Peptidomimetics That Inhibit and Partially Reverse ... - ACS Publications

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Peptidomimetics That Inhibit and Partially Reverse the Aggregation of Aβ1−42 Tina Stark,† Tobias Lieblein,‡ Maximilian Pohland,§ Elisabeth Kalden,† Petra Freund,‡ René Zangl,‡ Rekha Grewal,∥ Mike Heilemann,‡ Gunter P. Eckert,§,⊥ Nina Morgner,‡ and Michael W. Göbel*,† †

Institute of Organic Chemistry and Chemical Biology, ‡Institute of Physical and Theoretical Chemistry, and §Department of Pharmacology, Goethe University Frankfurt, Campus Riedberg, Max-von-Laue-Strasse 7-9, D-60438 Frankfurt am Main, Germany ∥ Institute of Nutritional Sciences, Justus-Liebig-University Giessen, Wilhelmstrasse 20, D-35392 Giessen, Germany S Supporting Information *

ABSTRACT: The peptide sequence KLVFF resembles the hydrophobic core of the Aβ peptide known to form amyloid plaques in Alzheimer’s disease. Starting from its retro-inverso peptide, we have synthesized three generations of peptidomimetics. Step by step natural amino acids have been replaced by aromatic building blocks accessible from the Pd-catalyzed Catellani reaction. The final compound 18 is stable against proteolytic decay and largely prevents the aggregation of Aβ1−42 over extended periods of time. The activity of the new inhibitors was tested first by fluorescence correlation spectroscopy. For closer examination of compound 18, additional techniques were also applied: laser-induced liquid bead ion desorption mass spectrometry, confocal laser scanning microscopy, thioflavin T fluorescence, and gel electrophoresis. Compound 18 not only retards the aggregation of chemically synthesized Aβ but also can partially dissolve the oligomeric structures. Thioflavin binding mature fibrils, however, seem to resist the inhibitor.

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peptide to RI-OR2 led to an analogue (RI-OR2-TAT) that was able to cross the blood−brain barrier and to reduce the plaque load, the level of oxidative damage, and the extent of inflammation in a transgenic mouse model of Alzheimer’s disease.17 Other inhibitors derived from the KLVFF sequence contain N-methylated amino acids,18,19 α,α-disubstituted amino acids,20,21 or alternating amide and ester bonds.22 It is also possible to combine several KLVFF sequences into a single molecule.23,24 Apart from KLVFF derivatives, β-sheet breaker peptides are quite prominent as Aβ aggregation inhibitors. These peptides are homologous to Aβ and likewise hydrophobic but have a weakened tendency to form β-sheets.25 One example is the peptide LPFFD26 and modifications thereof.27,28 The central hydrophobic part of Aβ is important for aggregation, as is the C-terminus. Peptides corresponding to this sequence were also tested as inhibitors.29−31 Examples of nonpeptidic aggregation inhibitors are molecular tweezers,32,33 aminopyrazoles,34,35 scyllo-inositol,36 tramiprosate,37 and polyphenols such as curcumin, resveratrol, and epigallocatechingallate.38 The aggregation of molecules may be analyzed by mass spectrometry (MS) provided the species distribution in the gas phase mirrors the situation of the liquid phase. This requires the mildest possible ionization conditions. Aβ aggregation has been analyzed previously by electrospray ionization (ESI) mass spectrometry.39−42 Such experiments are

lzheimer’s disease (AD) is the most common cause of dementia affecting around 47 million people worldwide in 2015.1,2 Despite the long search for therapies, still no cure for AD exists. Thus, developing concepts to counteract this illness is of great relevance.3−6 Pathologically, amyloid plaques composed of the Aβ peptide and neurofibrillary tangles of hyperphosphorylated Tau protein are found in the brains of AD patients. The Aβ peptide is formed from the amyloid precursor protein (APP) by proteolytic cleavage through β- and γsecretase. Because of different cleavage sites, various Aβ isoforms 38−43 amino acids in length exist, with Aβ1−40 and Aβ1−42 being the most common. According to the amyloid hypothesis, the aggregation of Aβ initiates a complex cascade that ultimately leads to neuronal cell death and loss of brain functions.7,8 Two recent studies by solid-phase nuclear magnetic resonance (NMR) have elucidated the three-dimensional structure of amyloid fibrils formed by Aβ1−42.9,10 It is well documented, however, that, not the Aβ fibrils themselves, but soluble oligomers of Aβ are the most cytotoxic agents.11,12 Thus, preventing the early phase of Aβ aggregation is a promising concept for impeding the progress of AD.6,12 It has been known for many years that the KLVFF sequence (Aβ16−20) plays an important role in the aggregation of Aβ.13 Many peptides derived from this sequence inhibit the aggregation process.14 Two examples are peptides OR1 and OR2 (RGKLVFFGR), where arginine was added to the hydrophobic core to enhance the solubility. The glycine units serve as spacers.15 As these inhibitors suffer from instability against proteases, a retro-inverso derivative (RI-OR2) was synthesized, which is not degraded by proteases and retains its inhibitory activity.16 Attachment of a retro-inverso TAT © XXXX American Chemical Society

Received: March 10, 2017 Revised: July 28, 2017

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DOI: 10.1021/acs.biochem.7b00223 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry challenging because of the fact that the mass spectra show mainly the monomer and some smaller peaks at very low intensities that can be assigned to the dimer and a few higher species. This challenge is even more pronounced with Aβ1−42, the amyloid peptide most prone to aggregation. Therefore, it was not possible to study the aggregation process over longer periods of time because the magnitudes of the small oligomer signals decreased further because of the formation of larger oligomers and fibrils.41 Nonetheless, the effect of inhibitor molecules on the aggregation of Aβ1−42 has been investigated by ESI.43−45 Recently, we established LILBID-MS, a technique that can detect weak molecular complexes, as a new tool to follow the aggregation kinetics of Aβ.31 Oligomers up to 20-mer as well as Aβ−inhibitor complexes can be observed and quantified. Larger aggregates were detected in this study by simultaneous application of fluorescence correlation spectroscopy (FCS) and transmission electron microscopy (TEM). Furthermore, we could show that these methods can be used to identify inhibitors of aggregation. Whereas FCS and TEM are sensitive to only large oligomers and fibrils, LILBID clearly demonstrates if an inhibitor can also stop the first steps of oligomerization. The best results were found for the KLVFF derivative OR2.31 In the study presented here, we modified its retro-inverso peptide in three steps by replacing hydrophobic amino acids with non-natural building blocks that can be accessed from the Catellani reaction.46,47 The final compound 18, stable against proteolytic degradation, retards the aggregation of chemically synthesized Aβ1−42 over 7 days at 37 °C and also partially dissolves pre-aggregated samples.

Scheme 1. Synthesis of Amino Acid 2a and of Precursors 1a−ha

(a) (1) KHMDS, dry THF, −78 °C; (2) trisyl-azide, dry THF, −78 °C; (3) HOAc, 30 °C; (b) H2O2, LiOH, THF/H2O, 0 °C; (c) Pd/C, H2, MeOH, room temperature (ref 47); (d) Boc2O, NaHCO3, dioxane/H2O, 0 °C, room temperature. a

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Scheme 2. Synthesis of Peptidomimetic 5fa

RESULTS The Catellani reaction is a Pd-catalyzed multicomponent reaction.46 It may be used to attach two or three side chains to an aromatic framework in a single synthetic step. Products can be converted stereoselectively into α-amino acids equipped with a second amino alkyl chain as shown for the 1h → 2a example (Scheme 1). In the same way, we have prepared αamino acids from intermediates 1b−g.47 However, in the work presented here, with exception of compound 2a, the α-amino residue was not required. Hence, compounds 1b−g were hydrolyzed to form the achiral Boc-protected amino acids 3b− h (Scheme 2 and the Supporting Information). In the first generation, the two N-terminal Phe residues of retro-inverso KLVFF were replaced by one of the non-natural amino acids (2a or 3b−h) to generate compounds 5a−h (Chart 1). Scheme 2 shows exemplarily how compound 5f was synthesized by a combination of solution- and solid-phase methods. Carboxylic acid 3f reacted first with D-valine methyl ester in solution. After saponification, this fragment was coupled to the D-Leu-D-Lys part, which had been assembled before on a solid support (Scheme 2). To evaluate compounds 5a−h, we used an assay based on FCS that is well suited for a fast and easy identification of inhibitors. Aβ (100 μM) and a Cy5-labeled analogue of this peptide (0.5 μM) are incubated at 4 °C in the presence of the potential inhibitor (400 μM).31 After different periods of time, samples are 10-fold diluted and analyzed by FCS. The Cy5labeled Aβ is integrated into the Aβ oligomers and fibrils, so that these can be detected by FCS. With the increasing size of aggregates, increasing diffusion times are expected. The instrument’s software allows the autocorrelation function to

(a) H2O2, LiOH, THF/H2O, 0 °C; (b) D-Val-OMe·HCl, DCC, HOBt, DIPEA, CH2Cl2, room temperature; (c) NaOH, MeOH/H2O, room temperature; (d) D-Lys-D-Leu-NH2 (resin-bound), DIC, HOBt, NMP, room temperature; (e) TFA/TIS/H2O, room temperature; HPLC purification. For all other compounds 3 and 4, see Scheme 1 for the aromatic residue. a

be formally fitted to a one-, two-, or three-component system. This is a crude approximation, as aggregating Aβ samples are composed of large numbers of individual oligomers. Nevertheless, the mixture can be roughly categorized as monomers and small oligomers (diffusion time of 80 μs), medium-sized oligomers (diffusion time of ∼5000 μs), and large aggregates (diffusion time of >50000 μs). We have shown that the fraction with the shortest diffusion time of ∼80 μs represents mainly monomeric Aβ with a few small oligomers (hereafter named “monomeric Aβ” for the sake of simplicity). During the aggregation of an Aβ sample, the fraction of monomeric Aβ decreases, while the amount of medium- and large-sized aggregates increases. Therefore, if a molecule inhibits aggregation, the percentage of the monomeric fraction will remain near 100% over the whole incubation time.31 Within the first generation of peptidomimetics, those containing small aromatic systems (5b−d) showed the fastest decay of the monomer fraction (Table 1). Stronger inhibition was seen with CF3 analogue 5e. The best structures were those B


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Biochemistry Chart 1. Inhibitors of the First Generation Compared to OR1, OR2, and the Retro-Inverso Peptide of KLVFF

Table 1. Inhibition of the Aβ Aggregation by 5a−h Measured with FCSa incubation time (h) controlc 5ad 5bd 5cd 5dd 5ed 5fd 5gd 5he OR2d,f

0 93 94 ± 8 100 ± 0 95 ± 7 100 ± 0 100 ± 0 97 ± 2 100 ± 0 100 ± 0 100 ± 0

6 79 94 ± 3 92 ± 6 78 ± 20 81 ± 11 94 ± 3 96 ± 3 86 ± 14 70 ± 7 100 ± 0g

24 36 100 ± 0 83 ± 12 60 ± 18 70 ± 11 87 ± 1 91 ± 2 97 ± 2 88 ± 2 96 ± 5

72 26 100 ± 0e ndh ndh ndh 96 ± 6 100 ± 1 99 ± 1 91 ± 0 ndh

192 20 96 ± 5 ndh ndh ndh ndh 100 ± 0 97 ± 4 ndh ndh

+48b 0 97 ± 0 ndh ndh ndh ndh 91 ± 9 89 ± 10 ndh ndh

a The percentage of the fastest fraction (diffusion time of ∼80 μs) is shown, corresponding mainly to Aβ monomers. Samples were incubated at 4 °C with a 4-fold excess of inhibitor. bFurther incubation at 37 °C, after 192 h at 4 °C. cSingle experiment. dMean value of three experiments. eMean value of two experiments. fData taken from ref 31. gMeasured after 4 h at 4 °C. hNot determined.

the C-terminal carboxamide. Pyrene analogue 13h was prepared in the same way. In the FCS assay, naphthalene 13f showed activity (89 ± 4% monomeric Aβ) that was higher than that of pyrene 13h (73 ± 1% monomeric Aβ) after incubation for 24 h at 4 °C. This became even more pronounced after subsequent incubation at 37 °C for 3 days. Whereas samples containing 13f still mainly consisted of monomeric Aβ (89 ± 3%), samples containing 13h showed an obvious decrease in the monomer fraction (49 ± 9%). In compounds 5a−h, 13f, and 13h, the FF part of the KLVFF sequence and its retro-inverso counterpart has been mimicked by the different aromatic ring systems, each equipped

with condensed aromatic systems: naphthalene 5f, phenanthrene 5g, and pyrenes 5a and 5h. Therefore, the incubation time of such samples was extended to 8 days at 4 °C (192 h), when the samples still consisted mainly of monomeric Aβ species. After additional incubation at 37 °C for 2 days, the monomeric fraction completely disappeared in the control. In contrast, samples with inhibitors 5a, 5f, and 5g still contained high levels of monomeric Aβ. These results also demonstrate that the α-amino group present in compound 5a may be helpful but is no requirement for good inhibitory activity. The promising effects of compounds 5a−h prompted us to further simplify the structures of the inhibitors. Scheme 3 shows the synthesis of compound 13f, derived from 5f by omission of C


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Biochemistry Scheme 3. Synthesis of Peptidomimetic 13fa

(a) Boc2O, Et3N, THF, 0 °C; (b) Cbz-D-Leu-OH, HATU, DIPEA, DMF, room temperature; (c) H2, Pd/C, THF, room temperature; (d) H2, Pd/ C, MeOH, room temperature; (e) (1) NaOH, MeOH/H2O, room temperature; (2) D-Val-OMe·HCl, HATU, DIPEA, DMF, room temperature; (f) (1) NaOH, MeOH/H2O, room temperature; (2) 9, HATU, DIPEA, DMF, room temperature; (g) TFA, CH2Cl2, room temperature; HPLC purification (as TFA salt). a

with a basic side chain. Our most promising candidate, 18, belongs to the third generation of inhibitors. In this structure, we also exchanged the KL part with a synthetic ring system. Its amino group should replace the side chain of lysine, whereas the aromatic residue was a hydrophobic substitute for leucine. The synthesis started from methyl ester 10f. After hydrogenation, the Boc protecting groups were replaced by Cbz. The ester was then reduced and the resulting alcohol converted into amine 16. The removal of Cbz was followed by coupling to naphthyl-D-Val building block 11f. Global deprotection with TFA resulted in molecule 18 (Scheme 4). In the first experiments, it turned out, however, that the presence of two naphthalene units reduced the solubility of 18 in water and caused precipitation at 400 μM. A slight adaptation of assay conditions was thus required. At

concentrations of 10 μM Aβ and 40 μM 18, precipitation could no longer be observed. Initial FCS experiments in this concentration range looked very promising. All subsequent experiments, therefore, were focused on this candidate. Three types of experiments have been conducted and analyzed by four independent analytical methods: FCS, LILBID mass spectrometry, thioflavin T (ThT) fluorescence, and confocal laser scanning microscopy (CLSM). First, we have studied the aggregation of Aβ in the absence of 18. In the next set of experiments, the inhibitor was present from the beginning on. In the third type of assays, compound 18 was added to a pre-aggregated sample to see if oligomers and fibrils of Aβ might be dissolved again. Aβ Aggregation in the Absence of Compound 18. Even at Aβ concentrations of 10 μM, fast aggregation could be observed with FCS (Table 2, control). As described previously,

Scheme 4. Synthesis of Peptidomimetic 18a

Table 2. Summary of FCS Measurementsa incubation time (days) control inhibition dissolution

0 μM 18 20 μM 18 40 μM 18 20 μM 18 40 μM 18

0 100 ± 0 ndb ndb ndb ndb

2 0±0 70 ± 16 88 ± 16 44 ± 30c 75 ± 24c

7 0±0 81 ± 26 99 ± 1 65 ± 30 63 ± 5

a The percentage of the fast-moving fraction (diffusion time of ∼80 μs) is shown, corresponding mainly to Aβ monomers. Samples were incubated at 37 °C. The mean values of three experiments are given. b Not determined. cMeasured after aggregation for 2 days and 1 h after addition of 18. Initial concentrations: 10 μM Aβ and 0.05 μM Cy5-Aβ.

the exact kinetics of Aβ aggregation is difficult to reproduce. Impurities from solid-phase synthesis that can hardly be removed by HPLC are known to retard the process.48 On the other hand, different samples may contain varying amounts of oligomeric species even at time zero. Such oligomers serve as nucleation sites, thereby enhancing the kinetics. This wellknown problem has led to the development of special protocols for the preparation of recombinant Aβ by size-exclusion

a

(a) H2, Pd/C, MeOH, room temperature; (b) (1) TFA, CH2Cl2, room temperature; (2) CbzCl, Na2CO3, THF/H2O, 0 °C, room temperature; (c) LiBH4, dry THF, Δ; (d) (1) PPh3, I2, imidazole, dry DCM, 0 °C; (2) HNBoc2, Cs2CO3, dry DMF, 90 °C; (e) (1) Pd/C, H2, MeOH, room temperature; (2) 11f, NaOH, MeOH/H2O, room temperature; (3) HATU, DIPEA, dry DMF, room temperature; (f) TFA, CH2Cl2, room temperature; HPLC purification (as TFA salt). D


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Biochemistry

Figure 1. Comparison between high and low mass settings. LILBID spectra of an Aβ sample (100 μM) incubated at 4 °C in the absence of an inhibitor. (A) Spectrum with high mass settings obtained directly after dissolving Aβ. (B) Spectrum of Aβ after incubation for 48 h using high mass settings. The different Aβ oligomers are indicated. (C) Spectrum of the same freshly dissolved sample as in panel A recorded using low mass settings. (D) Spectrum of the same incubated sample as in panel B using low mass settings.

Figure 2. LILBID spectra of Aβ (100 μM) with and without a 4-fold excess of compound 18. The sample was incubated for 48 h at 4 °C. The inlet shows the monomer/oligomer ratios for these spectra.

with individual concentrations that quickly fall below the detection limit for LILBID. Therefore, all experiments had to be performed at 100 μM and 4 °C as described previously.31 Panels A and B of Figure 1 depict Aβ directly after dissolution as well as after aggregation for 48 h, showing clearly the first oligomerization steps. Thus, LILBID can detect the early species in the Aβ aggregation process as seen here for (Aβ)1− (Aβ)17. Because the instrumental settings used for the analysis of these higher-order oligomers are not optimal for the investigation of the monomers and dimers, panels C and D of Figure 1 show the same samples at different settings, which improve the signal/noise ratio of the low-mass species. It is obvious that using the low mass settings increases the sensitivity for the monomers and dimers but still enables us to monitor the oligomerization process up to the decamers.

chromatography. Samples resulting from this method aggregate exceptionally fast with reproducible kinetics.6,49,50 In the study presented here, however, chemically synthesized Aβ has been used. Furthermore, determining the aggregation kinetics by FCS is difficult, as the sample is composed of so many different species that a meaningful mathematical analysis is nearly impossible. We therefore focus on the amount of the monomeric Aβ fraction that will decrease over time but should stay constant if aggregation is stopped by an inhibitor. After 2 days at 37 °C, this monomeric fraction consistently dropped to zero in the absence of 18 and the particle number declined by 1 order of magnitude. Unfortunately, at Aβ concentrations of 10 μM, the aggregation leads to a wide heterogeneity of oligomeric species E


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Biochemistry

Figure 3. LILBID spectra of an Aβ sample (100 μM) incubated for 48 h at 4 °C (black) and treated afterward with 400 μM inhibitor 18 for 70 min (blue) and 48 h (red). The different Aβ oligomers are indicated. The inlet shows the monomer/oligomer ratio for those three spectra.

ments, Aβ (10 μM) was incubated at 37 °C in the presence of a 2- or 4-fold molar excess of the inhibitor. Samples were then examined after 2 and 7 days. The results of the FCS assay clearly demonstrate the inhibitory effect. With a 4-fold excess of 18, the formation of larger oligomers was largely prevented, even after incubation for 7 days (see Table 2, inhibition). Results obtained with a 2-fold excess of 18 were still significant. To rule out other explanations, it is important to look at particle numbers. The dominance of the fast-moving fraction might result from precipitation of Aβ by compound 18 to levels below the aggregation threshold. Alternatively, higher-order oligomers might have been precipitated selectively so that only the fast-moving fraction remained in the sample. Both mechanisms would result in a depletion of dye-labeled Aβ. In these experiments, however, particle numbers stay constant over time. As a further control, samples of Aβ (10 μM) and compound 18 (40 μM) were centrifuged 24 h after incubation. Particle numbers before and after centrifugation were unchanged. In contrast, a major drop was seen when this test was repeated at higher concentrations (100 μM Aβ and 400 μM 18) when precipitation indeed occurs. LILBID experiments were conducted at laser desorption intensities below a level that would lead to dissociation of noncovalent complexes, to ensure that the mass spectra reflect the solution distribution of the Aβ oligomers. The concentrations employed were 100 μM Aβ and 400 μM 18 (4 °C). As already mentioned, Aβ and the inhibitor will partially precipitate under such conditions. The amount of Aβ remaining in solution, however, is sufficiently high for analysis by LILBID (>10 μM). Precipitation may also be the cause of difficulties with the LILBID droplet formation process when incubation temperatures increased to 37 °C. For the freshly dissolved sample (Figure 2, blue) as well as for the sample incubated with compound 18 (red), the spectra show mainly monomeric Aβ with a small amount of dimer, trimer, tetramer, and pentamer. In fact, the monomer/oligomer ratio in the presence of compound 18, calculated according to

These settings have been used for the inhibition studies (Figures 2 and 3), as we are interested specifically in the distribution of monomers and small oligomers. Via detection of the fluorescence of thioflavin T, which intercalates into sheets of Aβ, fast aggregation was also confirmed.51 After 2 days at 37 °C, the fluorescence reached 74% of the maximum value obtained after 7 days (see Table 3, control). Table 3. Summary of ThT Measurementsa incubation time (days) control inhibition dissolution

0 μM 18 20 μM 18 40 μM 18 20 μM 18 40 μM 18

0 0 ndb ndb ndb ndb

2 74 15 21 85 80

± ± ± ± ±

7 14 15 20c 20c

7 100 28 ± 34 ± 62 ± 75 ±

11d 5 10 7

a The relative ThT fluorescence with the starting value representing 0% and the maximum after 7 days representing 100% is shown. Samples were incubated at 37 °C. The mean values of three measurements are given. b Not determined. c Measured after aggregation for 2 days and 1 h after addition of 18. dMean value of two measurements.

In our FCS studies, a solution of Aβ was doped with a small quantity of its Cy5-labeled analogue. Such samples permit us to visualize peptide aggregation by confocal microscopy, as well. When the buffer also contains thioflavin, Aβ fibrils can be stained by Cy5-Aβ and ThT simultaneously (Figures S7− S11).51 At time zero, no aggregates could be found in the CLSM images. After incubation for 2 and 7 days, only a few aggregates were detected, with both Cy5 and ThT (Figure S7). The coincidence of red and green fluorescence in the pictures is important to demonstrate that Cy5-Aβ is integrated into Aβ fibrils and does not form aggregates by itself. If 100 μM Aβ is used, at time zero some larger structures can be seen after an extended search in the droplet. This confirms the findings of FCS and LILBID (Figure 1C) that even at the beginning of the aggregation process Aβ samples are not monomeric to 100%. Influence of Compound 18. To elucidate the activity of peptidomimetic 18, we examined its effect on the Aβ aggregation with the different methods. For FCS measure-

monomer/oligomer = F

I1 ∑ (Inn) DOI: 10.1021/acs.biochem.7b00223 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

Figure 4. Time dependence of the monomer/oligomer ratio measured via LILBID. Aβ (100 μM) was incubated for 48 h at 4 °C before the addition of 400 μM 18. The monomer/oligomer ratios determined 0 min, 70 min, and 48 h after the addition of 18 correspond to the spectra in Figure 3. The lines serve as only guides for the eye. The inset shows an enlargement of the first 75 min, revealing a lag time before dissolution.

(I is the intensity; n ≥ 2 for the size of the respective oligomer), increases from an initial value of 1.33 to a final number of 1.87, which shows an increase in the level of monomeric Aβ in relation to those of its oligomers. The largest oligomer size observed decreases from penta- to tetramer. In contrast, the control sample without the inhibitor (black) shows a monomer/oligomer value of 0.33 and (Aβ)8 as the largest oligomer. The ThT data show a distinct inhibition by 18 as the fluorescence intensity was reduced to ∼30% (see Table 3, inhibition). Both concentrations of 18 (20 or 40 μM; 10 μM Aβ) led to comparable decreases in fluorescence, but the fluorescence intensity was still higher than at time zero, indicating that some fibrillar forms must have built up. This is in accordance with the few structures observed with CLSM (see below). It can also be seen that the fluorescence intensity after 7 days is higher than after 2 days. We assume that the inhibitor massively slows Aβ oligomerization as implied by FCS and LILBID but cannot prevent the slow conversion of mediumsized oligomers into fibrillar forms. These may have arisen from minor nucleation sites still present after the preparation of monomeric Aβ. The confocal microscopy images showed some small structures, stained simultaneously by Cy5 and ThT (Figure S10). As in the Aβ samples incubated without inhibitor (Figure S7), the number of aggregates is low. A quantitative comparison by integrating over entire samples is not possible. Instead, we have analyzed several sectors (67.3 μm × 67.3 μm) where particles became visible after 7 days using the software package Fiji.52 The software superimposes the scans measured at 20 different axial positions within the sample droplet (distance between imaging planes of 0.5 μm) and integrates the fluorescence of Cy5 and ThT independently. In four different sectors, ratios of 10.5 ± 1.3 (intensity Cy5/intensity ThT) were measured in the sample containing 18. In contrast, samples of Aβ incubated without inhibitor showed a clear shift toward ThT emission with ratios of 5.2 ± 3.0 (see page S22). Accordingly, the fibrillar character of aggregates formed in the presence of 18 seems to be less pronounced. When samples

containing 100 μM Aβ and 400 μM 18 were analyzed by CLSM, red cloudy structures caused by co-precipitation of Cy5Aβ became visible (Figure S11). Such cloudy structures did not show the green emission of ThT. At lower concentrations (10 μM Aβ and 40 μM 18), precipitation effects could not be observed (Figure S9). Dissolution of Preformed Aggregates by Compound 18. Next we employed FCS to investigate the ability of 18 to dissolve preformed Aβ aggregates. Solutions of Aβ (10 μM) were kept at 37 °C for 2 days in the absence of the inhibitor. Then a 2- or 4-fold molar excess of 18 was added. Samples were examined 1 h after the addition and after an additional 5 days at 37 °C. Within the first hour of incubation with 40 μM inhibitor, the monomeric fraction of Aβ in the FCS assay increased from 0 to ∼70% (Table 2, dissolution). This effect was less pronounced for a 2-fold excess, but a significant effect was still seen. In parallel to the increase in the level of monomers, particle numbers also increased in these experiments by 1 order of magnitude. After 5 days in the presence of the inhibitor (7 day experiment), the fast-moving fraction of Aβ amounted to 60% for both concentrations of 18. This demonstrates that 18 can dissolve larger species to monomers and small oligomers. Nevertheless, the final amount of monomeric Aβ is larger when compound 18 is present over the whole period of incubation. In the ThT assay, no significant effect of molecule 18 was observed as the fluorescence intensity did not change after the addition of the inhibitor (Table 3, dissolution). Consistently, CLSM images showed some small aggregates. LILBID experiments were conducted as before with samples containing 100 μM Aβ and 400 μM 18 incubated at 4 °C. Samples were aggregated for 2 days in the absence of the inhibitor. Then a 4-fold excess of 18 was added, and spectra were recorded during the first 70 min and 2 days after the addition of the inhibitor. Already after 70 min, no oligomers larger than (Aβ)5 could be detected, and after an additional 2 days, the situation was comparable to that in which 18 was present during the whole aggregation period (Figure 3). The monomer/oligomer ratio increases from 0.33 before the G


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Biochemistry addition of the inhibitor to 0.97 70 min later, which is ∼50% of the final ratio of 1.62. The fast dissolution effect of 18 can be seen in Figure 4 where the largest increase in the monomer/oligomer ratio, after a short lag time, occurs within the first 70 min. For comparison, the known inhibitor OR115 was also examined, a peptide with an aqueous solubility higher than that of compound 18 (Figures S5 and S6). Distinct complexes of Aβ oligomers with one or two molecules of OR1 are visible in the spectra, and Aβ aggregates are dissolved, as well. Proteolytic Stability. We tested the resistance against proteolytic degradation by treatment with proteinase K at 37 °C. Peptide OR1 was completely degraded after incubation for 24 h, whereas 18 showed no sign of instability (Figures S2 and S3). Cytotoxicity. Cytotoxicity is an important issue when cell culture experiments are envisioned. For that reason, the proliferation of HEK-APPwt cells was examined in the presence of different concentrations of compound 18.53 In the concentration range of 30 nM to 10 μM, no toxic effects were observed. Cell viability, however, dropped to 50% at concentrations of 30 μM. In addition, we studied the toxicity of trifluoroacetate and chloride salts of 18 in human neuroblastoma cell line SH-SY5Y. Compared to the control, LDH release was significantly elevated after incubation for 24 h with 18-TFA (20 and 40 μM) and 18-Cl (20 and 40 μM). Metabolic activity was significantly reduced after incubation for 24 h with 10 μM 18-TFA. Although TFA ions are known for adverse effects, the impact of 18-Cl on metabolic activity was even slightly more pronounced (Figure S4).

Formation of a complex between Aβ and OR1 can be directly seen in LILBID spectra (Figures S5 and S6), whereas such signals are missing when Aβ is mixed with 18. This observation was surprising at first but can be understood in light of the interaction characteristics, which differ for OR1 and 18. The lack of signals representing Aβ−18 complexes does not necessarily indicate weaker binding in the case of 18: Aβ monomers have a net charge of −3. Thus, in addition to hydrophobic interactions, ligand binding also has a strong electrostatic component. Net charges of inhibitors fall from +4 (OR2) and +3 (OR1) to +2 (18). In the LILBID desorption process, the aqueous droplets undergo an explosive expansion upon laser irradiation, which sets the solvated ions free. With an increasing rate of loss of water molecules, ionic attraction more and more outperforms the hydrophobic effect. Accordingly, complexes of ligands with high opposite charges have a better chance to withstand dissociation in the gas phase: Even though the Arg-Arg-Arg peptide (net charge of +4) does not inhibit the aggregation of Aβ, the complexes are nicely visible.31 In contrast, the powerful aggregation inhibitor bexarotene,6 a lipophilic carboxylate with a net charge of −1, does not show signals for the Aβ complex in LILBID (data not shown). Given the increased lipophilicity of 18, we cannot strictly exclude sequestration of Aβ by hydrophobic aggregates of 18. This mechanism can be responsible for the nonspecific inhibition of enzymes by certain types of lipophilic compounds.54 Protein aggregation can be blocked in the same way.55 FCS data, however, provide evidence against hydrophobic sequestration: such effects should have increased the diffusion time and reduced particle numbers. At concentrations of compound 18 used in our assays, the viability of HEK-APPwt and SH-SY5Y cells is already compromised. It is important to note, however, that concentrations of Aβ used in such cell-free aggregation studies (10−100 μM) are also far above the physiological levels. Intracellular aggregation occurs in hydrophobic environments where both Aβ and inhibitor candidates will be locally concentrated. In fact, studies using HEK-APPwt cells in vitro showed beneficial effects of 1 and 10 μM 18. These results will be reported in due course.

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DISCUSSION Starting from the well-known sequence KLVFF, we have prepared new peptidomimetics that retard the aggregation of synthetic Aβ1−42. With the first generation of compounds, we showed that the FF part can be replaced by a condensed aromatic system and that the terminal amino group is not necessary for activity. In the second and third generations, the amount of amino acids in the inhibitor structure was further reduced. The most simplified candidate 18 resembles the starting peptide KLVFF only distantly. It has a largely reduced number of hydrogen bond donors and acceptors. As a consequence, 18 can hardly form β-sheet-like structures with Aβ as other peptidomimetics might. 18 bears some similarity to polyphenolic structures like curcumin and resveratrol, which also consist of two functionalized aromatic rings connected by a linker. Inhibitor 18 is stable against proteolytic degradation by proteinase K, and the inhibitory activity could be demonstrated by FCS, ThT fluorescence, LILBID, and CLSM. 18 can largely, but not completely, prevent Aβ aggregation over extended periods of time. A slight increase of ThT emission accompanies the formation of a few particles that can be viewed by CLSM. Existing aggregates of Aβ can be partially dissolved by both compound 18 and peptide OR1. However, no significant decrease in ThT emission is observed. Mature fibrillar structures thus seem to be more stable against the action of compound 18. This view is further supported by gel electrophoresis (Figure S12). The formation of large aggregates that cannot enter the gel is mostly prevented by 18. Once formed, however, this material does not disappear when 18 is added subsequently.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00223. Full description of synthetic methods, data for characterizing new compounds, proteolytic stability of OR1 and of compound 18, determination of solubility and toxicity, preparation and oligomerization of Aβ peptides, methods used to monitor Aβ aggregation in the absence and presence of OR1 and compound 18 (FCS, LILBID, ThT, CLSM, and gel electrophoresis), and 1H and 13C NMR spectra of all new compounds (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Mike Heilemann: 0000-0002-9821-3578 Gunter P. Eckert: 0000-0002-8002-9983 Nina Morgner: 0000-0002-1872-490X H


Article

Biochemistry

Arrest of beta-amyloid fibril formation by a pentapeptide ligand. J. Biol. Chem. 271, 8545−8548. (14) Re, F., Airoldi, C., Zona, C., Masserini, M., Ferla, B. L., Quattrocchi, N., and Nicotra, F. (2010) Beta amyloid aggregation inhibitors: small molecules as candidate drugs for therapy of Alzheimer’s disease. Curr. Med. Chem. 17, 2990−3006. (15) Austen, B. M., Paleologou, K. E., Ali, S. A. E., Qureshi, M. M., Allsop, D., and El-Agnaf, O. M. A. (2008) Designing peptide inhibitors for oligomerization and toxicity of Alzheimer’s beta-amyloid peptide. Biochemistry 47, 1984−1992. (16) Taylor, M., Moore, S., Mayes, J., Parkin, E., Beeg, M., Canovi, M., Gobbi, M., Mann, D. M. A., and Allsop, D. (2010) Development of a proteolytically stable retro-inverso peptide inhibitor of beta-amyloid oligomerization as a potential novel treatment for Alzheimer’s disease. Biochemistry 49, 3261−3272. (17) Parthsarathy, V., McClean, P. L., Hölscher, C., Taylor, M., Tinker, C., Jones, G., Kolosov, O., Salvati, E., Gregori, M., Masserini, M., and Allsop, D. (2013) A novel retro-inverso peptide inhibitor reduces amyloid deposition, oxidation and inflammation and stimulates neurogenesis in the APPswe/PS1ΔE9 mouse model of Alzheimer’s disease. PLoS One 8, e54769. (18) Gordon, D. J., Sciarretta, K. L., and Meredith, S. C. (2001) Inhibition of beta-amyloid(40) fibrillogenesis and disassembly of betaamyloid(40) fibrils by short beta-amyloid congeners containing Nmethyl amino acids at alternate residues. Biochemistry 40, 8237−8245. (19) Gordon, D. J., Tappe, R., and Meredith, S. C. (2002) Design and characterization of a membrane permeable N-methyl amino acidcontaining peptide that inhibits Aβ1−40 fibrillogenesis. J. Pept. Res. 60, 37−55. (20) Bett, C. K., Ngunjiri, J. N., Serem, W. K., Fontenot, K. R., Hammer, R. P., McCarley, R. L., and Garno, J. C. (2010) Structureactivity relationships in peptide modulators of β-amyloid protein aggregation: variation in α,α-disubstitution results in altered aggregate size and morphology. ACS Chem. Neurosci. 1, 608−626. (21) Bett, C. K., Serem, W. K., Fontenot, K. R., Hammer, R. P., and Garno, J. C. (2010) Effects of peptides derived from terminal modifications of the Aβ central hydrophobic core on Aβ fibrillization. ACS Chem. Neurosci. 1, 661−678. (22) Gordon, D. J., and Meredith, S. C. (2003) Probing the role of backbone hydrogen bonding in beta-amyloid fibrils with inhibitor peptides containing ester bonds at alternate positions. Biochemistry 42, 475−485. (23) Chafekar, S. M., Malda, H., Merkx, M., Meijer, E. W., Viertl, D., Lashuel, H. A., Baas, F., and Scheper, W. (2007) Branched KLVFF tetramers strongly potentiate inhibition of beta-amyloid aggregation. ChemBioChem 8, 1857−1864. (24) Ouberai, M., Dumy, P., Chierici, S., and Garcia, J. (2009) Synthesis and biological evaluation of clicked curcumin and clicked KLVFFA conjugates as inhibitors of beta-amyloid fibril formation. Bioconjugate Chem. 20, 2123−2132. (25) Soto, C., Kindy, M. S., Baumann, M., and Frangione, B. (1996) Inhibition of Alzheimer’s amyloidosis by peptides that prevent betasheet conformation. Biochem. Biophys. Res. Commun. 226, 672−680. (26) Soto, C., Sigurdsson, E. M., Morelli, L., Asok Kumar, R., Castano, E. M., and Frangione, B. (1998) Beta-sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: implications for Alzheimer’s therapy. Nat. Med. 4, 822−826. (27) Permanne, B., Adessi, C., Saborio, G. P., Fraga, S., Frossard, M.J., Dorpe, J. V., Dewachter, I., Banks, W. A., Leuven, F. V., and Soto, C. (2002) Reduction of amyloid load and cerebral damage in a transgenic mouse model of Alzheimer’s disease by treatment with a beta-sheet breaker peptide. FASEB J. 16, 860−862. (28) Adessi, C., Frossard, M.-J., Boissard, C., Fraga, S., Bieler, S., Ruckle, T., Vilbois, F., Robinson, S. M., Mutter, M., Banks, W. A., and Soto, C. (2003) Pharmacological profiles of peptide drug candidates for the treatment of Alzheimer’s disease. J. Biol. Chem. 278, 13905− 13911. (29) Fradinger, E. A., Monien, B. H., Urbanc, B., Lomakin, A., Tan, M., Li, H., Spring, S. M., Condron, M. M., Cruz, L., Xie, C.-W.,

Michael W. Göbel: 0000-0002-5694-4823 Present Address

⊥ G.P.E.: Institute of Nutritional Sciences, Justus-LiebigUniversity Giessen, Wilhelmstr. 20, D-35392 Giessen, Germany.

Funding

N.M. is supported by Cluster of Excellence Frankfurt (Macromolecular Complexes) and received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC Grant agreement n° 337567. T.L. is funded by the DFG-GRK 1986. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Prof. Martin Grininger (Goethe University Frankfurt) for providing access to his CCD camera. The solubility of compound 18-TFA was determined by Frank Kaiser.

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REFERENCES

(1) Jakob-Roetne, R., and Jacobsen, H. (2009) Alzheimer’s disease: from pathology to therapeutic approaches. Angew. Chem., Int. Ed. 48, 3030−3059. (2) World Alzheimer Report 2015: The Global Impact of Dementia (2015) Alzheimer’s Disease International, London. (3) Mohamed, T., Shakeri, A., and Rao, P. P. N. (2016) Amyloid cascade in Alzheimer’s disease: Recent advances in medicinal chemistry. Eur. J. Med. Chem. 113, 258−272. (4) Rajasekhar, K., Chakrabarti, M., and Govindaraju, T. (2015) Function and toxicity of amyloid beta and recent therapeutic interventions targeting amyloid beta in Alzheimer’s disease. Chem. Commun. 51, 13434−13450. (5) Sevigny, J., Chiao, P., Bussière, T., Weinreb, P. H., Williams, L., Maier, M., Dunstan, R., Salloway, S., Chen, T., Ling, Y., O’Gorman, J., Qian, F., Arastu, M., Li, M., Chollate, S., Brennan, M. S., QuinteroMonzon, O., Scannevin, R. H., Arnold, H. M., Engber, T., Rhodes, K., Ferrero, J., Hang, Y., Mikulskis, A., Grimm, J., Hock, C., Nitsch, R. M., and Sandrock, A. (2016) The antibody aducanumab reduces Aβ plaques in Alzheimer’s disease. Nature 537, 50−56. (6) Habchi, J., Arosio, P., Perni, M., Costa, A. R., Yagi-Utsumi, M., Joshi, P., Chia, S., Cohen, S. I. A., Müller, M. B. D., Linse, S., Nollen, E. A. A., Dobson, C. M., Knowles, T. P. J., and Vendruscolo, M. (2016) An anti cancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer’s disease. Sci. Adv. 2, e1501244. (7) Blennow, K., de Leon, M. J., and Zetterberg, H. (2006) Alzheimer’s disease. Lancet 368, 387−403. (8) Haass, C., and Selkoe, D. J. (2007) Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid betapeptide. Nat. Rev. Mol. Cell Biol. 8, 101−112. (9) Colvin, T. M., Silvers, R., Ni, Q. Z., Can, T. V., Sergeyev, I., Rosay, M., Donovan, K. J., Michael, B., Wall, J., Linse, S., and Griffin, R. G. (2016) Atomic resolution structure of monomorphic Aβ42 amyloid fibrils. J. Am. Chem. Soc. 138, 9663−9674. (10) Wälti, M. A., Ravotti, F., Arai, H., Glabe, C. G., Wall, J. S., Böckmann, A., Güntert, P., Meier, B. H., and Riek, R. (2016) Atomicresolution structure of a disease-relevant Aβ(1−42) amyloid fibril. Proc. Natl. Acad. Sci. U. S. A. 113, E4976−4984. (11) Walsh, D. M., and Selkoe, D. J. J. (2007) Aβ oligomers - a decade of discovery. J. Neurochem. 101, 1172−1184. (12) Hamley, I. W. (2012) The amyloid beta peptide: a chemist’s perspective. Role in Alzheimer’s and fibrillization. Chem. Rev. 112, 5147−5192. (13) Tjernberg, L. O., Näslund, J., Lindqvist, F., Johansson, J., Karlström, A. R., Thyberg, J., Terenius, L., and Nordstedt, C. (1996) I


Article

Biochemistry Benedek, G. B., and Bitan, G. (2008) C-terminal peptides coassemble into Abeta42 oligomers and protect neurons against Abeta42-induced neurotoxicity. Proc. Natl. Acad. Sci. U. S. A. 105, 14175−14180. (30) Li, H., Du, Z., Lopes, D. H. J., Fradinger, E. A., Wang, C., and Bitan, G. (2011) C-terminal tetrapeptides inhibit Aβ42-induced neurotoxicity primarily through specific interaction at the N-terminus of Aβ42. J. Med. Chem. 54, 8451−8460. (31) Cernescu, M., Stark, T., Kalden, E., Kurz, C., Leuner, K., Deller, T., Göbel, M., Eckert, G. P., and Brutschy, B. (2012) Laser-induced liquid bead ion desorption mass spectrometry: an approach to precisely monitor the oligomerization of the β-amyloid peptide. Anal. Chem. 84, 5276−5284. (32) Sinha, S., Lopes, D. H. J., Du, Z., Pang, E. S., Shanmugam, A., Lomakin, A., Talbiersky, P., Tennstaedt, A., McDaniel, K., Bakshi, R., Kuo, P.-Y., Ehrmann, M., Benedek, G. B., Loo, J. A., Klärner, F.-G., Schrader, T., Wang, C., and Bitan, G. (2011) Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid protein. J. Am. Chem. Soc. 133, 16958−16969. (33) Attar, A., Ripoli, C., Riccardi, E., Maiti, P., Li Puma, D. D., Liu, T., Hayes, J., Jones, M. R., Lichti-Kaiser, K., Yang, F., Gale, G. D., Tseng, C. H., Tan, M., Xie, C. W., Straudinger, J. L., Klärner, F.-G., Schrader, T., Frautschy, S. A., Grassi, C., and Bitan, G. (2012) Protection of primary neurons and mouse brain from Alzheimer’s pathology by molecular tweezers. Brain 135, 3735−3748. (34) Rzepecki, P., Nagel-Steger, L., Feuerstein, S., Linne, U., Molt, O., Zadmard, R., Aschermann, K., Wehner, M., Schrader, T., and Riesner, D. (2004) Prevention of Alzheimer’s disease-associated Abeta aggregation by rationally designed nonpeptidic beta-sheet ligands. J. Biol. Chem. 279, 47497−47505. (35) Hochdörffer, K., März-Berberich, J., Nagel-Steger, L., Epple, M., Meyer-Zaika, W., Horn, A. H. C., Sticht, H., Sinha, S., Bitan, G., and Schrader, T. (2011) Rational design of β-sheet ligands against Aβ42induced toxicity. J. Am. Chem. Soc. 133, 4348−4358. (36) McLaurin, J., Kierstead, M. E., Brown, M. E., Hawkes, C. A., Lambermon, M. H. L., Phinney, A. L., Darabie, A. A., Cousins, J. E., French, J. E., Lan, M. F., Chen, F., Wong, S. S. N., Mount, H. T. J., Fraser, P. E., Westaway, D., and George-Hyslop, P. S. (2006) Cyclohexanehexol inhibitors of Abeta aggregation prevent and reverse Alzheimer phenotype in a mouse model. Nat. Med. 12, 801−808. (37) Nie, Q., Du, X.-g., and Geng, M.-y. (2011) Small molecule inhibitors of amyloid β peptide aggregation as a potential therapeutic strategy for Alzheimer’s disease. Acta Pharmacol. Sin. 32, 545−551. (38) Porat, Y., Abramowitz, A., and Gazit, E. (2006) Inhibition of amyloid fibril formation by polyphenols: structural similarity and aromatic interactions as a common inhibition mechanism. Chem. Biol. Drug Des. 67, 27−37. (39) Bartolini, M., Naldi, M., Fiori, J., Valle, F., Biscarini, F., Nicolau, D. V., and Andrisano, V. (2011) Kinetic characterization of amyloidbeta 1−42 aggregation with a multimethodological approach. Anal. Biochem. 414, 215−225. (40) Bernstein, S. L., Wyttenbach, T., Baumketner, A., Shea, J.-E., Bitan, G., Teplow, D. B., and Bowers, M. T. (2005) Amyloid betaprotein: monomer structure and early aggregation states of Abeta42 and its Pro19 alloform. J. Am. Chem. Soc. 127, 2075−2084. (41) Bernstein, S. L., Dupuis, N. F., Lazo, N. D., Wyttenbach, T., Condron, M. M., Bitan, G., Teplow, D. B., Shea, J., Ruotolo, B. T., Robinson, C. V., and Bowers, M. T. (2009) Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer’s disease. Nat. Chem. 1, 326−331. (42) Chen, X. G., Brining, S. K., Nguyen, V. Q., and Yergey, A. L. (1997) Simultaneous assessment of conformation and aggregation of β-amyloid peptide using electro spray ionization mass spectrometry. FASEB J. 11, 817−823. (43) Young, L. M., Saunders, J. C., Mahood, R. A., Revill, C. H., Foster, R. J., Tu, L.-H., Raleigh, D. P., Radford, S. E., and Ashcroft, A. E. (2015) Screening and classifying small-molecule inhibitors of amyloid formation using ion mobility spectrometry-mass spectrometry. Nat. Chem. 7, 73−81.

(44) Young, L. M., Saunders, J. C., Mahood, R. A., Revill, C. H., Foster, R. J., Ashcroft, A. E., and Radford, S. E. (2016) ESI-IMS-MS: A method for rapid analysis of protein aggregation and its inhibition by small molecules. Methods 95, 62−69. (45) Woods, L. A., Radford, S. E., and Ashcroft, A. E. (2013) Advances in ion mobility spectrometry-mass spectrometry reveal key insights into amyloid assembly. Biochim. Biophys. Acta, Proteins Proteomics 1834, 1257−1268. (46) Catellani, M., Motti, E., and Della Ca’, N. (2008) Catalytic sequential reactions involving palladacycle-directed aryl coupling steps. Acc. Chem. Res. 41, 1512−1522. (47) Stark, T., Suhartono, M., Göbel, M. W., and Lautens, M. (2013) A palladium-catalyzed domino reaction as key step for the synthesis of functionalized aromatic amino acids. Synlett 24, 2730−2734. (48) Finder, V. H., Vodopivec, I., Nitsch, R. M., and Glockshuber, R. (2010) The recombinant amyloid-beta peptide Aβ1−42 aggregates faster and is more neurotoxic than synthetic Aβ1−42. J. Mol. Biol. 396, 9−18. (49) Walsh, D. M., Thulin, E., Minogue, A. M., Gustavsson, N., Pang, E., Teplow, D. B., and Linse, S. (2009) A facile method for expression and purification of the Alzheimer’s disease-associated amyloid betapeptide. FEBS J. 276, 1266−1281. (50) Meisl, G., Yang, X., Frohm, B., Knowles, T. P. J., and Linse, S. (2016) Quantitative analysis of intrinsic and extrinsic factors in the aggregation mechanism of Alzheimer-associated Aβ-peptide. Sci. Rep. 6, 18728. (51) Hossain, S., Hashimoto, M., Katakura, M., Al Mamun, A., and Shido, O. (2015) Medicinal value of asiaticoside for Alzheimer’s disease as assessed using single-molecule-detection fluorescence correlation spectroscopy, laser-scanning microscopy, transmission electron microscopy, and in silico docking. BMC Complementary Altern. Med. 15, 118. (52) Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P., and Cardona, A. (2012) Fiji: an open-source platform for biologicalimage analysis. Nat. Methods 9, 676−682. (53) Pohland, M., Hagl, S., Pellowska, M., Wurglics, M., SchubertZsilavecz, M., and Eckert, G. P. (2016) MH84: A novel γ-secretase modulator/PPARγ agonist improves mitochondrial dysfunction in a cellular model of Alzheimer’s disease. Neurochem. Res. 41, 231−242. (54) McGovern, S. L., Caselli, E., Grigorieff, N., and Shoichet, B. K. (2002) A common mechanism underlying promiscuous inhibitors from virtual and high- throughput screening. J. Med. Chem. 45, 1712− 1722. (55) Feng, B. Y., Toyama, B. H., Wille, H., Colby, D. W., Collins, S. R., May, B. C. H., Prusiner, S. B., Weissman, J., and Shoichet, B. K. (2008) Small-molecule aggregates inhibit amyloid polymerization. Nat. Chem. Biol. 4, 197−199.

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