Beyond Neddylation Inhibition: X‑ray Structures Reveal Carbonic Anhydrase Isoform Selectivity of Pevonedistat

Pevonedistat (MLN4924) is a first-in-class inhibitor of the NEDD8-activating enzyme that blocks protein neddylation and exhibits antitumor activity in multiple clinical phases. Here, we report a previously unrecognized and isoform-selective inhibitory profile of pevonedistat against the tumor-associated isoforms human carbonic anhydrase IX and XII (hCA IX and XII). To elucidate the structural basis of this selectivity, X-ray crystal structures were determined for pevonedistat in complex with hCA I, hCA II, and an engineered hCA II variant mimicking hCA XII. These findings provide a mechanistic explanation for the known preferential partitioning of pevonedistat into whole blood via binding to erythrocyte CAs and suggest that CA inhibition may contribute to its antitumor activity in hypoxic tumor microenvironments where hCA IX and XII are overexpressed. This study reveals a dual functional profile for pevonedistat, linking neddylation inhibition with selective targeting of tumor-associated CAs and offers to exploit this synergy in anticancer drug design.