Novel Phenoxyacetic Acid (4-Aminophenoacetic Acid) Shikonin Ester Kills KRAS Mutant Colon Cancer Cells via Targeting the Akt Allosteric Site

The PI3K-Akt axis is abnormally activated in KRAS-mutated colorectal cancer and is considered to be a potential therapeutic target. A novel series of phenoxyacetic acid (4-aminophenoacetic acid) shikonin esters was designed by computer-aided drug design (CADD) and synthesized as Akt allosteric inhibitors. Most compounds exhibited greater anti-proliferative activity compared to the positive control MK2206, while also demonstrating lower cytotoxicity against normal cells than shikonin. One of the promising candidates, L8, was selected for further biological evaluation. Docking studies indicated that L8 effectively bound to the allosteric site of Akt through hydrophobic and hydrogen interactions. Enzyme activity and kinetics assessments revealed that L8 bound to Akt with a Kd of 2.07 × 10⁻⁶ M and inhibited its activity. Further intracellular assays, including western blotting, enzyme activity assay, flow cytometry, etc., verified that L8 mediated the death of two KRAS-mutant colon cancer cell lines HCT116 (KRAS^G13D) and HCT-8 (KRAS^G12A) cells by inactivating Akt, causing tumor cell apoptosis, cell cycle arrest, and interfering with tumor cell invasion and metabolism. A 3D-QSAR model was constructed to understand the relationship between the structure of the shikonin derivatives and their anti-proliferative activity. The in silico ADMET and toxicity prediction studies revealed a few undesired pharmacokinetic attributes of our compounds.