Structure-Based Design of Small Peptide Ligands to Inhibit Early-Stage Protein Aggregation Nucleation

We
report a structure-based approach to design peptides that can
bind to aggregation-prone, partially folded intermediates (PFI) of
insulin, thereby inhibiting early stages of aggregation nucleation.
We account for the important role of the modular architecture of protein–protein
binding interfaces and tertiary structure heterogeneity of the PFIs
in the design of peptide inhibitors. The determination of association
hotspots revealed that two interface segments are required to capture
majority contribution to insulin homodimer binding energy. The selection
of peptides that will have a high probability to inhibit insulin self-association
was done on the basis of similarity in binding interface coverage
of PFI residues in the peptide–PFI complex and the native–PFI
dimer. Data on aggregate growth rate and secondary structure for formulations
incubated under amyloidogenic conditions show that designed peptides
inhibit insulin aggregation in a concentration-dependent manner. The
mechanism of aggregation inhibition was probed by determining the
enthalpy of peptide–insulin binding and peptide micellization
using isothermal titration calorimetry. Finally, the effect of designed
peptides on insulin activity was quantified using a spectrophotometric
assay for glucose uptake by HepG2 cells.