The tumor suppressor CYLD negatively regulates polyubiquitination-dependent cellular signaling such as nuclear factor (NF)-kappa B signaling. In addition to CYLD, multiple deubiquitinating enzymes (DUBs) are also involved in the regulation of this signaling pathway, and distinct role of CYLD is yet to be clarified. Here, we identified a small chemical named Subquinocin that inhibited the DUB activity of recombinant CYLD using a wheat cell-free protein synthesis and an AlphaScreen technology. In cells, Subquinocin increased the polyubiquitination of NEMO and RIP1 and enhanced NF-kappa B activation. Modeling and mutation analyses indicated that Subquinocin interacted with Y940 in CYLD, which locates close to catalytic center of CYLD, and is conserved among the USP-family DUBs. Further biochemical evaluation revealed that Subquinocin inhibited USP-family DUBs, but not other family DUBs including OTU. Although Subquinocin showed a broad specificity toward USP-family DUBs, the inhibitory effect of Subquinocin on NF-kappa B signaling was negligible in CYLD-KO cells, indicating that CYLD is a major target of Subquinocin on the suppression of NF-kappa B signaling. In conclusion, Subquinocin identified here is a useful tool to analyze the signal transduction mediated by USP-family DUBs. (C) 2019 The Authors. Published by Elsevier Inc.