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. 2024 Feb 20;22(1):139.
doi: 10.1186/s12964-023-01409-5.

Chemoproteomics-based profiling reveals potential antimalarial mechanism of Celastrol by disrupting spermidine and protein synthesis

Affiliations

Chemoproteomics-based profiling reveals potential antimalarial mechanism of Celastrol by disrupting spermidine and protein synthesis

Peng Gao et al. Cell Commun Signal. .

Abstract

Background: Malaria remains a global health burden, and the emergence and increasing spread of drug resistance to current antimalarials poses a major challenge to malaria control. There is an urgent need to find new drugs or strategies to alleviate this predicament. Celastrol (Cel) is an extensively studied natural bioactive compound that has shown potentially promising antimalarial activity, but its antimalarial mechanism remains largely elusive.

Methods: We first established the Plasmodium berghei ANKA-infected C57BL/6 mouse model and systematically evaluated the antimalarial effects of Cel in conjunction with in vitro culture of Plasmodium falciparum. The potential antimalarial targets of Cel were then identified using a Cel activity probe based on the activity-based protein profiling (ABPP) technology. Subsequently, the antimalarial mechanism was analyzed by integrating with proteomics and transcriptomics. The binding of Cel to the identified key target proteins was verified by a series of biochemical experiments and functional assays.

Results: The results of the pharmacodynamic assay showed that Cel has favorable antimalarial activity both in vivo and in vitro. The ABPP-based target profiling showed that Cel can bind to a number of proteins in the parasite. Among the 31 identified potential target proteins of Cel, PfSpdsyn and PfEGF1-α were verified to be two critical target proteins, suggesting the role of Cel in interfering with the de novo synthesis of spermidine and proteins of the parasite, thus exerting its antimalarial effects.

Conclusions: In conclusion, this study reports for the first time the potential antimalarial targets and mechanism of action of Cel using the ABPP strategy. Our work not only support the expansion of Cel as a potential antimalarial agent or adjuvant, but also establishes the necessary theoretical basis for the development of potential antimalarial drugs with pentacyclic triterpenoid structures, as represented by Cel. Video Abstract.

Keywords: Antimalarial; Celastrol; Protein synthesis; Spermidine.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Celastrol shows potential antimalarial effects in vivo in PbANKA infected C57BL/6 mice. A The scheme of animal modeling and drug handling. C57BL/6 mice were divided into five groups (Control; model; ATS treatment; Cel treatment; ATS + Cel treatment). B Daily parasitemia was monitored by Giemsa staining. C-D The behavioral observations and body temperature measurements on the first day and last day. E The histopathological observations of liver and spleen using H&E staining (scale bar = 50 μm). F-G The organ index of liver and spleen in each group after treatment. H-I The levels of serum ALT and AST in each group after treatment. All data were presented as mean ± standard error of the mean (SEM), ###P < 0.001 vs control; *P < 0.05, **P < 0.01, ***P < 0.001 vs model. ATS, artesunate; Cel, Celastrol
Fig. 2
Fig. 2
Identification of Cel potential antimalarial targets in P. falciparum based on the ABPP strategy. A The structure of Cel and Cel-P. B The antimalarial activity of Cel and Cel-P against P. falciparum 3D7 strain. C The workflow of ABPP for the profiling of Cel target proteins. D In situ fluorescence labeling of Cel-P in parasite proteins. E In situ competition experiment of Cel-P by Cel. F Confocal imaging showed the distribution of Cel-P in parasites with or without excess Cel (scale bar = 1 μm). G Heatmap representation of the target proteins identified by the Cel-P. H Gene ontology (GO) enrichment analysis for all potential targets of Cel-P. CBB, Coomassie brilliant blue; RBC, red blood cells; HZ, hemozoin; TAMRA, carboxy tetramethyl rhodamine
Fig. 3
Fig. 3
Integrated transcriptomics and proteomics analysis. A GO enrichment analysis for the differential proteins in proteomics after Cel treatment. B Venn diagrams showing the overlap of differential proteins in proteomics with the target proteins identified by Cel-P. C GO enrichment analysis for the overlapped proteins from (B). D Volcano plot showing the gene expression after Cel treatment. E-F GO enrichment analysis of the differential genes in transcriptomics
Fig. 4
Fig. 4
Validation of Cel binding to PfSpdsyn (PF3D7_1129,000). A Fluorescence labeling of recombinant PfSpdsyn protein with Cel-P in a dose-dependent manner. B Competition fluorescence labeling of Cel-P with excess Cel. C-D Immunofluorescence staining of co-localization of Cel-P with PfSpdsyn and quantitative analysis of colocalization. E Validation of the binding of Cel-P to PfSpdsyn using pull-down Western blotting. F Validation of the binding of Cel-P to PfSpdsyn using cellular thermal shift assay. G The UV absorbance spectra of Cel after incubation with PfSpdsyn. H The measurement of binding affinity of Cel with PfSpdsyn using the Bio-layer interferometry (BLI) assay. I Competition fluorescence labeling of IAA-P with Cel and IAA. (J) Docking simulation of Cel binding to Cys165 of PfSpdsyn. K Fluorescence labeling of Cel-P on the wild-type (WT), single-site mutants (C165A, C266A), and double-site mutant (C165A/C266A) of PfSpdsyn. L The spermidine level of parasites after treatment with different concentrations of Cel. All data were presented as mean ± standard error of the mean (SEM), **P < 0.01, ***P < 0.001
Fig. 5
Fig. 5
Validation of Cel binding to PfEGF1-α (PF3D7_1357000). A-J Similar validations as performed in Fig. 4(A-J). K The decrease in fluorescence of AHA-labeling after the treatment with Cel. CHX (cycloheximide) serves as a positive control for the inhibition of protein synthesis
Fig. 6
Fig. 6
The potential antimalarial mechanism of Celastrol

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