Cantharidin triggers cardiotoxicity via reactive anhydride-mediated covalent protein modification

Cantharidin (CTD), an anhydride-containing natural product from blister beetles, exhibits potent pharmacological activities in the treatment of molluscum contagiosum and tumors. However, CTD exposure induces multi-organ damage, among which CTD-triggered myocardial cell necrosis and electrophysiological abnormalities represent severe adverse reactions and are the primary causes of fatality. Our recent work revealed that the anhydride moiety of CTD possesses reactivity and can covalently modify proteins via lysine residues. This study investigated the mechanistic role of this covalent modification in CTD-induced cardiotoxicity. Single-dose CTD administration in mice caused acute cardiotoxicity manifested as bradycardia, arrhythmias, myocardial edema, and Na⁺/K⁺-ATPase (NKA) inhibition. Fourteen-day CTD exposure aggravated cardiac dysfunction and induced myocardial necrosis. In vitro studies identified 13 lysine residues on recombinant ATP1A1 protein (the NKA α1 subunit) that were covalently modified by CTD. Cardiac tissue exhibited the most abundant protein adduction by CTD. High-resolution mass spectrometric analysis further confirmed covalent modifications of cardiac ATP1A1 at lysine residues 476, 494, 508, and 629. In contrast to CTD, its anhydride ring-opened derivative cantharidic acid, which lacks reactivity, failed to modify cardiac ATP1A1 and did not produce cardiotoxic effects. The level of CTD-modified ATP1A1 was positively associated with the degree of cardiotoxic effects of CTD, thereby establishing the critical role of anhydride-mediated covalent modification in CTD-induced cardiac injury. This study provides novel mechanistic evidence that links CTD-induced cardiotoxicity to anhydride-dependent target engagement, particularly via ATP1A1 inactivation.