Structure-guided discovery of nitrobenzo-2-oxa-1,3-diazole (NBD) scaffold-based PFKFB4 inhibitors for cancer therapy.

Cancer remains a leading global cause of mortality, with treatment efficacy often compromised by drug resistance, highlighting the urgent need for novel targeted therapies. The enzyme fructose-2,6-bisphosphatase 4 (PFKFB4) governs glycolytic flux by modulating fructose-2,6-bisphosphate (F2,6BP) levels. PFKFB4 overexpression has been observed in various cancers and correlates with tumor growth, aggressiveness, and poor prognosis. Consequently, selective PFKFB4 inhibitors represent a promising therapeutic strategy for cancer treatment. In this study, we employed virtual screening combined with experimental validation to identify novel PFKFB4 inhibitors based on a nitrobenzo-2-oxa-1,3-diazole (NBD) scaffold. These compounds were systematically evaluated for antiproliferative effects in cancer cell lines with high PFKFB4 expression (MCF-7, A549, and HepG2) and for cytotoxicity in normal liver cells (HL7702). Among them, compound 2v, characterized by a 6-nitrofuran moiety, displayed the most potent antiproliferative activity. Mechanistic investigations confirmed that compound 2v effectively reduced intracellular PFKFB4 protein levels. Molecular docking analysis revealed favorable binding interactions between 2v and the ATP-binding site of PFKFB4. Moreover, compound 2v demonstrated robust antiproliferative, pro-apoptotic, and anti-migratory effects in MCF-7 breast cancer cells, accompanied by modulation of cell cycle- and apoptosis-related protein expression. In vivo, compound 2v achieved significant tumor growth inhibition in the MDA-MB-231 human breast cancer xenograft model in female BALB/c-nu nude mice, with a tumor growth inhibition (TGI) rate of 71.9 % at 30 mg/kg/day. Collectively, these findings establish the NBD scaffold as a valuable pharmacophore for developing novel PFKFB4 inhibitors for anticancer applications.