Computational and Experimental Characterization of Natural Compounds as Therapeutic Modulators of GFAT-1 in Diabetes

Abstract Background: Glucosamine-6-phosphate synthase catalyzes the critical step of hexosamine biosynthesis pathway, results production of UDP-N-acetylglucosamine, a key metabolite that modifies protein expression causing insulin resistance. This makes GFAT a promising target for diabetes. Methods: In this study, initially a structure-based virtual screening was done for 4,07,270 natural compounds from the COCONUT database using Schrödinger Suite 2024-2. 15 compounds were filtered based on their docking scores, binding free energy calculations and molecular dynamic simulations. A series of docking Glide docking protocol (SP and XP) followed by binding energy (MMGB/SA) calculations was performed. Later on, Cytoxicity assays were performed using L-6 and 3T3-L1 cell lines. addition to that, glucose uptake assay was also performed. Results: Among 4,07,270 compounds, compound 2542 (glide g score: -10.09 kcal/mol and MMGB/SA ΔG Bind: -93.01 kcal/mol) and compound 4636 (glide g score: -10.05 kcal/mol and MMGB/SA ΔG Bind: -90.65 kcal/mol) through high throughput virtual screening protocol. From 100ns molecular dynamics simulation it was concluded that compound 2542 was stabilized in the binding site of GFAT1 with minimal RMSD fluctuations. The compound 2542 formed hydrogen bonding and hydrophobic interactions with various interface amino acid residues between the two domains with further substantial pharmacological justification needed to validate these molecules as GFAT 1 inhibitors. Cytoxicity assays on L-6 and 3T3-L1 cell lines demonstrated IC₅₀ values of 50.58 and 35.20 µg/mL, respectively and Glucose uptake assay revealed a significant increase in glucose utilization with Biochanin A treatment. Conclusion: These findings suggest that Biochanin A can inhibit Glucosamine-6-phosphate synthase enzyme and serve as potential lead for the development of novel antidiabetic agents. Keywords: Glucosamine-6-phosphate synthase, Glutamine: D-fructose-6-phosphate amidotransferase (GFAT), hexosamine biosynthesis pathway, glucose uptake assay, Cytoxicity assay, virtual screening, molecular dynamic simulations.