Towards a Rational Design to Regulate Î²-Amyloid Fibrillation for

Subscriber access provided by READING UNIV

Review

Towards a Rational Design to Regulate #-Amyloid Fibrillation for Alzheimer’s Disease Treatment Xu Han, and Gefei He ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00477 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 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.

ACS Chemical Neuroscience 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 17 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

ACS Chemical Neuroscience

Towards a Rational Design to Regulate β-Amyloid Fibrillation for Alzheimer’s Disease Treatment Xu Han,a,* and Gefei Heb a Huston b East

Labs, 1951 NW 7th Ave, Suite 600, Miami, FL 33136, USA China Normal University, 3663 Zhongshan N Road, Putuo District, Shanghai 200062, China.

Contact: [email protected] Abstract. The last decades have witnessed a growing global burden of Alzheimer’s disease (AD). Evidence indicates the onset and progression of AD is associated with β-amyloid (Aβ) peptide fibrillation. As such, there is a strong passion with discovering potent Aβ fibrillation inhibitors that can be developed into antiamyloiddogenic agents for AD treatment. Current challenges arisen with this development involve with Aβ oligomer toxicity suppression and Blood Brain Barrier penetration capability. Considering most natural or biological events, one would observe that there is usually a “seed” to direct natural materials to assembly in response to a certain stimulation. Inspired by this, several materials or compounds, including nanoparticle, peptide or peptide mimics, and organic molecules, have been designed for the purpose of redirecting or impeding Aβ aggregation. Achieving these tasks requires comprehensive understanding on (1) how initial Aβ assembly into insoluble deposits, (2) main concerns arisen with fibrillation inhibition, and (3) current major methodologies to disrupt the aggregation. Herein, the objective of this review is to address these three areas, and enable the prompt for a promising therapeutic agent design for AD treatment. Keywords: Alzheimer’s disease • β-amyloid peptide • Nanoparticle • Peptide • Organic Molecules • Clinical Trial

Introduction. Natural materials guide a network of simple molecules or building blocks to assemble into hierarchical nanostructures to achieve structural complexity and intricate functions. Certain cellular functions, in turn, will be demonstrated by the efficiency of organizing such an assembly process in respond to the structure changes induced by either force1 or light2 or other incoming energy. One of the primary goals of biotechnology is to understand, mimic, and affect these nature assemblies so as to be applied in scientific and engineering areas.3 Within biological systems, the message that directs hierarchical assembly is ciphered within the Watson-Crick structure based genes. Peptide or protein, whereby directed by these genes, represent the major embodiment of bio-function. Research with regard to it can effectively help reveal the rules of life secrets. Amyloid is one of such products after several assembly processes.4 Extracellular deposition of these amyloid fibrils is believed to be pathological hallmark of several amyloidogenic diseases, such as Alzheimer’s, Parkinson’s, and diabetes type II.5 Among these amyloidosis, Alzheimer’s disease (AD) is the most common form of dementia and has become a major threat across the globe.6 Recent reports indicate 81.1

Figure 1. The formation of Aβ fibrils. Aβ is produced from amyloid precursor protein (APP) after sequentially proteolytic cleavage by β- and γ-secretases. Fibrils are formed via a variety of pathways by nucleation-dependent kinetics, which can be mainly categorized as lag, elongation, and saturation phase. There also exists fragmentation to catalyse the preformed fibrils to form new fibrils. The Aβ oligomers are believed to cause damages to neuron cells.

ACS Paragon Plus Environment

ACS Chemical Neuroscience 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

million people are expected to be living with it by the year 2040, and the financial burden was $236 billion in 2016 alone.7 Therefore, the development of therapeutic agents for AD, which can redirect the aggregation, is undoubtedly in dire need. It has been almost 30 years since AD was characterized as the accumulation of the extracellular plaques and intracellular tangles, of which β-amyloid (Aβ) peptide and tau protein are identified as their major component, respectively. 8 The explicit justification causing AD pathogenicity remains to be discovered, but Aβ and tau protein have been considered as primary suspects for AD. In a healthy cell, the main function of tau focus on transport system. But the twisted tau protein (deposit) at the tangle sites disintegrate the transporting system, resulting in the cell death. In most cases, intracellular tangles of tau protein were found to be deposited after Aβ “negative” metabolism.9 Most scientists hence believe the primary factor that underlies AD should be attributed to the accumulation of Aβ.10 Regarding the tau protein, the common pathogenesis starts with its detachment from microtubules by phosphatases, and subsequently to aggregate. The initiation of tau aggregation demands a binding to a non-specific site to expose the tau-tau binding domain. Thereafter the self-propagate would make result in the polymerization. To regulate this process, taubased therapeutic strategy hence involve with targeting appropriate kinase (such as GSK 3β) and interrupting the aggregation. Several potential drugs, such as the ones designed by Allon Therapeutics Inc. (NCT01110720) and Noscira (NCT01049399), have entered Phase II trials. Although it remains in the debate regarding how fibrils aggregates lead to cell death, the inhibition of fibril formation should still be the current and future goal of scientific communities. Because 1) fibrils cannot be dismissed as one of the possible cytotoxins, 2) inhibitors can help target different intermediates and provide invaluable insights. Up to now, anti-Aβ therapies have dominated clinical trials for US Food and Drug Administration (FDA) approval, whereby only five drugs that mainly target cholinesterase though not Aβ have been approved.11 One considerable molecular etiology towards fibrillation involves the aggregation of a 4-kDa Aβ peptide –40/42-residue fragments of Aβ produced from sequentially proteolytic cleavage of amyloid precursor protein (APP) by β- and γ-secretases under certain conditions (Figure 1).10 After the cleavage from APP, Aβ could assemble into insoluble fibers, which is structurally dominated by β-sheet. This process is initiated by the agglomeration of Aβ oligomers into clusters, which in turn aggregates into long chains named as fibrils. Accumulation of these fibrils will gradually turn into β-sheets, and finally develop into plaques that may comprise other substances apart from β-sheets. Despite the small difference in the produced Aβ 40 and 42, these two peptides present distinct clinical behaviors: (1) Aβ 42 is a more toxic form and

Scheme 1. Aβ sequence with subregions, whereby orange indicates the N-terminus, blue represents hydrophobic core, purple displays salt bridge, green shows the C-terminus, and HHQK presents a glycosaminoglycan (GAGs) binding site. “+” and “-” signs mean the amino acid is positively and negatively charged, respectively. Histidine (H) residue shows higher binding affinity to copper (Cu). predominating species in the plaques; 12 (2) The C-terminus region of Aβ 42 has stronger rigidity where the extra two residues can better promote a turn conformation.13 On a molecular level, both Aβ monomer does not have stabilized secondary structure but a few parallel folds. Dimerization starts with the hydrophobic core and subsequently continue with attachment to another monomer to form an anti-parallel β-sheet.14 Oligomer will be obtained after further packing of monomer to the dimer through hydrophobic C-terminus strand.15 After the primary nucleation, the conversion of spherical oligomer into larger aggregates will be mediated by the hydrophobic and hydrophilic (Glu22-Gly29) interactions.16 During this process, most oligomers can induce other Aβ peptides to track the former ones, further promoting the formation of Aβ fibrils in a tremendously effective catalytic cycle (Figure 1). It has been shown that different regions on Aβ 40 or 42 are associated with such actions in the process of Aβ fibrillation. For instance, HHQK fragment contains a binding site for glycosaminoglycans which can assist the conformation change of Aβ from α-helix to β-sheet (Scheme 1).17 It is this conformation alternation that lead to the nucleation of the Aβ monomers, wherein Aβ dimer can be stabilized by the salt bridge.17 The resulting hinge region along with hydrophobic patches enable further backbone-backbone interactions between Aβ peptides to contribute to the β-sheets assembly. Similar to crystal formation, it is “seed” that direct the format of the crystal eventually harbors. To successfully redirect or impede the accumulation of “unhealthy” assembly, the inhibitor design involves effective binding towards initial Aβ monomer, oligomer, filament, or other morphologies to re-guide the original encoded “misfolding” process, similar to the role of the seed on the crystal formation. Hydrophobic core acts as a main driving force behind Aβ polymerization. The inhibitor to target this region in order to break the β-sheet interaction, therefore, has been paid more attention as a potential drug candidate. Paradoxically, Aβ plays a negatively critical role on AD, while it is useful for some neurons, 18 so that it has been present at birth and continues to be moderately formed throughout the whole lifetime. To prevent over production of Aβ, an alternative method towards AD pathophysiology include targeting β- and γ-secretases. However, the design for γ-secretases inhibitor may not be a promising solution in spite of much attention, because γ-secretases are essential to regulate many other biological conditions and undesired side effects are inevitable with its deactivation.19 One of the most recent example is Semagacestat (LY-450139) developed by Eli Lilly and Company. The drug candidate was designed to target γ-secretases but failed in the Phase II clinical trial because of higher


Page 2 of 17

Page 3 of 17 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


percentage of incidents for skin cancer. Most groups focus more on developing β-secretases (BACE1) inhibitors.20 But the problem could arise with the large binding pocket in the BACE1. This will create another difficulty in blood-brain barrier (BBB) penetration.21 So far, one of the most successful BACE1 inhibitor is LY2886721, but the induced liver toxicity fails it in the Phase II trial.11 Potential antiamyloiddogenic agents can also create other challenges. They include: (1) The toxicity of Aβ 42 oligomer – this ~56 kDa oligomer is believed to be responsible for a dysfunction that is correlated to memory. 22 Both hydrophobic sequences, Leu17-Ala21 and Ala30-Ala42 in the N-terminus, were reported to account for the oligomerization at which steps the monomer assembles into toxic oligomer. 23 In addition, Aβ 42 tetramer adopts a bent structure.24 This distinct structure is able to give Aβ 42 more chances to form contacts with others, whereas ring-shaped Aβ 40 tetramer is less likely to further aggregate, which might be one reason behind its reduced toxicity. 25 Most scientists also think of the toxicity of Aβ 42 oligomer as the primary cause of AD, given the observation that soluble Aβ level is better correlated to the AD severity than insoluble fibrils.10, 26 Regarding toxic Aβ 42 oligomer, several studies have demonstrated that the toxicity might be mediated by different mechanisms. These include: a) Oxidative Poisoning,27 where the first 15 amino acids of Aβ contain metal binding sites which process abilities to reduce the metal and initiate sequential reactions, resulting in the over-expressed reactive species that is over the ability for the biological system to detoxify these intermediates; b) Neutron Damages,28 induced by negative regulation of agonists, enabling the activation of different signaling pathways inside the cell provoking neuronal damage; c) Membrane Interruption,29 wherein Aβ oligomer tends to bind to the cellular membrane quickly and strongly, leading to a rise in the amount of calcium within cells, which is intently related to cell damage and death; d) Special Structure30 – different oligomers derived from different peptides and proteins share the common conformation-dependent structure, and all of them show toxicity in vitro which can be abolished by an oligomer-specific antibody. The same agent can recognize different soluble oligomers, indicative of similar toxic structured mechanism. 30 (2) Capability to penetrate the BBB. Unfortunately, tight junctions between brain endothelial cells, which mainly constitute BBB, are notably immune to systemic chemotherapy, as many chemotherapeutic agents are impotent to efficiently transport across the BBB and arrive at the central nervous system , although intracerebroventricular injections could be an unfriendly solution. BBB typically only allows the passage of water and diffusion of small hydrophobic molecules, as well as the delivery via transporter mediated pathway. Overcoming the obstacle to deliver therapeutic agents to specific brain regions still remains a bottleneck towards AD treatment. Previous research has demonstrated several mechanisms for drugs to target inside the brain. It includes: a) To dilate blood vessels by using Bradykini, one of hyperosmotic agents;31 b) To utilize receptor-mediated approach, such as glucose and amino acid carriers. 32 In spite of the success in brain uptake of therapeutic agents, there are still risks arisen with the permeability changes of BBB. The entry of toxins and other unwanted molecules is inevitable by opening the barrier. To this end, nanoparticles (NPs) have been intensively reviewed to gain access through BBB in the past decades.33 Another similar means is the utilization of drug carrier systems, namely liposomes,34 which can entrap either hydrophilic or hydrophobic or lipophilic therapeutic agents inside its core. Nanoparticles. NP therapeutics are mainly particles in the range size of 1 nm - 1000 nm with remedial components. Most NPs feature self-assembly tendency, similar to the amyloid aggregation process. Directing on this area may reveal important insights into the fibrillation behavior. 35-37 Possible mechanism may come up with the methodology that NPs with proper geometry and tunable surface redirect the fibrillation pathway. In terms of Aβ fibrillation inhibition by NPs, recent investigations include inorganic NPs38 and polymeric NPs39. Despite being short of cellular model and in vivo observation, these nanomaterials typically have superior functions towards anti-amyloid due to more specific targeting function induced by the improved pharmacodynamics performance. It elucidates more molecular details to deepen our understanding of the peptide aggregation as well. The enhanced properties mainly rely on the unique functionalized surface and the size. However, potential risks from these NPs themselves deserve more attention for AD treatment or other biological applications. For example, when small enough, NPs will start to have an access to all parts of the body even including brain, giving rise to unexpected damages. 40 The Surface function, dominating interaction between NPs with biomolecules, has been one of major focus in the area of nanomedicine. Being in the stream of biological fluid, NPs can attract different proteins or other biological substances, resulting in the protein conformation changes with certain functions disrupted. 41 Poor understanding of this nature has been accomplished. Before the next generation design of novel nanomaterials to target amyloid peptide, it is critical to understand the basic kinetic and thermodynamics information regarding stoichiometry, binding affinity, association and dissociation rate, and others. To address these problems, a set of copolymers has been reported towards equilibrium and kinetic properties. 42 The research revealed how protein absorbance dependence and exchange rates are related to the NP hydrophobicity. Another interesting observation is that operation procedure such as incubation time and the concentrations of particles can also dictate their reactions with different proteins. Later on, the same group successfully used these co-polymeric NPs to target Aβ 40 at the nucleation stage without interrupting the elongation kinetics, by adjusting the hydrophobic character to its interaction with hydrophobic C-terminal portion of Aβ.43 The main concept herein is to apply hydrophobic surface character. In a similar view, fluorine is also known to modulate the physicochemical feature to be hydrophobic so as to affect molecule conformation. As a result, a fluorinated NP, instead of hydrogenated analogs, turns to induce α-helical structure in Aβ 40 and control Aβ 42 oligomerization and toxicity in a cellular model.44



Surface charge is another significant parameter to be acknowledged for the NPs-protein interaction. The bare, positively, and negatively charged AuNPs incredibly exhibited different Aβ inhibition mechanisms. 45, 46 Most recently, charged NPs were reported to be capable of inhibiting oppositely charged Aβ to further aggregate, irrespective of the NP material.46 Taking the Aβ sequence into account, there exists six negatively charged residues (Asp1, Glu3, Asp7, Glu11, Glu22, and Asp23) and three positively charged residues (Arg5, Lys16, and Lys28). For this reason, positively charged NPs might display stronger electrostatic interaction to the peptide. However, positively charged peptides were observed to be toxic by facilitating their interactions to the negatively charged phospholipid membrane bilayers, 47 indicating of potential damages caused by the positive NPs. The surface interaction is sophisticated, especially to the chemistry with protein or peptide. It is, hence, difficult to conclude one simple rule that has priority to other methods. The size of the NP might lead to controversial results in respect with fibrillation. Overall, the presence of some large NPs promotes Aβ polymerization – the attachment of peptides onto the NPs provides local high concentration and greatly accelerates the fibril formation because of nucleation-dependent kinetics. For example, a 16 nm TiO 2 was reported to promote Aβ fibrillation by lowering the energetic barriers and shortening the nucleation process.48 Also, molecular dynamics simulations demonstrated how NPs promote the peptides and proteins aggregation with a condensation dependent mechanism.49 The acceleration starts with immobilization of disordered oligomer onto a larger NP, and followed by the size growth of highly ordered β-sheet. However, the interaction with biomolecules is complex. On the contrary, smaller NPs, such as ~10 nm nanogels50 and an averaged 4 nm quantum dots 51, appeared to slow down the rate of Aβ fibrillation. The large size of the NPs does provide a platform to attract peptides and increase the concentration in

Figure 2. The effect of nanoparticle (NP) on aggregation. NP surface is capable of dominating its interactions with different targets including biomolecules. The large size of NP tends to provide high concentration of Aβ, resulting in the promotion of Aβ fibrillation. Whereas, the small size of NP can target subregions of Aβ and inhibit its further aggregation. local area whereas smaller size NPs are inclined to target fragments of peptides (Figure 2), but other conditions may also draw a different result. 40 nm polymeric NPs were found to be able to inhibit Aβ fibrillation 43, but the same NPs with diameters of 70 and 200 nm would promote rather than inhibit the amyloid protein of β2 microglobulin40. Recently, Kim et al. examined the consequences induced by different sizes of AuNPs on Aβ fibrillation using a total brain lipid extractbased supported lipid bilayer (brain SLB). 46 The research determined that more Aβ aggregation occurred with larger AuNPs (80 nm). Thus, smaller size might be preferentially selected for a potent inhibitor agent purpose. NPs in a distinctive shape can be obtained, when the size is extended differently in separate dimensions. It also concerns the effect of shape on aggregation because the complex biological conditions can alter the original shape of NPs by inevitable contacts with biomolecules. Indeed, Au nanocube promotes Aβ aggregation due to its isotropic structure and large surface interaction area, whereas spherical AuNPs and Au nanorod are both able to inhibit Aβ fibrillation as a β-sheet breaker.46 Furthermore, If the AuNP stretches in one dimension, the spherocylindrical NP reduces more β-sheet content.52 All of these observations well suggest the impact of the shape on the aggregation.


Page 4 of 17

Page 5 of 17 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


However, the inability to inhibit Aβ toxicity is the most critical bottleneck that has precluded most drug candidates from further potential in the AD treatment. To this end, Han et al. scrutinized the action of a non-toxic zero-dimensional carbon material (C-Dots) on Aβ fibrillation via experimental and computational methods.53 The prepared 4 nm C-Dots mainly feature hydrophilic surface after the top-down synthesis. Experimental observation and molecular dynamics simulations implied the substantial function achieved by the C-Dots hydrophilic surface with its interaction on Aβ monomer backbone. Moreover, a few Aβ residues, such as Phe, Ile, and Leu, have contacts with the C-Dots hydrophobic surface via side chain. In addition to directly quench the aggregation, the C-Dots turn out to restrict BACE1 activity to reduce Aβ production as well. More importantly, the C-Dots cannot merely inhibit Aβ 42 toxicity in vitro, but penetrate BBB in a Zebrafish model by covalently engineering transferrin onto it. However, a comprehensive in vivo experiments are still in need, because reactions inside organisms are complicated and human transferrin receptor was found to keep the drug remain entrapped in brain endothelial cells. 54 Unfortunately, few NPs with anti-aggregation strength have gone through clinical trials11, rather more of them served as a vehicle to deliver other potential pharmaceutical candidates. But it is still worth some considerations as therapeutic agents, because NPs are easy to synthesize, and have good biocompatibility and biodegradability. Table 1. Peptides selected for Aβ fibrillation inhibition. Name

Strategy

Description

Therapeutic Significance

Reference

None

KLVFF

Lys16, Leu17, and Phe20 are essential to Aβ fibrillation inhibition.

H2

GQKLVFFAE DVGGaKKKKKK N-Methyl Amino Acids in alternating positions of KLVFF hydrophilic moietyKLVFF

Oligolysine with three or more residues is effective on inhibiting Aβ toxicity.

Lay a good foundation to discover the KLVFF bearing inhibitor Solvent effect on the toxicity inhibition in a cellular model.

Tjernberg, L. O. et al. 1996.55 Ghanta, J. et al. 1996.56

N-Methyl Amino Acids assist to disassemble preformed fibrils

The method assist the discovery of Aspan, which has been in Phase II clinical trial.

Gordon, D. J. et al. 2001.57

Better performance to dissociate fibrils induced by hydrophilic moiety.

Akikusa, A. et al. 2003.58

D3

DRPRTRLHT HRNR

This sequence not derived from Aβ was identified as a highly specific ligand to Aβ 42

K4

DendrimerKLVFF

P1, P2

KLVF-∆A-I∆A and KF∆A-∆A-∆A-F -VVIA

Dendrimer provides a multivalent scaffold to effectively inhibit fibril formation and fibril disassembly. Dehydro amino acids containing peptides can help inhibit Aβ aggregation.

Cellular model indicates a better toxicity inhibition induced by the aid of hydrophilic moiety. It established the groundwork for D3 and its derivative to accomplish preclinical study. The multivalent scaffold help inhibit protofibrillar Aβ to from fibrils. A discovery of a motif to assist the inhibition. Cellular model as well as computer simulation demonstrate the oligomerization inhibition induced by the C-terminal peptides

Fradinger, E. A. et al. 2008.62

None

DDX

None

C-terminal peptides, especially Aβ (31–42) and Aβ (39–42), inhibit Aβ 42 toxicity and oligomerization.


Wiesehan, K. et al. 2003.59 Chafekar, S. M. et al. 2007.60 Rangachari, et al. 2009.61


None

N-methylated peptide based on IGLMVG

N-methylated C-terminal peptides inhibit Aβ 42 toxicity and oligomerization.

iAβ5

LPFFD-PEG

Proline can aid to inhibit Aβ fibrillation by preventing hydrogen bonding in the fibril extension. PEG reduces the immunogenicity but retains inhibition effect.

None

LPFFD LPFFN

LPFFN is a better motif to design good β-sheet breaker.

None

IGLMVG

and

Further demonstrate the effect of C-terminal peptides on Aβ aggregation, wherein IGLMVG can inhibit Aβ toxicity.

Page 6 of 17

A N-methylated peptide penta-peptide is better than partially N-methylated peptide at inhibiting Aβ 42 toxicity and oligomerization in a cellular model. Further study for the proline contained peptide, which aid NAP to go through Phase I and II clinical trial.

Arvidsson, P. I. et al. 2009.63

Further study for the proline contained peptide, which aid NAP to go through Phase I and II clinical trial. Cellular model shows toxicity inhibition by one of a C-terminal peptide

Minicozzi, V. et al. 2014.65

Rocha, S. et al. 2009.64

Bansal, S. et al. 2016.66

Peptides. Peptides typically refer to linear molecules comprised of at least two amino acids residues. This biological nature material or artificially manufactured substance is an excellent alternative to pharmaceutics, due to its essential rol e on regulating biological functions. However, the difficulty arisen with rapid degradation implies problems for administration and delivery. 67 To at least partially resolve the problem, it is fortunate that peptide chemistry allows diverse approaches for the modification via either the terminal group or the side chain. Nowadays has seen a globally growing peptide therapeutics market. As for the AD treatment in terms of fibrillation inhibition, the self-associating of the peptide could be utilized as another approach to interfere with fibril formation or poison the aggregation process. As early as 1996, the central hydrophobic sequence, Aβ 16-21 (KLVFFA), was observed to account for disrupting interactions to control its oligomerization, whereby typical antiparallel β-sheet structures were formed as a result of their interaction with full length Aβ. 55 Later, a series of Aβ sequence with KLVFF and LVFFA motifs were also found to be effective on inhibiting Aβ fibrillation in vitro.68 Since then, the fragment KLVFF or LVFFA has been added either to a scaffold, such as dendrimer or polymer, or a few motifs of hydrophilic amino acids (Table 1), to break β-sheet. Other than that, a proline residue, known to be a β-sheet breaker, was also inserted to obtain a proline based central hydrophobic sequence for the purpose of Aβ fibrillation. 64 The secret behind it involves the nitrogen in proline being lack of a proton, which can help prevent hydrogen bonding occurring in the fibril extension. Similarly, methylation of amide groups is also an impressive strategy to create new inhibitor categories, whereby N-methyl group is able to improve the solubility in aqueous and in turn reduce the Aβ toxicity. 63 A cellular model indicates a higher methylation level can reduce Aβ mediated toxicity.63 One of such peptides, named as Aspan, has even been in Phase II clinical trial. Another critical factor to predict Aβ aggregation is the ability to increase solvent tension. 56 Lysine (K) and glutamic acid (E) were identified as stabilizing kosmotropic agents by increasing the surface tension, whereas arginine (R) is categorized as a destabilizing chaotrope without correlating the solvent property. Considering this effect, poly K, E, or R can be added to each end of peptide recognition domain. As a result, both poly K and E enhanced Aβ fibrillation, and poly R facilitated to the inhibition process. On top of these strategies, Doig et al. selected SEN304 as a most potent inhibitor after customizing KLVFF by amidating C-terminal, acetylating N-terminal, varying the length and side chain identity. 69 Later on, one of SEN304 derivatives entered into preclinical trial, establishing a promising prospect for the central hydrophobic sequence against Aβ aggregation. Several substantial research has also suggested another β-sheet peptides breaker, the C-terminal fragments (CTFs) of Aβ 42 (Scheme 1). It is a vital region to target Aβ oligomerization. CTFs can regulate intermolecular interactions to control Aβ 42 oligomer formation. The two extra motifs from Aβ 42, Ile41 and Ala42, were suggested to contribute to the more rigid Aβ 42, enabling to stabilize a putative turn conformation. This brings about higher conformational stability for Aβ 42 so as to stabilize Aβ 42 oligomer with less neurotoxicity. 70 However, the improper addition to Ile41 and Ala42 bearing sequence could lead to controversial results. It abolished this protective effect by inserting Arginine (Arg) in an animal test.47 To further investigate the central role of Aβ 42 C-terminus, a series of Aβ 42 CTFs were derived to examine their potential to inhibit Aβ 42 oligomerization and toxicity, and of those, two lead compounds, Aβ 31-42 and Aβ 39-42, appeared to be more effective.62 Both experimental and computational studies indicated that, CTFs initially incorporated into a putative hydrophobic core of Aβ 42 oligomers, and thereafter co-assemble with Aβ 42 to form nontoxic heterooligomers in order to disrupt its further oligomerization and toxicity. These behaviors decrease their interaction with cellular targets. More details behind this mechanism were discovered afterwards. In addition to binding to C-terminus, in


Page 7 of 17 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


fact, CTFs reacts with several sites on Aβ 42, in particular with N-terminus. Pursuant to this knowledge, a multivalent scaffold is likely to manipulate the fibril assembly process effectively. Using multivalent polymer-peptide conjugates (mPPCs), researchers modified the oligomeric fibril aggregation of Aβ into discrete nanostructures.71 During Aβ nucleation, there is a formation of prefibrillar intermediates due to β-sheet interactions along the central hydrophobic sequence Aβ 17-21. The driving force behind mPPCs capability just took advantage of this weak and energetically disfavored noncovalent interaction between the monomers during the nucleation phase. Besides these, one recently prepared CTFs based peptide has been tested in cellular models.66 Both Rat PC-12 cells and MTT cell viability evaluated this hexapeptide derivative (Aβ 32-37, IGLMVG) as inhibitors of Aβ toxicity. Apart from the peptide inhibitors derived from Aβ sequence, tremendous efforts have also been dedicated to others that are not directly developed based on Aβ sequence. This idea descends from mimicking the nature materials. Most Aβ sequence derived peptide inhibitors feature similar characteristics inclusive of hydrophobicity and tendency to communicate with β sheet. Standing on this philosophy, multitudinous variations have been explored. 72 One of them covers an eight amino acid sequence, NAPVSIPQ (NAP). 73 The presence of polarized residues (Q and N), two proline residues (P), and the hydrophobic backbone, accounts for its inhibition and toxicity reduction capability. It failed in Phase III clinical trial because of unimpressive results for progressive supranuclear palsy. But the structure has set an excellent example for the following investigation. Together with the NAP, another peptide, D-RPRTRLHTHRNR (D3), was announced to exhibit a similar activity in the same year.59 Currently, both D3 and its derivatives have a remarkable opportunity to enter Phase III clinical trial, reasoning the completion of safety studies and the first two phases. 11 Given the comprehensive understanding on how peptide inhibit Aβ fibrillation, Protein, obtained when peptide “growing” (over approximately 50 amino acids) and being in much more defined structures, has been adding its appeal for many researchers. Previous results showed the clearance of plaques through Immunotherapy in a transgenic animal test, suggesting immunological factors being against production of Aβ deposition.74 Further research showed that there is a narrow and specific epitope in the monoclonal antibody which can recognize Aβ and inhibit its fibrillation and toxicity. 75 However, polyclonal antibodies seems not so good at performance as monoclonal antibody due to its unclear behavior. 76 The immunization can be classified into active and passive immunotherapy, where the active immunotherapy is identified to stimulate patient's immune system in response to AD and the passive immunotherapy attempts to provide vaccines. 77 The objective of both strategy is to mainly improve the ability for the clearance of Aβ accumulation. This year, Solanezumab (LY2062430), a humanized anti-Aβ peptide immunoglobulin G-1 (IgG1) monoclonal antibody has completed Phase III clinical trial. 11 This drug developed by Eli Lilly and Company aims to slow down cognitive and functional decline in AD by decreasing Aβ production. The detailed results are, to date, not available thus far. Other than this, numerous monoclonal antibody drug candidate has completed clinical trial, such as Ponezumab (PF-04360365) for Phase I and Albutein® 5% for Phase II.11

Figure 3. Strategies for using organic molecules to inhibit Aβ fibrillation. It includes: (a) a compound with a multi-functional groups to target subregions and a flexible linker, (b) using metal chelation chemistry to reduce the interactions between excess metal and Aβ.

Organic Molecules. Challenges, associated with using small synthetic molecule inhibitor, can be made by the weak interaction between small molecule and protein. The interaction region of protein-protein is approximately 20 nm 2, which is much larger than that of small molecule with protein (3-10 nm2).78 Small molecules, thus, cannot afford adequate steric hindrance to block the fibrillation. The relatively featureless protein-protein interaction region fails to provide small



molecule a spot to dock in for pharmacological intervention. Worse, the protein surface is so plastic that small molecules could be accommodated to avoid inhibition. In addition to this concern, the compound is better to be hydrophobic for BBB permeability. To tackle the barrier, one potential approach is to design a ligand to target multiple subregions, such as fragments containing KLVFF and CTFs, better with a suitable linker to extend the interaction towards larger areas in order to provide sufficient steric hindrance (Figure 3a). On the way to discover such a potent agent, natural products consistently inspire us the direction to the potential synthetic molecules, and of which, organic dyes, including congo red (CR), chrysamine G (CG), and curcumin, were described to bind to Aβ with high affinity (Scheme 2).26 One similar scaffold shared by these molecules contains aromatic or cyclohexance groups (Scheme 2). Likewise, natural polyphenolic compound, such as (-)-epigallocatechin gallate (EGCG) extracted from green tea, and natural phytoalexin like resveratrol, were also found to affect the fibrillation by using aromatic group to break the π-π stacking during polymerization (Scheme 2).79 To note, both EGCG and resveratrol and their analogues have entered into Phase II clinical trial.80 It is reasonable to predict such structures, because there are several aromatic groups (Phe 4, 19, 20) and other hydrophobic groups inside the Aβ residues accounting for the fibrillation. Further considering the effect hydrophobic groups, fluorinated groups were also found to bound hydrophobic subregion to retard Aβ fibrillation, 81 similar to the surface governed NPs case. In another study, the optimal length for the linker to connect these chemical scaffolds, in particular for curcumin, was identified between 0.8 nm and 1.6 nm.82 The study also presented that co-planar spatial rearrangement contributes to the inhibition effect. As a matter of fact, the spatial arrangement of atoms in molecules does play a substantial role in the pharmaceutical industry, since drug efficacy relates to chiral recognition. Usually, one enantiomer is pharmaceutically active and its mirror structure is inactive or exerts side effects. Previous studies have reported an α-helix in the 13-23 segment of Aβ.83 In light of the α-helix chiral structure, Aβ should be sensitive to a chiral inhibitor or environment. One example is associated with a metal-supramolecular model complex. 84 Taking advantage of the α/β discordance in Aβ fibril formation, researchers indicated that these chiral supramolecular complexes which are suitable for binding and targeting the α-helical form of the 16-23 region showed an enantioselective effect on inhibiting Aβ fibrillation. Given the thorough considerations of potential attacking target, flexible linkers, and spatial arrangement, the most astonishing strategy should be the one with


Page 8 of 17

Page 9 of 17 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


harnessing chaperones.85 Not only the conjugated chaperones offer the small-molecule sufficient steric bulk to break AβAβ interaction, but the flexible linker permits the terminated groups to scan the Aβ surface for an optimized arrangement. Phenylalanine (Phe) residues are of great importance in the hydrophobic interactions that take place during amyloid fibrillation. In fact, the Phe that mainly present in the central region is a key to the Aβ self-assembly as a result of its featured molecular recognition. 80 An alternative study to target Phe by using cucurbit[7]uril (CB[7]) presented a supramolecular strategy for inhibiting amyloid fibrillation. 86 Cucurbit[7]uril, as a synthetic receptor member of the family

Scheme 2. Chemical structures of natural materials used as fibrillation inhibitors and selected synthetic molecules under clinical trial as an Aβ fibrillation inhibitor. cucurbit[n]uril (n=5-8, 10, 14) was reported to bind specifically and tightly (Ka: 10 4-107M-1) to Phe residues in Aβ, which in turn initiates and enhances the hydrophobic clustering of amyloid proteins. Inspired by this supr amolecular method, Han et al. designed a resorcinarene inhibitor for Aβ aggregation degradation driven by non-polar interactions.87, 88 The resorcinarene has proved, by docking calculations and molecular dynamics simulations, to bind to the top and the bottom of the Aβ filament rather than target single Phe residue alone, thereby distributing the overall energy to a larger area. In addition, sea urchin toxicity test evidenced the toxic oligomer inhibition accomplished by the resorcinarene, suggesting an off-pathway mechanism towards anti-aggregation function.



Page 10 of 17

Table 2. Completed Phase IV clinical trial with amyloid treatment for AD. Entities

Interventions

Structure

Study Title

Sponsor

Drug: Lovostatin 1

The Effect of Short-Term Statin and NSAID Treatment on CSF Beta-Amyloid

National Institute of Mental Health (NIMH)

Do HMG CoA Reductase Inhibitors Affect Abeta Levels?

Seattle Institute for Biomedical and Clinical Research

Pilot Trial of Carvedilol in Alzheimer's Disease

Johns Hopkins University

Studying the Effects of Antihypertensives on Individuals at Risk for Alzheimer's

University of Wisconsin, Madison

Drug: Ibuprofen

Drug: Simvastatin

2

Drug: Pravastatin

Drug: Carvedilol 3

Drug: Placebo

-

Drug: Ramipril 4

Drug: Placebo

-

In recent years, one of the most advanced drug candidate for Aβ fibrillation inhibition is tramiprosate ( Scheme 2), which is prepared to target 1-28 subregion but mainly towards HHQK. 80 This interaction successfully manages to induce the anti-fibrillogenic a11ctivity as well as block the formation of neurotoxic aggregates. However, its activity in the clinical trial dose not exhibit significant better performance than the placebo, leading to the failure in the Phase III trial. The unsuccessful case does not curb the enthusiasm to use synthetic molecules to handle Aβ fibrillation for AD treatment. One intriguing example is brought by the research on bexarotene (Scheme 2), an anticancer drug approved by FDA, where bexarotene tends to selectively disturb the primary Aβ 42 nucleation steps and delay the production of toxic species


Page 11 of 17 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


in neuroblastoma cells.89 This candidate has completed Phase II clinical trial as an amyloid mediated drug candidate. Another one is Carvedilol sponsored by Johns Hopkins University (Scheme 2), which has completed Phase IV clinical trial.11 The design involves the bifunctional targeting group located at each end of the suitable linker to enhance the inhibition property.11 In addition to screens of chemical libraries to uncover direct interaction of organic molecules with amyloid, another potential strategy is to employ chelation method to interrupt the aberrant interactions between metal and Aβ (Figure 3b). The hypothesis involves the damage and fibrillation enhancement caused by the interaction of different Aβ morphologies with the excess zinc (Zn), iron (Fe), copper (Cu), and other metals. 79 The release of the metal from Aβ can enhance Aβ clearance, too.90 Previously, Fe content was reported to be abnormally concentrated in the Aβ plaques, whereas Cu concentration got depressed. 91 For the Zn level, it was found higher in the blood but lower in the cerebrospinal fluid in AD patients.92 Nature products, such as EGCG and Curcumin, contain several sites for metal ions coordination other than its ability to inhibit Aβ aggregation. 93 Several curcumin analogues has been patented as metal chelators. 79 However, one concern is that too strong chelators could cause risks by making more metal ions leave other important regulatory sites. An effective Kd to release Cu and Zn from Aβ was reported to be 10 -10 M and 10-8 M, respectively. 94 Clioquinol, one of chelators for Cu and Fe, has been studied for several years to release the metal from the Aβ, but it also has been one of suspects to cause serious neuro-problems.95 Alternatively, researchers focused on small multi-functional molecules that contain structural moieties for metal chelation along with Aβ recognition function. Basic 3-hydroxy-4-pyridinone framework of deferiprone (HL) is such a candidate due to its high affinity for Cu(II) and Zn(II), while having a low affinity for important biological electrolytes such as sodium, potassium, calcium and magnesium. 96 Brain uptake of a radiolabeled HL ligand successfully passed the BBB test using in situ rat brain perfusion technique. There are also other prominent chelators, such as lipophilic chelator DP-109, showing effective function with inhibiting Aβ accumulation in the animal test.97 One of them, which is named as PBT2, has completed Phase II clinical trial.11 Every technology is a two-edged sword. In addition to the negative effect, transition metal complexes, including copper(II), platinum(II) and ruthenium(II) complexes, were positively implicated to bind and inhibit the aggregation of Aβ peptides, as long as not being over-presence.98 These metal complexes might be another viable alternative to synthetic compounds described above, since they can be prepared in fewer and more easily modified steps. Table 3. Summary of different drug candidate under clinical trial. Category

Peptide

Immunization

Organic Molecule

Name SEN304 derivatives Aspan D3 derivatives iAβ5 NAPVSIPQ PF-04360365 Albutein® 5% LY2062430 EGCG analogues resveratrol analogues bexarotene PBT2 Carvedilol Lovostatin Simvastatin Ramipril

Status Preclinical Phase II Phase II Phase II Phase III Phase I Phase II Phase III Phase II Phase II Phase II Phase II Phase IV Phase IV Phase IV Phase IV

Clinical Trial. A complete clinical trial is commonly classified into five phases, from preclinical to Phase IV. The objectives for each stage include: (1) To collect preliminary data among a small amount of subjects, mostly animals. (2) To test the safety within a small group of human (~ 70) and determine the potential side effect. (3) To examine the efficacy with a larger group (~ 200 individuals). (4) To further confirm the safety and the efficiency with averaged 2000 people. (5) Postmarketing studies. In 2017 alone, 209 studies aiming at amyloid treatment for AD has gone through clinical trials in the US, and of those, 28 cases have failed, and 4 studies have completed Phase IV stage ( Table 2).11 The high failure rate may be associated with (1) the heterogeneous nature of the disease, whereby a) the amyloid is usually deposited even before clinical symptoms appear, and targeting amyloid may cause failure for patients with mild to moderate Alzheimer's disease, b) a late intervention could be arisen with the diagnostic methods used, leading to the difficulty with patients selection in the trial; (2) drug kinetics and dynamics – most companies, in order to rush into market, overlook basic pharmacokinetics and pharmacodynamics rather than appropriately consider preliminary evidence for dosing and efficacy design; (3) problems with inter-site variance and different operational protocols; (4) intellectual property, whereby if the



treatment target the early stage, trials would be extremely long, leading to the fact that patent has expired before the trial which requires new models and approaches. Up to now, only five drugs have been approved by FDA, four of which are cholinesterase inhibitors (tacrine, donepezil, rivastigmine, galantamine) and one of which is a NMDA receptor antagonist (memantine). In these, galantamine was recently announced to regulate Aβ aggregation as an alternative means for AD treatment.99 Nowadays has witnessed more and more research is devoting onto the Aβ inhibitor investigation. There are several novel studies with regard to Aβ fibrillation inhibition described in this review have completed different phases of clinical trials (Table 3). Following this idea, and considering the complicated mechanisms behind AD, the future development should be towards a goal with a multi-purpose drug candidate in addition to amyloid aggregation inhibition. Other hallmarks of AD include: decreasing level of Acetylcholine, and increasing level of Glutamate and Neurofibrillary Tangles and Neuroinflammation. However, one essential task to effectively test or screen the potential drug candidates is to develop efficient biomarker, giving indications on AD as a clinical standard. This can also at least guarantee relative enough time to avoid delayed treatment. 100 The development of new drugs to complete the clinical trial is costly and time consuming with a high failure rate, but novel therapies are in urgent need for AD treatment. Conclusions. AD is a progressive and irreversible neurodegenerative disease which is categorized into seven stages, from no impairment to very severe decline. The major hallmark of AD is associated with Aβ fibrillation and its subsequent deposition. During fibrillation, Aβ 42 oligomer toxicity is considered as another main causes of AD. As a result, it is better to inhibit Aβ fibrillation at an early stage before oligomerization. Comparing different kinds of inhibitors, advantages emerged with peptide based method over others are likely to come with (1) good biocompatible, (2) rational design to target specific subregions, (3) excellent tunability with simple chemistry for the modification, (4) a better solution to construct a combinatorial library for the screen process, (5) NPs are better to serve as a carrier to cross BBB due to the readily chemistry, or even to enhance the inhibition capability for the selected peptide or its mimics, and (6) the preparation for organic molecules is time-consuming and less efficient. However, it does not imply a discourage on exploring synthetic molecule or NPs inhibitors. Because this research is necessary to unveil the mechanism behind AD and achieve a better treatment. It is better to design a relative small and hydrophobic molecule in order to easily overcome BBB obstacle with regard to the organic molecules preparation, while NPs expect to take into account for the surface properties and size influence. Nevertheless, scientists still have to continue to explore different potential Aβ fibrillation inhibitor, to gain a better understanding regarding the role of Aβ fibrillation on the AD mechanism. Acknowledgement X.H. gratefully acknowledges the support from Catherine H. Lorié, Tom Huston, Jr, and Edward D. Miller.. Author contributions X.H. wrote the manuscript with contribution from G.F.H.. Additional information The authors declare no competing financial interests. Reference: 1. Han, X., Bian, S., Liang, Y., Houk, K. N., and Braunschweig, A. B. (2014) Reactions in elastomeric nanoreactors reveal the role of force on the kinetics of the Huisgen reaction on surfaces, J. Am. Chem. Soc. 136, 10553-10556. 2. Bian, S., Zieba, S. B., Morris, W., Han, X., Richter, D. C., Brown, K. A., Mirkin, C. A., and Braunschweig, A. B. (2014) Beam pen lithography as a new tool for spatially controlled photochemistry, and its utilization in the synthesis of multivalent glycan arrays, Chem. Sci. 5, 2023-2030. 3. Han, X., Zheng, Y., Munro, C. J., Ji, Y., and Braunschweig, A. B. (2015) Carbohydrate nanotechnology: hierarchical assembly using nature's other information carrying biopolymers, Curr. Opin. Biotechnol. 34, 41-47. 4. Knowles, T. P., Vendruscolo, M., and Dobson, C. M. (2014) The amyloid state and its association with protein misfolding diseases, Nat. Rev. Mol. Cell Biol. 15, 384-396. 5. Eisenberg, D., and Jucker, M. (2012) The amyloid state of proteins in human diseases, Cell 148, 1188-1203. 6. Mattson, M. P. (2004) Pathways towards and away from Alzheimer's disease, Nature 430, 631-639. 7. Association, A. s. (2016) 2016 Alzheimer's disease facts and figures, Alzheimer's & Dementia 12, 459-509. 8. Goedert, M., and Spillantini, M. G. (2006) A century of Alzheimer's disease, Science 314, 777-781. 9. Hardy, J., Duff, K., Hardy, K. G., Perez-Tur, J., and Hutton, M. (1998) Genetic dissection of Alzheimer's disease and related dementias: amyloid and its relationship to tau, Nat. Neurosci. 1, 355-358. 10. Hardy, J., and Selkoe, D. J. (2002) The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics, Science 297, 353-356. 11. clinicaltrials.gov. 12. Younkin, S. G. (1995) Evidence that A beta 42 is the real culprit in Alzheimer's disease, Ann. Neurol. 37, 287-288. 13. Urbanc, B., Cruz, L., Yun, S., Buldyrev, S. V., Bitan, G., Teplow, D. B., and Stanley, H. E. (2004) In silico study of amyloid beta-protein folding and oligomerization, Proc. Natl. Acad. Sci. USA 101, 17345-17350.


Page 12 of 17

Page 13 of 17 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


14. Losic, D., Martin, L. L., Mechler, A., Aguilar, M. I., and Small, D. H. (2006) High resolution scanning tunnelling microscopy of the beta-amyloid protein (Abeta1-40) of Alzheimer's disease suggests a novel mechanism of oligomer assembly, J. Struct. Biol. 155, 104-110. 15. Mastrangelo, I. A., Ahmed, M., Sato, T., Liu, W., Wang, C., Hough, P., and Smith, S. O. (2006) High-resolution atomic force microscopy of soluble Abeta42 oligomers, J. Mol. Biol. 358, 106-119. 16. Kayed, R., Pensalfini, A., Margol, L., Sokolov, Y., Sarsoza, F., Head, E., Hall, J., and Glabe, C. (2009) Annular protofibrils are a structurally and functionally distinct type of amyloid oligomer, J. Biol. Chem. 284, 4230-4237. 17. Bartolini, M., Bertucci, C., Bolognesi, M. L., Cavalli, A., Melchiorre, C., and Andrisano, V. (2007) Insight into the kinetic of amyloid beta (1-42) peptide self-aggregation: elucidation of inhibitors' mechanism of action, Chembiochem 8, 2152-2161. 18. Abramov, E., Dolev, I., Fogel, H., Ciccotosto, G. D., Ruff, E., and Slutsky, I. (2009) Amyloid-beta as a positive endogenous regulator of release probability at hippocampal synapses, Nat. Neurosci. 12, 1567-1576. 19. van Es, J. H., van Gijn, M. E., Riccio, O., van den Born, M., Vooijs, M., Begthel, H., Cozijnsen, M., Robine, S., Winton, D. J., Radtke, F., and Clevers, H. (2005) Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells, Nature 435, 959-963. 20. Ghosh, A. K., and Osswald, H. L. (2014) BACE1 (beta-secretase) inhibitors for the treatment of Alzheimer's disease, Chem. Soc. Rev. 43, 6765-6813. 21. Citron, M. (2010) Alzheimer's disease: strategies for disease modification, Nat. Rev. Drug Discov. 9, 387-398. 22. Walsh, D. M., Klyubin, I., Fadeeva, J. V., Cullen, W. K., Anwyl, R., Wolfe, M. S., Rowan, M. J., and Selkoe, D. J. (2002) Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo, Nature 416, 535-539. 23. Liu, R., McAllister, C., Lyubchenko, Y., and Sierks, M. R. (2004) Residues 17-20 and 30-35 of beta-amyloid play critical roles in aggregation, J. Neurosci. Res. 75, 162-171. 24. Bernstein, S. L., Dupuis, N. F., Lazo, N. D., Wyttenbach, T., Condron, M. M., Bitan, G., Teplow, D. B., Shea, J. E., Ruotolo, B. T., Robinson, C. V., and Bowers, M. T. (2009) Amyloid-beta protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer's disease, Nat. Chem. 1, 326-331. 25. Economou, N. J., Giammona, M. J., Do, T. D., Zheng, X., Teplow, D. B., Buratto, S. K., and Bowers, M. T. (2016) Amyloid beta-Protein Assembly and Alzheimer's Disease: Dodecamers of Abeta42, but Not of Abeta40, Seed Fibril Formation, J. Am. Chem. Soc. 138, 1772-1775. 26. Lee, S. J., Nam, E., Lee, H. J., Savelieff, M. G., and Lim, M. H. (2017) Towards an understanding of amyloid-beta oligomers: characterization, toxicity mechanisms, and inhibitors, Chem. Soc. Rev. 46, 310-323. 27. Kontush, A., Berndt, C., Weber, W., Akopyan, V., Arlt, S., Schippling, S., and Beisiegel, U. (2001) Amyloid-β is an antioxidant for lipoproteins in cerebrospinal fluid and plasma, Free Radic. Biol. Med. 30, 119-128. 28. Cerpa, W., Dinamarca, M. C., and Inestrosa, N. C. (2008) Structure-function implications in Alzheimer's disease: effect of Aβ oligomers at central synapses, Curr. Alzheimer Res. 5, 233-243. 29. Arispe, N., Rojas, E., and Pollard, H. B. (1993) Alzheimer disease amyloid beta protein forms calcium channels in bilayer membranes: blockade by tromethamine and aluminum, Proc. Natl. Acad. Sci. USA 90, 567-571. 30. Kayed, R., Head, E., Thompson, J. L., McIntire, T. M., Milton, S. C., Cotman, C. W., and Glabe, C. G. (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis, Science 300, 486-489. 31. Marcos-Contreras, O. A., Martinez de Lizarrondo, S., Bardou, I., Orset, C., Pruvost, M., Anfray, A., Frigout, Y., Hommet, Y., Lebouvier, L., Montaner, J., Vivien, D., and Gauberti, M. (2016) Hyperfibrinolysis increases blood-brain barrier permeability by a plasmin- and bradykinin-dependent mechanism, Blood 128, 2423-2434. 32. Klepper, J., and Voit, T. (2002) Facilitated glucose transporter protein type 1 (GLUT1) deficiency syndrome: impaired glucose transport into brain-- a review, Eur. J. Pediatr. 161, 295-304. 33. Koziara, J. M., Lockman, P. R., Allen, D. D., and Mumper, R. J. (2003) In situ blood-brain barrier transport of nanoparticles, Pharm. Res. 20, 1772-1778. 34. McNeeley, K. M., Karathanasis, E., Annapragada, A. V., and Bellamkonda, R. V. (2009) Masking and triggered unmasking of targeting ligands on nanocarriers to improve drug delivery to brain tumors, Biomaterials 30, 3986-3995. 35. Xia, Y. (2008) Nanomaterials at work in biomedical research, Nat. Mater. 7, 758. 36. Peng, Z., Han, X., Li, S., Al-Youbi, A. O., Bashammakh, A. S., El-Shahawi, M. S., and Leblanc, R. M. (2017) Carbon Dots: Biomacromolecule Interaction, Bioimaging and Nanomedicine, Coord. Chem. Rev. 37. Han, X., Li, S., Peng, Z., Al-Yuobi, A. O., Omar Bashammakh, A. S., El-Shahawi, M. S., and Leblanc, R. M. (2016) Interactions between Carbon Nanomaterials and Biomolecules, J Oleo Sci 65, 1-7. 38. Yoo, S. I., Yang, M., Vivekanandan Subramanian, D., Brender, J. R., Sun, K., Joo, N. E., Jeong, S.-H., Ramamoorthy, A., and Kotov, N. A. (2011) Mechanism of Fibrillation Inhibition of Amyloid Peptides by Inorganic Nanoparticles Reveal Functional Similarities with Proteins, Angew. Chem. Int. Ed. 50, 5110-5115. 39. Cabaleiro-Lago, C., Quinlan-Pluck, F., Lynch, I., Dawson, K. A., and Linse, S. (2010) Dual effect of amino modified polystyrene nanoparticles on amyloid beta protein fibrillation, ACS Chem. Neurosci. 1, 279-287. 40. Linse, S., Cabaleiro-Lago, C., Xue, W. F., Lynch, I., Lindman, S., Thulin, E., Radford, S. E., and Dawson, K. A. (2007) Nucleation of protein fibrillation by nanoparticles, Proc. Natl. Acad. Sci. USA 104, 8691-8696. 41. Peng, Z., Li, S., Han, X., Al-Youbi, A. O., Bashammakh, A. S., El-Shahawi, M. S., and Leblanc, R. M. (2016) Determination of the composition, encapsulation efficiency and loading capacity in protein drug delivery systems using circular dichroism spectroscopy, Anal. Chim. Acta 937, 113-118.



42. Cedervall, T., Lynch, I., Lindman, S., Berggard, T., Thulin, E., Nilsson, H., Dawson, K. A., and Linse, S. (2007) Understanding the nanoparticle-protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles, Proc. Natl. Acad. Sci. USA 104, 2050-2055. 43. Cabaleiro-Lago, C., Quinlan-Pluck, F., Lynch, I., Lindman, S., Minogue, A. M., Thulin, E., Walsh, D. M., Dawson, K. A., and Linse, S. (2008) Inhibition of amyloid beta protein fibrillation by polymeric nanoparticles, J. Am. Chem. Soc. 130, 1543715443. 44. Bitan, G., Kirkitadze, M. D., Lomakin, A., Vollers, S. S., Benedek, G. B., and Teplow, D. B. (2003) Amyloid beta -protein (Abeta) assembly: Abeta 40 and Abeta 42 oligomerize through distinct pathways, Proc. Natl. Acad. Sci. USA 100, 330-335. 45. Liao, Y. H., Chang, Y. J., Yoshiike, Y., Chang, Y. C., and Chen, Y. R. (2012) Negatively charged gold nanoparticles inhibit Alzheimer's amyloid-beta fibrillization, induce fibril dissociation, and mitigate neurotoxicity, Small 8, 3631-3639. 46. Kim, Y., Park, J.-H., Lee, H., and Nam, J.-M. (2016) How Do the Size, Charge and Shape of Nanoparticles Affect Amyloid β Aggregation on Brain Lipid Bilayer?, Sci. Rep. 6. 47. Li, H., Rahimi, F., and Bitan, G. (2016) Modulation of Amyloid beta-Protein (Abeta) Assembly by Homologous C-Terminal Fragments as a Strategy for Inhibiting Abeta Toxicity, ACS Chem. Neurosci. 7, 845-856. 48. Wu, W. H., Sun, X., Yu, Y. P., Hu, J., Zhao, L., Liu, Q., Zhao, Y. F., and Li, Y. M. (2008) TiO2 nanoparticles promote betaamyloid fibrillation in vitro, Biochem. Biophys. Res. Commun. 373, 315-318. 49. Auer, S., Trovato, A., and Vendruscolo, M. (2009) A condensation-ordering mechanism in nanoparticle-catalyzed peptide aggregation, PLOS Comput. Biol. 5, e1000458. 50. Ikeda, K., Okada, T., Sawada, S., Akiyoshi, K., and Matsuzaki, K. (2006) Inhibition of the formation of amyloid beta-protein fibrils using biocompatible nanogels as artificial chaperones, FEBS Lett. 580, 6587-6595. 51. Xiao, L., Zhao, D., Chan, W. H., Choi, M. M., and Li, H. W. (2010) Inhibition of beta 1-40 amyloid fibrillation with N-acetylL-cysteine capped quantum dots, Biomaterials 31, 91-98. 52. O'Brien, E. P., Straub, J. E., Brooks, B. R., and Thirumalai, D. (2011) Influence of Nanoparticle Size and Shape on Oligomer Formation of an Amyloidogenic Peptide, J. Phys. Chem. Lett. 2, 1171-1177. 53. Han, X., Jing, Z., Wu, W., Zou, B., Peng, Z., Ren, P., Wikramanayake, A., Lu, Z., and Leblanc, R. M. (2017) Biocompatible and blood-brain barrier permeable carbon dots for inhibition of Abeta fibrillation and toxicity, and BACE1 activity, Nanoscale 9, 12862-12866. 54. Wiley, D. T., Webster, P., Gale, A., and Davis, M. E. (2013) Transcytosis and brain uptake of transferrin-containing nanoparticles by tuning avidity to transferrin receptor, Proc. Natl. Acad. Sci. USA 110, 8662-8667. 55. Tjernberg, L. O., Naslund, J., Lindqvist, F., Johansson, J., Karlstrom, A. R., Thyberg, J., Terenius, L., and Nordstedt, C. (1996) Arrest of beta-amyloid fibril formation by a pentapeptide ligand, J. Biol. Chem. 271, 8545-8548. 56. Ghanta, J., Shen, C. L., Kiessling, L. L., and Murphy, R. M. (1996) A strategy for designing inhibitors of beta-amyloid toxicity, J. Biol. Chem. 271, 29525-29528. 57. Gordon, D. J., Sciarretta, K. L., and Meredith, S. C. (2001) Inhibition of beta-amyloid(40) fibrillogenesis and disassembly of beta-amyloid(40) fibrils by short beta-amyloid congeners containing N-methyl amino acids at alternate residues, Biochemistry 40, 8237-8245. 58. Akikusa, S., Watanabe, K. I., Horikawa, E., Nakamura, K., Kodaka, M., Okuno, H., and Konakahara, T. (2003) Practical assay and molecular mechanism of aggregation inhibitors of beta-amyloid, J. Pept. Res. 61, 1-6. 59. Wiesehan, K., Buder, K., Linke, R. P., Patt, S., Stoldt, M., Unger, E., Schmitt, B., Bucci, E., and Willbold, D. (2003) Selection of D-amino-acid peptides that bind to Alzheimer's disease amyloid peptide abeta1-42 by mirror image phage display, Chembiochem 4, 748-753. 60. 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. 61. Rangachari, V., Davey, Z. S., Healy, B., Moore, B. D., Sonoda, L. K., Cusack, B., Maharvi, G. M., Fauq, A. H., and Rosenberry, T. L. (2009) Rationally designed dehydroalanine (DeltaAla)-containing peptides inhibit amyloid-beta (Abeta) peptide aggregation, Biopolymers 91, 456-465. 62. 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., 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. USA 105, 14175-14180. 63. Pratim Bose, P., Chatterjee, U., Nerelius, C., Govender, T., Norstrom, T., Gogoll, A., Sandegren, A., Gothelid, E., Johansson, J., and Arvidsson, P. I. (2009) Poly-N-methylated amyloid beta-peptide (Abeta) C-terminal fragments reduce Abeta toxicity in vitro and in Drosophila melanogaster, J. Med. Chem. 52, 8002-8009. 64. Rocha, S., Cardoso, I., Borner, H., Pereira, M. C., Saraiva, M. J., and Coelho, M. (2009) Design and biological activity of betasheet breaker peptide conjugates, Biochem. Biophys. Res. Commun. 380, 397-401. 65. Minicozzi, V., Chiaraluce, R., Consalvi, V., Giordano, C., Narcisi, C., Punzi, P., Rossi, G. C., and Morante, S. (2014) Computational and experimental studies on beta-sheet breakers targeting Abeta1-40 fibrils, J. Biol. Chem. 289, 11242-11252. 66. Bansal, S., Maurya, I. K., Yadav, N., Thota, C. K., Kumar, V., Tikoo, K., Chauhan, V. S., and Jain, R. (2016) C-Terminal Fragment, Abeta32-37, Analogues Protect Against Abeta Aggregation-Induced Toxicity, ACS Chem. Neurosci. 7, 615-623. 67. Schubert, U., Anton, L. C., Gibbs, J., Norbury, C. C., Yewdell, J. W., and Bennink, J. R. (2000) Rapid degradation of a large fraction of newly synthesized proteins by proteasomes, Nature 404, 770-774. 68. Matsunaga, Y., Fujii, A., Awasthi, A., Yokotani, J., Takakura, T., and Yamada, T. (2004) Eight-residue Abeta peptides inhibit the aggregation and enzymatic activity of Abeta42, Regul. Pept. 120, 227-236.


Page 14 of 17

Page 15 of 17 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


69. Kokkoni, N., Stott, K., Amijee, H., Mason, J. M., and Doig, A. J. (2006) N-Methylated peptide inhibitors of beta-amyloid aggregation and toxicity. Optimization of the inhibitor structure, Biochemistry 45, 9906-9918. 70. Li, H., Du, Z., Lopes, D. H., Fradinger, E. A., Wang, C., and Bitan, G. (2011) C-terminal tetrapeptides inhibit Abeta42-induced neurotoxicity primarily through specific interaction at the N-terminus of Abeta42, J. Med. Chem. 54, 8451-8460. 71. Song, Y., Cheng, P. N., Zhu, L., Moore, E. G., and Moore, J. S. (2014) Multivalent macromolecules redirect nucleationdependent fibrillar assembly into discrete nanostructures, J. Am. Chem. Soc. 136, 5233-5236. 72. Goyal, D., Shuaib, S., Mann, S., and Goyal, B. (2017) Rationally Designed Peptides and Peptidomimetics as Inhibitors of Amyloid-beta (Abeta) Aggregation: Potential Therapeutics of Alzheimer's Disease, ACS Comb. Sci. 19, 55-80. 73. Ashur-Fabian, O., Segal-Ruder, Y., Skutelsky, E., Brenneman, D. E., Steingart, R. A., Giladi, E., and Gozes, I. (2003) The neuroprotective peptide NAP inhibits the aggregation of the beta-amyloid peptide, Peptides 24, 1413-1423. 74. Schenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Liao, Z., Lieberburg, I., Motter, R., Mutter, L., Soriano, F., Shopp, G., Vasquez, N., Vandevert, C., Walker, S., Wogulis, M., Yednock, T., Games, D., and Seubert, P. (1999) Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse, Nature 400, 173-177. 75. Du, Y., Wei, X., Dodel, R., Sommer, N., Hampel, H., Gao, F., Ma, Z., Zhao, L., Oertel, W. H., and Farlow, M. (2003) Human anti-beta-amyloid antibodies block beta-amyloid fibril formation and prevent beta-amyloid-induced neurotoxicity, Brain 126, 1935-1939. 76. Dodel, R. C., Du, Y., Depboylu, C., Hampel, H., Frolich, L., Haag, A., Hemmeter, U., Paulsen, S., Teipel, S. J., Brettschneider, S., Spottke, A., Nolker, C., Moller, H. J., Wei, X., Farlow, M., Sommer, N., and Oertel, W. H. (2004) Intravenous immunoglobulins containing antibodies against beta-amyloid for the treatment of Alzheimer's disease, J. Neurol. Neurosurg. Psychiatry 75, 1472-1474. 77. Hung, S. Y., and Fu, W. M. (2017) Drug candidates in clinical trials for Alzheimer's disease, J. Biomed. Sci. 24, 47. 78. Keskin, O., Gursoy, A., Ma, B., and Nussinov, R. (2008) Principles of protein-protein interactions: what are the preferred ways for proteins to interact?, Chem. Rev. 108, 1225-1244. 79. Kepp, K. P. (2012) Bioinorganic chemistry of Alzheimer's disease, Chem. Rev. 112, 5193-5239. 80. Belluti, F., Rampa, A., Gobbi, S., and Bisi, A. (2013) Small-molecule inhibitors/modulators of amyloid-beta peptide aggregation and toxicity for the treatment of Alzheimer's disease: a patent review (2010 - 2012), Expert Opin. Ther. Pat. 23, 581-596. 81. Vieira, E. P., Hermel, H., and Mohwald, H. (2003) Change and stabilization of the amyloid-beta(1-40) secondary structure by fluorocompounds, Biochim. Biophys. Acta Proteins Proteomics 1645, 6-14. 82. Reinke, A. A., and Gestwicki, J. E. (2007) Structure-activity relationships of amyloid beta-aggregation inhibitors based on curcumin: influence of linker length and flexibility, Chem. Biol. Drug Des. 70, 206-215. 83. Nerelius, C., Sandegren, A., Sargsyan, H., Raunak, R., Leijonmarck, H., Chatterjee, U., Fisahn, A., Imarisio, S., Lomas, D. A., Crowther, D. C., Stromberg, R., and Johansson, J. (2009) Alpha-helix targeting reduces amyloid-beta peptide toxicity, Proc. Natl. Acad. Sci. USA 106, 9191-9196. 84. Li, M., Howson, S. E., Dong, K., Gao, N., Ren, J., Scott, P., and Qu, X. (2014) Chiral metallohelical complexes enantioselectively target amyloid beta for treating Alzheimer's disease, J. Am. Chem. Soc. 136, 11655-11663. 85. Gestwicki, J. E., Crabtree, G. R., and Graef, I. A. (2004) Harnessing chaperones to generate small-molecule inhibitors of amyloid beta aggregation, Science 306, 865-869. 86. Lee, H. H., Choi, T. S., Lee, S. J., Lee, J. W., Park, J., Ko, Y. H., Kim, W. J., Kim, K., and Kim, H. I. (2014) Supramolecular inhibition of amyloid fibrillation by cucurbit[7]uril, Angew. Chem. Int. Ed. 53, 7461-7465. 87. Han, X., Park, J., Wu, W., Malagon, A., Wang, L., Vargas, E., Wikramanayake, A., Houk, K. N., and Leblanc, R. M. (2017) A resorcinarene for inhibition of Abeta fibrillation, Chem. Sci. 8, 2003-2009. 88. Han, X., Tian, C., Gandra, I., Eslava, V., Galindres, D., Vargas, E., and Leblanc, R. (2017) The Investigation on Resorcinarenes towards either Inhibiting or Promoting Insulin Fibrillation, Chem. Eur. J. 23, 1-6. 89. Habchi, J., Arosio, P., Perni, M., Costa, A. R., Yagi-Utsumi, M., Joshi, P., Chia, S., Cohen, S. I., Muller, M. B., Linse, S., Nollen, E. A., Dobson, C. M., Knowles, T. P., and Vendruscolo, M. (2016) An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Abeta42 aggregates linked with Alzheimer's disease, Sci. Adv. 2, e1501244. 90. Ritchie, C. W., Bush, A. I., Mackinnon, A., Macfarlane, S., Mastwyk, M., MacGregor, L., Kiers, L., Cherny, R., Li, Q. X., Tammer, A., Carrington, D., Mavros, C., Volitakis, I., Xilinas, M., Ames, D., Davis, S., Beyreuther, K., Tanzi, R. E., and Masters, C. L. (2003) Metal-protein attenuation with iodochlorhydroxyquin (clioquinol) targeting Abeta amyloid deposition and toxicity in Alzheimer disease: a pilot phase 2 clinical trial, Arch. Neurol. 60, 1685-1691. 91. Roberts, B. R., Ryan, T. M., Bush, A. I., Masters, C. L., and Duce, J. A. (2012) The role of metallobiology and amyloid-beta peptides in Alzheimer's disease, J. Neurochem. 120 Suppl 1, 149-166. 92. Molina, J. A., Jimenez-Jimenez, F. J., Aguilar, M. V., Meseguer, I., Mateos-Vega, C. J., Gonzalez-Munoz, M. J., de Bustos, F., Porta, J., Orti-Pareja, M., Zurdo, M., Barrios, E., and Martinez-Para, M. C. (1998) Cerebrospinal fluid levels of transition metals in patients with Alzheimer's disease, J. Neural Transm. 105, 479-488. 93. Gupta, S. C., Prasad, S., Kim, J. H., Patchva, S., Webb, L. J., Priyadarsini, I. K., and Aggarwal, B. B. (2011) Multitargeting by curcumin as revealed by molecular interaction studies, Nat. Prod. Rep. 28, 1937-1955. 94. Faller, P., and Hureau, C. (2009) Bioinorganic chemistry of copper and zinc ions coordinated to amyloid-beta peptide, Dalton Trans., 1080-1094.



95. Cahoon, L. (2009) The curious case of clioquinol, Nat. Med. 15, 356-359. 96. Scott, L. E., Telpoukhovskaia, M., Rodríguez-Rodríguez, C., Merkel, M., Bowen, M. L., Page, B. D., Green, D. E., Storr, T., Thomas, F., and Allen, D. D. (2011) N-Aryl-substituted 3-(β-D-glucopyranosyloxy)-2-methyl-4 (1 H)-pyridinones as agents for Alzheimer's therapy, Chem. Sci. 2, 642-648. 97. Lee, J. Y., Friedman, J. E., Angel, I., Kozak, A., and Koh, J. Y. (2004) The lipophilic metal chelator DP-109 reduces amyloid pathology in brains of human beta-amyloid precursor protein transgenic mice, Neurobiol. Aging 25, 1315-1321. 98. Kumar, A., Moody, L., Olaivar, J. F., Lewis, N. A., Khade, R. L., Holder, A. A., Zhang, Y., and Rangachari, V. (2010) Inhibition of Aβ42 peptide aggregation by a binuclear ruthenium (II)− platinum (II) complex: potential for multimetal organometallics as anti-amyloid agents, ACS Chem. Neurosci. 1, 691-701. 99. Takata, K., Kitamura, Y., Saeki, M., Terada, M., Kagitani, S., Kitamura, R., Fujikawa, Y., Maelicke, A., Tomimoto, H., Taniguchi, T., and Shimohama, S. (2010) Galantamine-induced amyloid-{beta} clearance mediated via stimulation of microglial nicotinic acetylcholine receptors, J. Biol. Chem. 285, 40180-40191. 100. Han, X., Li, S., Peng, Z., Othman, A. M., and Leblanc, R. (2016) Recent development of Cardiac Troponin I detection, ACS Sens. 1, 106-114.


Page 16 of 17

Page 17 of 17 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


For Table of Contents Use Only Title: Towards a Rational Design to Regulate β-Amyloid Fibrillation for Alzheimer’s Disease Treatment Authors: Xu Han and Gefei He


Towards a Rational Design to Regulate Î²-Amyloid Fibrillation for

Recommend Documents