Multivalent Polymer-Peptide Conjugates-A General Platform for Inhibiting Amyloid Beta Peptide Aggregation

Abstract

Protein aggregation is implicated in multiple deposition diseases including Alzheimer's Disease, which features the formation of toxic aggregates of amyloid beta (Aβ) peptides. Many inhibitors have been developed to impede or reverse Aβ aggregation. Multivalent inhibitors, however, have been largely overlooked despite the promise of high inhibition efficiency endowed by the multivalent nature of Aβ aggregates. In this work, we report the success of multivalent polymer-peptide conjugates (mPPCs) as a general class of inhibitors of the aggregation of Aβ₄₀. Significantly delayed onset of fibril formation was realized using mPPCs prepared from three peptide/peptoid ligands covering a range of polymer molecular weights (MWs) and ligand loadings. Dose dependence studies showed that the nature of the ligands is a key factor in mPPC inhibition potency. The negatively charged ligand LPFFD (LD) leads to more efficient mPPCs compared to the neutral ligands, and is most effective at 7% ligand loading across different MWs. Molecular dynamics simulations along with dynamic light scattering experiments suggest that mPPCs form globular structures in solution due to ligand-ligand interactions. Such interactions are key to the spatial proximity of ligands and thus to the multivalency effect of mPPC inhibition. Excess ligand-ligand interactions, however, reduce the accessibility of mPPC ligands to Aβ peptides, and impair the overall inhibition potency.

Figure 1.

The general design of mPPCs for inhibiting Aβ₄₀ aggregation. (A) mPPCs are synthesized by the modification of a poly (HPMA-co-NHSMA) backbone with an inhibitor ligand, e.g., LPFFD (LD). (B) The chemical structures of ligands LPFFN and G(mG)L(mG)F(mG)A with the N-terminal amines used in the polymer-peptide conjugation highlighted. (C) Three independent parameters, the peptide/peptoid ligands, the ligand loading, and the polymer MW are explored in this work for optimizing the inhibition of Aβ aggregation. (D) TEM and AFM images show Aβ₄₀ aggregation leading to fibril formation when incubated alone (left panels), but yielding aggregates of a few nanometers in the presence of an mPPC (right panels, see Figure 2B for details). Scale bar: 500 nm.

Figure 2.

The general efficacy of mPPC inhibitors. (A) When incubated alone at 10 μM, Aβ₄₀ quickly formed fibrils as detected by an increase of fluorescence intensity in ThT assays. Most mPPCs led to evident inhibition with no fluorescence increase within 24 h at 200 μg/mL, as is shown for (B) LD-mPPC of 77 kDa at 3% loading and (C) LN-mPPC of 43 kDa at 9% loading. (D) GA-mPPC of 18 kDa at 12% loading allowed partial fibril formation with a lag time approaching 24 h. The colored traces represent results from multiple replicas (n = 4). AFM (insets, B-D) confirmed the absence of fibrils for most of the tested mPPCs except for the GA-mPPC. Scale bar: 500 nm.

Figure 3.

Dose dependence of mPPC inhibition and the evaluation of inhibitory potency of all mPPCs. (A) ThT assays of 10 μM Aβ₄₀ in the presence of 10–200 μg/mL LN-mPPCs from 43 kDa polymer with 9% loading. Only one representative trace of four independent measurements at each concentration is shown for clarity. (B) The ratios of the lag time of the control (Aβ alone) to those in the presence of LN-mPPCs are plotted as a function of mPPC concentration. Error bars represent the standard deviation (n = 4). Critical concentration, c_1/3, defined as the concentration at which mPPCs extend the lag time to 3 times that of the control, is obtained directly from the plot. Plots summarizing this analysis for all mPPCs in both mass concentration (mPPCs) and molar concentration (peptide/peptoid ligand) are shown in Figure S2 and S3, respectively. (C) The relative potency of all mPPCs with the color of each bar representing the c_1/3 value of each mPPC (potency is inversely related to the c_1/3 value). The height of the grey triangles represents the corresponding ligand loading. The hydrodynamic size of mPPCs is determined by DLS, and the two highlighted LN-mPPCs feature diameters ca. 10 times largers than those of their analogs.

Figure 4.

MD simulations illustrate the importance of ligand–ligand contacts in mPPC globular structures. Three replicas of mPPC models (DP = 100) were generated with 3, 7, and 12 copies of LD and LN. The extended (A-C) and equilibrated (D-F) structures containg 3 (A,D), 7 (B,E), and 12 (C,F) copies of LD before and after simulation. The polymer backbone is shown in grey; LD ligands are shown in varied colors. (G) Radius of gyration (R_g) of the equilibrated mPPC models (averaged over three replicas in each case). (H) Atomic contacts (3.5 Å cut-off) extracted for polymer–polymer (violet), peptide–peptide (magenta) and polymer–peptide (brown) interactions in globular mPPC structures.