Targeting Trypanothione Metabolism in Trypanosomatids.

Infectious diseases caused by trypanosomatids, including African trypanosomiasis (sleeping sickness), Chagas disease, and different forms of leishmaniasis, are Neglected Tropical Diseases affecting millions of people worldwide, mainly in vulnerable territories of tropical and subtropical areas. In general, current treatments against these diseases are old-fashioned, showing adverse effects and loss of efficacy due to misuse or overuse, thus leading to the emergence of resistance. For these reasons, searching for new antitrypanosomatid drugs has become an urgent necessity, and different metabolic pathways have been studied as potential drug targets against these parasites. Considering that trypanosomatids possess a unique redox pathway based on the trypanothione molecule absent in the mammalian host, the key enzymes involved in trypanothione metabolism, trypanothione reductase and trypanothione synthetase, have been studied in detail as druggable targets. In this review, we summarize some of the recent findings on the molecules inhibiting these two essential enzymes for Trypanosoma and Leishmania viability.

Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline.

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1-2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly.

Targeting RNA Structure to Inhibit Editing in Trypanosomes.

Mitochondrial RNA editing in trypanosomes represents an attractive target for developing safer and more efficient drugs for treating infections with trypanosomes because this RNA editing pathway is not found in humans. Other workers have targeted several enzymes in this editing system, but not the RNA. Here, we target a universal domain of the RNA editing substrate, which is the U-helix formed between the oligo-U tail of the guide RNA and the target mRNA. We selected a part of the U-helix that is rich in G-U wobble base pairs as the target site for the virtual screening of 262,000 compounds. After chemoinformatic filtering of the top 5000 leads, we subjected 50 representative complexes to 50 nanoseconds of molecular dynamics simulations. We identified 15 compounds that retained stable interactions in the deep groove of the U-helix. The microscale thermophoresis binding experiments on these five compounds show low-micromolar to nanomolar binding affinities. The UV melting studies show an increase in the melting temperatures of the U-helix upon binding by each compound. These five compounds can serve as leads for drug development and as research tools to probe the role of the RNA structure in trypanosomal RNA editing.

Benzophenone Glucosides and B-Type Proanthocyanidin Dimers from Zambian Cassia abbreviata and Their Trypanocidal Activities.

Two benzophenone glucosides (1 and 2), five flavan-3-ol dimers (5-9), and 17 known compounds (3, 4, and 10-24) were identified from the bark extract of Cassia abbreviata. The chemical structures display two points of interest. First, as an unusual characteristic feature of the 1H NMR spectra of 1 and 2, the signals for the protons on glucosidic carbons C-2 are shielded as compared to those generally observed for glucosyl moieties. The geometrically optimized 3D structures derived from conformational analysis and density functional theory (DFT) calculations revealed that this shielding effect originates from intramolecular hydrogen bonds in 1 and 2. Additionally, 3-15 were identified as dimeric B-type proanthocyanidins, which have 2R,3S-absolute-configured C-rings and C-4-C-8″ linkages, as evidenced by X-ray crystallography and by NMR and ECD spectroscopy. These results suggest the structure-determining procedures for some reported dimers need to be reconsidered. The trypanocidal activities of the isolated compounds against Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, T. congolense, and T. evansi were evaluated, and the active compounds were identified.

Recent Progress in the Development of Indole-Based Compounds Active against Malaria, Trypanosomiasis and Leishmaniasis.

Human protozoan diseases represent a serious health problem worldwide, affecting mainly people in social and economic vulnerability. These diseases have attracted little investment in drug discovery, which is reflected in the limited available therapeutic arsenal. Authorized drugs present problems such as low efficacy in some stages of the disease or toxicity, which result in undesirable side effects and treatment abandonment. Moreover, the emergence of drug-resistant parasite strains makes necessary an even greater effort to develop safe and effective antiparasitic agents. Among the chemotypes investigated for parasitic diseases, the indole nucleus has emerged as a privileged molecular scaffold for the generation of new drug candidates. In this review, the authors provide an overview of the indole-based compounds developed against important parasitic diseases, namely malaria, trypanosomiasis and leishmaniasis, by focusing on the design, optimization and synthesis of the most relevant synthetic indole scaffolds recently reported.

Synthesis and evaluation of trypanocidal activity of derivatives of naturally occurring 2,5-diphenyloxazoles.

African trypanosomiasis is a zoonotic protozoan disease affecting the nervous system. Various natural products reportedly exhibit trypanocidal activity. Naturally occurring 2,5-diphenyloxazoles present in Oxytropis lanata, and their derivatives, were synthesized. The trypanocidal activities of the synthesized compounds were evaluated against Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, T. congolense, and T. evansi. Natural product 1 exhibited trypanocidal activity against all the species/subspecies of trypanosomes, exhibiting half-maximal inhibitory concentrations (IC50) of 1.1-13.5 µM. Modification of the oxazole core improved the trypanocidal activity. The 1,3,4-oxadiazole (7) and 2,4-diphenyloxazole (9) analogs exhibited potency superior to that of 1. However, these compounds exhibited cytotoxicity in Madin-Darby bovine kidney cells. The O-methylated analog of 1 (12) was non-cytotoxic and exhibited selective trypanocidal activity against T. congolense (IC50 = 0.78 µM). Structure-activity relationship studies of the 2,5-diphenyloxazole analogs revealed aspects of the molecular structure critical for maintaining selective trypanocidal activity against T. congolense.

Cysteine proteases as potential targets for anti-trypanosomatid drug discovery.

Leishmaniasis and trypanosomiasis are endemic neglected disease in South America and Africa and considered a significant public health problem, mainly in poor communities. The limitations of the current available therapeutic options, including the lack of specificity, relatively high toxicity, and the drug resistance acquiring, drive the constant search for new targets and therapeutic options. Advances in knowledge of parasite biology have revealed essential enzymes involved in the replication, survival, and pathogenicity of Leishmania and Trypanosoma species. In this scenario, cysteine proteases have drawn the attention of researchers and they are being proposed as promising targets for drug discovery of antiprotozoal drugs. In this systematic review, we will provide an update on drug discovery strategies targeting the cysteine proteases as potential targets for chemotherapy against protozoal neglected diseases.

Evolution, function and roles in drug sensitivity of trypanosome aquaglyceroporins.

Aquaglyceroporins (AQPs) are membrane proteins that function in osmoregulation and the uptake of low molecular weight solutes, in particular glycerol and urea. The AQP family is highly conserved, with two major subfamilies having arisen very early in prokaryote evolution and retained by eukaryotes. A complex evolutionary history indicates multiple lineage-specific expansions, losses and not uncommonly a complete loss. Consequently, the AQP family is highly evolvable and has been associated with significant events in life on Earth. In the African trypanosomes, a role for the AQP2 paralogue, in sensitivity to two chemotherapeutic agents, pentamidine and melarsoprol, is well established, albeit with the mechanisms for cell entry and resistance unclear until very recently. Here, we discuss AQP evolution, structure and mechanisms by which AQPs impact drug sensitivity, suggesting that AQP2 stability is highly sensitive to mutation while serving as the major uptake pathway for pentamidine.

Effects of trypanocidal drugs on DNA synthesis: new insights into melarsoprol growth inhibition.

Trypanothione is the primary thiol redox carrier in Trypanosomatids whose biosynthesis and utilization pathways contain unique enzymes that include suitable drug targets against the human parasites in this family. Overexpression of the rate-limiting enzyme, γ-glutamylcysteine synthetase (GSH1), can increase the intracellular concentration of trypanothione. Melarsoprol directly inhibits trypanothione and has predicted the effects on downstream redox biology, including ROS management and dNTP synthesis that require further investigation. Thus, we hypothesized that melarsoprol treatment would inhibit DNA synthesis, which was tested using BrdU incorporation assays and cell cycle analyses. In addition, we analysed the effects of eflornithine, which interfaces with the trypanothione pathway, fexinidazole, because of the predicted effects on DNA synthesis, and pentamidine as an experimental control. We found that melarsoprol treatment resulted in a cell cycle stall and a complete inhibition of DNA synthesis within 24 h, which were alleviated by GSH1 overexpression. In contrast, the other drugs analysed had more subtle effects on DNA synthesis that were not significantly altered by GSH1 expression. Together these findings implicate DNA synthesis as a therapeutic target that warrants further investigation in the development of antitrypanosomal drugs.

Antiparasitic Properties of Propolis Extracts and Their Compounds.

Propolis is a bee product that has been used in medicine since ancient times. Although its anti-inflammatory, antioxidant, antimicrobial, antitumor, and immunomodulatory activities have been investigated, its anti-parasitic properties remain poorly explored, especially regarding helminths. This review surveys the results obtained with propolis around the world against human parasites. Regarding protozoa, studies carried out with the protozoa Trypanosoma spp. and Leishmania spp. have demonstrated promising results in vitro and in vivo. However, there are fewer studies for Plasmodium spp., the etiological agent of malaria and less so for helminths, particularly for Fasciola spp. and Schistosoma spp. Despite the favorable in vitro results with propolis, helminth assays need to be further investigated. However, propolis has shown itself to be an excellent natural product for parasitology, thus opening new paths and approaches in its activity against protozoa and helminths.

Proposed Mechanism for the Antitrypanosomal Activity of Quercetin and Myricetin Isolated from Hypericum afrum Lam.: Phytochemistry, In Vitro Testing and Modeling Studies.

The in vitro activity of L. donovani (promastigotes, axenic amastigotes and intracellular amastigotes in THP1 cells) and T. brucei, from the fractions obtained from the hydroalcoholic extract of the aerial part of Hypericum afrum and the isolated compounds, has been evaluated. The chloroform, ethyl acetate and n-butanol extracts showed significant antitrypanosomal activity towards T. brucei, with IC50 values of 12.35, 13.53 and 12.93 µg/mL and with IC90 values of 14.94, 19.31 and 18.67 µg/mL, respectively. The phytochemical investigation of the fractions led to the isolation and identification of quercetin (1), myricitrin (2), biapigenin (3), myricetin (4), hyperoside (5), myricetin-3-O-ß-d-galactopyranoside (6) and myricetin-3'-O-ß-d-glucopyranoside (7). Myricetin-3'-O-ß-d-glucopyranoside (7) has been isolated for the first time from this genus. The chemical structures were elucidated by using comprehensive one- and two-dimensional nuclear magnetic resonance (1D and 2D NMR) spectroscopic data, as well as high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). These compounds have also been evaluated for their antiprotozoal activity. Quercetin (1) and myricetin (4) showed noteworthy activity against T. brucei, with IC50 and IC90 values of 7.52 and 5.71 µM, and 9.76 and 7.97 µM, respectively. The T. brucei hexokinase (TbHK1) enzyme was further explored as a potential target of quercetin and myricetin, using molecular modeling studies. This proposed mechanism assists in the exploration of new candidates for novel antitrypanosomal drugs.

Chemical and Pharmacological Potential of Coccoloba cowellii, an Endemic Endangered Plant from Cuba.

Coccoloba cowellii Britton (Polygonaceae) is an endemic and critically endangered plant that only grows in Camagüey, a province of Cuba. In this study, a total of 13 compounds were identified in a methanolic leaf extract, employing a dereplication of the UHPLC-HRMS data by means of feature-based molecular networking (FBMN) analysis in the Global Natural Products Social Molecular Network (GNPS), together with the interpretation of the MS/MS data and comparison with the literature. The major constituents were glucuronides and glycosides of myricetin and quercetin, as well as epichatechin-3-O-gallate, catechin, epicatechin and gallic acid, all of them being reported for the first time in C. cowellii leaves. The leaf extract was also tested against various microorganisms, and it showed a strong antifungal effect against Candida albicans ATCC B59630 (azole-resistant) (IC50 2.1 µg/mL) and Cryptococcus neoformans ATCC B66663 (IC50 4.1 µg/mL) with no cytotoxicity (CC50 > 64.0 µg/mL) on MRC-5 SV2 cells, determined by the resazurin assay. Additionally, the extract strongly inhibited COX-1 and COX-2 enzyme activity using a cell-free experiment in a dose-dependent manner, being significantly more active on COX-1 (IC50 4.9 µg/mL) than on COX-2 (IC50 10.4 µg/mL). The constituents identified as well as the pharmacological activities measured highlight the potential of C. cowellii leaves, increasing the interest in the implementation of conservation strategies for this species.

Phototemtide†A, a Cyclic Lipopeptide Heterologously Expressed from Photorhabdus temperata Meg1, Shows Selective Antiprotozoal Activity.

A new cyclic lipopeptide, phototemtide A (1), was isolated from Escherichia coli expressing the biosynthetic gene cluster pttABC from Photorhabdus temperata Meg1. The structure of 1 was elucidated by HR-ESI-MS and NMR experiments. The absolute configurations of amino acids and 3-hydroxyoctanoic acid in 1 were determined by using the advanced Marfey's method and comparison after total synthesis of 1, respectively. Additionally, three new minor derivatives, phototemtides B-D (2-4), were identified by detailed HPLC-MS analysis. Phototemtide A (1) showed weak antiprotozoal activity against Plasmodium falciparum, with an IC50 value of 9.8 µm. The biosynthesis of phototemtides A-D (1-4) was also proposed.

Identification of N-acyl quinolin-2(1H)-ones as new selective agents against clinical isolates of Acanthamoeba keratitis.

A collection of N-substituted quinolin-2(1H)-ones were screened against a panel of clinically relevant protozoa (Leishmania, Trypanosoma and Acanthamoeba). Three quinolin-2(1H)-one compounds were identified as selective anti-Acanthamoeba agents. Further assessment revealed that these compounds were active against both trophozoite and cyst forms of A. castellanii Neff, and caused protozoa death via apoptosis. The data presented herein identify N-acyl quinolin-2(1H)-ones as a promising new class of selective anti-Acanthamoeba agents.

Nanotechnology enabled the enhancement of antitrypanosomal activity of piperine against Trypanosoma evansi.

Nanoencapsulation is the promising approach to enhance the therapeutic potential of a drug. In the present investigation, piperine-loaded nanocapsules (NCs) was prepared and evaluated for antitrypanosomal activity against the parasite Trypanosoma evansi, a causative agent of trypanosomiasis. Piperine, a bioactive compound was selected as an alternative for drugs that have been used for the treatment of the disease from decades to overcome the toxic effects or drug resistance effect. Moreover, piperine has reported to possess therapeutic potential against other Trypanosoma spp. and has also been reported to cause reactive oxygen species (ROS) mediated effect in cancer cells that was the other reason for the selection. To date, piperine and its nanoformulations have not been evaluated for their growth inhibitory effect against T. evansi. Piperine-loaded NCs exhibited more significant antitrypanosomal effect at approximately three-times less IC50 value 5.04 µM as compared to piperine (IC50-14.45 µM). Moreover, increased production of reactive oxygen species observed in the case of piperine-loaded NCs as that of pure piperine in the axenic culture of T. evansi. Furthermore, different concentrations of piperine-loaded NCs showed less cytotoxicity on horse peripheral blood mononuclear cells as liken to pure piperine. In conclusion, our results demonstrated that piperine-loaded NCs induced more generation of ROS that contributed inhibitory effect on the growth of Trypanosoma evansi as compared to pure drug.

Drug-induced reactive oxygen species-mediated inhibitory effect on growth of Trypanosoma evansi in axenic culture system.

Trypanosoma evansi, an extracellular haemoflagellate, has a wide range of hosts receptive and susceptible to infection, in which it revealed highly inconsistent clinical effects. Drugs used for the treatment of trypanosomosis have been utilized for more than five decades and have several problems like local and systemic toxicity. In the present investigation, imatinib and sorafenib were selected as drugs as they are reported to have the potential to cause reactive oxygen species (ROS)-mediated effect in cancer cells. Both have also been reported to have potential against T. brucei, T. cruzi and Leishmania donovani. To date, imatinib and sorafenib have not evaluated for their growth inhibitory effect against T. evansi. Imatinib and sorafenib showed significant (p < 0.001) inhibition on parasite growth and multiplication with IC50 (50% inhibitory concentration) values 6.12 µM and 0.33 µM respectively against T. evansi. Both the drug molecules demonstrated for the generation of ROS in T. evansi and were found up to 65% increased level of ROS as compared with negative control in the axenic culture system. Furthermore, different concentrations of imatinib and sorafenib were found non-toxic on horse peripheral blood mononuclear cells and Vero cell lines. Also, in conclusion, our results demonstrated that imatinib- and sorafenib-induced generation of ROS contributed inhibitory effect on the growth of Trypanosoma evansi in an axenic culture system.

Alsinol, an arylamino alcohol derivative active against Plasmodium, Babesia, Trypanosoma, and Leishmania: past and new outcomes.

Malaria, babesiosis, trypanosomosis, and leishmaniasis are some of the most life-threatening parasites, but the range of drugs to treat them is limited. An effective, safe, and low-cost drug with a large activity spectrum is urgently needed. For this purpose, an aryl amino alcohol derivative called Alsinol was resynthesized, screened in silico, and tested against Plasmodium, Babesia, Trypanosoma, and Leishmania. In silico Alsinol follows the Lipinski and Ghose rules. In vitro it had schizontocidal activity against Plasmodium falciparum and was able to inhibit gametocytogenesis; it was particularly active against late gametocytes. In malaria-infected mice, it showed a dose-dependent activity similar to chloroquine. It demonstrated a similar level of activity to reference compounds against Babesia divergens, and against promastigotes, and amastigotes stages of Leishmania in vitro. It inhibited the in vitro growth of two African animal strains of Trypanosoma but was ineffective in vivo in our experimental conditions. It showed moderate toxicity in J774A1 and Vero cell models. The study demonstrated that Alsinol has a large spectrum of activity and is potentially affordable to produce. Nevertheless, challenges remain in the process of scaling up synthesis, creating a suitable clinical formulation, and determining the safety margin in preclinical models.

1,4-Naphthoquinones: Some Biological Properties and Application.

Over the past decade, a number of new 1,4-naphthoquinones have been isolated from natural sources and new 1,4-naphthoquinones with diverse structural features have been synthesized. Cardioprotective, anti-ischemic, hepatoprotective, neuroprotective and some other new properties were found for these compounds; their role in protecting against neurodegenerative diseases has been established. Their anti-inflammatory, antimicrobial and antitumor activities have been studied in more detail; new, previously unknown intracellular molecular targets and mechanisms of action have been discovered. Some compounds of this class are already being used as a medicinal drugs and some substances can be used as biochemical tools and probes for non-invasive detection of pathological areas in cells and tissues in myocardial infarction and neurodegenerative diseases using modern molecular imaging techniques.

Targeting Trypanothione Reductase, a Key Enzyme in the Redox Trypanosomatid Metabolism, to Develop New Drugs against Leishmaniasis and Trypanosomiases.

The protozoans Leishmania and Trypanosoma, belonging to the same Trypanosomatidae family, are the causative agents of Leishmaniasis, Chagas disease, and human African trypanosomiasis. Overall, these infections affect millions of people worldwide, posing a serious health issue as well as socio-economical concern. Current treatments are inadequate, mainly due to poor efficacy, toxicity, and emerging resistance; therefore, there is an urgent need for new drugs.

The Strong Anti-Kinetoplastid Properties of Bee Propolis: Composition and Identification of the Active Agents and Their Biochemical Targets.

The kinetoplastids are protozoa characterized by the presence of a distinctive organelle, called the kinetoplast, which contains a large amount of DNA (kinetoplast DNA (kDNA)) inside their single mitochondrion. Kinetoplastids of medical and veterinary importance include Trypanosoma spp. (the causative agents of human and animal African Trypanosomiasis and of Chagas disease) and Leishmania spp. (the causative agents of the various forms of leishmaniasis). These neglected diseases affect millions of people across the globe, but drug treatment is hampered by the challenges of toxicity and drug resistance, among others. Propolis (a natural product made by bees) and compounds isolated from it are now being investigated as novel treatments of kinetoplastid infections. The anti-kinetoplastid efficacy of propolis is probably a consequence of its reported activity against kinetoplastid parasites of bees. This article presents a review of the reported anti-kinetoplastid potential of propolis, highlighting its anti-kinetoplastid activity in vitro and in vivo regardless of geographical origin. The mode of action of propolis depends on the organism it is acting on and includes growth inhibition, immunomodulation, macrophage activation, perturbation of the cell membrane architecture, phospholipid disturbances, and mitochondrial targets. This gives ample scope for further investigations toward the rational development of sustainable anti-kinetoplastid drugs.