Withanolide A Inhibits hIAPP aggregation: An In silico, Biophysical, and Drosophila-Based In Vivo Validation

The aberrant aggregation of human islet amyloid polypeptide (hIAPP) or Amylin into toxic oligomers and fibrils leads to pancreatic β-cell dysfunction and progressive cell death, which is a key pathological feature of Type II diabetes mellitus (T2DM). In this study, we adopted a hybrid approach combining virtual screening and molecular dynamics (MD) simulation, with experimental validation, to identify inhibitors of hIAPP aggregation. Herein, we screened 2000 phytoconstituents from natural products using molecular docking, followed by in silico ADMET predictions. Withaferin A and Withanolide A (phytoconstituents of Ashwagandha) were found to be lead molecules with suitable drug-like properties. Next, we performed an 800 ns MD simulation to assess the stability and interaction dynamics of hIAPP-ligand complexes. Building on the computational screening, we further carried out a comprehensive experimental analysis to validate the inhibitory effects of lead molecules. The collective experimental results from the Thioflavin T (ThT) assays, combined with Confocal and Transmission Electron Microscopy (TEM), suggest that the ligands (preferably Withanolide A) have a potent inhibitory effect against hIAPP aggregation by increasing the lag phase and inhibiting fibril formation of hIAPP. For in vivo validation using Drosophila models, Withanolide A was found to mitigate hIAPP oligomer-induced toxicity by reducing apoptosis, necrosis, and oxidative stress in the Drosophila gut, as confirmed by multiple cell death staining assays and reactive oxygen species (ROS) analysis. Besides, in diabetic flies, Withanolide A lowered glucose levels, demonstrating anti-diabetic activity. Thus, this work, for the first time, suggests that Withanolide A may be a potential candidate for inhibiting hIAPP aggregation and as a T2DM drug.