EN67 is a cell-permeablethiol-reactive acrylamido compound th at preferentially and covalently binds E2 ubiquitin conjugating enzyme UBE2D family without affecting the enzymatic activity. EN67 is shown to selectively modifyyet reversiblyUBE2D2 allosteric cysteine-C111 without targeting the catalytic cysteine-C85. EN67 does not inhibit overall TP53 ubiquitination mediated by UBE2D2 and the whole ubiquitination machinery. PROTACs equipped with functionalized-EN67 warhead efficiently degrade the target proteins BRD4 and ARrespectivelyin a UBE2D-dependent manner.
