Development and Structural Characterization of UTE‐156, a Covalent Inhibitor of the VCP/p97 AAA+ ATPase

ABSTRACT The AAA+ ATPase valosin‐containing protein (VCP/p97) is a central regulator of protein homeostasis that is well characterized for its role in extracting and remodeling ubiquitinated substrates. Dysregulation of VCP activity contributes to the pathogenesis of neurodegenerative diseases and cancer, making it an important therapeutic target. Here, we report the development and characterization of UTE‐156, a novel covalent small‐molecule inhibitor that modifies Cys522 within the D2 ATPase domain of VCP. UTE‐156 potently inhibits VCP ATPase activity, while losing activity against a C522A mutant, supporting a covalent mechanism of action. High‐resolution cryo‐electron microscopy (cryo‐EM) structures reveal that UTE‐156 occupies the D2 nucleotide‐binding site, sterically blocking ATP binding and inducing conformational remodeling of the pocket. Biochemical and cell‐based assays demonstrate strong inhibitory potency but limited solubility and rapid metabolic turnover. These pharmacochemical limitations preclude immediate therapeutic use but underscore its value as a chemical probe. Together, these findings establish UTE‐156 as a powerful tool for dissecting VCP function and provide a framework for future optimization of covalent modulators of protein homeostasis.