Final Report

1. Project objective

This project aimed to screen approximately 200,000 compounds from the Drug Discovery Initiative (DDI) library, The University of Tokyo, against the asexual blood stage of Plasmodium falciparum. The goal of the project was to identify candidate compounds for further profiling needed for hit-to-lead development. The ultimate long-term goal is to deliver new antimalarial drugs with new chemical scaffold and the mechanism of action.

2. Project design

Screening of the DDI library was conducted in six phases. The primary screening was carried out against the blood stage of P. falciparum 3D7 drug-sensitive strain, based on the measurement of its lactate dehydrogenase (LDH) activity at a single final concentration (2 mM) of the compounds. This was followed by a secondary screening (SYBR Green) in order to eliminate false positive hits. For the tertiary screening, the remaining hits were tested against yeast dihydroorotate dehydrogenase (y-DHODH)-overexpressing 3D7 strain to exclude compounds targeting the mitochondrial electron transport chain. As a quaternary screening, Dd2 chloroquine and sulfadoxine-resistant strain was used. To further narrow down the quaternary hits, evaluation of structural novelty was performed by searching the hits against the malaria drug fragments of known anti-malarials and the Medicine of Malaria Venture (MMV) database. Finally, selectivity and safety were evaluated using human liver cancer line (HepG2). The whole screening was carried out at The University of Tokyo, whereas structure-related analyses were conducted by MMV.

3. Results, lessons learned

We successfully completed the screening of over 200,000 DDI compounds and identified hits that showed good potency against the asexual blood stage of Plasmodium falciparum. We selected compounds that have IC₅₀ < 2 mM in 3D7 strain, inhibited 70% growth of y-DHODH overexpressing strain and Dd2 strain at 2 mM. After removing compounds that contain fragments from the known antimalarials or those with similarity score higher than 0.7 with the MMV database, 105 compounds were selected. Furthermore, the compounds showing the selectivity index (the ratio of the IC₅₀ value against HepG2 cells over the IC₅₀ value against 3D7) of <10 were removed, a total of 82 hit compounds were finally selected. Further profiling of the physicochemical properties (lipophilicity, physiological solubility), ADME analysis, hit deconvolution using a resistant panel, are needed to prioritize compounds for further exploitation in the hit-to-lead development. During and after the project completion, such profiling was conducted to identify 12 hits that show reasonable physicochemical properties, initial ADME have a good probability to be further developed as hit-to-lead candidates in the R&D pipeline.