Identification of AKOS, a Chikungunya virus inhibitor, as a USP14 inhibitor for colorectal cancer treatment.

Ubiquitin-specific protease 14 (USP14) is a crucial modulator of proteasomal function and cellular proteostasis, which plays an important role in the development and progression of various cancers including colorectal cancer (CRC). In this study we screened 670 covalent compounds using the in vitro Ub-AMC hydrolysis assay, and identified AKOS, initially a Chikungunya virus inhibitor, as a novel small-molecule inhibitor of USP14. We showed that AKOS inhibiting USP14 deubiquitinase activity with an IC(50) value of 0.98 μM. AKOS directly bound to USP14, covalently modifying the active-site cysteine residue (Cys(114)), thereby effectively inhibiting its deubiquitinase activity. We demonstrated that inhibition of USP14 by AKOS might destabilize MEF2D, a critical substrate, resulting in downregulation of the expression and translation of ECM-related transcription factors such as ITGB4. AKOS exhibited potent anti-cancer effects: the USP14 inhibitor significantly inhibited the proliferation and metastasis of CRC cells in vitro with IC(50) values of 9.88 and 16.57 μM, respectively, in SW620 cells and HCT116 cells. Intratumoral injection of AKOS (15, 30 mg/kg, every 5 days) effectively suppressed the tumor growth in HCT116 xenograft mouse models in vivo. Collectively, we demonstrate that AKOS is a promising chemical probe for targeting USP14 in CRC, offering a novel strategy for disrupting the malignant progression of CRC.