Integrated In Silico and In Vitro Evaluation of Phloridzin for Dipeptidyl Peptidase-IV Inhibition and Glucose Uptake in Type 2 Diabetes Mellitus

Abstract

Dipeptidyl peptidase-IV (DPP-IV, CD26) inactivates the incretin hormones and several peptide mediators of tissue repair, and its pharmacological inhibition is an established strategy in type 2 diabetes mellitus. Plant-derived polyphenols are an attractive source of DPP-IV inhibitors, but few candidates have been carried from structure-based screening through to cell-based enzyme assays within a single study. In the present work a natural-product library was screened against the DPP-IV crystal structure 2P8S using a hierarchical Glide HTVS–SP–XP workflow, the highest-ranked poses were rescored by Prime MM-GBSA, and the leading compound was followed through a 100 ns Desmond molecular dynamics simulation. That compound was then evaluated experimentally in rat L6 myoblasts and mouse 3T3-L1 preadipocytes: for cytotoxicity by sulforhodamine B assay, for glucose uptake by the glucose oxidase–peroxidase method, and for DPP-IV inhibition by a chromogenic H-Gly-Pro-p-nitroanilide assay in both cell-free and cell-lysate formats, with sitagliptin as the reference inhibitor. Phloridzin ranked highest in extra-precision docking (Glide g-score −9.821 kcal/mol) and in MM-GBSA rescoring (ΔGbind −79.494 kcal/mol), ahead of the co-crystallised sitagliptin analogue (−8.384 and −20.289 kcal/mol, respectively), its advantage arising from hydrogen bonding (XP HBond −5.201 kcal/mol) and electrostatics (ΔGcoul −100.76 kcal/mol) at the canonical Glu205/Glu206 motif of the S2 subsite. Over 100 ns the protein Cα RMSD remained between 1.5 and 2.5 Å and ligand-contacting residues fluctuated below approximately 1 Å, but the ligand left its docked pose within the first 25 ns and thereafter occupied a stable alternative pose, maintaining hydrogen bonds to Glu205 (50% and 35% occupancy) and Asp556 (46%). Phloridzin inhibited DPP-IV concentration-dependently in both cell systems, reaching 70.41 ± 0.74% inhibition in L6 and 74.71 ± 1.75% in 3T3-L1 cells at 2.5 µM against 72.28 ± 2.45% and 78.16 ± 1.41% for sitagliptin. Glucose uptake increased in both lines. Phloridzin is therefore a credible natural DPP-IV inhibitor whose computationally predicted affinity is borne out functionally; the simulation indicates that its stable complex is a rearranged pose anchored at Glu205 and Asp556 rather than the docked pose, a distinction relevant to any future optimization of dihydrochalcone scaffolds.