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.

Parasitological, Serological and Molecular Prevalence of Trypanosoma evansi among Arabian Camels (Camelus dromedaries) with Evaluation of Antitrypanosomal Drugs.

PURPOSE: This study was carried out to assess the prevalence of Trypanosoma evansi infection in naturally diseased Dromedary camels in Dammam, Eastern region of Saudi Arabia. The detection of Trypanosoma evansi was performed using the parasitological, serological, and molecular diagnosis and a comparison between such methods were analyzed. In addition, evaluation of therapeutic efficacy of selected antitrypanosomal drugs, cymelarsan and quinapyrmine (aquin-1.5), was trialed for treatment of diagnosed infected cases. METHODS: A total 350 randomly selected camels were evaluated using thin blood smear (TBS), RoTat1.2 PCR and CATT/T. evansi techniques. RESULTS: The total prevalence was 6.9%, 7.7%, and 32.8% by TBS, RoTat1.2 PCR and CATT/T. evansi techniques, respectively. Although PCR detect T. evansi in more samples than TBS, the agreement was good (K = 0.9). Among the CATT/T. evansi results, PCR detect T. evansi in 12 and 15 CATT positive and negative camels, respectively, with low agreement (Kappa = 0.1). The use of cymelarsan and quinapyramine sulfate in the treatment of naturally infected cases demonstrated a very efficient therapeutic response. CONCLUSION: It was found that 1. Comparing the CATT/T. evansi and PCR results, the positivity of CATT was higher than PCR detection, while the agreement was poor (K = 0.1). 2. Cymelarsan and aquin-1.5 proved to be effective in the treatment of naturally infected camels, but cymelarsan presented with higher effectiveness (100%) than aquin-treated camels (83.3%). a 3. The use of cymelarsan and CATT is recommended for disease treatment and control.

Parasitic Protozoans: Exploring the Potential of N,N'-Bis[2-(5-bromo-7-azabenzimidazol-1-yl)-2-oxoethyl]ethylene-1,3-Diamine and Its Cyclohexyl-1,2-diamine Analogue as TryR and Pf-DHODH Inhibitors.

PURPOSE: Major human parasitic protozoans, such as Plasmodium falciparum and Trypanosoma brucei, cause malaria and trypanosomiasis also known as sleeping sickness. In anti-parasitic drug discovery research, trypanothione reductase (TryR) and P. falciparum dihydroorotate dehydrogenase (Pf-DHODH) enzymes are key drug targets in T. brucei and P. falciparum, respectively. The possibility of co-infection of single host by T. brucei and P. falciparum is because both parasites exist in sub-Saharan Africa and the problem of parasite drug resistance necessitates the discovery of new scaffolds, which are strange to the organisms causing these infectious diseases-new scaffolds may help overcome established resistance mechanisms of the organisms. METHOD: In this study, N,N'-bis[2-(5-bromo-7-azabenzimidazol-1-yl)-2-oxoethyl]ethylene-1,3-diamine and its cyclohexyl-1,2-diamine analogue were explored for their inhibitory potential against TryR and Pf-DHODH by engaging density functional study, molecular dynamic simulations, drug-likeness, in silico and in vitro studies RESULTS/CONCLUSION: Results obtained indicated excellent binding potential of the ligands to the receptors and good ADMET (adsorption, desorption, metabolism, excretion, and toxicity) properties.

Cyanotriazoles are selective topoisomerase II poisons that rapidly cure trypanosome infections.

Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo-electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.

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.

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.

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.

Update on the Biological Relevance of Lysine Acetylation as a Novel Drug Target in Trypanosomatids.

The number of acetylated proteins identified from bacteria to mammals has grown exponentially in the last ten years, and it is now accepted that acetylation is a key component in most eukaryotic signaling pathways and is as important as phosphorylation. The enzymes involved in this process are well described in mammals; acetyltransferases and deacetylases are found inside and outside the nuclear compartment and have different regulatory functions. In trypanosomatids, several of these enzymes have been described and are postulated to be novel antiparasitic targets for the rational design of drugs. In this review article, we present an update of the most important known acetylated proteins in trypanosomatids, analyzing the acetylomes available. Also, we summarize the information available regarding acetyltransferases and deacetylases in trypanosomes and their potential use as chemotherapeutic targets.

Differential activity on trypanosomatid parasites of a novel recombinant defensin type 1 from the insect Triatoma (Meccus) pallidipennis.

Defensins are one of the major families of antimicrobial peptides (AMPs) that are widely distributed in insects. In Triatomines (Hemiptera: Reduviidae) vectors of Trypanosoma cruzi the causative agent of Chagas disease, two large groups of defensin isoforms have been described: type 1 and type 4. The aim of this study was to analyze the trypanocidal activity of a type 1 recombinant defensin (rDef1.3) identified in Triatoma (Meccus) pallidipennis, an endemic specie from México. The trypanocidal activity of this defensin was evaluated in vitro, against the parasites T. cruzi, T. rangeli, and two species of Leishmania (L. mexicana and L. major) both causative agents of cutaneous leishmaniasis. Our data demonstrated that the defensin was active against all the parasites although in different degrees. The defensin altered the morphology, reduced the viability and inhibited the growth of T.cruzi. When tested against T. rangeli (a parasite that infects a variety of mammalian species), stronger morphological effects where observed. Surprisingly the greatest effects were observed against the two Leishmania species, of which L. major was the parasite most affected with 50% of dead cells or with damaged membranes, in addition of a reduction in its proliferative capacity in culture. These results suggest that rDef1.3 has an important antimicrobial effect against trypanosomatids which cause some of the more important neglected tropical diseases transmitted by insect vectors.

Amino Acid Based Antimicrobial Agents - Synthesis and Properties.

Structures of several dozen of known antibacterial, antifungal or antiprotozoal agents are based on the amino acid scaffold. In most of them, the amino acid skeleton is of a crucial importance for their antimicrobial activity, since very often they are structural analogs of amino acid intermediates of different microbial biosynthetic pathways. Particularly, some aminophosphonate or aminoboronate analogs of protein amino acids are effective enzyme inhibitors, as structural mimics of tetrahedral transition state intermediates. Synthesis of amino acid antimicrobials is a particular challenge, especially in terms of the need for enantioselective methods, including the asymmetric synthesis. All these issues are addressed in this review, summing up the current state-of-the-art and presenting perspectives fur further progress.

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.

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.

A store-operated Ca2+-entry in Trypanosoma equiperdum: Physiological evidences of its presence.

The Trypanosomatidae family encompasses many unicellular organisms responsible of several tropical diseases that affect humans and animals. Livestock tripanosomosis caused by Trypanosoma brucei brucei (T. brucei), Trypanosoma equiperdum (T. equiperdum) and Trypanosoma evansi (T. evansi), have a significant socio-economic impact and limit animal protein productivity throughout the intertropical zones of the world. Similarly, to all organisms, the maintenance of Ca2+ homeostasis is vital for these parasites, and the mechanism involved in the intracellular Ca2+ regulation have been widely described. However, the evidences related to the mechanisms responsible for the Ca2+ entry are scarce. Even more, to date the presence of a store-operated Ca2+ channel (SOC) has not been reported. Despite the apparent absence of Orai and STIM-like proteins in these parasites, in the present work we demonstrate the presence of a store-operated Ca2+-entry (SOCE) in T. equiperdum, using physiological techniques. This Ca2+-entry is induced by thapsigargin (TG) and 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ), and inhibited by 2-aminoethoxydiphenyl borate (2APB). Additionally, the use of bioinformatics techniques allowed us to identify putative transient receptor potential (TRP) channels, present in members of the Trypanozoon family, which would be possible candidates responsible for the SOCE described in the present work in T. equiperdum.

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.

Parasitic sirtuin 2 as an opportunity in drug discovery.

Infections caused by protozoans remain a public health issue, especially in tropical countries. Serious adverse events, lack of efficacy at the different stages of the infection and routes of administration that have a negative impact on treatment adherence are some of the problems with currently available therapy against these diseases. Here we describe an epigenetic target, sirtuin 2 and its related proteins, that is promising given the results in phenotypic assays and in vivo models against Sir2 of Plasmodium falciparum, Leishmania donovani, Leishmania infantum, Schistosoma mansoni, Trypanosoma brucei and Trypanosoma cruzi parasites. The results we present highlight how this target can be extensively explored and how its inhibitors might be employed in the clinic.

Synthesis, biological, and photophysical studies of molecular rotor-based fluorescent inhibitors of the trypanosome alternative oxidase.

We have recently reported on the development and trypanocidal activity of a class of inhibitors of Trypanosome Alternative Oxidase (TAO) that are targeted to the mitochondrial matrix by coupling to lipophilic cations via C14 linkers to enable optimal interaction with the enzyme's active site. This strategy resulted in a much-enhanced anti-parasite effect, which we ascribed to the greater accumulation of the compound at the location of the target protein, i.e. the mitochondrion, but to date this localization has not been formally established. We therefore synthesized a series of fluorescent analogues to visualize accumulation and distribution within the cell. The fluorophore chosen, julolidine, has the remarkable extra feature of being able to function as a viscosity sensor and might thus additionally act as a probe of the cellular glycerol that is expected to be produced when TAO is inhibited. Two series of fluorescent inhibitor conjugates incorporating a cationic julolidine-based viscosity sensor were synthesized and their photophysical and biological properties were studied. These probes display a red emission, with a high signal-to-noise ratio (SNR), using both single- and two-photon excitation. Upon incubation with T. brucei and mammalian cells, the fluorescent inhibitors 1a and 2a were taken up selectively in the mitochondria as shown by live-cell imaging. Efficient partition of 1a in functional isolated (rat liver) mitochondria was estimated to 66 ± 20% of the total. The compounds inhibited recombinant TAO enzyme in the submicromolar (1a, 2c, 2d) to low nanomolar range (2a) and were effective against WT and multidrug-resistant trypanosome strains (B48, AQP1-3 KO) in the submicromolar range. Good selectivity (SI > 29) over mammalian HEK cells was observed. However, no viscosity-related shift could be detected, presumably because the glycerol was produced cytosolically, and released through aquaglyceroporins, whereas the probe was located, virtually exclusively, in the trypanosome's mitochondrion.

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.

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.

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.