Abstract
INTRODUCTION: Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with limited treatment options. Phosphoglycerate kinase 1 (PGK1), with both glycolytic and kinase activities, has been implicated in tumor progression, but its therapeutic potential in ESCC remains unclear. METHODS: We assessed PGK1 by genetic knockdown and developed compd 25 − 4, a structure-based small-molecule inhibitor. Biochemical and cellular assays determined its activity against PGK1 and ESCC proliferation. Mechanisms were explored through cellular glycolysis analysis, autophagy assessment, reverse-phase protein array (RPPA) analysis, and signaling pathway characterization. RESULTS: PGK1 knockdown significantly impaired ESCC cell growth both in vitro and in vivo, supporting its role as a therapeutic target. Compd 25 − 4 inhibited PGK1 glycolytic activity with nanomolar potency (IC(50) = 41 nM) and demonstrated > 5-fold selectivity toward EGFR-positive ESCC cells compared to EGFR-negative cells. Beyond glycolysis inhibition, compd 25 − 4 suppressed PGK1 kinase-mediated PRAS40 signaling and induced autophagy-dependent degradation of EGFR. This dual mechanism of action simultaneously disrupted cancer metabolism and EGFR-driven oncogenic signaling, leading to enhanced therapeutic efficacy. CONCLUSIONS: Our findings establish PGK1 as a promising therapeutic target in ESCC and identify compd 25 − 4 as a potent chemical tool for probing PGK1’s dual enzymatic functions. By concurrently blocking glycolysis and promoting autophagy-mediated EGFR degradation, targeting PGK1 provides a novel therapeutic strategy for EGFR-positive ESCC. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13402-026-01215-4.