N-Amino peptide scanning reveals inhibitors of Aβ₄₂ aggregation

Abstract

The aggregation of amyloids into toxic oligomers is believed to be a key pathogenic event in the onset of Alzheimer's disease. Peptidomimetic modulators capable of destabilizing the propagation of an extended network of β-sheet fibrils represent a potential intervention strategy. Modifications to amyloid-beta (Aβ) peptides derived from the core domain have afforded inhibitors capable of both antagonizing aggregation and reducing amyloid toxicity. Previous work from our laboratory has shown that peptide backbone amination stabilizes β-sheet-like conformations and precludes β-strand aggregation. Here, we report the synthesis of N-aminated hexapeptides capable of inhibiting the fibrillization of full-length Aβ₄₂. A key feature of our design is N-amino substituents at alternating backbone amides within the aggregation-prone Aβ_16–21 sequence. This strategy allows for maintenance of an intact hydrogen-bonding backbone edge as well as side chain moieties important for favorable hydrophobic interactions. An N-amino scan of Aβ_16–21 resulted in the identification of peptidomimetics that block Aβ₄₂ fibrilization in several biophysical assays.

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Introduction

Amyloid protein aggregation has been linked to several neurodegenerative disorders, including Parkinson's and Alzheimer's Disease (AD).¹ In AD, amyloid precursor protein (APP) is sequentially cleaved by α and β secretase to release pathogenic amyloid beta (Aβ). Aβ is prone to aggregation and forms toxic β-sheet fibrils that are the main component of neuritic plaques in AD patients.² Of the two Aβ fragments formed during amyloidogenesis, the 42-residue variant (Aβ₄₂) is more prone to aggregation and displays greater neurotoxicity in comparison to the 40-residue variant (Aβ₄₀).³ Soluble oligomers of Aβ₄₂ have been shown to impair hippocampal synaptic plasticity,⁴ induce tau hyperphosphorylation,⁵ activate microglial inflammation,⁶ and impair memory.⁷ Interestingly, studies have shown that high molecular weight oligomers are relatively inactive. However, when allowed to dissociate into low molecular weight oligomers (8–70 kDa) severe toxicity is observed. Additionally, soluble oligomers (but not monomers) cause synaptic death in a dose dependent manner.⁸

Although Aβ₄₂ is considered to be intrinsically disordered, its fibrils are comprised of parallel β-sheets that feature edge–edge hydrogen-bonding between Aβ₄₂ monomers.⁹ This in-register association is driven in large part by a core hydrophobic sequence encompassing residues 16–21 (₁₆KLVFFA₂₁).¹⁰ Direct Aβ₄₂ aggregation blockers that have entered clinical trials for the treatment of AD include homotaurine, scyllo-inositol, and sodium oligomannurarate.¹¹ These and similar compounds face significant hurdles to advancement, as they are likely to interact in a non-specific manner with β-rich target proteins. In contrast, backbone modified peptides derived from aggregation-prone segments offer the prospect of sequence-specific inhibition of amyloidogenesis. Toward this end, several groups have reported Aβ_16–21 peptidomimetics harboring D-amino acids, unnatural residues, or backbone amide substituents.¹² In addition to optimizing affinity for Aβ₄₂, such peptide modifications may serve to enhance proteolytic stability, conformational rigidity, and prospects for passage through the blood–brain barrier. Rationally designed peptidomimetics targeting Aβ₄₂ fibrilization have shown efficacy in animal models of AD.¹²

Our laboratory has previously shown that peptide backbone N-amination stabilizes β-sheet-like conformation through cooperative non-covalent interactions.¹³ Like N-methylation, the incorporation of N-amino substituents within a peptide strand precludes canonical β-sheet associations along one of the hydrogen-bonding edges. This characteristic is useful for the design peptide derivatives that not only resist self-aggregation but also block β stacking of a target protein. Although backbone-methylated peptides are capable of disrupting Aβ aggregation,¹⁴ we viewed N-amination as an alternative strategy for the design of more conformationally rigid β-breaker peptides. Unlike N-methylation or proline incorporation, backbone amination does not promote a significant population of the cis amide rotamer. Given that Aβ₄₂ fibrils adopt a β-strand conformation with an all-trans amide geometry, backbone-substituted mimics with high trans amide propensity should exhibit enhanced affinity for the conformationally extended core sequence. The N-amino group is also able to engage in an intraresidue C6 H-bond, which in turn stabilizes ψ and ϕ torsions in the β-sheet region of Ramachandran space.^13a We sought to exploit these features in the design of β-strand peptidomimetics capable of blocking amyloid aggregation (Fig. 1). Here, we report the first example of N-amino peptide scanning and the identification of novel backbone-modified inhibitors of Aβ₄₂ fibrilization.

Fig. 1 Design of N-amino peptide (NAP) Aβ_16–21 mimics. (A) Conformational effects of peptide backbone N-amination. (B) Proposed model for inhibition of Aβ fibrilization by a representative NAP.

Results

Peptide N-amino scanning of the aggregation-prone ₁₆KLVFFA₂₁ sequence required the synthesis of 5 distinct α-hydrazino acid derivatives starting from commercially available α-amino esters. We previously demonstrated that monomers suitable for solid-phase peptide synthesis (SPPS) can be readily accessed via electrophilic amination of α-amino esters.¹⁵ As shown in Table 1, the requisite building blocks were thus prepared by treatment of 1 with (2-t-butyl-3,3-diethyl)-oxaziridine-2,3,3-tricarboxylate (TBDOT)¹⁶ under aqueous conditions, followed by alkaline ester hydrolysis to give 2a–e in good yields.

Table 1 Synthesis of α-hydrazino acid monomers for NAP scanning

	Residue	R	Yield
2a	aLys(Boc)	(CH2)4NHBoc	74
2b	aLeu	CH2CH(CH3)2	66
2c	aVal	CH(CH3)2	44
2d	aPhe	CH2Ph	66
2e	aAla	CH3	61

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Backbone-aminated analogues of Aβ_16–21 were prepared on rink amide MHBA resin using Fmoc chemistry (Scheme 1). Condensation of the Boc-protected α-hydrazino acids onto the growing peptide chain was achieved by HCTU/NMM-mediated activation of the carboxylic acid in the presence of the unprotected Nα. The poorly nucleophilic Nα is only acylated upon exposure to pre-formed Fmoc-protected amino acid chloride derivatives.¹⁷ These were prepared by reaction of Fmoc-protected amino acids with thionyl chloride, or by activation with Ghosez' reagent in the case of the Fmoc-Lys(Boc)-OH residue (due to the acid-labile side chain protecting group). Following sequence elongation, the peptidomimetics were N-terminally acetylated, cleaved from the resin, and purified by RP-HPLC.

Scheme 1 Solid-phase synthesis of NAP-based Aβ_16–21 mimics.

For the current study, we synthesized the 14 N-aminated Aβ_16–21 variants shown in Table 2, along with the parent hexapeptide (3). This positional scanning library is comprised of all possible mono-aminated peptides, as well as di- and tri-aminated sequences that harbor backbone substituents on the same edge of the putative β-strand. We excluded analogues featuring alternate edge N-aminations since these compounds are presumably devoid of a fully intact hydrogen-bonding edge. The identity of purified peptidomimetics was confirmed by HRMS and their purities assessed by analytical HPLC and NMR. The ¹H NMR spectra for 3–17 were recorded in DMSO-d₆ and proton resonance assignments were made on the basis of TOCSY spectra.

Table 2 Synthesized Aβ_16–21 mimics and HRMS data

Peptide	Sequence	HRMS [M + H]obs^+
3	Ac-Lys-Leu-Val-Phe-Phe-Ala-NH2	779.4701
4	Ac-aLys-Leu-Val-Phe-Phe-Ala-NH2	779.4697
5	Ac-Lys-aLeu-Val-Phe-Phe-Ala-NH2	779.4714
6	Ac-Lys-Leu-aVal-Phe-Phe-Ala-NH2	779.4711
7	Ac-Lys-Leu-Val-aPhe-Phe-Ala-NH2	779.4680
8	Ac-Lys-Leu-Val-Phe-aPhe-Ala-NH2	779.4687
9	Ac-Lys-Leu-Val-Phe-Phe-aAla-NH2	779.4690
10	Ac-aLys-Leu-aVal-Phe-Phe-Ala-NH2	794.4800
11	Ac-aLys-Leu-Val-Phe-aPhe-Ala-NH2	794.4802
12	Ac-Lys-aLeu-Val-aPhe-Phe-Ala-NH2	794.4811
13	Ac-Lys-aLeu-Val-Phe-Phe-aAla-NH2	794.4802
14	Ac-Lys-Leu-aVal-Phe-aPhe-Ala-NH2	794.4794
15	Ac-Lys-Leu-Val-aPhe-Phe-aAla-NH2	794.4810
16	Ac-aLys-Leu-Val-Phe-Phe-Ala-NH2	809.4920
17	Ac-Lys-aLeu-Val-aPhe-Phe-aAla-NH2	809.4912

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The aggregation of Aβ₄₂ was monitored in the presence of 1 molar equivalent of NAPs 4–17 using thioflavin T (ThT), a dye that fluoresces upon binding to the perpendicular axis of amyloid fibrils (Fig. 2).¹⁸ Compounds 5, 14 and 15 were identified as the most effective inhibitors of Aβ₄₂ aggregation in repeated experiments. Aβ₄₂ fibrillization is typically characterized by a sigmoidal curve comprised of a lag phase, an elongation phase, and a plateau phase. Over a 12 h experiment, Aβ₄₂ aggregation was inhibited by approximately 30–50% at an equimolar ratio for each inhibitor. Although the peak fluorescence values of all three inhibitors were comparable, the elongation/growth phase of 5 and 14 were slower than that of 15. In addition, 5 displayed increased lag times across all three runs (Fig. S1†). Although several other NAPs displayed some inhibitory effects on Aβ₄₂ aggregation kinetics, observed variations in repeated experiments led us to focus on compounds 5, 14, and 15 for further study. Consistent with previous reports, the parent hexapeptide 3 exhibited accelerated aggregation when incubated in the presence of Aβ₄₂ and ThT. Initial in vitro assay thus suggests that a single backbone amination, as in 5, is sufficient to convert a highly aggregation-prone peptide into an inhibitor of fibrilization.

Fig. 2 Effects of 1.0 equiv. of inhibitors 5, 14, and 15 on Aβ₄₂ aggregation (40 μM) as monitored by ThT fluorescence.

To gain further insight into the effects of NAPs 5, 14, and 15 on Aβ₄₂ structure we carried out circular dichroism (CD) measurements over several time points (Fig. 3). Incubation of Aβ₄₂ alone at 37 °C resulted in a transition from random coil to β-sheet structure over the course of 18 h, as evidenced by a pronounced minimum at 218 nm. Incubation of Aβ₄₂ in the presence of an equimolar concentration of 15 for 18 h resulted in a significantly attenuated β-sheet signature by CD, although some β-sheet character was still evident. In contrast, analogues 5 and 14 showed a similar CD signature at 18 h indicative of a random coil conformation. These spectra were also similar to those observed for Aβ₄₂ alone at 0 and 4 h timepoints. The Far UV-CD spectra of 40 μM 5, 14, and 15 revealed that each inhibitor showed irregular/random coil character in the absence of Aβ₄₂ (Fig. S2†).

Fig. 3 Circular dichroism spectra. (A) Time-dependent CD spectra of 40 μM Aβ₄₂ recorded in phosphate buffer. (B) CD spectrum of Aβ₄₂ at 18 h (black), and in the presence of an equimolar ratio of 5, 14, and 15.

Protofibrils, fibrillar precursors to amyloid formation, are preceded by the appearance of small Aβ oligomers.¹⁹ Transmission electron microscopy (TEM) can be used to visualize the morphology of these fibril and pre-fibrillar species at high resolution. In order to assess the ability of 5, 14, and 15 to alter the dominant morphology of Aβ₄₂, samples were taken directly from the ThT assays at the plateau phase (Fig. 4). Aβ₄₂ incubated on its own formed a dense network of mature fibrils when incubated without inhibitor. Aβ₄₂ incubated with 5 showed scattered, short, rod-like protofibrils. No evidence of mature fibril formation was visible. Aβ₄₂ incubated with 14 showed little to no evidence of fibril formation, however, small seeding was visible. Aβ₄₂ incubated with 15 showed sparse mature fibrils throughout the sample. These results are consistent with those from ThT assays and CD measurements showing that NAP 15 was not as effective as 5 or 14 in blocking the aggregation of Aβ₄₂ or its transition to β structure.

Fig. 4 Representative transmission electron microscopy images at t = 6 h. (A) Aβ₄₂ incubated alone, or in the presence of an equimolar amount of (B) 5, (C) 14, or (D) 15.

Based on results from TEM experiments, we investigated the ability of our most effective inhibitor to delay fibrilization in a dose-dependent manner by ThT. As shown in Fig. 5, 14 had little effect on aggregation lag time at 0.5 equiv. concentration, but delayed the aggregation of Aβ₄₂ by 80% and 220% at 1.0 equiv. and 4.0 equiv., respectively. Unlike 14, we did not observe a similar dose-dependency in the case of 5 or 15. Taken together, these data demonstrate that di-NAP 14 is effective in delaying Aβ₄₂ aggregation in a dose-dependent manner, inhibiting the formation of mature fibrils, and in blocking the transition of Aβ₄₂ from random coil to β structure in vitro.

Fig. 5 Effect of 0.5, 1.0 and 4.0 equiv. of 14 on 40 μM Aβ₄₂ aggregation lagtime as monitored by ThT fluorescence assay.

Conclusions

Here, we demonstrate the synthesis and evaluation of N-amino peptide inhibitors of Aβ₄₂ aggregation that are readily accessible from α-hydrazino acid monomers. The inhibitory activity of selected NAPs was confirmed through ThT assay, CD, and TEM. Di-aminated derivative 14 emerged from this study as an effective inhibitor of Aβ₄₂ aggregation in vitro. Interestingly, tri-aminated derivatives 16 and 17 were less effective by ThT assay, as were the majority of mono-aminated analogues. We previously observed that di-amination across a tripeptide strand is a particularly powerful approach for stabilizing β-sheet conformations within hairpin model systems.¹³ Peptidomimetics 14 and 15 likewise represent constrained β-strand mimics bearing two edge-blocking substituents in close proximity. This may explain their ability to engage Aβ₄₂ and shield one hydrogen-bonding edge from further association with Aβ monomers. The current work adds backbone N-amination to the repertoire of peptidomimetic strategies for targeting β-sheet assembly. We anticipate that this approach can be readily applied to other disease-relevant protein interactions and will provide useful lead compounds for structure-based refinement.

Experimental

Synthesis of compounds 3–17

Synthesis of all peptides/peptidomimetics was carried out on Fmoc-capped polystyrene rink amide MBHA resin (100–200 mesh). α-Hydrazino acid monomers were prepared from α-amino esters as previously described.¹⁵ Dry resin was washed with DMF three times and allowed to swell in DMF for 1 h prior to use. All reactions were carried out using gentle agitation. Fmoc deprotection steps were carried out by treating the resin with a solution of 20%piperidine/DMF (15 min × 2). Coupling of Fmoc-protected amino acids as well as (N′-Boc)-hydrazino acids was effected using 5 equiv. HCTU (0.5 M in DMF), 10 equiv. NMM (1.0 M in DMF), and 5 equiv. of the carboxylic acid in DMF at 50 °C (1 h). Coupling of residues N-terminal to the hydrazino acids was carried out with 30 equiv. collidine and 10 equiv. of pre-formed Fmoc amino acid chloride¹⁷ in THF at 45 °C (2 × 1 h). After each reaction the resin was washed with DMF three times. Peptides were cleaved from the resin by incubating with gentle stirring in 95 : 5 TFA : H₂O for 2 h. The cleavage mixture was filtered and the resin was rinsed with an additional portion of cleavage solution. The filtrate was concentrated to remove the bulk of the TFA and the remaining residue was treated with 8 mL of cold Et₂O to induce precipitation. The mixture was centrifuged and the supernatant was removed. The remaining solid was washed 2 more times with Et₂O and dried under vacuum. Peptides were analyzed and purified on C12 RP-HPLC columns (preparative: 4 μ, 90 Å, 250 × 21.2 mm; analytical: 4 μ, 90 Å, 150 × 4.6 mm) using linear gradients of MeCN/H₂O (with 0.1% formic acid), then lyophilized to afford white powders. All peptides were characterized by LCMS (ESI), HRMS (ESI-TOF), ¹H NMR, and TOCSY.

Thioflavin T fluorescence aggregation assay

ThT fluorescence assays were conducted in 96-well half area plates (black, flat bottom, non-binding surface) without shaking in a Biotek Synergy H1 microplate reader (444 nm excitation, 484 nm emission) at 37 °C for 12 h. Experiments were run in duplicate with 40 μM Aβ₄₂, 40 μM ThT, and 40 μM of inhibitor in 10 mM PBS buffer (pH 7.4) with a total reaction volume of 100 μL.

Composition of the wells of the 96-well plate. The ThT assay of Aβ_1–42 aggregation was performed by preparing stock solutions of 133.33 μM Aβ₄₂, 133.33 μM ThT, and 400 μM inhibitor and then adding 60, 30, and 10 μL portions, respectively, of these solutions into wells in a 96-well plate. Aβ₄₂ was added last. Prior to reading, the plate was incubated at 37 °C with orbital shaking for 3 min, then covered with an optically clear film.

Preparation of Aβ₄₂ stock solution. To disaggregate pre-formed Aβ₄₂ fibrils, lyophilized Aβ₄₂ was dissolved in HFIP and evaporated under vacuum to form a film. In order to delay further aggregation, the film was freshly dissolved in 10 mM aq. NaOH, followed by sonication for 30 s. This solution was further diluted into 10 mM PBS (pH 7.4) at a final concentration of 133.33 μM as a stock. This stock was then filtered through a pre-washed centricon filter (100 kD MW cutoff) at 6000 g, 4 °C for approximately 15 min.

Preparation of ThT stock solution. The ThT Stock solution was freshly prepared by dissolving 4 mg Thioflavin T powder in 20 mL 10 mM PBS (pH 7.4). This solution was filtered using a 13 mm syringe filter with 0.45 μM PTFE membrane. The concentration was determined by UV [ε = 36 000 M⁻¹ cm⁻¹ at 412 nm] and diluted to final concentration using 10 mM PBS (pH 7.4).

Preparation of inhibitor stock solution. The inhibitor stock solutions were prepared by diluting a 20 mM DMSO stock first to 100× desired concentration using DMSO, and second to 10× desired concentration using 10 mM PBS (pH 7.4).

Circular dichroism. Aβ₄₂ was prepared as previously described,²⁰ and further diluted into 10 mM PBS (pH 7.4) to a final concentration of 80 μM. This stock solution was further diluted to 40 μM in Millipore water and incubated at 37 °C for 0 h, 4 h, and 18 h; incubation at 37 °C with shaking (0 h timepoint was recorded immediately after dilution). The spectra of Aβ₄₂ were recorded at 1.0 nm intervals from 195 to 260 nm with an averaging time of 3.0 s and an average of three repeats on an Aviv Stopped Flow CD spectropolarimeter (model 215). Inhibitor stock solutions of 5, 14, and 15 were prepared by dissolving the lyophilized powders into Millipore water at a final concentration of 80 μM. The spectra of each inhibitor were recorded using the method above. Additionally, the spectra of an equimolar solution of the Aβ₄₂ stock and each inhibitor stock were recorded after incubation for 18 h at 37 °C with shaking.

Transmission electron microscopy

Samples for TEM studies were taken from the ThT assay during the delayed lag phase. Each grid was prepared by aliquoting 10 μL of sample from the appropriate wells at the 6 h timepoint. The samples were pipetted onto formvar and carbon coated electron microscopy grids (Ted Pella, catalog no. 01754-F) and incubated for 3 min. Capillary action was used to remove the remaining liquid from the grids using filter paper. The grids were rinsed with Millipore water and stained with 1% (w/v) uranyl acetate solution. Samples were imaged using FEI M268D; Morgagni Series TEM.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We thank Dr Kamlesh Makwana for assistance with data analysis and preparation of figures. This work was supported by a grant from the National Science Foundation (CHE1709927).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ra02009e

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