A New Strategy for the Rapid Identification and Validation of the Direct Targets of Aconitine-Induced Cardiotoxicity

Abstract

Background: The interaction of small molecules with direct targets constitutes the molecular initiation events of drug efficacy and toxicity. Aconitine, an active compound of the Aconitum species, has various pharmacological effects but is strongly toxic to the heart. The direct targets of aconitine-induced cardiotoxicity remain unclear.

Methods: We predicted the toxic targets of aconitine based on network pharmacology and followed a novel proteomic approach based on the "drug affinity responsive target stability" technology combined with LC-MS/MS to identify the direct targets of aconitine. The identified targets were analysed from the perspective of multilevel and multidimensional bioinformatics through a network integration method. The binding sites were investigated via molecular docking to explore the toxicity mechanism and predict the direct targets of aconitine. Finally, atomic force microscopy (AFM) imaging was performed to verify the affinity of aconitine to the direct targets.

Results: PTGS2, predicted by network pharmacology as a toxic target, encodes cyclooxygenase 2 (COX-2), which is closely related to myocardial injury. Furthermore, cytosolic phospholipase A2 (cPLA2) is the upstream signal protein of PTGS2, and it is a key enzyme in the metabolism of arachidonic acid during an inflammatory response. We determined cPLA2 as a direct target, and AFM imaging verified that aconitine could bind to cPLA2 well; thus, aconitine may cause the expression of PTGS2/COX-2 and release inflammatory factors, thereby promoting myocardial injury and dysfunction.

Conclusion: We developed a complete set of methods to predict and verify the direct targets of aconitine, and cPLA2 was identified as one. Overall, the novel strategy provides new insights into the discovery of direct targets and the molecular mechanism of toxic components that are found in traditional Chinese medicine.

Keywords: aconitine; atomic force microscopy; cardiotoxicity; direct targets; drug affinity responsive target stability technology.

Graphical abstract

Figure 1

Venn diagram of aconitine and cardiotoxicity-related targets (A); PPI network diagram (B); GO enrichment analysis: BP, Biological process; CC, cellular component; MF, molecular function. (C); Top 20 KEGG signalling pathways bubble chart (D); “Aconitine–Targets–Pathways” network diagram (E).

Figure 2

Effects of different concentrations of aconitine on H9c2 cell viability (A). All data are shown as mean ± SD (n = 6). **p <0.01 versus the normal group; SDS-PAGE results of protein digested with 1:100 pronase: protein ratio after incubation with aconitine (B–D); (B–D) represent three repetitions of the experiments (n = 3).

Figure 3

PPI network diagram of 152 core targets from network pharmacology and 15 direct targets from DARTS.

Figure 4

Molecular docking models of aconitine with 4 direct target proteins in 3D and 2D diagram. Aconitine with (A) cPLA2 (PDB: 1CJY); (B) GYS1 (PDB: 3CX4); (C) CTNNA1 (PDB: 4EHP); (D) ORC5 (PDB: 5UI7).

Figure 5

Potential toxicity mechanism of aconitine by acting on direct target cPLA2.

Figure 6

AFM analysis. AFM images (2D and 3D) of (A) cPLA2 and (B) aconitine acting on the protein.