Autophagy as a novel drug-development target for Chagas disease

Introduction and Background of the Project

1. Introduction

This project is a target-directed drug discovery project for Chagas disease. It is based on the outcome of previous research funded through the Target Research Platform (TRP, 2018-2019) where we conducted CRISPR/Cas9-aided exhaustive knockouts on over 500 Trypanosoma cruzi (T. cruzi, the causative protozoan of this disease) genes and identified 25 indispensable molecules for the parasite proliferation. Each of these 25 molecules was carefully examined for its potential as drug targets for Chagas disease, i.e., we investigated whether host cell-derived metabolites in the amastigote (the proliferating stage of this parasite in animals) will rescue dysfunction of the target molecules and whether the structural features of the active sites are suitable for discovery of T. cruzi-specific drugs. We finally selected an autophagy-regulating factor as a potential target of interest for this Chagas disease drug discovery project.


2. Project objective

The overall goal of this project is to obtain initial hit compounds amenable for further development as novel anti-T. cruzi drugs acting through inhibition of the parasite autophagy-regulating factor.


3. Project design

To achieve the goals of the project, we use two different but complementary screening approaches, a Fragment-Based Drug Discovery (FBDD) approach and a “classical” screening of an anti-T. cruzi compound library (the DNDi library) against the autophagy-regulating factor target. For the FBDD approach, we employ the DNA-encoded library (DEL) technology, where two fragments are intended to bind simultaneously to the protein in the same vicinity. On the other hand, the DNDi library consists of compounds already confirmed to have promising intracellular anti-T. cruzi activity in whole cell-based assays. In this project, we will require that both the identified hits and the target protein be druggable. We consider that the ideal initial hit compound should not only bind to the drug target and inhibit enzyme activity, but also have a protein-compound binding state that will facilitate subsequent modification during Hit-To-Lead and Lead Optimization steps. Since this requires a structural understanding of the compound-protein interactions, determination of the three-dimensional structures of the hit compound-target molecule complexes will be a critical part of this project, as well. Notably, hit compounds obtained from the FBDD approach will be initially selected based only on their target-binding and enzyme inhibition activity independently of their pharmacological activities. The identified compounds must have the potential to evolve and be optimized during the subsequent development stages to acquire the required properties.

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

Among the 8 Neglected Tropical Diseases (NTDs) which are targeted for elimination as a public health problem in the new WHO road map for NTDs, Chagas disease is still suffering from a relatively thin and fragile R&D portfolio. Our project will contribute to strengthening the current R&D portfolio for this disease. Under the previous GHIT TRP project, an exhaustive search for vital molecules addressing T. cruzi proliferation was undertaken and 25 indispensable molecules were identified. To take advantage of this outcome as a new starting point for Chagas disease drug discovery, in this project we aim at obtain initial hit compounds binding to the most promising drug target and pathway among our previously identified candidates. The novelty of the selected target has the potential to offer a breakthrough in the field of target validation as well as lead generation for Chagas disease.

What sort of innovation are you bringing in your project?

1) Reliability in the drug target selection

One of the main difficulties in attempting target-directed drug discovery approaches in Chagas disease is the lack of information on molecules vital to the survival and proliferation of the parasite T. cruzi. Genetic approaches to target validation have met significant obstacles: RNA interference does not work in this protozoan because it has no DICER activity, and direct genetic manipulations of amastigotes are quite difficult due to the intracellular nature of this parasite stage. In the previous TRP project, we performed an exhaustive gene knockout in protozoa, as described above. This was based on the construction of a CRISPR/Cas9-aided high-throughput gene knockout system in T. cruzi, and direct genetic manipulation of cultured extracellular amastigotes (Refs. 1 & 2). We believe that the potential of the present drug target is highly reliable based on our original technology and previous results.

2) Reliability in hit determination

An initial hit compound should have the potential to withstand the long subsequent drug development processes. In this project, we will determine the structural binding mode between the compounds and protein target by X-ray crystallography and validate whether the compounds/target are amenable to drug development.

Role and Responsibility of Each Partner

Close communication between the two institutions of this project will be critical for the management of this project. As the lead institution, AIST will be responsible for the overall progress of this project and is in charge of most of the experiments. DNDi will supply its compound library as well as apply its medicinal expertise to compound design and evaluation of hit compounds. DNDi is familiar with the global landscape of Chagas disease drug development and will ensure that this project contributes productively to progress drug discovery for Chagas disease at a global level.

Others (including references if necessary)


1. Takagi Y., Akutsu Y., Doi M., Furukawa K. Utilization of proliferable extracellular amastigotes for transient gene expression, drug sensitivity assay, and CRISPR/Cas9-mediated gene knockout in Trypanosoma cruzi. PLoS Negl Trop Dis. 2019 Jan 14;13(1):e0007088.

2. Akutsu Y., Doi M., Furukawa K., Takagi Y. Introducing a Gene Knockout Directly into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System. J Vis Exp. 2019 Jul 31;(149). doi: 10.3791/59962.