To achieve the ultimate goal of malaria elimination, a new generation of antimalarial therapeutics is required, that possess different mechanisms of action to drugs currently in clinical use. However, the identification of molecular targets and characterisation of mechanisms of action of antimalarial drug candidates to this day represents a critical bottleneck in the antimalarial drug development pipeline.
In order to provide novel avenues for the rational design of the next generation of antimalarial therapies, we carefully assembled a comprehensive library of over two dozens structurally diverse antimalarial drug candidates with different parasite-killing profiles and undetermined mechanisms of action. To identify their respective molecular targets and downstream cellular effectors of the drug-action, we used Cellular Thermal Shift Assay (MS-CETSA)1,2. MS-CETSA employs advanced quantitative mass spectrometry to interrogate the entire parasite proteome simultaneously and identify proteins thermally-stabilised under drug treatment, suggesting direct protein-ligand interaction. To maximise the information output, the MS-CETSA target engagement profiling was carried out for each drug in a multidimensional format including 10 compound concentrations, 4 thermal challenge temperatures and two sample types; assessing drug-protein interactions within intact Plasmodium falciparum infected red blood cells, as well as in the parasite lysate.
To further support MS-CETSA results, in parallel we carried out RNAseq-based drug-dose response transcriptional perturbation profiling for the assembled compound library, demonstrating highly specific transcriptional responses induced by different compounds. The molecular targets and drug mechanisms of action identified here provide novel directions for the development of future antimalarial intervention strategies and shed new light malaria parasite biology.