
==== Front
Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

38285951
202311733
10.1073/pnas.2311733121
research-articleResearch ArticlebiochemBiochemistrychemChemistry407
410
Biological Sciences
Biochemistry
Physical Sciences
Chemistry
Helical sulfonyl-γ-AApeptides modulating Aβ oligomerization and cytotoxicity by recognizing Aβ helix
Liu Heng a https://orcid.org/0000-0002-0456-4626

Cui Yunpeng a https://orcid.org/0009-0006-4865-0493

Zhao Xue a https://orcid.org/0009-0000-4872-1577

Wei Lulu a https://orcid.org/0000-0003-4657-9479

Wang Xudong b
Shen Ning a
Odom Timothy a
Li Xuming a
Lawless William a
Karunarathne Kanchana c https://orcid.org/0009-0008-8271-255X

Muschol Martin c
Guida Wayne a https://orcid.org/0000-0003-3353-4853

Cao Chuanhai d
Ye Libin b https://orcid.org/0000-0003-0818-2972

Cai Jianfeng jianfengcai@usf.edu
a 1 https://orcid.org/0000-0003-3106-3306

aDepartment of Chemistry, University of South Florida, Tampa, FL 33620
bDepartment of Molecular Biosciences, University of South Florida, Tampa, FL 33620
cDepartment of Physics, University of South Florida, Tampa, FL 33620
dTaneja College of Pharmacy, University of South Florida, Tampa, FL 33612
1To whom correspondence may be addressed. Email: jianfengcai@usf.edu.
Edited by Robert Tycko, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD; received July 11, 2023; accepted December 10, 2023

29 1 2024
6 2 2024
29 7 2024
121 6 e231173312111 7 2023
10 12 2023
Copyright © 2024 the Author(s). Published by PNAS.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Significance

Unlike general strategies to recognize Aβ aggregates and prevent Aβ fibrillar progression, we herein report the design of sulfonyl-γ-AApeptide helical foldamer that can recognize central Aβ sequence and stabilize Aβ helix, an off-pathway structure driving dissociation of Aβ aggregation. circular dichroism, 2D-NMR, and ESI-IMS mass spectrometry as well as the cell-based studies collectively support the mechanism of the foldamer for Aβ aggregation antagonism. The results may lead to a generation of agents combating Alzheimer’s disease.

In contrast to prevalent strategies which make use of β-sheet mimetics to block Aβ fibrillar growth, in this study, we designed a series of sulfonyl-γ-AApeptide helices that targeted the crucial α-helix domain of Aβ13-26 and stabilized Aβ conformation to avoid forming the neurotoxic Aβ oligomeric β-sheets. Biophysical assays such as amyloid kinetics and TEM demonstrated that the Aβ oligomerization and fibrillation could be greatly prevented and even reversed in the presence of sulfonyl-γ-AApeptides in a sequence-specific and dose-dependent manner. The studies based on circular dichroism, Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) spectra unambiguously suggested that the sulfonyl-γ-AApeptide Ab-6 could bind to the central region of Aβ42 and induce α-helix conformation in Aβ. Additionally, Electrospray ionisation-ion mobility spectrometry–mass spectrometry (ESI-IMS-MS) was employed to rule out a colloidal mechanism of inhibitor and clearly supported the capability of Ab-6 for inhibiting the formation of Aβ aggregated forms. Furthermore, Ab-6 could rescue neuroblastoma cells by eradicating Aβ-mediated cytotoxicity even in the presence of pre-formed Aβ aggregates. The confocal microscopy demonstrated that Ab-6 could still specifically bind Aβ42 and colocalize into mitochondria in the cellular environment, suggesting the rescue of cell viability might be due to the protection of mitochondrial function otherwise impaired by Aβ42 aggregation. Taken together, our studies indicated that sulfonyl-γ-AApeptides as helical peptidomimetics could direct Aβ into the off-pathway helical secondary structure, thereby preventing the formation of Aβ oligomerization, fibrillation and rescuing Aβ induced cell cytotoxicity.

Alzheimer’s disease
Aβ aggregation
foldamer
sulfonyl-γ-AApeptides
helix stabilization
HHS | NIH | National Institute on Aging (NIA) 100000049 2R01AG056569-06 Jianfeng Cai HHS | NIH | National Institute of General Medical Sciences (NIGMS) 100000057 1R01GM150196 Jianfeng Cai
==== Body
pmcThe transformation of misfolded peptides and proteins into amyloid aggregates and insoluble fibers is the common characteristic of neurodegenerative diseases, including Alzheimer’s disease (AD), Huntington’s disease, Parkinson’s disease, etc. (1–6). Additionally, the formation of amyloid aggregation also leads to other diseases such as type 2 diabetes and cancer (1). Among them, as the most prevalent neurodegenerative defect, AD is causing significant economic and social burden around the world. It is estimated that currently, 47 million people are suffering from AD and the number is expected to continue to surge to reach about 65 million by 2030 (7).

The aggregation and fibrillar formation of β-amyloid peptide Aβ42 has been considered as the hallmark of AD onset (6, 8) and is believed to play a critical role in the pathogenetic pathway of AD (9–12). Thus, it has been an active target for the development of therapeutic agents to treat AD or molecular probes to probe the mechanism of AD pathogenesis (13). To date, a large amount of work has been carried out to identify antagonists of Aβ aggregation, including protein antibodies (14–16), β sheet mimetics (17, 18), N-methylated and N-Amino peptide (19, 20), cyclic alpha peptide (21), and helix-mimicking foldamers (22–25). These efforts were focused on identifying the specific ligands which could either prevent β-sheet growth of Aβ aggregates or stabilize Aβ in its α-helix conformation.

We have recently been developing a unique class of foldameric peptidomimetics termed “sulfonyl-γ-AApeptide” (Fig. 1A). Their X-ray crystal structures reveal that this class of oligomers folds into 414-helices with a helical pitch of 5.1 Å (Fig. 1 B and C). Their well-defined and superior helical folding propensity to α-helix arises from sulfonamido moieties in the molecular backbone and the intramolecular hydrogen bonding (26). Together with proteolytic resistance and the enormous potential for diversifying side chain functional groups, sulfonyl-γ-AApeptides have been shown to be promising candidates for biomedical and material applications (27). For instance, to date a diverse set of helical sulfonyl-γ-AApeptide foldamers have been reported to disrupt medicinally relevant protein–protein interactions (28, 29) and to recognize protein targets with excellent binding affinity and specificity (30, 31). Although previous efforts were based on the mimicry of the helical domains of various targeted proteins, the highly predictable spatial distribution and positioning of side chains along the molecular framework prompted us to speculate that this class of helical foldamer, without mimicking any α-helix, could be rationally designed to recognize the surface of Aβ so as to stabilize its helical conformation (Fig. 1D), thereby driving Aβ aggregates into off-pathway helical secondary structures devoid of AD development.

Fig. 1. The structure of sulfonyl-γ-AApeptides and their use as ligands to stabilize helical structure of Aβ peptide. (A) Chemical structure of sulfonyl-γ-AApeptide. "a" and "b" represent the chiral side chain and sulfono side chain, respectively. (B and C) are the side view and top view of sulfonyl-γ-AApeptide foldamer. (D) The schematic illustration of helical Aβ peptide stabilized by ligands.

Herein, we describe our approach of sulfonyl-γ-AApeptides as the helical foldamer to interact with Aβ42 and induce its α-helical conformation, so as to drive Aβ into off-pathway secondary structure. The design led to the identification of a sulfonyl-γ-AApeptide sequence Ab-6 as one of the most potent inhibitors toward Aβ aggregation and neurotoxicity. Both fluorescence titration and MST assays reveal the nanomolar range of binding affinity Kd for sulfonyl-γ-AApeptides toward Aβ. In addition, the aggregation and fibrillation of Aβ42 were effectively inhibited by Ab-6 even up to 24 h. Seeding assays show that Ab-6 was equally effective toward inhibiting the Aβ aggregation process in the presence or absence of seeds. Both CD and HSQC 2D-NMR suggest that Ab-6 induced a α-helical conformation within Aβ and bound to the central sites in the α-helical domain. ESI-IMS-MS indicates that Ab-6 could bind to Aβ42 specifically, without multiple copies of ligand, which further confirms the specificity of their interaction. Furthermore, the cell cytotoxicity assay showed that Ab-6 could rescue mouse neuroblastoma cells from Aβ42-induced cytotoxicity dramatically even at a low molar ratio and in the presence of pre-formed Aβ42 aggregates strongly implying that Aβ42 aggregation could be prevented in the cellular environment. The targeting specificity Ab-6 of toward Aβ42 in cellular environment was also demonstrated by confocal microscopy, which delineates the colocalization of Ab-6 and Aβ42 in mitochondria, suggesting that the rescue of cell viability might be at least partially due to protection of mitochondria function from being damaged by Aβ42 aggregation.

Results and Discussion

Design and Antagonism of Sulfonyl-γ-AApeptide Foldamer Targeting Aβ.

Both Aβ40 and Aβ42 are unstructured in aqueous solution; however, they could adapt to certain secondary structures upon environmental stimuli (32). Although Aβ42 exhibits higher tendency for aggregation than Aβ40 (33), in the presence of sodium dodecyl sulfate (SDS) surfactants and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), both peptides were shown to form α-helical conformations within residues 15 to 24 and 29 to 35 (32, 34–36). As such, molecules could be designed to target the α-helical portion of Aβ and induce and stabilize the secondary helical structure within Aβ (22–24) Such interaction would thereafter alter the aggregation kinetics of Aβ into off-pathway helical structures devoid of AD pathogenesis (Fig. 2) and thereby reducing the Aβ-mediated cytotoxicity.

Fig. 2. Design of sulfonyl-γ-AApeptides targeting Aβ. (A) The potential binding sites on the helical Aβ40 peptide (PDB: 1BA4), which are named as cationic domain 1, hydrophobic domain 2, and anionic domain 3. (B) Chemical structures of sulfono-γ-AApeptides Ab-1, Ab-2, Ab-3. (C) Helical wheel of sulfonyl-γ-AApeptides to illustrate the distribution of side chains. (D–G) proposed PPI between Ab-3 and the Aβ helix. (D and E) Side view in which Aβ in stick or surface representation. (F) Closed view showing the interacting residues. (G) Hypothesized electrostatic and hydrophobic interactions between side chains. (H and I) Kinetic profile of 10 μM Aβ42 amyloid β-sheet formation in the absence (control) and presence of 10 µM Ab-1, Ab-2, and Ab-3, respectively.

It is widely known that the hydrophobic fragment LVFFA of Aβ is mostly responsible for the aggregation of Aβ and amyloid β-sheet formation (37, 38). It is thus speculated that molecules which are designed to target this region in the α-helical Aβ peptide could stabilize Aβ helix and prevent the formation of β-sheet and subsequent oligomerization and fibrillation. Through a close analysis of the α-helical Aβ40 (PDB: 1BA4) structure, we arbitrarily assigned three domains that might be critical for the design of ligands to recognize and stabilize Aβ helix (Fig. 2A). These include cationic domain 1 bearing mainly positively charged or polar residues (H13, H14, Q15, and K16), hydrophobic domain 2 containing hydrophobic fragment (L17, V18, F19, F20, and A21) crucial for aggregation, and anionic domain 3 consisting of negatively charged residues (E22 and D23). We speculated that ligands with complementary hydrophobic and charged groups, as well as the ability to simultaneously target multiple domains of the Aβ helix, could bind and stabilize the Aβ helix by forming both hydrophobic and electrostatic interactions with the residues in Aβ. To this end, based on the helical folding pattern of sulfonyl-γ-AApeptides, we designed a series of sulfono-γ-AApeptides projecting a range of side functional groups and investigated their ability to target the helical Aβ.

We first synthesized three sulfonyl-γ-AApeptide sequences Ab-1, Ab-2, and Ab-3 (Fig. 2B), which consist of five sulfonyl-γ-AA peptide building blocks and are comparable to a 10-mer peptide in length. Our previous findings demonstrated that sulfonyl-γ-AApeptides of this length are sufficiently forming the helical structure in solution (26). The helical wheel of sulfonyl-γ-AApeptides (Fig. 2C) which maps the distribution of the side chains of sulfonyl-γ-AApeptides suggested two helical faces (1a-3a-5a and 1b-3b-5b) may be utilized to engage the interaction with residues in the cationic domain 1 and hydrophobic domain 2 (13–21) in the Aβ helix. In brief, sulfonyl-γ-AApeptides bearing negatively charged groups near N-termini and hydrophobic groups on the C-termini should have good complementarity to the Aβ helix and thus are expected to display effective binding affinity. For instance, a modeling shown in Fig. 2 D–G suggested that all of six side chains of Ab-3 could form either charge–charge electrostatic attraction (1a↔H14, 1b↔Q15) or hydrophobic interaction (3a↔L17, 3b↔V18, 5a↔F20, 5b↔A21) with the Aβ helix (Fig. 2G). All of the three sequences exhibited antagonism toward Aβ aggregation with ThT-based assay (Fig. 2 H and I) (39). In the presence of equimolar Ab-1, the maximum fluorescence was reduced to 58.0% in comparison to the control after 24 h, indicating the aggregation of Aβ42 has been inhibited by 42%. Ab-2 revealed a very similar inhibitory effect to Ab-1, which is consistent with the fact that both Ab-1 and Ab-2 have exactly same side chains (1a, 1b, 3a, 3b, 5a, and 5b) to interact with Aβ42 according to our design (Fig. 2 B–G). Notably, Ab-3 (Fig. 2B) was demonstrated to be the most potent inhibitor toward the aggregation process of Aβ42 among these three sequences. Neither Ab-1 nor Ab-2 was capable of elongating the latency time (the time before aggregation is detected) of Aβ42 aggregation which is around 9 h in our assay condition. However, Ab-3 significantly increased the latency time to ~14 h and delayed the aggregation process. At 24 h, Ab-3 could inhibit Aβ42 aggregation by 60% compared with the control. The difference in the inhibition could be well rationalized by our design and modeling. Compared with both Ab-1 and Ab-2 projecting isopropyl and isobutyl groups as the side chains of 5a and 5b, Ab-3 has aromatic groups at those positions, which are speculated to have stronger hydrophobic interactions with F20 and A21 in Aβ.

Our initial success prompted us to design more potent inhibitors for Aβ aggregation and fibrillation. We speculated that targeting residues spanning all three domains in Aβ could enhance the binding affinity of our foldamers toward Aβ and thereby further stabilizing the Aβ helical structure. To this end, we noticed that residues Q15, F19, and E22 in the Aβ are located at the positions of i, i + 4, and i + 7, which are aligned on virtually one face of the Aβ helix. Intriguingly, residues K16, F20, and D23 also assume the same relationship of i, i + 4, and i + 7 and are lined on the face just adjacent to the helical face bearing Q15, F19, and E22. Therefore, a different series of sulfonyl-γ-AApeptides (Ab-4, Ab-5, and Ab-6) were designed (Fig. 3A). The helical wheel (Fig. 3B) suggested that 1a, 3a, and 5a could interact with Q15, F19, and E22, whereas 1b, 3b, and 5b on the adjacent face were speculated to target K16, F20, and D23, respectively. A close positioning of the Aβ helix with Ab-6 revealed that the side chains from the sulfonyl-γ-AApeptide could have excellent complementarity with the residues in the Aβ helix by potentially forming electrostatic or hydrophobic interactions (Fig. 3 C–F). One thing particularly noteworthy is that the side chain 3b could insert deeply into the hydrophobic pocket formed by F19, F20, and A21. Also, we observed that one more building block in sulfonyl-γ-AApeptides (comparable to 12-mer peptides in length) may confer additional hydrogen bond between their carboxyamide terminus with S26 (Fig. 3F). Since all three domains, cationic domain 1 (Q15, K16), hydrophobic domain 2 (F19 and F20), as well as anionic domain 3 (E22, D23) and the additional S26, were expected to be targeted with the design, we anticipated that this series of sulfonyl-γ-AApeptides would stabilize the Aβ helix and thereby inhibiting Aβ oligomerization and fibrillation more effectively. As expected, Ab-6 was demonstrated the most potent inhibitor to prevent the Aβ aggregation (Fig. 3 G and H), with virtually no aggregation being observed even up to 24 h. Ab-5, with isopropyl and isobutyl side chains at the positions of 3a and 3b, was believed to have weakened hydrophobic interaction with F19 and F20 in comparison to aromatic moieties in Ab-6 and thus only show 20% of inhibition for the Aβ oligomerization at 24 h. Nonetheless, Ab-5 was still noticed for its ability to slow down the aggregation process as. It is interesting that with an aromatic group at 5a compared to Ab-5, Ab-4 completely abolished inhibitory activity for Aβ aggregation, suggesting that the interaction with negative charged residues in domain 3 of Aβ is also crucial to stabilize the Aβ helix.

Fig. 3. Design of another series of sulfonyl-γ-AApeptides targeting Aβ. (A) Chemical structures of sulfono-γ-AApeptides Ab-4, Ab-5, Ab-6. (B) Helical wheel of sulfono-γ-AApeptides to illustrate the distribution of side chains. (C–F) Proposed PPI between Ab-6 and the Aβ helix. (C) Side view. (D) Closed view. (E and F) Hypothesized electrostatic and hydrophobic interactions between side chains. (G and H) Kinetic profile of 10 μM Aβ42 amyloid β-sheet formation in the absence (control) and presence of 10 µM Ab-4, Ab-5, and Ab-6, respectively.

Ab-6 Inhibited Aβ Aggregation by Stabilizing Aβ Helix.

As Ab-6 exhibited the most prominent antagonism for Aβ aggregation, it was next assessed for its binding affinity toward Aβ, which was first quantified by titrating Nα-fluorescein-labeled Aβ40 with Ab-6 (40). The fluorescence intensity diminished rapidly in the presence of increased concentration of Ab-6, revealing a binding affinity (Kd) of 0.32 μM for Ab-6 toward Aβ40 (Fig. 4A) that is in good agreement with its ability to inhibit Aβ aggregation determined by ThT kinetic assay. The binding affinity was also tested with the MST (microscale thermophoresis) assay (SI Appendix, Fig. S1), which revealed close binding affinity consistent with the fluorescence titration assay (41). It is known that preformed fibers of Aβ could catalyze the fibrillation via the nucleation (42), leading to key neurotoxic oligomers; the inhibition of such process is essential for therapeutic development. As shown in Fig. 4B, addition of preformed fibers (5%, v, v) to freshly aliquoted 10 μM Aβ42 accelerated the fibrillation by decreasing the lag time from 9 to 4 h. Notably, in the presence of Ab-6, the fibrillation of Aβ42 was almost completely inhibited with little formation of ThT-positive fibrillar aggregation up to 24 h. We next attempted to examine whether Ab-6 induced helical secondary structural arrangement within Aβ. As shown in Fig. 4C, the CD spectrum of the initial aliquot of Aβ42 (0 h) reveals a double minimum at 202 and 215 nm, suggesting a major α-helical confirmation. The blue shift from the characteristic double minimum of pure α-helix (208 and 222 nm) indicates the co-existence of β-sheets, which is consistent with the high tendency of Aβ42 aggregation and amyloid formation. As anticipated, the Aβ peptide completely transitioned into β-sheet after 24 h, as assessed by the CD spectrum which displays a characteristic single minimum at 214 nm (Fig. 4C). On the contrary, in the presence of Ab-6 at the equimolar ratio (Fig. 4D), Aβ42 shows a characteristic double minimum of pure α-helix at 208 and 222 nm even at 0 h, suggesting the rapid induction of the α-helical conformation in Aβ42 by Ab-6. Echoing with the findings obtained from ThT kinetic assay, in the presence of Ab-6, Aβ42 retained the α-helical conformation, preventing the transition into β-sheet and subsequent oligomerization and fibrillation.

Fig. 4. Binding characterization of Ab-6 toward Aβ peptides. (A) Binding curve of Ab-6 against Nα-fluorescein-labeled Aβ40 (50 nM). (B) Kinetic profile of 10 µM Aβ42 seed-induced aggregation in the absence (blue line) and presence of Ab-6 (orange line). (C) Circular dichroism (CD) spectra of Aβ42 (20 μM) in the absence of Ab-6. (D) CD spectra of Aβ42 (20 µM) in the presence of Ab-6 (20 µM). (E–J) TEM micrographs. (E) 10 µM Aβ42 at 0 h. (F) 10 µM Aβ42 at 24 h. (G) 10 µM Aβ42 with equimolar Ab-6 at 0 h. (H) 10 µM Aβ42 with equimolar Ab-6 at 24 h. (I) Seed-catalyzed fibrillation of 10 µM Aβ42 in the absence of Ab-6 at 24 h. (J) Seed-catalyzed fibrillation of 10 µM Aβ42 in the presence of Ab-6 at 24 h. (K) The particle size distribution of Ab42 (10 µM) after incubation for 24 h. (L) The correlation function of Aβ42 (10 µM) after incubation for 24 h. (M) Relative scattering intensities of Aβ42 (10 µM) after incubation for 24 h in the absence or presence of indicated molar ratios of Ab-6. Corresponding values for scattering from Ab-6 (10 µM) or buffer only are shown, as well.

The ability of Ab-6 to prevent and disrupt Aβ fibrillation was also visualized by TEM studies. No obvious aggregation of Aβ42 was observed at 0 h (Fig. 4E), and Aβ42 alone would aggregate to form fibrillar structures after 24 h, as observed in Fig. 4F. However, the aggregation process could be halted by equimolar Ab-6; no fiber morphology was observed (Fig. 4 G and H). Since the oligomerization of Aβ is associated with the pathology of AD (6), we also studied the effect of Ab-6 on preformed Aβ seed catalyzed process. As shown in Fig. 4I, in the presence of 5% seed (v/v) seed, a huge number of fibers were identified after 24 h. However, in the presence of 1.2 eq Ab-6, no amorphous aggregates were noticed (Fig. 4J). TEM analysis collectively supported that Ab-6 effectively inhibited Aβ oligomerization and fibrillation. To further confirm the anti-fibrillation effect of Ab-6, static (SLS) and dynamic (DLS) light scattering were next carried out. As seen in Fig. 4K, in the absence of Ab-6, fibril formation results in a well-defined correlation after 24 h of incubation. The corresponding size distribution (Fig. 4L) is bimodal with apparent radii of 60 and 300 nm (43). The highly elongated shape of fibrils, together with potential contributions from non-diffusive rotational and vibrational relaxation modes, prevents direct comparison of the apparent radii derived from DLS with actual fibril dimensions (44). However, after incubation of 10 μΜ Aβ with a fivefold excess of Ab-6 for 24 h, we found that the derived count rates were distinguishable from those of Ab-6 and buffer only (Fig. 4M). This is consistent with the inherent limitations of light scattering from detected small protein monomers (<6 kD) at sub-millimolar concentrations. Overall, our data indicated that Ab-6 could abrogate Aβ aggregation effectively. Therefore it is a potent inhibitor of Aβ aggregation.

2D-NMR and DOSY-NMR Studies to Investigate the Binding Mode of Ab-6 toward Aβ42.

To gain insight into the effect of Ab-6 on Aβ42 aggregation, a set of time-course NMR experiments were carried out. As shown in Fig. 5 A and B, there was no significant NMR spectral difference observed in the initial incubation of 6 h between Aβ42 alone and Ab-6 co-incubated Aβ42, consistent with the research of Wälti et al. (45) The result was also consistent with the ThT aggregation assay where no obvious ThT-positive signal was observed in the initial 8 h. However, a significant intensity of NMR resonances was observed for the apo sample (Fig. 5A) after 24 h incubation along with linewidth broadening whereas no substantial changes were observed in the Ab-6 co-incubated sample (Fig. 5B), suggesting that Ab-6 plays a role in inhibiting Aβ42 aggregation. We also employed 2D gNHSQC to further investigate the effect of Ab-6 on conformational changes of Aβ42. A structural perturbation was observed in Ab-6 co-incubated Aβ42 sample, reflected from the chemical shift differences of the Aβ42 in reference to free Aβ42 (Fig. 5C). The most prominent chemical shifts were observed from H13 to D23 (Fig. 5D), which is highly consistent with our initial design, in which Ab-6 was expected to interact with all three domains of Aβ42, cationic domain 1, hydrophobic domain 2, and negatively charged domain 3 (Fig. 3F). The diffusion-ordered NMR spectroscopy (DOSY-NMR) was also subsequently carried out to confirm the interaction between Aβ42 and Ab-6 (46, 47). As seen in Fig. 5 E–G, the decay rate of Aβ42 was significantly decreased upon the addition of Ab-6 in reference to free Aβ, indicating the interaction between Ab-6 and Aβ42.

Fig. 5. Investigation of Ab-6 binding to Aβ42 using NMR and ESI-MS. (A) Time-course 1H NMR spectra where the spectra were recorded at 0 h, 6 h, and 24 h, respectively, with 40 μM Aβ42 and (B) with 2.5 eq of Ab-6. (C) Overlay of the 1H–15N HSQC 2D-NMR spectra of 40 µM 15N- Aβ42 alone (black) and with Ab-6 at a stoichiometric ratio of 1:2.5 (15N-Aβ42:Ab-6). (D) Change of chemical shift in the presence of Ab-6. nd: not detected. (E) DOSY NMR of Aβ42 alone (40 μM). (F) DOSY NMR of Aβ42 with Ab-6 at a stoichiometric ratio of 1:2.5. (G) DOSY experimental analyses on the peak at 0.68 ppm derived from Aβ42 with and without Ab-6. (H) The sequence of Aβ42. (I) ESI-MS mass spectrum of Aβ42 alone at 0 h. (J) ESI-MS mass spectrum showing Ab-6 added at 110 μM to Aβ42 at 22 μM (denoted as ALx+, where A is Aβ42, L is Ab-6, x is the charge state). (K) ESI-IMS-MS Driftscope plot of Aβ42 at 22 μM in H2O showing IMS drift time versus m/z versus intensity. (L) ESI-IMS-MS Driftscope plot of Aβ42 (22 μM) and Ab-6 (110 μM) in H2O showing IMS drift time versus m/z versus intensity.

ESI-IMS-MS to Characterize the Ligand-Binding Capability of Helical Conformers of Aβ42.

Although extracellular plaques are considered as the hallmark of AD, the pre-fibrillar oligomers are believed to cause the major neurotoxic effect (48). However, the quantification of oligomers is a challenge due to the heterogeneity of the Aβ peptides. Electrospray ionization-ion mobility spectrometry (ESI-IMS) has been employed due to its potential to detect multiple ions present at the same mass-to-charge ratio (m/z) (49–56) as well as to detect Aβ-ligand complexes (57, 58). The mechanism of inhibition could be probed (59). Here, we also employed ESI-IMS-MS to explore Aβ42 aggregation and to investigate the mechanism behind the inhibition of Aβ42 by Ab-6.

Prior to performing inhibition studies, we first studied the oligomerization of Aβ42 alone (Fig. 5 I and K). As reported before (53–56, 60), Aβ42 is highly prone to aggregate (61). Consistently, the ESI-MS mass spectrum of Aβ42 shows that both monomer and dimer after Aβ42 were freshly dissolved into H2O (SI Appendix, Fig. S4). To have a direct observation of the interaction mode between Ab-6 and Aβ42, Ab-6 was incubated with Aβ42 (molar ratio of Ab-6 to Aβ42 is 5:1) and the mixture sample was applied to the ESI-MS. The Aβ42-Ab-6 complex was detected (Fig. 5 J and L), where multiple charge sates were observed, including 4+, 5+, 6+, and 7+ (SI Appendix, Fig. S5). Some Aβ42 monomers were still observed, which was assumed to be the dissociation of the Aβ42-Ab-6 complex in ionization process.

Ab-6 Rescued Fibrillation and Cytotoxicity.

The oligomerization of Aβ is linked to the pathogenesis of AD, and it is known that the aggregation exhibits significant cell cytotoxicity. Since Ab-6 has shown great potential to inhibit the process in vitro, we moved forward to investigate the cytotoxicity profile of Aβ42 upon treatment of Ab-6. The cell-based experiment was first conducted using N2a cells (mouse neuroblastoma cell line). Treatment with 3 µM fresh aliquot of Aβ42 reduced the viability of N2a cells to 78.6% after 24 h incubation (Fig. 6A). Even with the addition of 0.25 eq Ab-6, the cell viability boosted to 91.7% after 24 h. When 0.5 eq, 0.75 eq, and equimolar ratio were tested separately, the cell viability increased to 95.7, 98.2, and 99.6%. The cell toxicity assay demonstrated that Ab-6 effectively rescued cytotoxicity induced by Aβ42 aggregation in a dose-dependent manner, with the EC50 of 0.46 µM (Fig. 6B).

Fig. 6. Probing interaction between Ab-6 and Aβ42 in cellular assays. (A and B) Cytotoxicity of 3 μM Aβ42 applied to N2a cells with/without Ab-6 measured by the CCK-8 assay. (C and D) Cytotoxicity of 3 μM pre-aged Aβ42 toward N2a cells with/without Ab-6. Aβ42 was allowed to aggregate for 12 h before adding into cell culture. (E) The cell viability of N2a cell line at various concentrations of Aβ42. (F) In the presence of equimolar amount of Aβ42 and Ab-6, the cell viability of N2a cell line. (G) The cell viability of SH-SY5Y cell line at various concentrations of Aβ42. (H) In the presence of equimolar amount of Aβ42 and Ab-6, the cell viability of SH-SY5Y cell line. Full cell viability was normalized to 1 (100%). (I) Co-localization of Aβ42 and Ab-6 investigated using confocal microscope. N2a cells were incubated with 4 µM Aβ42 or Ab-6 and their fluorescent counterparts (1 µM AβHF647 or Ab-6F) for 24 h. (Scale bar, 20 µM.)

We next asked whether Ab-6 could disrupt preformed Aβ42 aggregation, as preformed seeds of Aβ not only are challenging to be disrupted but also accelerating the fibrillation to rapidly generate the key neurotoxic oligomers. Inhibition of such a process is considered more essential from a therapeutic perspective, as it could have profound significance by reversing the pathological progression of AD in addition to prevention at the early stage. To this end, Aβ42 was pretreated for 12 h before coincubation with the cell lines to allow the formation of prefibrillar structures, following with the addition of various concentrations of Ab-6. As shown in Fig. 6 C and D, in the absence of Ab-6, the cell viability dropped to 78.6%. However, after addition of only 0.5 eq of Ab-6, the viability went up to 86.1%. When 1 eq, 2 eq, and 4 eq of Ab-6 were tested, the viability was all above 90%, suggesting that Ab-6 could disrupt preformed Aβ42 aggregates and rescue the cell viability by reversing Aβ42 cytotoxicity. Overall, both prevention and disruption experiments demonstrated a potent antagonistic ligand toward the Aβ42 induced cytotoxicity in cellular milieu.

After confirming that equivalent Ab-6 could potentially mitigate the cell toxicity caused by Aβ aggregation, we set out to investigate the cell toxicity profile with a variety of Aβ42 concentrations with or without equivalent amount of Ab-6. As seen in Fig. 6E, the N2a cell viability decreased significantly in a dose-dependent manner, from 81 to 51%, as Aβ concentrations went up. Intriguingly, after co-incubation with equivalent Ab-6, the cell viability are all above 95% even when Aβ42 concentration reached 20 µM (Fig. 6F). The cell viability are still above 80% even in the presence of 50 µM Aβ42, demonstrating the strong potential of Ab-6 in inhibiting Aβ42 toxicity in various concentrations. Next, to verify that the toxicity profiles of Ab-6 were not artifactual to the model cell line used, we chose SH-SY5Y neuroblastoma cell line as the second model for the cell viability study. We first carried out the cell toxicity of Aβ alone with verifying concentrations. The cell viability also decreased from 88 to 46%, in the presence of increased concentration of Aβ42 from 1 to 50 µM (Fig. 6G). In the presence of equivalent Ab-6, the cell viability kept to be 90% or above even Aβ42 concentration increased to 20 µM, which is highly analogous to the behavior in N2a cells. When Aβ42 concentration reached to 50 µM, the presence of Ab-6 at the equimolar ratio could still enhance the cell by about 50%, suggesting could Ab-6 retain high potential antagonism of Aβ aggregation in the cellular conditions in different cell lines.

Ab-6 Interacts with Aβ42 in Cellular Condition.

While some ligands have demonstrated promising antagonist activity in solution studies, their mechanism of cellular activity was not known (62). To assess whether the cellular antagonism of Ab-6 toward Aβ42 was due to their direct interaction as observed in vitro, we subsequently employed confocal microscopy to investigate the interaction of Ab-6 with Aβ42 in the cellular environment. Aβ42 and Ab-6 were respectively labeled with HiLyte Fluor 647 (AβHF647) and fluorescein (Ab-6F). Confocal microscopy (Fig. 6I) revealed that N2a cells effectively took up both Aβ42 (1 μM AβHF647 + 4 μM Aβ42) and Ab-6 (4 μM Ab-6 + 1 μM Ab-6F) into mitochondria within 24 h of incubation. The uptake of Aβ42 by the cells is similar to previous reports which suggest that Aβ may exert its neurotoxicity by causing mitochondrial dysfunction (63–65). The favorable cell permeability of Ab-6 may be attributed to the net positive charges it carries. The fact that the Aβ42-Ab-6 complex can effectively penetrate the cell membrane and co-localize in the cells indicates that there is a strong binding interaction between the two molecules even in the complex intracellular environment, providing important insight into the inhibitory mechanism of Ab-6 as a potential antagonist of Aβ42. The results may suggest that the ability of Ab-6 to rescue the cell viability is due to its protection of mitochondrial function which may be impaired by Aβ42 aggregation.

Discussion

Amyloid fibril formation, characterized by the self-assembly and aggregation of peptides or proteins into insoluble cross-β deposits (66, 67), is a complicated process. The resulted human disorders, range from AD, Parkinson’s disease, to type II diabetes and others (13, 68–72). From therapeutic point of view, it could be a potential treatment option by modulating the oligomerization process. In this article, we illustrated our strategy of inhibiting Aβ42 aggregation through the rational design and optimization of helical sulfonyl-γ-AApeptide-based ligand toward Aβ42 aggregation. Unlike previously reported peptides designed to mimic β-sheets and prevent Aβ sheet growth and fibrillation, herein we proposed to design helical peptidomimetics that recognize Aβ helix and therefore modulate Aβ conformation into off-pathway structure to avoid forming the neurotoxic Aβ oligomeric fibrils. To facilitate the design of the ligands, we arbitrarily assigned three domains on Aβ that might be critical for the design of ligands to recognize and stabilize Aβ helix (Fig. 2A), including cationic domain 1, hydrophobic domain 2, and anionic domain 3; such a design was subsequently proved to be successful. We initially focused on cationic domain 1 (Q15, K16) and hydrophobic domain 2 (F19 and F20). Based on the two domains, Ab-3 was identified as potential antagonist. After we set out to design molecules targeting all the three domains, we found Ab-6 as the most potent ligand toward Aβ42 aggregation. Ab-6 was found to not only prevent Aβ aggregation but also disrupt the preformed Aβ fibrillar nucleates. 1H–15N HSQC NMR spectrum clearly confirms the design by showing obvious shifts in all the three domains of Aβ. Circular dichroism spectrum analysis was carried to characterize the secondary structure induction because of Ab-6. Without Ab-6, conformation transformation from α-helix to β-sheets was clearly detected. In the presence of equimolar Ab-6, α-helix conformation becomes the main species throughout the course of the assay. In addition, ESI-IMS-MS analysis helped to investigate the details of the Aβ42 aggregation and inhibition, which demonstrated that Ab-6 and Aβ42 interact specifically in multiple charge status rather than random and non-specific interaction. The interaction between Ab-6 and Aβ42 retained in the cellular milieu. Again, Ab-6 was found to rescue cytotoxicity-induced Aβ42 aggregation with the ability of both prevention and reverse the cytotoxic effect of preformed Aβ42 aggregates, analogous to in vitro findings. Last but not least, confocal microscopy demonstrated that Ab-6 and Aβ42 could co-localize in mitochondria, which may account for its capability to interact with Aβ42 in cellular environment and rescue cytotoxicity due to the mitochondrial damage caused by Aβ42.

Conclusion.

In this article we describe an alternative strategy in which sulfonyl-γ-AApeptide foldamer was designed to induce and stabilize an α-helical conformation within Aβ. By exploiting a series of SAR studies and biophysical assays such as CD, DLS, 2D and DOSY NMR, and ESI-IMS-MS, we were able to identify a lead compound, Ab-6, which could specifically interact with several domains within Aβ and stabilize the Aβ as predominantly α-helical conformation. The Aβ structure stabilized by Ab-6 is nontoxic and does not exhibit prion-like property. The studies illustrate potential sites on Aβ for binding, providing insight into designing more efficient Aβ antagonists for Aβ recognition. The ability to disrupt pre-fibril Aβ in vitro and in cellular milieu makes Ab-6 a potential therapeutic agent for the treatment of AD. In addition, we believe the strategy to induce secondary structure in Aβ, and the design strategy of sulfonyl-γ-AApeptides for protein/peptide recognition could be applied to combat other amyloid proteins.

Materials and Methods

Materials.

Aβ42 were purchased at >95% purity from NovoPep and used without further purification. N15-labeled wild-type human Aβ42 was purchased from rPeptide. ThT was obtained from TCI. The 96-well plates (black, flat bottom) were purchased from Thermal Scientific. All chemicals were purchased from commercial suppliers.

Peptide Preparation.

Aβ42 and N15-labeled Aβ42 were dissolved in 1,1,1, 3, 3, 3-hexafluoroisopropanol (HFIP) (1 mg/mL) and kept at 37 °C overnight. Peptides were then aliquoted and lyophilized and stored at −80 °C until use.

Synthesis of Sulfono-γ-AApeptide Helical Mimetics.

The synthesis protocol and the characterization of the relevant compounds are presented in detail in SI Appendix.

Supplementary Material

Appendix 01 (PDF)

Click here for additional data file.

This work was supported by NIH 2R01AG056569-06 and NIH 1R01GM150196. We thank the Chemical Purification Analysis and Screening core facility for using the Agilent 6120 Mass Spectrometer and Agilent 6540 Mass Spectrometer. We thank University of South Florida (USF) Electron Microscopy Core Lab for helping withTransmission Electron Microscopy analysis. We thank USF Lisa Muma Weitz Laboratory for Advanced Microscopy & Cell Imaging core facility for help with confocal microscopy.

Author contributions

J.C. designed research; H.L., Y.C., X.Z., X.W., W.L., K.K., and L.Y. performed research; T.O., X.L., and W.G. contributed new reagents/analytic tools; L.W., T.O., and X.L. helped with discussion; W.G. helped with suggestion; H.L., Y.C., X.Z., L.W., X.W., N.S., W.L., K.K., M.M., C.C., L.Y., and J.C. analyzed data; and H.L. and J.C. wrote the paper.

Competing interests

The authors declare no competing interest.

Data, Materials, and Software Availability

All study data are included in the article and/or SI Appendix.

Supporting Information

This article is a PNAS Direct Submission.
==== Refs
1 J. L. Silva, E. A. Cino, I. N. Soares, V. F. Ferreira, G. A. P. de Oliveira, Targeting the prion-like aggregation of mutant p53 to combat cancer. Acc. Chem. Res. 51 , 181–190 (2018).29260852
2 L. S. Wolfe , Protein-induced photophysical changes to the amyloid indicator dye thioflavin T. Proc. Natl. Acad. Sci. U.S.A. 107 , 16863–16868 (2010).20826442
3 J. W. Kelly, The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways. Curr. Opin. Struct. Biol. 8 , 101–106 (1998).9519302
4 J. Hardy, D. J. Selkoe, The amyloid hypothesis of Alzheimer’s disease: Progress and problems on the road to therapeutics. Science 297 , 353–356 (2002).12130773
5 J. A. Hardy, G. A. Higgins, Alzheimer’s disease: The amyloid cascade hypothesis. Science 256 , 184–185 (1992).1566067
6 C. Haass, D. J. Selkoe, Soluble protein oligomers in neurodegeneration: Lessons from the Alzheimer’s amyloid beta-peptide. Nat. Rev. Mol. Cell. Biol. 8 , 101–112 (2007).17245412
7 R. Jakob-Roetne, H. Jacobsen, Alzheimer’s disease: From pathology to therapeutic approaches. Angew. Chem. Int. Ed. 48 , 3030–3059 (2009).
8 C. G. Glabe, Structural classification of toxic amyloid oligomers. J. Biol. Chem. 283 , 29639–29643 (2008).18723507
9 D. E. Ehrnhoefer , EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nat. Struct. Mol. Biol. 15 , 558–566 (2008).18511942
10 D. Maity, M. Howarth, M. C. Vogel, M. Magzoub, A. D. Hamilton, Peptidomimetic-based vesicles inhibit amyloid-beta fibrillation and attenuate cytotoxicity. J. Am. Chem. Soc. 143 , 3086–3093 (2021).33600171
11 H. F. Wu , gamma-AApeptide-based small-molecule ligands that inhibit A beta aggregation. Chem. Commun. 50 , 5206–5208 (2014).
12 C. Haass, D. J. Selkoe, Soluble protein oligomers in neurodegeneration: Lessons from the Alzheimer’s amyloid beta-peptide. Nat. Rev. Mol. Cell Biol. 8 , 101–112 (2007).17245412
13 R. Kayed , Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300 , 486–489 (2003).12702875
14 B. Solomon, R. Koppel, D. Frankel, E. Hanan-Aharon, Disaggregation of Alzheimer beta-amyloid by site-directed mAb. Proc. Natl. Acad. Sci. U.S.A. 94 , 4109–4112 (1997).9108113
15 P. Sormanni, F. A. Aprile, M. Vendruscolo, Rational design of antibodies targeting specific epitopes within intrinsically disordered proteins. Proc. Natl. Acad. Sci. U.S.A. 112 , 9902–9907 (2015).26216991
16 J. Delrieu, P. J. Ousset, T. Voisin, B. Vellas, Amyloid beta peptide immunotherapy in Alzheimer disease. Rev. Neurol. (Paris) 170 , 739–748 (2014).25459121
17 P. N. Cheng, C. Liu, M. L. Zhao, D. Eisenberg, J. S. Nowick, Amyloid beta-sheet mimics that antagonize protein aggregation and reduce amyloid toxicity. Nat. Chem. 4 , 927–933 (2012).23089868
18 S. A. Sievers , Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation. Nature 475 , 96–100 (2011).21677644
19 L. M. Yan , Selectively N-methylated soluble IAPP mimics as potent IAPP receptor agonists and nanomolar inhibitors of cytotoxic self-assembly of both IAPP and Abeta40. Angew. Chem. Int. Ed. 52 , 10378–10383 (2013).
20 K. C. Tillett, J. R. Del Valle, N-Amino peptide scanning reveals inhibitors of Abeta(42) aggregation. RSC Adv. 10 , 14331–14336 (2020).35498502
21 M. Richman , In vitro and mechanistic studies of an antiamyloidogenic self-assembled cyclic D, L-alpha-peptide architecture. J. Am. Chem. Soc. 135 , 3474–3484 (2013).23360549
22 S. Kumar, A. Henning-Knechtel, M. Magzoub, A. D. Hamilton, Peptidomimetic-based multidomain targeting offers critical evaluation of Abeta structure and toxic function. J. Am. Chem. Soc. 140 , 6562–6574 (2018).29648815
23 S. Kumar, A. D. Hamilton, alpha-helix mimetics as modulators of abeta self-assembly. J. Am. Chem. Soc. 139 , 5744–5755 (2017).28273416
24 S. Kumar, A. Henning-Knechtel, I. Chehade, M. Magzoub, A. D. Hamilton, Foldamer-mediated structural rearrangement attenuates Abeta oligomerization and cytotoxicity. J. Am. Chem. Soc. 139 , 17098–17108 (2017).29058422
25 J. Kaffy , Helical gamma-peptide foldamers as dual inhibitors of amyloid-beta peptide and islet amyloid polypeptide oligomerization and fibrillization. Chemistry 26 , 14612–14622 (2020).32542806
26 F. She , De novo left-handed synthetic peptidomimetic foldamers. Angew. Chem. Int. Ed. 57 , 9916–9920 (2018).
27 Y. Shi , Helical sulfono-gamma-AApeptides with aggregation-induced emission and circularly polarized luminescence. J. Am. Chem. Soc. 141 , 12697–12706 (2019).31335135
28 P. Sang , alpha-helix-mimicking sulfono-gamma-AApeptide inhibitors for p53-MDM2/MDMX protein–protein interactions. J. Med. Chem. 63 , 975–986 (2020).31971801
29 P. Sang , Inhibition of beta-catenin/B cell lymphoma 9 protein–protein interaction using alpha-helix-mimicking sulfono-gamma-AApeptide inhibitors. Proc. Natl. Acad. Sci. U.S.A. 116 , 10757–10762 (2019).31088961
30 P. Sang , The activity of sulfono-gamma-AApeptide helical foldamers that mimic GLP-1. Sci. Adv. 6 , eaaz4988 (2020).32440547
31 S. Abdulkadir , Modulating angiogenesis by proteomimetics of vascular endothelial growth factor. J. Am. Chem. Soc. 144 , 270–281 (2022).34968032
32 R. Riek, P. Guntert, H. Dobeli, B. Wipf, K. Wuthrich, NMR studies in aqueous solution fail to identify significant conformational differences between the monomeric forms of two Alzheimer peptides with widely different plaque-competence, A beta(1–40)(ox) and A beta(1–42)(ox). Eur. J. Biochem. 268 , 5930–5936 (2001).11722581
33 Y. Yan, C. Wang, Abeta42 is more rigid than Abeta40 at the C terminus: Implications for Abeta aggregation and toxicity. J. Mol. Biol. 364 , 853–862 (2006).17046788
34 J. Jarvet, J. Danielsson, P. Damberg, M. Oleszczuk, A. Graslund, Positioning of the Alzheimer Abeta(1–40) peptide in SDS micelles using NMR and paramagnetic probes. J. Biomol. NMR 39 , 63–72 (2007).17657567
35 L. C. Serpell, Alzheimer’s amyloid fibrils: Structure and assembly. Biochim. Biophys. Acta. 1502 , 16–30 (2000).10899428
36 M. Coles, W. Bicknell, A. A. Watson, D. P. Fairlie, D. J. Craik, Solution structure of amyloid beta-peptide(1–40) in a water-micelle environment. Is the membrane-spanning domain where we think it is? Biochemistry 37 , 11064–11077 (1998).9693002
37 P. N. Cheng, R. Spencer, R. J. Woods, C. G. Glabe, J. S. Nowick, Heterodivalent linked macrocyclic beta-sheets with enhanced activity against Abeta aggregation: Two sites are better than one. J. Am. Chem. Soc. 134 , 14179–14184 (2012).22827298
38 C. Liu , Out-of-register beta-sheets suggest a pathway to toxic amyloid aggregates. Proc. Natl. Acad. Sci. U.S.A. 109 , 20913–20918 (2012).23213214
39 A. Jan, O. Gokce, R. Luthi-Carter, H. A. Lashuel, The ratio of monomeric to aggregated forms of Abeta40 and Abeta42 is an important determinant of amyloid-beta aggregation, fibrillogenesis, and toxicity. J. Biol. Chem. 283 , 28176–28189 (2008).18694930
40 E. Andreetto , Identification of hot regions of the Abeta-IAPP interaction interface as high-affinity binding sites in both cross- and self-association. Angew. Chem. Int. Ed. 49 , 3081–3085 (2010).
41 M. Brockhaus , Thermodynamic studies on the interaction of antibodies with beta-amyloid peptide. J. Phys. Chem. B 111 , 1238–1243 (2007).17266280
42 S. I. Cohen , Proliferation of amyloid-beta42 aggregates occurs through a secondary nucleation mechanism. Proc. Natl. Acad. Sci. U.S.A. 110 , 9758–9763 (2013).23703910
43 J. Foley , Structural fingerprints and their evolution during oligomeric vs. oligomer-free amyloid fibril growth. J. Chem. Phys. 139 , 121901 (2013).24089713
44 C. Niyangoda , Origin, toxicity and characteristics of two amyloid oligomer polymorphs. RSC Chem. Biol. 2 , 1631–1642 (2021).34977578
45 M. A. Walti , Probing the mechanism of inhibition of amyloid-beta(1–42)-induced neurotoxicity by the chaperonin GroEL. Proc. Natl. Acad. Sci. U.S.A. 115 , E11924–E11932 (2018).30509980
46 X. Li, S. E. Rios, J. S. Nowick, Enantiomeric beta-sheet peptides from Abeta form homochiral pleated beta-sheets rather than heterochiral rippled beta-sheets. Chem. Sci. 13 , 7739–7746 (2022).35865901
47 Y. Wang, N. L. Truex, N. D. P. Vo, J. S. Nowick, Effects of charge and hydrophobicity on the oligomerization of peptides derived from IAPP. Bioorg. Med. Chem. 26 , 1151–1156 (2018).29074350
48 M. Sakono, T. Zako, Amyloid oligomers: Formation and toxicity of Abeta oligomers. FEBS J. 277 , 1348–1358 (2010).20148964
49 L. M. Young, P. Cao, D. P. Raleigh, A. E. Ashcroft, S. E. Radford, Ion mobility spectrometry-mass spectrometry defines the oligomeric intermediates in amylin amyloid formation and the mode of action of inhibitors. J. Am. Chem. Soc. 136 , 660–670 (2014).24372466
50 M. Kloniecki , Ion mobility separation coupled with MS detects two structural states of Alzheimer’s disease Abeta1-40 peptide oligomers. J. Mol. Biol. 407 , 110–124 (2011).21237171
51 C. Bleiholder, N. F. Dupuis, T. Wyttenbach, M. T. Bowers, Ion mobility-mass spectrometry reveals a conformational conversion from random assembly to beta-sheet in amyloid fibril formation. Nat. Chem. 3 , 172–177 (2011).21258392
52 S. L. Bernstein , Amyloid-beta protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer’s disease. Nat. Chem. 1 , 326–331 (2009).20703363
53 D. P. Smith , Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies. Eur. J. Mass. Spectrom. (Chichester) 15 , 113–130 (2009).19423898
54 D. P. Smith, S. E. Radford, A. E. Ashcroft, Elongated oligomers in beta2-microglobulin amyloid assembly revealed by ion mobility spectrometry-mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 107 , 6794–6798 (2010).20351246
55 N. F. Dupuis, C. Wu, J. E. Shea, M. T. Bowers, Human islet amyloid polypeptide monomers form ordered beta-hairpins: A possible direct amyloidogenic precursor. J. Am. Chem. Soc. 131 , 18283–18292 (2009).19950949
56 L. M. Young , ESI-IMS-MS: A method for rapid analysis of protein aggregation and its inhibition by small molecules. Methods 95 , 62–69 (2016).26007606
57 L. A. Woods , Ligand binding to distinct states diverts aggregation of an amyloid-forming protein. Nat. Chem. Biol. 7 , 730–739 (2011).21873994
58 A. C. Susa , Defining the molecular basis of amyloid inhibitors: Human islet amyloid polypeptide–insulin interactions. J. Am. Chem. Soc. 136 , 12912–12919 (2014).25144879
59 L. M. Young , Screening and classifying small-molecule inhibitors of amyloid formation using ion mobility spectrometry-mass spectrometry. Nat. Chem. 7 , 73–81 (2015).25515893
60 N. J. Economou , Amyloid beta-protein assembly and Alzheimer’s disease: Dodecamers of Abeta42, but not of Abeta40, seed fibril formation. J. Am. Chem. Soc. 138 , 1772–1775 (2016).26839237
61 C. A. Scarff, A. E. Ashcroft, S. E. Radford, Characterization of amyloid oligomers by electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS). Methods Mol. Biol. 1345 , 115–132 (2016).26453209
62 L. M. Young, A. E. Ashcroft, S. E. Radford, Small molecule probes of protein aggregation. Curr. Opin. Chem. Biol. 39 , 90–99 (2017).28649012
63 S. D. Yan , An intracellular protein that binds amyloid-beta peptide and mediates neurotoxicity in Alzheimer’s disease. Nature 389 , 689–695 (1997).9338779
64 S. D. Yan , Role of ERAB/L-3-hydroxyacyl-coenzyme A dehydrogenase type II activity in Abeta-induced cytotoxicity. J. Biol. Chem. 274 , 2145–2156 (1999).9890977
65 J. W. Lustbader , ABAD directly links Abeta to mitochondrial toxicity in Alzheimer’s disease. Science 304 , 448–452 (2004).15087549
66 F. Chiti, C. M. Dobson, Protein misfolding, amyloid formation, and human disease: A summary of progress over the last decade. Annu. Rev. Biochem. 86 , 27–68 (2017).28498720
67 M. G. Iadanza, M. P. Jackson, E. W. Hewitt, N. A. Ranson, S. E. Radford, A new era for understanding amyloid structures and disease. Nat. Rev. Mol. Cell. Biol. 19 , 755–773 (2018).30237470
68 M. Bucciantini , Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416 , 507–511 (2002).11932737
69 B. Winner , In vivo demonstration that alpha-synuclein oligomers are toxic. Proc. Natl. Acad. Sci. U.S.A. 108 , 4194–4199 (2011).21325059
70 S. W. Chen , Structural characterization of toxic oligomers that are kinetically trapped during alpha-synuclein fibril formation. Proc. Natl. Acad. Sci. U.S.A. 112 , E1994–E2003 (2015).25855634
71 A. Abedini , Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics. eLife 5 , e12977 (2016).27213520
72 N. Cremades , Direct observation of the interconversion of normal and toxic forms of alpha-synuclein. Cell 149 , 1048–1059 (2012).22632969
