Final Report

1. Project objective

The objective of this project is to screen a structurally defined compound library from the Drug Discovery Initiative (DDI) against Leishmania donovani extracellular amastigotes to identify progressable hit series that fulfill GHIT/DNDi criteria. This is to our knowledge first-of-its-kind high throughput screening systems established in Japan to screen against Leishmania. In addition to delivering hits for further optimization and mechanisms of action exploration, this project also supports capacity building in anti-leishmanial drug screening, validation, and advancing Leishmania biology in Japan, fostering regional expertise and collaboration toward new drug discovery against leishmaniasis.

2. Project design

We screened over 210 thousand compounds from the Drug Discovery Initiative (DDI) library at The University of Tokyo to identify hits against Leishmania donovani axenic amastigotes. The criteria of the primary hits were defined as showing >50% growth inhibition at 2 μM against extracellular amastigotes and lacking cytotoxicity against HepG2 and THP-1 cells at 10 µM, leading to 593 confirmed hits with acceptable toxicity profile. Further testing using THP-1-derived macrophages followed by manual inspection of the novelty and druglikeness of the hits allowed prioritization. Eighty eight commercially available analogues of three most promising chemical series were selected and reevaluated for activity in axenic and intracellular amastigote assays at the Swiss Tropical Public Health Institute and The University of Tokyo for potential lead identification.

3. Results, lessons learned

Results
Approximately 210 thousand compounds from the Drug Discovery Initiative (DDI) at The University of Tokyo were screened by high throughput screening to identify hits against Leishmania donovani extracellular amastigotes. At 10 μM, 6,810 compounds showed >75% inhibition of parasite growth in the axenic amastigote assay. Those primary hits were reevaluated at 2 μM, narrowing down to 2,088 compounds showing >50% inhibition, representing about 1% of the total library. Exclusion of the compounds that showed cytotoxicity to human HepG2 and THP-1 cell lines, 593 compounds were found to be safe. Further testing by the intra-macrophage amastigote assay narrowed the hits to 11 compounds. After manual inspection, 7 compounds with new chemical groups were selected for further analysis. We purchased 88 analogues related to three major scaffolds. These analogues were tested at both the Swiss Tropical and Public Health Institute (STPH) and The University of Tokyo for efficacy against intracellular amastigotes. Unfortunately, none of the compounds showed high activity against intracellular amastigotes that meets the GHIT/DNDi criteria for bringing hits to the hit-to-lead development stage.

Lessons Learned

Poor translation of activity from the axenic into the intracellular Leishmania donovani assays

The lack of robust translatability of qualified Leishmania donivani extracellular axenic hits to the intra-macrophage hits was observed. The reasons for this poor translation could be explained by different hypotheses (e.g. low permeability of the compounds, chemical or metabolic instability, cellular efflux, compound identity and purity, various expression levels of protein targets between 2 assay systems). Although the issue was not further investigated, our finding suggests the need for a model that better mimics intracellular environments earlier in the pipeline.

Collaboration: Independent confirmatory testing at The University of Tokyo and STPH reinforced cross-validation to ensure reproducibility. The project fostered strong collaboration between only a single Japanese institution that can perform Leishmania donivani axenic and intracellular amastigote assays and foreign institutions, which can be exploited in future drug discovery campaigns.