Malaria is a parasitic disease that continues to be a global health burden. Drug resistance and the lack of mechanistic diversity in approved and emerging antimalarials are significant issues facing efforts to control and eradicate the disease. A major barrier to the development of new antimalarial candidates with novel mechanisms of action is drug target deconvolution. To address this, we have established a mass spectrometry-based metabolomics and proteomics pipeline to understand drug mechanisms on a system-wide scale. This includes metabolomics, to provide an unbiased assessment of the impact of compounds on parasite metabolism, and thermal stability proteomics and limited proteolysis coupled mass spectrometry (LiP-MS), to map drug-protein interactions in complex proteomes.
As an example, we previously discovered inhibitors of the malaria parasite M1 and M17 metalloaminopeptidases. Biochemical assays revealed that one of these compounds, MIPS2673, potently inhibits recombinant parasite M1 metalloaminopeptidase, with excellent selectivity over other parasite aminopeptidases. To validate that MIPS2673 selectively engages M1 within parasites, we applied our unbiased drug target deconvolution methods. Both thermal stability proteomics and LiP-MS analyses reproducibly identified M1 as the sole target of MIPS2673 from approximately 2,000 detected proteins. Furthermore, we found that structurally perturbed M1 peptides identified with LiP-MS were located in very close proximity to the ligand binding site. This peptide data enabled estimation of the binding site on M1 to within ~5 Å of that determined by X-ray crystallography, as well as mapping of functionally important drug interaction sites on the M1 protein with peptide-level resolution. Our functional investigation by untargeted metabolomics revealed significant accumulation of short peptides in treated parasites. The profile of elevated peptides (enriched basic peptides) was consistent with specific inhibition of M1 and disruption of its key role in parasite haemoglobin digestion. Combined, our target deconvolution strategies provided unbiased confirmation of the on-target activity of a novel malaria parasite M1 aminopeptidase inhibitor.
Our study demonstrates the power of MS-based techniques to simultaneously probe whole proteomes in an unbiased manner and identify direct drug-protein interactions. In an effort to expand the antimalarial drug target landscape and dive deeper into the malaria parasite druggable proteome we are combining our target deconvolution pipeline with the new Orbitrap Astral MS to map ligand-protein interactions of other promising antimalarial candidates with unknown modes of action.