Screening, designing, and validation of novel siRNAs to target GLUT-3: new avenue of targeted therapy for solid tumors

Abstract Glucose transporters-3 (GLUT-3) are implicated in the altered glucose uptake of cancer cells, and the study investigates the potential of silencing GLUT-3 using small interfering RNA (siRNA). A set of in silico tools was employed to design and validate seven optimal siRNAs (G1, G2, G3, G4, G5, G6, G7) against GLUT-3. The off-target analysis was performed using the Basic Local Alignment Search Tool (BLASTn), while secondary structure prediction was performed using RNAfold. The designed siRNAs' thermodynamic stability and GC content were studied using the OligoCalc web server. Binding energies between siRNAs and human argonaute-2 (hAgo2) were calculated through the HDOCK web server. Based on the off-target elimination, secondary structure formation, thermodynamic stability, and binding with hAgo2 protein, we considered G1 and G7 as potential candidates for further evaluation. The AMBER99SB force field was employed to investigate the structural stability of the siRNA–hAgo2 complex via molecular dynamics (MD) simulations. Post-simulation analyses included: Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Radius of Gyration (Rg), and hydrogen bond evaluations. The molecular mechanics Poisson–Boltzmann surface area (MMPBSA) assay was used to assess the binding free energy of the siRNA–hAgo2 complex with the selected siRNA candidates. The findings suggested G5 (AGAAAUAGAAACUACAGUG) as a promising siRNA candidate for targeting GLUT-3. Given its potential effectiveness, further evaluation is warranted to assess its suitability for therapeutic applications.