Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as a cross-species strategy to treat malaria

All current treatments for malaria are threatened by drug resistance, and new drug candidates that act via novel mechanisms are urgently needed. Here, we describe MIPS2673, an inhibitor of the Plasmodium M1 alanyl metalloaminopeptidase, which displays excellent in vitro antimalarial activity with no significant host cell toxicity. Biochemical assays revealed potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (Pv-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases. Orthogonal chemoproteomic methods based on thermal stability and limited proteolysis reproducibly identified PfA-M1 as the sole target of MIPS2673 in parasites from approximately 2,000 detected proteins. Furthermore, the limited proteolysis approach enabled estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Functional investigation by untargeted metabolomics further demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our unbiased target deconvolution strategies confirmed the on-target activity of a PfA-M1 inhibitor, and validated selective inhibition of this enzyme as a promising multi-stage and cross-species antimalarial strategy.