β-Hairpin mimics containing a piperidine–pyrrolidine scaffold modulate the β-amyloid aggregation process preserving the monomer species

Acyclic β-hairpins designed on oligomeric and fibril structures of Aβ1–42 disrupt protein–protein interactions mediating amyloid β-peptide aggregation.


Computational Methods
The parameters for the piperidine-pyrrolidine semi-rigid scaffold were derived by following the same protocol previously reported for similar studies. [1,2] Briefly, the scaffold geometry was built using MOE,[3] capped with an acetyl (Ac) and a NHMe group at the N-and Ctermini, respectively, and sub f L w method implemented in MOE (MMFF94x force field, Born solvation, iteration limit = 40000, MM iteration limit = 2500, rejection limit = 500). The two lowest energy conformations, selected for partial charges parameterization with the R.E.D.IV, [4] were optimized at the HF/6-31G(d) level. Two different spatial orientations were used to derive RESP-A1 charges.
REMD simulations were performed on G1a and G2a peptides, built with the tLEaP module of AMBER 14 by p x f (φ = ψ = ω = 180°) 5 with the AMBER ff96 force field and GB-OBC(II) solvent model (igb = 5, mbondi2 set of radii). [6,7] Structures were initially geometry-optimized by 500 steps of steepest-descendant followed by 500 steps of conjugated-gradient minimization. For the REMD simulation, 20 replicas were distributed over the following temperatures 260. 00 REMD simulations were conducted with pmemd on each peptide for 50 ns at constant temperature by using Langevin dynamics (ntt = 3) with different seeds (ig) for every simulation. [5] A time step of 0.002 ps and an infinite cut-off for electrostatic were requested, and the SHAKE algorithm was used in order to constrain all bonds involving hydrogens. [9] Exchanges were attempted every 2 ps and were on average accepted with a 55% probability.
The trajectories at 302.76 K of the REMD simulations were extracted and analyzed. To evaluate convergence, H-bonds, DSSP, [10] cluster population, and geometry of cluster   Figure S1. Comparison of the RMSD vs simulation time patterns obtained by RMSD analyses of the 302.76 K REMD trajectories (25 -50 ns) of peptides G1a and G2a. The most representative structure obtained by cluster analysis was used as a reference (backbone heavy atoms).

Experimental Materials and Methods
Solvents, reagents were purchased from commercial sources. All peptides were purified using RP-HPLC and a C-18 column (10 µm, 250 22 mm). ESI mass spectra were recorded on a LCQ Advantage spectrometer.

Synthesis of Fmoc-protected scaffold 4
The Fmoc-protected compound 4 was synthesized in solution starting from the known compound S1. 12 (Scheme S1). Hydrogenolysis of S1 was performed in toluene affording free amino compound 2 (99%  Operating in a sealed tube, compound 2 (0.

Circular Dichroism of compounds G1a and G2a
Solutions of G1a and G2a were prepared in MeOH (50 µM, 1.5 mL). CD spectra were obtained from 195 to 250 nm with a 0.1 nm step and 1 s collection time per step, taking three averages. The spectrum of the solvent was subtracted to eliminate interference from cell, solvent, and optical equipment. The CD spectra were plotted p y θ (degree x cm 2 x dmol -1 ) w λ ( ) N -reduction was obtained using a Fourier-transform filter program. Figure S2. CD spectra of G1a and G2a (50 μM in MeOH)

NMR discussion for compounds G1a and G2a
All experiments ( 1 H, COSY, TOCSY, NOESY and ROESY) were recorded in MeOH (4 mM) at 500 MHz. As the piperidine scaffold is present as a mixture of conformers, many signals are overlapped in the aliphatic region. Only chemical shifts of the piperidine main conformer are reported.
Compound G1a is present in solution as a dynamic equilibrium between two different hairpin structures (2:1 ratio), characterized by a different alignment of the two peptide arms (compounds G1a-1 is the main isomer, and G1a-2 is the minor one). On the other hand, compound G2a is present in solution as a stable single -hairpin conformation characterized by a peptide arms alignment similar to G1a-2.  Figure S3. Structures of compounds G1a-1, G1a-2, and G2a

NMR data and discussion for peptide G1a
NMR characterization of G1a has been very complex but the chemical shifts of two main isomers (2:1 ratio), named G1a-1 (Table S6) and G1a-2 (Table S7), were assigned.
The presence of two conformers of G1a is proved by several negative NH/NH ROEs ( Figure   FS4 in comparison with Figure FS13 related to G2a). This observation indicates a dynamic equilibrium between different conformers. 15 Interestingly, a strong intrastrand ROE was found between NH Val4 of G1a-2 and CH Val4 of G1a-1 one ( Figure FS6B), suggesting that Val-4 is probably involved in the dynamic switch between the two isomers.
ROESY experiments confirmed the presence of a turn structure in both G1a-1 and G1a-2 isomers. Spatial proximity was indeed observed between the piperidine moiety of scaffold S1 (H-2 and H-6) with both proline and Gly-5 ( Figure FS5), confirming the reported data for model sequences. 12 A complete set of CH/NH(i, i+1) ROEs is present for isomer G1a-1 (except for Val-4/Met-3 and Leu-2/Gly-1; Figures FS6A and FS6C The presence of a -hairpin structure is further confirmed by positive H shift values. 16 ( Table TS8 and Figure FS8). These values are smaller with respect to G1a-2 and G2a indicating that the hairpin conformation of G1a-1 is not very stable. Only Met-3 is characterized by a negative H value. This is probably due to the anisotropic effect of the aromatic ring of Phe-11, 17 that faces Met-3, as demontrated by Roesy experiment.
The minor conformer G1a-2 showed higher  values with respect to those of G1a-1 (Table TS8). Of relevance, the positive value of methionine, indicating its different sterical environment. A complete set of CH/NH(i, i+1) ROEs are present, except for NH Val9 and CH Leu8 (Figures FS6B and FS6C). Interstrand ROEs were found between NH Val4 /CH Leu8 , NH Phe11 /MeCO and NH Leu2 /CH Val9 , indicating the formation of a -hairpin characterized by the same H-bond network proposed for G2a (see below, Figures FS6B and FS7).      16 and the values determined experimentally for G2a (blue) and isomers G1a-1 (red) and G1a-2 (green) in CD 3 OH at 298 K. 5:E. 18

NMR data and discussion for peptide G2a
NOESY and ROESY experiments on compound G2a evidenced strong CH/NH(i, i+1)ROEs ( Figure FS10) and the formation of a turn in the region containing S1 scaffold. Spatial proximity between the piperidine moiety of scaffold S1 (H-2 and H-6) with both proline and Glu-5 ( Figure FS11) was observed.
A cross-strand Noe was detected between CH Gly-Gly1?? and the phenyl ring of Phe-10 ( Figure FS12). However, no other not ambiguous NOEs were observed between cross-strand residues due to peaks overlapping among them or overlapped with the solvent signals.
The profiles of H conformational shift values ( observed - random coil , ppm; Table TS10 and Figure FS8), as well as a deviation of more than 0.1 ppm from random coil for several successive residues exhibited by peptide G2a, are consistent with those of the target stranded antiparallel -sheet. 18 Furthermore the separation of the Gly resonances indicates a -hairpin structure as reported in the literature. 19 A further confirmation is given by 3 J HN/CH coupling constant distributions that are commonly used for identifying secondary structure in the NMR structure determination. 20 Values higher Gly-1 Val-2/Leu-2 Val-3/Met-3 Ile-4/Val-4 Glu-5/Gly-5 Pro-6 Lys-7 Leu-8 Val-9 Phe-10 Phe-11 than 8 Hz characterize a  strand, as observed for G2a peptide. As shown in Figure FS9 and Table TS10, positive difference between 3 J HN/CH values in the random coil and the values determined experimentally were found. As an exception, Glu-5 is characterized by a lower J value that is justified by a sharp change in backbone direction indicative of a -turn. 21       1.17±0.01 ne NA = no aggregation observed, ne = no effect, parameters are expressed as mean ± SE, n=3.
[a] Sat means that a saturation of the fluorescence signal is observed because G2a selfaggregates at 100 M.

Thioflavin-T assay (IAPP)
IAPP was purchased from Bachem. IAPP was dissolved in hexafluoro-isopropanol (HFIP) at a concentration of 1 mM and incubated for 1 hour to dissolve any preformed aggregates.
Next, HFIP was evaporated with dry nitrogen gas followed by vacuum desiccation for 3 hours. The resulting peptide film was then dissolved in DMSO to obtain stock solutions of In the presence of G1a In the presence of G1b In the presence of G2b  We focused our attention on three kinds of species: (1) the monomer (peak ES), (2) different small metastable oligomers grouped under peak ES I and (3) transient species formed later and which correspond to species larger than dodecamers and still soluble (peak LS). Aggregation kinetics of Aβ 1-42 peptide alone ( Figure S18) showed that overtime, the monomer ES peak decreased in favor of the oligomer peaks ES I and LS, and that insoluble species, forming spikes in the profile, appeared after 8 hours.
In the presence of G1b, the aggregation kinetics of Aβ 1-42 peptide was greatly modified ( Figure S19). Noteworthy, the monomeric species (peak ES) was dramatically stabilized. 86% of the monomer remained after 24 h in the presence of G1b, while it was no more detected in the control sample. Moreover, the larger aggregated species LS (> dodecamers) were not detected but new aggregated forms of Aβ 1-42 b w E ' and LS migration times were observed on each electrophoretic profile. We checked that these new aggregated forms of Aβ 1-42 were not due to G1b degradation or self-assemblies (Figure S17A.) They were probably aggregated forms with a different morphology than both LS and those giving spikes observed in Aβ  control. This observation is in accordance with the TEM images where globular aggregates were observed instead of the classical dense network of fibers (Figures 5c) and S16). In ThT-assays, no fluorescence was detected, indicating that the globular species were not characterized by a highly ordered β-structures (Figure 5a)). Considering the intensities of ES and ES I peaks over time it seems that G1b induces the formation of metastable species at the beginning of the kinetics that are then transformed into monomeric state (Figures S19 and S21). Remarkably, the presence of the monomer was maintained even after 4 days ( Figure S19B). We concluded that G1b is able to prevent the formation of toxic soluble oligomers of Aβ 1-42 peptide and to maintain the presence of the non toxic monomer overtime.
The electrophoretic profile of Aβ 1-42 in the presence of G2b (G2b/Aβ 1-42 ratio of 1/1) was very different from the one observed in the presence of G1b. The oligomerization process was dramatically delayed in comparison with the kinetics control ( Figure S20). G2b also dramatically maintained the presence of the monomer (peak ES). 80% of the monomer remained after 24 h ( Figure 6c) and S21). New aggregated forms were only transiently observed (after 8h) but were not longer detectable after 24h. This result was also in accordance with the TEM images where we observed a much less dense network of fibers, however the typical morphology was retained.   Cell toxicity SH-SY5Y neuroblastoma cells were grown in low serum Optimem (Life Technologies) for 24 37˚ 5% 2 96 w p 20 000 p w β 1-42 was dissolved in sterile PBS at 50 µM concentration in the presence of 1, 5, 10 and 50 µM of the four compounds for 24 hours at room temperature, along with a control incubation with no inhibitor. After the 24 hour period, media was removed from the cells and replaced with Optimem containing the pre-b β 1-42 plus inhibitor diluted o (5 µ β f concentration) in quadruplicate. The cells were incubated for a further 24 hours as before and the cell viability (MTS assay) and cell proliferation (LDH assay) assessed using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega) and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) respectively. The assays were repeated twice and representative samples are shown.