Miltefosine (hexadecylphosphocholine) inhibits cytochrome c oxidase in Leishmania donovani promastigotes

Abstract

Miltefosine (hexadecylphosphocholine [HePC]) is currently on trial as a first-choice, orally active drug for the treatment of visceral leishmaniasis when resistance to organic pentavalent antimonials becomes epidemic. However, data on the targets involved in its leishmanicidal mechanism have, until now, been only fragmentary. We have carried out a systematic study of the alterations induced on the bioenergetic metabolism of Leishmania donovani promastigotes by HePC. Overnight incubation with HePC caused a significant decline in the intracellular ATP levels of the parasites, together with a reduction in the oxygen consumption rate and mitochondrial depolarization, while the integrity of the plasma membrane remained undamaged. In a further step, the effects of HePC on the respiratory chain were addressed in digitonized parasites. The inhibition of the oxygen consumption rate caused by HePC was not reverted either with the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone or with tetramethyl-p-phenylenediamine plus ascorbate, which feeds the electron transport chain at the level of cytochrome c. These results suggest that cytochrome c oxidase is a likely target in the complex leishmanicidal mechanism of HePC. This was further confirmed from the finding that this enzyme was specifically inhibited in a dose-dependent manner by HePC, but not the cytochrome c reductase, ruling out an unspecific effect of HePC on the respiratory chain.

FIG. 1.

Plasma membrane permeabilization and viability of L. donovani promastigotes after incubation for 14 h with HePC. The entry of SYTOX green after incubation with HePC was monitored by determination of the increase in fluorescence (excitation λ, 485 nm; emission λ, 520 nm). The results were normalized as the percentage of fluorescence relative to that obtained by maximal parasite permeabilization with 0.1% TX-100. The inhibition of parasite viability was assessed by determination of the inhibition of MTT reduction.

FIG. 2.

Variation of in vivo luminescence of strain 3-Luc L. donovani promastigotes treated with HePC. Promastigotes incubated for different times with HePC at the indicated concentrations were loaded with 25 μM DMNPE-luciferin. The variation in luminescence was normalized relative to that for the untreated parasites. Maximal inhibition of luminescence was achieved with 10 mM KCN.

FIG. 3.

Variation of ΔΨ_m of L. donovani promastigotes, as monitored by rhodamine 123 accumulation. The geometric mean from the fluorescence histograms was plotted against the HePC concentration. Parasites preincubated for 14 h with HePC were loaded with 0.3 μg/ml rhodamine 123, and the fluorescence level was measured by cytofluorometry. (Inset) Fluorescence histograms for the respective experiments. The drug concentration is shown at the side of each trace. Fully depolarized parasites were obtained by incubation with 10 mM KCN.

FIG. 4.

Oxygen consumption rates of digitonin-permeabilized L. donovani promastigotes. The oxygen consumption rates before HePC addition were 13.1 nmol × min⁻¹ × 10⁻⁸ cells when 5 mM succinate was used as the substrate. Other substrates and inhibitors were added at their respective final concentrations, as follows: 60 μM digitonin (Dgt), 100 μM ADP, 12.6 μM oligomycin (Olg), 10 μM FCCP, 25 μM HePC, 1 μM antimycin A (Ant), and 0.1 mM TMPD plus 1.7 mM ascorbate (TMPD).

FIG. 5.

CcO activity with HePC. The mean CcO activity ± standard deviation was monitored by determination of the decrease in the absorbance at 550 nm that a reduced cytochrome c solution (32 μM) underwent when it was oxidized by CcO at 37°C. The spontaneous oxidation rate was determined in samples previously incubated with 10 mM KCN. The following HePC concentrations (μM) were tested: 40 (▪), 25 (▿), 15 (▾), and 0 (no treatment) (•). ○, 10 mM KCN.