Design, synthesis, and evaluation of indole-pyridine chalcone derivatives as novel HK2 inhibitors to modulate macrophage glycolysis and inflammation for IBD treatment

Hexokinase 2 (HK2) promotes M1 macrophage polarization through aerobic glycolysis, so as to increase the release of inflammatory factors and to maintain the pro-inflammatory phenotype. HK2 inhibitors may regulate the phenotype of immune cells and exert anti-inflammatory effects by modulating glycolysis. Building upon our prior discovery that indole-chalcone derivatives specifically bind to HK2 and inhibit its enzymatic activity, a focused library of novel indole-pyridine chalcone derivatives was rationally designed and facilely synthesized via Claisen-Schmidt condensation of corresponding indole-3-carboxaldehydes with 3-acetylpyridine. Systematic screening of HK2 enzyme inhibitory activity identified compound ZSW as a potent lead candidate, exhibiting significant HK2 inhibition with an IC₅₀ value of 0.48 ± 0.13 μM. Both molecular docking and biotin probe pull-down experiments confirmed ZSW-HK2 binding. Further experiments indicated that ZSW alleviated inflammatory bowel disease primarily via two possible mechanisms: one is modulating macrophage phenotypes through glycolysis inhibition, and the Other is inhibiting NF-κB nuclear translocation by preventing IκBα phosphorylation and degradation. Animal experiments demonstrated that ZSW improved the intestinal inflammatory state of IBD mice. This study not only lays the research foundation for HK2 as a target for the development of anti-IBD drugs, but also provides theoretical support for expanding the application of HK2 inhibitors in the development of anti-inflammatory drugs.