Inhibition of Î²-Amyloid aggregation Through a Designed Î²-hairpin

Subscriber access provided by READING UNIV

Article

Inhibition of #-Amyloid aggregation Through a Designed #-hairpin Peptide Anjali Jha, Mothukuri Ganesh Kumar, Hosahudya N Gopi, and Kishore M Paknikar Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03617 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Inhibition of β-Amyloid aggregation Through a Designed β-hairpin Peptide

Anjali Jha1*, Mothukuri Ganesh Kumar2, Hosahudya N. Gopi2*, Kishore M. Paknikar1*

1

Nanobioscience Group, Agharkar Research Institute, G. G. Agarkar road, Pune 411004, India 2

Department of Chemistry, Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pune 411008, India

*To

whom

correspondence

should

be

addressed.

E-mail:[email protected],

[email protected], [email protected]

ACS Paragon Plus Environment

1

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 34

Abstract Designing peptide based drugs to target the β-sheet rich toxic intermediates during the aggregation of amyloid-β 1-42 (Aβ1-42) has been a major challenge. In general, β-sheet breaker peptides (BSBPs) are designed to complement the enthalpic interactions with the aggregating protein and entropic effects are usually ignored. Here, we have developed a conformationally constrained cyclic BSBP by the use of an unnatural amino acid and a disulfide bond. We show that our peptide strongly inhibits the aggregation of Aβ1-42 in a concentration-dependent manner. It stabilizes the random coil conformation of Aβ1-42 monomers and inhibits the secondary structural transition to a β−sheet rich conformation which allows Aβ1-42 to oligomerize in an ordered assembly during its aggregation. Our cyclic peptide also rescues the toxicity of soluble aggregates of Aβ1-42 towards neuronal cells. However, it significantly loses its potency in the conformationally relaxed acyclic form. It appears that limiting the loss of conformational entropy of the BSBP ligand can play a very important role in the attainment of conformations for precise and tight binding, making them a potent inhibitor for Aβ1-42 amyloidosis.

Keyword: Amyloid beta (Aβ), protein aggregation, cyclic peptide, disulfide, peptide conformation, BSBPs, β-hairpin, conformational entropy.


2

Page 3 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Introduction Alzheimer's disease (AD) is characterized by the cerebral extracellular deposition of insoluble amyloid fibrils formed predominantly by the 42 residue protein, amyloid-β 1-42 (Aβ1-42)1-3. Progressive accumulation of amyloid fibrils leads to the decay of neurons, cause the deterioration of the brain and eventually death of an AD patient. Current medication regimen available for AD are highly inadequate and can only temporarily treat the symptoms of AD4-5. Moreover, these drug formulations are associated with a variety of complications like dose-dependent side effects, bioavailability, etc.4. Therefore, there lies an immediate need for safe and efficient therapeutic molecules which can directly target amyloid or amyloid-like protofibrils and fibrils, the root cause of AD, and can cure the progression of the disease6-9. The aggregation of natively disordered Aβ1-42 to cross-β sheeted fibrils proceeds via nucleation polymerization mechanism10 with the formation of neurotoxic soluble intermediates, rich in βsheet content11-12. An attractive and biocompatible strategy is to design peptide molecules which can bind the β-sheet rich intermediates and inhibit the amyloid protein assembly13-14. Extensive efforts have been made to design these therapeutic β-sheet breaker peptides (BSBPs)14-22. Studies have shown that small fragments of Aβ1-42 itself, for example, pentapeptides Aβ17-21(LPFFD)2325

and Aβ16-20(KLVFF)26-27can act as promising BSBPs22, 28-29. Classical features of these peptide

inhibitors are their hydrophobicity and their tendency to incorporate into β-sheets24, 30. Although this approach is elegant and powerful, it suffers from many limitations. As a result, most of the currently available BSBPs have poor efficacy and are generally prone to proteolytic degradation and have a relatively short half-life in vivo28, 31-32. To overcome these limitations and enhance their pharmacological properties, these sequences have undergone several chemical modifications including N-methylation, incorporation of unnatural amino acids, retro-inverso sequences, cyclization etc. 18, 33-35. These modifications result in better inhibition activity and enhanced serum stability of the inhibitor peptides30-32 However, we still do not fully understand the basic principles of designing BSBPs and as a result, none of the BSBPs are yet approved for AD medication 36-37.

In general, BSBPs are designed to enhance the enthalpic contributions to the free energy of binding and entropic contributions are usually ignored

13, 38-39

. However, limiting the loss of

conformational entropy of the BSBP ligand upon binding could also favorably increase the binding affinity

40-41

. For example, constraining the structure of BSBPs by backbone cyclization and


3

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 34

restricting their conformational entropy can favour conformations which can precisely complement the β-sheet rich intermediate states and bind them with much higher affinity. Recently, Teplow and colleagues examined a series of θ-defensins against aggregation of Aβ1-42 and showed the significant amyloid inhibition activity from a cyclic β-hairpin analogue of θ-defensins42. Other studies have also proposed that conformationally constrained peptides can attain higher chemical, thermal and proteolytic stability and could be much better drugs as compared to their linear counterparts 43-46. However, their exploitation for AD medication has been very limited. We have been working on the design of β-hairpins and three-stranded β-sheets consisting of various non-natural amino acids using DPro-Gly as a β-turn inducing segment 47. The DPro-Gly dipeptide segment is known to induce either type I′ or II′ β-turns in the peptide sequences48-49. Recently, we have demonstrated the stable solution conformation of a decapeptide β-hairpin containing DPro-Gly at the turn and Cys residues mediated disulfide bond at the terminals of the anti-parallel β-strands 50. The sequence of the β-hairpin and its CD spectrum is shown in the Figure 1. As many hydrophobic β-sheet type fragments derived from the Aβ1-42 peptide28 and synthetic analogues of θ-defensins42 have been shown to inhibit the amyloid aggregation, we sought to investigate whether the conformationally stabilized β-hairpin peptide can be used to inhibit the Aβ1-42 amyloid aggregation. Here we show that our peptide actively inhibits Aβ1-42 aggregation and does not allow the secondary structural transition which is required for Aβ1-42 monomers to grow into an ordered aggregate form. We also show that our cyclic peptide can remarkably rescue the cell toxicity of soluble aggregates of Aβ1-42. Interestingly, it significantly loses its potency when the conformational constraint is relaxed by the disruption of the disulfide bond.

Experimental procedures Chemicals and buffers All the chemicals used were of the highest purity grade. Black flat bottom polystyrene nonbinding 96-well plates were purchased from Corning, USA. Fmoc amino acids were purchased from GL Biochem, China. MBHA amide resin was obtained from Anaspec, USA. Aβ1-42 peptide (purity > 95%) were purchased from Proteogenix, France. SH-SY5Y cell line was procured from the National Cell Repository of National Centre for Cell Sciences (NCCS), Pune, India. Sodium phosphate buffer contains 10 mM sodium phosphate with 150 mM NaCl and 1 mM EDTA, pH


4

Page 5 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

adjusted to 7.4.

BHdS peptide synthesis BHdS was synthesized on MBHA amide resin (0.2 mmol) using standard Fmoc-chemistry. N-terminal of the peptide was protected with acetyl group. The detailed method of synthesis, disulfide formation and its purification using RP-HPLC is described elsewhere

50

. Purity of the

peptide was confirmed by MALDI TOF/TOF. Lyophilized peptide was stored at -20ºC. Prior to the experiment stock solution of peptide was prepared by resuspension of the lyophilized peptide in 10 mM sodium phosphate buffer (pH 7.4) followed by a brief sonication. Preparation of the soluble aggregates of Aβ1-42 Aβ1-42 peptides were dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). The resultant solution was kept overnight in a laminar flow in order to evaporate HFIP. Complete evaporation of HFIP was achieved by lyophilization of the sample before storing Aβ1-42 peptide at -80ºC. Stock solutions of monomeric Aβ1-42 were made by resuspension of the lyophilized Aβ1-42 in dimethyl sulfoxide (DMSO) at a concentration of 2 mM. It was sonicated for 1 min in a bath sonicator. Aggregates of Aβ1-42 were prepared by diluting the stock solution with 10 mM sodium phosphate buffer (pH 7.4) to 100 µM, which was further incubated at 37 ºC in a dry bath. Aggregates of Aβ142

pre-incubated with BHdS were prepared by diluting the mixture of Aβ1-42 and BHdS with 10

mM sodium phosphate buffer (pH 7.4). Final concentration of Aβ1-42 in the mixture was kept at 100 µM. This was further incubated at 37 ºC in a dry bath and the final pH was maintained at 7.4.

Congo Red (CR) binding assay Protein aggregates were prepared from 100 μM of Aβ1-42 and from the mixture of 100 μM of Aβ1-42 and 1 mM BHdS as described above. CR binding was measured in a transparent 96 well plate using a microplate reader (Bio-Tek Instruments, VT, USA) at 37 °C. 10 µM stock solution of CR dye was prepared in the sodium phosphate buffer (pH 7.4). 100 μL of aggregated samples were mixed with 100 μL of CR, and incubated at 37 °C for at least 10 min prior to absorbance measurement. The spectrum of BHdS alone was measured by adding it to 100 μL of CR stock solution. The spectra of CR (5 μM) alone was also measured for control reference. Three scans


5

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 34

each of triplicate samples were measured and averaged and background measurements for buffer containing only CR were subtracted from aggregated samples spectra.

Thioflavin T (ThT) fluorescence monitored kinetics of aggregation Thioflavin T fluorescence measurements were carried out as described previously42. In brief, the measurements were carried out in situ in a black polystyrene non-binding 96-well plate using a Synergy Biotek fluorescence plate reader, by exciting the sample at 440 nm and monitoring the fluorescence emission at 482 nm. A fresh stock solution of 400 μM ThT in 10 mM sodium phosphate buffer (pH 7.4) was prepared. For the control assay, an aliquot of 10 μL of Aβ1-42 (from a stock solution of 2 mM in DMSO) was added to the 10 μL of the ThT assay solution in sodium phosphate buffer (pH 7.4) such that the final Aβ1-42 and ThT concentrations in the assay solution were 100 μM and 20 μM, respectively. For the inhibition assay, 180 μL of BHdS from the respective stock solutions were added to the wells containing 10 μL of Aβ1-42 and 10 μL of the ThT. The final concentration of BHdS in the wells was kept at 1, 2 and 2.5 mM along with 100 μM of Aβ1-42 and 20 μM of ThT. The plate was incubated at 37 ºC and the final pH of each well was maintained at 7.4. Fluorescence readings were recorded for a period of 24 h at 37 ºC with orbital shaking for 20 s prior to each reading for more homogeneous results. Measurements were performed as independent triplicates. Three recorded values were averaged and background measurements for buffer containing only ThT were subtracted.

Disruption of the disulfide linkage of BHdS peptide DTT (Dithiothreitol) was used to disrupt the disulfide linkage of the BHdS peptide 50. DTT stock was prepared at a concentration of 13 mg/ml in 0.22 µm filtered water. Disulfide reduced BHdS peptide was obtained by the addition of 13 µl of DTT stock solution to 187 μL of 3 mg/ml BHdS peptide solution and incubated at 37 °C for 12 h. Final peptide and DTT concentration was kept at 2.8 mM and 5.6 mM, respectively. This solution was further used for co-incubation with 100 µM Aβ1-42 at 37 °C in order to measure the aggregation kinetics of Aβ1-42 in the presence of disulfide reduced BHdS using thioflavin-T assay as described above.

Circular dichroism 100 μM of Aβ1-42 was incubated (i) alone, (ii) in the presence of 2.5 mM of cyclic BHdS, and ACS Paragon Plus Environment

6

Page 7 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(iii) in the presence of 2.5 mM of disulfide reduced BHdS at 37 οC. Far-UV CD measurements were carried out using a Jasco J-720 spectropolarimeter. The cuvette used for the measurement was of 1 mm path length. Spectra were collected from 190 nm to 260 nm, with a step resolution of 1 nm, a scan speed of 100 nm/min, and a bandwidth of 1 nm. All the spectra were averaged over 5 scans and background measurements for buffer were subtracted. The CD spectrum of Aβ1-42 in the presence of cyclic BHdS was analyzed by subtracting the spectrum of BHdS peptide alone from that of Aβ1-42 and BHdS mixture. Similarly, the CD spectrum of Aβ1-42 in the presence of disulfide reduced BHdS was analyzed by subtracting the spectrum of disulfide reduced BHdS peptide alone from that of the mixture.

Transmission electron microscopy (TEM) 100 μM of Aβ1-42 was incubated (i) alone, (ii) in the presence of 2.5 mM of cyclic BHdS, and (iii) in the presence of 2.5 mM of disulfide reduced BHdS at 37 οC in 0.22 micron filtered water for 96 h. One μL aliquots were taken from each sample, diluted ten times, and placed on individual carbon-coated 200-mesh copper grids and were air-dried. Further, the grids were negatively stained with 2 % uranium acetate in milliQ water, and examined under an electron microscope.

Cell viability assay MTT (3, (4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide) reduction assay was used to measure the cell viability of human neuroblastoma cell line SH-SY5Y. Briefly, cells were cultured in DMEM/F12 (Sigma, USA) medium in humidified 5% (v/v) CO2/air at 37 °C in 10% (v/v) (fetal bovine serum) FBS (Invitrogen, USA) and 100 U/ml penicillin. 104 numbers of cells per well were seeded in a 96-well plates and incubated for 24 hours at 37 °C. Thereafter, the medium was replaced with the solution containing 10 µM of Aβ1-42 aggregates (diluted in DMEM/F12 medium from a stock solution of 100 µM Aβ1-42 incubated over a period of 12 hours at 37 °C). To study the effect of BHdS on the toxicity of Aβ1-42 aggregates, cells were incubated with 10 µM Aβ1-42 and 0.25 mM BHdS aggregated solution (diluted ten times from the stock solution of 100 µM Aβ1-42 and 2.5 mM BHdS mixture incubated over a period of 12 hours at 37 °C). To study the effect of disulfide reduced BHdS on the toxicity of Aβ1-42 aggregates, cells were incubated with 10 µM Aβ1-42 and 0.25 mM disulfide reduced BHdS aggregated solution


7

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 34

(diluted ten times from the stock solution of 100 µM Aβ1-42 and 2.5 mM disulfide reduced BHdS mixture incubated over a period of 12 hours at 37 °C). To study the effect of BHdS alone, cells were incubated with 2.5 mM BHdS. The plates were incubated in humidified 5% (v/v) CO2/air at 37 °C and MTT reduction assay was performed after 24 h of treatment. MTT was added to the wells at a final concentration of 0.5 mg/ml and incubated for 4 h at 37 °C in CO2 incubator. Thereafter, supernatant from each well was removed and 200 μL of DMSO was added to the cells to solubilize the formazan and mixed well. This was incubated in a humidified CO2 incubator for 15-20 min. The absorbance was read at 570 nm using a microplate reader (Synergy HT, Bio-Tek Instruments, VT, USA). Cell viability was compared to the control cells without any peptide treatment.

Results Design of the β-hairpin containing cyclic peptide The primary sequence of our decapeptide is CVKVDPGLKVC (Figure 1A). We have introduced the DPro-Gly motif, consisting of the unnatural amino acid DPro, in the middle of the peptide sequence, which is known to impart enhanced proteolytic stability and induce a turn in βhairpin (BH) structure51. In order to make it cyclic and constrained, we have clipped the ends of the BH by a disulfide (dS) bond between the first and the last Cys residues. The synthesis of this peptide, called BHdS here onwards, has been described in detail in a previous study50. The cyclic BH structure of BHdS has been well characterized 50. The three-dimensional structure of BHdS consists of two antiparallel β-strands and a reverse β-turn and its structure is stabilized by hydrogen bond interactions and one disulfide bond between the two opposite β-strands and the hydrophobic effect. Figure 1B compares the far-UV CD spectra of BHdS in the cyclic and the disulfide reduced form. Cyclic BHdS shows a single absorbance band centered at 217 nm indicating a predominantly β-hairpin containing secondary structure (Figure 1B). However, when the disulfide bond of BHdS is reduced by the addition of DTT, there is a dramatic loss of absorbance at 217 nm (Figure 1B). These results suggest that the presence of the disulfide bond is crucial to maintain the cyclic βhairpin conformation of BHdS. The β-hairpin conformation of BHdS peptide remains stable at elevated temperature up to 60 ºC 50.

Inhibition of amyloid aggregation by BHdS peptide


8

Page 9 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

We investigated the anti-amyloidogenic activity of BHdS and observed that it acts as a potent inhibitor of Aβ1-42 aggregation.Aβ1-42 undergoes a series of structural changes during its aggregation11,

52

. In the monomeric form it adopts a random-coil like conformation52. Upon

incubation at 37 οC for 24 h, it forms soluble protofibrillar aggregates that have predominantly βsheet structure11. Upon further incubating it for a few days it forms large insoluble amyloid-like fibrils11, 53. We first examined the inhibition effect of cyclic BHdS peptide on the early random coil to soluble protofibril step of Aβ1-42 aggregation. We prepared the soluble protofibrillar aggregates of Aβ1-42 by incubating 100 µM of the protein at 37 ºC for 24 h at pH 7.4, as described previously42. We identified the formation of protofibrillar structures by their binding to the congo red (CR) and thioflavin-T (ThT) dyes (Figure 2), which are known to bind the substantial β-sheet content of these protofibrils 54. CR absorbs maximally at 490 nm, and upon binding the aggregates of Aβ1-42, the wavelength of maximum absorbance shifts to 540 nm with a huge increase in absorbance intensity (Figure 2A). We observed that when Aβ1-42 is incubated with BHdS at 37 ºC for 24 h in 1:10 molar ratio, the resultant Aβ1-42 + BHdS sample binds CR very weakly and there is a drastic reduction in the absorbance intensity (Figure 2A). BHdS alone did not show any binding to CR (Figure 2A). ThT monitored inhibition of the Aβ1-42 aggregation showed similar results. ThT exhibits maximum fluorescence emission at 440 nm upon excitation at 385 nm, where it absorbs maximally (Figure 2B). We observed that upon binding to β-sheet rich Aβ1-42 amyloid aggregates, ThT displays a considerable shift of the excitation peak (from 385 nm to 450 nm) and the emission peak (from 440 nm to 482 nm) that is also accompanied by a dramatic enhancement in the emission intensity (Figure 2B), as reported previously 55. When Aβ1-42 is incubated with BHdS at 37 ºC for 24 h in 1:10 molar ratio, we observed that the resultant Aβ1-42 + BHdS sample binds ThT very weakly and there is a drastic reduction in the fluorescence emission intensity (Figure 2B). We also observed that BHdS alone did not show any binding to ThT (Figure 2B). Above results indicate that in the presence of BHdS peptide there is a significant reduction in the extent of formation of the β-sheet rich Aβ1-42 protofibrils. Most importantly, BHdS alone did not show any binding to either CR or ThT indicating that the presence of the peptide is unlikely to alter the binding of the dyes to Aβ1-42 aggregates.


9

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 34

Concentration dependence of BHdS inhibition on Aβ1-42 aggregation kinetics To understand the inhibitory effect of BHdS in a systematic manner, we investigated the concentration dependence of BHdS inhibition on the kinetics of aggregation of Aβ1-42 by monitoring the time-dependent change in ThT fluorescence. When 100 µM of Aβ1-42 is incubated alone at 37 ºC, we observed that the kinetics of its aggregation is characterized by a huge single exponential increase in the ThT fluorescence without any lag phase (Figure 3A), as has been reported previously42,

56

. Generally, amyloid aggregation has been shown to be nucleation-

dependent process and proceeds via three major steps: formation of initial nucleus or seed, which constitutes a lag phase, followed by a rapid growth phase of different intermediates and eventually the formation of fibrils

10, 57-59

. The kinetic parameters of each phase depend on various physio-

chemical environment like concentration, pH, temperature etc.60-61. For example, the lag phase or nucleation phase could be absent for higher concentration of amyloid peptide 42, 53, 62. Hence, the non-appearance of lag phase in our results is not surprising42. The amplitude of the kinetic curve monitored by ThT fluorescence is directly proportional to the amount of the β-sheet rich aggregates present in the solution. We observed that, when Aβ1-42 is incubated with BHdS in 1:10, 1:20 and 1:25 molar ratios of their concentration, the amplitude of ThT fluorescence respectively decreased to ~ 70 %, ~ 40% and ~ 20% relative to the amplitude observed in the absence of the BHdS peptide (Figure 3A and 3B). This result indicates that incubation of BHdS with Aβ1-42 in 25:1 molar ratio can reduce ~ 80 % of the β-sheet rich soluble aggregates, and hence BHdS peptide is a potent inhibitor of Aβ1-42 aggregation. It is important to note that 2.5 mM BHdS when incubated alone at 37 °C does not form any aggregates. Figure 3A shows the ThT assay for 2.5 mM BHdS peptide alone when incubated at 37 °C for 24 hrs. The fluorescence intensity of ThT remains almost zero for 24 hrs. We observed that BHdS also decreases the rate of aggregation of Aβ1-42. The aggregation of Aβ1-42 at the concentration of 100 µM occurs in a single exponential manner with an apparent first order rate constant (kobs) of 0.0314 ± 0.0023 min−1 (Figure 3A and 3C). Upon incubation of Aβ1-42 with BHdS in 1:10, 1:20 and 1:25 molar ratios of their concentration, the kobs decreased to 0.0283 ± 0.003 min−1, 0.0176 ± 0.0009 min−1 and 0.0123 ± 0.0012 min−1 respectively (Figure 3A and 3C). Figure 3C shows that kobs depends linearly on the concentration of the inhibitor peptide BHdS. The slope yields a second-order rate constant of - 0.0078 ± 0.0007 mM-1min-1. These results indicate that BHdS actively inhibits the aggregation of Aβ1-42 in a concentration-dependent manner.


10

Page 11 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

The role of the constrained conformation in the inhibitory effect of BHdS In order to understand the significance of the constrained conformation produced by the disulfide stapling, we investigated the aggregation kinetics of Aβ1-42 in the presence of disulfidereduced BHdS. Addition of DTT reduces the disulfide bond and relaxes the conformational constraint produced by it50. We observed that when Aβ1-42 is incubated with disulfide reduced BHdS in 1:25 molar ratio, the amount of β-sheet rich soluble aggregates as monitored by the amplitude of ThT fluorescence remains similar to the Aβ1-42 alone sample (Figure 3A and 3B). However, the apparent rate constant of aggregation under these conditions was observed to be 0.0123 ± 0.004 min−1, which is ~ 3 fold lower than Aβ1-42 alone sample (Figure 3A and 3C). These results indicate that primary sequence of BHdS (in the disulfide reduced form) does not have a significant inhibitory effect even at 25-fold higher concentration than Aβ1-42. Hence, the interaction observed for the cyclic BHdS with Aβ1-42 is very specific and its inhibition activity is primarily due to its cyclic conformation. BHdS stabilizes the random coil structure of Aβ1-42 During aggregation, within 24 h of incubation at 37 οC, Aβ1-42 undergoes a transformation in its internal secondary structure from a random coil conformation to a soluble protofibrillar aggregate form with β-sheet rich structure11-12. In order to understand the effect of BHdS on the structural transition of Aβ1-42 during its aggregation, we employed far-UV CD spectroscopy. We observed that the far-UV CD spectrum of monomeric Aβ1-42, immediately after dissolution in buffer, shows almost zero absorbance in the wavelength range of 215-250 nm and a huge negative absorbance band centered at 195 nm, which is typical of a random coil conformation 11 (Figure 4). The far-UV CD spectrum of the aggregates of Aβ1-42 formed after incubating the protein at 37 ºC for 24 h showed a huge absorbance band centered at 218 nm and a positive band at 190-200 nm, indicating a predominantly β-sheet conformation (Figure 4). However, when Aβ1-42 was incubated with BHdS, the far-UV CD spectrum obtained after subtracting the absorption of BHdS from the measured spectrum shows a random coil conformation, very similar to what was observed for monomeric Aβ1-42 (Figure 4). These results strongly indicate that BHdS prevents structural conversion of Aβ1-42 from adopting a β-sheet conformation required for amyloid assembly by


11

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 34

stabilizing monomeric Aβ1-42. These results also indicate that co-incubation of BHdS with Aβ1-42 does not result in the formation of any co-aggregates. Interestingly, when Aβ1-42 was incubated at 37 ºC in the presence of disulfide reduced BHdS, the far-UV CD spectrum obtained after subtracting the absorption of disulfide reduced BHdS from the measured spectrum shows a broad negative absorbance band centered at 218 nm. This result shows the formation of β-sheet rich conformation in the presence of the disulfide reduced BHdS, very similar to what was observed for the protofibrillar aggregates of Aβ1-42 formed without any inhibitor peptide (Figure 4). Hence, the BHdS peptide loses its inhibition activity in the disulfide reduced form and the loss of cyclic conformation in the disulfide reduced BHdS allows the structural conversion of Aβ1-42 into a β-sheet rich conformation. BHdS does not allow Aβ1-42 to assemble into ordered aggregates We next examined whether BHdS remains active during the soluble protofibril to the insoluble fibril step of Aβ1-42 aggregation as well. We prepared the insoluble fibrils of Aβ1-42 by incubating 100 µM of the protein at 37 ºC for 96 h at pH 7.4, as described previously42. We examined the external morphology of the fibrils formed in the absence and the presence of BHdS by transmission electron microscopy (TEM) (Figure 5). The external morphology of the protofibrils of Aβ1-42 formed after 24 h of incubation (in the presence or the absence of the inhibitor peptide) remained indistinct in TEM due to their small size. The fibrils of Aβ1-42 (formed in the absence of BHdS) show a twisted morphology and appear as several micrometer long unbranched thread-like structures with a diameter ranging from 20-80 Å (Figure 5A). These characteristics are typical of amyloid fibrils63-65. On the contrary, when Aβ1-42 was incubated at 37 ºC for 96 h in the presence of 2.5 mM cyclic BHdS, the TEM images of the resultant sample show that the fibrils of Aβ1-42 were not formed (Figure 5B). These results indicate that the β-hairpin containing cyclic BHdS peptide inhibits the assembly of Aβ1-42 into ordered fibrillar aggregates. Interestingly, when Aβ1-42 was incubated at 37 ºC for 96 h in the presence of 2.5 mM disulfide reduced BHdS, the TEM images of the resultant sample show the formation of Aβ1-42 fibrils (Figure 5C). These results strongly show that the disulfide reduced BHdS peptide fails to inhibit the formation of ordered fibrillar aggregates of Aβ1-42.


12

Page 13 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Prevention of toxicity of Aβ1-42 aggregates by BHdS The soluble aggregates formed on the pathway of amyloid fibrils are known to be toxic to neuronal cells 66-69.We observed that BHdS can rescue the toxicity of soluble aggregates of Aβ1-42 towards neuronal cells. We investigated the toxicity of the aggregates of Aβ1-42 formed in the absence and the presence of BHdS using MTT (3, (4, 5-dimethylthiazol-2-yl) 2, 5diphenyltetrazolium bromide) reduction assay70 on SH-SY5Y human neuroblastoma cell lines. The assay is based on the ability of the live cells to convert MTT into insoluble formazan crystals. Therefore, the amount of formazan produced is proportional to the number of live cells. We observed that the soluble aggregates of Aβ1-42 exhibited cytotoxicity to SH-SY5Y cells and reduce the cell viability to ~ 30-35 % relative to the control in which the cells are not subjected to any Aβ1-42 treatment (Figure 6). However, when Aβ1-42 was incubated at 37 οC in the presence of BHdS in 1:25 ratio, the resultant sample showed striking improvement in cell survival with nearly complete protection against Aβ1-42 toxicity. On the contrary, when Aβ1-42 was incubated at 37 οC in the presence of disulfide reduced BHdS in 1:25 ratio, the resultant sample showed the percentage of cell survival similar to the Aβ1-42 alone sample. Figure 6 also shows that BHdS peptide alone did not show any cytotoxicity towards SH-SY5Y cells. These results strongly indicate that BHdS peptide in its cyclic conformation could be a powerful candidate for therapeutic intervention against AD.

Discussion In this study, we have shown that the cyclic and conformationally constrained BHdS peptide is a potent inhibitor of Aβ1-42 aggregation. Congo red and ThT binding experiments suggested that BHdS can significantly inhibit the aggregation of Aβ1-42 (Figure 2). We investigated the dependence of the kinetics of aggregation of Aβ1-42 on the concentration of BHdS and observed that 1:25 ratio of Aβ1-42: BHdS results in 80 % reduction in the amount of β-sheet rich soluble aggregates (Figure 3A and 3B). Concentration dependence experiments also revealed that 1:25 ratio of Aβ1-42: BHdS can significantly reduce the rate of aggregation of Aβ1-42 (Figure 3A and 3C). Far-UV CD experiments revealed that BHdS stabilizes the random coil conformation of Aβ1-42 and inhibits the random coil to β-sheet conformational conversion which occurs during the aggregation of Aβ1-4252 (Figure 4). Due to this, Aβ1-42 could not aggregate into an ordered assembly as revealed


13

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 34

by TEM measurements (Figure 5). Cell toxicity experiments showed that when Aβ1-42 is incubated with of BHdS, the resultant conformational ensemble are not toxic to the neuronal cells (Figure 6). It has been challenging to design peptide-based inhibitors of Aβ1-42 aggregation. One of the least understood parameter in BSBP design is the knowledge of exclusive contribution of structural conformation of inhibitor peptide to its activity. Gain in the inhibition activity due to cyclization of peptide where the sequences are selected directly from self-recognition region of amyloid full length peptide has been recently studied18, 71-73. However, experimental studies on the effect of cyclization on the activity of non-specific peptide sequences are scarce. Recently, using a cyclic D, L-α-peptide, the effect of backbone cyclization on the anti-amyloidogenic activity has been studied 74. It was proposed that the cyclic inhibitor peptide populated an off-pathway aggregation mechanism for Aβ1-42 to yield lesser cytotoxic aggregates. For β-hairpin conformation, ppleu is a unique example of a θ–defensin peptide containing non-amyloid sequence that has shown remarkable activity against Aβ1-4242. However, the role of constrained conformation in its activity was not quantified. Our study provides a direct experimental measurement of the role played by the cyclic βhairpin conformation of a non-amyloidogenic peptide sequence in inhibiting the aggregation of Aβ1-42. We reduced the disulfide bond of BHdS to populate its acyclic conformation. When Aβ1-42 was incubated with disulfide reduced BHdS in 1:25 molar ratio, we observed that the amount of β-sheet rich soluble aggregates remains similar to the Aβ1-42 alone sample (Figure 3A and 3B). The loss of amyloid inhibition activity for the disulfide reduced BHdS peptide was confirmed using CD and TEM (Figure 4 and 5C). The disulfide reduced BHdS peptide also failed to protect neuronal cells against Aβ1-42 toxicity (Figure 6). These results indicate that the presence of constrained conformation is crucial for the efficient activity of the BHdS peptide. However, the apparent rate constant of aggregation of Aβ1-42, when incubated with disulfide reduced BHdS in 1:25 molar ratio, was reduced by 3-fold compared to the Aβ1-42 alone sample (Figure 3A and 3C). It appears that the disulfide reduced BHdS could bind to Aβ1-42 to some extent and slows down the initial rate of aggregation, but because of the lack of complementarity in shapes at the binding interface, it is unable to eliminate the formation of final aggregated form (Figure 7). Since we incubated BHdS with Aβ1-42 prior to the initiation of aggregation process, our inhibition results suggest that constrained, rigid, highly stable and preformed β-hairpin structure of BHdS can


14

Page 15 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

strongly bind to early β-sheet rich monomeric or neurotoxic intermediates and almost permanently block the sites of Aβ1-42 for further stacking and assembly (Figure 7). It should be noted that the Aβ1-42: peptide ratio observed for BHdS for effective inhibition is similar to the cyclic θ–defensin peptide, examined previously42. This result is also comparable with inhibition activity of LPFFD peptide, where incubation of Aβ1-42 with 20-fold molar excess of inhibitor peptide, results in nearly 80% inhibition24. In another study, Tjernberg and co-workers have shown a hexapeptide KLVFF active at equimolar concentration 26. Similarly other inhibitor peptides with sequence modification also have shown greater inhibition with molar or sub-molar equivalents of inhibitor relative to Aβ142

13, 25, 28

. We believe that like many inhibitors of amyloid aggregation, β-hairpin forms non-

fibrillar assemblies that inhibit the fibril formation from Aβ1-42. Both the residue composition and β-hairpin structure of the peptide play crucial role in the sequestration of amyloid fibrils. Considering the experimental evidence obtained in the present study, the possible mechanism of inhibition of Aβ1-42 aggregation by BHdS is shown in Figure 7. Our results also indicate that restricting the loss in conformational entropy during binding can play an important role in the activity of BSBP and have important implications for future design of novel BSBP drugs. The effectiveness of the inhibitor BHdS peptide is yet to be examined in in-vivo studies. We observed that the required concentration of BHdS peptide for 80% inhibition activity is ~25 fold higher than Aβ1-42. This concentration is slightly higher for in-vivo studies. Thus, to enhance the efficacy of BHdS for therapeutic applications, further sequence optimizations are needed, while preserving its cyclic β-hairpin structure. For example, one simple strategy could be to modify a few residues of BHdS to mimic the hydrophobic central region of Aβ1-42 sequence. In future studies, it will also be interesting to test the efficacy of BHdS peptide encapsulated in nanoparticles for assisted delivery across the blood brain barrier. These studies are currently underway in our laboratory. However, considering that the most potent pharmacologically active naturally occurring peptides, like knottins, conotoxins, chlorotoxin and cyclotides have cyclic and constrained conformation75-77, β-hairpin peptide presents itself as a powerful candidate for therapeutic intervention against AD and other aggregation related neurodegenerative diseases.

Acknowledgements


15

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 34

AJ is recipient of DST-Inspire Faculty award. This work was funded by DST-Inspire Grant by Department of Science & Technology, India.

Conflict of interest The authors declare that they have no conflict of interest with the contents of this article.

Author contributions A.J., M.G.K., H.N.G. and K.M.P. designed the experiments and wrote the manuscript. A.J. performed and analyzed the experiments.

References

1.

Cohen, F. E.; Kelly, J. W., Therapeutic approaches to protein-misfolding diseases. Nature

2003, 426 (6968), 905-909. 2.

Selkoe, D. J., Folding proteins in fatal ways. Nature 2003, 426 (6968), 900-904.

3.

Masliah, E., Neuropathology - Alzheimer's in real time. Nature 2008, 451 (7179), 638-639.

4.

Kavirajan, H.; Schneider, L. S., Efficacy and adverse effects of cholinesterase inhibitors

and memantine in vascular dementia: a meta-analysis of randomised controlled trials. Lancet Neurol 2007, 6 (9), 782-792. 5.

Karran, E.; Mercken, M.; De Strooper, B., The amyloid cascade hypothesis for Alzheimer's

disease: an appraisal for the development of therapeutics. Nat Rev Drug Discov 2011, 10 (9), 698712. 6.

Klafki, H. W.; Staufenbiel, M.; Kornhuber, J.; Wiltfang, J., Therapeutic approaches to

Alzheimer's disease. Brain 2006, 129, 2840-2855. 7.

Mangialasche, F.; Solomon, A.; Winblad, B.; Mecocci, P.; Kivipelto, M., Alzheimer's

disease: clinical trials and drug development. Lancet Neurol 2010, 9 (7), 702-716. 8.

Atwal, J. K.; Chen, Y. M.; Chiu, C.; Mortensen, D. L.; Meilandt, W. J.; Liu, Y. C.; Heise,

C. E.; Hoyte, K.; Luk, W.; Lu, Y. M.; Peng, K.; Wu, P.; Rouge, L.; Zhang, Y. N.; Lazarus, R. A.; Scearce-Levie, K.; Wang, W. R.; Wu, Y.; Tessier-Lavigne, M.; Watts, R. J., A Therapeutic Antibody Targeting BACE1 Inhibits Amyloid-beta Production in Vivo. Sci Transl Med 2011, 3 (84), 84ra43.


16

Page 17 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

9.

Schneider, L. S.; Mangialasche, F.; Andreasen, N.; Feldman, H.; Giacobini, E.; Jones, R.;

Mantua, V.; Mecocci, P.; Pani, L.; Winblad, B.; Kivipelto, M., Clinical trials and late-stage drug development for Alzheimer's disease: an appraisal from 1984 to 2014. J Intern Med 2014, 275 (3), 251-283. 10.

Jarrett, J. T.; Lansbury, P. T., Seeding One-Dimensional Crystallization of Amyloid - a

Pathogenic Mechanism in Alzheimers-Disease and Scrapie. Cell 1993, 73 (6), 1055-1058. 11.

Chimon, S.; Shaibat, M. A.; Jones, C. R.; Calero, D. C.; Aizezi, B.; Ishii, Y., Evidence of

fibril-like beta-sheet structures in a neurotoxic amyloid intermediate of Alzheimer's beta-amyloid. Nat Struct Mol Biol 2007, 14 (12), 1157-1164. 12.

Ono, K.; Condron, M. M.; Teplow, D. B., Structure-neurotoxicity relationships of amyloid

beta-protein oligomers. Proc Natl Acad Sci U S A 2009, 106 (35), 14745-14750. 13.

Sievers, S. A.; Karanicolas, J.; Chang, H. W.; Zhao, A.; Jiang, L.; Zirafi, O.; Stevens, J. T.;

Munch, J.; Baker, D.; Eisenberg, D., Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation. Nature 2011, 475 (7354), 96-100. 14.

Takahashi, T.; Mihara, H., Peptide and Protein Mimetics Inhibiting Amyloid beta-Peptide

Aggregation. Accounts Chem Res 2008, 41 (10), 1309-1318. 15.

Rajasekhar, K.; Chakrabarti, M.; Govindaraju, T., Function and toxicity of amyloid beta

and recent therapeutic interventions targeting amyloid beta in Alzheimer's disease. Chem Commun (Camb) 2015, 51 (70), 13434-13450. 16.

Rajasekhar, K.; Madhu, C.; Govindaraju, T., Natural Tripeptide-Based Inhibitor of

Multifaceted Amyloid beta Toxicity. ACS chemical neuroscience 2016, 7 (9), 1300-1310. 17.

Rajasekhar, K.; Suresh, S. N.; Manjithaya, R.; Govindaraju, T., Rationally designed

peptidomimetic modulators of abeta toxicity in Alzheimer's disease. Scientific reports 2015, 5, 8139. 18.

Arai, T.; Araya, T.; Sasaki, D.; Taniguchi, A.; Sato, T.; Sohma, Y.; Kanai, M., Rational

Design and Identification of a Non-Peptidic Aggregation Inhibitor of Amyloid-beta Based on a Pharmacophore Motif Obtained from cyclo[-Lys-Leu-Val-Phe-Phe-]. Angew Chem Int Edit 2014, 53 (31), 8236-8239. 19.

Adessi, C.; Soto, C., Beta-sheet breaker strategy for the treatment of Alzheimer's disease.

Drug Develop Res 2002, 56 (2), 184-193.


17

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

20.

Page 18 of 34

Taylor, M.; Moore, S.; Mayes, J.; Parkin, E.; Beeg, M.; Canovi, M.; Gobbi, M.; Mann, D.

M. A.; Allsop, D., Development of a Proteolytically Stable Retro-Inverso Peptide Inhibitor of betaAmyloid Oligomerization as a Potential Novel Treatment for Alzheimer's Disease. BiochemistryUs 2010, 49 (15), 3261-3272. 21.

Paul, A.; Nadimpally, K. C.; Mondal, T.; Thalluri, K.; Mandal, B., Inhibition of Alzheimer's

amyloid-beta peptide aggregation and its disruption by a conformationally restricted alpha/beta hybrid peptide. Chem Commun 2015, 51 (12), 2245-2248. 22.

Permanne, B.; Adessi, C.; Fraga, S.; Frossard, M. J.; Saborio, G. P.; Soto, C., Are beta-

sheet breaker peptides dissolving the therapeutic problem of Alzheimer's disease? J Neural Transm Suppl 2002, (62), 293-301. 23.

Soto, C.; Kindy, M. S.; Baumann, M.; Frangione, B., Inhibition of Alzheimer's amyloidosis

by peptides that prevent beta-sheet conformation. Biochem Bioph Res Co 1996, 226 (3), 672-680. 24.

Soto, C.; Sigurdsson, E. M.; Morelli, L.; Kumar, R. A.; Castano, E. M.; Frangione, B., beta-

sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: Implications for Alzheimer's therapy. Nat Med 1998, 4 (7), 822-826. 25.

Martin-Jones, Z.; Chen, D.; Gilbertson, S.; Schein, C.; Kayed, R.; Soto, C., beta-sheet

breaker and small molecule inhibitors or amyloids in Alzheimer's disease. Ann Neurol 2008, 64, S48-S48. 26.

Tjernberg, L. O.; Naslund, J.; Lindqvist, F.; Johansson, J.; Karlstrom, A. R.; Thyberg, J.;

Terenius, L.; Nordstedt, C., Arrest of beta-amyloid fibril formation by a pentapeptide ligand. J Biol Chem 1996, 271 (15), 8545-8548. 27.

Austen, B. M.; Paleologou, K. E.; Ali, S. A.; Qureshi, M. M.; Allsop, D.; El-Agnaf, O. M.,

Designing peptide inhibitors for oligomerization and toxicity of Alzheimer's beta-amyloid peptide. Biochemistry-Us 2008, 47 (7), 1984-1992. 28.

Funke, S. A.; Willbold, D., Peptides for Therapy and Diagnosis of Alzheimer's Disease.

Curr Pharm Design 2012, 18 (6), 755-767. 29.

Minicozzi, V.; Chiaraluce, R.; Consalvi, V.; Giordano, C.; Narcisi, C.; Punzi, P.; Rossi, G.

C.; Morante, S., Computational and experimental studies on beta-sheet breakers targeting Abeta140 fibrils. J Biol Chem 2014, 289 (16), 11242-11252.


18

Page 19 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

30.

Adessi, C.; Permanne, B.; Saborio, G.; Frossard, M. J.; Fraga, S.; Soto, C.; Banks, W., beta-

sheet breaker peptides: Pharmacological profile of drug candidates for the treatment of Alzheimer disease. Neurobiol Aging 2002, 23 (1), S270-S271. 31.

Poduslo, J. F.; Curran, G. L.; Kumar, A.; Frangione, B.; Soto, C., beta-sheet breaker peptide

inhibitor of Alzheimer's amyloidogenesis with increased blood-brain barrier permeability and resistance to proteolytic degradation in plasma. J Neurobiol 1999, 39 (3), 371-382. 32.

Adessi, C.; Soto, C., Converting a peptide into a drug: Strategies to improve stability and

bioavailability. Curr Med Chem 2002, 9 (9), 963-978. 33.

Bett, C. K.; Serem, W. K.; Fontenot, K. R.; Hammer, R. P.; Garno, J. C., Effects of Peptides

Derived from Terminal Modifications of the A beta Central Hydrophobic Core on A beta Fibrillization. ACS chemical neuroscience 2010, 1 (10), 661-678. 34.

Chalifour, R. J.; McLaughlin, R. W.; Lavoie, L.; Morissette, C.; Tremblay, N.; Boule, M.;

Sarazin, P.; Stea, D.; Lacombe, D.; Tremblay, P.; Gervais, F., Stereoselective interactions of peptide inhibitors with the beta-amyloid peptide. J Biol Chem 2003, 278 (37), 34874-34881. 35.

Sinopoli, A.; Giuffrida, A.; Tomasello, M. F.; Giuffrida, M. L.; Leone, M.; Attanasio, F.;

Caraci, F.; De Bona, P.; Naletova, I.; Saviano, M.; Copani, A.; Pappalardo, G.; Rizzarelli, E., AcLPFFD-Th: A Trehalose-Conjugated Peptidomimetic as a Strong Suppressor of AmyloidOligomer Formation and Cytotoxicity. Chembiochem 2016, 17 (16), 1541-1549. 36.

Rafferty, J.; Nagaraj, H.; McCloskey, A. P.; Huwaitat, R.; Porter, S.; Albadr, A.; Laverty,

G., Peptide Therapeutics and the Pharmaceutical Industry: Barriers Encountered Translating from the Laboratory to Patients. Curr Med Chem 2016, 23 (37), 4231-4259. 37.

Fosgerau, K.; Hoffmann, T., Peptide therapeutics: current status and future directions. Drug

Discov Today 2015, 20 (1), 122-128. 38.

Kumar, S.; Miranker, A. D., A foldamer approach to targeting membrane bound helical

states of islet amyloid polypeptide. Chem Commun 2013, 49 (42), 4749-4751. 39.

Kulikov, O. V.; Kumar, S.; Magzoub, M.; Knipe, P. C.; Saraogi, I.; Thompson, S.; Miranker,

A. D.; Hamilton, A. D., Amphiphilic oligoamide alpha-helix peptidomimetics inhibit islet amyloid polypeptide aggregation. Tetrahedron Lett 2015, 56 (23), 3670-3673. 40.

Heller, G. T.; Sormanni, P.; Vendruscolo, M., Targeting disordered proteins with small

molecules using entropy. Trends Biochem. Sci 2015, 40 (9), 491-496.


19

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

41.

Page 20 of 34

Kumar, S.; Birol, M.; Schlamadinger, D. E.; Wojcik, S. P.; Rhoades, E.; Miranker, A. D.,

Foldamer-mediated manipulation of a pre-amyloid toxin. Nat Commun 2016, 7, 11412-11423. 42.

Yamin, G.; Ruchala, P.; Teplow, D. B., A Peptide Hairpin Inhibitor of Amyloid beta-Protein

Oligomerization and Fibrillogenesis. Biochemistry-Us 2009, 48 (48), 11329-11331. 43.

Craik, D. J.; Du, J., Cyclotides as drug design scaffolds. Curr Opin Chem Biol 2017, 38,

8-16. 44.

Bhardwaj, G.; Mulligan, V. K.; Bahl, C. D.; Gilmore, J. M.; Harvey, P. J.; Cheneval, O.;

Buchko, G. W.; Pulavarti, S. V. S. R. K.; Kaas, Q.; Eletsky, A.; Huang, P. S.; Johnsen, W. A.; Greisen, P. J.; Rocklin, G. J.; Song, Y. F.; Linsky, T. W.; Watkins, A.; Rettie, S. A.; Xu, X. Z.; Carter, L. P.; Bonneau, R.; Olson, J. M.; Coutsias, E.; Correnti, C. E.; Szyperski, T.; Craik, D. J.; Baker, D., Accurate de novo design of hyperstable constrained peptides. Nature 2016, 538 (7625), 329-335. 45.

Gongora-Benitez, M.; Tulla-Puche, J.; Albericio, F., Multifaceted Roles of Disulfide

Bonds. Peptides as Therapeutics. Chem Rev 2014, 114 (2), 901-926. 46.

Robinson, J. A., beta-Hairpin Peptidomimetics: Design, Structures and Biological

Activities. Accounts Chem Res 2008, 41 (10), 1278-1288. 47.

Bandyopadhyay, A.; Misra, R.; Gopi, H. N., Structural features and molecular aggregations

of designed triple-stranded beta-sheets in single crystals. Chem Commun (Camb) 2016, 52 (27), 4938-4941. 48.

Karle, I. L.; Awasthi, S. K.; Balaram, P., A designed beta-hairpin peptide in crystals. Proc

Natl Acad Sci U S A 1996, 93 (16), 8189-8193. 49.

Haque, N. S.; Gellman, S. H., Insights on β-Hairpin Stability in Aqueous Solution from

Peptides with Enforced Type I‘ and Type II‘ β-Turns. J Am Chem Soc 1997, 119 (9), 2303-2304. 50.

Kumar, M. G.; Mali, S. M.; Raja, K. M. P.; Gopi, H. N., Design of Stable beta-Hairpin

Mimetics through Backbone Disulfide Bonds. Org Lett 2015, 17 (2), 230-233. 51.

Espinosa, J. F.; Gellman, S. H., A designed beta-hairpin containing a natural hydrophobic

cluster. Angew Chem Int Edit 2000, 39 (13), 2330-2333. 52.

Novick, P. A.; Lopes, D. H.; Branson, K. M.; Esteras-Chopo, A.; Graef, I. A.; Bitan, G.;

Pande, V. S., Design of beta-amyloid aggregation inhibitors from a predicted structural motif. J Med Chem 2012, 55 (7), 3002-3010.


20

Page 21 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

53.

Fezoui, Y.; Teplow, D. B., Kinetic studies of amyloid beta-protein fibril assembly -

Differential effects of alpha-helix stabilization. J Biol Chem 2002, 277 (40), 36948-36954. 54.

Jha, A.; Udgaonkar, J. B.; Krishnamoorthy, G., Characterization of the Heterogeneity and

Specificity of Interpolypeptide Interactions in Amyloid Protofibrils by Measurement of SiteSpecific Fluorescence Anisotropy Decay Kinetics. J Mol Biol 2009, 393 (3), 735-752. 55.

Levine, H., Thioflavine-T Interaction with Synthetic Alzheimers-Disease Beta-Amyloid

Peptides - Detection of Amyloid Aggregation in Solution. Protein Sci 1993, 2 (3), 404-410. 56.

Hortschansky, P.; Schroeckh, V.; Christopeit, T.; Zandomeneghi, G.; Fandrich, M., The

aggregation kinetics of Alzheimer's beta-amyloid peptide is controlled by stochastic nucleation. Protein Sci 2005, 14 (7), 1753-1759. 57.

Harper, J. D.; Lansbury, P. T., Models of amyloid seeding in Alzheimier's disease and

scrapie: Mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Annu. Rev. Biochem 1997, 66, 385-407. 58.

Chiti, F.; Stefani, M.; Taddei, N.; Ramponi, G.; Dobson, C. M., Rationalization of the

effects of mutations on peptide and protein aggregation rates. Nature 2003, 424 (6950), 805-808. 59.

Lomakin, A.; Chung, D. S.; Benedek, G. B.; Kirschner, D. A.; Teplow, D. B., On the

nucleation and growth of amyloid beta-protein fibrils: Detection of nuclei and quantitation of rate constants. P Natl Acad Sci USA 1996, 93 (3), 1125-1129. 60.

Morel, B.; Varela, L.; Azuaga, A. I.; Lara, F. C., Environmental Conditions Affect the

Kinetics of Nucleation of Amyloid Fibrils and Determine Their Morphology. Biophys J 2010, 99 (11), 3801-3810. 61.

Nielsen, L.; Khurana, R.; Coats, A.; Frokjaer, S.; Brange, J.; Vyas, S.; Uversky, V. N.; Fink,

A. L., Effect of environmental factors on the kinetics of insulin fibril formation: Elucidation of the molecular mechanism. Biochemistry-Us 2001, 40 (20), 6036-6046. 62.

Cohen, S. I. A.; Linse, S.; Luheshi, L. M.; Hellstrand, E.; White, D. A.; Rajah, L.; Otzen,

D. E.; Vendruscolo, M.; Dobson, C. M.; Knowles, T. P. J., Proliferation of amyloid-beta 42 aggregates occurs through a secondary nucleation mechanism. P Natl Acad Sci USA 2013, 110 (24), 9758-9763. 63.

Smith, J. F.; Knowles, T. P. J.; Dobson, C. M.; MacPhee, C. E.; Welland, M. E.,

Characterization of the nanoscale properties of individual amyloid fibrils. P Natl Acad Sci USA 2006, 103 (43), 15806-15811.


21

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

64.

Page 22 of 34

Khurana, R.; Ionescu-Zanetti, C.; Pope, M.; Li, J.; Nielson, L.; Ramirez-Alvarado, M.;

Regan, L.; Fink, A. L.; Carter, S. A., A general model for amyloid fibril assembly based on morphological studies using atomic force microscopy. Biophys J 2003, 85 (2), 1135-1144. 65.

Serpell, L. C., Alzheimer's amyloid fibrils: structure and assembly. Bba-Mol Basis Dis

2000, 1502 (1), 16-30. 66.

Nichols, M. R.; Colvin, B. A.; Hood, E. A.; Paranjape, G. S.; Osborn, D. C.; Terrill-Usery,

S. E., Biophysical Comparison of Soluble Amyloid-beta(1-42) Protofibrils, Oligomers, and Protofilaments. Biochemistry-Us 2015, 54 (13), 2193-2204. 67.

Walsh, D. M.; Hartley, D. M.; Kusumoto, Y.; Fezoui, Y.; Condron, M. M.; Lomakin, A.;

Benedek, G. B.; Selkoe, D. J.; Teplow, D. B., Amyloid beta-protein fibrillogenesis - Structure and biological activity of protofibrillar intermediates. J Biol Chem 1999, 274 (36), 25945-25952. 68.

Lashuel, H. A.; Hartley, D.; Petre, B. M.; Walz, T.; Lansbury, P. T., Neurodegenerative

disease - Amyloid pores from pathogenic mutations. Nature 2002, 418 (6895), 291-291. 69.

Shankar, G. M.; Li, S. M.; Mehta, T. H.; Garcia-Munoz, A.; Shepardson, N. E.; Smith, I.;

Brett, F. M.; Farrell, M. A.; Rowan, M. J.; Lemere, C. A.; Regan, C. M.; Walsh, D. M.; Sabatini, B. L.; Selkoe, D. J., Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat Med 2008, 14 (8), 837-842. 70.

Liu, M. L.; Hong, S. T., Early phase of amyloid beta 42-induced cytotoxicity in neuronal

cells is associated with vacuole formation and enhancement of exocytosis. Exp Mol Med 2005, 37 (6), 559-566. 71.

Arai, T.; Sasaki, D.; Araya, T.; Sato, T.; Sohma, Y.; Kanai, M., A Cyclic KLVFF-Derived

Peptide Aggregation Inhibitor Induces the Formation of Less-Toxic Off-Pathway Amyloid-beta Oligomers. Chembiochem 2014, 15 (17), 2577-2583. 72.

Kino, R.; Araya, T.; Arai, T.; Sohma, Y.; Kanai, M., Covalent modifier-type aggregation

inhibitor of amyloid-beta based on a cyclo-KLVFF motif. Bioorg. Med. Chem. Lett. 2015, 25 (15), 2972-2975. 73.

Kapurniotu, A.; Buck, A.; Weber, M.; Schmauder, A.; Hirsch, T.; Bernhagen, J.; Tatarek-

Nossol, M., Conformational restriction via cyclization in beta-amyloid peptide A beta(1-28) leads to an inhibitor of A beta(1-28) amyloidogenesis and cytotoxicity. Chem Biol 2003, 10 (2), 149159.


22

Page 23 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

74.

Richman, M.; Wilk, S.; Chemerovski, M.; Warmlander, S. K. T. S.; Wahlstrom, A.;

Graslund, A.; Rahimipour, S., In Vitro and Mechanistic Studies of an Antiamyloidogenic SelfAssembled Cyclic D,L-alpha-Peptide Architecture. J Am Chem Soc 2013, 135 (9), 3474-3484. 75.

Zorzi, A.; Deyle, K.; Heinis, C., Cyclic peptide therapeutics: past, present and future. Curr

Opin Chem Biol 2017, 38, 24-29. 76.

Craik, D. J.; Fairlie, D. P.; Liras, S.; Price, D., The future of peptide-based drugs. Chem

Biol Drug Des 2013, 81 (1), 136-147. 77.

Craik, D. J.; Cemazar, M.; Daly, N. L., The cyclotides and related macrocyclic peptides as

scaffolds in drug design. Curr Opin Drug Disc 2006, 9 (2), 251-260.


23

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 34

Figure legends

Figure 1. Sequence and structure of the cyclic peptide BHdS. (A) Primary sequence and (B) Far-UV CD spectrum of the cyclic (red broken line) and the disulfide reduced (black solid line) BHdS peptide. Figure 2. BHdS inhibits Aβ1-42 aggregation. (A) Absorbance spectrum of 5 µM congo red dye alone (red dotted line) and upon addition of (i) 50 μM of Aβ1-42 aggregates drawn from a sample of 100 μM Aβ1-42 that had been incubated at 37 °C for 24 h (blue dashed line), (ii) a mixture of 50 μM of Aβ1-42 and 0.5 mM of BHdS drawn from a sample of 100 μM Aβ1-42 co-incubated with 1 mM BHdS that had been incubated at 37 °C for 24 h (green dashed line), (iii) 0.5 mM of BHdS drawn from a sample of 1 mM BHdS that had been incubated at 37 °C for 24 h (black line). (B) Fluorescence emission spectrum of 20 µM thioflavin T (ThT) dye alone (red dotted line) and upon addition of (i) 50 μM of Aβ1-42 aggregates drawn from a sample of 100 μM Aβ1-42 that had been incubated at 37 °C for 24 h (blue dashed line), (ii) a mixture of 50 μM of Aβ1-42 and 0.5 mM of BHdS drawn from a sample of 100 μM Aβ1-42 co-incubated with 1 mM BHdS that had been incubated at 37 °C for 24 h (green dashed line), (iii) 0.5 mM of BHdS drawn from a sample of 1 mM BHdS that had been incubated at 37 °C for 24 h (black line).

Figure 3. Dependence of the kinetics of aggregation of Aβ1-42 on the concentration of the BHdS peptide. (A) The kinetics of aggregation of Aβ1-42 was monitored in the absence and in the presence of different concentration of BHdS at 37 ºC using the change in thioflavin-T fluorescence at 480 nm upon excitation at 440 nm. (i) 100 µM Aβ1-42 only (black circles), (ii) 100 µM Aβ1-42 + 1mM BHdS (red circles), (iii) 100 µM Aβ1-42 + 2mM BHdS (green circles), (iv) 100µM Aβ1-42 + 2.5 mM BHdS (blue circles), (v) 100 µM Aβ1-42 + 2.5 mM BHdS + 5 mM DTT (blue diamonds), (vi) 2.5 mM BHdS only (pink circles). The solid lines through the data in panel A are fits to a single exponential equation. The relative amplitudes and the rate constants of the kinetic curves obtained from the fit are plotted against the concentration of BHdS, respectively, in panel B and C. The blue bar in panel B and the blue diamond in panel C represent the data for the sample containing 100 µM Aβ1-42 + 2.5 mM BHdS + 5 mM DTT.


24

Page 25 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 4. Effect of BHdS on the secondary structural transition during the aggregation of Aβ1-42.The dotted red line shows the far-UV CD spectrum of Aβ1-42 taken immediately after dissolution of the monomer. The blue dashed line represents the far-UV CD spectrum of the aggregates formed by incubation of 100 µM of Aβ1-42 at 37 ºC for 24 h. 100 µM of Aβ1-42 was also incubated at 37 ºC for 24 h in the presence 2.5 mM BHdS. The green dashed line denotes the farUV CD spectrum of the resultant sample after subtraction of the CD signal of the BHdS peptide. 100 µM of Aβ1-42 was also incubated at 37 ºC for 24 h in the presence 2.5 mM disulfide reduced BHdS. The black solid line denotes the far-UV CD spectrum of the resultant sample after subtraction of the CD signal of the disulfide reduced BHdS peptide.

Figure 5. Effect of BHdS on the changes in external morphology of Aβ1-42 fibrils. The aggregates of Aβ1-42 were prepared by incubation of (i) 100 µM Aβ1-42 alone and (ii) 100 µM Aβ142 co-incubated

with 2.5 mM BHdS, and (iii) 100 µM Aβ1-42 co-incubated with 2.5 mM BHdS + 5

mM DTT at 37 ºC. Transmission electron microscopy (TEM) image of the protein aggregates formed by (A) Aβ1-42 alone (i); (B) Aβ1-42 + cyclic BHdS (ii); and (C) Aβ1-42 + disulfide reduced BHdS (iii). The black scale bar in the lower left corner of the panels A-C is100 nm.

Figure 6. Effect of BHdS on the cell toxicity of soluble aggregates of Aβ1-42. The cell viability of the SHSY5Y neuronal cells was measured by MTT reduction assay upon exposure to: (i) 10 μM of Aβ1-42 aggregates drawn from a sample of 100 μM Aβ1-42 that had been incubated at 37 °C for 12 h (1), (ii) a mixture of 10 μM of Aβ1-42 co-incubated with BHdS drawn from a sample of 100 μM Aβ1-42 co-incubated with 2.5mM BHdS that had been incubated at 37 °C for 12 h (2), (iii) ) a mixture of 10 μM of Aβ1-42 co-incubated with disulfide reduced BHdS drawn from a sample of 100 μM Aβ1-42 co-incubated with 2.5 mM BHdS + 5 mM DTT that had been incubated at 37 °C for 12 h (3), (iv) 2.5 mM of BHdS sample that had been incubated at 37 °C for 12 h (4). The data represent the percentage of viable cells compared to the control (cells only without any treatment).

Figure 7. Schematic model for the mechanism of action of the BHdS peptide.Aβ1-42 monomers have a random coil structure (as seen by CD in Figure 4). Upon incubating it at 37οC for 24 h, it


25

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 34

changes to soluble protofibrillar aggregates which has significant β-sheet content (as seen by CD in Figure 4) and binds to thioflavin T (Figure 3). Upon further incubation of the sample for another 72 h (total 96 h of incubation for monomers) at 37 οC, Aβ1-42 forms insoluble fibrils which has an ordered assembly typical of amyloid fibrils (as seen by TEM in Figure 5). The model shows that the pre-formed cyclic structure of BHdS peptide containing the constrained β-hairpin motif is complementary to the β-sheet rich structure of Aβ1-42 soluble aggregates. It increases the binding affinity and makes it a potent inhibitor. The inhibitor activity is lost when the disulfide stapling of BHdS is reduced by the addition of DTT. Failure of the inhibition activity results into the formation of amyloid-like fibrils (as seen by TEM in Figure 5C).


26

Page 27 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 1


27

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 34

Figure 2


28

Page 29 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 3


29

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 34

Figure 4


30

Page 31 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 5


31

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 34

Figure 6


32

Page 33 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 7


33

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 34

TOC graphic


34

Inhibition of Î²-Amyloid aggregation Through a Designed Î²-hairpin

Recommend Documents