Selenosugars targeting the infective stage of Trypanosoma brucei with high selectivity.

Earlier evidences showed that diglycosyl diselenides are active against the infective stage of African trypanosomes (top hits IC50 0.5 and 1.5 µM) but poorly selective (selectivity index <10). Here we extended the study to 33 new seleno-glycoconjugates with the aim to improve potency and selectivity. Three selenoglycosides and three glycosyl selenenylsulfides displayed IC50 against bloodstream Trypanosoma brucei in the sub-µM range (IC50 0.35-0.77 µM) and four of them showed an improved selectivity (selectivity index >38-folds vs. murine and human macrohages). For the glycosyl selenylsulfides, the anti-trypanosomal activity was not significantly influenced by the nature of the moiety attached to the sulfur atom. Except for a quinoline-, and to a minor extent a nitro-derivative, the most selective hits induced a rapid (within 60 min) and marked perturbation of the LMWT-redox homeostasis. The formation of selenenylsulfide glycoconjugates with free thiols has been identified as a potential mechanism involved in this process.

Ascofuranone antibiotic is a promising trypanocidal drug for nagana.

Trypanosomosis is a disease complex which affects both humans and animals in sub-Saharan Africa, transmitted by the tsetse fly and distributed within the tsetse belt of Africa. But some trypanosome species, for example, Trypanosoma brucei evansi, T. vivax, T. theileri and T. b. equiperdum are endemic outside the tsetse belt of Africa transmitted by biting flies, for example, Tabanus and Stomoxys, or venereal transmission, respectively. Trypanocidal drugs remain the principal method of animal trypanosomosis control in most African countries. However, there is a growing concern that their effectiveness may be severely curtailed by widespread drug resistance. A minimum number of six male cattle calves were recruited for the study. They were randomly grouped into two (T. vivax and T. congolense groups) of three calves each. One calf per group served as a control while two calves were treatment group. They were inoculated with 105 cells/mL parasites in phosphate buffered solution (PBS) in 2 mL. When parasitaemia reached 1 × 107.8 cells/mL trypanosomes per mL in calves, treatment was instituted with 20 mL (25 mg/kg in 100 kg calf) ascofuranone (AF) for treatment calves, while the control ones were administered a placebo (20 mL PBS) intramuscularly. This study revealed that T. vivax was successfully cleared by AF but the T. congolense group was not cleared effectively.Contribution: There is an urgent need to develop new drugs which this study sought to address. It is suggested that the AF compound can be developed further to be a sanative drug for T. vivax in non-tsetse infested areas like South Americas.

Lead Optimization of the 5-Phenylpyrazolopyrimidinone NPD-2975 toward Compounds with Improved Antitrypanosomal Efficacy.

Human African trypanosomiasis (HAT) still faces few therapeutic options and emerging drug resistance, stressing an urgency for novel antitrypanosomal drug discovery. Here, we describe lead optimization efforts aiming at improving antitrypanosomal efficacy and better physicochemical properties based on our previously reported optimized hit NPD-2975 (pIC50 7.2). Systematic modification of the 5-phenylpyrazolopyrimidinone NPD-2975 led to the discovery of a R4-substituted analogue 31c (NPD-3519), showing higher in vitro potency (pIC50 7.8) against Trypanosoma brucei and significantly better metabolic stability. Further, in vivo pharmacokinetic evaluation of 31c and experiments in an acute T. brucei mouse model confirmed improved oral bioavailability and antitrypanosomal efficacy at 50 mg/kg with no apparent toxicity. With good physicochemical properties, low toxicity, improved pharmacokinetic features, and in vivo efficacy, 31c may serve as a promising candidate for future drug development for HAT.

Identification of a potent and selective LAPTc inhibitor by RapidFire-Mass Spectrometry, with antichagasic activity.

BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 < 34 µM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with Ki 0.27 µM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of >500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination. CONCLUSIONS/SIGNIFICANCE: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.

Antitrypanosomal Chloronitrobenzamides.

Human African trypanosomiasis (HAT), a neglected tropical disease caused by Trypanosoma brucei gambiense (Tbg) or Trypanosoma brucei rhodesiense (Tbr), remains a significant public health concern with over 55 million people at risk of infection. Current treatments for HAT face the challenges of poor efficacy, drug resistance, and toxicity. This study presents the synthesis and evaluation of chloronitrobenzamides (CNBs) against Trypanosoma species, identifying previously reported compound 52 as a potent and selective orally bioavailable antitrypanosomal agent. 52 was well tolerated in vivo and demonstrated favorable oral pharmacokinetics, maintaining plasma concentrations surpassing the cellular EC50 for over 24 h and achieving peak brain concentrations exceeding 7 µM in rodents after single oral administration (50 mg/kg). Treatment with 52 significantly extended the lifespan of mice infected with Trypanosoma congolense and T. brucei rhodesiense. These results demonstrate that 52 is a strong antitrypanosomal lead with potential for developing treatments for both human and animal African trypanosomiasis.

In vitro and in vivo antitrypanosomal activity of the fresh leaves of Ranunculus Multifidus Forsk and its major compound anemonin against Trypanosoma congolense field isolate.

BACKGROUND: Animal trypanosomiasis is a major livestock problem due to its socioeconomic impacts in tropical countries. Currently used trypanocides are toxic, expensive, and the parasites have developed resistance to the existing drugs, which calls for an urgent need of new effective and safe chemotherapeutic agents from alternative sources such as medicinal plants. In Ethiopian traditional medicine fresh leaves of Ranunculus multifidus Forsk, are used for the treatment of animal trypanosomiasis. The present study aimed to evaluate the antitrypanosomal activity of the fresh leaves of R. multifidus and its major compound anemonin against Trypanosoma congolense field isolate. METHODS: Fresh leaves of R. multifidus were extracted by maceration with 80% methanol and hydro-distillation to obtain the corresponding extracts. Anemonin was isolated from the hydro-distilled extract by preparative TLC. For the in vitro assay, 0.1, 0.4, 2 and 4 mg/ml of the test substances were incubated with parasites and cessation or drop in motility of the parasites was monitored for a total duration of 1 h. In the in vivo assay, the test substances were administered intraperitoneally daily for 7 days to mice infected with Trypanosoma congolense. Diminazene aceturate and 1% dimethylsulfoxide (DMSO) were used as positive and negative controls, respectively. RESULTS: Both extracts showed antitrypanosomal activity although the hydro-distilled extract demonstrated superior activity compared to the hydroalcoholic extract. At a concentration of 4 mg/ml, the hydro-distilled extract drastically reduced motility of trypanosomes within 20 min. Similarly, anemonin at the same concentration completely immobilized trypanosomes within 5 min of incubation, while diminazene aceturate (28.00 mg/kg/day) immobilized the parasites within 10 min. In the in vivo antitrypanosomal assay, anemonin eliminates parasites at all the tested doses (8.75, 17.00 and 35.00 mg/kg/day) and prevented relapse, while in diminazene aceturate-treated mice the parasites reappeared on days 12 to 14. CONCLUSIONS: The current study demonstrated that the fresh leaves of R. multifidus possess genuine antitrypanosomal activity supporting the use of the plant for the treatment of animal trypanosomiasis in traditional medicine. Furthermore, anemonin appears to be responsible for the activity suggesting its potential as a scaffold for the development of safe and cost effective antitrypanosomal agent.

Efficacy of short-course treatment for prevention of congenital transmission of Chagas disease: A retrospective cohort study.

BACKGROUND: In regions with controlled vector transmission of T. cruzi, congenital transmission is the most frequent route of infection. Treatment with benznidazole (BZ) or nifurtimox (NF) for 60 days in girls and women of childbearing age showed to be effective in preventing mother to child transmission of this disease. Reports on short-course treatment (≤30 days) are scarce. METHODS: Retrospective cohort study. Offspring of women with Chagas disease who received short-course treatment (≤30 days) with BZ or NF, attended between 2003 and 2022, were evaluated. Parasitemia (microhaematocrit and/or PCR) was performed at <8 months of age, and serology (ELISA and IHA) at ≥8 months to rule out congenital infection. RESULTS: A total of 27 women receiving ≤30 days of treatment and their children were included in this study. NF was prescribed in 17/27 (63%) women, and BZ in 10/27 (37%). The mean duration of treatment was 29.2 days. None of the women experienced serious adverse events during treatment, and no laboratory abnormalities were observed. Forty infants born to these 27 treated women were included. All newborns were full term, with appropriate weight for their gestational age. No perinatal infectious diseases or complications were observed. DISCUSSION: Several studies have shown that treatment of infected girls and women of childbearing age for 60 days is an effective practice to prevent transplacental transmission of T. cruzi. Our study demonstrated that short-duration treatment (≤30 days) is effective and beneficial in preventing transplacental transmission of Chagas disease.

In silico toxicologic profile and in vivo trypanocidal activity of estafietin, a sesquiterpene lactone isolated from Stevia alpina Griseb.

Chagas disease is an infection caused by the protozoan Trypanosoma cruzi, affecting 6-8 million people worldwide. Only two drugs are available for its treatment, having a limited efficacy and adverse side-effects. Estafietin is a sesquiterpene lactone isolated from Stevia alpina with in vitro activity against T. cruzi and low cytotoxicity against mammalian cells. The aim of this work was to predict the toxicologic profile of estafietin by in silico methods and assess its in vivo activity on a murine model of Chagas disease. Estafietin showed low toxicity according to pkCSM web tool and passed the PAINS filter from PAINS-remover web server. The treatment of infected mice with 1 mg/Kg/day of estafietin for five consecutive days administrated by intraperitoneal route significatively decreased parasitemia levels and reduced inflammatory infiltrates and myocyte damage on muscle tissue. These results suggest that estafietin had effect both on acute and chronic stages of the infection.

Discovery of an orally active nitrothiophene-based antitrypanosomal agent.

Human African Trypanosomiasis (HAT), caused by Trypanosoma brucei gambiense and rhodesiense, is a parasitic disease endemic to sub-Saharan Africa. Untreated cases of HAT can be severely debilitating and fatal. Although the number of reported cases has decreased progressively over the last decade, the number of effective and easily administered medications is very limited. In this work, we report the antitrypanosomal activity of a series of potent compounds. A subset of molecules in the series are highly selective for trypanosomes and are metabolically stable. One of the compounds, (E)-N-(4-(methylamino)-4-oxobut-2-en-1-yl)-5-nitrothiophene-2-carboxamide (10), selectively inhibited the growth of T. b. brucei, T. b. gambiense and T. b. rhodesiense, have excellent oral bioavailability and was effective in treating acute infection of HAT in mouse models. Based on its excellent bioavailability, compound 10 and its analogs are candidates for lead optimization and pre-clinical investigations.

Five-year serological and clinical evolution of chronic Chagas disease patients in Cochabamba, Bolivia.

BACKGROUND: Chagas disease, caused by the parasite Trypanosoma cruzi, is a neglected infectious disease that exerts the highest public health burden in the Americas. There are two anti-parasitic drugs approved for its treatment-benznidazole and nifurtimox-but the absence of biomarkers to early assess treatment efficacy hinders patients´ follow-up. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a longitudinal, observational study among a cohort of 106 chronically T. cruzi-infected patients in Cochabamba (Bolivia) who completed the recommended treatment of benznidazole. Participants were followed-up for five years, in which we collected clinical and serological data, including yearly electrocardiograms and optical density readouts from two ELISAs (total and recombinant antigens). Descriptive and statistical analyses were performed to understand trends in data, as well as the relationship between clinical symptoms and serological evolution after treatment. Our results showed that both ELISAs documented average declines up to year three and slight inclines for the following two years. The recorded clinical parameters indicated that most patients did not have any significant changes to their cardiac or digestive symptoms after treatment, at least in the timeframe under investigation, while a small percentage demonstrated either a regression or progression in symptoms. Only one participant met the "cure criterion" of a negative serological readout for both ELISAs by the final year. CONCLUSIONS/SIGNIFICANCE: The study confirms that follow-up of benznidazole-treated T. cruzi-infected patients should be longer than five years to determine, with current tools, if they are cured. In terms of serological evolution, the single use of a total antigen ELISA might be a more reliable measure and suffice to address infection status, at least in the region of Bolivia where the study was done. Additional work is needed to develop a test-of-cure for an early assessment of drugs´ efficacy with the aim of improving case management protocols.

Predictors of Trypanosoma cruzi PCR positivity in patients with chronic Chagas disease.

BACKGROUND: A positive Trypanosoma cruzi polymerase chain reaction (PCR) is associated with a worse prognosis in patients with chronic Chagas disease (CD). OBJECTIVES: To study the association of clinical, electrocardiographic, and echocardiographic characteristics and biomarker blood levels with positive T. cruzi PCR in chronic CD. METHODS: This is a single-centre observational cross-sectional study. Positive T. cruzi PCR association with clinical, electrocardiographic, and echocardiographic characteristics, and biomarker blood levels were studied by logistic regression analysis. p values < 0.05 were considered significant. FINDINGS: Among 333 patients with chronic CD (56.4% men; 62 ± 10 years), T. cruzi PCR was positive in 41.1%. Stepwise multivariate logistic regression showed an independent association between positive T. cruzi PCR and diabetes mellitus {odds ratio (OR) 0.53 [95% confidence interval (CI) 0.30-0.93]; p = 0.03}, right bundle branch block [OR 1.78 (95% CI 1.09-2.89); p = 0.02], and history of trypanocidal treatment [OR 0.13 (95% CI 0.04-0.38); p = 0.0002]. Among patients with a history of trypanocidal treatment (n = 39), only four (10%) patients had a positive T. cruzi PCR. MAIN CONCLUSIONS: Among several studied parameters, only diabetes mellitus, right bundle branch block, and history of trypanocidal treatment showed an independent association with positive T. cruzi PCR. History of trypanocidal treatment was a strong protective factor against a positive T. cruzi PCR.

Lychnopholide loaded in surface modified polylactide nanocapsules (LYC-PLA-PEG-NC) cure mice infected by Trypanosoma cruzi strain a prototype of resistance to benznidazole and nifurtimox: First insights of its mechanism of action.

Chagas disease (CD) remains neglected and causes high morbidity and mortality. The great difficulty is the lack of effective treatment. The current drugs cause side effects and have limited therapeutic efficacy in the chronic phase. This study aims to fulfil some gaps in studies of the natural substance lychnopholide nanoencapsulated LYC-PLA-PEG-NC (LYC-NC) and free (Free-LYC): the activity in epimastigotes and amastigotes to determine its selectivity index (SI), the therapeutic efficacy in mice infected with Colombian Trypanosoma cruzi strain and insight of the mechanism of LYC-NC action on T. cruzi. The SI was obtained by calculation of the ratio between the IC50 value toward H9c2 cells divided by the IC50 value in the anti-T. cruzi test. Infected Swiss mice were treated with 2 and 12 mg/kg/day via intravenous and oral, respectively, and the therapeutic efficacy was determined. The IC50 of LYC-NC and Free-LYC for epimastigotes of T. cruzi were similar. Both were active against amastigotes in cell culture, particularly Free-LYC. The SI of LYC-NC and Free-LYC were 45.38 and 32.11, respectively. LYC-NC 2 and 12 mg/kg/day cured parasitologically, 62.5% and 80% of the animals, respectively, infected with a strain resistant to treatment. The fluorescent NC was distributed in the cardiomyocyte cytoplasm, infected or not, and interacted with the trypomastigotes. Together, these results represent advances in demonstrating LYC as a potent new therapeutic option for treating CD.

In vitro evaluation of 2-(1H-pyrazol-1-yl)-1,3,4-thiadiazole derivatives against replicative and infective stages of Trypanosoma cruzi.

Current treatment of Chagas disease (CD) is based on two substances, nifurtimox (NT) and benzonidazole (BZ), both considered unsatisfactory mainly due to their low activities and high toxicity profile. One of the main challenges faced in CD management concerns the identification of new drugs active in the acute and chronic phases and with good pharmacokinetic profiles. In this work, we studied the bioactivity of twenty 2-(1H-pyrazol-1-yl)-1,3,4-thiadiazole derivatives against Trypanosoma cruzi epimastigotes and trypomastigotes. We identified seven derivatives with promising activity against epimastigote forms with IC50 values ranging from 6 µM to 44 µM. Most of the compounds showed no significant toxicity against murine macrophages. Our initial investigation on the mechanism of action indicates that this series of compounds may exert their anti-parasitic effect, inducing cell membrane damage. The results in trypomastigotes showed that one derivative, PDAN 78, satisfactorily inhibited metabolic alteration at all concentrations. Moreover, we used molecular modeling to understand how tridimensional and structural aspects might influence the observed bioactivities. Finally, we also used in silico approaches to assess the potential pharmacokinetic and toxicological properties of the most active compounds. Our initial results indicate that this molecular scaffold might be a valuable prototype for novel and safe trypanocidal compounds.

Benznidazole treatment decreases IL-6 levels in Trypanosoma cruzi-infected human adipocytes differentiated from adipose tissue-derived stem cells.

BACKGROUND: Trypanosoma cruzi, which causes Chagas disease (CD), is a versatile haemoparasite that uses several strategies to evade the host's immune response, including adipose tissue (AT), used as a reservoir of infection. As it is an effective barrier to parasite evasion, the effectiveness of the drug recommended for treating CD, Benznidazole (BZ), may be questionable. OBJECTIVE: To this end, we evaluated the parasite load and immunomodulation caused by BZ treatment in the culture of adipocytes differentiated from human adipose tissue-derived stem cells (ADSC) infected with T. cruzi. METHODS: The ADSC were subjected to adipogenic differentiation. We then carried out four cultures in which we infected the differentiated AT with trypomastigote forms of the Y strain of T. cruzi and treated them with BZ. After the incubation, the infected AT was subjected to quantitative polymerase chain reaction (qPCR) to quantify the parasite load and transmission electron microscopy (TEM) to verify the infection. The supernatant was collected to measure cytokines, chemokines, and adipokines. FINDINGS: We found elevated secretion of IL-6, CXCL-10/IP-10, CCL2/MCP-1, CCL5/RANTES, and leptin in infected fat cells. However, treatment with BZ promoted a decrease in IL-6. MAIN CONCLUSION: Therefore, we believe that BZ has a beneficial role as it reduces inflammation in infected fat cells.

Localized cardiac small molecule trajectories and persistent chemical sequelae in experimental Chagas disease.

Post-infectious conditions present major health burdens but remain poorly understood. In Chagas disease (CD), caused by Trypanosoma cruzi parasites, antiparasitic agents that successfully clear T. cruzi do not always improve clinical outcomes. In this study, we reveal differential small molecule trajectories between cardiac regions during chronic T. cruzi infection, matching with characteristic CD apical aneurysm sites. Incomplete, region-specific, cardiac small molecule restoration is observed in animals treated with the antiparasitic benznidazole. In contrast, superior restoration of the cardiac small molecule profile is observed for a combination treatment of reduced-dose benznidazole plus an immunotherapy, even with less parasite burden reduction. Overall, these results reveal molecular mechanisms of CD treatment based on simultaneous effects on the pathogen and on host small molecule responses, and expand our understanding of clinical treatment failure in CD. This link between infection and subsequent persistent small molecule perturbation broadens our understanding of infectious disease sequelae.

Persistent Biofluid Small-Molecule Alterations Induced by Trypanosoma cruzi Infection Are Not Restored by Parasite Elimination.

Chagas disease (CD), caused by Trypanosoma cruzi (T. cruzi) protozoa, is a complicated parasitic illness with inadequate medical measures for diagnosing infection and monitoring treatment success. To address this gap, we analyzed changes in the metabolome of T. cruzi-infected mice via liquid chromatography tandem mass spectrometry of clinically accessible biofluids: saliva, urine, and plasma. Urine was the most indicative of infection status across mouse and parasite genotypes. Metabolites perturbed by infection in urine include kynurenate, acylcarnitines, and threonylcarbamoyladenosine. Based on these results, we sought to implement urine as a tool for the assessment of CD treatment success. Strikingly, it was found that mice with parasite clearance following benznidazole antiparasitic treatment had an overall urine metabolome comparable to that of mice that failed to clear parasites. These results provide a complementary hypothesis to explain clinical trial data in which benznidazole treatment did not improve patient outcomes in late-stage disease, even in patients with successful parasite clearance. Overall, this study provides insights into new small-molecule-based CD diagnostic methods and a new approach to assess functional responses to treatment.

Bis-6-amidino-benzothiazole Derivative that Cures Experimental Stage 1 African Trypanosomiasis with a Single Dose.

We designed and synthesized a series of symmetric bis-6-amidino-benzothiazole derivatives with aliphatic central units and evaluated their efficacy against bloodstream forms of the African trypanosome Trypanosoma brucei. Of these, a dicationic benzothiazole compound (9a) exhibited sub-nanomolar in vitro potency with remarkable selectivity over mammalian cells (>26,000-fold). Unsubstituted 5-amidine groups and a cyclohexyl spacer were the crucial determinants of trypanocidal activity. In all cases, mice treated with a single dose of 20 mg kg-1 were cured of stage 1 trypanosomiasis. The compound displayed a favorable in vitro ADME profile, with the exception of low membrane permeability. However, we found evidence that uptake by T. brucei is mediated by endocytosis, a process that results in lysosomal sequestration. The compound was also active in low nanomolar concentrations against cultured asexual forms of the malaria parasite Plasmodium falciparum. Therefore, 9a has exquisite cross-species efficacy and represents a lead compound with considerable therapeutic potential.

Structure-Guided Design and Synthesis of a Pyridazinone Series of Trypanosoma cruzi Proteasome Inhibitors.

There is an urgent need for new treatments for Chagas disease, a parasitic infection which mostly impacts South and Central America. We previously reported on the discovery of GSK3494245/DDD01305143, a preclinical candidate for visceral leishmaniasis which acted through inhibition of the Leishmania proteasome. A related analogue, active against Trypanosoma cruzi, showed suboptimal efficacy in an animal model of Chagas disease, so alternative proteasome inhibitors were investigated. Screening a library of phenotypically active analogues against the T. cruzi proteasome identified an active, selective pyridazinone, the development of which is described herein. We obtained a cryo-EM co-structure of proteasome and a key inhibitor and used this to drive optimization of the compounds. Alongside this, optimization of the absorption, distribution, metabolism, and excretion (ADME) properties afforded a suitable compound for mouse efficacy studies. The outcome of these studies is discussed, alongside future plans to further understand the series and its potential to deliver a new treatment for Chagas disease.