Investigation of antimalarial activity and molecular targets of natural compounds identified by chemoinformatics and genetic analysis of Plasmodium falciparum parasites

Abstract

With approximately 229 million cases and over 400 thousand deaths reported in 2019, malaria still imposes a heavy burden upon developing countries. One of the biggest challenges in fighting malaria is the selection and expansion of parasite strains resistant to available antimalarial drugs, which has already been reported even for frontline treatments such as artemisinin-based combined therapies (ACTs). Hence, this scenario of rapid emergence of drug-resistant parasite strains calls for the urgent development of new antimalarial drugs coupled to the understanding of their modes of action. Strategies such as in vitro evolution and whole genome analysis (IVIEWGA) and P. falciparum genetic crosses in a humanized mouse model have been applied to explore genomic changes involved in genome-encoded potential drug targets and genetically map resistance-associated genes, respectively. Given the lengthy drug discovery process, we used machine learning models to predict the antimalarial activity of molecules in a natural-compound based commercial library using quantitative structure-activity relationships (QSAR). We have successfully identified a 4-aminoquinoline (LDT-623) that presents strong antimalarial activity against chloroquine-sensitive (3D7) as well as multidrug-resistant (Dd2) P. falciparum strains in vitro (EC50: 23 and 37 nM, respectively), while also showing considerable selectivity towards the parasite compared with treatment of COS-7 and HepG2 cells (Selective Index: 21.3 and 54.8, respectively). LDT-623 shares several features with chloroquine: both are fast-acting compounds in vitro and inhibit ²-hematin formation. However, we performed an in vitro transmission-blocking assay in a P. berghei model and found a critical difference between LDT-623 and other established 4-aminoquinolines: unexpectedly, we observed activity of LDT-623 against parasite sexual stages. Further, we sought to explore the combination of LDT-623 with a known gametocidal molecule, methylene blue, and found synergy between them in P. falciparum blood stages. The dual activity seen for LDT-623 - inhibition of both asexual blood stages and ookinetes - has not been widely reported for 4-aminoquinolines. For these reasons, we believe that LDT-623 shares a mode of action not exclusive to that observed for other 4-aminoquinolines. Considering the transmission-blocking activity observed with LDT-623, the goal of this BEPE Proposal is to investigate the P. falciparum molecular targets involved in the activity of LDT-623. We believe that the assays we propose have the potential to reveal new information about these targets as well as inform further investigations into the antimalarial activity of this dual-stage 4-aminoquinoline derivative. (AU)