Chemoproteomics validates selective targeting of <i>Plasmodium</i> M1 alanyl aminopeptidase as an antimalarial strategy.

Abstract

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of <i>Plasmodium</i> M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant <i>Plasmodium falciparum</i> (<i>Pf</i>A-M1) and <i>Plasmodium vivax</i> (<i>Pv</i>A-M1) M1 metalloaminopeptidases, with selectivity over other <i>Plasmodium</i> and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets <i>Pf</i>A-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on <i>Pf</i>A-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of <i>Pf</i>A-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.