USP7-IN-18 (Compound X21) serves as a novel USP7 inhibitor by directly suppressing enzyme activity and modulating downstream pathways, including the newly identified PCLAF target. At concentrations of 0.25-1 μM for 24 hours, it exhibits inhibitory effects. USP7-IN-18 effectively reduces proliferation in leukemia (RS4;11) and colon cancer (MC38/CT26.WT) cells at 0.01-100 μM over 72 hours. It demonstrates high selectivity for USP7 at 2.5 μM within 0.5 hours, surpassing eight other deubiquitinating enzymes. SPR analysis shows that at 1.95-2000 nM for 3 minutes, the compound binds the catalytic domain of USP7 with a KD value of 4.9 μM.

USP7-IN-18 binds to the catalytic domain of USP7 with a K D value of 4.9 μM, as determined by SPR analysis within the concentration range of 1.95-2000 nM over 3 minutes. Also known as Compound X21, USP7-IN-18 (0.25-1 μM, 24 h) acts as an innovative USP7 inhibitor by directly hindering enzyme activity and modulating downstream pathways, which include the newly identified target PCLAF. Moreover, at concentrations between 0.01-100 μM over 72 hours, USP7-IN-18 significantly curbs the proliferation of RS4;11 leukemia and MC38/CT26.WT colon cancer cells. It demonstrates high selectivity for USP7 over eight other deubiquitinating enzymes at a concentration of 2.5 μM for 0.5 hours.

USP7-IN-18 (Compound X21), administered intraperitoneally at doses of 5 mg/kg and 10 mg/kg once daily for 16 consecutive days, significantly inhibits tumor growth in C57BL/6J mice with MC38 tumors.