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

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 Plasmodium falciparum. 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 P. falciparum (Pf) lysate. At first, we profiled
the solvent-induced denaturation behavior of the Pf proteome, generating
denaturation curves and determining the melting concentration (CM) 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.