A TXNIP-driven bioluminescent reporter for high-throughput discovery of glycolytic inhibitors against renal cell carcinoma

The glycolytic inhibitor 2-deoxy-D-glucose (2-DG) has demonstrated consistent preclinical antitumor efficacy; however, the discovery of novel 2-DG-like agents for renal cell carcinoma (RCC) remains challenging due to the lack of specific, high-throughput screening (HTS) tools. In this study, RNA-seq analysis identified Thioredoxin-interacting protein (TXNIP) as a gene markedly upregulated by 2-DG in A498 RCC cells. We further confirmed that 2-DG transcriptionally upregulates the expression of both TXNIP and its transcription factor, MLX-interacting protein (MLXIP). Leveraging this mechanism, we engineered a bioluminescent reporter system by constructing a TXNIP promoter-driven luciferase construct (TXNIP-Pro-Luc2) and generating a stable A498-TXNIP-Pro-Luc2 cell line. In this system, 2-DG and its functional analogs activate the TXNIP promoter, resulting in a concentration-dependent increase in bioluminescence that serves as a direct functional readout for 2-DG-like activity. Collectively, we developed a novel reporter system based on the MLXIP/TXNIP pathway, which shows promise as a high-throughput screening platform for identifying glycolysis-targeting anti-RCC drug candidates.