Chemo-proteomics in antimalarial target identification and engagement

Abstract

Humans have lived in tenuous battle with malaria over millennia. Today, while much of the world is free of the disease, areas of South America, Asia, and Africa still wage this war with substantial impacts on their social and economic development. The threat of widespread resistance to all currently available antimalarial therapies continues to raise concern. Therefore, it is imperative that novel antimalarial chemotypes be developed to populate the pipeline going forward. Phenotypic screening has been responsible for the majority of the new chemotypes emerging in the past few decades. However, this can result in limited information on the molecular target of these compounds which may serve as an unknown variable complicating their progression into clinical development. Target identification and validation is a process that incorporates techniques from a range of different disciplines. Chemical biology and more specifically chemo-proteomics have been heavily utilized for this purpose. This review provides an in-depth summary of the application of chemo-proteomics in antimalarial development. Here we focus particularly on the methodology, practicalities, merits, and limitations of designing these experiments. Together this provides learnings on the future use of chemo-proteomics in antimalarial development.

Keywords: antimalarial; chemical probe; malaria; target engagement; target identification.

Figure 1

Structures and workflow of chemical probes used for target deconvolution. A range of chemical probe types can be employed for target elucidation, including resin immobilized probes, biotin‐streptavidin‐linked probes, fluorescent tag‐linked probes, and finally, probes with click chemistry and photoreactive tags. Chemical probes are constructed by linking the drug moiety to a solid support resin. The cellular lysate is applied to the resin to identify binding proteins. Rigorous washing steps reduce the levels of nonspecific, leaving only high‐affinity binders attached to the resin. The proteins are separated by SDS‐PAGE and are characterized either by western blot analysis or mass spectrometry. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Common bioorthogonal reactions used in the construction of chemical probes. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Common photoaffinity ligands. Benzophenones, diazirines, and aryl azides generate highly reactive species upon excitation with UV light which facilitate photocrosslinking to adjacent proteins when included in a probe structure.

Figure 4

Resin immobilized probes of the known antimalarials primaquine and hydroxychloroquine for the identification of cellular targets. Pulldown of the resin immobilized probes in infected erythrocyte lysate resulted in the enrichment of two human proteins, ALDH1 and QR2. Biochemical validation confirmed QR2 as a probable target and indicated that inhibition of ALDH1 may be the result of an off‐target effect. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

A resin immobilized chemical probe of MMV048 used in the identification of Plasmodium falciparum cellular targets. An active analog of MMV048 with an amine functionality was chosen to link to the Sepharose resin. Phosphatidylinositol 4‐kinase (PI4K) was identified as a probable target, confirmed with competition experiments with MMV048 and subsequent in vitro resistance evolution. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

Summary of the human mTOR inhibitor Torin 2 P. falciparum activities and AfBPP design. An equipotent and structurally related compound WWH030 was used to construct a chemical probe for Torin 2 as it possessed a suitable handle. The negative control was constructed from the significantly less active Torin 2. Pulldown in P. falciparum gametocytes revealed putative targets, including phosphoribosyl pyrophosphate synthetase, aspartate carbamoyltransferase, ATCase, and (PF3D7_0914700). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 7

A resin immobilized chemical probe used in the identification of Plasmodium falciparum targets of the human cyclin‐dependent kinase 2 (CDK2) inhibitor purvalanol B. Purvalanol B and related inactive controls were immobilized via a PEG linker to an agarose resin for target identification in P. falciparum. Pulldown identified only one potential target, P. falciparum casein kinase 1 (PfCK1). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 8

A resin immobilized chemical probe for validation of the cellular targets of purfalcamine. Pulldown with this probe identified several proteins with PfCDPK1 as the likely candidate. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 9

Imidazopyridazine compounds identified using a target‐based screen against PfCDPK1. Two classes of imidazopyridazine compounds were identified, differing in their aromatic linker. Class 1 imidazopyridazines possessed a pyrimidine linker and arrested parasites at the late schizont stage. Class 2 imidazopyridazines are linked via nonpyrimidine aromatic rings and arrest at the trophozoite stage. Biotinylation of compound D enabled streptavidin affinity pulldown for the identification of cellular targets. The probe identified the molecular chaperone PfHSP90 as a potential target for the compound. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 10

SPAAC probes used in the target validation of WM382 against plasmepsin X. An azide functionalized derivative of the lead compound (WM‐853) was used to attach the compound of interest to a Sepharose resin using SPAAC copper‐free conditions. These probes were incubated with lysate from an HA‐tagged PMX parasite line where PMX was identified as a binder by western blot with an anti‐HA antibody. Pulldown of PMX was competitively inhibited by the addition of the parent compound WM382. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 11

A resin immobilized chemical probe for the novel endoperoxide N‐251. The novel endoperoxide N‐251 was linked to an azlactone Sepharose resin via a lysine linker to create the probe N‐346. Treatment with cellular lysate resulted in the enrichment of PfERC, Pf14‐3‐3, and PfHSP70. Weak binding of N‐251 and N‐89 to PfERC was confirmed subsequently by surface plasmon resonance (SPR). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 12

Artemisinin‐based click chemistry probes. (A) Alkyne and azide click chemistry probes by Hemingway and Ward et al. identified 42 common proteins in an affinity pulldown, with a majority containing a glutathione binding motif which may be particularly susceptible to radical alkylation. When P1 was retested by Maser et al. with additional controls far fewer proteins were pulled down, none of which were found in the original study. (B) Alkyne probe AP1 pulled down 125 high‐confidence proteins with similar pathway coverage to the P1 and P2 probes. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 13

Structures of ozonide click‐chemistry probes TP1 and TP2 and their inactive nonperoxidic control compounds CTP1 and CPT2 synthesized by O'Neill et al. Probes based on an alkyne handle (above) were optimized for a copper‐mediated click reaction, whereas probes with an azide handle (below) use copper‐free methods. 53 common proteins were identified between the two probes with diverse roles, although the majority were glutathionylated. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 14

Structures of bioorthogonal ozonide probes by Maser et al. The alkyne‐based copper click chemistry probes identified stochastically alkylated targets with little overlap between similarly structured probes OZ726 and OZ727. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 15

Multi‐functional click chemistry probes of Marine natural product Salinipostin A (Sal A). This multi‐functional probe helped to identify 10 enriched proteins with a common α/β serine hydrolase domain, 4 of which were found to be essential for parasite survival. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 16

YnMyr probe developed for the recognition of P. falciparum N‐myristoylated protein targets. An analog of the MyrCoA with an alkyne handle (YnMyr) was constructed for capture with a trifunctional capture reagent. The terminal azide reagent contains a TAMRA fluorophore for in‐gel fluorescence, a biotin moiety for affinity capture, and a trypsin cleavable linker capable of acting as a tag for the identification of myristoylated proteins by tandem mass spectrometry (MS/MS). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 17

ACT‐186128 chemical probe. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 18

Structure of multifunctional hydroxyethyl chemical probes used for the target identification. Pulldown identified all four known plasmepsins (I–IV) as targets for the hydroxyethyl warhead. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 19

Structure of multifunctional (Z‐LL)₂ probe used for the target validation study. A benzophenone moiety enabled photoaffinity labeling, while the biotin moiety enabled affinity pulldown which could be detected via western blot for PfSPP. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 20

Structure of Albitiazolium bifunctional probe. Photo‐crosslinking and click chemistry affinity purification resulted in the identification of choline/ethanolamine phosphotransferase (CEPT) as a promising target. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 21

Structure of BIX‐01294 probe. 104 enriched protein targets were identified following photo‐crosslinking and affinity purification, only 35 of which were found to be essential. These targets included those with roles in translational and transcriptional regulation. Notably, histone lysine methyl transferases were absent from the list of targets, which are commonly inhibited by diaminoquinazoline compounds in humans.

Figure 22

Experimental workflow for CETSA‐MS. Thermal challenge is applied to the samples of interest, modifying either temperature or drug concentration between samples. The soluble protein fraction is isolated, digested, and analyzed by MS/MS or western blot. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 23

Chemical structures of antimalarials assessed by CETSA. Pyrimethamine, quinine, and mefloquine were used as examples to develop and validate CETSA‐MS as a target deconvolution method in P. falciparum. As expected, CETSA‐MS identified the target engagement of dihydrofolate reductase‐thymidylate synthase (PfDHFR‐TS) as the target for pyrimethamine whose target was known. CETSA‐MS identified purine nucleoside phosphorylase (PfPNP) as a probable target for quinine and a potential weak target for mefloquine.

Figure 24

Structures of plasmepsin inhibitors and their specific targets. CETSA‐WB was used to confirm WM382 targets plasmepsin IX and X.

Figure 25

ICAT reagent and experiment workflow. The ICAT reagent contains a biotin tag for purification of labeled peptides, a heavy/light labeled PEG reporter region for mass spectral identification, as well as a sulfhydryl (cysteine) reactive group. ICAT is capable of differentially labeling two different samples which are first digested into tryptic peptides and reduced to expose sulfhydryl groups. The peptides are labeled with reagent and purified via streptavidin binding. The samples are then mixed and analyzed by mass spectrometry, where the relative intensity of the samples can be measured using differences in their mass. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 26

iTRAQ and TMT are methods of isobaric labeling for quantitative proteomic measurements. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 27

Mechanism of action of proteolysis‐targeting chimeras (PROTACs). PROTACs are heterodimeric bifunctional molecules that link an E3 ligand to a drug molecule. By doing this, they bring into proximity a target with complexes that polyubiquitylate it and target it for degradation by the proteasome. Coupled with mass spectrometry, these molecules can detect the targets of drug molecules identified by phenotypic screening. [Color figure can be viewed at wileyonlinelibrary.com]