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

Abstract

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.

A Multiscale Model for Quantitative Prediction of Insulin Aggregation Nucleation Kinetics

“…57 The reduced interaction strength was chosen such that more than half of rigid-body docked N . A dimers, found to be metastable in earlier all-atomistic simulations, 30 stay in an associated state with a stable binding interface during CG simulation (see details in Table S1). CG-MD simulations were carried out using GROMACS at aggregation-prone experimental conditions of 338 K, pH 2, and 600 µM insulin.…”

“…Details of the protocol to obtain a total charge of +7 e on the dimer, in accordance with ion mobility mass spectroscopy measurements, are provided in our previous work. 30 The final structure from CG-MD simulations was refined by backmapping to the AA representation followed by a 20 ns MD run using the CHARMM36 force-field. 60 The central structure of the largest cluster from the stable portion of this AA MD trajectory was taken as the diffusional association complex.…”

“…A set of 16 most stable (lowest binding free energy) N.A insulin dimer conformations were taken from our earlier study wherein extensive rigid-body docking was used to generate a large ensemble of possible dimer conformations, followed by conformation ranking on basis of binding free energies calculated using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) implicit solvent model. 30 We carried out an extensive set of molecular dynamics simulations starting with these 16 structures with a specific aim of allowing reorientation of binding partners under influence of long-range electrostatics and refinement of contacts determined from rigid-body docking. To allow for the determination of an electrostatically optimal binding mode, five replicate simulations were done for each rigid-body docked structure wherein monomer A in any given N.A dimer was moved perpendicular to the binding interface by a distance equal to twice the Debye length (2λ D =2.2 nm at 100 mm NaCl) to ensure a negligible effect of any specific proteinprotein interactions (PPIs) at this initial separation (see Figure S1).…”

Template-based design of peptides to inhibit SARS-CoV-2 RNA-dependent RNA polymerase complexation

“…Following the work of Mishra et al,(17) se-365 quences of peptides were built by sewing a chain of spatially 366 close hotspot residues from two different binding segments of 367 the hotspot as given in Table1. A segment is defined as a 368 continuous sequence of residues on the NSP12 binding inter-369 face.…”

OUP accepted manuscript

“…Small peptide ligands have been highlighted over the past few decades on account of their particular advantages, such as less cost, little immunogenic responses and more stable physicochemical properties ( 1 , 2 ). Especially, peptide ligands’ chemical structures are highly compatible with those of the target proteins ( 3 ).…”