Integral Solvent-Induced Protein Precipitation for Target-Engagement Studies in <i>Plasmodium falciparum</i>

The limited understanding of the mechanism of action (MoA) of several antimalarials and the rise of drug resistance toward existing malaria therapies emphasizes the need for new strategies to uncover the molecular target of compounds in <i>Plasmodium falciparum</i>. Integral solvent-induced protein precipitation (iSPP) is a quantitative mass spectrometry-based (LC-MS/MS) proteomics technique. The iSPP leverages the change in solvent-induced denaturation of the drug-bound protein relative to its unbound state, allowing identification of the direct drug-protein target without the need to modify the drug. Here, we demonstrate proof-of-concept of iSPP in <i>P. falciparum</i> (Pf) lysate. At first, we profiled the solvent-induced denaturation behavior of the Pf proteome, generating denaturation curves and determining the melting concentration (<i>C</i>_M) of 2712 proteins. We then assessed the extent of stabilization of three antimalarial target proteins in multiple organic solvent gradients, allowing for a rational selection of an optimal solvent gradient. Subsequently, we validated iSPP by successfully showing target-engagement of several standard antimalarials. The iSPP assay allows the testing of multiple conditions within reasonable LC-MS/MS measurement time. Furthermore, it requires a minimal amount of protein input, reducing culturing time and simplifying protein extraction. We envision that iSPP will be useful as a complementary tool for MoA studies for next-generation antimalarials.