The Impact of Activity-Based Protein Profiling in Malaria Drug Discovery

Abstract

Activity-based protein profiling (ABPP) is an approach used at the interface of chemical biology and proteomics that uses small molecular probes to provide dynamic fingerprints of enzymatic activity in complex proteomes. Malaria is a disease caused by Plasmodium parasites with a significant death burden and for which new therapies are actively being sought. Here, we compile the main achievements from ABPP studies in malaria and highlight the probes used and the different downstream platforms for data analysis. ABPP has excelled at studying Plasmodium cysteine proteases and serine hydrolase families, the targeting of the proteasome and metabolic pathways, and in the deconvolution of targets and mechanisms of known antimalarials. Despite the major impact in the field, many antimalarials and enzymatic families in Plasmodium remain to be studied, which suggests ABPP will be an evergreen technique in the field.

Keywords: Drug Discovery; Fluorescent probes; Malaria; Mass spectrometry; Proteomics.

Figure 1

Activity‐based Protein Profiling. A) Activity‐based Probe general structure. B) General ABPP experiment where proteomes are labeled with an ABP and downstream analysis by in‐gel fluorescence reading or LC‐MS/MS. C) Competitive ABPP. Proteomes are pre‐treated with a competitor molecule or DMSO and then general proteome labeling is performed in both samples with a broad reactivity ABP. Comparison between both samples identifies labeling events where the competitor prevented probe labeling, meaning the competitor is bound to the active site of the target.

Figure 2

Malaria life cycle. 1) An infected female Anopheles mosquito infects the host with sporozoites, which move into the blood and home into liver hepatocytes. 2) The parasite multiplies within hepatocytes until cell lysis releases merozoites in the bloodstream. 3) The parasites infect erythrocytes and develop into ring‐stage parasites. 4) The early trophozoites develop into late trophozoites. 5) Trophozoite gradually develops into a fully mature schizont. Erythrocytes rupture, releasing the parasite into the bloodstream. 6) Some parasites differentiate into female and male gametocytes. 7) Mosquitos are infected upon taking a blood meal from an infected host.

Figure 3

Cysteine protease targeting probes. A) I¹²⁵‐DCG04, a radioactive cysteine protease reactive probe selective for clan CA/papain family cysteine proteases based on the natural product E‐64. B) FY01, a cysteine protease reactive probe selective for DPAPs. C−D) Examples of the vinyl sulfone probes developed by Tan et al., including cy dyes (C, cy dye represented as “tag”), and a trifunctional probe (D, TAMRA tag and desthiobiotin tag).

Figure 4

Serine hydrolase targeting reactive groups and probes. A) Model isocoumarin‐ and phosphonate‐based compounds used by Arastu‐Kapur et al. to identify serine hydrolases associated with erythrocyte rupture. B) JCP‐01, a biotinylated chloroisocoumarin compound that emerged as the optimal serine hydrolase hit in the rupture assay screening performed by Arastu‐Kapur et al. C) Example of FP probe used by Elahi et al. to target serine hydrolases.

Figure 5

A) Ubiquitin probe developed by Artavanis‐Tsakonas et al. B) PR3 proteasome inhibitor. C) Proteasome ABP developed by Li and co‐workers to target al.l three catalytic sites of the Plasmodium proteasome.

Figure 6

Affinity‐based Probe (AfBP) and Affinity‐based Protein Profiling. In this ABPP variation, the ligand/reactive group does not directly react with the target protein, but rather promotes proximity between probe and target. A photo‐crosslinker is included in the AfBP and, upon UV irradiation, forms a non‐specific, irreversible covalent bond with a neighboring amino acid, thus allowing enzymes that do not react by covalent bond formation to be addressed by ABPP.

Figure 7

A) AfBP for plasmepsin targeting developed by Liu et al., including a hydroxyethyl‐based warhead (red), a rhodamine tag (blue) and a benzophenone photo‐crosslinker (green). B) MH01 AfBP used by Harbut and colleagues based on the bestatin scaffold (red), containing a biotin tag (blue) and a benzophenone photo‐crosslinker (green).

Figure 8

Endoperoxides studied by ABPP. A) Examples of endoperoxide ABPs developed by Stocks et al. B) Alkynylated artemisinin probe developed by Wang et al. C) Example of alkyne and azide artemisinin derivatives used by Ismail et al. D) Alkynylated artemisinin ABP developed by Zhang et al. containing an A triphenylphosphoniumbromide moiety to direct the probe to the mitochondria. E) Ozonide compounds studied by Giannangelo et al. using competitive ABPP.

Figure 9

General ABPP experiments for deconvolution of the mechanisms of artemisinin. A. Artemisinin‐alkyne probes are incubated with parasite‐infected cultures or other relevant Plasmodium proteomes. Iron‐mediated activation of the endoperoxide bridge generates radicals, which have been shown to be mostly secondary radicals. Extensive protein alkylation affects multiple essential pathways, overwhelms the parasite's protein repair systems, and leads to parasite death. B) Alkylated proteins were enriched with streptavidin after appending biotin to the probe‐protein complexes by click chemistry. Trypsinization of enriched proteins, followed by LC‐MS/MS analysis identified the alkylated proteins, which participate in essential parasite functions like antioxidant defense, the glycolytic pathway, DNA and protein synthesis, among others.

Figure 10

Antimalarials studied by ABPP and their respective ABPs. A) Symplostatin 4. B) Albitiazolium. C) BIX‐01294. D) Salinipostin A.

Figure 11

A) Example of an azaglycine O‐acylhydroxamate compound developed by Verhelst et al. B) Model compound incorporated in the ABPP microarray developed by Wu et al. C) Carbamate‐linked hybrid molecule developed by Mahajan et al. and general mechanism of the fragmenting hybrid approach developed by the authors for drug release. D) YnMyr. Myristate surrogate probe used by Wright et al. to study targeting of N‐myristoylation in P. falciparum. E) Bicyclononyne probe used by Schipper et al. to study sulfenylation in P. falciparum. F) Example of plasmodione‐based ABP developed by Cichocki et al. and one of the mechanisms of activation, highlighting the benzophenone‐like photoreactive group (green).