Development of an OTUD1 ubiquitin variant inhibitor

OTUD1 (Ovarian tumor domain-containing deubiquitinase 1) is a member of the OTU domain-containing deubiquitinase family of enzymes involved in immunoregulation and tumorigenesis pathways. OTUD1 consists of three distinct regions: an unstructured N-terminal region, an OTU-fold catalytic domain, and a ubiquitin-interacting motif (UIM) containing region. Enhanced enzymatic activity and a strong preference for K63-linked substrates are imparted by the UIM containing region. We used phage display with a ubiquitin variant (UbV) library to identify binders for OTUD1 lacking the unstructured N-terminal region (OTUD1OTU + UIM) in an attempt to identify inhibitors bridging the catalytic domain and the UIM containing region. Two UbVs were identified (UbVOD.1 and UbVOD.2) with high affinity and specificity for OTUD1. Of the UbVs identified, UbVOD.1 inhibited OTUD1 activity towards mono-Ub and K63-linked di-Ub substrates in vitro with single-digit nanomolar IC50 and potently inhibited deubiquitinase activity with poly-Ub chains of other linkages. In vivo expression of UbVOD.1 alone was unstable, however as a di-UbV, global deubiquitination and deubiquitinase activity with the OTUD1 substrate RIPK1 were inhibited. Herein we describe the development of molecular tools for exploring the activity of OTUD1 in a cellular context, towards protein-based therapeutics.