Ent-kaurane diterpenes isolated from n-hexane extract of Baccharis sphenophylla by bioactivity-guided fractionation target the acidocalcisomes in Trypanosoma cruzi.

BACKGROUND: In the present work the bioactivity-guided fractionation of n-hexane extract from aerial parts of Baccharis sphenophylla (Asteraceae) against trypomastigote forms of Trypanosoma cruzi was performed. PURPOSE: To evaluate the antitrypanosomal potential of diterpenes ent­kaurenoic (1), grandifloric (2). and 15ß-tiglinoyloxy­ent-kaurenoic (3) acids, isolated from n-hexane extract from aerial parts of B. sphenophylla, and elucidate their mechanism of action against T. cruzi. METHODS/STUDY DESIGN: n-Hexane and MeOH extracts from aerial parts of B. sphenophylla were prepared and caused, respectively, 100% and 50% of death of trypomastigote forms of T. cruzi. Based on these results, the n-hexane extract was subjected to bioactivity-guided fractionation procedures to afford three related ent­kaurane diterpenoids (1-3). Based on spectrofluorometric assays and flow cytometry analysis, the mechanism of action of compounds 1 and 3 was investigated. RESULTS: Compounds 1 and 3, isolated from n-hexane extract from aerial parts of B. sphenophylla, showed potent activity against parasites with EC50 values of 10.6 µM (SI > 18.8) and 2.4 µM (SI = 34.8), respectively. On the other hand, compound 2 was inactive against trypomastigotes. In mechanism of action studies using the fluorescent probe SYTOX Green, the plasma membrane permeability was unaltered after treatment with compounds 1 and 3, but compound 1 induced a depolarization of the plasma membrane electric potential (ΔΨp). No substantial alterations were observed in the mitochondria after treatment with compound 3, but a transient hyperpolarization of the mitochondrial membrane potential (ΔΨm) by compound 1. Despite the increased ATP levels induced by compounds 1 and 3, no alterations of ROS and Ca2+ levels were registered. However, both compounds promoted a time-dependent alkalinization of the acidocalcisomes, probably contributing to an osmotic imbalance of the cell. In silico physicochemical studies of compounds 1-3 suggested that lipophilicity and molecular complexity may play an important role in the antitrypanosomal activity. Moreover, no pan-assay interference compounds (PAINS) alerts were detected for compounds 1-3. CONCLUSION: Obtained data indicated that the isolated ent­kaurane diterpenes from n-hexane extract from aerial parts of B. sphenophylla, especially compound 3, could be considered interesting prototypes for further modifications aiming the discovery of new hits against T. cruzi.

SQ109 inhibits proliferation of Leishmania donovani by disruption of intracellular Ca2+ homeostasis, collapsing the mitochondrial electrochemical potential (ΔΨm) and affecting acidocalcisomes.

Leishmania donovani is the causative agent of visceral leishmaniasis. Annually, 500 million new cases of infection are reported mainly in poor communities, decreasing the interest of the pharmaceutical industries. Therefore, the repositioning of new drugs is an ideal strategy to fight against these parasites. SQ109, a compound in phase IIb/III of clinical trials to treat resistant Mycobacterium tuberculosis, has a potent effect against Trypanosoma cruzi, responsible for Chagas' disease, and on Leishmania mexicana, the causative agent of cutaneous and muco-cutaneous leishmaniasis. In the latter, the toxic dose against intramacrophagic amastigotes is very low (IC50 ~ 11 nM). The proposed mechanism of action on L. mexicana involves the disruption of the parasite intracellular Ca2+ homeostasis through the collapse of the mitochondrial electrochemical potential (ΔΨm). In the present work, we show a potent effect of SQ109 on L. donovani, the parasite responsible for visceral leishmaniasis, the more severe and uniquely lethal form of these infections, obtaining a toxic effect on amastigotes inside macrophages even lower to that obtained in L. mexicana (IC50 of 7.17 ± 0.09 nM) and with a selectivity index > 800, even higher than in L. mexicana. We also demonstrated for first time that SQ109, besides collapsing ΔΨm of the parasite, induced a very rapid damage to the parasite acidocalcisomes, essential organelles involved in the bioenergetics and many other important functions, including Ca2+ homeostasis. Both effects of the drug on these organelles generated a dramatic increase in the intracellular Ca2+ concentration, causing parasite death.

Antiproliferative effect of a benzofuran derivate based on the structure of amiodarone on Leishmania donovani affecting mitochondria, acidocalcisomes and intracellular Ca2+ homeostasis.

Leishmaniasis is a parasitic disease representing an important problem of public health. Visceral leishmaniasis, resulting from infection with Leishmania donovani, causes considerable mortality and morbidity in the poorest region of the word. At present there is no current effective treatment, since the approved, drugs are expensive and are not free of undesirable side effects. Therefore, there is a need for the identification of new drugs. In this context, the parasite Ca2+ regulatory mechanisms in which mitochondria and acidocalcisomes are involved have been postulated as important targets for several trypanocidal drugs. Thus, amiodarone and dronedarone, common human antiarrythmics, exert its known action on these parasites through the disruption of the intracellular Ca2+ homeostasis. AMIODER is a benzofuran derivate based on the structure of amiodarone that recently demonstrates a significant effect on Trypanosoma cruzi. We now report the effect of AMIODER on Leishmania donovani demonstrating that it inhibit the growth of promastigotes and also of amastigotes inside macrophages, the clinically relevant stage of the parasite, obtaining IC50 values significantly lower than those reported for T. cruzi. We also show that this compound disrupted Ca2+ homeostasis in L. donovani, through its action on two organelles involved in the intracellular Ca2+ regulation and on the bioenergetics of the parasite. AMIODER totally collapsed the electrochemical membrane potential of the unique giant mitochondrion and simultaneously induced the alkalinization of acidocalcisomes, driving together to a large increase in the intracellular Ca2+ concentration of the parasite as the main mechanism of action of this benzofurane derivative.

Prokaryotic cells: structural organisation of the cytoskeleton and organelles

For many years, prokaryotic cells were distinguished from eukaryotic cells based on the simplicity of their cytoplasm, in which the presence of organelles and cytoskeletal structures had not been discovered. Based on current knowledge, this review describes the complex components of the prokaryotic cell cytoskeleton, including (i) tubulin homologues composed of FtsZ, BtuA, BtuB and several associated proteins, which play a fundamental role in cell division, (ii) actin-like homologues, such as MreB and Mb1, which are involved in controlling cell width and cell length, and (iii) intermediate filament homologues, including crescentin and CfpA, which localise on the concave side of a bacterium and along its inner curvature and associate with its membrane. Some prokaryotes exhibit specialised membrane-bound organelles in the cytoplasm, such as magnetosomes and acidocalcisomes, as well as protein complexes, such as carboxysomes. This review also examines recent data on the presence of nanotubes, which are structures that are well characterised in mammalian cells that allow direct contact and communication between cells.