Chemoproteomics for Plasmodium Parasite Drug Target Discovery

Abstract

Emerging Plasmodium parasite drug resistance is threatening progress towards malaria control and elimination. While recent efforts in cell-based, high-throughput drug screening have produced first-in-class drugs with promising activities against different Plasmodium life cycle stages, most of these antimalarial agents have elusive mechanisms of action. Though challenging to address, target identification can provide valuable information to facilitate lead optimization and preclinical drug prioritization. Recently, proteome-wide methods for direct assessment of drug-protein interactions have emerged as powerful tools in a number of systems, including Plasmodium. In this review, we will discuss current chemoproteomic strategies that have been adapted to antimalarial drug target discovery, including affinity- and activity-based protein profiling and the energetics-based techniques thermal proteome profiling and stability of proteins from rates of oxidation. The successful application of chemoproteomics to the Plasmodium blood stage highlights the potential of these methods to link inhibitors to their molecular targets in more elusive Plasmodium life stages and intracellular pathogens in the future.

Keywords: Plasmodium; chemoproteomics; malaria; mechanism of action; target identification.

Figure 1.

Label-based chemoproteomic methods for malaria drug target identification. A) Affinity-based protein profiling (AfBPP). Plasmodium parasite lysates are incubated with bead-immobilized compounds. After washes, the ligand-binding proteins can be eluted for MS analysis. B) Activity-based protein profiling (ABPP). Enriched Plasmodium-infected RBCs or the parasite lysates are incubated with an ABPP probe in the presence or absence of drugs. The RBCs are lysed (for intact cells), and a reporter tag (e.g., biotin or fluorescent tags) can be conjugated to the reactive head group via click chemistry. Proteins covalently modified by the probe are enriched using an avidin or streptavidin column for MS analysis. Fluorescently tagged proteins can be visualized by gel electrophoresis. Proteins targeted by the drug will show a deceased signal as the ABPP probe is competed out by the compound.

Figure 2.

Energetics-based chemoproteomics to deconvolute malaria drug targets. A) Thermal proteome profiling (TPP). Enriched Plasmodium-infected RBCs or the parasite lysates are subjected to a temperature gradient in the presence or absence of drugs. The samples are lysed (for intact cells) and centrifuged to precipitate denatured protein aggregates. The soluble fractions (native proteins) are digested for quantitative bottom-up proteomics analysis and isobaric mass tag labeled for mass spectrometry. The abundance of each protein as a function of temperature can be plotted to create a melting curve, and the altered melting profile and melting temperature shift (ΔTm) indicates a drug-induced thermal stability change of the corresponding protein target. For the ITDR experiment, proteins are incubated at a fixed temperature with drugs at different concentrations. As such, the protein thermostability change can be measured in a dose-dependent manner. B) Stability of proteins from rates of oxidation (SPROX). The infected RBCs are permeabilized with saponin to remove host proteins. Enriched Plasmodium parasite lysate with and without drugs are incubated in denaturant-containing buffers (e.g., 0.5–6 M urea). After reaching the protein folding and unfolding equilibrium, the samples are treated with hydrogen peroxide, subsequently quenched, digested for quantitative bottom-up proteomics analysis and isobaric mass tag labeled for mass spectrometry. The abundance of wild-type as opposed to the oxidized methionine-containing peptide is then plotted against the denaturant concentration to generate the chemical denaturation curves and the transition midpoint shifts (ΔC_1/2) upon ligand binding.