Squalene synthase as a target for Chagas disease therapeutics.

Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease.

SQ109, a new drug lead for Chagas disease.

We tested the antituberculosis drug SQ109, which is currently in advanced clinical trials for the treatment of drug-susceptible and drug-resistant tuberculosis, for its in vitro activity against the trypanosomatid parasite Trypanosoma cruzi, the causative agent of Chagas disease. SQ109 was found to be a potent inhibitor of the trypomastigote form of the parasite, with a 50% inhibitory concentration (IC50) for cell killing of 50 ± 8 nM, but it had little effect (50% effective concentration [EC50], ∼80 µM) in a red blood cell hemolysis assay. It also inhibited extracellular epimastigotes (IC50, 4.6 ± 1 µM) and the clinically relevant intracellular amastigotes (IC50, ∼0.5 to 1 µM), with a selectivity index of ∼10 to 20. SQ109 caused major ultrastructural changes in all three life cycle forms, as observed by light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). It rapidly collapsed the inner mitochondrial membrane potential (Δψm) in succinate-energized mitochondria, acting in the same manner as the uncoupler FCCP [carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone], and it caused the alkalinization of internal acidic compartments, effects that are likely to make major contributions to its mechanism of action. The compound also had activity against squalene synthase, binding to its active site; it inhibited sterol side-chain reduction and, in the amastigote assay, acted synergistically with the antifungal drug posaconazole, with a fractional inhibitory concentration index (FICI) of 0.48, but these effects are unlikely to account for the rapid effects seen on cell morphology and cell killing. SQ109 thus most likely acts, at least in part, by collapsing Δψ/ΔpH, one of the major mechanisms demonstrated previously for its action against Mycobacterium tuberculosis. Overall, the results suggest that SQ109, which is currently in advanced clinical trials for the treatment of drug-susceptible and drug-resistant tuberculosis, may also have potential as a drug lead against Chagas disease.

Inhibition of NAD+-dependent histone deacetylases (sirtuins) causes growth arrest and activates both apoptosis and autophagy in the pathogenic protozoan Trypanosoma cruzi.

Chagas disease, which is caused by the parasite Trypanosoma cruzi, affects approximately 7-8 million people in Latin America. The drugs available to treat this disease are ineffective against chronic phase disease and are associated with toxic side effects. Therefore, the development of new compounds that can kill T. cruzi at low concentrations is critically important. Herein, we report the effects of a novel 3-arylideneindolin-2-one that inhibits sirtuins, which are highly conserved proteins that are involved in a variety of physiological processes. The compound KH-TFMDI was tested against the epimastigote, trypomastigote and amastigote forms of T. cruzi, and its effects were evaluated using flow cytometry, light and electron microscopy. KH-TFMDI inhibited the replication of T. cruzi intracellular amastigotes with an IC50 of 0.5 ± 0.2 µM, which is significantly lower than the IC50 of benznidazole. The compound also lysed the highly infectious bloodstream trypomastigotes (BST) with LC50 values of 0.8 ± 0.3 µM at 4 °C and 2.5 ± 1.1 µM at 37 °C. KH-TFMDI inhibited cytokinesis and induced several morphological changes in the parasite, leading to its death by apoptosis and autophagy. This study highlights sirtuins as a potential new target for Chagas disease therapy.

Effect of elatol, isolated from red seaweed Laurencia dendroidea, on Leishmania amazonensis.

In the present study, we investigated the antileishmanial activity of sesquiterpene elatol, the major constituent of the Brazilian red seaweed Laurencia dendroidea (Hudson) J.V. Lamouroux, against L. amazonensis. Elatol after 72 h of treatment, showed an IC(50) of 4.0 µM and 0.45 µM for promastigote and intracellular amastigote forms of L. amazonensis, respectively. By scanning and transmission electron microscopy, parasites treated with elatol revealed notable changes compared with control cells, including: pronounced swelling of the mitochondrion; appearance of concentric membrane structures inside the organelle; destabilization of the plasma membrane; and formation of membrane structures, apparently an extension of the endoplasmic reticulum, which is suggestive of an autophagic process. A cytotoxicity assay showed that the action of the isolated compound is more specific for protozoa, and it is not toxic to macrophages. Our studies indicated that elatol is a potent antiproliferative agent against promastigote and intracellular amastigote forms, and may have important advantages for the development of new anti-leishamanial chemotherapies.

The natural compounds piperovatine and piperlonguminine induce autophagic cell death on Trypanosoma cruzi.

The currently available treatments for Chagas disease show limited therapeutic potential and are associated with serious side effects. Our group has been attempting to find alternative drugs isolated from natural products as a potential source of pharmacological agents against Trypanosoma cruzi. Here, we demonstrate the antitrypanosomal activity of the amides piperovatine and piperlonguminine isolated from Piper ovatum against epimastigotes and intracellular amastigotes. We also investigated the mechanisms of action of these compounds on extracellular amastigote and epimastigote forms of T. cruzi. These amides showed low toxicity to LLCMK(2) mammalian cells. By using transmission and scanning electron microscopy, we observed that the compounds caused severe alterations in T. cruzi. These alterations were mainly located in plasma membrane and mitochondria. Furthermore, the study of treated parasites labeled with Rh123, PI and MDC corroborate with our TEM data. These mitochondrial dysfunctions induced by the amides might trigger biochemical alterations that lead to cell death. Altogether, our data evidence a possible autophagic process.

Effect of thiazolidine LPSF SF29 on the growth and morphology of Trypanosoma cruzi.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is an endemic illness in Latin America. Efforts have been made by several groups to develop new effective and safe anti-T. cruzi drugs. In the present work, we show that thiazolidine LPSF SF29 inhibited growth of the epimastigote and amastigote forms and caused lysis in the trypomastigote form of T. cruzi, leading to death of the protozoan. Mitochondrial dysfunction was also observed. The thiazolidine induced ultrastructural alterations such as detachment of the flagellar membrane, intense mitochondrial swelling, formation of myelin-like figures and the appearance of autophagosomes. Taken together, these results suggest that this new thiazolidine is active against T. cruzi and constitutes a promising drug for the therapy of Chagas disease.

Effects of amiodarone and posaconazole on the growth and ultrastructure of Trypanosoma cruzi.

The antifungal posaconazole (PCZ) is the most advanced candidate for the treatment of Chagas disease, having potent anti-Trypanosoma cruzi activity in vitro and in animal models of the disease as well as an excellent safety profile in humans. Amiodarone (AMD) is the antiarrhythmic drug most frequently used for the symptomatic treatment of chronic Chagas disease patients, but it also has specific anti-T. cruzi activity. When used in combination, these drugs exhibit potent synergistic activity against the parasite. In the present work, electron microscopy was used to analyse the effects of both compounds, acting individually or in combination, against T. cruzi. The 50% inhibitory concentration (IC(50)) against epimastigote and amastigote forms was 25 nM and 1.0 nM for PCZ and 8 µM and 5.6 µM for AMD, respectively. The antiproliferative synergism of the drugs (fractional inhibitory concentration<0.5) was confirmed and the ultrastructural alterations in the parasite induced by them, leading to cell death, were characterised using electron microscopy. These alterations include intense wrinkling of the protozoan surface, swelling of the mitochondrion, shedding of plasma membrane vesicles, the appearance of vesicles in the flagellar pocket, alterations in the kinetoplast, disorganisation of the Golgi complex, accumulation of lipid inclusions in the cytoplasm, and the formation of autophagic vacuoles, the latter confirmed by immunofluorescence microscopy. These findings indicate that the association of PCZ and AMD may constitute an effective anti-T. cruzi therapy with low side effects.

Trypanocidal action of eupomatenoid-5 is related to mitochondrion dysfunction and oxidative damage in Trypanosoma cruzi.

Because of its severe side effects and variable efficacy, the current treatment for Chagas disease is unsatisfactory. Natural compounds are good alternative chemotherapeutic agents for the treatment of this infection. Recently, our group reported the antiproliferative activity and morphological alterations in epimastigotes and intracellular amastigotes of Trypanosoma cruzi treated with eupomatenoid-5, a neolignan isolated from leaves of Piper regnellii var. pallescens. Here, we demonstrate that eupomatenoid-5 exhibited activity against trypomastigotes, the infective form of T. cruzi (EC50 40.5 µM), leading to ultrastructural alteration and lipoperoxidation in the cell membrane. Additionally, eupomatenoid-5 induced depolarization of the mitochondrial membrane, lipoperoxidation and increased G6PD activity in epimastigotes of T. cruzi. These findings support the possibility that different mechanisms may be targeted, according to the form of the parasite, and that the plasma membrane and mitochondria are the structures that are most affected in trypomastigotes and epimastigotes, respectively. Thus, the trypanocidal action of eupomatenoid-5 may be associated with mitochondrial dysfunction and oxidative damage, which can trigger destructive effects on biological molecules of T. cruzi, leading to parasite death.