SUMOylation is really a reversible publish-translational modification that regulates protein function through covalent attachment of small ubiquitin-like modifier (SUMO) proteins. The entire process of SUMOylating proteins involves an enzymatic cascade, the initial step which entails the activation of the SUMO protein with an ATP-dependent process catalyzed by SUMO-activating enzyme (SAE). Here, we describe the identification of TAK-981, a mechanism-based inhibitor of SAE which forms a SUMO-TAK-981 adduct because the inhibitory species inside the enzyme catalytic site. Optimization of selectivity against related enzymes in addition to enhancement of mean residence duration of the adduct were important to the identification of compounds with potent cellular path inhibition and eventually an extended pharmacodynamic effect and effectiveness in preclinical tumor models, culminating within the identification from the clinical molecule TAK-981.