Malaria causes extensive morbidity and mortality in many of the world’s poorest counties, and the rapid emergence of resistance to current antimalarials underscores a critical need for new antimalarial agents. The 2-Aminobenzimidazole (ABI) class has emerged as a promising antimalarial chemotype, exhibiting potent activity against drug-resistant malaria parasites with low nanomolar IC50 values. To elucidate the molecular targets of ABI, we employed advanced proteomics-based target deconvolution strategies using the Orbitrap Astral mass spectrometer. Specifically, we applied Solvent Profiling Proteomics (SPP) and Limited Proteolysis–Mass Spectrometry (LiP-MS) to detect drug–protein interactions by examining ligand-induced structural changes in proteins. These studies were conducted using both first-generation (MIPS-2862) and second-generation (MIPS-3459) ABI compounds.
In two independent SPP experiments, over 3,000 parasite proteins were identified. Analysis of their denaturation curves revealed 10 proteins consistently stabilised in the presence of ABIs compared to controls—primarily proteins linked to translation and protein folding, including multiple 60S ribosomal proteins and protein disulfide isomerase (PF3D7_0827900). Complementary LiP-MS analysis quantified over 70,000 peptides from more than 2,800 parasite proteins, and differential profiling of fully tryptic and half-tryptic peptides identified 29 shared potential targets of both ABI compounds. Notably, among these targets were the 60S acidic ribosomal protein P2 (PF3D7_0309600) and protein disulfide isomerase (PF3D7_0827900). These results suggest that ABI compounds interfere with the parasite’s protein translation and folding machinery.
These findings are further supported by untargeted proteomics performed on ABI-treated parasite cultures, which identified over 3,000 parasite proteins. Among these, 100 proteins showed significant changes in abundance, including 60S ribosomal proteins L12 (PF3D7_0517000) and L28 (PF3D7_1142500) previously implicated in the SPP experiments. Future research will focus on validating the impact of ABI’s on protein translation, with the ultimate goal of designing novel antimalarials that act via unique mechanisms of action.