Chagas disease (CD), also known as American trypanosomiasis, is a potentially life-threatening Neglected Tropical Disease (NTD) caused by the protozoan parasite Trypanosoma cruzi (T. cruzi). An estimated 6 to 7 million people worldwide are infected with T. cruzi (WHO, 2023). The disease is found mainly in endemic areas of 21 Latin American countries but with climate change and population movement the impact is spreading to other regions of the globe (Figure 1). CD places a significant direct financial burden on healthcare system and society that has been estimated to exceed $625 million in healthcare costs and 806,170 DALYs (Gómez-Ochoa et al., 2022). However, the full economic cost to endemic communities is much more significant, resulting in a loss of 752,000 workdays annually due to premature deaths, which carries an average financial burden of 1.2 billion dollars per year in the southern countries of Latin America (Ramsey et al., 2014). Additionally, it is currently estimated that <1% of those infected with CD have access to care and treatment (Cucunubá et al., 2017).

There is no vaccine for CD and there are only two drugs’ treatments approved for this illness. These are benznidazole 1 (BZN), in use since 1971, and nifurtimox 2 (NFX), which was first approved in 1965 (Figure 2). While these old medications remain effective in preventing or curbing disease progression in infected adults, especially those with no symptoms and during the acute infection, they are not without challenges (Crespillo-Andújar et al., 2022). The duration of treatment is long (up to 2 months) and possible adverse reactions can occur in up to 40% of treated adult patients (Jackson et al., 2020), potentially requiring additional treatment for cardiac, digestive, or neurological manifestations. The drugs are contraindicated for certain populations, such as pregnant women and people with kidney or liver failure (Meymandi et al., 2018). Collectively, these factors can contribute to poor patient adherence, which may result in relapse and the development of resistance.

Given this, there is an urgent need for the development of new cost-effective, efficacious drugs. This is challenged by the global resources dedicated to ameliorating the burden of the NTDs. Moreover, even within this limited space, research into CD received only 0.67% of the overall funding allocated to all NTDs over a span of 10 years (2010–2020) (Miranda-Arboleda et al., 2021) reinforcing its reputation as the “most neglected of the neglected tropical diseases” (Zaidel and Sosa Liprandi, 2021).

Drug repurposing (the process of finding new indications for existing drugs) presents a promising approach to addressing this challenge. In this review, we discuss the use of drug repositioning as a cost-effective strategy for the development of new solutions to this devastating disease. We not only explore the latest advances in this approach but also shed light on the challenges that remain for T. cruzi chemotherapies.

Identifying and developing new drugs against T. cruzi is complicated by the parasite’s complex life cycle (Figure 3). The life cycle has four developmental stages, involving, metacyclic trypomastigotes, epimastigotes, blood-form trypomastigotes, and intracellular amastigotes, which span two host organisms; a triatomine insect, commonly known as a kissing bug, and the mammalian host (Nagajyothi et al., 2012). In humans and other mammals, transmission primarily occurs through insect vectors. This happens when an infected triatomine insect feeds on the blood of a host. Trypomastigotes are released in its faeces near the site of the bite wound. The trypomastigotes can then enter the host’s body through the wound or through intact mucosal membranes, such as the conjunctiva. Alternative modes of transmission can occur through ingestion of contaminated food, mother-to-child transmission and infected blood transfusions and organ transplantation.

Once inside the host, the trypomastigotes invade nearby cells and differentiate into intracellular amastigotes. These amastigotes multiply by binary fission and differentiate back into trypomastigotes, which are then released into the bloodstream. The trypomastigotes can infect cells from various tissues and transform into intracellular amastigotes in new infection sites. Unlike African trypanosomes (T. brucei), bloodstream trypomastigotes of T. cruzi do not replicate. Replication only resumes when the parasites enter another cell or are ingested by another vector. The cycle is propagated when an uninfected kissing bug feeds on human or animal blood that contains circulating parasites. The ingested trypomastigotes transform into epimastigotes in the vector’s midgut, where they multiply and differentiate. The parasites then differentiate into infective metacyclic trypomastigotes in the hindgut, ready to be transmitted to another host. This life cycle leads to a disease with two distinct phases (PAHO, 2023). Following first infection there is an initial acute phase, which can last for several months, during which a high number of parasites circulate in the blood. This can lead to a range of visible signs, initially a skin lesion or swelling of the eye lids but may involve fever, headache, enlarged lymph glands, muscle pain, breathing difficulties, and abdominal and chest discomfort. However, challenging early diagnosis for the majority of individuals, these symptoms are often mild and not easily attributed to a T. cruzi infection. During the chronic phase, the blood population of the parasite falls dramatically, and the parasite reside mainly in the heart and digestive muscles. One to three decades later, up to a third of patients suffer from cardiac disorders (CD is the second leading cause of chronic heart failure in Latin America) and up to 1 in 10 suffer from digestive (typically enlargement of the esophagus or colon), neurological or mixed alterations. Then, the infection can lead to progressive damage to the nervous system and heart muscle, resulting in cardiac arrhythmias, heart failure, and sudden death. This unusually slow progression, often without noticeable symptoms has led CD to be labelled as a “silent and silenced disease” and challenges diagnosis and a recognition of the severity of the problem (WHO, 2023).

De novo drug discovery, spanning from biochemical concept to the clinic, is an intricate, time-consuming, and expensive journey encompassing several crucial stages. These stages include compound identification, formulation, verification of efficacy and safety in animal models and human volunteers, numerous rounds of pre-clinical and clinical trials, and, ultimately, registration and regulatory approval. Collectively, this is estimated to take between 12–18 years and cost $1–2B to develop a new drug (Figure 4A). Moreover, the probability of failures, most commonly at later more expensive clinical trial stages, are high and this is reflected in the high cost of patented drugs. Consequently, the discovery of new drugs for CD, and other NTDs, for which the average patient exists on an income of less than $2 per day (Clark et al., 2014), is simply not viable for pharmaceutical companies without significant public cross-subsidy.

Drug repurposing, also known as drug repositioning or drug reprofiling, in which compounds developed for one indication are then utilized to provide solutions to an alternative disease, offers various advantages over developing an entirely new drug for a given indication (Pushpakom et al., 2019; Jourdan et al., 2020). Firstly, the risk of failure is lower because the repurposed drug has, if early-stage trials have been completed, already been found to be sufficiently safe in preclinical models and humans. Secondly, the drug development time frame is commonly both shorter and cheaper (Figure 4B) since some preclinical testing, safety assessments, and sometimes even formulation development, will have been completed. Given the reduced costs, drug repurposing is a particularly attractive approach for NTD (Krishnamurthy et al., 2022). Whilst repurposing approaches has been successful applied to afford approved drugs for other diseases, including NTDs such as Human African Trypanosomiasis and Leishmaniasis (where the majority of the current chemotherapy are repurposed drug (Charlton et al., 2018; Braga, 2019), there has yet not been a successful outcome for CD.

Strategies for drug repurposing follow traditional models for other drug discovery processes and encompass a combination of molecular and empirical approaches to enhance effectiveness and efficiency. Molecular approaches, commonly known as target-based strategies, primarily rely on hypothesis-driven methods at the single protein level. On the other hand, empirical approaches, known as phenotypic strategies, are centered around evaluating observable measures of response in a whole cell, organism, or system (Schenone et al., 2013; Swinney and Lee, 2020). Both strategies have their advantages and disadvantages that have been discussed in numerous review articles (Swinney, 2013; Moffat et al., 2017).

The process of discovering drugs through phenotypic screening is also referred to as “forward (or classical) pharmacology” or “forward chemical biology”. For instance, a sensitive assay to identify compound activity against T. cruzi was developed by Sykes and Avery (2015). This used an image-based assay to estimate the effect of compound treatment on T. cruzi amastigotes in 3T3 fibroblasts and host cells. This assay identified active compounds from an in-house FDA-approved drug library and the MMV Malaria Box collection. Prominent compounds include camptothecin 3, clemastine 4, crystal violet 5, and clotrimazole 6 (Figure 5A), all with sub-micromolar activities against this protozoan. In another phenotypic-based assay using a library of 100 registered drugs with drug repositioning potential for NTDs, two compounds with in vitro activity against T. cruzi were discovered (Kaiser et al., 2015). These compounds were tadalafil 7, a phosphodiesterase type 5 inhibitor used to treat erectile dysfunction, with an EC₅₀ of 8.6 μM and a selectivity index >26 (selectivity index or SI is defined as the ratio of the EC₅₀ of host cell versus the EC₅₀ of T. cruzi), and the antispasmodic mebeverine 8 with an EC₅₀ of 3.89 μM and a SI = 18.

In contrast, target-based drug discovery, also known as ‘reverse pharmacology,’ begins with the formulation of a hypothesis that modulation of a specific protein target, believed to be crucial in disease modification, will yield favorable therapeutic effects (Swinney, 2013). The initial step involves the identification and validation of a molecular target, such as an enzyme or receptor, that plays a significant role in the disease process. Subsequently, a range of strategies is employed to discover and develop drugs capable of modulating the target’s activity. These strategies encompass diverse techniques including simple biochemical and biophysical assays, in both single and high-throughput formats (vide infra), as well as virtual screening and other in silico methods. In the spirit of reducing cost, in silico methods which can screen large numbers of drug candidate compounds in a virtual manner using computer generated models of targets or known ligands are ideally suited to repurposing previously reported structures. By employing these techniques, researchers strive to identify innovative therapeutic interventions for CD, aiming to tackle the disease at its molecular roots. Over the past decade, numerous successful cases of drug repurposing for the treatment of CD have been reported using in silico approaches (Figure 5B). As an example, a novel virtual screening approach was developed by Juárez-Saldivar et al. (2020) to identify drug repositioning opportunities against T. cruzi infection by targeting the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS). Ten putative TcDHFR-TS inhibitors were identified, including nilotinib 9, glipizide 10, glyburide 11, and gliquidone 12. These compounds showed growth inhibitory activity against T. cruzi epimastigotes. In other study (Valera-Vera et al., 2020), a virtual screening strategy was employed to identify potential inhibitors of enolase, a key enzyme and potential drug target for CD. This in silico study proposed etidronate 13, a bisphosphonate inhibitor of bone resorption, as a potential inhibitor of this molecular target. Due to its safety profile, etidronate 13 provides valuable insights for the development of new drugs targeting T. cruzi enolase (TcENO). However, further exploration of its potential through in vitro and in vivo studies is necessary.

An important technique in both approaches is high-throughput screening (HTS), which allows researchers to rapidly screen thousands to millions of compounds in a relatively short period of time, greatly accelerating the drug discovery process. In a typical HTS experiment, a library of compounds is screened against a particular molecular target (target-based) or whole cell (phenotypic-based) assay (Blay et al., 2020). For instance, in the search for drug targets against T. cruzi, a HTS campaign focused on TcGlcK, a glucokinase enzyme was performed by Mercaldi et al. (2019). Glucokinase and hexokinase are crucial in T. cruzi’s metabolic pathways, making their inhibition a promising strategy for drug discovery. Out of 13,040 compounds screened, the HTS campaign found 25 enzyme inhibitors from nine chemical classes. Thirteen compounds displayed low micromolar IC₅₀ values for enzyme inhibition, with four showing low toxicity to NIH-3T3 murine host cells and notable in vitro trypanocidal activity. These four compounds, belonging to three chemical classes (3-nitro-2-phenyl-2H-chromene, N-phenyl-benzenesulfonamide, and gossypol scaffolds), include two potential hit-to-lead candidates (Figure 6A) from the 3-nitro-2-phenyl-2H-chromene class (namely, GLK2-003 14 and GLK2-004 15), holding promise for further exploration in drug discovery.

However, it is important to note that drug repurposing strategies often incorporate both molecular and empirical approaches in complementary ways. This integrative approach maximizes the chances of success by leveraging the strengths of each strategy. By combining target-based and phenotypic methods, researchers can gain a more comprehensive understanding of the underlying biology, discover new targets or pathways, and optimize drug candidates for efficacy and safety. For instance, cysteine proteases are a class of enzymes that have been validated as drug targets for the development of safe and effective pharmacological agents for CD. One such cysteine protease is cruzain, the major cysteine protease of T. cruzi. It has been shown to be essential in various stages of the T. cruzi life cycle and is a target of rational drug design for chemotherapy of CD (Ferreira et al., 2022). A study screened the ZINC database for compounds with lead-like properties to find new inhibitors of the cruzain protease (Wiggers et al., 2013). Using consensus scoring, target-based molecular docking, and ligand-based similarity searching, 8,600 lead structures were identified from an initial library of 8.5 million compounds. These were then screened through Glide XP and HQSAR, and the top 5% were visually inspected. 23 compounds were selected for in vitro phenotypic-based testing against T. cruzi-infected cells, with the top two hits (NEQ176 16 with an EC₅₀ of 108 μM and NEQ42 17 with an EC₅₀ of 10.6 μM—Figure 6B) being structurally characterized by X-ray crystallography to understand their binding modes with cruzain. In a second virtual screening campaign, 163 FDA-approved and investigational drugs identified from the DrugBank database, were screened against cruzipain, another major cysteine protease and validated molecular target of T. cruzi (Bellera et al., 2014). In this case, using a combination of target-based and phenotypic-based approaches, levothyroxine 18 (Figure 6B), a drug used in hormone replacement therapy for hypothyroidism, had dose-dependent inhibition of cruzipain (IC₅₀ of 38.43 μM) and antiproliferative activity on the T. cruzi epimastigotes (EC₅₀ of 121 mM).

An intriguing strategy for drug repositioning involves the use of combination drug therapy. Combination therapy or polytherapy is a therapeutic intervention in which more than one medication or modality is used. This approach has become increasingly common in medicine, particularly in the treatment of cancer (Bayat Mokhtari et al., 2017) and infectious diseases (Shyr et al., 2021), as it can offer several benefits over single-drug therapies including enhanced efficacy, commonly greater than simple additive effects (synergistic effect), delaying the development of drug resistance, and reducing the risk of side effects and toxicity. Combining two or more drugs with different mechanisms of action can increase the success rate of drug repositioning by providing an alternative approach to treatment (Sun et al., 2016). For example, disulfiram 19, a medicine used to treat chronic alcoholism, is currently undergoing Phase I/II clinical trials as combined chemotherapy with BZN 1 for CD (Saraiva et al., 2021). In vitro and in vivo experiments of BZN 1 with disulfiram 19 and/or its metabolite diethyldithiocarbamate 20 (Figure 6C) demonstrated synergistic effects, inhibiting parasite proliferation in infected macrophages and improved survival rates in infected mice compared to BZN 1 alone (Almeida-Silva et al., 2022). Microscopic analysis revealed structural alterations in the parasites, whilst diethyldithiocarbamate 20 treatment increased reactive oxygen species production.

Given the pressing need for new treatments for CD, significant progress has been made over the past years through drug repositioning efforts. In this review, we present an overview of these works based on the therapeutic origin of the drugs used (for a comprehensive list of the compounds discussed and their biological activities, please refer to Supplementary Table S1; Supplementary Figures S1–S3, in Supplementary Material).

Many natural products exhibit significant therapeutic potential and possess a diverse array of biological properties, including antibacterial, antiprotozoal, antimycobacterial, antileishmanial, antitumor, and anti-human immunodeficiency virus (HIV) activities and represent a valuable and fruitful resource for the discovery and development of potent drugs against a variety of diseases (Álvarez-Bardón et al., 2020; Tempone et al., 2021; Lazarin-Bidóia et al., 2022). As such, there are numerous review studies focusing on the potential use of natural products with effects on T. cruzi, providing valuable insights for further investigation (Scotti et al., 2016; Nekoei et al., 2022; Barbosa et al., 2023). For instance, antibiotics such as echinomycin derived from Streptomyces echinatus, exhibit notable anti-T. cruzi activity at nanomolar concentrations (EC₅₀ of 1.1 nM) (Annang et al., 2015). However, reflecting the complexities associated with the use of natural products as repurposing therapies, notably the lack of comprehensive in vivo studies and limited progress through clinical phases that hinder their immediate application, they will not be further explored in this review.

In these next sections, we delve into the exciting frontier of FDA-approved drug repositioning towards CD, investigating the rationale behind this approach, the challenges encountered, and the successful examples that have paved the way for the identification of novel therapeutic options. Although the majority of drugs discussed in this review are FDA-approved, we have also included, in certain instances, noteworthy molecules that are currently or have been undergoing clinical assessments, expanding the scope of potential therapeutic options for CD. The ultimate target product profile for CD is a multifactorial issue, but for simplicity in this review is described in terms of enhance efficacy and selectivity, safety and tolerability, and is realized by comparison to BZN 1 (DNDi, 2023). Additionally, we classified the selected examples of drug repurposing for CD in two ways: by approach (target-based or phenotypic-based strategies) and by technique. Within the technique category, we further subcategorize our findings into single drug trials (or small libraries), high-throughput screening, virtual screening, and combination drug therapy.

In line with the WHO’s roadmap 2021–2030 for eradicating NTDs, and especially in the context of CD, there is an urgent need to expand the range of chemotherapeutic options (Casulli, 2021). The current therapy for this condition is limited, inefficient, and insufficient, consisting of only two outdated drugs. As such, finding a rapid solution is crucial. As evidenced by successes in treating other diseases (Charlton et al., 2018; Hua et al., 2022), coupled with the encouraging results achieved over the past years, summarized in this article, to develop to develop new and improved therapies for CD, drug repurposing offers hope for making progress in the fight against one of the most neglected NTDs.

Despite its promise, drug repositioning (and drug discovery in general) in T. cruzi faces several challenges, both technical and applied. One major challenge is the lack of comprehensive knowledge of T. cruzi biology and drug targets (Jansen et al., 2020). This is exemplified by the extensive morphological and metabolic transformations that occur throughout the various developmental stages of T. cruzi, which challenge efforts to develop effective drugs against this parasite (Teixeira et al., 2012). Most notably, the intracellular amastigote can enter in a metabolically quiescent or dormant state which can be non-responsive to otherwise effective trypanocidal drugs (Sánchez-Valdéz et al., 2018; Bhattacharya et al., 2020). As such, there is a pressing need for compounds that can address this chronic state or even restore the sensitivity of the dormant parasite. In this respect, a very recent study by Rial et al. (2023) suggest that isotretinoin 72 (Figure 15), an FDA-approved drug used for severe acne that has previously shown reduction of the trypomastigote burst from infected mammal cells at nanomolar concentrations (Reigada et al., 2017), can also reduce blood parasitemia, prevent negative chronotropic effects and lead to reduced anti-T. cruzi antibody levels in murine models of chronic CD.

The success of drug repurposing remains reliant on the availability of existing drugs with well-established safety profiles, which can limit the pool of potential drug candidates. Moreover, this limited pool, and the very nature of repurposing, can mean that innovation and new modes of action are less likely to be revealed. Despite this, there are multitude of libraries available for testing and it is possible to initiate large-scale screening programs that can effectively harness these resources and unlock new treatment options. However, HTS is not cheap and for repurposing it is expected that in silico drug-target evaluation will have a growing role. By leveraging computational tools, researchers can predict the activity of existing drugs against specific targets in T. cruzi, allowing for the prioritization of compounds for further testing (Trevisan et al., 2020). While this approach is well-established in other diseases, alternative rational methods such as network-based and signature-based approaches have yet to be applied in the context of CD (Bellera et al., 2020). The future application of artificial intelligence (AI) and machine learning to this field holds great promise (Villalta and Rachakonda, 2019).

CD chemotherapy is further challenged by the extensive tissue distribution of T. cruzi which requires a similarly wide drug distribution profile in order to clear all parasites (De Rycker et al., 2023). Furthermore, the pharmacological management of this disease is further complicated by the resistance of different parasite strains to currently available drugs, potentially leading to treatment failures (García-Huertas and Cardona-Castro, 2021). One increasingly popular solution to these issues is the use of combination drug therapy, which involves administering multiple drugs with different profiles and modes of action. This can also help address challenges due to drug toxicity. Reflecting this, most combinations that have been reported involve BZN 1 or NFX 2 as the currently approved therapies. However, other repurposing combinations have been explored (Machado et al., 2020; Rocha-Hasler et al., 2021). For instance, combinations of Posaconazole 52 with either amlodipine 73 (Figure 15) or clemastine 4 demonstrated synergistic activity and greater effectiveness in reducing parasitemia levels in mice (Planer et al., 2014). Although attractive for these reasons, developing drug combinations does have hurdles that may not exist for a single repurposed drug, particularly in the assessment of pharmacokinetic and pharmacodynamic parameters as well as efficacy and safety within patients.

In conclusion, repurposing approved drugs presents a fast and appealing approach to address the challenge of CD (Supplementary Table S1, Supplementary Material), with a number of advantages compared to conventional bottom-up drug discovery. Firstly, it significantly reduces the time and cost associated with drug development by leveraging the known safety profiles and pharmacokinetic properties of these drugs, streamlining the regulatory approval process. Secondly, repositioning can rapidly expand the limited treatment options currently available for CD, potentially uncovering additional effective therapies. By exploring diverse mechanisms of action, repurposed drugs may address drug resistance and target different stages of the disease, leading to improved treatment outcomes. Moreover, combining repurposed drugs with existing treatments can result in synergistic effects, enhancing their efficacy in a safer manner. Lastly, utilizing existing drug supply chains facilitates broader access to medications for CD, particularly in resource-constrained regions. Despite these advantages, rigorous research and clinical trials remain essential to confirm the safety, efficacy, and optimal usage of repurposed drugs for CD. Furthermore, the regulatory landscape for drug repurposing is complex, with challenges related to intellectual property, regulatory approval, and commercialization (Halabi, 2018). Despite these minor limitations, whilst not the sole solution, drug repositioning represents a valuable, relatively fast, and cost-effective strategy for developing essential new therapies, particularly for NTDs such as CD.