HK2-IN-4

HK2-IN-4 is a selective hexokinase 2 (HK2) inhibitor with an IC50 value of 0.79 μM and a Kd value of 0.41 μM. It disrupts the interaction between HK2 and the voltage-dependent anion channel 1 (VDAC1). HK2-IN-4 reduces lactate and ATP levels in cancer cells and induces apoptosis markers, including increased p-AMPK/AMPK ratio and Bax levels, along with decreased Bcl2 levels. Additionally, HK2-IN-4 inhibits the proliferation of cancer cells with high HK2 expression and can be utilized in research related to colorectal cancer and non-small cell lung cancer.

HK2-IN-4 (Compound 106) effectively and selectively inhibits the activity of recombinant HK2 enzyme with an IC50 of 0.79 μM, while its IC50 values for HK1, HK3, and HK4 are 78.47 μM, 60.86 μM, and 46.14 μM, respectively. At a concentration of 10-100 μM for 24-72 hours, HK2-IN-4 selectively reduces the viability of colorectal cancer cells (SW480, LOVO, HCT116) with high HK2 expression, achieving an IC50 as low as 5.00 μM under hypoxic conditions. Its efficacy is enhanced under hypoxia compared to normoxia, and its cytotoxicity is dependent on HK2 expression. HK2-IN-4 at 0-40 μM for 6 hours decreases mitochondrial HK2 levels in HCT116 and SW480 cells in a dose-dependent manner under hypoxic conditions. Treatment with 50 μM HK2-IN-4 for 7 hours significantly reduces HK2 and VDAC1 colocalization in SW480 cells, disrupting their interaction. At concentrations of 50-100 μM for 2 hours, the compound inhibits glycolysis in hypoxic SW480 and HCT116 WT cells, but not in HK2 KO cells, indicating HK2-dependent glycolytic inhibition. Finally, HK2-IN-4 at 2.5-20 μM for 12-24 hours decreases lactate and ATP levels in hypoxic SW480 colorectal cancer cells, increases the p-AMPK/AMPK ratio, and modulates Bax/Bcl2 expression levels, inducing bioenergetic stress and apoptosis.