Aberrations in the ubiquitin–proteasome system — which maintains cellular protein homeostasis — are implicated in tumour development and progression. Here, Hyer et al. report the identification of TAK-243, a first-in-class inhibitor of the ubiquitin-activating enzyme. In vitro, TAK-243 induced defective protein turnover and proteotoxic stress, impaired cell cycle progression and inhibited DNA repair, leading to cytotoxicity in a panel of human cancer cell lines. In mice bearing subcutaneous xenograft tumours, representing both solid and haematological cancers, intravenous TAK-243 treatment for 3 weeks induced a marked and robust antitumour effect.