Introduction and Background of the Project

1. Introduction

Chagas also known as American trypanosomiasis is a live-threatening infectious disease caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), affecting more than 10 million of people, mainly in Latin America. However, due to human migration, this disease can be found in Europe, Oceania and also in Japan. The disease is primarily transmitted by the triatomine bugs’ faeces excreted during blood feeding. The disease can also be transmitted by contaminated blood transfusion, organ transplantation, and from mother to child during childbirth and breastfeeding. Symptoms of the initial phase of the Chagas disease include flu-like symptoms and it could be unnoticed, however if not treated, it can progress to a chronic stage where it leads to serious heart and gastrointestinal complications and eventually death.

2. Project objective

The main objective of this research project is to identify novel and potent antichagasic compounds from selected chemical library subsets from the Center for Supporting Drug Discovery and Life Science Research, Osaka University that meets the GHIT/DNDi criteria (1).

3. Project design

A total of seven chemical library subsets from the Center for Supporting Drug Discovery and Life Science Research, Osaka University (Osaka Library, total number 62,029 compounds) will be tested in a cell-based high-throughput screening system against intracellular amastigote stage of a T. cruzi strain that were genetically engineered to express the firefly luciferase gene. The cytotoxicity against human cell lines as well as the biological activity against four major T. cruzi strains of the primary hits will be evaluated.

How can your partnership (project) address global health challenges?

Chagas is one of the neglected tropical diseases that historically received limited attention and funding for drug development. Despite the discovery of Chagas disease since 1909, still there are only two medications (benznidazole and nifurtimox) for the treatment of the disease with limited efficacy in the chronic stage, and few compounds in the developmental pipeline. We aim to discover novel hits series with pertinent mechanisms of action that would potentially be developed into novel effective and safe drugs to combat Chagas disease.

What sort of innovation are you bringing in your project?

In the past, one of the challenges in Chagas drug discovery field was the lack of sensitive drug screening tools. Here we took advantages of the recent advance in the genetic engineering of T. cruzi parasite and established transgenic T. cruzi parasites (four genetically different reference strains) expressing luciferase which will facilitate rapid and accurate evaluation of active compounds in cell-based assay systems. Additionally the screening libraries from Osaka University which will be evaluated in this project have not been assayed against T. cruzi parasite, the causative agent responsible for Chagas disease, and we therefore anticipate novel antichagasic series will be identified from this collaboration.

Role and Responsibility of Each Partner

To ensure effective project management, and successful outcomes, a screening collaboration agreement has been established between Nagasaki University (NU) and Drugs for Neglected Diseases initiative (DNDi), where at NU, Prof. Daniel Inaoka’s group will conduct the HTS, which has been already established and validated, as well as data analysis and interpretation, report writing and collaboration with other partners of the joint research. DNDi will provide coordination and support to initiate the project as well as, data analysis and hit prioritization support to the project.

Others (including references if necessary)

(1) https://dndi.org/scientific-articles/2015/hit-lead-infectious-diseases-nature-2015

Final Report

1. Project objective

The objective of this project is to identify and validate novel chemical starting points for drug discovery against Trypanosoma cruzi, the causative agent of Chagas disease, a neglected tropical disease with currently limited and suboptimal treatment options. By applying high-throughput screening (HTS) to diverse compound libraries, the project aims to discover potent, selective, and non-cytotoxic hits suitable for progression into hit-to-lead development.

2. Project design

The project follows a staged HTS workflow. After acquisition of compound libraries and optimization of the primary screening assay, a comprehensive HTS campaign was conducted to identify primary hits. Due to delays in compound dispensing and limited library resources, the screening strategy was adapted to include a one-dose cytotoxicity assay (10 μM) in HeLa cells to prioritize non-toxic compounds. Compounds passing this filter were progressed to dose–response testing across multiple T. cruzi strains, with assay performance monitored using established reference controls.

3. Results, lessons learned

A total of 1,986 primary hits were initially identified from the screening of 63,680 compounds sourced by the Osaka University (OU) compound library,. To ensure selection of compounds with acceptable safety profiles, a single-dose cytotoxicity filter was applied, resulting in 860 non-cytotoxic compounds being advanced to dose–response testing against four Trypanosoma cruzi strains: CL Brener, Sylvio-X10, Tulahuen, and Esmeraldo. Assay robustness and reproducibility were confirmed across all experiments using benznidazole and posaconazole as reference compounds, with consistent EC₅₀ values observed for each T. cruzi strain.

Dose–response data were analyzed using a standard EC₅₀ cutoff of ≤10 µM across all four strains. Following this multi-strain evaluation, 403 compounds remained active against all T. cruzi strains, corresponding to an overall hit rate of 0.63% from the 63,680 compounds screened. This stringent filtering strategy was essential to exclude any strain-specific or weakly active compounds and to prioritize broadly active antitrypanosomal candidates for further development.

Chemical structures of the 403 validated hits were obtained from the OU library and integrated with phenotypic potency and cytotoxicity data. The curated dataset was subsequently shared with DNDi for clustering, comparative analysis, and expert-driven prioritization. Concurrently, confirmatory assays were conducted to identify any compounds interfering with the luciferase-2 readout used in the phenotypic assay. This step led to the exclusion of 14 compounds, corresponding to a low false-positive rate of approximately 3.5%.

Based on drug-like properties, structural novelty, and overlap with previous DNDi screening programs, a final set of 15 priority hit compounds was selected. These hits cover seven distinct chemical classes, including four multi-member series and three singletons, providing both chemical diversity and opportunities for structure–activity relationship exploration.

All planned screening, evaluation, and reporting activities have now been completed, and the project has successfully achieved its primary objective of identifying validated hit compounds for Chagas disease. The program has advanced to the next phase, with compound plates for the selected hits requested for follow-up testing using the newly developed High-Throughput Rate-of-Kill (HT-RoK) assay. This assay, performed using co-funding support, enabled the identification of one compound cluster of three compounds that demonstrated consistently cidal intracellular amastigote killing activity.