Discovery of novel indole-chalcone derivatives as potent hexokinase 2 inhibitors for triple negative breast cancer therapy.

Accelerated glucose metabolism is one of the important characteristics of cancer cells. Hexokinase 2 (HK2) is the rate-limiting enzyme that catalyzes the first step reaction of the glycolytic pathway and it is always found overexpressed in various tumor cells. In tumor cells, the overexpression of HK2 not only promotes aerobic glycolysis to provid energy and metabolite support for the rapid proliferation of tumor cells, but also inhibits apoptosis by binding to the outer mitochondrial membrane protein VDAC. It is recognized as important target of tumor metabolic reprogramming and it owns significant potential for anti-cancer treatment. This project was based on the structure of HK2 protein (PDB: 2NZT). Through virtual screening and molecular docking, twelve potential inhibitors were screened out from the ourself-established compound library. In vitro enzymatic activity assays revealed that compound 12 exerted potent inhibitory activity with an IC₅₀ value of 1.17 μM. It also significantly suppressed the proliferation of MDA-MB-231 tumor cells, with an IC₅₀ of 4.04 μM. Notably, this compound exhibited notable selectivity, as its IC50 against normal Vero cells was 137.21 μM and against NCM460 cells was 35.12 μM, corresponding to selectivity indices of 34 and 8.7, respectively, compared with MDA-MB-231 cells (IC50 = 4.04 μM). The target binding ability was supported by the cell thermal migration assay (CETSA), and its anti-tumor mechanism was preliminarily explored through ROS detection. The research provides candidate molecules and multi-dimensional validation strategies for the development of anti-tumor drugs targeting HK2.