Dronedarone, an amiodarone analog with improved anti-Leishmania mexicana efficacy

Abstract

Dronedarone and amiodarone are cationic lipophilic benzofurans used to treat cardiac arrhythmias. They also have activity against the parasitic protozoan Trypanosoma cruzi, the causative agent of Chagas' disease. They function by disrupting intracellular Ca2+ homeostasis of the parasite and by inhibiting membrane sterol (ergosterol) biosynthesis. Amiodarone also has activity against Leishmania mexicana, suggesting that dronedarone might likewise be active against this organism. This might be of therapeutic interest, since dronedarone is thought to have fewer side effects in humans than does amiodarone. We show here that dronedarone effectively inhibits the growth of L. mexicana promastigotes in culture and, more importantly, has excellent activity against amastigotes inside infected macrophages (the clinically relevant form) without affecting the host cell, with the 50% inhibitory concentrations against amastigotes being 3 orders of magnitude lower than those obtained previously with T. cruzi amastigotes (0.65 nM versus 0.75 μM). As with amiodarone, dronedarone affects intracellular Ca2+ homeostasis in the parasite, inducing an elevation of intracellular Ca2+ levels. This is achieved by rapidly collapsing the mitochondrial membrane potential and inducing an alkalinization of acidocalcisomes at a rate that is faster than that observed with amiodarone. We also show that dronedarone inhibits parasite oxidosqualene cyclase, a key enzyme in ergosterol biosynthesis known to be vital for survival. Overall, our results suggest the possibility of repurposing dronedarone as a treatment for cutaneous, and perhaps other, leishmaniases.

FIG 1

Structures of molecules of interest.

FIG 2

Inhibition of L. mexicana promastigotes growth by dronedarone. (A) Susceptibility of promastigotes to dronedarone (Droned). Each point represents the mean ± SD of at least three independent experiments. (Inset) Dose-response curve from panel A (IC₅₀, 115 nM). (B) Percentages of growth inhibition induced by dronedarone in promastigotes. All of the values were taken from the data of panel A, using the values obtained at 10 days after addition of the corresponding drug concentration. Each point represents the mean ± SD.

FIG 3

Effect of dronedarone against intracellular amastigotes. Macrophages (J774 cells) infected with L. mexicana amastigotes were exposed to different concentrations of dronedarone. The percentages of infected macrophages and the effects on noninfected macrophages were determined at 72 h after the addition of the drug. Around 100 cells were counted in each experiment. Each point represents the mean ± SD of at least three independent experiments.

FIG 4

Effect of dronedarone (Droned) on the [Ca²⁺]_i of L. mexicana promastigotes. Promastigotes were loaded as described under Materials and Methods. (A) Effect of 2.5 μM dronedarone (arrow) on the parasite cytoplasmic Ca²⁺ concentration in the presence of 2 mM external Ca²⁺. (B) Effect of 2.5 μM dronedarone (arrow) on fura 2-loaded promastigotes in the absence of external Ca²⁺ (EGTA). (C) Effect of 0.65 nM dronedarone (arrow) on the parasite cytoplasmic Ca²⁺ concentration in the presence of 2 mM external Ca²⁺. The figures shown are representative of at least three independent experiments. (D) Initial slopes (300 s) of the curves obtained in the presence of 2 mM external Ca²⁺. The first and second slopes were taken from at least three independent experiments similar to that shown in panel A. The slope named IC₅₀ was taken from at least three independent experiments similar to that shown in panel C. Each point represents the mean ± SD. *, significant difference (measured using Student's t test, P < 0.01); nd, not determined.

FIG 5

Effect of dronedarone on acidocalcisomes from L. mexicana promastigotes. Parasites were loaded with acridine orange (2 μM) as described in Materials and Methods. The excitation wavelength was 488 nm, and the emission wavelength was 530 nm. (A) Upper black trace, effect of dronedarone (Droned, 2.5 μM), followed by the addition of nigericin (Nig, 2 μM) on the acidic level of acidocalcisomes. Lower gray trace, effect of amiodarone (Amiod, 10 μM) and then of nigericin (2 μM) on the acidic level of acidocalcisomes. The additions in panel B are in reverse order versus those in panel A. (C) Initial slopes (250 s) of the curves from experiments similar to that of panels A and B. The slopes corresponding to dronedarone and amiodarone were taken from at least three independent experiments similar to that shown in panel A. The slope corresponding to nigericin was taken from at least three independent experiments similar to that shown in panel B. Each point represents the mean ± SD. *, significant difference (measured using Student's t test, P < 0.01). (D) Top, effect of dronedarone (6.5 nM) followed by the addition of nigericin (2 μM) on the acidic level of acidocalcisomes; bottom, effects of nigericin (2 μM) and then dronedarone (6.5 nM) on the acidic level of acidocalcisomes. The figures shown are representative of at least three independent experiments.

FIG 6

Action of dronedarone on the mitochondrial electrochemical potential of L. mexicana promastigotes. Parasites were incubated in the presence of rhodamine 123 for 45 min at room temperature, as indicated under Materials and Methods. (A) Effect of dronedarone (Droned, 2.5 μM), followed by the addition of FCCP (1 μM) on the mitochondrial electrochemical potential (upper black trace). Effect of amiodarone (Amiod, 10 μM) followed by the addition of FCCP (1 μM) on the mitochondrial electrochemical potential (lower gray trace). The additions in panel B are in reverse order versus those in panel A. Arrows indicate the different additions. (C) Initial slopes (300 s) of the curves from experiments similar to those of panels A and B. The slopes corresponding to dronedarone and amiodarone were taken from at least three independent experiments similar to that shown in panel A. The slope corresponding to FCCP was taken from at least three independent experiments similar to that shown in panel B. Each point represents the mean ± SD. *, Significant difference (measured using Student's t test, P < 0.01). (D) Top, effect of dronedarone (6.5 nM), followed by the addition of FCCP (1 μM) on the release of rhodamine 123 from the mitochondrion; bottom, effects of FCCP (1 μM) and then dronedarone (6.5 nM) on the release of rhodamine 123 from the mitochondrion. The figures are representative of at least three independent experiments.

FIG 7

Kinetics and inhibition of L. mexicana microsomal OSC. (A) Double-reciprocal plot of LmOSC enzyme activity as a function of the [2,3-¹⁴C]oxidosqualene concentration in the presence of the different fixed concentrations of dronedarone. (Inset) Plot of the intercepts as a function of the dronedarone concentration which yields a K_i value of ∼0.7 μM. Activity was measured in the presence of 0.1% Triton X-100. (B) Effects of dronedarone on the activity of L. mexicana microsomal OSC in the presence of saturating substrate concentrations.

FIG 8

Suggested binding site for dronedarone in L. mexicana oxidosqualene cyclase. (A) Glide docking pose of dronedarone (compound 2) bound to a Phyre2-predicted structure model (magenta) superimposed on the X-ray structure of Ro-48-8071 (compound 3, white) bound to human OSC (PDB code 1W6J). (B) Expanded view of panel A, showing the most highly conserved Asp residues in the catalytic site. Dronedarone is in magenta, and Ro-48-8071 (compound 3) is in white. The cationic centers likely interact with the conserved Asp. (C) Superimposed structures of Ro-48-8071 (compound 3) bound to HsOSC and amiodarone (compound 1) docked to the T. cruzi OSC model. (D) Comparison between dronedarone (compound 2) and amiodarone (compound 1) docked poses. (E) Common feature pharmacophore for potent HsOSC inhibitors (compounds 3 to 7) superimposed on dronedarone (compound 2) showing common cationic (red) and aromatic/hydrophobic (orange) features.