Β-Turn Mimetic Synthetic Peptides As Amyloid-Β

Bioorganic Chemistry 101 (2020) 104012

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Bioorganic Chemistry

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β-Turn mimetic synthetic peptides as amyloid-β aggregation inhibitors T Stefanie Deikea, Sven Rothemundb, Bruno Voigte, Suman Samantrayc, Birgit Strodelc,d, ⁎ Wolfgang H. Bindera, a Department of Chemistry, Martin Luther University Halle-Wittenberg, Von-Danckelmann-Platz 4, 06120 Halle, Germany b Core Unit Peptid-Technologien, University Leipzig, Liebigstr. 21, 04103 Leipzig, Germany c Institute of Biological Information Processing (IBI-7: Structural Biochemistry), Forschungszentrum Jülich, 52428 Jülich, Germany d Institute of Theoretical and Computational Chemistry, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany e Department of Physics, Martin Luther University Halle-Wittenberg, Betty-Heimannstrasse 7 4, 06120 Halle, Germany

ARTICLE INFO ABSTRACT

Keywords: Aggregation of amyloid peptides results in severe neurodegenerative diseases. While the fibril structures of Aβ40

Beta-turn mimetics and Aβ42 have been described recently, resolution of the aggregation pathway and evaluation of potent in- Amyloid-fibrillation hibitors still remains elusive, in particular in view of the hairpin-region of Aβ40. We here report the preparation Hybrid-peptide of beta-turn mimetic conjugates containing synthetic turn mimetic structures in the turn region of Aβ40 and Aβ16- Oligomerization 35, replacing 2 amino acids in the turn-region G25 – K28. The structure of the turn mimic induces both, acceleration of fibrillation and the complete inhibition of fibrillation, confirming the importance of theturnregion on the aggregation. Replacing position G25-S26 provided the best inhibition effect for both beta-turn mimetics, the bicyclic BTD 1 and the aromatic TAA 2, while positions N27-K28 and V24-G25 showed only weaker or no inhibitory effects. When comparing different turn mimetics at the same position (G25-S26), conjugate 1a bearing

the BTD turn showed the best inhibition of Aβ40 aggregation, while 5-amino-valeric acid 4a showed the weakest effect. Thus there is a pronounced impact on fibrillation with the chemical nature of the embedded beta-turn- mimic: the conformationally constrained turns 1 and 2 lead to a significantly reduced fibrillation, even inhibiting

fibrillation of native40 Aβ when added in amounts down to 1/10, whereas the more flexible beta-turn-mimics 4- amino-benzoic acid 3a and 5-amino-valeric acid 4a lead to enhanced fibrillation. Toxicity-testing of the most successful conjugate showed only minor toxicity in cell-viability assays using the N2a cell line. Structural

downsizing lead to the short fragment BTD/peptide Aβ16-35 as inhibitor of the aggregation of Aβ40, opening large potential for further small peptide based inhibitors.

1. Introduction often a large structural heterogeneity is observed [10]. Hydrophobic β- strands are connected by a hydrophilic region, which shows a loop/ Peptide- and protein fibrillation [1] represents an important prin- bend structure, both in oligomers and fibrils. Inhibition of fibrillation ciple in supramolecular assembly, pointing towards a range of diseases by artificial systems and molecules is a longlasting challenge, identi- such as Alzheimeŕs disease or Parkinsońs disease, but also responsible fying several types of small and large molecules as inhibitors. Of par- for reversible assembly of hormones such as the parathyroid-hormone ticular interest is the structural modification of the of Aβ40 protein with (PTH) [2]. Being guided by a careful balance of inter- and in- artificial elements, able to change the overall aggregation profile ofthis tramolecular interactions, a variety of artificial, [3] biomimetic and protein. Two approaches were reported using either 4-(2-aminoethyl)- conjugate structures [4] displays fibrillation in vivo and in vitro. 6-dibenzofuranopropionic acid or diazo-phenyl-systems [11,12], Amyloid β (Aβ) peptides in particular are important model systems, leading to only small reduction of fibrillation, but no catalytic effects on being studied widely in terms of aggregation, their underlying nuclea- the fibrillation of native of Aβ40. tion-dependent aggregation pathways [5,6], as well as potential in- We here investigate the influence of artificial turn elements (see hibitors against aggregation [7–9]. Structural analysis of Aβ has re- Scheme 1) introduced into the Aβ sequence on the aggregation and vealed a cross-beta-sheet morphology, which comprises β-hairpin units structure of Aβ40. Turn mimics of different size, hydrophilicity and ri- aligned in parallel, stabilized by intermolecular interactions, although gidity were chosen to replace two amino acids each in the putative turn

⁎ Corresponding author. E-mail address: [email protected] (W.H. Binder). https://doi.org/10.1016/j.bioorg.2020.104012 Received 5 May 2020; Received in revised form 9 June 2020; Accepted 10 June 2020 Available online 16 June 2020 0045-2068/ © 2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). S. Deike, et al. Bioorganic Chemistry 101 (2020) 104012

Scheme 1. (A) Primary sequence of Aβ40. Arrows illustrate the position of β-strands and the substituted turn-region is highlighted in blue. (B) Chemical structures of β-turn mimetics 1, 2, 3 and 4 used in this study to replace two amino acids of Aβ40 to generate the beta-turn mimetic conjugates. (C) Synthetic pathway towards artificial peptides.

region of Aβ40. As only few investigations have been dealing with hy- Table 1 brid-amyloid sequences, the synthetic elements we focus here on are Overview of synthesized peptide conjugates replacing the respective amino- designed to understand the structure/activity relationship of several acids by the turn mimetics 1 – 4, indicating the corresponding replaced amino acids in native Aβ . artificial beta-turns, placed within Aβ40. Previous investigations have 40 1 concentrated on the replacement of amino acids by mutation [13–17] at Conjugate Turn Aβ sequence Replaced Mth [g Yield Purity position E22 (resulting in an enhanced fibrillation for the arctic mutant mimetic position mol−1] E22G [18]), while the opposite effect was observed for the Osaka mu- 1a BTD 1 Aβ G25-S26 4456.06 13.6 99.7 tant E22Δ [13]. Short peptide fragments as models for the full length Aβ 40 1b BTD 1 Aβ40 N27-K28 4357.91 8.0 98.8 can modulate aggregation [19–23], but without deeper understanding 1c BTD 1 Aβ16-35 G25-S26 2235.44 4.4 97.2 of the underlying interactions. Mutations of the turn region, such as 2a TAA 2 Aβ40 G25-S26 4448.03 14.8 100 removal of Gly25 [24] or substitution of two adjacent amino acids with 2b TAA 2 Aβ40 V24G25 4435.73 14.0 99.7 a turn-nucleator [25] have altered the fibrillation propensities and 2c TAA 2 Aβ16-35 G25-S26 2227.46 5.0 95.8 3a ABA 3 Aβ40 G25-S26 4304.82 6.0 97.0 toxicity of Aβ, indicating that the hairpin is an important structural 4a AVA 4 Aβ40 G25-S26 4284.77 5.7 99.8 motif on the aggregation pathway of Aβ [26,27]. We here report aggregation studies combined with molecular dy- 1 Determined by HPLC. namics simulations, demonstrating the ability to accelerate or eliminate the fibrillation process depending on the artificial turn motif andthe incorporated at two different positions, while turn mimetics 3 and 4 position of substitution. Furthermore, we observe that some specific were introduced solely to replace the position G25-S26 in comparison. artificial peptides induce a reduced fibrillation in mixture with native All peptide conjugates 1a – 4a displayed high purity of the final pep- Aβ40. tide-conjugates, as judged by HPLC and MS-analysis (see Table 1 and SI).

2. Results and discussion 2.2. Aggregation kinetics 2.1. Chemistry In order to investigate the fibrillation behavior of the beta-turn Beta-turn mimetic conjugates (1–4) with different propensities to mimetic conjugates, ThT-assays were performed. In a first attempt the induce turns and variable hydrophilicity were synthesized, in order to pure conjugates were investigated and the results are shown in Fig. 1. be able to investigate the influence of size, rigidity and hydrophilicity Two characteristic times were taken as indicators for the aggregation: on the aggregation behavior (Scheme 1). As analysis of solid state NMR the lag time, indicated in the inset of Fig. 2, is the time in which no data of fibrillated Aβ40 indicated a break of the β-sheet at residues G25- fluorescence signal occurs, while the characteristic half-time t1/2 is the S26 [28] we therefore have concentrated on chemical modifications time at which 50% of the protein is aggregated. Interestingly, con- around this region. jugates 1a and 1b exhibited no fibrillation in the ThT-assay after 40h, We have selected the beta-turn mimetic 1 (BTD), known to induce and even after one week of thermoshaking at 37 °C no fibrillar struc- beta-turns [29] and possessing a bicyclic structure mimicking two ad- tures were obtained. These findings were confirmed by CD measure- jacent amino acids, while in the hydrophobic beta-turn mimetic 2 ments, indicating random coil structures. In addition, TEM investiga- (TAA) the triazole ring acts as a peptide surrogate, displaying a similar tions revealed neither fibrillar structures nor aggregates (Supporting dipolar character as the trans amide bond and the ability to form hy- Information, Fig. S11). All other beta-turn mimetic conjugates exhibited drogen bonds [30–32]. Two other (nonbiogenic) amino acids, 3 (ABA) an increase in fluorescence intensity upon time with a behavior strongly and 4 (AVA), bearing different spacer groups in between the carboxylic depending on the nature of the turn mimetic. While conjugate 3a, and the amine functionality were chosen to investigate the influence of bearing the rigid 4-ABA amino acid, showed an increase in lag time rigidity on fibrillation. All selected structures were introduced into the compared to Aβ40 (tlag ~ 3 h), conjugates 2b and 4a exhibited similar synthetic pathway via their Fmoc-protected amine groups and methyl behavior with a decreased lag time (tlag < 1 h) and a high fluorescence ester protected carboxylic acids (Scheme 1). Furthermore, three dif- intensity. The position of substitution apparently has a strong impact on ferent positions in the probable turn region of Aβ40 have been chosen fibrillation, as conjugate 2a displays a different behavior compared to for the replacement of two amino acids by the turn mimetics 1–4 as conjugate 2b. summarized in Table 1. The synthesized turn mimetics 1 and 2 were Next we were interested in the overall inhibitory behavior which

2 S. Deike, et al. Bioorganic Chemistry 101 (2020) 104012

observable at a concentration of 3 μM (tlag ~ 7 h), further addition of conjugate results in a decrease of lag time and thus an acceleration of fibrillation. Beta-turn mimetic conjugates 3a and 4a showed quite similar behavior with slight differences. Upon addition of a small amount of the conjugates (3a: 2–3 μM; 4a: 2–5 μM) an increased lag time is observed, which even reaches a value which is more than ten-fold

higher compared to Aβ40 upon addition of 3a, while only a tripling is attained when using 4a. Upon further increase of the amount of additive, the lag time decreases again. Thus, similarly to conjugate 2a, also 3a and 4a show their best inhibition effect only when they are used in mixture with

native Aβ40. Taking all these measurements into account we concluded: replacing position G25-S26 provided the best inhibition effect for both beta- turn mimetics BTD 1 and TAA 2, while positions N27-K28 and V24-G25 showed only weaker or no inhibitory effects. When comparing different Fig. 1. Aggregation kinetics of beta-turn mimetic conjugates 1a, 1b, 2a, 2b, 3a, turn mimetics at the same position (G25-S26), conjugate 1a bearing the

4a (10 μM) in comparison to native Aβ40 (10 μM) as monitored by thioflavin T BTD turn mimetic showed the best inhibition of Aβ40 aggregation, (ThT, 10 μM) fluorescence at 37 °C in PBS (pH 7.4). Curves exhibiting sigmoidal while 4a showed the weakest effect. These findings correlate with the behavior were fitted with a sigmoidal function with the curve fit shown. observed ThT-curves of the pure conjugates, confirming that conjugate 1a, which does not undergo fibrillation, can inhibit the fibrillation of the beta-turn mimetic conjugates would exert in mixture with native Aβ40, very efficiently, even at low mixing ratios (amyloid/con- Aβ40 [11,12]. Based on our first results further ThT-assays were per- jugate = 1/0.2). formed using the conjugates as additives for the inhibition of Aβ40 fibrillation (Supporting Information Fig. S9). The results concerning lag 2.3. Circular dichroism studies and TEM images time and half-time are summarized in Fig. 2. Mixtures containing 10 μM of Aβ and varying concentrations of the conjugates ranging from 1 μM 40 We were then interested whether conformational structures prior to to 10 μM were probed, comparing the conjugates containing beta-turn fibrillation are responsible for the observed inhibitory effect ofthe mimetic 1. In both cases already 0.2 equivalents of the conjugates in- amyloid-beta-turn mimetic conjugates. From literature it is known that duced an increased lag time. The effect was more pronounced for native (unfibrillated) Aβ40 displays a random coil conformation, with a conjugate 1a, replacing positions G25-S26, while it was less pro- marked cross-beta-structure after fibrillation [33]. Disaggregated pep- nounced for 1b (N27-K28). Further increase of the amount of additive tides and BTD-conjugates 1a and 1b at pH 7.4 also exhibit a random resulted in a more than ten-fold increase of the original lag time of Aβ 40 coil structure, with a strong negative signal at 200 nm (Supporting (tlag ~ 4 h) upon addition of 0.3 equivalents (1a) and 0.8 equivalents Information, Fig. S12), while Aβ40 reveals a β-sheet structure with a (1b) respectively. Next, we compared the conjugates containing beta- minimum at 218 nm after fibrillation. Beta-turn mimetic conjugate 1a turn mimetic 2. Adding very low amounts of 2a (1 μM) already results was successfully fibrillated at pH 6.5 with a higher salt concentration in a strong inhibition of fibrillation increasing the lag time to 24 hand (500 μM) (Fig. 3A). Peptides 2a and 2b showed a similar behavior the half-time to 27 h. Increasing the amount of the added conjugate to without any visible influence of the different positions, whereas 2 and 3 μM results in a further increase of lag time by six and eleven changes were observed for conjugates 3a (215 nm) and 4a (227 nm). hours respectively. Thus, the maximum lag time increase is observed Investigations of mixtures of Aβ40 and beta-turn mimetic conjugates 3a upon addition of 0.3 equivalents of conjugate 2a, resulting in a lag time confirmed the influence of the conjugate on the fibrillar structure, asa of around 35 h, whereas upon addition of an increased amount of 2a shift of the minimum from 218 nm for pure Aβ40 to about 215 nm for a (5 μM, 10 μM) the lag time decreases again, resulting in a similar 1:1 mixture occurs, similarly to the signal found for pure peptide 3a curvature as the pure artificial peptide 2a. Conjugate 2b reveals a lar- (Supporting Information, Fig. S10). Negatively stained TEM images gely different behavior. While only a small increase in lag timeis confirmed the absence of regular fibrillar structure for conjugates 1a

Fig. 2. Summary of aggregation kinetics of mixtures of native Aβ40 (10 μM) in mixture with the conjugates (1a-b, 2a-b, 3a, 4a) (1–10 μM) as monitored by thioflavin

T (ThT, 10 μM) fluorescence at 37 °C in PBS (pH 7.4). Lag-timeslag (t ) and characteristic half-times (t1/2) were determined from the sigmoidal fit curves.

3 S. Deike, et al. Bioorganic Chemistry 101 (2020) 104012

Fig. 3. A) CD spectra of 10 μM Aβ40 and of 10 μM beta-turn mimetic conjugates after fibrillation at pH7.4 or pH6.5. B) CD spectra 40of 10μMAβ and of mixtures with conjugate 2c (5–20 μM). C-F) Negatively stained TEM images of beta-turn mimetic conjugates. C) 3a, D) 4a, E) Aβ40/2a 2:1, F) Aβ40/2a 1:1. and 1b treated at pH7.4, while fibrils with a diameter of 12.5 nmwere and 2c, while the lowest one resulted for conjugate 4a bearing the obtained for mixtures thereof with Aβ40, slightly higher than the one of flexible AVA linker. Only weak differences occurred between the pure Aβ40 (11.5 nm; Supporting Information, Fig. S11). monomeric and fibrillar samples, supporting previous investigations, The TAA-conjugate 2a formed the thinnest fibrils with a diameter of which showed that oligomeric species are the most toxic aggregates 8.0 nm, while the fibrils of the 4-ABA-conjugate 3a are nearly twice as [34,35]. However, finding a defined oligomeric state for the different thick with a diameter of 14.7 nm, while the 5–AVA-conjugate 4a pos- peptide conjugates is challenging and needs to be investigated in future sesses an intermediate thickness of 9.8 nm (Fig. 3C-D). work. With these promising results in mind, a structure reduction of the best conjugates was performed, resulting in the short peptides 1c and 2c, bearing only residues 16–35 of Aβ, thus containing the important β- 2.5. Theoretical investigations sheet regions and the turn region as the beta-turn mimetic (Table 1). Similar to full-length conjugate 1a, also the fragment peptide 1c did not In order to complement the experimental findings with theoretical show fibrillation and revealed inhibition properties when usedin calculations, molecular dynamics (MD) simulations of the short pep- mixture with native Aß40 (Fig. 2 and Fig. S13). While the lag time in- tides 1c and 2c as well as Aβ16-35 as reference system were performed. creased upon addition of 0.5 and 1.0 equivalents to 15 and 20 h re- Fig. 5 shows the oligomerization state, i.e., monomer or dimer over spectively, it was shortened to five hours upon addition of 2.0 time, averaged over the three simulations performed per system. Con- equivalents. Fragmented peptide 2c, bearing the TAA turn mimic jugate 2c forms a stable dimer quicker than conjugate 1c and Aβ16-35. lacked the ability of its full-length analogue to induce an increased lag In fact, 2c dimerized within 100 ns and apart from a short period be- time (Fig. S13). tween 250 and 375 ns 2c remained in the associated state. The aggregation speeds of 1c and Aβ16-35 appear to be similar, with Aβ16-35 2.4. Cytotoxicity testing forming a dimer somewhat faster than 1c. It should be noted that oligomer formation does not need to lead to fibril formation - as discussed In order to investigate whether modification of the turn region has in detail below -, i.e., the formation of 1c dimers in our simulations is an impact on the toxicity of the peptides, cell viability was determined not in contradiction to the experimental findings of the current work. using N2a cells. The peptides were applied both as monomers and fi- In order to understand the driving forces of the aggregation process brils and cell viability was determined after 72 h as shown in Fig. 4. observed for each system, we calculated the (normalized) inter-peptide The highest viability was observed for the short peptide conjugates 1c contacts between residues (Fig. 6A) and contrasted them with the

Fig. 5. Average oligomerization state (“1” for monomer and “2” for dimer) as a

Fig. 4. Cell viability in response to treatment with Aβ40 and peptide conjugates function of time obtained from MD simulations of 1c (top), 2c (middle), and

1a, 1c, 2a, 2c, 3a, 4a as monomeric and fibrillar samples (20 μM). Aβ16-35 (bottom).

4 S. Deike, et al. Bioorganic Chemistry 101 (2020) 104012

Fig. 6. (A) Inter- and (B) intra-peptide contact maps calculated from the MD trajectories of 1c (left), 2c (middle), Aβ16-35 (right). The contacts are averaged over the three MD simulations per system and normalized with respect to the most prominent contact per system. For the sake of clarity, the diagonal and first off diagonal in the intra-peptide contact map corresponding to self-contacts and contacts with direct neighbors are not shown. The intra-peptide contacts were averaged over both peptides composing the system.

Fig. 7. Representative structures of 1c (left), 2c

(middle), and Aβ16-35 (right) taken from the MD simulations at t = 0.7 μs. The two peptides composing each system are shown as green and violet cartoon. The N- and C-termini are indicated as blue and red spheres, respectively. The turn mimetics 1 and 2 are shown with sticks and balls using cyan for C, red for O, blue for N, and yellow for S atoms. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) corresponding intra-peptide contacts (Fig. 6B). Clear differences can be 1 keeps the N- and C-terminal sides of the peptide somewhat further observed between the inter-peptide contacts of the three systems, apart from each other than the residues G25-S26 in Aβ16-35 do, ex- especially 2c is considerably different from 1c and Aβ16-35. The latter is plaining the weaker contacts in 1c, which includes a less stable D23- characterized by contacts between the terminal residues, involving each K28 salt bridge. of NeN, NeC and CeC contacts, but is devoid of noteworthy inter- In 2c the situation is markedly different. The inter-peptide contacts peptide contacts around the middle of the peptide, including residues reveal an antiparallel alignment of both peptides in the dimer, with a G25-S26. The corresponding intra-peptide contact map shows that pronounced contact between the two turn mimetics 2. This hydro- these residues are involved in the typical intra-peptide hairpin struc- phobic interaction is strengthened by a few more hydrophobic inter- ture, which is confirmed by the representative snapshot shown in Fig. 7. actions, such as L17-L34 and F20-I32. The intra-peptide interactions, on

Both Aβ16-35 peptides composing this dimer are in a hairpin con- the other hand, are less strong compared to those of the other two formation with the terminal residues of both peptides interacting with systems, indicating more elongated peptide structures. This is con- each other, while the turns, which are stabilized by a salt bridge be- firmed by the structural representation of the dimer of 2c in Fig. 7, tween D23 and K28, are solvent exposed. A similar structure pattern as which shows that the aromatic turn mimetics 2 stack on top of each for Aβ16-35 is observed for 1c, yet the turn formation is not as pro- other as a result of π-π interactions, while their rigidity keeps the N- nounced. The inter- and intra-peptide contacts of 1c bear great simi- and C-terminal halves per peptide mostly apart from each other. In- larity to the corresponding contacts of Aβ16-35, and also the two re- stead, they interact with the neighboring peptide strands in an anti- presentative structures look similarly. However, the beta-turn mimetic parallel manner. It is easy to imagine how this can lead to β-sheets if

5 S. Deike, et al. Bioorganic Chemistry 101 (2020) 104012 more peptides were present in the system, eventually yielding fibrils as trifluoroacetic acid (TFA) and sodium ascorbate were purchased from seen in the experiments (Fig. 3). The beta-turn mimetic 1, on the other Sigma Aldrich. Trifluoromethanesulfonic anhydride (Tf2O) was received hand, enforces a hairpin structure in monomeric 1c, which is not able to from Alfa Aesar. 2-Iodoxybenzoic acid (IBX) and Fmoc-N-hydro- form fibrils (Fig. 3) in agreement with another Aβ system were the β- xysuccinimide ester (Fmoc-ONSu) were purchased from Fluorochem. hairpin was stabilized. While 1c and Aβ16-35 behaved similarly in our Sodium sulfate, Ce(SO4)2·4H2O, (NH4)6Mo7O24·4H2O and copper(II) sulfate simulations, residues G25-S26 guarantee sufficient structural flexibility pentahydrate were purchased from VEB, N,N–Dimethylformamide (DMF) to allow the peptide to also adopt other structures as shown by both was received from Gruessing, pyridine from Acros Organics and lithium experiments [36] and simulations [37], which eventually should lead to hydroxide from Lachema. All these chemicals were used without further fibril formation. However, it should be mentioned that despite the purification. wealth of studies on the Aβ peptide, no study was found that in- NMR spectra were recorded on a Varian Gemini 400 or 500 spec- vestigated the amyloid fibril formation of Aβ16-35. In our simulations trometer (400 MHz or 500 MHz) at 27 °C in CDCl3 (Chemotrade, only limited β-sheet formation was observed (less than 10% in each 99.8 Atom%D), DMSO‑d6 (Chemotrade, 99.8 Atom%D) or CD3OD system), which can be explained that our simulations started from a (Chemotrade, 99.8 Atom%D). Chemical shifts are given in ppm and helical peptide structure (PDB ID: 1bA4), which is not stable in water referred to the solvent residual signal (CDCl3: δ = 7.26 ppm and but takes a while (~300–400 ns) to transform into other structures, and δ = 77.0 ppm; DMSO‑d6: δ = 2.50 ppm and δ = 39.5 ppm; CD3OD: that the simulation time of 1 μs is short compared to experimental time δ = 3.31 ppm and δ = 49.0 ppm). The following abbreviations were scales. The β-sheet formation that was observed took place within the used for 1H- and 13C NMR peaks assignment: s = singlet, d = doublet, peptide in the case of 1c and Aβ16-35, while it was between peptides in t = triplet, dd = doublet of doublet, m = multiplet. MestReNova 2c (Fig. 7). 6.0.2–5475 was used for data interpretation. ESI-ToF mass spectroscopy was performed on a Bruker Daltonics microTOF. Samples were dis- 3. Conclusion solved in HPLC grade solvents (MeOH, THF or mixtures; purchased from Sigma Aldrich) at concentrations of 0.1 mg/mL and measured via In conclusion, we have demonstrated that the structure and the direct injection with a flow rate of 180 μL/h using the positive mode nature of the turn region in Aβ peptides has a huge impact on their with a capillary voltage of 4.5 kV. The spectra were analyzed with aggregation behavior. Four different synthetic β-turns were successfully Bruker Data Analysis 4.0. introduced into the amyloid beta sequence and their influence on fi- MALDI-ToF mass spectroscopy measurements were carried out on a brillation was investigated. While introduction of the aromatic turn Bruker Autoflex III system equipped with a smart beam laser (355nm, mimetic TAA 2 resulted in an about four times enhanced fibrillation, 532 nm, 808 nm and 1064 nm ± 5 nm; 3 ns pulse width; up to peptides containing the bicyclic turn mimic BTD 1 lack aggregation. 2500 Hz repetition rate) accelerated by a voltage of 20 kV and detected Insertion of small linker moieties, namely 5–aminovaleric acid 3 and as positive ions in reflectron or linear mode. The data evaluation was 4–aminobenzoic acid 4, enhanced flexibility in the turn region and performed on flexAnalysis software (version 3.0). Samples were dis- increased fibrillation, whereas rigidity within the bicyclic turn mimic solved in 0.1% TFA at a concentration of 0.1 mg/mL. α-Cyano-4-hy- BTD and the sterically constrained turn mimetic 2 diminished ag- droxycinnamic acid was used as matrix and dissolved in acetonitrile / gregation. These observations are supported by MD simulations per- 0.1% TFA (1 : 1) at a concentration of 20 mg/mL. The solutions of formed in this work. The simulations revealed that the aromatic turn matrix and sample were mixed in a volume ratio of 1 : 1 and 1 μL of the mimetic 2 can self-assemble resulting from π-π stacking interactions, solution mixture was spotted on the MALDI-target plate. The instru- while at the same time reducing intra-peptide contacts between the N- ment was externally calibrated with a poly(ethylene glycol) mono- and C-terminal peptide halves. Instead, inter-peptide contacts with β- methyl ether (PEG) standard (Mn = 4200 g/mol, Mw/Mn = 1.05) ap- sheet formation are encouraged, giving rise to fibril formation on the plying a quadratic calibration method. experimental time scale. Turn mimetic 1, on the other hand, stabilizes Fibrillation kinetics of artificial peptides and mixtures with WTAβ40 the intra-peptide β-hairpin, which does not form amyloid fibrils. were investigated by fluorescence intensity measurements using Further investigations using these beta-turn mimetic conjugates as ad- Thioflavin T (ThT) as fluorescent dye. Lyophilized peptides were dis- ditives during the aggregation of Aβ40 revealed a strong inhibitory ef- solved in 10 mM NaOH at a concentration of 1 mg/mL. The samples fect upon replacement of position G25-S26. This is valid for both the were left to stand for 10 min and applied to ultrasound for 1 min for bicyclic BTD and the aromatic triazole TAA turn mimetics, corre- complete dissolution of the peptides. The solutions were centrifuged at sponding to conjugates 1a, 2a, confirming the importance of the small 10,000 rpm (…g) for 1 h at 4 °C, the supernatant was transferred to glycine group for the formation of β-sheets in Aβ fibrils. Reduction of another tube and the sample was kept on ice in the next steps. Finally full length Aβ conjugates yielded the Aβ-fragment 16–36 with the turn the samples were diluted with 50 mM phosphate buffer (pH 7.4, mimetics at positions G25-S26 (1c, 2c), which as additive also reduced 150 mM NaCl) to obtain final concentrations of 10 μM WT Aβ40, 10 μM the fibrillation of40 Aß as demonstrated by a nearly 10–fold increased ThT and different concentrations of peptide conjugate (1–10 μM). For lag time. each sample, a total volume of 480 μL was prepared and 3 × 150 μL

In summary, the here generated Aβ40 turn mimetic conjugates were pipetted to a 96-well plate. The plate was sealed with a microplate bearing the turns 1 and 2 can have a huge impact on the aggregation of cover. The fluorescence intensity was monitored at 37 °C using aBMG amyloid-β peptides with large implications for inhibitory design to FLUOStar Omega multi-mode plate reader using fluorescence excitation modulate amyloid-β assembly. The toxicity of the short conjugates (1c, and emission wavelengths at 440 nm and 482 nm respectively. One 2c) was slightly lower than for the full-length conjugates, allowing for a measurement cycle of 5 min consisted of double-orbital shaking for future design of peptides as inhibitors for amyloid-fibrillation. 240 s and waiting for 60 s. Concentrations of WT Aβ40 and conjugates 1a, 1b, 1c, 4a were determined by absorbance at 280 nm at a Jasco V- 3.1. Experimental 660 absorbance spectrometer using the molar extinction coefficient of −1 −1 Aβ40 (ε280 = 1490 cm M ). The concentrations of conjugates 2a, 3.1.1. General 2b, 2c and 3a were estimated by weight. All technical solvents were distilled prior use. Air- and moisture-sensi- Circular dichroism (CD) spectra were recorded on a Jasco J-815 tive reactions were carried out in flame-dried glassware under atmospheric spectropolarimeter using a 2 mm path length cell and a concentration of pressure of nitrogen or argon. L-Cysteine methyl ester hydrochloride, so- 10 – 20 μM in buffer. Ellipticity values were normalized to the mean dium azide, palladium on charcoal, N,N–diisoproylethylamine (DIPEA), residue weight. N,N-dicyclohexylcarbodiimide, N,N-dimethylaminopyridine (DMAP), TEM images were taken with an electron microscope (EM 900; Zeiss)

6 S. Deike, et al. Bioorganic Chemistry 101 (2020) 104012 at 80 kV acceleration voltage. For preparation, 5 μL of the peptide so- simulations of the conjugates 1c and 2c. GaussView (version 5) [40] lution (2 – 5 μM) was dropped on Formvar/Cu grids (mesh 200). After was used to create the Gaussian input files for BTD (1) and TAA (2), three minutes waiting, the grids were gently cleaned with water for one which were then used for geometry optimization of the structures of 1 minute and then negatively stained using uranylacetate (1%, w/v) for and 2 at the HF/6-31G* level of theory and calculation of atomic partial one minute. charges with MP2/6-31G* using Gaussian [40]. The bonded and non- Cell viability assay with MTT reduction assay: N2a cells were cultured bonded parameters obtained were converted into GROMACS [41] in a RPMI medium containing L-Glutamine, FBS (10%) and 1% peni- compatible file formats, adopting the naming convention for atom types cillin–streptomycin at 37 °C in 5% CO2. After 2–3 days, the cells were in CHARMM [43] realized with the Antechamber package [42] of trypsinized for five minutes, then diluted with the medium and plated AmberTools (version 16) [43,44]. The resulting topology and para- onto 96-well plates (5000 cells/well). After 24 h of incubation, mono- meter data are provided in the Supporting Information (beta-turn mi- meric or fibrillar Aβ was added. Fibrillar samples were re-suspended in metic 1: Fig. S15 and Tables S3-S7; beta-turn mimetic 2: Fig. S16 and the medium at a concentration of 100 μM and 30 μL were added to the Tables S8-S12). cells. Monomeric samples were obtained by a previously described BTD and TAA were then incorporated into Aβ16-35 by replacing protocol. [56-58] Briefly, peptide conjugates were dissolved inTFA residues 25 and 26 with each of the turn mimetics. This was accom- (1 mg/mL) and sonicated for 10 min at room temperature. After eva- plished with the PyMOL software package [45] and using Aβ co- poration under a stream of nitrogen, HFIP was added, followed by ordinates available in the NMR structure with PDB code 1BA4 [46]. evaporation using nitrogen to yield a peptide film at the wall of the These structures were relaxed by an energy minimization in vacuum tube. This process was repeated twice. Afterwards, the sample was using CHARMM36m [47] to model the peptides including turn mi- subjected to high vacuum for 30 min and was afterwards kept under dry metics and the steepest descent method until a maximum force of −1 −1 nitrogen atmosphere. The samples were treated with NaOH (60 mM, 500 kJ mol nm was reached. Two molecules of Aβ16-35, serving as 5 μL), PBS (30 μL), HCl (60 mM, 5 μL) and RPMI medium (40 μL) in this reference, 1c and 2c, respectively, were then inserted into a simulation order to yield a concentration of 100 μM. 30 μL were added to the cells box of size 10 nm × 10 nm × 10 nm with a distance of > 1 nm from to reach a final concentration of 20 μM. The measurements were per- each other using the molecule packing optimization software formed as triplicates. Cell viability was determined after 72 h using a PACKMOL [48] for this purpose. Aβ16-35, 1c and 2c were described MTT reduction assay. Therefore MTT was added to each well to a final using CHARMM36m with a modified Lennard-Jones well-depth value concentration of 0.5 mg/mL. After 3.5 h incubation at 37 °C, the for interactions with water, which was modeled using the TIP3P po- medium was removed and the blue crystals were dissolved in DMSO tential. The charges of the ionisable residues were set to correspond to (100 μL/well). The plate was read on a micro plate reader at a wave- pH 7. Na+ and Cl- ions were added to neutralize the system and model a length of 570 nm. Cell viability was related to the 100% control. concentration of ~ 150 mM NaCl. The solvated systems were equili- brated by an energy minimization and a set of short MD simulations as 3.1.2. Synthesis of β-turn mimetic synthetic peptides 1a – 4a described in detail in ref. [49]. The production MD simulations for each

The synthesis of β-turn mimetics BTD 1 and TAA 2 was performed of the dimeric systems of Aβ16-35, 1c and 2c were performed for one with slight modifications according to literature [29,38] and the results microsecond (μs) at 300 K using the Nosé-Hoover thermostat [49,50] are given in the Supporting information (Scheme S1, S2; Figs. S1 – S4). for temperature control and 1.0 bar using the Parrinello-Rahman Solid phase peptide synthesis was performed on a CEM microwave barostat [51] for pressure control. To obtain MD data of statistical peptide synthesizer LibertyBlue (CEM GmbH, Kamp-Lintfort, Germany) significance, each system was simulated in triplicate. All MD simula- using standard Fmoc-chemistry and preloaded PHB-TentaGel (Rapp tions reported in this work, including prior energy minimization and Polymere GmbH, Germany) resins on a 0.05 mmolar scale. Standard equilibration were accomplished with GROMACS 2016.4 [52]. coupling of protected natural amino acids were performed as single GROMACS was also used for determining the secondary structure of the couplings in DMF using the resprective amino acid (3 eq.), N, N’-dii- peptide sequences in each system, for which GROMACS invokes the DSSP sopropylcarbodiimid (DIC, 3 eq.) as coupling reagent and ethyl 2- algorithm [53]. Here, we include both β-sheets and β-bridges to report on cyano-2-(hydroxyimino)acetate (Oxyma, 3 eq.) as additive at elevated the β-sheet content. The aggregation behavior and inter-residue contacts temperatures (3 min, 90 °C; His, Gly 10 min, 50 °C). Arg couplings were were studied using in-house Python scripts in combination with the MDA- performed as double couplings (2x3 min, 90 °C). For couplings of turn nalysis [54] and MDTraj [55] Python libraries. Two residues were con- building blocks, 2–3 equivalents where dissolved in DMF and coupled sidered to be in contact if the distance between any atom from residue A with DIC (3 eq.) and 1-hydroxybenzotriazole (HOBT) (3 eq.) outside the and any atom from residue B was not larger than 0.4 nm. The various synthesizer with gentle stirring (12 h, room temperature). Fmoc re- quantities calculated were averaged over the three simulations run per moval was carried out with piperidine / DMF (1:5, 0.1% Oxyma, system and in the case of the intra-peptide contacts the were also averaged 1.5 min, 90 °C). The final side chain deprotection and cleavage from the over both peptides composing each system. The scripts for the calculation of resin was performed using a mixture of TFA, triisopropylsilane, water the oligomer size and contact are available at https://github.com/strodel- and phenol (92.5 : 2.5 : 2.5 : 2.5 vol%) with gentle agitation for three group/Oligomerization-State_and_Contact-Map.git. hours at room temperature. Crude peptides were purified to > 95% purity using preparative Declaration of Competing Interest RP-HPLC (Gilson, Limburg, Germany). For both analytical and preparative use, the mobile phases were water (A) and acetonitrile (B), each containing 0.1% TFA. Samples were dissolved in DMSO and eluted The authors declare that they have no known competing financial with a linear gradient from 5% B to 90% B in 15 min for analytical runs interests or personal relationships that could have appeared to influ- and in 90 min for preparative runs. Finally, all peptides and peptide- ence the work reported in this paper. turn conjugates were characterized by analytical HPLC Dionex Ultimate 3000 (Thermo Scientific, Germany) using a PLRP-S column (Agilent Acknowledgements Technologies, 150x3 mm, 3 μm) and MALDI-MS (Bruker Microflex LT, Bremen, Germany), which gave the expected [M + H]+ mass peaks. We thank the DFG, Project ID 189853844 - TRR 102, TP A03 and A12 for financial support; Prof. Dr. Jochen Balbach (Martin Luther 3.1.3. Molecular dynamics simulations University Halle-Wittenberg, Department of Physics) for the use of The beta-turn mimetics needed to be parameterized for the force equipment, their advice, and for discussions. We acknowledge Dr. Gerd field (FF) of choice, here CHARMM36m [39], for the subsequent MD Hause (Martin Luther University Halle-Wittenberg, Biozentrum) for

7 S. Deike, et al. Bioorganic Chemistry 101 (2020) 104012 providing access to the TEM equipment. We acknowledge the com- Alters the Tertiary Fold of Amyloid-β18–35 and Makes It Soluble, J. Biol. Chem. puting provided at the high-performance computing cluster IANVS at 290 (50) (2015) 30099–30107. [25] T.M. Doran, E.A. Anderson, S.E. Latchney, L.A. Opanashuk, B.L. Nilsson, Turn Martin Luther University Halle-Wittenberg. Nucleation Perturbs Amyloid β Self-Assembly and Cytotoxicity, J. Mol. Biol. 421 (2) (2012) 315–328. Appendix A. Supplementary data [26] W. Hoyer, C. Grönwall, A. Jonsson, S. Ståhl, T. Härd, Stabilization of a β-hairpin in monomeric Alzheimer's amyloid-β peptide inhibits amyloid formation, Proc. Natl. Acad. Sci. 105 (13) (2008) 5099–5104. Supplementary data to this article can be found online at https:// [27] A. Abelein, J.P. Abrahams, J. Danielsson, A. Gräslund, J. Jarvet, J. Luo, A. Tiiman, doi.org/10.1016/j.bioorg.2020.104012. S.K.T.S. Wärmländer, The hairpin conformation of the amyloid β peptide is an important structural motif along the aggregation pathway, J. Biol. Inorg. Chem. 19 (4) (2014) 623–634. References [28] I. Bertini, L. Gonnelli, C. Luchinat, J. Mao, A. Nesi, A New Structural Model of Aβ40 Fibrils, J. Am. Chem. Soc. 133 (40) (2011) 16013–16022. [29] B. Eckhardt, W. Grosse, L.-O. Essen, A. Geyer, Structural characterization of a β-turn [1] I.W. Hamley, Peptide Fibrillization, Angew. Chem. Int. Ed. 46 (43) (2007) mimic within a protein–protein interface, Proc. Natl. Acad. Sci. 107 (43) (2010) 8128–8147. 18336–18341. [2] M. Gopalswamy, A. Kumar, J. Adler, M. Baumann, M. Henze, S.T. Kumar, [30] W.S. Horne, M.K. Yadav, C.D. Stout, M.R. Ghadiri, Heterocyclic Peptide Backbone M. Fändrich, H.A. Scheidt, D. Huster, J. Balbach, Structural characterization of Modifications in an α-Helical Coiled Coil, J. Am. Chem. Soc. 126 (47) (2004) amyloid fibrils from the human parathyroid hormone, Biochim. Biophys. Acta, 15366–15367. Proteins Proteomics 1854 (4) (2015) 249–257. [31] A. Brik, J. Alexandratos, Y.-C. Lin, J.H. Elder, A.J. Olson, A. Wlodawer, [3] M.P. Hendricks, K. Sato, L.C. Palmer, S.I. Stupp, Supramolecular Assembly of D.S. Goodsell, C.-H. Wong, 1,2,3-Triazole as a Peptide Surrogate in the Rapid Peptide Amphiphiles, Acc. Chem. Res. 50 (10) (2017) 2440–2448. Synthesis of HIV-1 Protease Inhibitors, ChemBioChem 6 (7) (2005) 1167–1169. [4] I.W. Hamley, V. Castelletto, Self-Assembly of Peptide Bioconjugates: Selected [32] A.A.H. Ahmad Fuaad, F. Azmi, M. Skwarczynski, I. Toth, Peptide Conjugation via Recent Research Highlights, Bioconjug. Chem. 28 (3) (2017) 731–739. CuAAC ‘Click’ Chemistry, Molecules 18 (11) (2013) 13148–13174. [5] I.W. Hamley, The Amyloid Beta Peptide: A Chemist’s Perspective. Role in [33] Y. Fezoui, D.M. Hartley, J.D. Harper, R. Khurana, D.M. Walsh, M.M. Condron, Alzheimer’s and Fibrillization, Chem. Rev. 112 (10) (2012) 5147–5192. D.J. Selkoe, P.T. Lansbury, A.L. Fink, D.B. Teplow, An improved method of pre- [6] P. Arosio, T.P.J. Knowles, S. Linse, On the lag phase in amyloid fibril formation, paring the amyloid β-protein for fibrillogenesis and neurotoxicity experiments, Phys. Chem. Chem. Phys. 17 (12) (2015) 7606–7618. Amyloid 7 (3) (2000) 166–178. [7] N.C. Thomas, G.J. Bartlett, D.N. Woolfson, S.H. Gellman, Toward a Soluble Model [34] C. Manzoni, L. Colombo, P. Bigini, V. Diana, A. Cagnotto, M. Messa, M. Lupi, System for the Amyloid State, J. Am. Chem. Soc. 139 (46) (2017) 16434–16437. V. Bonetto, M. Pignataro, C. Airoldi, E. Sironi, A. Williams, M. Salmona, The [8] S. Kumar, A.D. Hamilton, α-Helix Mimetics as Modulators of Aβ Self-Assembly, J. Molecular Assembly of Amyloid Aβ Controls Its Neurotoxicity and Binding to Am. Chem. Soc. 139 (16) (2017) 5744–5755. Cellular Proteins, PLoS One 6 (9) (2011) e24909. [9] J. Habchi, P. Arosio, M. Perni, A.R. Costa, M. Yagi-Utsumi, P. Joshi, S. Chia, [35] P. Krotee, S.L. Griner, M.R. Sawaya, D. Cascio, J.A. Rodriguez, D. Shi, S. Philipp, S.I.A. Cohen, M.B.D. Müller, S. Linse, E.A.A. Nollen, C.M. Dobson, T.P.J. Knowles, K. Murray, L. Saelices, J. Lee, P. Seidler, C.G. Glabe, L. Jiang, T. Gonen, M. Vendruscolo, An anticancer drug suppresses the primary nucleation reaction that D.S. Eisenberg, Common fibrillar spines of amyloid-β and human Islet Amyloid initiates the production of the toxic Aβ42 aggregates linked with Alzheimer’s dis- Polypeptide revealed by Micro Electron Diffraction and inhibitors developed using ease, Sci. Adv. 2 (2) (2016) e1501244. structure-based design, J. Biol. Chem. (2017). [10] B.H. Meier, A. Böckmann, The structure of fibrils from ‘misfolded’ proteins, Curr. [36] M. Grimaldi, M. Scrima, C. Esposito, G. Vitiello, A. Ramunno, V. Limongelli, Opin. Struct. Biol. 30 (2015) 43–49. G. D'Errico, E. Novellino, A.M. D'Ursi, Membrane charge dependent states of the β- [11] Y. Hamada, N. Miyamoto, Y. Kiso, Novel β-amyloid aggregation inhibitors posses- amyloid fragment Aβ (16–35) with differently charged micelle aggregates, sing a turn mimic, Bioorg. Med. Chem. Lett. 25 (7) (2015) 1572–1576. Biochimica et Biophysica Acta (BBA) -, Biomembranes 1798 (3) (2010) 660–671. [12] T.M. Doran, E.A. Anderson, S.E. Latchney, L.A. Opanashuk, B.L. Nilsson, An [37] Y. Chebaro, N. Mousseau, P. Derreumaux, Structures and Thermodynamics of Azobenzene Photoswitch Sheds Light on Turn Nucleation in Amyloid-β Self- Alzheimer’s Amyloid-β Aβ(16–35) Monomer and Dimer by Replica Exchange Assembly, ACS Chem. Neurosci. 3 (3) (2012) 211–220. Molecular Dynamics Simulations: Implication for Full-Length Aβ Fibrillation, The [13] T. Tomiyama, T. Nagata, H. Shimada, R. Teraoka, A. Fukushima, H. Kanemitsu, Journal of Physical Chemistry B 113 (21) (2009) 7668–7675. H. Takuma, R. Kuwano, M. Imagawa, S. Ataka, Y. Wada, E. Yoshioka, T. Nishizaki, [38] S.S. Bag, S. Jana, A. Yashmeen, S. De, Triazolo-β-aza-ε-amino acid and its aromatic Y. Watanabe, H. Mori, A new amyloid β variant favoring oligomerization in analogue as novel scaffolds for b-turn peptidomimetics, Chem. Commun. 51(25) Alzheimer's-type dementia, Ann. Neurol. 63 (3) (2008) 377–387. (2015) 5242–5245. [14] O. Bugiani, G. Giaccone, G. Rossi, M. Mangieri, R. Capobianco, M. Morbin, [39] J. Huang, S. Rauscher, G. Nawrocki, T. Ran, M. Feig, B.L. de Groot, H. Grubmüller, G. Mazzoleni, C. Cupidi, G. Marcon, A. Giovagnoli, A. Bizzi, G. Di Fede, G. Puoti, A.D. MacKerell, CHARMM36m: an improved force field for folded and intrinsically F. Carella, A. Salmaggi, A. Romorini, G.M. Patruno, M. Magoni, A. Padovani, disordered proteins, Nature Methods 14 (1) (2017) 71–73. F. Tagliavini, Hereditary Cerebral Hemorrhage With Amyloidosis Associated With [40] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, the E693K Mutation of APPHereditary Cerebral Hemorrhage and APP Mutation, G. Scalmani, V. Barone, G.A. Petersson, H. Nakatsuji, Gaussian 16, Gaussian, Inc.,, Arch. Neurol. 67 (8) (2010) 987–995. Wallingford CT, 2016. [15] C. Nilsberth, A. Westlind-Danielsson, C.B. Eckman, M.M. Condron, K. Axelman, [41] D. Van Der Spoel, E. Lindahl, B. Hess, G. Groenhof, A.E. Mark, H.J.C. Berendsen, C. Forsell, C. Stenh, J. Luthman, D.B. Teplow, S.G. Younkin, J. Näslund, L. Lannfelt, GROMACS: Fast, flexible, and free, J. Comput. Chem. 26 (16) (2005) 1701–1718. The 'Arctic' APP mutation (E693G) causes Alzheimer's disease by enhanced Aβ [42] R. Duke, T. Giese, H. Gohlke, A. Goetz, N. Homeyer, S. Izadi, P. Janowski, J. Kaus, protofibril formation, Nat. Neurosci. 4 (2001) 887. A. Kovalenko, U.o.C. T. Lee, San Francisco (2016), AmberTools 16, (2016). [16] E. Levy, M. Carman, I. Fernandez-Madrid, M. Power, I. Lieberburg, S. van Duinen, [43] W.L. DeLano, 2002, CCP4 Newsletter on protein crystallography 40(1) (Pymol: An G. Bots, W. Luyendijk, B. Frangione, Mutation of the Alzheimer's disease amyloid open-source molecular graphics tool,) 82–92. gene in hereditary cerebral hemorrhage, Dutch type, Science 248 (4959) (1990) [44] J. Wang, W. Wang, P. Kollman, D. Case, ANTECHAMBER: an accessory software 1124–1126. package for molecular mechanical calculations, J. Chem. Inform. Comput. Sci. - [17] T.J. Grabowski, H.S. Cho, J.P.G. Vonsattel, G.W. Rebeck, S.M. Greenberg, Novel JCISD 222 (2000). amyloid precursor protein mutation in an Iowa family with dementia and severe [45] M. Coles, W. Bicknell, A.A. Watson, D.P. Fairlie, D.J. Craik, Solution Structure of cerebral amyloid angiopathy, Ann. Neurol. 49 (6) (2001) 697–705. Amyloid β-Peptide(1–40) in a Water−Micelle Environment. Is the Membrane- [18] I.H. Cheng, K. Scearce-Levie, J. Legleiter, J.J. Palop, H. Gerstein, N. Bien-Ly, Spanning Domain Where We Think It Is? Biochemistry 37 (31) (1998) J. Puoliväli, S. Lesné, K.H. Ashe, P.J. Muchowski, L. Mucke, Accelerating Amyloid-β 11064–11077. Fibrillization Reduces Oligomer Levels and Functional Deficits in Alzheimer Disease [46] L. Martínez, R. Andrade, E.G. Birgin, J.M. Martínez, PACKMOL: A package for Mouse Models, J. Biol. Chem. 282 (33) (2007) 23818–23828. building initial configurations for molecular dynamics simulations, J. Comput. [19] B. Ma, R. Nussinov, Stabilities and conformations of Alzheimer's β-amyloid peptide Chem. 30 (13) (2009) 2157–2164. oligomers (Aβ16–22, Aβ16–35, and Aβ10–35): Sequence effects, Proc. Natl. Acad. Sci. [47] R. Dennington, T.A. Keith, Shawnee Mission, KS GaussView, Version 5, John M. 99 (22) (2002) 14126–14131. Semichem Inc., Millam, 2016. [20] T.L.S. Benzinger, D.M. Gregory, T.S. Burkoth, H. Miller-Auer, D.G. Lynn, R.E. Botto, [48] M. Schwarten, O.H. Weiergräber, D. Petrović, B. Strodel, D. Willbold, Structural S.C. Meredith, Two-Dimensional Structure of β-Amyloid(10–35) Fibrils, Studies of Autophagy-Related Proteins, in: N. Ktistakis, O. Florey (Eds.), Autophagy: Biochemistry 39 (12) (2000) 3491–3499. Methods and Protocols, Springer, New York, New York, NY, 2019, pp. 17–56. [21] N.-V. Buchete, R. Tycko, G. Hummer, Molecular Dynamics Simulations of [49] S. Nosé, A molecular dynamics method for simulations in the canonical ensemble, Alzheimer's β-Amyloid Protofilaments, J. Mol. Biol. 353 (4) (2005) 804–821. Mol. Phys. 52 (2) (1984) 255–268. [22] Y. Miller, B. Ma, R. Nussinov, Polymorphism of Alzheimer's Aβ17-42 (p3) [50] W.G. Hoover, Canonical dynamics: Equilibrium phase-space distributions, Phys. Oligomers: The Importance of the Turn Location and Its Conformation, Biophys. J. Rev. A 31 (3) (1985) 1695–1697. 97 (4) (2009) 1168–1177. [51] M. Parrinello, A. Rahman, Polymorphic transitions in single crystals: A new mole- [23] J.-P. Colletier, A. Laganowsky, M. Landau, M. Zhao, A.B. Soriaga, L. Goldschmidt, cular dynamics method, J. Appl. Phys. 52 (12) (1981) 7182–7190. D. Flot, D. Cascio, M.R. Sawaya, D. Eisenberg, Molecular basis for amyloid-β [52] M.J. Abraham, T. Murtola, R. Schulz, S. Páll, J.C. Smith, B. Hess, E. Lindahl, polymorphism, Proc. Natl. Acad. Sci. 108 (41) (2011) 16938–16943. GROMACS: High performance molecular simulations through multi-level paralle- [24] M. Chandrakesan, D. Bhowmik, B. Sarkar, R. Abhyankar, H. Singh, M. Kallianpur, lism from laptops to supercomputers, SoftwareX 1–2 (2015) 19–25. S.P. Dandekar, P.K. Madhu, S. Maiti, V.S. Mithu, Steric Crowding of the Turn Region [53] W. Kabsch, C. Sander, Dictionary of protein secondary structure: Pattern

8 S. Deike, et al. Bioorganic Chemistry 101 (2020) 104012

recognition of hydrogen-bonded and geometrical features, Biopolymers 22 (12) [56] B. O'Nuallain, A.K., Thakur, A.D. Williams, A.M. Bhattacharyya, S. Chen, G. (1983) 2577–2637. Thiagarajan, R. Wetzel, in: Methods Enzymol., Vol. 413, Academic Press, 2006, pp. [54] N. Michaud-Agrawal, E.J. Denning, T.B. Woolf, O. Beckstein, MDAnalysis: A toolkit 3474. for the analysis of molecular dynamics simulations, J. Comput. Chem. 32 (10) [57] D. Schlenzig, S. Manhart, Y. Cinar, M. Kleinschmidt, G. Hause, D. Willbold, (2011) 2319–2327. S.A. Funke, S. Schilling, H.-U. Demuth, Biochemistry 48 (29) (2009) 7072–7078. [55] Robert T. McGibbon, Kyle A. Beauchamp, Matthew P. Harrigan, C. Klein, Jason [58] A. Piechotta, C. Parthier, M. Kleinschmidt, K. Gnoth, T. Pillot, I. Lues, H.- M. Swails, Carlos X. Hernández, Christian R. Schwantes, L.-P. Wang, Thomas U. Demuth, S. Schilling, J.-U. Rahfeld, M.T. Stubbs, J. Biol. Chem. 292 (30) (2017) J. Lane, S. Vijay, Pande, MDTraj: A Modern Open Library for the Analysis of 12713–12724. Molecular Dynamics Trajectories, Biophys. J. 109 (8) (2015) 1528–1532.