An amphiphilic small molecule drives insulin aggregation inhibition and amyloid disintegration.

The aggregation of proteins into ordered fibrillar structures called amyloids, and their disintegration represent major unsolved problems that limit the therapeutic applications of several proteins. For example, insulin, commonly used for the treatment of diabetes, is susceptible to amyloid formation upon exposure to non-physiological conditions, resulting in a loss of its biological activity. Here, we report a novel amphiphilic molecule called PAD-S, which acts as a chemical chaperone and completely inhibits fibrillation of insulin and its biosimilars. Mechanistic investigations and molecular docking lead to the conclusion that PAD-S binds to key hydrophobic regions of native insulin, thereby preventing its self-assembly. PAD-S treated insulin was biologically active as indicated by its ability to phosphorylate Akt, a protein in the insulin signalling pathway. PAD-S is non-toxic and protects cells from insulin amyloid induced cytotoxicity. The high aqueous solubility and easy synthetic accessibility of PAD-S facilitates its potential use in commercial insulin formulations. Notably, PAD-S successfully disintegrated preformed insulin fibrils to non-toxic smaller fragments. Since the structural and mechanistic features of amyloids are common to several human pathologies, the understanding of the amyloid disaggregation activity of PAD-S will inform the development of small molecule disaggregators for other amyloids.

Synthesis and Evaluation of Arylamides with Hydrophobic Side Chains for Insulin Aggregation Inhibition.

Insulin, a peptide hormone, forms fibrils under aberrant physiological conditions leading to a reduction in its biological activity. To ameliorate insulin aggregation, we have synthesized a small library of oligopyridylamide foldamers decorated with different combination of hydrophobic side chains. Screening of these compounds for insulin aggregation inhibition using a Thioflavin-T assay resulted in the identification of a few hit molecules. The best hit molecule, BPAD2 inhibited insulin aggregation with an IC50 value of 0.9 µM. Mechanistic analyses suggested that BPAD2 inhibited secondary nucleation and elongation processes during aggregation. The hit molecules worked in a mechanistically distinct manner, thereby underlining the importance of structure-activity relationship studies in obtaining a molecular understanding of protein aggregation.

Amphiphilic Small-Molecule Assemblies to Enhance the Solubility and Stability of Hydrophobic Drugs.

Amphiphilic assemblies made from diverse synthetic building blocks are well known for their biomedical applications. Here, we report the synthesis of gemini-type amphiphilic molecules that form stable assemblies in water. The assembly property of molecule M2 in aqueous solutions was first inferred from peak broadening observed in the proton NMR spectrum. This was supported by dynamic light scattering and transmission electron microscopy analysis. The assembly formed from M2 (M2agg) was used to solubilize the hydrophobic drugs curcumin and doxorubicin at physiological pH. M2agg was able to effectively solubilize curcumin as well as protect it from degradation under UV irradiation. Upon solubilization in M2agg, curcumin showed excellent cell permeability and higher toxicity to cancer cells over normal cells, probably because of enhanced cellular uptake and increased stability. M2agg also showed pH-dependent release of doxorubicin, resulting in controlled toxicity on cancer cell lines, making it a promising candidate for the selective delivery of drugs to cancer cells.

Small-Molecule Inhibitor Prevents Insulin Fibrillogenesis and Preserves Activity.

Amyloidosis is a well-known but poorly understood phenomenon caused by the aggregation of proteins, often leading to pathological conditions. For example, the aggregation of insulin poses significant challenges during the preparation of pharmaceutical insulin formulations commonly used to treat diabetic patients. Therefore, it is essential to develop inhibitors of insulin aggregation for potential biomedical applications and for important mechanistic insights into amyloidogenic pathways. Here, we have identified a small molecule M1, which causes a dose-dependent reduction in insulin fibril formation. Biophysical analyses and docking results suggest that M1 likely binds to partially unfolded insulin intermediates. Further, M1-treated insulin had lower cytotoxicity and remained functionally active in regulating cell proliferation in cultured Drosophila wing epithelium. Thus, M1 is of great interest as a novel agent for inhibiting insulin aggregation during biopharmaceutical manufacturing.