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.