EN67 is a cell-permeable, thiol-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 modify, yet reversibly, UBE2D2 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 AR, respectively, in a UBE2D-dependent manner.
