Chagas Disease Drug Discovery Research Articles

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

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. 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. Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.

Novel Diarylthioether Compounds as Agents for the Treatment of Chagas Disease.

Herein, we describe the hit optimization of a novel diarylthioether chemical class found to be active against Trypanosoma cruzi; the parasite responsible for Chagas disease. The hit compound was discovered through a whole-cell phenotypic screen and as such, the mechanism of action for this chemical class is unknown. Our investigations led to clear structure-activity relationships and the discovery of several analogues with high in vitro potency. Furthermore, we observed excellent activity during acute in vivo efficacy studies in mice infected with transgenic T. cruzi. These diarylthioether compounds represent a promising new chemotype for Chagas disease drug discovery and merit further development to increase oral exposure without increasing toxicity.

Avoiding Clinical Trial Failures in Neglected Tropical Diseases: The Example of Chagas Disease.

Human clinical trials are expensive, and when they fail, they create the impression that a problem is intractable, thus depressing interest in future attempts. For neglected tropical diseases, where there are likely limited numbers of "shots on goal," such failures need to be assiduously avoided. Chagas disease drug discovery efforts have experienced more than its share of human clinical trial failures. Here are some guidelines, many specific for Chagas, but some that might also have application for other neglected tropical diseases. Chagas disease has major challenges (eg, the lack of a definitive test of cure) but also has outstanding advantages, among these the unmatched multi-species natural infection systems that can be exploited to de-risk compounds before human trials. Fully utilizing these advantages while frankly acknowledging and addressing the challenges should bring better options to patients, sooner.

Biological characteristics of the Trypanosoma cruzi Arequipa strain make it a good model for Chagas disease drug discovery

Trypanosoma cruzi, the causative agent of Chagas disease (CD), is a genuine parasite with tremendous genetic diversity and a complex life cycle. Scientists have studied this disease for more than 100 years, and CD drug discovery has been a mainstay due to the absence of an effective treatment. Technical advances in several areas have contributed to a better understanding of the complex biology and life cycle of this parasite, with the aim of designing the ideal profile of both drug and therapeutic options to treat CD. Here, we present the T. cruzi Arequipa strain (MHOM/Pe/2011/Arequipa) as an interesting model for CD drug discovery. We characterized acute-phase parasitaemia and chronic-phase tropism in BALB/c mice and determined the in vitro and in vivo benznidazole susceptibility profile of the different morphological forms of this strain. The tropism of this strain makes it an interesting model for the screening of new compounds with a potential anti-Chagas profile for the treatment of this disease.

Novel trypanocidal thiophen-chalcone cruzain inhibitors: structure- and ligand-based studies.

Background: Chagas disease is a neglected tropical disease that affects millions of people worldwide and for which no effective treatment is available. Materials & methods: 17chalcones were synthesized, for which the inhibition of cruzain and trypanocidal activity were investigated. Results: Chalcone C8 showed the highest cruzain inhibitory (IC50=0.536μm) and trypanocidal activity (IC50=0.990μm). Molecular docking studies showed interactions involving Asp161 and the thiophen group interacting with the S2 subsite. Furthermore, quantitative structure-activity relationship (q2=0.786; r2=0.953) and density functional theory studies were carried out, and a correlation between the lowest unoccupied molecular orbital surface and trypanocidal activity was observed. Conclusion: These results demonstrate that these chalcones are worthwhile hits to be further optimized in Chagas disease drug discovery programs.

Bioactivity of Novel Pyrazole-Thiazolines Scaffolds against Trypanosoma cruzi: Computational Approaches and 3D Spheroid Model on Drug Discovery for Chagas Disease.

Chagas disease, a century-old disease that mainly affects the impoverished population in Latin America, causes high morbidity and mortality in endemic countries. The available drugs, benznidazole (Bz) and nifurtimox, have limited effectiveness and intense side effects. Drug repurposing, and the development of new chemical entities with potent activity against Trypanosoma cruzi, are a potential source of therapeutic options. The present study describes the biological activity of two new series of pyrazole-thiazoline derivatives, based on optimization of a hit system 5-aminopyrazole-imidazoline previously identified, using structure–activity relationship exploration, and computational and phenotype-based strategies. Promising candidates, 2c, 2e, and 2i derivatives, showed good oral bioavailability and ADMET properties, and low cytotoxicity (CC50 > 100 µM) besides potent activity against trypomastigotes (0.4–2.1 µM) compared to Bz (19.6 ± 2.3 µM). Among them, 2c also stands out, with greater potency against intracellular amastigotes (pIC50 = 5.85). The selected pyrazole-thiazoline derivatives showed good permeability and effectiveness in the 3D spheroids system, but did not sustain parasite clearance in a washout assay. The compounds’ mechanism of action is still unknown, since the treatment neither increased reactive oxygen species, nor reduced cysteine protease activity. This new scaffold will be targeted to optimize in order to enhance its biological activity to identify new drug candidates for Chagas disease therapy.

Design and synthesis of Mannich base-type derivatives containing imidazole and benzimidazole as lead compounds for drug discovery in Chagas Disease.

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, the most important parasitic infection in Latin America. The only treatments currently available are nitro-derivative drugs that are characterised by high toxicity and limited efficacy. Therefore, there is an urgent need for more effective, less toxic therapeutic agents. We have previously identified the potential for Mannich base derivatives as novel inhibitors of this parasite. To further explore this family of compounds, we synthesised a panel of 69 new analogues, based on multi-parametric structure-activity relationships, which allowed optimization of both anti-parasitic activity, physicochemical parameters and ADME properties. Additionally, we optimized our invitro screening approaches against all three developmental forms of the parasite, allowing us to discard the least effective and trypanostatic derivatives at an early stage. We ultimately identified derivative 3c, which demonstrated excellent trypanocidal properties, and a synergistic mode of action against trypomastigotes in combination with the reference drug benznidazole. Both its druggability and low-cost production make this derivative a promising candidate for the preclinical, invivo assays of the Chagas disease drug-discovery pipeline.

Diaminomaleonitrile derivatives as new potential antichagasic compounds: a study of structure-activity relationships.

Background: Schiff bases are synthetically accessible compounds that have been used in medicinal chemistry. Methods & results: In this work, 27 Schiff bases derived from diaminomaleonitrile were synthesized in high yields (80-98%). Molecular docking studies suggested that the Schiff bases interact with the catalytic site of cruzain. The most active cruzain inhibitor, analog 13 (IC50=263nM), was predicted to form an additional hydrophobic contact with Met68 in the binding site of the enzyme. A strong correlation between the IC50 values and ChemScore binding energies was observed (R=0.99). Kernel-based 2D quantitative structure-activity relationship models for the whole dataset yielded sound correlation coefficients (R2=0.844; Q2=0.719). Conclusion: These novel and potent cruzain inhibitors are worthwhile starting points in further Chagas disease drug discovery programs.

A Novel High-Content Screening-Based Method for Anti-Trypanosoma cruzi Drug Discovery Using Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Chagas disease is caused by Trypanosoma cruzi infection and remains a relevant cause of chronic heart failure in Latin America. The pharmacological arsenal for Chagas disease is limited, and the available anti-T. cruzi drugs are not effective when administered during the chronic phase. Cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) have the potential to accelerate the process of drug discovery for Chagas disease, through predictive preclinical assays in target human cells. Here, we aimed to establish a novel high-content screening- (HCS-) based method using hiPSC-CMs to simultaneously evaluate anti-T. cruzi activity and cardiotoxicity of chemical compounds. To provide proof-of-concept data, the reference drug benznidazole and three compounds with known anti-T. cruzi activity (a betulinic acid derivative named BA5 and two thiazolidinone compounds named GT5A and GT5B) were evaluated in the assay. hiPSC-CMs were infected with T. cruzi and incubated for 48 h with serial dilutions of the compounds for determination of EC50 and CC50 values. Automated multiparametric analyses were performed using an automated high-content imaging system. Sublethal toxicity measurements were evaluated through morphological measurements related to the integrity of the cytoskeleton by phalloidin staining, nuclear score by Hoechst 33342 staining, mitochondria score following MitoTracker staining, and quantification of NT-pro-BNP, a peptide released upon mechanical myocardial stress. The compounds showed EC50 values for anti-T. cruzi activity similar to those previously described for other cell types, and GT5B showed a pronounced trypanocidal activity in hiPSC-CMs. Sublethal changes in cytoskeletal and nucleus scores correlated with NT-pro-BNP levels in the culture supernatant. Mitochondrial score changes were associated with increased cytotoxicity. The assay was feasible and allowed rapid assessment of anti-T. cruzi action of the compounds, in addition to cardiotoxicity parameters. The utilization of hiPSC-CMs in the drug development workflow for Chagas disease may help in the identification of novel compounds.

Rate-of-Kill (RoK) assays to triage large compound sets for Chagas disease drug discovery: Application to GSK Chagas Box.

Chagas disease (CD) is a human disease caused by Trypanosoma cruzi. Whilst endemic in Latin America, the disease is spread around the world due to migration flows, being estimated that 8 million people are infected worldwide and over 10,000 people die yearly of complications linked to CD. Current chemotherapeutics is restricted to only two drugs, i.e. benznidazole (BNZ) and nifurtimox (NIF), both being nitroaromatic compounds sharing mechanism of action and exerting suboptimal efficacy and serious adverse effects. Recent clinical trials conducted to reposition antifungal azoles have turned out disappointing due to poor efficacy outcomes despite their promising preclinical profile. This apparent lack of translation from bench models to the clinic raises the question of whether we are using the right in vitro tools for compound selection. We propose that speed of action and cidality, rather than potency, are properties that can differentiate those compounds with better prospect of success to show efficacy in animal models of CD. Here we investigate the use of in vitro assays looking at the kinetics of parasite kill as a valuable surrogate to tell apart slow- (i.e. azoles targeting CYP51) and fast-acting (i.e. nitroaromatic) compounds. Data analysis and experimental design have been optimised to make it amenable for high-throughput compound profiling. Automated data reduction of experimental kinetic points to tabulated curve descriptors in conjunction with PCA, k-means and hierarchical clustering provide drug discoverers with a roadmap to guide navigation from hit qualification of a screening campaign to compound optimisation programs and assessment of combo therapy potential. As an example, we have studied compounds belonging to the GSK Chagas Box stemmed from the HTS campaign run against the full GSK 1.8 million compounds collection [1].

In Vitro Image-Based Assay for Trypanosoma cruzi Intracellular Forms.

The advances in development and popularization of automated fluorescence microscopes and pipetting robots allowed scientists to establish high-throughput compound screening using image-based assays for Trypanosoma cruzi intracellular forms, which are associated to chronic Chagas disease. An intracellular T. cruzi image-based assay is a valuable tool to early stage drug discovery for Chagas disease, because it allows scientists to assess a compound's efficacy and safety in the same experiment. During the last 10 years, several improvements have been incorporated into intracellular T. cruzi assay protocols to make them more predictable in what happens with parasites within an infected organism. In the present chapter, a protocol will be presented for an intracellular T. cruzi assay, but at a low-throughput scale, more compatible with facilities in many academic laboratories.

Current Approaches to Drug Discovery for Chagas Disease: Methodological Advances.

In recent years, there has been an improvement in the in vitro and in vivo methodology for the screening of anti-chagasic compounds. Millions of compounds can now have their activity evaluated (in large compound libraries) by means of high throughput in vitro screening assays. Current approaches to drug discovery for Chagas disease. This review article examines the contribution of these methodological advances in medicinal chemistry in the last four years, focusing on Trypanosoma cruzi infection, obtained from the PubMed, Web of Science, and Scopus databases. Here, we have shown that the promise is increasing each year for more lead compounds for the development of a new drug against Chagas disease. There is increased optimism among those working with the objective to find new drug candidates for optimal treatments against Chagas disease.

Why hasn’t there been more progress in new Chagas disease drug discovery?

ABSTRACTIntroduction: Chagas disease (CD) is a neglected disease caused by the protozoan parasite Trypanosoma cruzi. In terms of novel drug discovery, there has been no progress since the 1960s with the same two drugs, benznidazole and nifurtimox, still in use. The complex life cycle, genetic diversity of T. cruzi strains, different sensitivities to the available drugs, as well as little interest from pharmaceutical companies and inadequate methodologies for translating in vitro and in vivo findings to the discovery of new drugs have all contributed to the lack of progress.Areas covered: In this perspective, the authors give discussion to the relevant points connected to the lack of developments in CD drug discovery and provide their expert perspectives.Expert opinion: There are few drugs currently in the preclinical pipeline for the treatment of CD. Only three classes of compounds have been shown to achieve high cure rates in mouse models of infection: nitroimidazoles (fexinidazole), oxaborole DNDi-6148 and proteasome inhibitors (GNF6702). New biomarkers for Chagas’ disease are urgently needed for the diagnosis and detection of cure/treatment efficacy. Efforts from academia and pharmaceutical companies are in progress and more intense interaction to accelerate the process of new drugs development is necessary.

Drug Discovery for Chagas Disease: Impact of Different Host Cell Lines on Assay Performance and Hit Compound Selection.

Cell-based screening has become the major compound interrogation strategy in Chagas disease drug discovery. Several different cell lines have been deployed as host cells in screening assays. However, host cell characteristics and host-parasite interactions may play an important role when assessing anti-T. cruzi compound activity, ultimately impacting on hit discovery. To verify this hypothesis, four distinct mammalian cell lines (U2OS, THP-1, Vero and L6) were used as T. cruzi host cells in High Content Screening assays. Rates of infection varied greatly between different host cells. Susceptibility to benznidazole also varied, depending on the host cell and parasite strain. A library of 1,280 compounds was screened against the four different cell lines infected with T. cruzi, resulting in the selection of a total of 82 distinct compounds as hits. From these, only two hits were common to all four cell lines assays (2.4%) and 51 were exclusively selected from a single assay (62.2%). Infected U2OS cells were the most sensitive assay, as 55 compounds in total were identified as hits; infected THP-1 yielded the lowest hit rates, with only 16 hit compounds. Of the selected hits, compound FPL64176 presented selective anti-T. cruzi activity and could serve as a starting point for the discovery of new anti-chagasic drugs.

Dehydrodieugenol B derivatives as antiparasitic agents: Synthesis and biological activity against Trypanosoma cruzi

Chagas disease is a neglected protozoan disease that affects more than eight million people in developing countries. Due to the limited number and toxicity profiles of therapies in current use, new drugs are urgently needed. In previous studies, we reported the isolation of two related antitrypanosomal neolignans from Nectandra leucantha (Lauraceae). In this work, a semi-synthetic library of twenty-three neolignan derivatives was prepared to explore synthetically accessible structure activity relationships (SAR) against Trypanosoma cruzi. Five compounds demonstrated activity against trypomastigotes (IC50 values from 8 to 64 μM) and eight showed activity against intracellular amastigotes (IC50 values from 7 to 16 μM). Eighteen derivatives demonstrated no mammalian cytotoxicity up to 200 μM. The phenolic acetate derivative of natural dehydrodieugenol B was effective against both parasite forms and eliminated 100% of amastigotes inside macrophages. This compound caused rapid and intense depolarization of the mitochondrial membrane potential, with decreased levels of intracellular reactive oxygen species being observed. Fluorescence assays demonstrated that this derivative affected neither the permeability nor the electric potential of the parasitic plasma membrane, an effect also corroborated by scanning electron microscopy studies. Structure-activity relationship studies (SARs) demonstrated that the presence of at least one allyl side chain on the biaryl ether core was important for antitrypanosomal activity, and that the free phenol is not essential. This set of neolignan derivatives represents a promising starting point for future Chagas disease drug discovery studies.

Challenges in Chagas Disease Drug Discovery: A Review.

Chagas disease or American trypanosomiasis is a neglected tropical disease caused by the parasite Trypanosoma cruzi. Although the number of infected individuals has decreased, about 6-7 million people are infected worldwide. The chemotherapy drugs currently used are limited to benznidazole and nifurtimox. They are effective in acute phase, congenital transmission and children with chronic infection; however, recent clinical trials have shown limitations in adults with chronic infection, presenting drawbacks during the treatment. Thus, there is an urgent need for new effective, safe and affordable drugs to fight against this complex disease. There were high expectations for azole derivatives as they appeared to be the most promising drugs for the treatment of Chagas disease during the last decade; however, the disappointing results obtained so far in clinical trials evidenced the lack of correlation between preclinical and clinical development. Therefore, the feedback obtained from these studies should define the starting point for addressing a roadmap for the drug discovery process in the fight against this disease. To tackle this challenge, it is important to keep in mind the drug target profile, already defined by panels of experts, and the coordinated work involving multi-disciplinary networks focusing not only on the discovery of new drugs but also on the standardization of the protocols that would allow acceleration in the Chagas disease drug discovery process.

Advances and Progress in Chagas Disease Drug Discovery.

Chagas disease represents a serious burden for millions of people worldwide. Transmitted by the protozoan parasite Trypanosoma cruzi, this neglected tropical disease causes more than 10,000 deaths each year and is the main cause of heart failure in Latin America, where it is endemic. Although most cases are concentrated in Latin American countries, Chagas disease has been increasingly reported in non-endemic regions, where the low level of public awareness on the subject contributes to the growing prevalence of the disease. The available medicines are characterized by several safety and efficacy drawbacks that prevent millions of people, particularly those with advanced disease, from receiving adequate treatment. This urgent need has stimulated the emergence of diverse initiatives dedicated to the research and development (R&D) of novel therapeutic agents for Chagas disease. Public-private partnerships have been responsible for a significant increase in the investments in R&D programs and major advancements have been achieved over the past ten years. A number of collaborative projects have been leveraged by this organizational model, which privileges sharing of data, expertise, and resources between research institutions and pharmaceutical companies. Among the current strategies employed by these consortia, target-based and phenotypic screenings have achieved the most promising results. This article provides an overview on the current status and recent advances in Chagas disease drug discovery.

Translational challenges of animal models in Chagas disease drug development: a review.

Chagas disease, or American trypanosomiasis, caused by Trypanosoma cruzi parasite infection is endemic in Latin America and presents an increasing clinical challenge due to migrating populations. Despite being first identified over a century ago, only two drugs are available for its treatment, and recent outcomes from the first clinical trials in 40 years were lackluster. There is a critical need to develop new drugs to treat Chagas disease. This requires a better understanding of the progression of parasite infection, and standardization of animal models designed for Chagas disease drug discovery. Such measures would improve comparison of generated data and the predictability of test hypotheses and models designed for translation to human disease. Existing animal models address both disease pathology and treatment efficacy. Available models have limited predictive value for the preclinical evaluation of novel therapies and need to more confidently predict the efficacy of new drug candidates in clinical trials. This review highlights the overall lack of standardized methodology and assessment tools, which has hampered the development of efficacious compounds to treat Chagas disease. We provide an overview of animal models for Chagas disease, and propose steps that could be undertaken to reduce variability and improve predictability of drug candidate efficacy. New technological developments and tools may contribute to a much needed boost in the drug discovery process.

Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease.

Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.

A new era for chagas disease drug discovery?

Recent clinical trials investigating treatment of chronic indeterminate Chagas disease with two re-purposed azole anti-fungal drugs, posaconazole and ravuconazole, revealed their inferiority to the current standard-of-care benznidazole and highlighted the inadequacy of the existing pre-clinical testing paradigm for this disease. A very limited number of controlled clinical trials for Chagas disease have been conducted to date. The selection of these compounds for clinical evaluation relied heavily on pre-clinical data obtained from in vitro screens and animal studies. This chapter reviews the evolution of CYP51 as a target for Trypanosoma cruzi growth inhibition and also explores the impact of clinical trial data on contemporary Chagas disease drug discovery. Advances in pre-clinical profiling assays, the current compound landscape and progress towards the identification of new drug targets to re-invigorate research are reviewed.