The clickable activity-based probe of anti-apoptotic calenduloside E.

CONTEXT: Calenduloside E (CE), one of the primary natural products found in Aralia elata (Miq.) Seem. (Araliaceae), possesses prominent anti-apoptotic potential. A previous study found that one of the anti-apoptotic CE targets is heat shock protein 90 AB1 (Hsp90AB1) by probe CE-P, while the other targets of CE still need to be identified with more efficient probes.

OBJECTIVE

This study investigates CE analogue (CEA) as one clickable activity-based probe for use in exploring anti-apoptotic CE targets.

MATERIALS AND

METHODS

Pretreatment of HUVECs with CEA (1.25 μM) for 8 hr, followed by ox-LDL stimulation for 24 h. Flow cytometry analysis and JC-1 staining assays were performed The kinetic constant measurements were tested by the Biacore T200, CM5 Sensor Chip which was activated by using sulpho-NHS/EDC. Ligands were dissolved and injected with a concentration of 12.5, 6.25, 3.125, 1.56, 0.78 and 0 μM.

RESULTS

CEA was confirmed to possess an anti-apoptotic effect. The probable targets of CE/CEA were calculated, and as one of the higher scores proteins (Fit values: 0.88/0.86), Hsp90 properly got our attention. Molecular modelling study showed that both CE and CEA could bind to Hsp90 with the similar interaction, and the docking scores (S value) were -7.61 and -7.33. SPR assay provided more evidence to prove that CEA can interact with Hsp90 with the KD value 11.7 µM.

DISCUSSION AND

CONCLUSIONS

Our results suggest that clickable probe CEA could alleviate ox-LDL induced apoptosis by a similar mechanism of anti-apoptotic CE, and afforded the possibility of identifying additional anti-apoptotic targets of CE.