Multi-Level High-Throughput Screening for Discovery of Ligands That Inhibit Insulin Aggregation.

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Insulin aggregation is implicated in various diseases.
• Developing inhibitors for insulin aggregation is crucial for therapeutic interventions.
• Non-native protein-protein interactions present unique challenges for drug design.

Purpose of the Study:

• To develop a virtual screening protocol to identify inhibitors of insulin aggregation.
• To screen the FDA inactives database for potential lead molecules.
• To validate the efficacy of identified lead molecules in preventing insulin aggregation.

Main Methods:

• Multi-level virtual screening protocol including pharmacophore screening and ensemble docking.
• Molecular dynamics simulations and binding affinity calculations.
• Experimental validation using size exclusion chromatography, Thioflavin T assay, and circular dichroism spectroscopy.

Main Results:

• Identified five potential lead molecules from the FDA inactives database.
• Riboflavin demonstrated significant inhibition of insulin aggregation, retaining 85% native monomer after 48 hours.
• Screened inactives, including riboflavin and aspartame, helped maintain insulin's alpha-helical structure, preventing beta-sheet rich conformations.

Conclusions:

• The developed virtual screening protocol is effective in identifying insulin aggregation inhibitors.
• Riboflavin is a potent inhibitor of insulin aggregation and a promising therapeutic candidate.
• Inhibiting insulin aggregation preserves its native alpha-helical structure, preventing pathological conformational changes.