Identification of Putative Equilibrative Nucleoside Transporter Inhibitors Through Dual-Pharmacophore Virtual Screening and Validation in a Gemcitabine-Based Cell Assay

Pharmacological inhibition of the nucleoside transporter hENT1 is a promising therapeutic target across a range of diseases, including cardiovascular disorders, neurodegenerative conditions, and cancer. However, current inhibitors lack drug-like properties, necessitating the development of new inhibitors with improved pharmacological profiles. We employed a dual-pharmacophore virtual screening protocol to identify putative hENT1 inhibitors from a library of over 2 million compounds, followed by structure-based molecular docking. To validate the inhibition effect of the lead compounds, we established a functional assay using gemcitabine (GEM)-induced cytotoxicity as a readout of hENT transport activity using eight cancer cell lines. H292 was the optimal cancer cell line for the validation assay based on its high GEM sensitivity (IC50 = 28 nM) and the concentration-dependent cytotoxicity inhibition of the reference inhibitor NBTI, a hENT1 inhibitor. Of the 19 candidate compounds, two leads (compounds 2 and 3) demonstrated potency comparable to NBTI, increasing GEM IC50 values by 2.2- and 2.9-fold at 5 µM, respectively. Both compounds were non-cytotoxic to normal fibroblasts, exhibited favorable ADME properties, displayed superior docking scores of -12.63 and -12.49 kcal/mol compared to NBTI (-9.06 kcal/mol), and displayed a novel vertical binding orientation within the hENT1 binding pocket distinct from NBTI's horizontal mode. This study established a validated non-radioactive, gemcitabine-based functional assay for hENT inhibitor discovery and identified two putative inhibitors with therapeutic potential for cancer chemosensitization, pain management, and cardio- and neuroprotection. The non-radioactive functional assay overcomes the limitations of traditional radiolabeled methods, enabling scalable, broader screening applications.