Integral Solvent-Induced Protein Precipitation for\nTarget-Engagement Studies in Plasmodium falciparum

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