Selection and Characterization of Tau Binding ᴅ-Enantiomeric Peptides with Potential for Therapy of Alzheimer Disease

Abstract

A variety of neurodegenerative disorders, including Alzheimer disease (AD), are associated with neurofibrillary tangles composed of the tau protein, as well as toxic tau oligomers. Inhibitors of pathological tau aggregation, interrupting tau self-assembly, might be useful for the development of therapeutics. Employing mirror image phage display with a large peptide library (over 109 different peptides), we have identified tau fibril binding peptides consisting of d-enantiomeric amino acids. d-enantiomeric peptides are extremely protease stable and not or less immunogenic than l-peptides, and the suitability of d-peptides for in vivo applications have already been demonstrated. Phage display selections were performed using fibrils of the d-enantiomeric hexapeptide VQIVYK, representing residues 306 to 311 of the tau protein, as a target. VQIVYK has been demonstrated to be important for fibril formation of the full lengths protein and forms fibrils by itself. Here, we report on d-enantiomeric peptides, which bind to VQIVYK, tau isoforms like tau3RD (K19) as well as to full lengths tau fibrils, and modulate the aggregation of the respective tau form. The peptides are able to penetrate cells and might be interesting for therapeutic and diagnostic applications in AD research.

Fig 1. Tau constructs and principle of the mirror image phage display selection.

(A) The largest human tau isoform in the central nervous systems contains four microtubule binding repeats, whereas the tau construct tau^3RD (K19) lacks repeat 2 [42]. PHF6, consisting of amino acids 306 to 311, is located at the beginning of repeat three. (B) The d-enantiomeric form of PHF6 was synthesized. (C) After fibrillization, the d-enantiomeric fibrils were used for phage display (D). (E) An l-peptide, binding to the d-enantiomeric PHF6 fibrils, was selected. (F) The d-enantiomeric version of the selected l-peptide was synthesized, which (G) will bind to the l-enantiomeric form of the target, regular PHF6 fibrils.

Fig 2. ThT assays for characterization of the peptides abilities to inhibit fibril formation.

(A) PHF6 fibrillizes spontaneously within 2 hours. The assay was performed using 25 μM PHF6 peptide in PBS with 10 μM ThT (black line). PBS and 10 μM ThT without addition of PHF6 was used as control (grey line). Fluorescence was measured at 490 nm in relative units (mean +/- standard deviations of results, five replicates per run. (B) PHF6 fibrillization was inhibited by the selected peptides. The respective peptide was added in concentrations of 25 or 250 μM to 25 μM PHF6 samples, respectively. Upon addition of ThT, fluorescence was measured at 490 nm in relative units (mean +/- standard deviations of results, five replicates per run). The relative fluorescence for the PHF6 only control after 2 hours incubation (saturation level) was set at 100%. *: p < 0.05; **: p < 0.0005; students t-test. (C) Recombinant full lengths tau protein fibrillizes under assay conditions. 5 μM tau, 100 μM AA and 10 μM ThT were incubated in PBS (black line). As a negative control, 5 μM tau was incubated in PBS with ThT, without addition of AA. Fluorescence was measured at 490 nm in relative units (mean +/- standard deviations of results, five replicates per run). The relative fluorescence of a PBS sample with 100 mM AA was subtracted. (D) ThT assays were employed to characterize the abilities of peptides to inhibit full lengths tau fibril formation. The respective peptide was added in concentrations of 50 μM peptide to 5 μM tau and 100 M AA. Upon addition of ThT, fluorescence was measured at 490 nm in relative units (mean +/- standard deviations of results, five replicates per run). The relative fluorescence for the tau and AA control without peptides and after 2.5 hours incubation (saturation level) was set at 100%. *: p < 0.05; **: p < 0.0005; students t-test.

Fig 3. ThS inhibition assays were performed in vitro using the tau^3RD construct K19.

Dose-response curves for inhibition of tau aggregation were plotted as percentage of the untreated control. Samples treated with d-peptides show a decreasing ThS signal at increasing concentrations, indicating an inhibition of tau aggregation.

Fig 4. Effect of the selected peptides on tau aggregation, measured by DLS.

Recombinant tau was dissolved in PBS to a concentration of 21.8 μM (1 mg/ml). As positive control, AA was added to a concentration of 100 μM. The peptides were added in a ratio of 1:10 (tau:peptide) and 100 μM AA. All samples were incubated for 24 hours. DLS was performed with an acquisition time of 5 seconds for 3 minutes at room temperature. The ratio of the hydrodynamic radii of tau protein in the presence of the peptides to tau fibrils (positive control) was calculated. The error was below 10% for all measurements, the mass was ≥ 99%.

Fig 5. Inducible N2aTau^4RDΔK280 cells were incubated with 60 μM of the respective, FAM-labeled peptide for two hours and four days.

After incubation, the cells were fixed on glass cover slips and the locations of the respective peptide were detected by the FAM-label (green). In addition, cell nuclei were stained with TOPRO3 dye (red). Here, representative pictures for APT-FAM, KNT-FAM and TD28-FAM are shown in 60 times magnification. Scale bar: 30 μm.

Fig 6. Structure of the tau fibril and binding mode of different d-peptides.

(A) Crystal structure of the steric zipper formed by the PHF6 peptide. The first and last residue for each of the two stacks of interacting β-sheet is labeled. The individual layers of β-sheet within each stack are gradually colored from red to blue. (B) Binding mode of the TLKIVW peptide according to Sievers et al. [19]. The side chain interaction between K3 and Q307 is marked by a green arrow. The red ellipse denotes steric repulsion between L2 of TLKIVW and V306/I308 of the second stack of β-strands. Peptide residues are labeled in italic. (C) Binding mode of the TD28 peptide. Same type of presentation as in (B); an additional repulsive interaction formed between L4 and Y310 is highlighted by a magenta ellipse. (D) Binding mode of the APT peptide. Same type of presentation as in (B); an additional repulsive interaction formed between T3 and Y310 is highlighted by a magenta ellipse.