USP25/28 inhibitor AZ1 is an orally active, selective, noncompetitive, dual ubiquitin specific protease (USP) 25/28 inhibitor with IC50 values of 0.62μM and 0.7μM, respectively[1]. USP25/28 inhibitor AZ1 blocks USP28-dependent stabilization of ΔNp63, causing an imbalance in the Fanconi anemia (FA)-DNA damage response (DDR) signal, and enhancing the sensitivity of squamous cell carcinoma cells to chemotherapy[2]. USP25/28 inhibitor AZ1 has been widely used to enhance the immune response and inhibit the activity of USP25 in promoting intestinal cancer[3].
In vitro, USP25/28 inhibitor AZ1 (20μM) treatment for 48 hours significantly inhibited the proliferation of A549 and H460 cells, and induced cell cycle arrest and the expression of P27[4]. Treatment with 10μM of the USP25/28 inhibitor AZ1 for 48 hours significantly reduced the protein expression levels of SMARCA4, UIMC1 and SLX4 in SKOV3 cells, and induced DNA double-strand break damage. Inhibit homologous recombination repair[5].
In vivo, USP25/28 inhibitor AZ1 treatment via intraperitoneal injection at a dose of 20mg/kg/day for 4 weeks improved amyloid plaque deposition in Alzheimer's disease mice[6]. Subcutaneous intraperitoneal injection of USP25/28 inhibitor AZ1 (5mg/kg/day) for 7 consecutive weeks inhibited the development of renal cysts in mice with Autosomal dominant polycystic kidney disease (ADPKD), and down-regulated the expression levels of STAT3 as well as the transcriptional targets c-Myc, Bcl-xL and CyclinD2[7]. Intragastric gavage of the USP25/28 inhibitor AZ1 at a dose of 60mg/kg every other day for 24 days inhibited tumor growth in the 786-O xenograft mouse model without affecting body weight[8].
References:
[1] Wrigley J D, Gavory G, Simpson I, et al. Identification and characterization of dual inhibitors of the USP25/28 deubiquitinating enzyme subfamily[J]. ACS chemical biology, 2017, 12(12): 3113-3125.
[2] Prieto-Garcia C, Hartmann O, Reissland M, et al. Inhibition of USP28 overcomes Cisplatin-resistance of squamous tumors by suppression of the Fanconi anemia pathway[J]. Cell Death & Differentiation, 2022, 29(3): 568-584.
[3] Patzke J V, Sauer F, Nair R K, et al. Structural basis for the bi-specificity of USP25 and USP28 inhibitors[J]. EMBO reports, 2024, 25(7): 2950.
[4] Song Y, Wang L, Zheng Y, et al. Deubiquitinating enzyme USP28 inhibitor AZ1 alone and in combination with cisplatin for the treatment of non-small cell lung cancer[J]. Apoptosis, 2024, 29(9): 1793-1809.
[5] Han F, Qi G, Li R, et al. USP28 promotes PARP inhibitor resistance by enhancing SOX9-mediated DNA damage repair in ovarian cancer[J]. Cell Death & Disease, 2025, 16(1): 305.
[6] Zheng Q, Song B, Li G, et al. USP25 inhibition ameliorates Alzheimer’s pathology through the regulation of APP processing and Aβ generation[J]. The Journal of clinical investigation, 2022, 132(5).
[7] Ren Y, Zhu X, Fu K, et al. Inhibition of deubiquitinase USP28 attenuates cyst growth in autosomal dominant polycystic kidney disease[J]. Biochemical Pharmacology, 2023, 207: 115355.
[8] Ren Y, Yang Y, Zhu X, et al. Targeting USP28 inhibits clear cell renal cell carcinoma growth[J]. Cellular Signalling, 2025: 112344.