Peptides, Peptidomimetics and Carbohydrate-peptide conjugates as

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Review

Peptides, Peptidomimetics and Carbohydrate-peptide conjugates as Amyloidogenic Aggregation Inhibitors for Alzheimer’s Disease Philip Ryan, Bhautikkumar Patel, Vivek Makwana, Hemant Jadhav, Milton John Kiefel, Andrew Davey, Tristan Reekie, Santosh Rudrawar, and Michael Kassiou ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00185 • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 22, 2018

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ACS Chemical Neuroscience

Peptides, Peptidomimetics and Carbohydrate-peptide conjugates as Amyloidogenic Aggregation Inhibitors for Alzheimer’s Disease Philip Ryan1, Bhautikkumar Patel1, Vivek Makwana1, Hemant R. Jadhav2, Milton Kiefel3, Andrew Davey1,4,5, Tristan A. Reekie6, Santosh Rudrawar1,4,5,6*†, Michael Kassiou6* 1

School of Pharmacy and Pharmacology, Griffith University, Gold Coast, 4222, Australia

Department of Pharmacy, Birla Institute of Technology and Science, Pilani Campus, Pilani-333031, Rajasthan, India 3Institute for Glycomics, Griffith University, Gold Coast 4222, Australia 2

Menzies Health Institute Queensland, Griffith University, Gold Coast 4222, Australia Quality Use of Medicines Network, Griffith University, Gold Coast 4222, Australia 6School of Chemistry, The University of Sydney, NSW 2006, Australia 4 5

KEYWORDS: Beta-amyloid, tau, Alzheimer’s disease, neurodegenerative disease, peptidomimetics, glycopeptides, aggregation inhibitors, GlcNAc, glycosylation. ABSTRACT: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder accounting for 60-80% of dementia cases. For many years, AD causality was attributed to amyloid-β (Aβ) aggregated species. Recently, multiple therapies that target Aβ aggregation have failed in clinical trials, since Aβ aggregation is found in AD and healthy patients. Attention has therefore shifted towards the aggregation of the tau protein as a major driver of AD. Numerous inhibitors of tau-based pathology have recently been developed. Diagnosis of AD has shifted from measuring late stage senile plaques to early-stage biomarkers, amyloid-β and tau monomers and oligomeric assemblies. Synthetic peptides and some derivative structures are being explored for use as theranostic tools as they possess the capacity to both bind the biomarkers, and to inhibit their pathological self-assembly. Several studies have demonstrated that Olinked glycan addition can significantly alter amyloid aggregation kinetics. Furthermore, natural O-glycosylation of amyloid-forming proteins, including amyloid precursor protein (APP), tau and α-synuclein, promote alternative nonamyloidogenic processing pathways. As such, glycopeptides and related peptidomimetics are being investigated within the AD field. Here we review advancements made in the last five years, as well as the arrival of sugar-based derivatives.

INTRODUCTION Alzheimer’s Disease (AD) is a progressive degenerative disease characterized by the destruction of nerve cells and neural connections resulting in impaired memory, cognition and behavior and a loss of independence.1 Worldwide, over 46 million people currently suffer from dementia with over 9.9 million cases arising annually.2 This number is expected to reach 131.5 million by 2050.2 The global societal cost of dementia is estimated at US$818 billion, a value corresponding to 1% of the world’s gross domestic product (GDP).2 The most common form of dementia, AD, currently accounts for 6080% of dementia cases. By 2050, it is expected that a new case of AD will arise every 33 seconds, leading to a tripling of medical costs. These staggering numbers outline

our urgent need to better understand, diagnose and treat AD. There are number of emerging strategies being developed for use as tools to diagnose and treat AD. Recently, Radford and co-workers reviewed non-proteinaceous macromolecules such as carbohydrates, nucleic acids, lipids and metal ions as modulators of amyloid-beta (Aβ) protein aggregation.3 In continuation of our interest in AD drug discovery4-6 we embarked on our task to evaluate and summarize the evidence that exists for amyloidogenic aggregation inhibition for Aβ and tau protein. In this review, we focused on small peptides, peptidomimetics and carbohydrate-peptide conjugates that selectively bind Aβ and tau monomers/oligomers and inhibit their aggregation and toxicity. This comparison is designed to aid the reader considering rational design of 1

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anti-amyloidogenic peptide-based scaffolds. Within the review we have also highlighted the critical interaction between the O-GlcNAc transferase (OGT) enzyme and amyloidogenic proteins; the effects that O-glycosylation has on protein self-assembly; and how rational design of glycopeptides that mimic these interactions may yield effective inhibitors of protein aggregation. This review may provide an excellent starting point for further studies and facilitate efforts to develop new effective anti-AD therapy. The identified number of neurodegenerative diseases associated with aberrant protein aggregation is growing (e.g. α-synuclein,7 prion8). The common structural and pathogenic features of these diverse diseases may provide opportunity for development of a unified treatment approach. Early detection of AD is key in its prevention and treatment. Currently, diagnosis follows the analysis of cognitive function and behavioral patterns, an unreliable practice often leading to misdiagnosis.1 Recently, Hampel and co-wokers reported diagnostic and classificatory performance of AD biomarkers (Aβ, total tau, phosphorylated tau, neurofilament light chain protein, neurogranin, and YKL-40) from cerebrospinal fluid (CSF).9 However, further longitudinal studies are required before we use biomarkers for AD diagnosis. Moreover, analysis of CSF is an expensive and invasive procedure.10 A true diagnosis is only currently available postmortem.1 As such, endeavors to aid ex vivo imaging of the AD biomarkers are being made.11-14 Beyond diagnosis, no true therapy has arisen, though there are four drugs (donepezil, galantamine, rivastigmine, and memantine) currently used in the treatment of AD-related cognitive decline.15 These only modestly effect the downstream neuropsychiatric symptoms, with limited evidence that they prevent, or even alter the course of the underlying dementing process.16 There have been no drugs approved for the treatment, nor prevention of AD in its preclinical nor presymptomatic stages. The chief pathological hallmarks of AD are amyloid plaques and neurofibrillary tangles (NFTs) in an individual’s brain (see Figure 1). The plaques are dense, insoluble deposits of the Aβ peptide, small (39-43 amino acids) truncated amyloid precursor protein (APP) segments arising from γ-secretase- and β-secretasemediated processing. Similarly, NFTs arise from the aggregation of paired helical filaments (PHFs) which, in turn, are fibrous deposits of the intraneuronal, microtubule-associated tau protein. The widely-accepted theories had attributed AD pathology to neurotoxicity from the Aβ plaques, and so they earned a majority of empirical attention. The plaques offered a simple solution for curing AD: their clearance would halt nerve cell death, thus halting dementia. Whilst removal of amyloid deposits from AD mice model brains is restorative,17 amy-

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loid removal from the human AD brain is not, nor has it prevented the continued advance of AD.18 This represents current limitations with translation of AD mouse models. Findings since have suggested that AD neurotoxicity may occur irrespective of the presence of Aβ plaques.19-21

Figure 1. Amyloid- and tau-cascade hypotheses and AD therapeutic approaches.

Today the cascade hypothesis has shifted focus, designating the soluble precursors and their accumulation the source of NFT formation, synaptic damage, neurotoxicity and dementia.22, 23 Thus oligomeric Aβ species have become major therapeutic targets toward treatment of AD. Current strategies toward inhibiting Aβ-derived toxicity include i) targeting primary and secondary nucleation, and the progressive aggregation, of Aβ oligomers; ii) targeting the metal ions that stabilize and accelerate Aβ oligomer formation;24 iii) halting Aβ production by inhibiting β-secretase25 or γ-secretase activities;26 iv) introducing Aβ-specific compounds that promote Aβ clearance and activate autophagy27-29 (see Figure 1). Unlike some of the other approaches, there is seemingly no mechanism-based toxicity associated with the direct binding of Aβ precursors by drug-like molecules nor the inhibition of their aggregation. As such, Aβ-targeted therapy is theoretically free from side-effects. Furthermore, direct binding to the soluble Aβ-precursors per2


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mits their direct quantification with ex vivo imaging techniques and hence may facilitate identification of AD in its earliest-stages.

structures that arise during the process. Peptide-based strategies circumvent these issues and have become prevalent as a result.

Controversially, a temporal inconsistency exists between the appearance and accrual of Aβ and its correlation with the pathological symptoms of AD that goes unexplained by the amyloid cascade hypothesis.30 Additionally, the hypothesis does not account for the discrepancy between the locations of highest amyloid deposition and the locations most affected by NFT formation and consequent neuronal death.31, 32 Furthermore, it has been shown that the levels of Aβ do not appear to discriminate between some AD and healthy brains.33 Recent evidence even suggests Aβ-related clinical decline may only occur with elevated tau pathology. It has been observed that decreasing endogenous tau levels addresses AD symptoms without altering Aβ levels or plaque load in APP tg-mice.34 Also postulated is that toxicity might be inferred through a pas de deux, whereby either Aβ drives tau pathology, tau mediates Aβ toxicity or toxicity is synergistically derived.35 Nevertheless, it is presently emphasized that tau plays a central role in AD pathogenesis, with tau pathology potentially occurring independent of Aβ. As such, the tau protein has been recognized as another key therapeutic objective. Strategies focused on inhibiting tau pathology include: i) addressing tau hyperphosphorylation;36-40 ii) inhibiting tau selfassembly and propagation; iii) microtubule network stabilization;41 and iv) anti-tau immunotherapy.42 General strategies toward treating downstream AD pathology includes the rescuing of the synaptic plasticity deficit and enhancement of neurogenesis (see Figure 1).

Peptides offer biologically active compounds boasting high selectivity due to their ability to establish multiple points of contact with their target. Their highly selective interactions translate to a reduction of side-effects and toxicity. There is a growing interest in peptide science due to an increase in: i) therapeutic targets;49 ii) improvements on delivery strategies;50 iii) manufacture of large peptide libraries; iv) synthetic viability and practicality;51, 52 and v) high-throughput screening processes.53 Though there are drawbacks to peptide-based approaches, notably their lack of physiological stability, these hurdles are progressively being overcome.54 Currently, Aβ- and tau-binding peptide candidates are rated by their ability to inhibit pathogenic self-assembly and toxicity, disrupt pathologically relevant conformational transformations, or to promote alternative, non-toxic aggregation pathways. Their strong, reversible binding also qualifies peptides as model ex vivo imaging agents, and so their potential for diagnostic application is also investigated.

Due to the marked empirical attention garnered by the amyloid cascade hypothesis, the AD therapeutic field is enriched with Aβ-focused strategies. As such, there exists a smorgasbord of small molecules43, 44 probing the Aβ peptide and its derivative structures. Conversely, tau has received significantly less attention, having only recently gained traction as a therapeutic target. While a number of agents targeting tau-associated pathology have been developed,45-48 the consequences of selectively, directly binding pathologically relevant tau species and inhibiting their aggregation in vivo have remained largely unexplored. PEPTIDES TARGETING AD BIOMARKERS Most small-molecules that directly bind Aβ or tau also bind non-selectively to a variety of biomolecules and are consequently burdened with a potential for inflicting numerous side-effects. Increasing selectivity through structural optimization is complicated by the nature of the amyloidogenic aggregation process. Intermediate structural conformations are highly unstable and there are a multitude of currently unknown three-dimensional

AMYLOID-β The 695 residue APP is sequentially cleaved by aspartyl proteases, beta-site APP-cleaving enzyme 1 (BACE1) and γ-secretase respectively, to form the 39-43 residue long Aβ-peptides. Aβ40 constitutes the majority of Aβ present in a normal human brain, however it is the excess Aβ42 produced that predominantly accumulates into amyloids.55 Though Aβ40 is more prevalent, the production rate of both peptides does not differ in the AD brain, and so high levels of Aβ are suggested to be due to an impairment of efflux mechanisms.56 The aggregation of either peptide induces cytotoxicity, however many studies have shown Aβ42 is significantly more predisposed to self-assembly and that its aggregation results in more severe neurotoxic consequences. In fact, even minor increases in the Aβ42/Aβ40 ratio are observed to stabilize oligomeric species and increase neurotoxicity.57 Aβ42 has promptly become the main culprit in AD-associated amyloidogenesis. The natively unfolded Aβ peptide transforms slowly into partially folded β-sheets, conferring a considerable initial lag time prior to aggregation. Secondary nucleation via addition of aggregate seed structures (higher order oligomers) significantly accelerates the process. Partially folded units then associate via hydrophobic interactions and H-bonding to form higher order protofibril structures. These structures are elongated to form longer fibrillar structures, along with the formation of stabilized, bent β-hairpin structures. These fibrils also accelerate the aggregation process, facilitating oligomer 3



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formation on their surfaces. Hence it is observed that primary nucleation guides Aβ oligomerization and that secondary nucleation enriches it (see Figure 2). Numerous strategies toward inhibiting Aβ aggregation and its associated pathology include the blocking of β-sheet formation, prevention of fibrillation, destabilizing oligomeric species and the

4


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ACS Chemical Neuroscience (segment 22-27), and C-terminal (segment 31-40/42) outlined.55

cation of the CHD and its key residues constituents (see Table 1).60 By mimicking the self-recognition interactions occurring naturally, the peptide Ac-QKLVFF-NH2, a truncated analogue of the CHD sequence, was able to bind Aβ42 and inhibit its assembly into fibrils.61 Following this logic, numerous CHD-derived sequences62-66 have been probed for activity against Aβ.

Figure 2. Amyloid-β aggregation pathway. Proteolytic processing of APP yields monomeric Aβ peptides. These then transform structurally into conformations that promote their self-recognition into low-n oligomeric species (these are currently thought to be associated with AD toxicity). Oligomers then elongate into fibrillar structures and eventually characteristic senile plaques.55

promotion of alternative assembly pathways that yield non-toxic structures. The Aβ sequence itself has amphipathic character, with a hydrophilic N-terminal segment and relatively high hydrophobic character moving towards the C-terminal segment (see Figure 3). The predicted localization of Aβ within APP places residues 1-28 within a soluble, extracellular APP domain while the remaining segment, Aβ(29-42), exists within a transmembrane helix of APP. In organic solvent mixtures, Aβ typically adopts αhelical conformation interrupted by a kinking, disordered β-turn.58 In aqueous solution, Aβ40 adopts a random coil structure while, in contrast, Aβ42 rapidly adopts β-sheet structure.59 Studies have suggested β-sheet conformation is initiated by the β-turn formed prior to oligomerization. The oligomerization process is initiated by dimeric interactions spanning residues L17 to F20, a segment described as the Aβ peptides’ central hydrophobic domain (CHD) and the central core of Aβ self-recognition. Attachment occurs at this site, unifying the monomer hairpins in an antiparallel β-sheet conformation. A majority of Aβ monomer- and oligomer-binding inhibitor substrates have been designed to target the CHD, with numerous peptide-based designs employing this segment of the Aβ sequence to mimic the self-recognition interactions occurring naturally. PEPTIDIC INHIBITORS OF Aβ AGGREGATION The first Aβ-sequence derived aggregation inhibitors were generated over 20 years ago following the identifi DAEFRHDSGY10 EVHHQKLVFF20 AEDVGSNKGA30IIGLMVGGVV 40IA 42

Figure 3. Aβ1-42 peptide with N-terminal (segment 1-15), central hydrophobic domain (CHD) (segment 16-20), β-turn region

Almost immediately, the novel ‘β-sheet breaker’ class emerged, composed of peptides inferring their inhibitory activity through high-affinity binding modes coupled with a disruptively low propensity for adopting β-sheet conformation.67 The popular 5-mer iAβ5 (LPFFD) exhibited notable inhibitory activity against Aβ aggregation while conjunctively disaggregating preformed fibrils in vitro.68 This class inflicts its activity by mimicking the CHD interactions but with a low propensity to adopt the β-sheet conformation. Since then, modifications upon the iAβ5 sequence have yielded more peptides83-84 including, most recently, the Ac-LPFFN-NH2 sequence (see Table 1). Molecular dynamics simulations and far UV CD correlating with experimental evidence suggested that the peptide exhibited stronger binding and enhanced activity against Aβ40 aggregation relative to both the N-terminally acetylated iAβ5p (Ac-LPFFD-NH2) and its taurine-substituted derivative (tau-LPFFD-NH2).69, 85-86 The LPFFD sequence has also contributed to the design of nanoparticle-based Aβ-targeted strategies.87-89 Further modification of the CHD segment led to the development of 5-mer RIVFF. The K16R mutation is designed to enhance the electrostatic interaction between amyloid peptides, hence disrupting aggregation, and the L17I mutation probes the influence of a non-polar amino acid on the propensity of self-assembly of the segment. Surface tension measurements, Thioflavin T (ThT) fluorescence assays and Selected Area Electron Diffraction (SAED) measurements showed that RIVFF was selfassembling into β-sheet structures with the potential to reduce surface tension. It was determined that due to its lowered surface tension, it would not be an efficient chaotropic agent and instead may actually promote cytotoxicity, rather than offer any therapeutic effects.70 Jagota and Rajadas developed three short, synthetic, Dpeptides derived from mutations to the CHD of Aβ42 designed to inhibit Aβ aggregation and its associated toxicity.71 ThT and turbidity assays implied peptides kklvffa and klvffarrrra reduced fibrillation, by promoting aggregation into insoluble amorphous globules. Peptide pgklvya seemingly had no effect on fibrillization during ThT studies, however fluorescence images suggested it was triggering an alternative mechanism of assembly of Aβ42, leading to a mixture of aggregates with different morphologies. Although ThT assays are 5



Table 1. Aβ sequence-derived Aβ-targeting peptides

widely

used

Name

Sequence details*

Effects

none69 none70

Strong binding to Aβ40; inhibits aggregation Self-assembling; potentially promotes cytotoxicity without therapeutic effects

none72

Ac-LPFFN-NH2 RIVFF Kklvffa-OH Klvffarrrra-OH Pgklvya-OH KLVFWAK

LK7

Ac-LVFFARK-NH2

none71

66

to

Two inhibited fibrillation, the other altered fibrillogenesis mechanisms; reduced β-sheet content; two prolonged survival of transgenic-AD C. elegans

LK7-HH73

Ac-LVFFARKHH-NH2

OR174 OR2

RGKLVFFGR-OH (OR1) RGKLVFFGR-NH2 (OR2)

RI-OR275

NH2-rGffvlkGr

RI-OR2-TAT76

AcrGffvlkgrrrrqrrkkrGyNH2

Aβ1-6A2V77

DVEFRH

Aβ1-6A2V(D)77

dvefrh

Aβ1-6A2VTAT(D)78-79

grkkrrqrrrggggdvefrh

C-Terminal Fragments (CTFs)80

IIGLMGGVVIA (Aβ31-42) VVIA (Aβ39-42) VVIA-NH2 Ac-VVIA-OH Ac-IGLMVG-NH2 and IGLMVG-NH2

none81

Ac-Aβ32-37-NH2 and Aβ32-37-NH282

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Binds Aβ oligomers and fibrils Dose-dependently inhibited fibrillogenesis; self-assembly promoted cytotoxicity; conjugated onto fibrillogenesis inhibiting nanoparticles Enhanced inhibition against Aβ aggregation relative to LK7; chelated Cu2+, KD = 5.50 µM; arrests generation of reactive oxygen species rescuing PC12 cells Effective aggregation inhibitors; OR2 blocked oligomer formation and inhibited amyloidogenic toxicity Effective aggregation inhibitor, resists proteolytic degradation; binds Aβ42 monomers, KD = 9 µM, and fibrils, KD = 12 µM Increased inhibitory activity relative to RI-OR2; enhanced binding of Aβ42 fibrils, KD = 58-125 nM; permeates cell membranes and BBB

Demonstrates better binding to Aβ40 than synthetic Aβ1-6; inhibited fibril formation and elongation Designed to resist proteolytic degradation Inhibits Aβ oligomerization, fibrillation and Aβ-mediated neurotoxicity; short-term treatment prevents Aβ-deposition in brain; long-term treatment endorses Aβ-deposition in brain Inhibited toxicity and rescued synaptic activity at micromolar concentrations.

Both bind monomers and inhibit dodecamer formation. VVIA-NH2 disaggregates dodecamers and inhibits Aβ-toxicity Aβ32-37-NH2 effectively inhibits both Aβ species’ aggregation at 1:1 molar ratio; fully rescues PC12 cells from Aβ-toxicity

*Upper case denotes L-amino acids, lower case denotes D-amino acids; C-terminal and N-terminal modifications as indicated, where not shown C-terminal and N-terminal peptides are not capped/modified.

investigate amyloid formation, there are several limitations to their application. Thus ThT should be complemented with fixed point assays and other in situ assays independent of ThT fluorescence (e.g. EM, AFM, turbidity measurement, CD, FTIR).90 CD suggested that all Dpeptide inhibitors conferred Aβ42 aggregates possessing reduced β-sheet content. Only peptides kklvffa and pgklvya had any prolonging effect on the survival of transgenic-AD C. elegans. The authors hypothesized that pgklvya and kklvffa therefore work to inhibit oligomeric Aβ42 whilst kklvffarrrra inhibits only smaller (~20 kDa) oligomeric species, instead favoring the formation of the larger (~50 kDa) species that are associated with AD pathology.71 Point mutations to Aβ16-22 yielded the novel ligand, KLVFWAK. It possesses minimal self-aggregation propensity and preferentially binds to Aβ oligomers and fibrils. The E22K mutation was designed to encourage solubility and discourage self-assembly via intermolecular electrostatic repulsion. The peptide exhibited a 10fold stronger binding to aggregates rather than mono-

mers. A competitive binding assay conducted using a panel of antibodies implied that the peptide binds at the C-terminus in Aβ oligomers but elsewhere in fibrils.72 KLVFWAK required higher sensitivity for reliable in vivo or ex vivo detection, and is presented as a potential alternative Aβ aggregate probe to be evolved for enhanced affinity and specificity.72 In 2015, Sun and co-workers developed a novel aggregation inhibiting peptide, LK7 (Ac-LVFFARK-NH2) by incorporating two positively charged residues into the CHD of Aβ42. LK7 demonstrated dose-dependent inhibition of Aβ42 fibrillogenesis but possessed a strong selfassembly characteristic promoting cytotoxicity. To eliminate self-assembly of LK7, the peptide was conjugated onto poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PLGA-NPs) and the resulting LK7@PLGA-NPs complexes inhibited Aβ42 fibrillation significantly.66 Sequence modifications gave rise to LK7-HH (Ac-LVFFARKHHNH2), which exhibited enhanced anti-aggregative effects against Aβ but also chelated Cu2+ with KD = 5.50 μM, enabling it to arrest the Cu2+ or Cu2+-Aβ catalyzed genera6


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tion of reactive oxygen species (ROS) and increase PC12 cell viability.73 Additionally, the HH modification led to a reduction in self-assembly propensity.73 Another derivative, the head-to-tail cyclized LK7 analogue cLK7 had reduced self-assembly propensity besides increased proteolytic stability, reduced cytotoxicity and a 6-fold increase in binding affinity for Aβ40.91 The cLK analogue is shown to stabilize the secondary structure of Aβ40, promote the formation of amorphous aggregates, and inhibit fibrillation and toxicity. LK7 was also conjugated to β-cyclodextrin, significantly improving its solubility suppressing its self-assembly propensity and increasing its binding to Aβ and inhibition potency against aggregation.92 El-Agnaf and co-workers designed a series of Aβ16-20 derived peptides with enhanced water solubility, OR1 (RGKLVFFGR) and OR2 (RGKLVFFGR-NH2).74 It was observed that both were effective fibrillation inhibitors, though only OR2 inhibited oligomer formation and blocked amyloidogenic cytotoxicity.74 The retro-inverso derivative RI-OR2 exhibited high resistance to proteolysis and was therefore assumed stable in vivo.75 Surface Plasmon Resonance (SPR) studies outlined the inhibitors modest affinity, a KD of 9 and 12 µM for Aβ42 monomers and fibrils respectively, values in close agreement with results from kinetic analysis.75 Enhancement of RI-OR2 with a retro-inverted segment of the HIV protein transduction domain,93 “TAT” was expected to promote blood-brain barrier (BBB) permeation.76 ThT assays suggested ‘RI-OR2-TAT’ (Ac-rGffvlkGrrrrqrrkkrGy-NH2) was a more proficient inhibitor than RI-OR2 with AFM and Aβ aggregation time-course experiments confirming inhibition of oligomer formation at a 1:2 molar ratio of RI-OR2-TAT:Aβ42. SPR gave an apparent affinity value for RI-OR2-TAT with KD = 58 - 125 nM with Aβ42 fibrils. Indeed RI-OR2-TAT was capable of permeating cell membranes and crossing the BBB, while RI-OR2 was not. Furthermore, attachment of RI-OR2-TAT to nanoliposomal carrier nanoparticles has produced a multivalent inhibitor of Aβ42 aggregation.94 In 2009 a familial mutation in APP was identified, exhibiting AD promoting properties in homozygous carriers and AD inhibiting properties in heterozygous.77 The mutation describes an alanine-to-valine substitution of residue 673 of APP (A673V), the residue corresponding to residue 2 of Aβ (A2V).77 Additionally, an A2T variant has been shown to protect against AD.95 Recent studies surrounding these mutations have reported conflicting observations surrounding their effects on Aβ peptides’ (Aβ40/Aβ42) aggregation and their associated toxicities.77, 96-98 A short synthetic peptide, Aβ1-6A2V (DVEFRH) has demonstrated better binding to wt Aβ1-40 fibrils than wt Aβ1-6. Furthermore, Aβ1-6A2V inhibits the formation of fibrils and elongation of pre-formed fibrils. The D-

peptide derivative, Aβ1-6A2V(D) (D-DVEFRH), was designed to resist proteolytic degradation and was shown to exhibit enhanced anti-fibrillogenic activity. From there, the D-peptide was linked to an all-D-form of the TAT sequence yielding Aβ1-6A2VTAT(D) (DGRKKRRQRRR-GGGG-DVEFRH), an inhibitor with increased anti-amyloidogenic effects including its inhibition of oligomerization, fibrillation and Aβ-mediated neurotoxicity.78, 79, 99 Preclinical studies showed that while short-term treatment with Aβ1-6A2VTAT(D) prevents Aβ aggregation and deposition in the brain, longer treatment endorses an increase in amyloid burden.79 Additionally, a series of N-terminal fragments of A2T with sequences analogous to the Aβ1-10 stretch were assessed for inhibitory activity against wt Aβ.100 The peptides were able to retard Aβ fibrillization and rescue cells from toxicity within the MTT assay. Sequences derived from fragments of the C-terminus of Aβ42, including Aβ31-42 (IIGLMGGVVIA) and Aβ39-42 (VVIA), have also been identified as potent Aβ aggregation inhibitors.80, 82 Both reportedly inhibited Aβ42 induced toxicity whilst rescuing synaptic activity at micromolar concentrations.80 These ‘C-terminal fragments’ (CTFs) worked to stabilize oligomeric assemblies, altering their size and abundance by forming non-toxic hetero-oligomeric structures.101, 102 The tetrameric Aβ39-42 peptide is shown to bind Aβ42 monomers and smaller oligomeric assemblies102 at several locations, though preferentially at the N-terminus.103 Recently, two of the Aβ39-42 derivatives, VVIA-NH2 and Ac-VVIA, that prescribed different effects on Aβ activity103 were investigated, with results demonstrating unique binding modes for both candidates.81 While both CTFs bind monomeric Aβ42 and inhibit formation of Aβ dodecamers, VVIANH2 displayed an ability to disaggregate preformed dodecamers.81 Neither peptide had any effect on fibril formation, however VVIA-NH2 displayed inhibitory activity against Aβ-derived toxicity while Ac-VVIA did not. Notably, VVIA-NH2 bound exclusively to the C-terminal whilst Ac-VVIA demonstrated disperse binding.81 The VVIA sequence has since been employed in the construction of Aβ-targeting nanoparticles.88 The I32-G37 segment derived Ac-Aβ32-37-NH2 (AcIGLMVG-NH2) sequence exhibited mild activity against Aβ-derived toxicity with some poly-N-methylated analogues exhibiting potent inhibition of fibrillation.104 A recent library screen showed N-terminally free Aβ32-37NH2 (IGLMVG-NH2) was able to fully rescue PC12 cells from Aβ40 induced cytotoxicity at stoichiometric concentrations.82 Furthermore it rescued PC12 cells from Aβ42 aggregation related toxicity. The peptide exhibited 92.5% inhibition of Aβ40 aggregation at a 1:1 molar ratio, and when co-incubated with Aβ42, exhibited complete inhibition. Aβ32-37-NH2 conferred a significant reduction in Aβ42 7



β-sheet content when co-incubated, with particles taking on a spherical morphology observed by Transmission Electron Microscopy (TEM).82 PEPTIDES IDENTIFIED via HIGH THROUGHPUT TECHNIQUES Technologies that can be used to interrogate large numbers of substrates have contributed significantly toward the identification of high-affinity Aβ-binding peptides.105-119 Phage display is one such technique frequently employed as it permits the creation of huge libraries containing up to 1010 different sequence variants in a relatively short time.120 Phage display traditionally only produces peptides composed of natural L-amino acids, but certain techniques, such as mirror-image phage display121 used to produce D-peptides, have contributed toward expanding its applications. Additionally, scanning of peptide combinatorial libraries is employed to produce lead peptide sequences unrestricted by the naturally occurring amino acid residues. The field has been enriched with inhibitor sequences identified via high throughput means, and as such only representative substrates are described in detail here (see Table 2). Nishigaki and co-workers generated two libraries of novel 8-9 residue peptide aptamers with the most promising candidate, P5105 (CGILDPIPW) exhibiting a stronger P84 (CGILDPIPWGGSGGSCGILDPIPW) and P131(GCPCIGIIGGSGGSDCSSDLTPS), which exhibited strong binding for Aβ42 (KD = 536 nM by SPR) than any other candidates previously reported.105 A novel ‘paired peptide library’ was subsequently generated, with candidates composed of linked pairs of Aβ42 binding sequences.122 Peptides binding to monomeric Aβ42 with KD values of 20 nM and 12 nM respectively. ThT assays and Atomic Force Microscopy (AFM) analysis found that both peptides each inhibited fibrillization of Aβ42 (100 µM) when co-incubated (10 µM) over 48 hours. Interestingly, P84 was a more effective inhibitor than P131, also demonstrating inhibitory activity against oligomer formation and was more effective at protecting PC12 cells from toxicity. Currently P84 and P131 are undergoing in vivo investigation on mouse models and are also being used to study Aβ oligomer formation under different conditions.122 A multifunctional heptapeptide XD4 (PIKTLPM) was developed following phage screening.107 The sequence was present in 18 of 30 positive clones that bound with high affinity to Aβ42. XD4 dose-dependently binds Aβ42 and demonstrates significant inhibition of Aβ42-induced cytotoxicity in SH-SY5Y cells at a 1:10 (Aβ42:XD4) molar ratio. XD4 also decreased toxic ROS, NO (1:10 molar ratio) and Ca2+ (1:1 or 1:10 molar ratios) concentrations. Furthermore, XD4 significantly improves memory deficits and Aβ clearance in APP/PS1 transgenic mice.

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By generating and screening a pair of peptide libraries and coupling them with a protein-fragment complementation assay (PCA)130, 131 Mason and co-workers were able to generate a series of Aβ-binding substrates.108 The lead from the first PCA, KAT (Ac-GAKATLM-NH2) was used to create the second library, resulting in an additional two sequences, L2P1 (Ac-FSKATSN-NH2) and L2P2 (Ac-PVKATTA-NH2). The ThT assay conducted against all peptides, including iAβ5 employed as a control, showed all could bind Aβ42 and modestly reduce aggregation levels with activity consistent with a general dose dependency. Additionally, ThT assays with preformed fibrils showed peptides were able to bind Aβ42 and effectively reverse its aggregation.108 All PCAderived peptides exhibited inhibitory activity comparing favorably with iAβ5, however during MTT assays, none of the peptides improved cell viability to any significant amount.108 Retro-inverso sequences were synthesized along with RI TAT fused sequences, and their inhibition and reversal of Aβ42 aggregation was assessed by ThT.109 Dose-dependent decreases in bound ThT were observed in each experiment, suggesting increased binding and βsheet content was reduced, however the peptides were ineffective at decreasing Aβ42 induced toxicity during MTT studies. In an attempt to screen compounds that bind to toxic, oligomeric Aβ42, Mason and co-workers reported the generation and screening of an additional three libraries, again coupled with PCA.110 This time, a disulfidebridged mutant of full-length Aβ42, Aβ42cc (A21C/A30C) was targeted, a peptide forced into the β-hairpin conformation associated with toxic Aβ42 species.132 Each library consisted of candidates targeting specific components of the β-hairpin structure of Aβ42cc, with the top hit of each being studied further. ThT and CD experiments confirmed that the peptides were able to inhibit aggregation of monomeric Aβ42 into larger structures by as much as 80%, additional studies proved that a combined mixture of peptides cys1521 and cys2935a boosted such activity.110 The peptides only modestly reduced Aβ42-induced toxicity however. An index of natural and non-natural amino acids (NNAAIndex)111 constructed by combining two experimentally verified databases of amyloidogenic hexapeptides, Waltz133 and Amylhex,134-135 coupled with a highthroughput quantitative structure-activity relationship (QSAR) screening method was used to identify a series of highly active Aβ-targeted sequences.123 It was hypothesized that self-assembling peptides that form βstructure-rich aggregates might competitively interact with structurally similar Aβ motifs, inhibiting crucial Aβ-Aβ interactions.123 Of the randomly selected series, ThT assays identified peptides CTIYWG, CTLWWG and GTVWWG as strong inhibitors of Aβ42 oligomerization 8


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with GTVWWG exhibiting notably high-affinity binding (KD = 841 ± 78 nM by SPR). Of the selected sequences, only five

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Table 2. Aβ-targeting peptides identified using high-throughput techniques Name P5105105 P84122 P131

Sequence details* CGILDPIPW CGILDPIPWGGSGGSCGI LDPIPW (P84) GCPCIGIIGGSGGSDCSSD LTPS (P131) PIKTLPM

KAT108 L2P1 L2P2 PCA-derived retroinversed and RITAT-fused KAT series109 self-assembling hexapeptides123

7-mers e.g. Ac-GAKATLM-NH2 (KAT) e.g. mltakag-NH2 (KAT-RI) rrrqrrkkrmltakag-NH2 (Tat-KAT-RI) e.g. CTIYWG; CTILWWG; GTVWWG e.g. WWW-OH; PWW-OH; WPW-OH Ac-QKVLLFA-NH2 (cys1521) Ac-AGKATGL-NH2 (cys2935a) Ac-GAKATAN-NH2 (cys2935b) Ac-RWGVVWG-NH2 (cys3642) GLFKVLGSVAKHLLPHV VPVIAEKL-NH2

cys1521110 cys2935a cys2935b cys3642

Caerin 1.8115

D1116

qshyrhispaqv

D3117

rprtrlhthrnr-NH2

D3D3124

rprtrlhthrnrrprtrlhthrnrNH2

RD2125

ptlhthnrrrr-NH2

RD2D3126

ptlhthnrrrrrprtrlhthrnrNH2

DB3127

rpitrlrthqnr-NH2

DB3DB3127

rpitrlrthqnrrpitrlrthqnrNH2

Mosd1118

ysyltsyhmvwr-NH2

AEOP2128

FDYKAEFMPWDT

Carnosine129

βAH

‘Paired peptide aptamers’; binds Aβ42 monomer; KD = 20 nM and 12 nM respectively; inhibits fibrillation and toxicity; P84 inhibits oligomerization

Dose-dependently binds Aβ42; rescues SH-SY5Y cells from toxicity; reduces NO, ROS and Ca2+ concentration; improves memory deficit; improves Aβ clearance

XD4107

tripeptides112

Effects Binds Aβ42 with KD = 536 nM

Bind Aβ42; inhibits aggregation dose-dependently; reverse aggregation of preformed fibrils

Bind Aβ42; enhanced activity relative to precursors (KAT; L2P1; L2P2)

Binds monomeric Aβ42, best KD = 841 ± 78 nM; strong inhibition of aggregation/oligomerization; some candidates inhibited toxicity dose-dependently. Calculated nanomolar affinity binding; strong depolymerization ability.

Target β-hairpin structure of disulfide bridged mutant Aβ42cc; inhibit aggregation of monomeric Aβ42 by as much as 80%; modest reduction of toxicity.

Inhibited Aβ42 fibrillation at molar ratio 1:4 (Aβ42:caerin 1.8); Caerin 1.8(1-13) retained 85% activity. High affinity binding to oligomers/fibrils but not monomers; KD = 0.4 µM; increases concentration of aggregates but alters their size; reduces fibrillation and cytotoxicity; stained Aβ42 plaques in brain tissue (AD patients & AD-tg mice); selective for Aβ42 among other plaques Binds oligomers; encourages their precipitation into amorphous, non-toxic aggregates; modulates toxicity; cell permeable; reduces plaque-load in AD transgenic mice; improves impaired cognitive function; high proteolytic stability Twice as efficient at reducing oligomer concentration as D3; larger size jeopardizes pharmacokinetic properties compared to D3 Scrambled D3; strong binding of Aβ; reduction of monomeric & fibrillar Aβ concentrations; high metabolic stability; high bioavailability More efficient precipitation of Aβ42 oligomers than RD2 but not as bioavailable Binds Aβ42 monomers, KD = 75 µM; inhibits Aβ42 aggregation, EC50 = 6 µM; disaggregates preformed structures. Binds Aβ42 monomers, KD = 1 µM; inhibits Aβ42 aggregation, EC50 = 8 nM; disaggregates preformed structures; inhibits toxicity dose-dependently Minimizes Aβ42 oligomer concentration; encourages precipitation of non-toxic amorphous aggregates; reduces preformed Aβ seeds’ inductive effect; rescues PC12 cells from toxicity; no inhibition of γ-secretase activity. Aβ oligomer mimotope; binds Aβ42 concentration dependently; binds monomeric, oligomeric and fibrillar Aβ; inhibits toxicity; decreases production of proinflammatory cytokines dose-dependently Inhibited organized structure of Aβ42 aggregates; encouraged formation of amorphous globules; dose-dependent inhibition of Aβ42 polymerization

10


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of the thirty-six exhibited a dose-dependent increase in SH-5YSY cell viability during the MTT assay with the others being near inactive. Li and co-workers conducted molecular dynamics simulations on all possible natural tripeptide sequences (203 = 8000 tripeptides) against truncated Aβ peptide derived protofibril structures (6Aβ9-40) with the aim of identifying potential high-affinity Aβ40 fibril binding candidates.112 In silico experiments calculated the lead sequences (WWW, PWW, WPW and WWP) as having estimated binding constants in the nanomolar range. In vitro experiments, including ThT assays and AFM, demonstrated the candidates’ strong binding and depolymerization abilities with DC50 values of half-maximal disassembly in the low micromolar range, yielding extensive reduction of overall concentration of Aβ40 fibril structures.112 The authors hypothesized the inclusion of proline enhances the structural rigidity of the tripeptides, encouraging tighter binding to fibrils while a high tryptophan concentration interrupts key π-stacking interactions between fibrils. A series of antimicrobial peptides isolated from skin secretions of Australian frogs and toads were tested for inhibition of Aβ42 aggregation using ThT.115 Caerin 1.8 from Litoria chloris, exhibited inhibitory activity against fibrillation at a molar ratio of 1:4 (Aβ42:caerin 1.8), and has been proposed to form an [Aβ42/caerin 1.8] H+ complex as observed during positive-ion Matrix-assisted laser desorption/ionization Mass Spectrometry (MALDIMS) with an Aβ42/caerin 1.8 mixture. Molecular dynamics studies outlined the major interactions leading to the formation of this complex as being hydrogen bonding throughout the sequences and hydrophobic interactions at the CHD of Aβ42. These interactions block the addition of a second Aβ42 molecule inhibiting aggregation. Truncated caerin 1.8 (1-13) demonstrated 85% of the inhibitory activity of full caerin 1.8 (1-25), suggesting the most important interactions occur toward caerin 1.8 peptides N-terminal.115 A randomized mirror image phage display was searched for 12-mers with high binding affinity for fibrillary Aβ42. The peptide D1 (QSHYRHISPAQV) was selected and, once synthesized, demonstrated specific binding to Aβ deposits in human brain tissue.116 Surface Plasmon Resonance (SPR) determined D1 binds Aβ42 structures with high affinity (KD = 0.4 µM), interacting with oligomers and fibrils but not monomers.136 Further investigation with FCS, ThT and EM indicated D1 increases the number of Aβ aggregates while significantly reducing their size. At higher concentrations, D1 reduced fibril formation and Aβ cytotoxicity.137 D1 stained Aβ42 plaques in

sections of brain tissue from AD patients,116 and again in vivo in transgenic APP/PS1-model mice, whilst ignoring non-Aβ amyloidogenic deposits and other diffuse plaques containing Aβ40.138 The highly specific D3 (D-RPRTRLHTHRNR) was identified as the dominant sequence of a mirror image phage display with a large randomized 12-mer library.117 D3 was shown to modulate Aβ aggregation and toxicity by binding to oligomers, altering their morphology, and facilitating their precipitation to form non-toxic amorphous aggregates lacking in ‘seeding’ properties.117 FITC-labelled D3 was useful for in vivo and in vitro staining of Aβ42 structures in the brains of transgenic Aβmodel mice.138 Furthermore, in vitro BBB cell culture models validated D3’s cell permeability, and oral treatments of both old and young transgenic AD-model mice with D3 has yielded significant reduction of plaque load and related inflammation, whilst improving impaired cognitive function.139-140 D3 has also demonstrated high proteolytic stability, efficient permeation of the BBB, and high oral bioavailability.141 Further investigation yielded the head-to-tail tandem peptide D3D3 (DRPRTRLHTHRNRRPRTRLHTHRNR), an inhibitor almost twice as efficient at reducing Aβ42 oligomer concentration as D3.124 Both were active in vivo and were seen to display the same mechanism of action, eliminating Aβ oligomers via precipitation to larger, non-toxic, amorphous aggregates.124 It was reported that the larger size of D3D3 jeopardizes its pharmacokinetic properties compared to D3, resulting in a comparatively decreased concentration in examined mice brains.126 Recently, a sequence-scrambled derivative of D3, RD2 (D-PTLHTHNRRRR-NH2), has exhibited strong binding to Aβ, significant reduction in monomeric and fibrillar Aβ concentrations in ThT assays,125 a high stability in mouse plasma and organ homogenates, a long plasma half-life, exposure to the brain, and high oral and subcutaneous bioavailability.142 RD2D3, a head-to-tail tandem peptide derivative, exhibited a more efficient precipitation of Aβ42 oligomers, however RD2’s favorable pharmacokinetic properties were not translated to the larger derivative.126 A library screening on more than 600 D3 derivatives was screened for their ability to bind monomeric Aβ. The most promising candidate, DB3 (D-RPITRLRTHQNRNH2), and a head-to-tail tandem peptide derived from it, DB3DB3 (D- RPITRLRTHQNRRPITRLRTHQNR-NH2) exhibited high binding affinity to Aβ42 monomers, with KD values of 75 µM and 1 µM respectively, determined using biolayer interferometry (BLI).127 DB3 and DB3DB3 inhibited aggregation with EC50 values of 6 µM and 8 11



nM respectively and additionally were able to disaggregate preformed Aβ42 structures with high efficiency. In fact, an EC50 for DB3DB3 could not be obtained due to such low peptide concentrations required. Both DB3 and DB3DB3 eliminated oligomeric Aβ, triggering their precipitation into larger Aβ aggregates. DB3DB3 was able to inhibit Aβ-induced cytotoxicity in a dose-dependent manner but DB3 could not.127 Both are currently subject to further in vitro and in vivo investigation. Similarly, Willbold and co-workers conducted a mirror image phage display to identify peptides targeting Nterminally biotinylated, SEC-derived Aβ42 monomers.118 The most interesting candidate, clone 5.60, was used to produce the corresponding D-enantiomeric Mosd1 (ysyltsyhmvwr-NH2) which was investigated in vitro. Mosd1 was able to selectively bind monomeric Aβ42 in a concentration-dependent manner and minimizes concentrations of toxic Aβ42 oligomers, encouraging their precipitation into larger non-toxic, amorphous aggregates. Additionally, Mosd1 cripples the inductive effects of preformed Aβ42 seeds on monomeric Aβ42, minimizing their conversion into toxic aggregates. Mosd1 also demonstrated an ability to rescue PC12 cells from Aβ42 induced toxicity, and was demonstrated not to have an inhibitory effect on γ–secretase activity.118 Mosd1 is currently subject to further investigation. Liu and co-workers identified a mimotope of the Aβ oligomer from a phase-display library using oligomer specific antibodies.128 AOEP2 (FDYKAEFMPWDT) bound Aβ42 concentration-dependently during ELISA studies and, once spotted onto nitrocellulose membrane, exhibited binding to all three of monomeric, oligomeric and fibrillar Aβ. AOEP2 also lowered Aβ42 β-sheet content and attenuated Aβ42-induced cytotoxicity concentration-dependently. Additionally, AOEP2 significantly decreased production of proinflammatory cytokines TNF-α and IL-6 dose-dependently.128 Carnosine, a naturally occurring imidazole dipeptide, has exhibited inhibitory properties against toxicity and aggregation of multiple amyloidogenic species including Aβ, natural143 and glycated α-crystallin,144 hen egg-white lysozyme (HEWL),145 and prion protein.146 It has exhibited protection of rat brain endothelial cells and rat PC12 cells against toxicity mediated by Aβ peptides147-148 and, when orally administered, reduced interneuronal Aβ accumulation, improved AD-related mitochondrial dysfunction and recovered the activity of hippocampal and cerebral complexes within AD-transgenic mice.149 It attenuates in vitro fibrillogenesis of Aβ42129, 144 with coincubation yielding a dose-dependent arrest of Aβ42 polymerization, encouraging the production of shorter fibrils and globular aggregates, reducing mean fibril length and halting growth of pre-existing fibrils.129 Addi-

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tionally, sub-fibrillar aggregates become disordered, lacking any structural organization. Docking simulations suggest carnosine prevents formation of the intermolecular salt bridge that works to link Aβ42 monomers during fibrillization.129 RATIONALLY DESIGNED PEPTIDES Here we discuss peptide sequences that have arisen as a result of rational design, rather than derivations of the Aβ sequence, though some too are developed using rational design (see Table 3). The design and construction of specific sequences produces naturally occurring amino acid sequences at a slower rate than high-throughput methods, however rational design often produces highly functional compounds, and has led to the production of a larger amount of non-naturally occurring amino acid sequences, peptidomimetics and peptide-conjugate derivatives. Many designs replicate useful characteristics of the Aβ sequence, such as hydrophobicity and propensity to adopt β-sheet conformations to promote selectivity and binding affinity for Aβ (see Table 3). Yuan and co-workers developed a novel β-sheet breaking decapeptide designed to synergistically bind multiple recognition sites along the Aβ40 sequence.150 Specifically, RR (Ac-RYYAAFFAARR-NH2) targeted Aβ’s His13 and His14 residues, its CHD and the “salt bridge” formed between Asp23 and Lys28. SPR studies found RR bound monomeric Aβ40 with affinity roughly 100fold greater than the Soto peptide (KD = 1.10 µM vs 156 µM respectively) with a high degree of specificity. The ThT assay proved RR effectively inhibits Aβ40 aggregation dose-dependently, promoting the formation of disordered, amorphous aggregates as illustrated by TEM. During MTT assays, RR demonstrated an ability to rescue PC12 cells from Aβ40 induced cytotoxicity, increasing cell viability to ~95% at a molar ratio of 1:4 (Aβ40:RR).150 N-terminal modification of RR with a GGH-stretch, designed to encourage chelation to Cu(II) ions,163 yielded the bifunctional GR (GGHRYYAAFFARR-NH2) sequence.151 ThT fluorescence and MTT assays showed GR was equally as proficient as RR at inhibiting Aβ40 aggregation and improving PC12 cell viability respectively.151 ThT assays against an Aβ-Cu(II) complex showed GR was able to achieve inhibition of Cu(II)-induced aggregation of Aβ more effectively than RR. GR also inhibited Aβ-Cu(II) complex-induced toxicity on PC12 cells, improving cell viability by 88%. Faller and co-workers developed two bifunctional peptide inhibitors of toxic Aβ aggregate formation.152 The peptides sequences were composed of copper chelating and β-sheet breaking moieties. Both peptides could inhibit Aβ1-40 aggregation but were unable to disassemble preformed aggregates. Only one inhibitor, Aβ12-20 12


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(VHHGKLVFF), was partially able to rescue MTT cells from Aβ induced cytotoxicity.138 In 2013, Charkravarthy et al., reported that rPK-4, a protein kinase-C inhibitor identified by phage display,164 binds oligomeric Aβ42 with nanomolar affinity.153 A smaller ~5 kDa peptide, denoted ABP-p4-5, was con-

structed and identified to bind Aβ42 at nM levels. The core of the sequence KDKTPKSKSK was suggested to play a key role in the peptide’s selective and strong binding.153 Further studies showed microinjected FITCABP targeted

Table 3. Rationally designed Aβ-targeting peptides Name RR150

GR151

Sequence details* Ac-RYYAAFFAARRNH2

Aβ12-20152

GGHRYYAAFFAARRNH2 VHHGKLVFF-OH

ABP153

KDKTPKSKSK

I1-I14154

Peptides based upon RxTxExKx/xGxFxGxF architecture e.g. RGTWEGKW-NH2 (I10) RGTFEGKF-OH

AIP155 ANA 1156 ANA 2 ANA 3 15M S.A.157

a1 or AP90158

G16159 mTTR160 cG8160

a1 or AP90158

TNPNRRNRTPQMLKR (ANA 1) PLPQML (ANA 2) MTMPTM (ANA 3) Ac-rklmqptrnrrnpntNH2 AcRGEmNlSwMNEYSGWtMnLkMGRNH2 AcPRRYTIAALLSPYSWSNH2 TTR mutant (F87M/L110M) cyclo[-SSPYSYSQTKVVTpPRYTIAKL-]

NF11161

AcRGEmNlSwMNEYSGWtMnLkMGRNH2 NAVRWSLMRPF-NH2

IAPP162

37 residue peptide

Effects Binds monomeric Aβ40, KD = 1.10 µM; highly specific for Aβ40; inhibits Aβ40 aggregation dose-dependently; promotes formation of disordered, amorphous aggregates; rescues PC12 cells from Aβ-derived toxicity As effective as RR at inhibiting aggregation and toxicity; inhibits Cu(II)induced Aβ aggregation and toxicity more proficiently than RR Inhibits Aβ40 aggregation, but unable to disaggregate preformed assemblies; partially able to rescue MTT cells from Aβ-induced toxicity rPK-4 derivative playing key role in selective, strong binding; FITC-ABP targets Aβ42 containing aggregates in hippocampi of AD-model mice and human brain tissue Inhibitors target groove and ridge regions of β-sheet structure; inhibit Aβ fibrillation; depolymerize adult Aβ40 fibrils; rescue neurons from Aβ42induced cell death dose-dependently. Targets low-n Aβ42 oligomers; inhibits aggregation into larger structure; inhibits toxicity ANA 1 and 2 reduce H2O2 production by Aβ42 by almost 50%; ANA 1 inhibited SOD activity, IC50 < 10 nM; ANA 1 is human specific; peptides inhibit toxicity Primarily binds Aβ42 fibrils concentration-dependently; retained ANA 1 activity and potency; enhanced proteolytic stability; encourages formation of amorphous non-toxic aggregates. Adopts α-sheet conformation; binds both Aβ and TTR oligomers; Inhibits both Aβ and TTR aggregation; Inhibits both Aβ- and TTR-derived cytotoxicity; G16 oligomers scavenge Aβ monomers/oligomers; encourage Aβ assembly into globular oligomeric structures; cripple fibrillogenesis; inhibits Aβ-toxicity at substoichiometric concentration Lacks self-assembly propensity, more effective at binding Aβ40/42 than TTR, inhibits Aβ aggregation at 20:1 and 50:1 molar ratio and inhibits Aβ toxicity Reduced self-assembly propensity, inhibits Aβ aggregation at 20:1 and 50:1 molar ratio and inhibits Aβ toxicity, stable against proteolysis Adopts α-sheet conformation; binds both Aβ and TTR oligomers; Inhibits both Aβ and TTR aggregation; Inhibits both Aβ- and TTR-derived cytotoxicity Inhibits Aβ40 aggregation dose-dependently, increasing lag-time; dissolves preformed oligomers and fibrils; reduces toxicity in neuronal cells; binds near CHD and C-terminal hydrophobic region Strong binding to Aβ40, KD = 48.5 ± 4.2 nM; co-incubation inhibits selfassembly and toxicity associated with either peptide species


13



Aβ42 containing aggregates in the hippocampi of transgenic-AD model mice and similar amyloid deposits in cortices and hippocampi of ex-vivo human brain tissue.165 The 8-mer AIP (RGTFEGKF), similarly based off the framework xGxFxGxF, was designed to disrupt Aβ40 sheet-to-sheet packing, but was observed to target low-n Aβ42 oligomers specifically, preventing their aggregation into larger structures and consequently abrogating associated neurotoxicity.155 It was observed that oligomers maintained stability in the presence of excess AIP, suggesting that rather than causing dissociation of Aβ complexes or enhancing aggregation into non-toxic amorphous structures, AIP simply competes with the addition of further monomers or oligomers, preventing the growth of Aβ42 oligomers into larger structures.155 This ‘chaperone’ characteristic classes AIP as a unique Aβ aggregation inhibitor. Data obtained suggests AIP contacts Aβ42 through the Gly33- or Gly37- groove and interferes with the salt bridge, thus inhibiting Aβ-induced neurotoxicity. Further investigation into defining the precise mechanisms by which AIP and associated inhibitors interact with amyloidogenic peptides is still required for rational therapeutic design.155

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S.A. was observed to decrease the concentration of Aβ42 oligomers whilst increasing the concentration of nontoxic amorphous Aβ aggregates.157 Currently the diagnostic and therapeutic potentials of the 15M S.A. peptide are being explored further.157 Buxbaum and co-workers presented evidence that Aβ binds to transport protein transthyretin (TTR) and protects from toxicity in vivo.168 Further study of their binding interaction led to the development of G16 (PRRYTIAALLSPYSWS), a peptide corresponding to residues 102-117 of TTR with a Y116W mutation used for concentration determination.159 ThT aggregation assays and TEM studies implied G16 oligomers were scavenging smaller Aβ monomer and oligomer species and encouraging their assembly into larger soluble globular oligomeric structures. G16 was observed impeding genesis of Aβ fibrils while having minimal effect on those already present. Curiously, MTS assays showed G16 inhibits Aβ toxicity at substoichiometric ratios. This ability of G16, to inhibit toxicity whilst accelerating aggregation, supports the hypothesis that Aβ-derived toxicity may be linked to the actual mechanism by which prefibrillar structures aggregate rather than linked to their mere presence.169 Further modification on the G16 peptides structure has led to the development of cyclic peptidomimetics with similar, accelerant effects on Aβ aggregation.170

In 2010, Martins and co-workers screened two phage display libraries to identify 6-mers and 15-mers that selectively bind soluble human Aβ at a region previously untargeted.156 The region, referred to as Aβ’s ‘SOD-like site’, is where Aβ coordinates with redox-active metal ions to generate H2O2 and encourages oxidative stress and ultimately death of neuronal cells.166 During a superoxide dismutase (RANSOD) assay, two of the three most enriched sequences, 15-mer ANA 1 (TNPNRRNRTPQMLKR) and 6-mer ANA-2 (PLPQML) were able to reduce Aβ42 H2O2 production by almost 50%. ANA 1 was human specific, and inhibited SOD-like activity with an IC50 of less than 10 nM.156 Additionally, MTT and LDH assays revealed an effective protection of cell viability by prevention of Aβ42 derived cytotoxicity, rendering ANA 1 equipotent to clioquinol, a known inhibitor of Aβ’s SOD-like activity that has been shown to modestly improve cognition in AD patients.167

The Murphy group engineered a TTR mutant (F87M/L110M) mTTR, which does not aggregate and is more effective than TTR at binding Aβ40/42 and inhibiting their aggregation and toxic effects.160 Furthermore, mTTR does not bind TTR substrates and so does not interfere with wt TTR functions. They also designed a cyclic cG8 (cyclo[-SPPYSQTKVVTpPRYTIAKL-]) which had decreased self-assembly propensity. Both mTTR and cG8 reduced Aβ40 aggregation at 20:1 and 50:1 (Aβ:peptide) molar ratios however neither disaggregated preformed fibrils. Relative to mTTR, the cG8 peptide was less susceptible to proteolysis and interference from biological materials, however mTTR had a stronger impact on fiber morphology.160

To increase ANA-1’s utility, Martins and co-workers designed novel analogues to enhance metabolic stability and therapeutic potential. To discourage proteolytic degradation, they designed the retro-inverso analogue, 15M S.A. (Ac-rklmqptrnrrnpnt-NH2), and to encourage BBB permeation, the shortened analogue 9M S.A. (Acrklmqptrn-NH2).157 The peptide 15M S.A. interacted with all monomeric, oligomeric and fibrillar Aβ42 species in a concentration-dependent manner with the highest magnitude of binding being observed for fibrils. This observation was attributed to the multiple interaction sites provided by the fibrils repeated monomeric units. 15M

Since as early as 2004, the Daggett group have been interrogating a novel secondary structure element observed in their studies of amyoidogenic proteins.171 These structures, referred to as α-sheets, are proposed to be a defining feature of the misfolded, amyloidogenic Aβ oligomers. In 2014, a series of computationally designed α-sheet peptides were developed and proficiently inhibited both Aβ and TTR aggregation by binding intermediate oligomeric species. The most promising inhibitor, AP90, a 23-mer hairpin peptide possessing alternating Land D- residues, adopted an α-sheet conformation and demonstrated strong inhibition of both TTR and Aβ de14


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rived cytotoxicity during a MTT assay.158 Investigation into the structural components of AP90 revealed the chief contributor to its inhibitory activity was credited to the L/D template. It was determined that the order of residues was inconsequential, and that the L/D template alone augments both inhibitory activity and selectivity between toxic and nontoxic conformers, even when lacking a turn component or high α-strand forming propensity.172 Furthermore, destabilization of the α-sheet structure was shown to reduce the inhibitory characteristics of the peptide.172 The Daggett group are currently using the proposed inhibitory mechanisms associated with αsheet peptides to develop diagnostic assays for binding oligomeric species from solution. Modification of SARS coronavirus E-protein-derived, insulin fibrillation inhibitor NK9 yielded NF11 (NAVRWSLMRPF), a nontoxic peptide capable of inhibiting Aβ40 aggregation dose-dependently while increasing aggregation lag-time.161 Furthermore, NF11 was observed to dissolve preformed Aβ oligomers and fibrils in the ThT assays and reduce amyloid-associated toxicity in neuronal cells. A combination of Nuclear Magnetic Resonance (NMR), Saturation-Transfer Difference (STD)NMR spectroscopy and docking studies suggested that NF11 preferred to bind near the Aβ CHD and C-terminal hydrophobic region (I31-V40).161 Recently, links and similarities between AD and Type 2 diabetes (T2D) have arisen, on both pathophysiological and molecular levels.173-174 T2D is linked with amyloid formation in the pancreas by the 37-residue human islet amyloid polypeptide (IAPP).175 IAPP is a peptide that shares a high degree of sequence similarity and identity with Aβ, notably at locations within their respective βstrand forming regions.174 Co-incubation of IAPP and Aβ40 produces a significant delay, but not prevention, of assembly into their respective oligomeric and fibrillar structures.162 The binding affinity between the peptides has been measured, with an apparent KD = 48.5 ± 4.2 nM. Enhanced effects were observed by the conformationally restricted, nonamyloidogenic, di-N-methylated mimic IAPP-GI.162 Furthermore IAPP, when intraperitoneally injected into AD murine mice models, translocates Aβ out of the brain.176 The IAPP segments involved in binding Aβ were identified,177 and subsequently a new class of inhibitors, IAPP cross-amyloid interaction surface mimics (ISMs) were developed.178 These ISMs are synthetic peptidomimetics, designed to emulate the combination of ‘hot segments’ involved in the binding interaction, with candidates exhibiting nanomolar inhibition against the cytotoxic aggregation of Aβ40 and IAPP.178

TAU Human tau is located in neurons and modulates the stability of axonal microtubules by interacting with tubulin, promoting its assembly into microtubules and stabilizing pre-established microtubule structures. Tau’s modulation of tubulin assembly and stability is regulated by its degree of phosphorylation. Hyperphosphorylation of tau (P-tau) suppresses this activity, detaching it from microtubules, and secondarily promoting tau’s selfassembly into larger aggregate structures. These structures and their formation are tightly associated with taurelated diseases, termed tauopathies, such as Alzheimer’s disease. The patterns by which tau aggregates are distributed within the brain correlate well with ADrelated cognitive decline, and so its monitoring is proposedly key to gauging AD progression.179 P-tau is regularly misconceived as constituting a majority of the PHF structure.180-181 Although tau hyperphosphorylation is proposedly a key step in the tau-related pathogenesis of AD, the notion that it plays a critical role in the actual aggregation of tau is unconvincing. While the PHF garbs a ‘fuzzy coat’ composed of extended phosphorylated N-terminal tau portions, the structural core (and majority of the mass of the PHF) is composed of truncated tau, restricted to the repeat domain units.182183 Consequently, the fuzzy coat is suggested to reflect merely an ancillary phase of sequestration noncritical to the nucleation and assembly of tau.184-185 Supporting this, it has been observed that less than 5% of PHF-tau is phosphorylated,184, 186 that phosphorylation is not required for fibril propagation,187 and that hyperphosphorylation rather uniformly inhibits tau-tau binding.188 Thus, although full-length tau is able to aggregate in vitro, it may be of little consequence regarding pathogenic PHF formation. Rather it is the truncated core sequences of the PHF structure that retain prion-like properties in vitro,189 and are able to catalyze and broadcast the transformation of regular soluble tau into the aggregated and truncated oligomeric species189 (see Figure 4). These seeding effects would be self-limited, however proteolytically stable tau oligomeric species possess the ability to move between neurons, initiating this cascade within healthy neighbors.190-192 The most effective aggregation inhibitors will therefore not only inhibit oligomerization, but also disassemble preformed oligomers. This would assault the seeding process that catalyzes the cascade while concomitantly promoting the release of soluble, monomeric tau that is susceptible to proteolytic removal.

15



Crucially, the PHF core-situated repeat domain fragment is phase-shifted with respect to regular repeats, with key segments being reversely positioned. The PHF core fragment is the subject of structural constraints that

make

the

Figure 4. Tau protein aggregation pathway. Hyperphosphorylation of tau inhibits its interaction with tubulin leading to microtubule destabilization. Detached P-tau is processed and the truncated core sequence is released; truncated tau, consisting of the repeat domain units, nucleates into oligomers – these retain prion-like properties, are proteolytically stable and propagate interneuronally sequestering healthy tau in seeding events; Oligomers elongate into fibrils – the presence of these fibrils correlate better with AD symptoms than amyloid-β plaques.193194

tau-tau binding mode distinguishable from that of tautubulin binding. This feature theoretically permits selectiv ity among tau-targeting substrates via discrimination between the two binding modes. Selectivity among tautargeting substrates is paramount since the impairment of tau-tubulin binding is a major component of the tau cascade hypothesis. Numerous studies have come to identify two key segments in tau sequences, 306VQIVYK311 (termed PHF6) and 275VQIINK280 (termed PHF6*) (see Figure 5), located within the Microtubule-binding domain repeat (MTBR) domain that represent the primary and secondary sites of oligomer nucleation respectively.195-196 It has been demonstrated that short peptides retaining one or both of these self-recognition motifs stack in parallel β-sheets, and form fibrils and ‘steric-zippers’.197-199 The term ‘steric-zipper’ describes the union of a pair of β-sheets associating via the interdigitation of their side chains.199 Self-assembling PHF6-PHF6 interactions are stabilized by a highly ordered H-bonding network, complementary electrostatic interactions, and strong π

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interactions that also aid in establishing the dry interface required to form the steric zipper motif.200 Notably, PHF6* in some cases hosts the ΔK280 mutation, causing a severe form of hereditary neurodegeneration201 likely, in part, due to a subsequent promotion of parallel βsheet arrangement.200 Similarly, a P301S mutation nearby to PHF6 gives rise to disinhibition-like behavior and early NFT-pathology in tg mice.202 It is due to their implications in tau self-assembly that PHF6, and to a lesser extent PHF6*, has become the primary target in tau aggregation inhibitor design. A recent study has found that selected inhibitors targeting PHF6 in tau have a minimal preference for monomers over fibrils, suggesting the site may be obfuscated within the fibril structure, further complicating its targeting.203 Furthermore, the ability of the truncated hexapeptide derivative (VQIVYK) to form fibrils that retain biophysical properties akin to fulllength PHF fibrils199, 204-205 has qualified it as a simplified amyloidogenic tau model.187-188 As such, numerous studies probe their designs’ effects on the peptides aggregation propensity in vitro, with positive results translating to activity against full length tau. Additionally, an Nacetylated and C-amidated PHF6 analogue (AcVQIVYK-NH2) has exhibited pro-aggregative effects on both Aβ40/42, significantly reducing lag phase, increasing fibril growth rate and promoting formation of spherical aggregates while reducing Aβ-derived toxicity in mouse hippocampal cells. To date, only a handful of peptides are known to address tau pathology. One such peptide, davunetide (NAP) (NAPVSIPQ), effectually protects cells from death and microtubule disruption and improves tau deposits within transgenic mice model brains, improving cognitive performance.45 Though NAP decreases P-tau levels, it does not work to inhibit its amyloidogenic aggregation. Although progressive supranuclear palsy (PSP) is linked to tau pathology, davunetide failed to demonstrate efficacy in clinical trials with PSP. Thus, it is not an effective treatment option for PSP.206 The comparatively limited exploration of peptidic tau aggregation inhibitors reflects how little research and clinical development has been conducted. PEPTIDIC INHIBITORS OF TAU AGGREGATION In 2011, the first series of aggregation inhibitor peptides were developed following computational structurebased design. These sequences were designed to target the PHF6 segment and block fibril ends, thus inhibiting the formation of the steric zipper.207 Supported by their results, it was proposed that the steric zipper structure of the VQIVYK segment represented the spine of fibrils formed by tau. Use of Rosetta software afforded Dpeptides, notably D-TLKIVW, that packed tightly along the top of the VQIVYK steric-zipper structure. D16


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TLKIVW delays fibril formation of PHF6 and other tau models, K12 and K19, even in sub-equimolar concentrations. The dissociation constant between D-TLKIVW and VQIVYK fibrils was determined by NMR to be ~2 µM. NMR binding experiments also suggest that the peptide

1: 61: 121: 181: 241: 301: 361: 421:

MAEPRQEFEV SETSDAKSTP HVTQARMVSK TPPSSGEPPK SRLQTAPVPM PGGGSVQIVY THVPGGGNKK DSPQLATLAD

MEDHAGTYGL TAEDVTAPLV SKDGTGSDDK SGDRSGYSSP PDLKNVKSKI KPVDLSKVTS IETHKLTFRE EVSASLAKQG

GDRKDQGGYT DEGAPGKQAA KAKGADGKTK GSPGTPGSRS GSTENLKHQP KCGSLGNIHH NAKAKTDHGA L

inhibitor does not interact with monomers, rather with structured, fibril-like species. Inhibition was not translated to a scrambled sequence confirming their design. Notably the D-peptide

MHQDQEGDTD AQPHTEIPEG IATPRGAAPP RTPSLPTPPT GGGKVQIINK KPGGGQVEVK EIVYKSPVVS

AGLKESPLQT PTEDGSEEPG TTAEEAGIGD TPSLEDEAAG GQKGQANATR IPAKTPPAPK REPKKVAVVR TPPKSPSSAK KLDLSNVQSK CGSKDNIKHV SEKLDFKDRV QSKIGSLDNI GDTSPRHLSN VSSTGSIDMV

Figure 5. Human tau isoform 40 (2N4R). In human brains, tau’s pre-RNA undergoes alternative splicing which produces six molecular isoforms containing either zero (0N), one (1N) or two (2N) amino-terminal, 29-residue inserts with either three (3R) or four (4R) microtubule-binding domain repeats (MTBRs) They differ in terms of structure, relative levels of expression and rates and extents of fibrillization.208-209 Known sites of O-phosphorylation (Ser46, Thr123, Ser199/Ser202, Thr231, Ser235, Ser396, Ser404) and OGlcNAcylation (Ser208, 238, 356, 400 and 409/412/413)

did not interact with Aβ fibril formation, suggesting the inhibitor is not generally applicable to amyloid systems and is specific to tau fibril formation. Inspired by the β-sheet breaker iAβ5p, Segal and coworkers designed novel tau inhibitors from introducing point mutations along the Ac-PHF6-NH2 sequence.210 Six peptides were explored via Pro substitution with all six exhibiting decreased self-assembly propensity and two, P2 (VPIVYK) and P3 (VQPVYK), displaying inhibitory activity against Ac-PHF6-NH2 aggregation, even dissembling preformed aggregates. Unlike P3, the P2 peptide increased lag time of the aggregation process and rescued neuronal PC12 cells from Ac-PHF6-NH2induced cytotoxicity.210 Another series of D-peptides was generated following mirror image phage display against D-PHF6.203 Selected peptides reduced PHF6 fibril formation significantly, especially in a 1:10 ratio, without exhibiting selffibrillization propensity. This activity was translated to inhibitory activity against full length tau fibrillization, with subsequently examined 6-carboxylfuorescein (FAM)-labeled peptides confirming binding to monomers and fibrils with a slight preference for monomers. This was suggested to be due to an increased relative accessibility of the PHF6 motif, perhaps obfuscated within the fibril structure. Thioflavin S (ThS) assays showed the peptides inhibited tau model K19 fibrillation, with two of the most effective, TD28 (ttslqmrlyypp; halfmaximal concentration = 7.9 µM) and TD28rev (ppyylrmqlstt; half-maximal concentration = 96.2 µM), also exhibiting self-aggregation propensity at concentrations >100 µM under the experimental conditions and during DLS experiments. FAM-Labeled peptides also demonstrated cell-permeation against tau expressing N2a neuroblastoma cells, visibly accumulating in the cytoplasm

after 4 days, with TD28-FAM, TD28rev-FAM and APTFAM (aptllrlhslga-FAM) potentially entering the nucleus. Fluorescence-activated cell sorting (FACS) was used to quantify ThS fluorescence of peptide-treated inducible N2aTau cells, exhibiting a decrease in fluorescence for those treated with APT and KNT (kntpqhrklrls), implying inhibition of tau fibril formation within the cells. Using electron diffraction and biochemical studies, Eisenberg et al. found the tau VQIINK stretch formed tightly interdigitated steric-zipper interfaces that appeared more adhesive than the VQIVYK stretch.211 Furthermore, they found tau constructs containing only VQIINK aggregate more rapidly than both wild type tau and constructs containing only VQIVYK. They suggest VQIINK drives tau aggregation and so designed sequence derivatives MINK (DVQMINKKRK) and WINK (DVQWINKKRK) for use as tau aggregation inhibitors. Both peptides were at-least 20-fold less aggregative than full-length tau and both reduced tau aggregation by near 50% at a two-fold molar excess. They were also observed inhibiting the prion-like spread of seeding tau between cells concentration-dependently with IC50 values of 22.6 and 28.9 µM respectively. Re-engineering of MINK produced W-MINK which was shown to block seeding more effectively than MINK with a 20-fold lower IC50 value of 1.1 µM.211 The PHF6 segment remains the prime target among rationally designed, non-natural peptidomimetics targeting tau. Nowick and co-workers incorporated AcPHF6related sequences (VQIVY; QIVYK) into a novel macrocyclic scaffold to generate PHF6-specific aggregation inhibitors.212 The cyclic peptides were designed to target the edges of AcPHF6 β-sheets via distinct H-bonding interactions. Inhibitor 1 (see Figure 6) demonstrated a successful concentration-dependent delay in AcPHF6 17



aggregation during ThS fluorescence assays. The group confirmed the importance of i) hydrophobic residues via substitution with hydrophilic residues; ii) the macrocyclic structure; iii) the recognition motif and preorganized β-sheet structure; iv) facial hydrophobicity and v) the stereochemistry of key residues. X-ray crystallography and NMR spectroscopy were used to confirm a macrocyclic β-sheet structure. Naturally-occurring cyclic disulfide-rich peptides were also investigated for inhibitory activity against PHF6 aggregation.213 SFT-1, a potent inhibitor of trypsin, and kB1, a cyclotide boasting anti-viral and anti-insecticidal activity, were shown to effectively inhibit PHF6 fibril formation during ThS fluorescence assays. Although kB1 was more effective, the SFT-1 structure was used as a template for the development of a series of peptides due to its relative synthetic accessibility. Several of the novel sunflower aggregation inhibitor (SFAI) peptides inhibited PHF6 aggregation more effectively than SFT-1. NMR spectroscopy was used to confirm that the SFAI template adopted a similar conformation to SFT-1. Notably, the removal of the disulfide bridge, and thus destabilization of the inhibitor’s β-sheet conformation, worked to diminish inhibitor efficacy and serum stability. Cyclic peptides have also demonstrated activity against Aβ-inhibition.214-217 Apart from these structures, numerous other peptidomimetics, such as peptoids,218 Nmethylated peptides,104, 219-220 and α,α-disubstituted amino acid bearing,221 and γ-amino acid bearing-peptides,222 have been identified to bind and inhibit amyloids and their pathogenic activity. So too have a number of peptide-conjugate structures bearing moieties such as ferrocene,223-225 extended polyethylene glycol (PEG) chains,226 and glycans.227 APP and tau peptides bearing particular glycosides co-exist with their respective unmodified counter

Figure 6. Macrocycle 1 binds PHF6 and inhibits its aggregation.

parts naturally. It is possible that these glycopeptides interact with the pathogenic peptides under natural conditions, potentially modulating their aggregation and toxicity. The glycopeptide class represents a relatively unex plored avenue toward the generation of novel aggregation inhibitors. PROTEIN O-GLYCOSYLATION AND AD In nature, amyloid forming proteins are subject to multiple post-translational modifications including phosphorylation, proteolysis, acetylation, glycation, and glycosylation. Numerous amyloidogenic proteins are naturally glycosylated, including α-synuclein (α-syn), prion protein (PrP), APP and tau. Tau is modified with O-

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linked N-acetyl-D-glucosamine (O-GlcNAc) at numerous Ser/Thr residues by a single glycosyltransferase, OGlcNAc transferase (OGT), and similarly are refunded by only one enzyme, O-GlcNAc hydrolase (OGA). OGT and numerous kinases competitively modify tau protein at identical or nearby sites, modulating its function and processes. In fact, it has been found that tau phosphorylation is inversely regulated by O-GlcNAc addition (OGlcNAcylation),228 and that downregulation of OGlcNAcylation by either OGT knockout or hexosamine biosynthetic pathway (HBP) inhibition yields increased tau phosphorylation as seen in AD.229 Additionally, inhibition of OGA (and thus O-GlcNAc removal) in both cells230 and transgenic mice231 has been shown to reduce tau-associated neuronal cell death and block cognitive decline. Enhancing OGT activity has been presented as a therapeutic target, with increased O-GlcNAcylation of APP232 promoting non-amyloidogenic processing and of tau enhancing stabilization against aggregation,230 concomitantly slowing neurodegeneration. While the role of O-phosphorylation in tau aggregation has been investigated in detail, the role of O-GlcNAcylation is still being uncovered. Studies have demonstrated that simple O-linked glycoside addition can significantly alter amyloid aggregation mechanisms.233-235 Recently, the addition of O-GlcNAc to recombinant, full-length tau 441 in vitro demonstrated the sugars ability to inhibit tau aggregation.234 Notably, it was found that inhibition was not due to global changes in the monomers structural conformation. This reflected a previous finding, that aggregation and toxicity of an OGlcNAc modified α-synuclein is completely blocked without effects to membrane binding or bending.235 The inhibitory effect on tau aggregation was also translated to a truncated tau fragment (residues 353-408), with data collectively suggesting that the lack of aggregation resulted from enhanced monomer solubility or destabilization of fibrils or soluble aggregates, rather than by altering monomeric conformational properties.234 Designing small sugar-based molecule inhibitors that mimic these phenomena could yield potent AD therapeutic and diagnostic tools. Membrane permeation and proteolytic processing represent significant limitations of this approach, however these may be combatted by the recent advancements surrounding delivery systems such as nanoparticle transport.236 Sugars and sugar-based substrates have exhibited amyloid binding properties in previous studies.237-238 These include cis-glyco-fused benzopyran derivatives,239 sulfated and non-sulfated sugars and glycopolymers,240 proteoglycans and glycosaminoglycans241-242 and glycopeptides. The field has also seen the arrival of amyloidbinding glyco-conjugated nanoparticles, allowing Aβ detection ex vivo by MRI.11 Short sequence glycopeptides 18


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represent an emerging class of compounds that retain the binding affinity provided by peptides, coupled with the aggregation blocking potential of the sugar moiety. Specifically, even just a single sugar unit covalently attached to a peptide side chain can introduce steric interactions along the peptide-linkage, modulating its flexibility. Furthermore, sugar addition can attune the peptides hydrophilicity, H-bonding opportunity and charge balancing, all while providing a unique platform for isosteric substitution. Glycosylation is therefore qualified to attenuate the therapeutic potential of pre-existing peptidic anti-amyloidogenic candidates.

were comprised of two short hydrophobic dipeptide segments branching from the C1 and C6 positions of a central D-glucosylpyranose scaffold via amide and carboxyethyl linker moieties respectively (see Figure 7). This structure was inspired by previous β-sheet breaker pseudopeptides and was proposed to facilitate amyloidogenic inhibition through similar means. ThT fluorescence assays showed the compounds inhibited Aβ1-40 fibrillization at molar ratios as low as 1:0.1 (Aβ1-40:2), significantly increasing initial lag time and reducing equilibrium values. The results indicated that the length of the aminoalkyl link and the sugar’s anomeric configuration had only minor influence over the inhibitory properties of the compounds. NMR studies conducted upon peptidomimetic 2 confirmed the

While not strictly glycopeptides, a novel class of small, water soluble, glycan-based peptidomimetic Aβ1-40 inhibitors were designed in 2011.243 Compounds of this class

RO RO

O

H N

O O

O

O 2 3 4 5 6

R = H R' R = H R' R = H R' R = H R' R = H R'

= Boc n = = Boc n = = Boc n = = Boc n = =H n=

3 C1 3 C1 2 C1 2 C1 2 C1

O

O C1

HO O

R H N

N H

O N H

O

H N

N H

O

HO

C4

HO

N H

O

OH O

O HO

O OH

OH OH

O HO

NH 2 OH O

H 2N O

R

H N O

NH 2

N H HO HO

15

HN

O

O

H N

NHR'

7 R = H R' = Boc n = 3 C1 = β 8 R = Ac R' = Boc n = 2 C1 = α 9 R = Ac R' = Boc n = 3 C1 = β 10 R = Bn R' = Boc n = 2 C1 = α 11 R = Bn R' = Boc n = 3 C1 = β

O

H N

O

HO HO

=β =α =β =α =β

O

O

N H

n

H N

N

O

H N

O

O

HN

O

C1

RO

O

O

N H

O

O

O N H

H H N O

O N H

H N O

O N H

NH 2 O OH

O 12 C1 = α, β R = iPr 13 C1 = α, β R = (CH 2) 4NH 2 14 C1 = β R = (CH 2) 4NH 2

16 C 4 = β R = OH 17 C 4 = α R = NH 2 18 C 4 = α R = NHCOCH 3

Figure 7. Sugar-based inhibitors of protein aggregation top left: inhibitors 2-11; top right: 15 (Th-CT); bottom left: 12-14; bottom right: sugar-modified PHF6 sequences 16-18.

compounds’ preference for aggregated Aβ1-40 species rather than the monomer. These compounds also demonstrated a lack of inhibitory activity against IAPP fibril formation, implying selectivity for the Aβ sequence. Only β-anomers (2 and 4) exhibited activity against the more aggressive Aβ1-42 however, and only at a higher molar ratio, 1:10 (Aβ1-42:compound).244 Novel Capillary Electrophoresis (CE) techniques conducted upon 2 implied interaction with large, insoluble aggregates rather than oligomeric intermediates.244 Removal of the C1 dipeptide’s terminal Boc group (6 and 7) gave mixed results during ThT assays.244 Compound 7 seemed to exhibit enhanced anti-Aβ1-42 activity compared

to 1 at high molar ratios of 1:5 and 1:10 (Aβ1-42:7), however it did not demonstrate activity at a 1:1 ratio. In contrast, compound 6 retained no anti-Aβ1-42 activity, even at a higher ratio of 1:10 (Aβ1-42:6). CE studies indicated that 7 did stabilize monomeric Aβ1-42 however, clearly slowing down the kinetics of oligomerization even at a molar ratio of 1:1 (Aβ1-42:7).244 Per-acetylated derivatives (8 and 9) exhibited no activity, demonstrating the importance of the sugar’s H-bond donating capability. Benzylated derivatives 10 and 11 also lacked inhibitory activity, rather accelerating Aβ1-42 fibril formation likely through increased beneficial, intermolecular π-stacking interactions. CE studies of 11 confirmed this acceleration. 19



The lead structure, 7, was explored further by the development of 5-amino-2-methoxybenzhydrazidesubstituted 12, 13 and 14 (see Figure 7).245 This functionality was incorporated as it has been shown to promote proteolytic stability.246 ThT assays found 12 and 13 were significantly more effective at inhibiting Aβ1-42 aggregation than 7, in particular at lower molar ratios of 1:1 and 1:0.1 (Aβ1-42:compounds). Lysine-containing 13 and 14 consistently outperformed valine-containing 11, yielding an extended aggregation lag-time as well as decreased relative equilibrium values across all examined molar ratios (1:10, 1:1, 1:0.1, Aβ1-42:compounds). TEM experiments confirmed 12 had a greater effect on aggregation than 12. CE outlined a superior effect on aggregation by 14 relative to the mixture 13, stabilizing monomeric and oligomeric species as 7 had previously. STD experiments on 14 implied that the dipeptide groups were directly involved in the interaction with Aβ1-42. In contrast, NMR suggested transient binding of 13 to Aβ1-42 oligomeric species, and SPR suggested the substrates only had a low affinity for Aβ1-42. Substrates 7 and 13 were also shown to protect SH-SY5Y neuroblastoma cells from Aβ1-42-induced cytotoxicity during MTT assays. Additionally, 13 demonstrated significant plasma stability. Trehalose-modification of the LPFFD sequence afforded three glycopeptides with inhibitory activity against Aβ142 aggregation.227 Though the inhibitors lacked potency in PBS buffer during ThT assays, they were relatively effective in NEM buffer at a 5-fold molar excess. Single treatment with any glycopeptide during MTT cytotoxicity assays did not prevent Aβ-induced toxicity, however a second treatment was seen to protect the cells. Further investigation found 15, Ac-LPFFD-Th (Th-CT), (see Figure 7) almost completely blocked Aβ1-42 fibrillogenesis at 20-fold molar excess.247 The glycopeptide was reported to be a more effective inhibitor than both the iAβ5p peptide and trehalose, validating its design. Molecular dynamics simulations suggested the glycopeptide destabilized the protofibril structure, and efficiently bound both ends of the protofibril, hindering the association of successive peptides.248 CD confirmed that the trehalosemodification did not significantly modify the conformational features of the peptides.227 Notably, the trehalosemodification increased the peptides’ stability toward enzymatic degradation. To date, a single study has evaluated the inhibitory activity of glycosylated peptides against tau aggregation.249 A series of sugar-modified, truncated PHF6 peptide derivatives (see Figure 7) were examined for inhibitory activity against the model, amyloidogenic PHF6 peptide (Ac-VQIVYK-NH2). All glycopeptides exhibited a significant decrease in relative self-assembly propensity, with CD suggesting inhibition of the structural transformation from random coil to β-sheet exhibited by sub-

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strates 16 and 17. Curiously the transformations of 18 remained largely unchanged relative to that of PHF6. TEM showed all glycopeptide substrates formed fewer fibrils than PHF6. Upon co-incubation only the OGlcNAc modified peptide seemed effective at inhibiting PHF fibril formation. CONCLUSIONS AND FUTURE PERSPECTIVES Peptide based strategies have been useful in more accurately characterizing Alzheimer’s pathology. Despite their in vitro efficacy, only few have proven effective in one of either cellular or rodent mouse models with fewer examined in clinical trials.141, 206, 250 Despite the benefits associated with peptides, namely high affinity binding and selectivity among biomolecules, their poor serum stability and membrane permeability threatens their application. These obstacles are swiftly overcome via chemical modifications such as D-amino acid incorporation or glycoside-addition. Protein misfolding and self-assembly into amyloids characterize a multitude of diseases and so targeted intervention at stages along the amyloid pathways has remained a primary therapeutic focus. Aberrant Oglycosylation has been causally linked to a number of diseases and is situated at both the Aβ and tau cascades starting points, while upregulation of glycosylation is presented as a potential therapeutic strategy. Sitespecific O-GlcNAc substitution along the amyloidforming sequences significantly hinders pathological aggregation and toxicity. Rationally designed glycoconjugates and glycopeptides often boast modulated flexibility, hydrophilicity and steric bulk and, as described here, are reported to inhibit amyloid formation and pathological progression. It is expected that rationally designed short sequence glycopeptides that selectively bind the proteins, introduce a sugar moiety, and hence artificially reproduce the natural inhibition may yield potent AD therapeutic and diagnostic tools. Glycosylation is therefore qualified to attenuate the therapeutic potential of pre-existing peptidic anti-amyloidogenic candidates. Moreover, the amyloid forming proteins’ common molecular aetiology, their common O-GlcNAc modification and the protective effects supplied via the modification, infers some possibility that a common strategy may be applied toward the development of therapeutic molecular entities that may disrupt pathological protein-protein interactions or stabilize monomeric units. It is crucial that the link between tau and Aβ and other related factors is more clearly discerned if a successful theranostic strategy for AD is to be developed. More work is necessary to elucidate the nature of these neurotoxic species so that their pathological effects may be more rapidly halted or prevented. 20


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AUTHOR INFORMATION Corresponding Author * [email protected]; *[email protected]

Present Addresses †School of Pharmacy and Pharmacology, Griffith University, Gold Coast 4222, Australia.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources Australian Research Council – Discovery Early Career Research Award (ARC DECRA: DE140101632).

Notes The authors declare no competing financial interest.

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Peptides, Peptidomimetics and Carbohydrate-peptide conjugates as Amyloidogenic Aggregation Inhibitors for Alzheimer’s Disease

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Peptides, Peptidomimetics and Carbohydrate‐peptide conjugates as Amyloidogenic Aggregation Inhibitors for Alzheimer’s Disease


Peptides, Peptidomimetics and Carbohydrate-peptide conjugates as

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