Caffeic acid phenethyl ester (CAPE) revisited: Covalent modulation of XPO1/CRM1 activities and implication for its mechanism of action

Caffeic acid phenethyl ester (CAPE) is the bioactive constituent of propolis from honeybee hives and is well known for its anti-inflammatory, anticarcinogenic, antioxidant, and immunomodulatory properties. Herein, we revisited the cellular mechanism underlying the diverse biological effects of CAPE. We demonstrated that XPO1/CRM1, a major nuclear export receptor, is a cellular target of CAPE. Through nuclear export functional assay, we observed a clear shift of XPO1 cargo proteins from a cytoplasmic localization to nucleus when treated with CAPE. In particular, we showed that CAPE could specifically target the non-catalytic and conserved Cys⁵²⁸ of XPO1 through the means of mass spectrometric analysis. In addition, we demonstrated that the mutation of Cys⁵²⁸ residue in XPO1 could rescue the nuclear export defects caused by CAPE. Furthermore, we performed position-restraint molecular dynamics simulation to show that the Michael acceptor moiety of CAPE is the warhead to enable covalent binding with Cys⁵²⁸ residue of XPO1. The covalent modulation of nuclear export by CAPE may explain its diverse biological effects. Our findings may have general implications for further investigation of CAPE and its structural analogs.