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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 Ca(2+) homeostasis by releasing organelle Ca(2+) into the cytoplasm, and as such, it is an interesting new leishmaniasis drug hit candidate., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The disruption of the intracellular Ca(2+) homeostasis of Leishmania mexicana represents a major target for the action of drugs, such as amiodarone and miltefosine. However, little is known about the mechanism of Ca(2+) entry to these cells. Here we show the presence of a Ca(2+) channel in the plasma membrane of these parasites. This channel has many characteristics similar to the human L-type voltage-gated Ca(2+) channel. Thus, Ca(2+) entry is blocked by verapamil, nifedipine and diltiazem while Bay K 8644 opened this channel. However, different to its human counterpart, sphingosine was able to open this channel, while other well known sphingolipids had no effect. This fact could have important pharmacological implications., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Leishmaniasis represents a serious public health problem worldwide. The first line of treatment is based on glucantime and pentostan, which generate toxic effects in treated patients. We have recently shown that amiodarone, frequently used as an antiarrhythmic, possesses activity against Trypanosoma cruzi through the disruption of mitochondrial Ca(2+) homeostasis and the inhibition of parasite ergosterol biosynthesis, specifically at the level of oxidosqualene cyclase activity (G. Benaim, J. Sanders, Y. Garcia-Marchan, C. Colina, R. Lira, A. Caldera, G. Payares, C. Sanoja, J. Burgos, A. Leon-Rossell, J. Concepcion, A. Schijman, M. Levin, E. Oldfield, and J. Urbina, J. Med. Chem. 49:892-899, 2006). Here we show that at therapeutic concentrations, amiodarone has a profound effect on the viability of Leishmania mexicana promastigotes. Additionally, its effect on the viability of the parasite was greater against intracellular amastigotes than against promastigotes, and it did not affect the host cell. Using fluorimetric and confocal microscopy techniques, we also demonstrated that the mechanism of action of amiodarone was related to the disruption of intracellular Ca(2+) homeostasis through a direct action not only on the mitochondria but also on the acidocalcisomes. On the other hand, analysis of the free sterols in promastigotes incubated with amiodarone showed that this drug also affected the biosynthesis of 5-dehydroepisterol, which results in squalene accumulation, thus suggesting that amiodarone inhibits the squalene epoxidase activity of the parasite. Taken together, the results obtained in the present work point to a more general effect of amiodarone in trypanosomatids, opening potential therapeutic possibilities for this infectious disease.

Calmodulin (CaM), a major intracellular Ca2+ receptor protein, has been identified and partially characterized in several trypanosomatids. The amino acid sequences of CaM from Trypanosoma cruzi and Trypanosoma brucei are known, while that from Leishmania mexicana is not. CaM from T. cruzi contains 18 amino acid substitutions, as compared with CaM from bovine brain. In addition, CaM from bovine brain contains two tyrosine residues (Tyr-99 and Tyr-138), while CaM from T. cruzi only contains Tyr-138. In the present work we show that a monoclonal antibody developed against the carboxyl-terminal region of bovine brain CaM fails to recognize CaM from both T. cruzi and L. mexicana. CaM from both parasites and from bovine brain were phosphorylated in vitro by a preparation of CaM-binding protein kinases enriched in the epidermal growth factor (EGF) receptor. Phosphoamino acids analysis demonstrated EGF-dependent phosphorylation of tyrosine residues in bovine brain CaM, while only trace amounts of tyrosine phosphorylation were detected in CaM from both trypanosomatids. These results demonstrate that the EGF receptor tyrosine kinase targets Tyr-99, but not Tyr-138, as the single major phosphorylatable residue of CaM. On the other hand, and in contrast to bovine brain CaM, there is a significant phosphorylation of serine residues in CaM from trypanosomatids which is activated by the EGF receptor via a protein-serine/threonine kinase cascade.

Some general features of the respiratory chain and respiratory control were characterized in coupled mitochondrial preparations from Leishmania mexicana promastigotes. O2 uptake was sensitive to the electron-transfer inhibitors rotenone, flavone, malonate, 4,4,4-trifluoro-1-(2-thienyl) 1.3 butanedione (TTFA), antimycin A, 2n-nonyl-4-hydroxyquinoline-N-oxide (HQNO), myxothiazol, cyanide and azide. A high concentration of rotenone (60 microM) was required to inhibit O2 uptake effectively. Difference spectra revealed the presence of cytochromes (a + a3), b and c. Respiratory control was stimulated 2-fold by ADP with different exogenous oxidizable substrates. Calculated ADP/O ratios were consistent with the notion that ascorbate/N,N,N',N'-tetramethylphenylenediamine (TMPD)-linked and FAD-linked respiration proceeds, respectively, with one third and two thirds of the ATP producing capacity of NADH-linked respiration. State 3 was suppressed by the ATP synthase inhibitors oligomycin and aurovertin and by the adenine nucleotide translocator inhibitors atractyloside and carboxy atractyloside. The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) provoked state 3u respiration. The mitochondrial preparation was capable of Ca2+ uptake and Ca2+ stimulated respiration. Data obtained suggests strongly that mitochondrial complexes I, II, III and IV are present in a major pathway of electron-transfer and that oxidative phosphorylation might proceed with high bioenergetic efficiency.

The mechanism responsible for the regulation of intracellular Na+ and K+ concentrations in trypanosomatids is unknown. In higher eukaryotes a ouabain-sensitive Na+,K(+)-ATPase located in the plasma membrane is the main mechanism for the regulation of the intracellular concentrations of Na+ and K+, while in trypanosomatids there are conflicting evidences about the existence of this type of ATPase. By the use of a highly enriched plasma membrane fraction, we showed that an ouabain-sensitive Na+,K(+)-ATPase is present in L. mexicana. The affinity of the enzyme for Na+ and K+ is similar to that reported for the mammalian Na+,K(+)-ATPase, showing also the same kinetic parameters regarding the relative concentration of those cations that give the optimal activity. Vanadate (10 microM) fully inhibits the ATPase activity, suggesting that the enzyme belongs to the P-type family of ionic pumps. The enzyme is sensitive to ouabain and other cardiac glycosides. These cardiac glycosides do not show any appreciable effect on the higher Mg(2+)-ATPase activity present in the same preparation. By the use of [3H]ouabain, we also show in this report that the binding of the inhibitor to the enzyme was specific. Taken together, these results demonstrate that an ouabain-sensitive Na+,K(+)-ATPase is present in the plasma membrane of Leishmania mexicana. Therefore, this Na+,K(+)-ATPase should participate in the intracellular regulation of these cations in Leishmania.

The intracellular Ca2+ concentration in different trypanosomatids is about 50 nanomolar, which concentration in different trypanosomatids is about 50 nanomolar, which is 4 orders of magnitude lower than in the extracellular milieu. This fact implies the existence of well developed mechanisms for the maintenance of such a high calcium gradient. In higher eukaryotics a number of different structures have been implicated in this function. Some of them are located in intracellular organelles, and others in the plasma membrane. Since intracellular organelles are limited by their storage capacity, long-term Ca2+ homeostasis resides solely in the plasma membrane. In higher eukaryotics, a calcium pump or Ca(2+)-ATPase located in the plasma membrane, because of its high Ca2+ affinity, has been proposed as the structure responsible for the maintenance of the cytoplasmic Ca2+ concentration at the submicromolar level. The presence of a Ca(2+)-ATPase in trypanosomatids has been debated. While some groups have reported its absence, others have reported the existence of an enzyme which is Mg(2+)-independent or even inhibited by Mg2+. On the other hand, in none of these reports any correlation was shown between the Ca(2+)-ATPase activity observed and the Ca2+ transport function attributed to this enzyme. We have previously shown that a calmodulin-stimulated Mg(2+)-dependent Ca(2+)-ATPase is present in the plasma membrane of Leishmania braziliensis and of Trypanosoma cruzi. Plasma membrane vesicles from these parasites are able to accumulate Ca2+ in the presence of the ATP-Mg complex. The similarities found between the kinetics parameters and other properties of the Ca(2+)-ATPase and the Ca2+ transport activity strongly suggest a common molecular entity. The stoichiometry calculated from these parameters approaches the 1:1 stoichiometry for Ca2+ and ATP, as reported for the Ca2+ pump from higher eukaryotic cells. In this report we show that plasma membrane vesicles from Leishmania mexicana possess a Ca(2+)-ATPase with characteristics which are similar to that reported by us for other trypanosomatids. Thus, the enzyme has a high Ca2+ affinity which is further increased upon addition of calmodulin. The maximal velocity is also increased by calmodulin. As it has been found in the Ca(2+)-ATPase from human erythrocytes, trypsin proteolysis stimulates the ATPase activity mimicking the effect of calmodulin. On the other hand, antibodies raised against the isolated Ca(2+)-ATPase from human erythrocytes are effective in recognizing the enzyme from Leishmania mexicana, thus supporting a stronger homology between both Ca(2+)-ATPases.