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

Abstract

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 (C_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.

Keywords: antimalarial; drug discovery; iSPP; proteomics; solvents; target-engagement.

Figure 1

Schematic workflow of iSPP in Plasmodium falciparum (created with BioRender.com).

Figure 2

Solvent profiling of the Plasmodium falciparum proteome. (A) Denaturation curve of the P. falciparum proteome. For each data point, the median value among all quantified proteins is shown. The fold change of the relative abundance was calculated relative to 0% A.E.A. The curve was obtained for proteins with high-quality fitting curves (2712; R² ≥ 0.8, Plateau < 0.3, C_M ≥ 4). The inset shows the calculated C_M, R², Hillslope and bottom plateau; (B) distribution profiles of high confidence C_M values calculated in P. falciparum and Escherichia coli proteomes. (C) Correlation between C_M values (y-axis) vs protein abundance (x-axis, log₁₀ intensity), displaying a weak relationship between the two variables (r = 0.26); (D) heatmap representation of all proteins quantified (n = 3492). For each protein, the relative abundance (fold-change) was calculated relative to 0% A.E.A. Protein fold-changes were then clustered.

Figure 3

Solvent gradient has an impact on the extent of stabilization for each expected target (A) schematic figure of the iSPP solvent gradients used and the different increments (i) 1.5% A.E.A, 2% A.E.A, and 3% A.E.A. (v/v) (B) log₂FC for the expected targets across solvent gradient used. Red inline displays the threshold log₂FC = 0.3.

Figure 4

The solvent gradient 14–28% stabilized the majority of the expected protein targets with the least nonspecific proteins (de)stabilized. P. falciparum lysates were incubated with vehicle control or 50 μM of pyrimethamine, DSM265, and fosmidomycin (mixed drug approach). Lysates were then exposed to either of the five % A.E.A. gradient v/v: (A) 11–25%, (B) 12–33%, the broadest gradient which resulted in the most (de)stabilization of unexpected proteins, (C) 14–28%, (D) 16–26.5%, the narrowest increment and (E) 17–31%. Data is shown as volcano plots where the threshold criteria for identification of proteins exhibiting statistically significant changes in response to the compound treatment was set to a log₂ fold change (log₂FC) > |0.3| and p-value < 0.05. Red shows stabilized proteins with log₂FC > 0.3 and p-value < 0.05; Blue shows destabilized proteins with log₂FC < −0.3 and p-value < 0.05; gray shows proteins with −0.3 < log₂FC < 0.3 and p-value < 0.05 and proteins with p-value > 0.05.

Figure 5

The iSPP assay in Plasmodium falciparum was able to identify the majority of the expected targets of the standard antimalarials. P. falciparum lysates were incubated with vehicle control or 100 μM of the compounds. The lysates were then exposed to 14–28% A.E.A. gradient (v/v). The threshold criteria for identification of proteins exhibiting statistically significant changes in response to the compound treatment was set to a log₂ fold change (log₂FC) > |0.3| and p-value < 0.05. Data is shown as volcano plots that highlights changes in abundance vs statistical significance for treatment with compounds (A) pyrimethamine (B) DSM265 (C) fosmidomycin (D) MMV390048, (E) sulfadoxine, and (F) Mefloquine. Red shows stabilized proteins with log₂FC > 0.3 and p-value <0.05; Blue shows destabilized proteins with log₂FC < −0.3 and p-value < 0.05; gray shows proteins with −0.3 < log₂FC < 0.3 and p-value < 0.05 and proteins with p-value > 0.05.