The expression of immune response genes in patients with chronic Chagas disease is shifted toward the levels observed in healthy subjects as a result of treatment with Benznidazole.

Introduction: Chagas disease, caused by the Trypanosoma cruzi parasite infection, is a potentially life-threatening neglected tropical disease with a worldwide distribution. During the chronic phase of the disease, there exists a fragile balance between the host immune response and parasite replication that keeps patients in a clinically-silent asymptomatic stage for years or even decades. However, in 40% of patients, the disease progresses to clinical manifestations mainly affecting and compromising the cardiac system. Treatment is recommended in the chronic phase, although there are no early markers of its effectiveness. The aim of this study is to identify differential expression changes in genes involved in the immune response in antigen-restimulated PBMC from chronic patients with Chagas disease due to benznidazole treatment. Methods: Thus, high-throughput real-time qPCR analysis has been performed to simultaneously determine global changes in the expression of 106 genes involved in the immune response in asymptomatic (IND) and early cardiac manifestations (CCC I) Chagas disease patients pre- and post-treatment with benznidazole. Results and discussion: The results revealed that 7 out of the 106 analyzed genes were differentially expressed (4 up- and 3 downregulated) after treatment in IND patients and 15 out of 106 (3 up- and 12 downregulated) after treatment of early cardiac Chagas disease patients. Particularly in CCC I patients, regulation of the expression level of some of these genes towards a level similar to that of healthy subjects suggests a beneficial effect of treatment and supports recommendation of benznidazole administration to early cardiac Chagas disease patients. The data obtained also demonstrated that both in asymptomatic patients and in early cardiac chronic patients, after treatment with benznidazole there is a negative regulation of the proinflammatory and cytotoxic responses triggered as a consequence of T. cruzi infection and the persistence of the parasite. This downregulation of the immune response likely prevents marked tissue damage and healing in early cardiac patients, suggesting its positive effect in controlling the pathology.

Potential anti-HIV and antitrypanosomal components revealed in Sorindeia nitidula via LC-ESI-QTOF-MS/MS.

Sorindeia nitidula (Anacardiaceae) is used by traditional practitioners to treat influenza illnesses with cephalgia and febrile aches. However, the potential active ingredients for its remarkable antioxidant, anti-HIV and antitrypanosomal activities remain unexplored. The present study aims to evaluate the antioxidant, anti-HIV and antitrypanosomal activities of the ethyl acetate extract of S. nitidula (SN) in order to screen out the bioactive compounds and to analyze their possible mechanisms of action. Overall, 21 phenolic compounds were annotated, by using the MS and MS/MS information provided by the QTOF-MS. In vitro assays on the extract revealed potent antioxidant (IC50 = 0.0129 ± 0.0001 mg/mL), anti-HIV (IC50 = 1.736 ± 0.036 µM), antitrypanosomal (IC50 = 1.040 ± 0.010 µM) activities. Furthermore, SN did not present cytotoxic effect on HeLa cancer cell lines. The integrated strategy based on LC-ESI-QTOF-MS/MS provided a powerful tool and a multidimensional perspective for further exploration of active ingredients in S. nitidula responsible for the antioxidant, anti-HIV and antitrypanosomal activities.

Synthesis and Anti-Trypanosoma cruzi Activity of New Pyrazole-Thiadiazole Scaffolds.

Chagas disease, a silent but widespread disease that mainly affects a socioeconomically vulnerable population, lacks innovative safe drug therapy. The available drugs, benznidazole and nifurtimox, are more than fifty years old, have limited efficacy, and carry harmful side effects, highlighting the need for new therapeutics. This study presents two new series of pyrazole-thiadiazole compounds evaluated for trypanocidal activity using cellular models predictive of efficacy. Derivatives 1c (2,4-diCl) and 2k (4-NO2) were the most active against intracellular amastigotes. Derivative 1c also showed activity against trypomastigotes, with the detachment of the flagellum from the parasite body being a predominant effect at the ultrastructural level. Analogs have favorable physicochemical parameters and are predicted to be orally available. Drug efficacy was also evaluated in 3D cardiac microtissue, an important target tissue of Trypanosoma cruzi, with derivative 2k showing potent antiparasitic activity and a significant reduction in parasite load. Although 2k potentially reduced parasite load in the washout assay, it did not prevent parasite recrudescence. Drug combination analysis revealed an additive profile, which may lead to favorable clinical outcomes. Our data demonstrate the antiparasitic activity of pyrazole-thiadiazole derivatives and support the development of these compounds using new optimization strategies.

New insights into the pro-oxidant mechanism of dehydroleucodine on Trypanosoma cruzi.

Chagas disease, caused by Trypanosoma cruzi (T. cruzi), is one of the most important neglected diseases in Latin America. The limited use of the current nitro-derivative-based chemotherapy highlights the need for alternative drugs and the identification of their molecular targets. In this study, we investigated the trypanocidal effect of the sesquiterpene lactone dehydroleucodine (DhL) and its derivatives, focusing on the antioxidative defense of the parasites. DhL and two derivatives, at lesser extent, displayed antiproliferative effect on the parasites. This effect was blocked by the reducing agent glutathione (GSH). Treated parasites exhibited increased intracellular ROS concentration and trypanothione synthetase activity, accompanied by mitochondrial swelling. Although molecular dynamics studies predicted that GSH would not interact with DhL, 1H-NMR analysis confirmed that GSH could protect parasites by interacting with the lactone. When parasites overexpressing mitochondrial tryparedoxin peroxidase were incubated with DhL, its effect was attenuated. Overexpression of cytosolic tryparedoxin peroxidase also provided some protection against DhL. These findings suggest that DhL induces oxidative imbalance in T. cruzi, offering new insights into potential drug targets against this parasite.

Screening of synthetic 1,2,3-triazolic compounds inspired by SRPIN340 as anti-Trypanosoma cruzi agents.

BACKGROUND: The current treatments for Chagas disease (CD) include benznidazole and nifurtimox, which have limited efficacy and cause numerous side effects. Triazoles are candidates for new CD treatments due to their ability to eliminate T. cruzi parasites by inhibiting ergosterol synthesis, thereby damaging the cell membranes of the parasite. METHODS: Eleven synthetic analogs of the kinase inhibitor SRPIN340 containing a triazole core (compounds 6A-6K) were screened in vitro against the Tulahuen strain transfected with ß-galactosidase, and their IC50, CC50, and selectivity indexes (SI) were calculated. Compounds with an SI > 50 were further evaluated in mice infected with the T. cruzi Y strain by rapid testing. RESULTS: Eight compounds were active in vitro with IC50 values ranging from 0.5-10.5 µg/mL. The most active compounds, 6E and 6H, had SI values of 125.2 and 69.6, respectively. These compounds also showed in vivo activity, leading to a reduction in parasitemia at doses of 10, 50, and 250 mg/kg/day. At doses of 50 and 250 mg/kg/day, parasitemia was significantly reduced compared to infected untreated animals, with no significant differences between the effects of 6E and 6H. CONCLUSIONS: This study identified two new promising compounds for CD chemotherapy and confirmed their activity against T. cruzi.

Supramolecular eutectogel as new oral paediatric delivery system to enhance benznidazole bioavailability.

Benznidazole (BNZ) serves as the primary drug for treating Chagas Disease and is listed in the WHO Model List of Essential Medicines for Children. Herein, a new child-friendly oral BNZ delivery platform is developed in the form of supramolecular eutectogels (EGs). EGs address BNZ's poor oral bioavailability and provide a flexible twice-daily dose in stick-pack format. This green and sustainable formulation strategy relies on the gelation of drug-loaded Natural Deep Eutectic Solvents (NaDES) with xanthan gum (XG) and water. Specifically, choline chloride-based NaDES form stable and biocompatible 5 mg/mL BNZ-loaded EGs. Rheological and Low-field NMR investigations indicate that EGs are viscoelastic materials comprised of two co-existing regions in the XG network generated by different crosslink distributions between the biopolymer, NaDES and water. Remarkably, the shear modulus and relaxation spectrum of EGs remain unaffected by temperature variations. Upon dilution with simulated gastrointestinal fluids, EGs results in BNZ supersaturation, serving as the primary driving force for its absorption. Interestingly, after oral administration of EGs to rats, drug bioavailability increases by 2.6-fold, with a similar increase detected in their cerebrospinal fluid. The noteworthy correlation between in vivo results and in vitro release profiles confirms the efficacy of EGs in enhancing both peripheral and central BNZ oral bioavailability.

Identification of Innovative Folate Inhibitors Leveraging the Amino Dihydrotriazine Motif from Cycloguanil for Their Potential as Anti-Trypanosoma brucei Agents.

Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles (1) and 2-guanidino benzimidazoles (2), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of TbDHFR and TbPTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against T. brucei and L. infantum and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole 2g resulted to be the most potent (Ki = 9 nM) and highly selective TbDHFR inhibitor, 6000-fold over TbPTR1 and 394-fold over hDHFR. The 5,6-dimethyl tricyclic analogue 1g, despite showing a lower potency and selectivity profile than 2g, shared a comparable antiparasitic activity against T. brucei in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles 2c and 2d revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of T. brucei and L. infantum parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.

The activities of suaveolol and other compounds from Hyptis suaveolens and Momordica charantia against the aetiological agents of African trypanosomiasis, leishmaniasis and malaria.

African trypanosomiasis and malaria are among the most severe health challenges to humans and livestock in Africa and new drugs are needed. Leaves of Hyptis suaveolens Kuntze (Lamiaceae) and Momordica charantia L. (Cucurbitaceae) were extracted with hexane, ethyl acetate, and then methanol, and subjected to silica gel column chromatography. Structures of six isolated compounds were elucidated through NMR and HR-EIMS spectrometry. Callistrisic acid, dehydroabietinol, suaveolic acid, suaveolol, and a mixture of suaveolol and suaveolic acid (SSA) were obtained from H. suaveolens, while karavilagenin D and momordicin I acetate were obtained from M. charantia. The isolated biomolecules were tested against trypomastigotes of Trypanosoma brucei brucei and T. congolense, and against Plasmodium falciparum. The most promising EC50 values were obtained for the purified suaveolol fraction, at 2.71 ± 0.36 µg/mL, and SSA, exhibiting an EC50 of 1.56 ± 0.17 µg/mL against T. b. brucei trypomastigotes. Suaveolic acid had low activity against T. b. brucei but displayed moderate activity against T. congolense trypomastigotes at 11.1 ± 0.5 µg/mL. Suaveolol and SSA were also tested against T. evansi, T. equiperdum, Leishmania major and L. mexicana but the antileishmanial activity was low. Neither of the active compounds, nor the mixture of the two, displayed any cytotoxic effect on human foreskin fibroblast (HFF) cells at even the highest concentration tested, being 200 µg/mL. We conclude that suaveolol and its mixture possessed significant and selective trypanocidal activity.

DNA G-quadruplexes in the genome of Trypanosoma cruzi as potential therapeutic targets for Chagas disease: Dithienylethene ligands as effective antiparasitic agents.

Chagas disease is caused by the parasite Trypanosoma cruzi and affects over 7 million people worldwide. The two actual treatments, Benznidazole (Bzn) and Nifurtimox, cause serious side effects due to their high toxicity leading to treatment abandonment by the patients. In this work, we propose DNA G-quadruplexes (G4) as potential therapeutic targets for this infectious disease. We have found 174 PQS per 100,000 nucleotides in the genome of T. cruzi and confirmed G4 formation of three frequent motifs. We synthesized a family of 14 quadruplex ligands based in the dithienylethene (DTE) scaffold and demonstrated their binding to these identified G4 sequences. Several DTE derivatives exhibited micromolar activity against epimastigotes of four different strains of T. cruzi, in the same concentration range as Bzn. Compounds L3 and L4 presented remarkable activity against trypomastigotes, the active form in blood, of T. cruzi SOL strain (IC50 = 1.5-3.3 µM, SI = 25-40.9), being around 40 times more active than Bzn and displaying much better selectivity indexes.

Wajeepeptin, a Cytotoxic and Antitrypanosomal Cyclic Depsipeptide from a Marine Moorena sp. Cyanobacterium.

Here, we report wajeepeptin (1), a new cyclic depsipeptide isolated from a marine Moorena sp. cyanobacterium. The structure was elucidated by a combination of spectroscopic analyses, X-ray diffraction analysis, and degradation reactions. Wajeepeptin (1) showed moderate cytotoxicity (IC50 = 3.7 µM against HeLa cells) and potent antitrypanosomal activity (IC50 = 0.73 ± 0.14 µM against Trypanosoma brucei rhodesiense).

Drug screening and development cascade for Chagas disease: an update of in vitro and in vivo experimental models.

Chagas disease is a tropical neglected disease that affects millions of people worldwide, still demanding a more effective and safer therapy, especially in its chronic phase which lacks a treatment that promotes substantial parasitological cure. The technical note of Romanha and collaborators published in 2010 aimed establish a guideline with the set of minimum criteria and decision gates for the development of new agents against Trypanosoma cruzi with the focus on developing new antichagasic drugs. In this sense, the present review aims to update this technical note, bringing the state of the art and new advances on this topic in recent years.

Identification of novel bromodomain inhibitors of Trypanosoma cruzi bromodomain factor 2 (TcBDF2) using a fluorescence polarization-based high-throughput assay.

Bromodomains are structural folds present in all eukaryotic cells that bind to other proteins recognizing acetylated lysines. Most proteins with bromodomains are part of nuclear complexes that interact with acetylated histone residues and regulate DNA replication, transcription, and repair through chromatin structure remodeling. Bromodomain inhibitors are small molecules that bind to the hydrophobic pocket of bromodomains, interfering with the interaction with acetylated histones. Using a fluorescent probe, we have developed an assay to select inhibitors of the bromodomain factor 2 of Trypanosoma cruzi (TcBDF2) using fluorescence polarization. Initially, a library of 28,251 compounds was screened in an endpoint assay. The top 350-ranked compounds were further analyzed in a dose-response assay. From this analysis, seven compounds were obtained that had not been previously characterized as bromodomain inhibitors. Although these compounds did not exhibit significant trypanocidal activity, all showed bona fide interaction with TcBDF2 with dissociation constants between 1 and 3 µM validating these assays to search for bromodomain inhibitors.

Synthesis and activity of benzimidazole N-Acylhydrazones against Trypanosoma cruzi, Leishmania amazonensis and Leishmania infantum.

In this study, we present the design, synthesis, and cytotoxic evaluation of a series of benzimidazole N-acylhydrazones against strains of T. cruzi (Y and Tulahuen) and Leishmania species (L. amazonensis and L. infantum). Compound (E)-N'-((5-Nitrofuran-2-yl)methylene)-1H-benzo[d]imidazole-2-carbohydrazide demonstrated significant activity against both trypomastigote and amastigote forms (Tulahuen strain), with an IC50/120 h of 0.033 µM and a selectivity index (SI) of 7680. This represents a potency 46 times greater than that of benznidazole (IC50/120 h = 1.520 µM, SI = 1390). Another compound (E)-N'-(2-Hydroxybenzylidene)-1H-benzo[d]imidazole-2-carbohydrazide showed promising activity against both trypomastigote and amastigote forms (Tulahuen strain), with an IC50/120 h of 3.600 µM and an SI of 14.70. However, its efficacy against L. infantum and L. amazonensis was comparatively lower. These findings provide valuable insights for the development of more effective treatments against Trypanosoma cruzi.

Revisiting the dipeptidyl carboxypeptidase inhibitor captopril as a source of pan anti-trypanosomatid agents.

The protozoan parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for continued propagation of neglected tropical diseases such as African sleeping sickness, Chagas disease and leishmaniasis respectively. Following a report that captopril targets Leishmania donovani dipeptidyl carboxypeptidase, a series of simple proline amides and captopril analogues were synthesized and found to exhibit 1-2 µM in vitro inhibition and selectivity against Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. The results were corroborated with computational docking studies. Arguably, the synthetic proline amides represent the structurally simplest examples of in vitro pan antiprotozoal compounds.

A Case of Chagas Disease in South Dakota: Diagnosis and Treatment.

Chagas disease is a chronic, systemic parasitic infection caused by the protozoan Trypanosoma cruzi. The primary mode of transmission to humans is by the Reduviid insect, endemic to South America. Recent migration of the vector has led to increased cases in the southern United States and has prompted increased surveillance and blood donation screening. It is unusual to diagnose and treat individuals with Chagas disease in the northern United States. This case describes an immigrant female from El Salvador that was informed she had Chagas disease from a blood bank screening. Confirmation and treatment of the disease were performed by her South Dakota primary care provider thus demonstrating the importance of identifying Chagas disease in the immigrant population in regions where Chagas disease infection is uncommon.

Structure-Activity Study on Substituted, Core-Extended, and Dyad Naphthalene Diimide G-Quadruplex Ligands Leading to Potent Antitrypanosomal Agents.

Several G-quadruplex nucleic acid (G4s) ligands have been developed seeking target selectivity in the past decade. Naphthalene diimide (NDI)-based compounds are particularly promising due to their biological activity and red-fluorescence emission. Previously, we demonstrated the existence of G4s in the promoter region of parasite genomes, assessing the effectiveness of NDI-derivatives against them. Here, we explored the biological activity of a small library of G4-DNA ligands, exploiting the NDI pharmacophore, against both Trypanosoma brucei and Leishmania major parasites. Biophysical and biological assays were conducted. Among the various families analyzed, core-extended NDIs exhibited the most promising results concerning the selectivity and antiparasitic effects. NDI 16 emerged as the most potent, with an IC50 of 0.011 nM against T. brucei and remarkable selectivity vs MRC-5 cells (3454-fold). Fascinating, 16 is 480-fold more potent than the standard drug pentamidine (IC50 = 5.3 nM). Cellular uptake and parasite localization were verified by exploiting core-extended NDI red-fluorescent emission.

Novel 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidine derivatives and their antitrypanosomal activities against T.brucei.

Human African trypanosomiasis, or sleeping sickness, is a neglected tropical disease caused by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense and is invariably fatal unless treated. Current therapies present limitations in their application, parasite resistance, or require further clinical investigation for wider use. Our work, informed by previous findings, presents novel 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidine derivatives with promising antitrypanosomal activity. In particular, 32 exhibits an in vitro EC50 value of 0.5 µM against Trypanosoma brucei rhodesiense, and analogues 29, 30 and 33 show antitrypanosomal activities in the <1 µM range. We have demonstrated that substituted 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidines present promising antitrypanosomal hit molecules with potential for further preclinical development.

Venturicidin A affects the mitochondrial membrane potential and induces kDNA loss in Trypanosoma brucei.

Neglected tropical diseases caused by trypanosomatid parasites have devastating health and economic consequences, especially in tropical areas. New drugs or new combination therapies to fight these parasites are urgently needed. Venturicidin A, a macrolide extracted from Streptomyces, inhibits the ATP synthase complex of fungi and bacteria. However, its effect on trypanosomatids is not fully understood. In this study, we tested venturicidin A on a panel of trypanosomatid parasites using Alamar Blue assays and found it to be highly active against Trypanosoma brucei and Leishmania donovani, but much less so against Trypanosoma evansi. Using fluorescence microscopy, we observed a rapid loss of the mitochondrial membrane potential in T. brucei bloodstream forms upon venturicidin A treatment. Additionally, we report the loss of mitochondrial DNA in approximately 40%-50% of the treated parasites. We conclude that venturicidin A targets the ATP synthase of T. brucei, and we suggest that this macrolide could be a candidate for anti-trypanosomatid drug repurposing, drug combinations, or medicinal chemistry programs.

Listen to what the animals say: a systematic review and meta-analysis of sterol 14-demethylase inhibitor efficacy for in vivo models of Trypanosoma cruzi infection.

Sterol 14-demethylase (CYP51) inhibitors, encompassing new chemical entities and repurposed drugs, have emerged as promising candidates for Chagas disease treatment, based on preclinical studies reporting anti-Trypanosoma cruzi activity. Triazoles like ravuconazole (RAV) and posaconazole (POS) progressed to clinical trials. Unexpectedly, their efficacy was transient in chronic Chagas disease patients, and their activity was not superior to benznidazole (BZ) treatment. This paper aims to summarize evidence on the global activity of CYP51 inhibitors against T. cruzi by applying systematic review strategies, risk of bias assessment, and meta-analysis from in vivo studies. PubMed and Embase databases were searched for original articles, obtaining fifty-six relevant papers meeting inclusion criteria. Characteristics of animal models, parasite strain, treatment schemes, and cure rates were extracted. Primary outcomes such as maximum parasitaemia values, survival, and parasitological cure were recorded for meta-analysis, when possible. The risk of bias was uncertain in most studies. Animals treated with itraconazole, RAV, or POS survived significantly longer than the infected non-treated groups (RR = 4.85 [3.62, 6.49], P < 0.00001), and they showed no differences with animals treated with positive control drugs (RR = 1.01 [0.98, 1.04], P = 0.54). Furthermore, the overall analysis showed that RAV or POS was not likely to achieve parasitological cure when compared with BZ or NFX treatment (OD = 0.49 [0.31, 0.77], P = 0.002). This systematic review contributes to understanding why the azoles had failed in clinical trials and, more importantly, how to improve the animal models of T. cruzi infection by filling the gaps between basic, translational, and clinical research.