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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

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

by
Sedra Kremesh
1,2,†,
Azza Ramadan
1,2,*,†,
Sedq Ahmad Moutraji
1,2,†,
Shaima Hasan
1,2,
Radwa E. Mahgoub
1,2,
Imogen R. Coe
3,4,
Nour Sammani
1,2,
Lama Abuamer
1,2,
Noor Atatreh
1,2 and
Mohammad A. Ghattas
1,2
1
College of Pharmacy, Al Ain University, Abu Dhabi 64141, United Arab Emirates
2
AAU Health and Biomedical Research Center, Al Ain University, Abu Dhabi 112612, United Arab Emirates
3
Institute for Biomedical Engineering, Science and Technology (iBEST), Toronto, ON M5B 1T8, Canada
4
Department of Chemistry and Biology, Toronto Metropolitan University, Toronto, ON M5B 2K3, Canada
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2026, 31(8), 1293; https://doi.org/10.3390/molecules31081293
Submission received: 25 February 2026 / Revised: 29 March 2026 / Accepted: 3 April 2026 / Published: 15 April 2026
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Abstract

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.
Keywords: hENT; hENT inhibitors; gemcitabine; cancer cell lines; MTT assay; virtual screening; pharmacophore modeling; drug discovery hENT; hENT inhibitors; gemcitabine; cancer cell lines; MTT assay; virtual screening; pharmacophore modeling; drug discovery
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MDPI and ACS Style

Kremesh, S.; Ramadan, A.; Moutraji, S.A.; Hasan, S.; Mahgoub, R.E.; Coe, I.R.; Sammani, N.; Abuamer, L.; Atatreh, N.; Ghattas, M.A. Identification of Putative Equilibrative Nucleoside Transporter Inhibitors Through Dual-Pharmacophore Virtual Screening and Validation in a Gemcitabine-Based Cell Assay. Molecules 2026, 31, 1293. https://doi.org/10.3390/molecules31081293

AMA Style

Kremesh S, Ramadan A, Moutraji SA, Hasan S, Mahgoub RE, Coe IR, Sammani N, Abuamer L, Atatreh N, Ghattas MA. Identification of Putative Equilibrative Nucleoside Transporter Inhibitors Through Dual-Pharmacophore Virtual Screening and Validation in a Gemcitabine-Based Cell Assay. Molecules. 2026; 31(8):1293. https://doi.org/10.3390/molecules31081293

Chicago/Turabian Style

Kremesh, Sedra, Azza Ramadan, Sedq Ahmad Moutraji, Shaima Hasan, Radwa E. Mahgoub, Imogen R. Coe, Nour Sammani, Lama Abuamer, Noor Atatreh, and Mohammad A. Ghattas. 2026. "Identification of Putative Equilibrative Nucleoside Transporter Inhibitors Through Dual-Pharmacophore Virtual Screening and Validation in a Gemcitabine-Based Cell Assay" Molecules 31, no. 8: 1293. https://doi.org/10.3390/molecules31081293

APA Style

Kremesh, S., Ramadan, A., Moutraji, S. A., Hasan, S., Mahgoub, R. E., Coe, I. R., Sammani, N., Abuamer, L., Atatreh, N., & Ghattas, M. A. (2026). Identification of Putative Equilibrative Nucleoside Transporter Inhibitors Through Dual-Pharmacophore Virtual Screening and Validation in a Gemcitabine-Based Cell Assay. Molecules, 31(8), 1293. https://doi.org/10.3390/molecules31081293

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