AKT-IN-28 is an allosteric inhibitor of Akt and a derivative of Shikonin. It effectively binds to Akt's allosteric site through hydrophobic and hydrogen bond interactions, with a Kd value of 2.07 μM. AKT-IN-28 significantly inhibits Akt activity, inducing apoptosis in KRAS-mutant colorectal cancer cells, arresting the cell cycle at the G2/M phase, and suppressing cell proliferation, migration, and metabolism.

Akt:2.07 μM (Kd)

AKT-IN-28 (Compound L8) exhibits potent antiproliferative effects with IC50 values of 4.51, 4.57, 5.07, 8.94, and 6.59 μM against HCT-8, HCT-116, PC-3, MDA-MB-231, and HeLa cells, respectively, while showing relatively low cytotoxicity towards normal cells (MCF-10A and HCoEpiC cells with IC50 values of > 100 and 67 μM, respectively). At concentrations of 1-4 μM for 24 hours, AKT-IN-28 reduces Akt enzymatic activity in a dose-dependent manner in HCT-8 and HCT-116 cells, significantly suppressing protein expression of total Akt, p-Akt, PARP, and β-catenin, as well as cell proliferation and migration, while increasing levels of cleaved-PARP protein. Additionally, at 0.25-1 μM over 7 days, it significantly inhibits colon cancer cell viability and reduces colony formation in HCT-116 cells. AKT-IN-28 induces apoptosis and G2/M phase cell cycle arrest in HCT-116 and HCT-8 cells at 2-8 μM for 24 hours, as well as decreases expression of cell cycle proteins CDH1, CDK1, and CDC20, while enhancing caspase3 activity. Furthermore, at concentrations of 2-8 μM for 2-24 hours, AKT-IN-28 significantly reduces glucose consumption, lactate production, and ATP levels in HCT-116 cells.