruzi and L. mexicana (1–5). Here, as shown in Fig. 3A, we find that SQ109 collapses the PMF in L. mexicana promastigotes, as determined using rhodamine 123. The addition of the known uncoupler FCCP [carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazine] after the addition of SQ109 (5 μM) has no further effect, since the PMF is already collapsed, but if FCCP (2 μM) is added first, there is a partial collapse in the PMF, which is complete after the addition of SQ109 (5 μM) (Fig. 3B). These results are similar to the effects of dronedarone on L. mexicana (5). Likewise, we find that SQ109 causes a release of Ca²⁺ from internal Ca²⁺ (Ca²⁺_i) stored in organelles (Fig. 3C and D). In the presence of 2 mM external Ca²⁺, the same amount of Ca²⁺ is released by SQ109 (as determined using fura 2) as in the absence of external Ca²⁺ (i.e., in the presence of 8 mM ethylene glycol tetraacetic acid [EGTA], a high-affinity Ca²⁺-specific chelator), which means that it is not the external Ca²⁺ that is involved in the increase in Ca²⁺_i. Rather, Ca²⁺ is released from internal stores, mitochondria and acidocalcisomes, again, just as found for T. cruzi and L. mexicana with amiodarone and dronedarone (1–5). This disruption of Ca²⁺ homeostasis in addition to the effects on the proton motive force (as seen also in mycobacteria [10–12]) are likely to make major contributions to L. mexicana cell killing.

FIG 3.

Effects of SQ109 and FCCP on the mitochondrial electrochemical potential and SQ109 on Ca²⁺ flux. (A) SQ109 at 5 μM, followed by 2 μM FCCP using rhodamine 123 as a probe of the electrochemical potential. (B) Same as panel A but FCCP added first and then SQ109. (C) Ca²⁺_i as determined using the radiometric fluorescent Ca²⁺ indicator fura 2 in the presence of 2 mM external Ca²⁺. (D) Same as panel C but no external Ca²⁺ (8 mM EGTA). Methods are as described in reference 5.

Overall, the results we described above are of interest, since we find that the tuberculosis drug SQ109, currently in clinical trials, has potent activity (IC₅₀, 11 ± 0.9 nM) against the clinically relevant amastigote form of Leishmania mexicana, inside macrophages, with a selectivity index of >500. SQ109 appears to act, at least in part, as a protonophore uncoupler (as it does in mycobacteria), in addition to releasing Ca²⁺ from intracellular stores, basically the same mechanism as that found with amiodarone and dronedarone, and as such, it may represent a potential new hit candidate for treating leishmanial diseases.

Funding Statement

This work was supported by the Fondo Nacional de Ciencias, Tecnología e Investigación, Venezuela (FONACIT; grant 2011000884 to G.B.), by the Consejo de Desarrollo Científico y Humanístico-Universidad Central de Venezuela (CDCH-UCV; grant PG-03-8728-2013/2 to G.B.), and in part by the U.S. Public Health Service (NIH grants CA158191 and GM065307 to E.O.), a Harriet A. Harlin Professorship (E.O.), and the University of Illinois Foundation/Oldfield Research Fund.