Rational DESIGN and Structure-activity relationship study of novel JOSD2 inhibitor against colorectal cancer

Deubiquitinases (DUBs) catalyze the removal of ubiquitin moieties from substrate proteins, playing pivotal roles in regulating protein homeostasis. Targeting DUBs with small-molecule inhibitors to induce substrate protein degradation has emerged as a compelling strategy for addressing traditionally "undruggable" targets. Notably, multiple DUB inhibitors have advanced to preclinical and clinical stages. In our preliminary research, we discovered that the deubiquitinating enzyme Josephin domain-containing 2 (JOSD2) played a significant role in the development and progression of colorectal Cancer (CRC). In this study, we identified a novel hit compound 1 targeting the JOSD2 through high-throughput screening of an internal compound library. Subsequently, guided by structure-activity relationship (SAR) analysis, compound 31 was synthesized, a JOSD2 inhibitor featuring a cyanamide warhead that selectively engaged the catalytic cysteine residue. Preliminary biological mechanism studies revealed a covalent binding mode between compound 31 and JOSD2. Further mechanistic studies have shown that 31 could induce the downregulation of KRAS protein expression in HCT116 cells, thereby exerting proliferation inhibitory activity (IC₅₀ = 0.93 ± 0.01 μM). Collectively, our research identified that targeting JOSD2 with small-molecule inhibitors could represent a potential future therapeutic strategy for CRC.