Peptides, Peptidomimetics, and CarbohydrateâPeptide Conjugates as

Review Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

pubs.acs.org/chemneuro

Peptides, Peptidomimetics, and Carbohydrate−Peptide Conjugates as Amyloidogenic Aggregation Inhibitors for Alzheimer’s Disease Philip Ryan,† Bhautikkumar Patel,† Vivek Makwana,† Hemant R. Jadhav,‡ Milton Kiefel,§ Andrew Davey,†,∥,⊥ Tristan A. Reekie,# Santosh Rudrawar,*,†,∥,⊥,# and Michael Kassiou*,# †

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 § Institute for Glycomics, Griffith University, Gold Coast 4222, Australia ∥ Menzies Health Institute Queensland, Griffith University, Gold Coast 4222, Australia ⊥ Quality Use of Medicines Network, Griffith University, Gold Coast 4222, Australia # School of Chemistry, The University of Sydney, NSW 2006, Australia

Downloaded via KAOHSIUNG MEDICAL UNIV on July 17, 2018 at 08:29:39 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

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 toward 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 both to bind the biomarkers and to inhibit their pathological self-assembly. Several studies have demonstrated that Olinked glycoside addition can significantly alter amyloid aggregation kinetics. Furthermore, natural O-glycosylation of amyloid-forming proteins, including amyloid precursor protein (APP), tau, and α-synuclein, promotes 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 5 years, as well as the arrival of sugar-based derivatives. KEYWORDS: Beta-amyloid, tau, Alzheimer’s disease, neurodegenerative disease, peptidomimetics, glycopeptides, aggregation inhibitors, GlcNAc, glycosylation

■

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 60−80% of dementia cases. By 2050, it is expected that a new case of AD will arise every 33 s, 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 nonproteinaceous 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 discovery,4−6 we © XXXX American Chemical Society

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 antiamyloidogenic 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 Received: April 17, 2018 Accepted: May 21, 2018 Published: May 21, 2018 A

DOI: 10.1021/acschemneuro.8b00185 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Review

ACS Chemical Neuroscience

precursor protein (APP) segments arising from γ-secretaseand β-secretase-mediated processing. Similarly, NFTs arise from the aggregation of paired helical filaments (PHFs) which, in turn, are fibrous deposits of the intraneuronal, microtubuleassociated 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. While removal of amyloid deposits from AD mice model brains is restorative,17 amyloid 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 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 permits their direct quantification with ex vivo imaging techniques and hence may facilitate identification of AD in its earliest stages. 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 self-assembly 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).

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-workers 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 or prevention of AD in its preclinical or 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

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


Review


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 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

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 tauassociated 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 nonselectively 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 structures that arise during the process. Peptide-based strategies circumvent these issues and have become prevalent as a result. 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) highthroughput 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, to disrupt pathologically relevant conformational transformations, or to promote alternative, nontoxic 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.

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

toward inhibiting Aβ aggregation and its associated pathology include the blocking of β-sheet formation, prevention of fibrillation, destabilizing oligomeric species and the promotion of alternative assembly pathways that yield nontoxic structures. The Aβ sequence itself has amphipathic character, with a hydrophilic N-terminal segment and relatively high hydrophobic character moving toward the C-terminal segment (see Figure 3). The predicted localization of Aβ within APP places

■

AMYLOID-β The 695 residue APP is sequentially cleaved by aspartyl proteases, beta-site APP-cleaving enzyme 1 (BACE1) and γsecretase respectively, to form the 39−43 residue long Aβpeptides. 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

Figure 3. Aβ1−42 peptide with N-terminal (segment 1−15), central hydrophobic domain (CHD) (segment 16−20), β-turn region (segment 22−27), and C-terminal (segment 31−40/42) outlined.55

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 α-helical 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. AttachC


Review

ACS Chemical Neuroscience Table 1. Aβ Sequence-Derived Aβ-Targeting Peptides name 69

none none70 none71

sequence detailsa

none72 LK766

Ac-LPFFN-NH2 RIVFF kklvffa-OH kklvffarrrra-OH pgklvya-OH KLVFWAK Ac-LVFFARK-NH2

LK7-HH73

Ac-LVFFARKHH-NH2

OR174 OR2 RI-OR275

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

RI-OR2-TAT76

Ac-rGffvlkgrrrrqrrkkrGy-NH2

Aβ1−6A2V77 Aβ1−6A2V(D)77 Aβ1−6A2VTAT(D)78,79

DVEFRH dvefrh grkkrrqrrrggggdvefrh

C-terminal fragments (CTFs)80

IIGLMVGGVVIA (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

effects strong binding to Aβ40; inhibits aggregation self-assembling; potentially promotes cytotoxicity without therapeutic effects two inhibited fibrillation, the other altered fibrillogenesis mechanisms; reduced β-sheet content; two prolonged survival of transgenic-AD C. elegans 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

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

sequence has also contributed to the design of nanoparticlebased 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 nonpolar 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 self-assembling 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 widely used to 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 D-peptide 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

ment occurs at this site, unifying the monomer hairpins in an antiparallel β-sheet conformation. A majority of Aβ monomerand 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 selfrecognition interactions occurring naturally.

■

PEPTIDIC INHIBITORS OF Aβ AGGREGATION The first Aβ-sequence derived aggregation inhibitors were generated over 20 years ago following the identification 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. 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 Nterminally acetylated iAβ5p (Ac-LPFFD-NH2) and its taurinesubstituted derivative (tau-LPFFD-NH2).69,85,86 The LPFFD D


Review


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 preformed fibrils. The Dpeptide derivative, Aβ1−6A2V(D) (D-DVEFRH), was designed to resist proteolytic degradation and was shown to exhibit enhanced antifibrillogenic activity. From there, the D-peptide was linked to an all-D-form of the TAT sequence yielding Aβ1−6A2VTAT(D) (D-GRKKRRQRRR-GGGG-DVEFRH), an inhibitor with increased antiamyloidogenic 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 Aβ 31−42 (IIGLMGGVVIA) and Aβ39−42 (VVIA), have also been identified as potent Aβ aggregation inhibitors.80,82 Both reportedly inhibited Aβ42 induced toxicity while rescuing synaptic activity at micromolar concentrations.80 These “C-terminal fragments” (CTFs) worked to stabilize oligomeric assemblies, altering their size and abundance by forming nontoxic 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, VVIA-NH2 displayed an ability to disaggregate preformed dodecamers.81 Neither peptide had any effect on fibril formation, however VVIANH2 displayed inhibitory activity against Aβ-derived toxicity while Ac-VVIA did not. Notably, VVIA-NH2 bound exclusively to the C-terminal while 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 (Ac-IGLMVG-NH2) sequence exhibited mild activity against Aβderived toxicity with some poly-N-methylated analogues exhibiting potent inhibition of fibrillation.104 A recent library screen showed N-terminally free Aβ32−37-NH2 (IGLMVGNH2) 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

that pgklvya and kklvffa therefore work to inhibit oligomeric Aβ42 while 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 10-fold stronger binding to aggregates rather than monomers. 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 self-assembly 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@PLGANPs complexes inhibited Aβ42 fibrillation significantly.66 Sequence modifications gave rise to LK7-HH (Ac-LVFFARKHH-NH2), which exhibited enhanced antiaggregative effects against Aβ but also chelated Cu2+ with KD = 5.50 μM, enabling it to arrest the Cu2+ or Cu2+-Aβ catalyzed generation 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 selfassembly 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 E


Review

ACS Chemical Neuroscience Table 2. Aβ-Targeting Peptides Identified Using High-Throughput Techniques sequence detailsa

name 105

P5105 P84122 P131 XD4107

CGILDPIPW CGILDPIPWGGSGGSCGILDPIPW (P84) GCPCIGIIGGSGGSDCSSDLTPS (P131) PIKTLPM

KAT108 L2P1 L2P2 PCA-derived retro-inversed and RI-TATfused KAT series109 self-assembling hexapeptides123 tripeptides112 cys1521110 cys2935a cys2935b cys3642 Caerin 1.8115

7-mers, e.g., Ac-GAKATLM-NH2 (KAT)

e.g., WWW-OH; PWW-OH; WPW-OH Ac-QKVLLFA-NH2 (cys1521) Ac-AGKATGL-NH2 (cys2935a) Ac-GAKATAN-NH2 (cys2935b) Ac-RWGVVWG-NH2 (cys3642) GLFKVLGSVAKHLLPHVVPVIAEKL-NH2

D1116

qshyrhispaqv

D3117

rprtrlhthrnr-NH2

D3D3124

rprtrlhthrnrrprtrlhthrnr-NH2

RD2

125

dose-dependently binds Aβ42; rescues SH-SY5Y cells from toxicity; reduces NO, ROS and Ca2+ concentration; improves memory deficit; improves Aβ clearance bind Aβ42; inhibits aggregation dose-dependently; reverse aggregation of preformed fibrils

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

e.g., CTIYWG; CTILWWG; GTVWWG

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 Aβ42 cm3; inhibit aggregation of monomeric Aβ42 by as much as 80%; modest reduction of toxicity.

RD2D3126 DB3127

ptlhthnrrrrrprtrlhthrnr-NH2 rpitrlrthqnr-NH2

DB3DB3127

rpitrlrthqnrrpitrlrthqnr-NH2

Mosd1118

ysyltsyhmvwr-NH2

AEOP2128

FDYKAEFMPWDT

carnosine

binds Aβ42 with KD = 536 nM “Paired peptide aptamers”; binds Aβ42 monomer; KD = 20 and 12 nM, respectively; inhibits fibrillation and toxicity; P84 inhibits oligomerization

e.g., mltakag-NH2 (KAT-RI); rrrqrrkkrmltakag-NH2 (Tat-KAT-RI)

ptlhthnrrrr-NH2

129

effects

βAH

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, nontoxic 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 and 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 nontoxic amorphous aggregates; reduces preformed Aβ seeds’ inductive effect; rescues PC12 cells from toxicity; no inhibition of γsecretase 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

a

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

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 and 12 nM, respectively. ThT assays and atomic force microscopy (AFM) analysis found that both peptides each inhibited fibrillization of Aβ42 (100 μM) when coincubated (10 μM) over 48 h. 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.

peptide exhibited 92.5% inhibition of Aβ40 aggregation at a 1:1 molar ratio, and when coincubated with Aβ42, exhibited complete inhibition. Aβ32−37-NH2 conferred a significant reduction in Aβ42 β-sheet content when coincubated, 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 Lamino 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 F


Review


GTVWWG exhibiting notably high-affinity binding (KD = 841 ± 78 nM by SPR). Of the selected sequences, only 5 of the 36 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 highaffinity 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 (MALDI-MS) 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, while 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 nontoxic amorphous aggregates lacking in “seeding” properties.117 FITC-labeled D3 was useful for in vivo and in vitro staining of Aβ42 structures in the brains of transgenic Aβ-model 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

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. 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 (AcFSKATSN-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 PCA-derived 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 β-sheet 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 disulfide-bridged 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 high-throughput quantitative structure−activity relationship (QSAR) screening method was used to identify a series of highly active Aβtargeted sequences.123 It was hypothesized that self-assembling peptides that form β-structure-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 with G


Review

ACS Chemical Neuroscience Table 3. Rationally Designed Aβ-Targeting Peptides name RR

150

sequence detailsa Ac-RYYAAFFAARR-NH2

GR151

GGHRYYAAFFAARR-NH2 152

Aβ12−20

VHHGKLVFF-OH

ABP153

KDKTPKSKSK

I1−I14154

peptides based upon RxTxExKx/ xGxFxGxF architecture, e.g., RGTWEGKW-NH2 (I10) RGTFEGKF-OH TNPNRRNRTPQMLKR (ANA 1) PLPQML (ANA 2) MTMPTM (ANA 3) Ac-rklmqptrnrrnpnt-NH2

AIP155 ANA 1156 ANA 2 ANA 3 15M S.A.157 a1 or AP90158 G16159

Ac-RGEmNlSwMNEYSGWtMnLkMGRNH2 Ac-PRRYTIAALLSPYSWS-NH2

mTTR160

TTR mutant (F87M/L110M)

cG8

160

a1 or AP90158

cyclo[-SSPYSYSQTKVV-TpPRYTIAKL-]

NF11161

Ac-RGEmNlSwMNEYSGWtMnLkMGRNH2 NAVRWSLMRPF-NH2

IAPP162

37 residue peptide

effects binds monomeric Aβ40, KD = 1.10 μM; highly specific for Aβ40; inhibits Aβ40 aggregation dosedependently; 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 Aβ42-induced 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 nontoxic 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; coincubation inhibits self-assembly and toxicity associated with either peptide species

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

binding affinity to Aβ42 monomers, with KD values of 75 and 1 μM, respectively, determined using biolayer interferometry (BLI).127 DB3 and DB3DB3 inhibited aggregation with EC50 values of 6 μM and 8 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 N-terminally 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 nontoxic, 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.

with D3 has yielded significant reduction of plaque load and related inflammation, while 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 (D-RPRTRLHTHRNRRPRvTRLHTHRNR), 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, nontoxic, 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 (DPTLHTHNRRRR-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-RPITRLRTHQNR-NH2), and a head-to-tail tandem peptide derived from it, DB3DB3 (DRPITRLRTHQNRRPITRLRTHQNR-NH2) exhibited high H


Review


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 Aβ 1−40 aggregation but were unable to disassemble preformed aggregates. Only one inhibitor, Aβ12−20 (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 constructed 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 FITC-ABP targeted 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-tosheet 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 nontoxic 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 Gly33or 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 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 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, 15 M S.A. (Ac-

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 Aβ 42 polymerization, encouraging the production of shorter fibrils and globular aggregates, reducing mean fibril length and halting growth of pre-existing fibrils.129 Additionally, subfibrillar 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 (AcRYYAAFFAARR-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 100-fold greater than the Soto peptide (KD = 1.10 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 NTerminal 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 I


Review


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 α-sheet 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 dosedependently while increasing aggregation lag-time.161 Furthermore, NF11 was observed to dissolve preformed Aβ oligomers and fibrils in the ThT assays and reduce amyloidassociated 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 β-strand 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

rklmqptrnrrnpnt-NH2), and to encourage BBB permeation, the shortened analogue 9M S.A. (Ac-rklmqptrn-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 S.A. was observed to decrease the concentration of Aβ42 oligomers while 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 while 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 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 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 L- and D-residues, adopted an α-sheet conformation and demonstrated strong inhibition of both TTR and Aβ derived 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

■

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 self-assembly 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 AD-related 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 J


Review


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. 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 tau−tau binding mode distinguishable from that of tau−tubulin binding. This feature theoretically permits selectivity among tau-targeting substrates via discrimination between the two binding modes. Selectivity among tau-targeting 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 275 VQIINK280 (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 βsheets, 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 Selfassembling PHF6−PHF6 interactions are stabilized by a highly ordered H-bonding network, complementary electrostatic interactions, and strong π 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 β-sheet 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 full-length 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 N-

PHF) is composed of truncated tau, restricted to the repeat domain units.182,183 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 fulllength 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-

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.193,194

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

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, and Ser404) and O-GlcNAcylation (Ser208, 238, 356, 400, and 409/ 412/413). K


Review


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 APT-FAM (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 2-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 WMINK 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 AcPHF6-related 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)

acetylated and C-amidated PHF6 analogue (Ac-VQIVYKNH2) 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 structure-based 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 D-peptides, notably D-TLKIVW, that packed tightly along the top of the VQIVYK steric-zipper structure. D-TLKIVW delays fibril formation of PHF6 and other tau models, K12 and K19, even in subequimolar 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 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 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 co-workers 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-PHF6NH2 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 self-fibrillization 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; half-maximal concentration = 7.9 μM) and TD28rev (ppyylrmqlstt; half-maximal concentration = 96.2

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

demonstrated a successful concentration-dependent delay in AcPHF6 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 antiviral and anti-insecticidal activity, were shown to effectively inhibit PHF6 fibril formation during ThS fluorescence assays. Although kB1 was more effective, the SFT1 structure was used as a template for the development of a series of peptides due to its relative synthetic accessibility. L


Review


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

tion),228 and that downregulation of O-GlcNAcylation 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 OGlcNAc 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 OGlcNAcylation of APP232 promoting nonamyloidogenic 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 O-GlcNAc 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-

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 Aβinhibition.214−217 Apart from these structures, numerous other peptidomimetics, such as peptoids,218 N-methylated peptides,104,219,220 and α,α-disubstituted amino acid bearing221 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 coexist with their respective unmodified counter 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 unexplored 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-linked N-acetyl-D-glucosamine (OGlcNAc) at numerous Ser/Thr residues by a single glycosyltransferase, O-GlcNAc 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 (O-GlcNAcylaM


Review

ACS Chemical Neuroscience glyco-fused benzopyran derivatives,239 sulfated and nonsulfated sugars and glycopolymers,240 proteoglycans and glycosaminoglycans241,242 and glycopeptides. The field has also seen the arrival of amyloid-binding glyco-conjugated nanoparticles, allowing Aβ detection ex vivo by MRI.11 Short sequence glycopeptides 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 peptide’s hydrophilicity, Hbonding opportunity, and charge balancing, all while providing a unique platform for isosteric substitution. Glycosylation is therefore qualified to attenuate the therapeutic potential of preexisting peptidic antiamyloidogenic candidates. While not strictly glycopeptides, a novel class of small, watersoluble, glycan-based peptidomimetic Aβ1−40 inhibitors were designed in 2011.243 Compounds of this class 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 βsheet 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 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 βanomers (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 Peracetylated 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. The lead structure, 7, was explored further by the development of 5-amino-2-methoxybenzhydrazide-substituted 12−14 (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). Lysinecontaining 13 and 14 consistently outperformed their valinecontaining analogue (not depicted) 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 13 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 Aβ1−42 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-LPFFDTh (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 trehalose-modification did not significantly modify the conformational features of the peptides.227 Notably, the trehalose-modification 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 selfassembly propensity, with CD suggesting inhibition of the structural transformation from random coil to β-sheet exhibited by substrates 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 coincubation only the O-GlcNAc 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. N


Review


(5) Chua, S., Kassiou, M., and Ittner, L. (2014) The translocator protein as drug target in Alzheimer’s disease. Expert Rev. Neurother. 14, 439−448. (6) Giboureau, N., Aumann, K., Beinat, C., and Kassiou, M. (2010) Development of vesicular acetylcholine transporter ligands: Molecular probes for Alzheimer’s disease. Curr. Bioact. Compd. 6, 129−155. (7) Ruzza, P., Gazziero, M., De Marchi, M., Massalongo, G., Marchiani, A., Autiero, I., Tessari, I., Bubacco, L., and Calderan, A. (2015) Peptides as modulators of α-synuclein aggregation. Protein Pept. Lett. 22, 354−361. (8) Srivastava, A., Sharma, S., Sadanandan, S., Gupta, S., Singh, J., Gupta, S., Haridas, V., and Kundu, B. (2017) Modulation of prion polymerization and toxicity by rationally designed peptidomimetics. Biochem. J. 474, 123−147. (9) Hampel, H., Toschi, N., Baldacci, F., Zetterberg, H., Blennow, K., Kilimann, I., Teipel, S. J., Cavedo, E., Melo dos Santos, A., Epelbaum, S., Lamari, F., Genthon, R., Dubois, B., Floris, R., Garaci, F., and Lista, S. (2018) Alzheimer’s disease biomarker-guided diagnostic workflow using the added value of six combined cerebrospinal fluid candidates: Aβ1−42, total-tau, phosphorylated-tau, NFL, neurogranin, and YKL-40. Alzheimer's Dementia 14, 492−501. (10) Toledo, J. B., Xie, S. X., Trojanowski, J. Q., and Shaw, L. M. (2013) Longitudinal change in CSF Tau and Aβ biomarkers for up to 48 months in ADNI. Acta Neuropathol. 126, 659−670. (11) Kouyoumdjian, H., Zhu, D. C., El-Dakdouki, M. H., Lorenz, K., Chen, J., Li, W., and Huang, X. (2013) Glyconanoparticle aided detection of β-amyloid by magnetic resonance imaging and attenuation of β-amyloid induced cytotoxicity. ACS Chem. Neurosci. 4, 575−584. (12) Shah, M., and Catafau, A. M. (2014) Molecular imaging insights into neurodegeneration: focus on tau PET radiotracers. J. Nucl. Med. 55, 871−874. (13) Cagnin, A., Kassiou, M., Meikle, S. R., and Banati, R. B. (2007) Positron emission tomography imaging of neuroinflammation. Neurotherapeutics 4, 443−452. (14) Kassiou, M., Banati, R., Holsinger, D. R. M., and Meikle, S. (2009) Challenges in molecular imaging of Parkinson’s disease: a brief overview. Brain Res. Bull. 78, 105−108. (15) Reisberg, B., Doody, R., Stöffler, A., Schmitt, F., Ferris, S., and Mobius, H. J. (2003) Memantine in moderate-to-severe Alzheimer’s disease. N. Engl. J. Med. 348, 1333−1341. (16) Schneider, L. S., Dagerman, K. S., Higgins, J. P., and McShane, R. (2011) Lack of evidence for the efficacy of memantine in mild Alzheimer disease. Arch. Neurol. 68, 991−998. (17) Buttini, M., Masliah, E., Barbour, R., Grajeda, H., Motter, R., Johnson-Wood, K., Khan, K., Seubert, P., Freedman, S., Schenk, D., and Games, D. (2005) β-amyloid immunotherapy prevents synaptic degeneration in a mouse model of Alzheimer’s disease. J. Neurosci. 25, 9096−9101. (18) Boche, D., Denham, N., Holmes, C., and Nicoll, J. A. (2010) Neuropathology after active Aβ42 immunotherapy: implications for Alzheimer’s disease pathogenesis. Acta Neuropathol. 120, 369−384. (19) Harrington, C. R., Louwagie, J., Rossau, R., Vanmechelen, E., Perry, R. H., Perry, E. K., Xuereb, J. H., Roth, M., and Wischik, C. M. (1994) Influence of apolipoprotein E genotype on senile dementia of the Alzheimer and Lewy body types: significance for etiological theories of Alzheimer’s disease. Am. J. Pathol. 145, 1472−1484. (20) Holmes, C., Boche, D., Wilkinson, D., Yadegarfar, G., Hopkins, V., Bayer, A., Jones, R. W., Bullock, R., Love, S., Neal, J. W., Zotova, E., and Nicoll, J. A. R. (2008) Long-term effects of Aβ42 immunisation in Alzheimer’s disease: follow-up of a randomised, placebo-controlled phase I trial. Lancet 372, 216−223. (21) Chui, D.-H., Tanahashi, H., Ozawa, K., Ikeda, S., Checler, F., Ueda, O., Suzuki, H., Araki, W., Inoue, H., Shirotani, K., Takahashi, K., Gallyas, F., and Tabira, T. (1999) Transgenic mice with Alzheimer presenilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation. Nat. Med. 5, 560−564.

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 O-glycosylation 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. Site-specific O-GlcNAc substitution along the amyloid-forming 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 preexisting peptidic antiamyloidogenic candidates. Moreover, the amyloid forming proteins’ common molecular etiology, their common O-GlcNAc modification, and the protective effects supplied via the modification infer 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.

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: s.rudrawar@griffith.edu.au. ORCID

Michael Kassiou: 0000-0002-6655-0529 Author Contributions

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

Australian Research Council − Discovery Early Career Research Award (ARC DECRA: DE140101632). Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Alzheimer’s Association (2018) Alzheimer’s disease facts and figures. Alzheimer’s Dementia 14, 367−429. (2) World Alzheimer Report 2016, Alzheimer’s Disease International. (3) Stewart, K. L., and Radford, S. E. (2017) Amyloid plaques beyond Aβ: a survey of the diverse modulators of amyloid aggregation. Biophys. Rev. 9, 405−419. (4) Chaney, A., Bauer, M., Bochicchio, D., Smigova, A., Kassiou, M., Davies, K., Williams, S., and Boutin, H. (2018) Longitudinal investigation of neuroinflammation and metabolite profiles in the APPswe×PS1Δe9 transgenic mouse model of Alzheimer’s disease. J. Neurochem. 144, 318−335. O


Review

ACS Chemical Neuroscience (22) Ferreira, S. T., and Klein, W. L. (2011) The Aβ oligomer hypothesis for synapse failure and memory loss in Alzheimer’s disease. Neurobiol. Learn. Mem. 96, 529−543. (23) Hardy, J. A., and Higgins, G. A. (1992) Alzheimer’s disease: the amyloid cascade hypothesis. Science 256, 184−185. (24) Matlack, K. E. S., Tardiff, D. F., Narayan, P., Hamamichi, S., Caldwell, K. A., Caldwell, G. A., and Lindquist, S. (2014) Clioquinol promotes the degradation of metal-dependent amyloid-β (Aβ) oligomers to restore endocytosis and ameliorate Aβ toxicity. Proc. Natl. Acad. Sci. U. S. A. 111, 4013−4018. (25) Yan, R., and Vassar, R. (2014) Targeting the β secretase BACE1 for Alzheimer’s disease therapy. Lancet Neurol. 13, 319−329. (26) Wolfe, M. S. (2012) γ-Secretase as a target for Alzheimer’s disease. Adv. Pharmacol. 64, 127−153. (27) Deane, R., Sagare, A., and Zlokovic, B. V. (2008) The role of the cell surface LRP and soluble LRP in blood-brain barrier Aβ clearance in Alzheimer’s disease. Curr. Pharm. Des. 14, 1601−1605. (28) Liu, B., Frost, J. L., Sun, J., Fu, H., Grimes, S., Blackburn, P., and Lemere, C. A. (2013) MER5101, a novel Aβ1−15:DT conjugate vaccine, generates a robust anti-Aβ antibody response and attenuates Aβ pathology and cognitive deficits in APPswe/PS1ΔE9 transgenic mice. J. Neurosci. 33, 7027−7037. (29) Panza, F., Solfrizzi, V., Imbimbo, B. P., Tortelli, R., Santamato, A., and Logroscino, G. (2014) Amyloid-based immunotherapy for Alzheimer’s disease in the time of prevention trials: the way forward. Expert Rev. Clin. Immunol. 10, 405−419. (30) Lesne, S. E., Sherman, M. A., Grant, M., Kuskowski, M., Schneider, J. A., Bennett, D. A., and Ashe, K. H. (2013) Brain amyloid-β oligomers in ageing and Alzheimer’s disease. Brain 136, 1383−1398. (31) Giannakopoulos, P., Herrmann, F. R., Bussière, T., Bouras, C., Kövari, E., Perl, D. P., Morrison, J. H., Gold, G., and Hof, P. R. (2003) Tangle and neuron numbers, but not amyloid load, predict cognitive status in Alzheimer’s disease. Neurology 60, 1495−1500. (32) Gold, G., Kövari, E., Corte, G., Herrmann, F. R., Canuto, A., Bussière, T., Hof, P. R., Bouras, C., and Giannakopoulos, P. (2001) Clinical validity of Aβ-protein deposition staging in brain aging and Alzheimer disease. J. Neuropathol. Exp. Neurol. 60, 946−952. (33) Corrada, M. M., Berlau, D. J., and Kawas, C. H. (2012) A population-based clinicopathological study in the oldest-old: the 90+ study. Curr. Alzheimer Res. 9, 709−717. (34) Roberson, E. D., Scearce-Levie, K., Palop, J. J., Yan, F., Cheng, I. H., Wu, T., Gerstein, H., Yu, G. Q., and Mucke, L. (2007) Reducing endogenous tau ameliorates amyloid β-induced deficits in an Alzheimer’s disease mouse model. Science 316, 750−754. (35) Ittner, L. M., and Götz, J. (2011) Amyloid-β and tau − a toxic pas de deux in Alzheimer’s disease. Nat. Rev. Neurosci. 12, 67−72. (36) van Eersel, J., Ke, Y. D., Liu, X., Delerue, F., Kril, J. J., Götz, J., and Ittner, L. M. (2010) Sodium selenate mitigates tau pathology, neurodegeneration, and functional deficits in Alzheimer’s disease models. Proc. Natl. Acad. Sci. U. S. A. 107, 13888−13893. (37) Corcoran, N. M., Martin, D., Hutter-Paier, B., Windisch, M., Nguyen, T., Nheu, L., Sundstrom, L. E., Costello, A. J., and Hovens, C. M. (2010) Sodium selenate specifically activates PP2A phosphatase, dephosphorylates tau and reverses memory deficits in an Alzheimer’s disease model. J. Clin. Neurosci. 17, 1025−1033. (38) Lovestone, S., Boada, M., Dubois, B., Hüll, M., Rinne, J. O., Huppertz, H. J., Calero, M., Andrés, M. V., Gómez-Carrillo, B., León, T., and del Ser, T. (2015) A phase II trial of tideglusib in Alzheimer’s disease. J. Alzheimer's Dis. 45, 75−88. (39) de la Torre, A. V., Junyent, F., Folch, J., Pelegri, C., Vilaplana, J., Auladell, C., Beas-Zarate, C., Pallàs, M., Verdaguer, E., and Camins, A. (2012) GSK3β inhibition is involved in the neuroprotective effects of cyclin-dependent kinase inhibitors in neurons. Pharmacol. Res. 65, 66−73. (40) Resnick, L., and Fennell, M. (2004) Targeting JNK3 for the treatment of neurodegenerative disorders. Drug Discovery Today 9, 932−939.

(41) Butler, D., Bendiske, J., Michaelis, M. L., Karanian, D. A., and Bahr, B. A. (2007) Microtubule-stabilizing agent prevents protein accumulation-induced loss of synaptic markers. Eur. J. Pharmacol. 562, 20−27. (42) Pedersen, J. T., and Sigurdsson, E. M. (2015) Tau immunotherapy for Alzheimer’s disease. Trends Mol. Med. 21, 394− 402. (43) Doig, A. J., and Derreumaux, P. (2015) Inhibition of protein aggregation and amyloid formation by small molecules. Curr. Opin. Struct. Biol. 30, 50−56. (44) Belluti, F., Rampa, A., Gobbi, S., and Bisi, A. (2013) Smallmolecule inhibitors/modulators of amyloid-β peptide aggregation and toxicity for the treatment of Alzheimer’s disease: a patent review (2010 - 2012). Expert Opin. Ther. Pat. 23, 581−596. (45) Shiryaev, N., Jouroukhin, Y., Giladi, E., Polyzoidou, E., Grigoriadis, N. C., Rosenmann, H., and Gozes, I. (2009) NAP protects memory, increases soluble tau and reduces tau hyperphosphorylation in a tauopathy model. Neurobiol. Dis. 34, 381−388. (46) Baddeley, T. C., McCaffrey, J., Storey, J. M., Cheung, J. K., Melis, V., Horsley, D., Harrington, C. R., and Wischik, C. M. (2015) Complex disposition of methylthioninium redox forms determines efficacy in tau aggregation inhibitor therapy for Alzheimer’s disease. J. Pharmacol. Exp. Ther. 352, 110−118. (47) Chua, S. W., Cornejo, A., van Eersel, J., Stevens, C. H., Vaca, I., Cueto, M., Kassiou, M., Gladbach, A., Macmillan, A., Lewis, L., Whan, R., and Ittner, L. M. (2017) The polyphenol altenusin inhibits in vitro fibrillization of tau and reduces induced tau pathology in primary neurons. ACS Chem. Neurosci. 8, 743−751. (48) Moir, M., Chua, S. W., Reekie, T., Martin, A. D., Ittner, A., Ittner, L. M., and Kassiou, M. (2017) Ring-opened aminothienopyridazines as novel tau aggregation inhibitors. MedChemComm 8, 1275−1282. (49) Sierra, J. M., Fusté, E., Rabanal, F., Vinuesa, T., and Viñas, M. (2017) An overview of antimicrobial peptides and the latest advances in their development. Expert Opin. Biol. Ther. 17, 663−676. (50) Patel, M. M., and Patel, B. M. (2017) Crossing the blood-brain barrier: recent advances in drug delivery to the brain. CNS Drugs 31, 109−133. (51) Behrendt, R., White, P., and Offer, J. (2016) Advances in Fmoc solid-phase peptide synthesis. J. Pept. Sci. 22, 4−27. (52) Ryan, P., Koh, A. H. W., Lohning, A. E., and Rudrawar, S. (2017) Solid-phase O-glycosylation with a glucosamine derivative for the synthesis of a glycopeptide. Aust. J. Chem. 70, 1151−1157. (53) Prashanth, J. R., Hasaballah, N., and Vetter, I. (2017) Pharmacological screening technologies for venom peptide discovery. Neuropharmacology 127, 4−19. (54) Bruno, B. J., Miller, G. D., and Lim, C. S. (2013) Basics and recent advances in peptide and protein drug delivery. Ther. Delivery 4, 1443−1467. (55) Selkoe, D. J. (2001) Alzheimer’s disease: genes, proteins, and therapy. Physiol. Rev. 81, 741−766. (56) Mawuenyega, K. G., Sigurdson, W., Ovod, V., Munsell, L., Kasten, T., Morris, J. C., Yarasheski, K. E., and Bateman, R. J. (2010) Decreased clearance of CNS β-amyloid in Alzheimer’s disease. Science 330, 1774. (57) Kuperstein, I., Broersen, K., Benilova, I., Rozenski, J., Jonckheere, W., Debulpaep, M., Vandersteen, A., Segers-Nolten, I., Van Der Werf, K., Subramaniam, V., Braeken, D., Callewaert, G., Bartic, C., D’Hooge, R., Martins, I. C., Rousseau, F., Schymkowitz, J., and De Strooper, B. (2010) Neurotoxicity of Alzheimer’s disease Aβ peptides is induced by small changes in the Aβ42 to Aβ40 ratio. EMBO J. 29, 3408−3420. (58) Serpell, L. C. (2000) Alzheimer’s amyloid fibrils: structure and assembly. Biochim. Biophys. Acta, Mol. Basis Dis. 1502, 16−30. (59) Sgourakis, N. G., Yan, Y., McCallum, S. A., Wang, C., and Garcia, A. E. (2007) The Alzheimer’s peptides Aβ40 and 42 adopt distinct conformations in water: a combined MD/NMR study. J. Mol. Biol. 368, 1448−1457. P


Review

ACS Chemical Neuroscience (60) Tjernberg, L. O., Näslund, J., Lindqvist, F., Johansson, J., Karlström, A. R., Thyberg, J., Terenius, L., and Nordstedt, C. (1996) Arrest of β-amyloid fibril formation by a pentapeptide ligand. J. Biol. Chem. 271, 8545−8548. (61) Tjernberg, L. O., Lilliehöök, C., Callaway, D. J. E., Näslund, J., Hahne, S., Thyberg, J., Terenius, L., and Nordstedt, C. (1997) Controlling amyloid β-peptide fibril formation with protease-stable ligands. J. Biol. Chem. 272, 12601−12605. (62) Pallitto, M. M., Ghanta, J., Heinzelman, P., Kiessling, L. L., and Murphy, R. M. (1999) Recognition sequence design for peptidyl modulators of β-amyloid aggregation and toxicity. Biochemistry 38, 3570−3578. (63) Gibson, T. J., and Murphy, R. M. (2005) Design of peptidyl compounds that affect β-amyloid aggregation: importance of surface tension and context. Biochemistry 44, 8898−8907. (64) 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 (ΔAla)-containing peptides inhibit amyloid-β (Aβ) peptide aggregation. Biopolymers 91, 456−465. (65) Chalifour, R. J., McLaughlin, R. W., Lavoie, L., Morissette, C., Tremblay, N., Boulé, M., Sarazin, P., Stéa, D., Lacombe, D., Tremblay, P., and Gervais, F. (2003) Stereoselective interactions of peptide inhibitors with the β-amyloid peptide. J. Biol. Chem. 278, 34874− 34881. (66) Xiong, N., Dong, X. Y., Zheng, J., Liu, F. F., and Sun, Y. (2015) Design of LVFFARK and LVFFARK-functionalized nanoparticles for inhibiting amyloid β-protein fibrillation and cytotoxicity. ACS Appl. Mater. Interfaces 7, 5650−5662. (67) Soto, C., Kindy, M. S., Baumann, M., and Frangione, B. (1996) Inhibition of Alzheimer’s amyloidosis by peptides that prevent β-sheet conformation. Biochem. Biophys. Res. Commun. 226, 672−680. (68) Soto, C., Sigurdsson, E. M., Morelli, L., Kumar, R. A., Castano, E. M., and Frangione, B. (1998) β-Sheet breaker peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: implications for Alzheimer’s therapy. Nat. Med. 4, 822−826. (69) Minicozzi, V., Chiaraluce, R., Consalvi, V., Giordano, C., Narcisi, C., Punzi, P., Rossi, G. C., and Morante, S. (2014) Computational and experimental studies on β-sheet breakers targeting Aβ1−40 fibrils. J. Biol. Chem. 289, 11242−11252. (70) Ramaswamy, K., Kumaraswamy, P., Sethuraman, S., and Krishnan, U. M. (2014) Self-assembly characteristics of a structural analogue of Tjernberg peptide. RSC Adv. 4, 16517−16523. (71) Jagota, S., and Rajadas, J. (2013) Synthesis of D-amino acid peptides and their effect on beta-amyloid aggregation and toxicity in transgenic Caenorhabditis elegans. Med. Chem. Res. 22, 3991−4000. (72) Aoraha, E., Candreva, J., and Kim, J. R. (2015) Engineering of a peptide probe for β-amyloid aggregates. Mol. BioSyst. 11, 2281−2289. (73) Zhang, H., Zhang, C., Dong, X. Y., Zheng, J., and Sun, Y. (2018) Design of nonapeptide LVFFARKHH: a bifunctional agent against Cu2+ -mediated amyloid β-protein aggregation and cytotoxicity. J. Mol. Recognit. 31, e2697. (74) Austen, B. M., Paleologou, K. E., Ali, S. A., Qureshi, M. M., Allsop, D., and El-Agnaf, O. M. (2008) Designing peptide inhibitors for oligomerization and toxicity of Alzheimer’s β-amyloid peptide. Biochemistry 47, 1984−1992. (75) Taylor, M., Moore, S., Mayes, J., Parkin, E., Beeg, M., Canovi, M., Gobbi, M., Mann, D. M., and Allsop, D. (2010) Development of a proteolytically stable retro-inverso peptide inhibitor of β-amyloid oligomerization as a potential novel treatment for Alzheimer’s disease. Biochemistry 49, 3261−3272. (76) Parthsarathy, V., McClean, P. L., Hölscher, C., Taylor, M., Tinker, C., Jones, G., Kolosov, O., Salvati, E., Gregori, M., Masserini, M., and Allsop, D. (2013) A novel retro-inverso peptide inhibitor reduces amyloid deposition, oxidation and inflammation and stimulates neurogenesis in the APPswe/PS1ΔE9 mouse model of Alzheimer’s disease. PLoS One 8, e54769. (77) Di Fede, G., Catania, M., Morbin, M., Rossi, G., Suardi, S., Mazzoleni, G., Merlin, M., Giovagnoli, A. R., Prioni, S., Erbetta, A.,

Falcone, C., Gobbi, M., Colombo, L., Bastone, A., Beeg, M., Manzoni, C., Francescucci, B., Spagnoli, A., Cantù, L., Del Favero, E., Levy, E., Salmona, M., and Tagliavini, F. (2009) A recessive mutation in the APP gene with dominant-negative effect on amyloidogenesis. Science 323, 1473−1477. (78) Cimini, S., Sclip, A., Mancini, S., Colombo, L., Messa, M., Cagnotto, A., Di Fede, G., Tagliavini, F., Salmona, M., and Borsello, T. (2016) The cell-permeable Aβ1−6A2VTAT(D) peptide reverts synaptopathy induced by Aβ1−42wt. Neurobiol. Dis. 89, 101−111. (79) Di Fede, G., Catania, M., Maderna, E., Morbin, M., Moda, F., Colombo, L., Rossi, A., Cagnotto, A., Virgilio, T., Palamara, L., Ruggerone, M., Giaccone, G., Campagnani, I., Costanza, M., Pedotti, R., Salvalaglio, M., Salmona, M., and Tagliavini, F. (2016) Tackling amyloidogenesis in Alzheimer’s disease with A2V variants of Amyloidβ. Sci. Rep. 6, 20949. (80) 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 Aβ42 oligomers and protect neurons against Aβ42-induced neurotoxicity. Proc. Natl. Acad. Sci. U. S. A. 105, 14175−14180. (81) Zheng, X., Wu, C., Liu, D., Li, H., Bitan, G., Shea, J. E., and Bowers, M. T. (2016) Mechanism of C-Terminal Fragments of Amyloid β-Protein as Aβ Inhibitors: Do C-Terminal Interactions Play a Key Role in Their Inhibitory Activity? J. Phys. Chem. B 120, 1615− 1623. (82) Bansal, S., Maurya, I. K., Yadav, N., Thota, C. K., Kumar, V., Tikoo, K., Chauhan, V. S., and Jain, R. (2016) C-Terminal fragment, Aβ32−37, analogues protect against Aβ aggregation-induced toxicity. ACS Chem. Neurosci. 7, 615−623. (83) Loureiro, J. A., Crespo, R., Borner, H., Martins, P. M., Rocha, F. A., Coelho, M., Pereira, M. C., and Rocha, S. (2014) Fluorinated betasheet breaker peptides. J. Mater. Chem. B 2, 2259−2264. (84) Datki, Z., Papp, R., Zádori, D., Soós, K., Fülöp, L., Juhász, A., Laskay, G., Hetényi, C., Mihalik, E., and Zarándi, M. (2004) In vitro model of neurotoxicity of Aβ 1−42 and neuroprotection by a pentapeptide: irreversible events during the first hour. Neurobiol. Dis. 17, 507−515. (85) Giordano, C., Masi, A., Pizzini, A., Sansone, A., Consalvi, V., Chiaraluce, R., and Lucente, G. (2009) Synthesis and activity of fibrillogenesis peptide inhibitors related to the 17−21 β-amyloid sequence. Eur. J. Med. Chem. 44, 179−189. (86) Giordano, C., Sansone, A., Masi, A., Masci, A., Mosca, L., Chiaraluce, R., Pasquo, A., and Consalvi, V. (2012) Inhibition of amyloid peptide fragment Aβ25−35 fibrillogenesis and toxicity by Nterminal β-amino acid-containing esapeptides: Is taurine moiety essential for in vivo effects? Chem. Biol. Drug Des. 79, 30−37. (87) Gao, N., Sun, H., Dong, K., Ren, J., and Qu, X. (2015) Goldnanoparticle-based multifunctional amyloid-β inhibitor against Alzheimer’s disease. Chem. - Eur. J. 21, 829−835. (88) Xiong, N., Zhao, Y., Dong, X., Zheng, J., and Sun, Y. (2017) Design of a molecular hybrid of dual peptide inhibitors coupled on AuNPs for enhanced inhibition of amyloid β-protein aggregation and cytotoxicity. Small 13, 1601666. (89) Ruff, J., Hüwel, S., Kogan, M. J., Simon, U., and Galla, H. J. (2017) The effects of gold nanoparticles functionalized with βamyloid specific peptides on an in vitro model of blood-brain barrier. Nanomedicine 13, 1645−1652. (90) Gade Malmos, K., Blancas-Mejia, L. M., Weber, B., Buchner, J., Ramirez-Alvarado, M., Naiki, H., and Otzen, D. (2017) ThT 101: a primer on the use of thioflavin T to investigate amyloid formation. Amyloid 24, 1−16. (91) Ma, S., Zhang, H., Dong, X., Yu, L., Zheng, J., and Sun, Y. (2018) Head-to-tail cyclization of a heptapeptide eliminates its cytotoxicity and significantly increases its inhibition effect on amyloid β-protein fibrillation and cytotoxicity. Front. Chem. Sci. Eng. 12, 283− 295. (92) Zhang, H., Dong, X., Liu, F., Zheng, J., and Sun, Y. (2018) AcLVFFARK-NH2 conjugation to β-cyclodextrin exhibits significantly Q


Review

ACS Chemical Neuroscience enhanced performance on inhibiting amyloid β-protein fibrillogenesis and cytotoxicity. Biophys. Chem. 235, 40−47. (93) Green, M., and Loewenstein, P. M. (1988) Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell 55, 1179−1188. (94) Sherer, M., Fullwood, N. J., Taylor, M., and Allsop, D. (2015) A preliminary electron microscopic investigation into the interaction between Aβ1−42 peptide and a novel nanoliposome-coupled retroinverso peptide inhibitor, developed as a potential treatment for Alzheimer’s disease. J. Phys.: Conf. Ser. 644, 012040. (95) Jonsson, T., Atwal, J. K., Steinberg, S., Snaedal, J., Jonsson, P. V., Bjornsson, S., Stefansson, H., Sulem, P., Gudbjartsson, D., Maloney, J., Hoyte, K., Gustafson, A., Liu, Y., Lu, Y., Bhangale, T., Graham, R. R., Huttenlocher, J., Bjornsdottir, G., Andreassen, O. A., Jönsson, E. G., Palotie, A., Behrens, T. W., Magnusson, O. T., Kong, A., Thorsteinsdottir, U., Watts, R. J., and Stefansson, K. (2012) A mutation in APP protects against Alzheimer’s disease and age-related cognitive decline. Nature 488, 96−99. (96) Maloney, J. A., Bainbridge, T., Gustafson, A., Zhang, S., Kyauk, R., Steiner, P., van der Brug, M., Liu, Y., Ernst, J. A., Watts, R. J., and Atwal, J. K. (2014) Molecular mechanisms of Alzheimer disease protection by the A673T allele of amyloid precursor protein. J. Biol. Chem. 289, 30990−31000. (97) Benilova, I., Gallardo, R., Ungureanu, A. A., Castillo Cano, V., Snellinx, A., Ramakers, M., Bartic, C., Rousseau, F., Schymkowitz, J., and De Strooper, B. (2014) The Alzheimer disease protective mutation A2T modulates kinetic and thermodynamic properties of amyloid-β (Aβ) aggregation. J. Biol. Chem. 289, 30977−30989. (98) Meinhardt, J., Tartaglia, G. G., Pawar, A., Christopeit, T., Hortschansky, P., Schroeckh, V., Dobson, C. M., Vendruscolo, M., and Fandrich, M. (2007) Similarities in the thermodynamics and kinetics of aggregation of disease-related Aβ(1−40) peptides. Protein Sci. 16, 1214−1222. (99) Diomede, L., Romeo, M., Cagnotto, A., Rossi, A., Beeg, M., Stravalaci, M., Tagliavini, F., Di Fede, G., Gobbi, M., and Salmona, M. (2016) The new beta amyloid-derived peptide Aβ1−6A2V-TAT(D) prevents Aβ oligomer formation and protects transgenic C. elegans from Aβ toxicity. Neurobiol. Dis. 88, 75−84. (100) Lin, T. W., Chang, C. F., Chang, Y. J., Liao, Y. H., Yu, H. M., and Chen, Y. R. (2017) Alzheimer’s amyloid-β A2T variant and its Nterminal peptides inhibit amyloid-β fibrillization and rescue the induced cytotoxicity. PLoS One 12, e0174561. (101) Li, H., Monien, B. H., Lomakin, A., Zemel, R., Fradinger, E. A., Tan, M., Spring, S. M., Urbanc, B., Xie, C. W., Benedek, G. B., and Bitan, G. (2010) Mechanistic investigation of the inhibition of Aβ42 assembly and neurotoxicity by Aβ42 C-terminal fragments. Biochemistry 49, 6358−6364. (102) Gessel, M. M., Wu, C., Li, H., Bitan, G., Shea, J. E., and Bowers, M. T. (2012) Aβ(39−42) modulates Aβ oligomerization but not fibril formation. Biochemistry 51, 108−117. (103) Li, H., Du, Z., Lopes, D. H., Fradinger, E. A., Wang, C., and Bitan, G. (2011) C-terminal tetrapeptides inhibit Aβ42-induced neurotoxicity primarily through specific interaction at the N-terminus of Aβ42. J. Med. Chem. 54, 8451−8460. (104) Pratim Bose, P., Chatterjee, U., Nerelius, C., Govender, T., Norström, T., Gogoll, A., Sandegren, A., Göthelid, E., Johansson, J., and Arvidsson, P. I. (2009) Poly-N-methylated amyloid β-peptide (Aβ) C-terminal fragments reduce Aβ toxicity in vitro and in Drosophila melanogaster. J. Med. Chem. 52, 8002−8009. (105) Tsuji-Ueno, S., Komatsu, M., Iguchi, K., Takahashi, M., Yoshino, S., Suzuki, M., Nemoto, N., and Nishigaki, K. (2011) Novel high-affinity Aβ-binding peptides identified by an advanced in vitro evolution, progressive library method. Protein Pept. Lett. 18, 642−650. (106) Schwarzman, A. L., Tsiper, M., Gregori, L., Goldgaber, D., Frakowiak, J., Mazur-Kolecka, B., Taraskina, A., Pchelina, S., and Vitek, M. P. (2005) Selection of peptides binding to the amyloid βprotein reveals potential inhibitors of amyloid formation. Amyloid 12, 199−209.

(107) Xue, D., Zhao, M., Wang, Y. J., Wang, L., Yang, Y., Wang, S. W., Zhang, R., Zhao, Y., and Liu, R. T. (2012) A multifunctional peptide rescues memory deficits in Alzheimer’s disease transgenic mice by inhibiting Aβ42-induced cytotoxicity and increasing microglial phagocytosis. Neurobiol. Dis. 46, 701−709. (108) Acerra, N., Kad, N. M., and Mason, J. M. (2013) Combining intracellular selection with protein-fragment complementation to derive Aβ interacting peptides. Protein Eng., Des. Sel. 26, 463−470. (109) Acerra, N., Kad, N. M., Griffith, D. A., Ott, S., Crowther, D. C., and Mason, J. M. (2014) Retro-inversal of intracellular selected βamyloid-interacting peptides: implications for a novel Alzheimer’s disease treatment. Biochemistry 53, 2101−2111. (110) Acerra, N., Kad, N. M., Cheruvara, H., and Mason, J. M. (2014) Intracellular selection of peptide inhibitors that target disulphide-bridged Aβ42 oligomers. Protein Sci. 23, 1262−1274. (111) Liang, G., Liu, Y., Shi, B., Zhao, J., and Zheng, J. (2013) An index for characterization of natural and non-natural amino acids for peptidomimetics. PLoS One 8, e67844. (112) Viet, M. H., Siposova, K., Bednarikova, Z., Antosova, A., Nguyen, T. T., Gazova, Z., and Li, M. S. (2015) In silico and in vitro study of binding affinity of tripeptides to amyloid β fibrils: Implications for Alzheimer’s disease. J. Phys. Chem. B 119, 5145− 5155. (113) Larbanoix, L., Burtea, C., Laurent, S., Van Leuven, F., Toubeau, G., Vander Elst, L., and Muller, R. N. (2010) Potential amyloid plaque-specific peptides for the diagnosis of Alzheimer’s disease. Neurobiol. Aging 31, 1679−1689. (114) Larbanoix, L., Burtea, C., Ansciaux, E., Laurent, S., Mahieu, I., Vander Elst, L., and Muller, R. N. (2011) Design and evaluation of a 6-mer amyloid-beta protein derived phage display library for molecular targeting of amyloid plaques in Alzheimer’s disease: Comparison with two cyclic heptapeptides derived from a randomized phage display library. Peptides 32, 1232−1243. (115) Liu, Y., Wang, T., Calabrese, A. N., Carver, J. A., Cummins, S. F., and Bowie, J. H. (2015) The membrane-active amphibian peptide caerin 1.8 inhibits fibril formation of amyloid β1−42. Peptides 73, 1− 6. (116) 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 Aβ1−42 by mirror image phage display. ChemBioChem 4, 748− 753. (117) van Groen, T., Wiesehan, K., Funke, S. A., Kadish, I., NagelSteger, L., and Willbold, D. (2008) Reduction of Alzheimer’s disease amyloid plaque load in transgenic mice by D3, a D-enantiomeric peptide identified by mirror image phage display. ChemMedChem 3, 1848−1852. (118) Rudolph, S., Klein, A. N., Tusche, M., Schlosser, C., Elfgen, A., Brener, O., Teunissen, C., Gremer, L., Funke, S. A., Kutzsche, J., and Willbold, D. (2016) Competitive mirror image phage display derived peptide modulates amyloid beta aggregation and toxicity. PLoS One 11, e0147470. (119) Kawasaki, T., Onodera, K., and Kamijo, S. (2010) Selection of peptide inhibitors of soluble Aβ1−42 oligomer formation by phage display. Biosci., Biotechnol., Biochem. 74, 2214−2219. (120) Bazan, J., Całkosiński, I., and Gamian, A. (2012) Phage display − a powerful technique for immunotherapy: 1. introduction and potential of therapeutic applications. Hum. Vaccines Immunother. 8, 1817−1828. (121) Sun, N., Funke, S. A., and Willbold, D. (2012) Mirror image phage display - generating stable therapeutically and diagnostically active peptides with biotechnological means. J. Biotechnol. 161, 121− 125. (122) Ghimire Gautam, S., Komatsu, M., and Nishigaki, K. (2015) Strong inhibition of beta-amyloid peptide aggregation realized by twosteps evolved peptides. Chem. Biol. Drug Des. 85, 356−368. (123) Wang, Q., Liang, G., Zhang, M., Zhao, J., Patel, K., Yu, X., Zhao, C., Ding, B., Zhang, G., Zhou, F., and Zheng, J. (2014) De novo R


Review

ACS Chemical Neuroscience design of self-assembled hexapeptides as β-amyloid (Aβ) peptide inhibitors. ACS Chem. Neurosci. 5, 972−981. (124) Brener, O., Dunkelmann, T., Gremer, L., van Groen, T., Mirecka, E. A., Kadish, I., Willuweit, A., Kutzsche, J., Jürgens, D., Rudolph, S., Tusche, M., Bongen, P., Pietruszka, J., Oesterhelt, F., Langen, K. J., Demuth, H. U., Janssen, A., Hoyer, W., Funke, S. A., Nagel-Steger, L., and Willbold, D. (2015) QIAD assay for quantitating a compound’s efficacy in elimination of toxic Aβ oligomers. Sci. Rep. 5, 13. (125) Olubiyi, O. O., Frenzel, D., Bartnik, D., Glück, J. M., Brener, O., Nagel-Steger, L., Funke, S. A., Willbold, D., and Strodel, B. (2014) Amyloid aggregation inhibitory mechanism of arginine-rich Dpeptides. Curr. Med. Chem. 21, 1448−1457. (126) Leithold, L. H., Jiang, N., Post, J., Niemietz, N., Schartmann, E., Ziehm, T., Kutzsche, J., Shah, N. J., Breitkreutz, J., Langen, K. J., Willuweit, A., and Willbold, D. (2016) Pharmacokinetic properties of tandem D-peptides designed for treatment of Alzheimer’s disease. Eur. J. Pharm. Sci. 89, 31−38. (127) Klein, A. N., Ziehm, T., Tusche, M., Buitenhuis, J., Bartnik, D., Boeddrich, A., Wiglenda, T., Wanker, E., Funke, S. A., Brener, O., Gremer, L., Kutzsche, J., and Willbold, D. (2016) Optimization of the all-D peptide D3 for Aβ oligomer elimination. PLoS One 11, e0153035. (128) Zhang, Y. X., Wang, S. W., Lu, S., Zhang, L. X., Liu, D. Q., Ji, M., Wang, W. Y., and Liu, R. T. (2017) A mimotope of Aβ oligomers may also behave as a β-sheet inhibitor. FEBS Lett. 591, 3615−3624. (129) Aloisi, A., Barca, A., Romano, A., Guerrieri, S., Storelli, C., Rinaldi, R., and Verri, T. (2013) Anti-aggregating effect of the naturally occurring dipeptide carnosine on Aβ1−42 fibril formation. PLoS One 8, e68159. (130) Remy, I., Campbell-Valois, F. X., and Michnick, S. W. (2007) Detection of protein-protein interactions using a simple survival protein-fragment complementation assay based on the enzyme dihydrofolate reductase. Nat. Protoc. 2, 2120−2125. (131) Mason, J. M., Schmitz, M. A., Müller, K. M., and Arndt, K. M. (2006) Semirational design of Jun-Fos coiled coils with increased affinity: universal implications for leucine zipper prediction and design. Proc. Natl. Acad. Sci. U. S. A. 103, 8989−8994. (132) Sandberg, A., Luheshi, L. M., Söllvander, S., Pereira de Barros, T., Macao, B., Knowles, T. P., Biverstal, H., Lendel, C., EkholmPetterson, F., Dubnovitsky, A., Lannfelt, L., Dobson, C. M., and Härd, T. (2010) Stabilization of neurotoxic Alzheimer amyloid-β oligomers by protein engineering. Proc. Natl. Acad. Sci. U. S. A. 107, 15595− 15600. (133) Maurer-Stroh, S., Debulpaep, M., Kuemmerer, N., Lopez de la Paz, M., Martins, I. C., Reumers, J., Morris, K. L., Copland, A., Serpell, L., Serrano, L., Schymkowitz, J. W., and Rousseau, F. (2010) Exploring the sequence determinants of amyloid structure using position-specific scoring matrices. Nat. Methods 7, 237−242. (134) Thompson, M. J., Sievers, S. A., Karanicolas, J., Ivanova, M. I., Baker, D., and Eisenberg, D. (2006) The 3D profile method for identifying fibril-forming segments of proteins. Proc. Natl. Acad. Sci. U. S. A. 103, 4074−4078. (135) Goldschmidt, L., Teng, P. K., Riek, R., and Eisenberg, D. (2010) Identifying the amylome, proteins capable of forming amyloidlike fibrils. Proc. Natl. Acad. Sci. U. S. A. 107, 3487−3492. (136) Bartnik, D., Funke, S. A., Andrei-Selmer, L. C., Bacher, M., Dodel, R., and Willbold, D. (2010) Differently selected Denantiomeric peptides act on different Aβ species. Rejuvenation Res. 13, 202−205. (137) Wiesehan, K., Stöhr, J., Nagel-Steger, L., van Groen, T., Riesner, D., and Willbold, D. (2008) Inhibition of cytotoxicity and amyloid fibril formation by a D-amino acid peptide that specifically binds to Alzheimer’s disease amyloid peptide. Protein Eng., Des. Sel. 21, 241−246. (138) van Groen, T., Kadish, I., Wiesehan, K., Funke, S. A., and Willbold, D. (2009) In vitro and in vivo staining characteristics of small, fluorescent, Aβ42-binding D-enantiomeric peptides in transgenic AD mouse models. ChemMedChem 4, 276−282.

(139) Funke, S. A., van Groen, T., Kadish, I., Bartnik, D., NagelSteger, L., Brener, O., Sehl, T., Batra-Safferling, R., Moriscot, C., Schoehn, G., Horn, A. H., Muller-Schiffmann, A., Korth, C., Sticht, H., and Willbold, D. (2010) Oral treatment with the D-enantiomeric peptide D3 improves the pathology and behavior of Alzheimer’s Disease transgenic mice. ACS Chem. Neurosci. 1, 639−648. (140) van Groen, T., Kadish, I., Funke, S. A., Bartnik, D., and Willbold, D. (2013) Treatment with D3 removes amyloid deposits, reduces inflammation, and improves cognition in aged AβPP/PS1 double transgenic mice. J. Alzheimer's Dis. 34, 609−620. (141) Jiang, N., Leithold, L. H. E., Post, J., Ziehm, T., Mauler, J., Gremer, L., Cremer, M., Schartmann, E., Shah, N. J., Kutzsche, J., Langen, K. J., Breitkreutz, J., Willbold, D., and Willuweit, A. (2015) Preclinical pharmacokinetic studies of the tritium labelled Denantiomeric peptide D3 developed for the treatment of Alzheimer’s disease. PLoS One 10, e0128553. (142) Leithold, L. H. E., Jiang, N., Post, J., Ziehm, T., Schartmann, E., Kutzsche, J., Shah, N. J., Breitkreutz, J., Langen, K. J., Willuweit, A., and Willbold, D. (2016) Pharmacokinetic properties of a novel Dpeptide developed to be therapeutically active against toxic β-amyloid oligomers. Pharm. Res. 33, 328−336. (143) Attanasio, F., Cataldo, S., Fisichella, S., Nicoletti, S., Nicoletti, V. G., Pignataro, B., Savarino, A., and Rizzarelli, E. (2009) Protective effects of L- and D-carnosine on α-Crystallin amyloid fibril formation: implications for cataract disease. Biochemistry 48, 6522−6531. (144) Attanasio, F., Convertino, M., Magno, A., Caflisch, A., Corazza, A., Haridas, H., Esposito, G., Cataldo, S., Pignataro, B., Milardi, D., and Rizzarelli, E. (2013) Carnosine inhibits Aβ42 aggregation by perturbing the H-bond network in and around the central hydrophobic cluster. ChemBioChem 14, 583−592. (145) Wu, J. W., Liu, K. N., How, S. C., Chen, W. A., Lai, C. M., Liu, H. S., Hu, C. J., and Wang, S. S. (2013) Carnosine’s effect on amyloid fibril formation and induced cytotoxicity of lysozyme. PLoS One 8, e81982. (146) Li, Q. Q., Sun, Y. P., Ruan, C. P., Xu, X. Y., Ge, J. H., He, J., Xu, Z. D., Wang, Q., and Gao, W. C. (2011) Cellular prion protein promotes glucose uptake through the Fyn-HIF-2α-Glut1 pathway to support colorectal cancer cell survival. Cancer Sci. 102, 400−406. (147) Preston, J. E., Hipkiss, A. R., Himsworth, D. T., Romero, I. A., and Abbott, J. N. (1998) Toxic effects of β-amyloid(25−35) on immortalised rat brain endothelial cell: protection by carnosine, homocarnosine and β-alanine. Neurosci. Lett. 242, 105−108. (148) Fu, Q., Dai, H., Hu, W., Fan, Y., Shen, Y., Zhang, W., and Chen, Z. (2008) Carnosine protects against Aβ42-induced neurotoxicity in differentiated rat PC12 cells. Cell. Mol. Neurobiol. 28, 307− 316. (149) Corona, C., Frazzini, V., Silvestri, E., Lattanzio, R., La Sorda, R., Piantelli, M., Canzoniero, L. M., Ciavardelli, D., Rizzarelli, E., and Sensi, S. L. (2011) Effects of dietary supplementation of carnosine on mitochondrial dysfunction, amyloid pathology, and cognitive deficits in 3xTg-AD mice. PLoS One 6, e17971. (150) Liu, J., Wang, W., Zhang, Q., Zhang, S., and Yuan, Z. (2014) Study on the efficiency and interaction mechanism of a decapeptide inhibitor of β-amyloid aggregation. Biomacromolecules 15, 931−939. (151) Zhang, Q., Hu, X., Wang, W., and Yuan, Z. (2016) Study of a bifunctional Aβ aggregation inhibitor with the abilities of antiamyloidβ and copper chelation. Biomacromolecules 17, 661−668. (152) Jensen, M., Canning, A., Chiha, S., Bouquerel, P., Pedersen, J. T., Østergaard, J., Cuvillier, O., Sasaki, I., Hureau, C., and Faller, P. (2012) Inhibition of Cu-amyloid-β by using bifunctional peptides with β-sheet breaker and chelator moieties. Chem. - Eur. J. 18, 4836− 4839. (153) Chakravarthy, B., Ménard, M., Brown, L., Hewitt, M., Atkinson, T., and Whitfield, J. (2013) A synthetic peptide corresponding to a region of the human pericentriolar material 1 (PCM-1) protein binds β-amyloid (Aβ1−42) oligomers. J. Neurochem. 126, 415−424. (154) Sato, T., Kienlen-Campard, P., Ahmed, M., Liu, W., Li, H., Elliott, J. I., Aimoto, S., Constantinescu, S. N., Octave, J.-N., and S


Review

ACS Chemical Neuroscience Smith, S. O. (2006) Inhibitors of amyloid toxicity based on β-sheet packing of Aβ40 and Aβ42. Biochemistry 45, 5503−5516. (155) Barucker, C., Bittner, H. J., Chang, P. K., Cameron, S., Hancock, M. A., Liebsch, F., Hossain, S., Harmeier, A., Shaw, H., Charron, F. M., Gensler, M., Dembny, P., Zhuang, W., Schmitz, D., Rabe, J. P., Rao, Y., Lurz, R., Hildebrand, P. W., McKinney, R. A., and Multhaup, G. (2015) Aβ42-oligomer interacting peptide (AIP) neutralizes toxic amyloid-β42 species and protects synaptic structure and function. Sci. Rep. 5, 15410. (156) Taddei, K., Laws, S. M., Verdile, G., Munns, S., D’Costa, K., Harvey, A. R., Martins, I. J., Hill, F., Levy, E., Shaw, J. E., and Martins, R. N. (2010) Novel phage peptides attenuate beta amyloid-42 catalysed hydrogen peroxide production and associated neurotoxicity. Neurobiol. Aging 31, 203−214. (157) Barr, R. K., Verdile, G., Wijaya, L. K., Morici, M., Taddei, K., Gupta, V. B., Pedrini, S., Jin, L., Nicolazzo, J. A., Knock, E., Fraser, P. E., and Martins, R. N. (2016) Validation and characterization of a novel peptide that binds monomeric and aggregated β-amyloid and inhibits the formation of neurotoxic oligomers. J. Biol. Chem. 291, 547−559. (158) Hopping, G., Kellock, J., Barnwal, R. P., Law, P., Bryers, J., Varani, G., Caughey, B., and Daggett, V. (2014) Designed α-sheet peptides inhibit amyloid formation by targeting toxic oligomers. eLife 3, e01681. (159) Cho, P. Y., Joshi, G., Johnson, J. A., and Murphy, R. M. (2014) Transthyretin-derived peptides as β-amyloid inhibitors. ACS Chem. Neurosci. 5, 542−551. (160) Pate, K. M., Kim, B. J., Shusta, E. V., and Murphy, R. M. (2018) Transthyretin mimetics as anti-β-amyloid agents: a comparison of peptide and protein approaches. ChemMedChem 13, 968−979. (161) Ghosh, A., Pradhan, N., Bera, S., Datta, A., Krishnamoorthy, J., Jana, N. R., and Bhunia, A. (2017) Inhibition and degradation of amyloid beta (Aβ40) fibrillation by designed small peptide: a combined spectroscopy, microscopy, and cell toxicity study. ACS Chem. Neurosci. 8, 718−722. (162) Yan, L. M., Velkova, A., Tatarek-Nossol, M., Andreetto, E., and Kapurniotu, A. (2007) IAPP mimic blocks Aβ cytotoxic selfassembly: cross-suppression of amyloid toxicity of Aβ and IAPP suggests a molecular link between Alzheimer’s disease and type II diabetes. Angew. Chem., Int. Ed. 46, 1246−1252. (163) Yang, W., Jaramillo, D., Gooding, J. J., Hibbert, D. B., Zhang, R., Willett, G. D., and Fisher, K. J. (2001) Sub-ppt detection limits for copper ions with Gly-Gly-His modified electrodes. Chem. Commun., 1982−1983. (164) Chakravarthy, B., Menard, M., Brown, L., Atkinson, T., and Whitfield, J. (2012) Identification of protein kinase C inhibitory activity associated with a polypeptide isolated from a phage display system with homology to PCM-1, the pericentriolar material-1 protein. Biochem. Biophys. Res. Commun. 424, 147−151. (165) Chakravarthy, B., Ito, S., Atkinson, T., Gaudet, C., Ménard, M., Brown, L., and Whitfield, J. (2014) Evidence that a synthetic amyloid-β oligomer-binding peptide (ABP) targets amyloid-β deposits in transgenic mouse brain and human Alzheimer’s disease brain. Biochem. Biophys. Res. Commun. 445, 656−660. (166) Bush, A. I. (2003) The metallobiology of Alzheimer’s disease. Trends Neurosci. 26, 207−214. (167) Adlard, P. A., Cherny, R. A., Finkelstein, D. I., Gautier, E., Robb, E., Cortes, M., Volitakis, I., Liu, X., Smith, J. P., Perez, K., Laughton, K., Li, Q. X., Charman, S. A., Nicolazzo, J. A., Wilkins, S., Deleva, K., Lynch, T., Kok, G., Ritchie, C. W., Tanzi, R. E., Cappai, R., Masters, C. L., Barnham, K. J., and Bush, A. I. (2008) Rapid restoration of cognition in Alzheimer’s transgenic mice with 8hydroxy quinoline analogs is associated with decreased interstitial Aβ. Neuron 59, 43−55. (168) Buxbaum, J. N., Ye, Z., Reixach, N., Friske, L., Levy, C., Das, P., Golde, T., Masliah, E., Roberts, A. R., and Bartfai, T. (2008) Transthyretin protects Alzheimer’s mice from the behavioral and biochemical effects of Aβ toxicity. Proc. Natl. Acad. Sci. U. S. A. 105, 2681−2686.

(169) Jan, A., Adolfsson, O., Allaman, I., Buccarello, A. L., Magistretti, P. J., Pfeifer, A., Muhs, A., and Lashuel, H. A. (2011) Aβ42 neurotoxicity is mediated by ongoing nucleated polymerization process rather than by discrete Aβ42 species. J. Biol. Chem. 286, 8585−8596. (170) Cho, P. Y., Joshi, G., Boersma, M. D., Johnson, J. A., and Murphy, R. M. (2015) A cyclic peptide mimic of the β-amyloid binding domain on transthyretin. ACS Chem. Neurosci. 6, 778−789. (171) Armen, R. S., Alonso, D. O., and Daggett, V. (2004) Anatomy of an amyloidogenic intermediate: conversion of β-sheet to α-sheet structure in transthyretin at acidic pH. Structure 12, 1847−1863. (172) Kellock, J., Hopping, G., Caughey, B., and Daggett, V. (2016) Peptides composed of alternating L- and D-amino acids inhibit amyloidogenesis in three distinct amyloid systems independent of sequence. J. Mol. Biol. 428, 2317−2328. (173) Sims-Robinson, C., Kim, B., Rosko, A., and Feldman, E. L. (2010) How does diabetes accelerate Alzheimer disease pathology? Nat. Rev. Neurol. 6, 551−559. (174) Andreetto, E., Yan, L. M., Tatarek-Nossol, M., Velkova, A., Frank, R., and Kapurniotu, A. (2010) Identification of hot regions of the Aβ-IAPP interaction interface as high-affinity binding sites in both cross- and self-association. Angew. Chem., Int. Ed. 49, 3081−3085. (175) Paulsson, J. F., and Westermark, G. T. (2005) Aberrant processing of human proislet amyloid polypeptide results in increased amyloid formation. Diabetes 54, 2117−2125. (176) Zhu, H., Wang, X., Wallack, M., Li, H., Carreras, I., Dedeoglu, A., Hur, J. Y., Zheng, H., Li, H., Fine, R., Mwamburi, M., Sun, X., Kowall, N., Stern, R. A., and Qiu, W. Q. (2015) Intraperitoneal injection of the pancreatic peptide amylin potently reduces behavioral impairment and brain amyloid pathology in murine models of Alzheimer’s disease. Mol. Psychiatry 20, 252−262. (177) Andreetto, E., Yan, L. M., Caporale, A., and Kapurniotu, A. (2011) Dissecting the role of single regions of an IAPP mimic and IAPP in inhibition of Aβ40 amyloid formation and cytotoxicity. ChemBioChem 12, 1313−1322. (178) Andreetto, E., Malideli, E., Yan, L. M., Kracklauer, M., Farbiarz, K., Tatarek-Nossol, M., Rammes, G., Prade, E., Neumuller, T., Caporale, A., Spanopoulou, A., Bakou, M., Reif, B., and Kapurniotu, A. (2015) A hot-segment-based approach for the design of cross-amyloid interaction surface mimics as inhibitors of amyloid self-assembly. Angew. Chem., Int. Ed. 54, 13095−13100. (179) Braak, H., and Braak, E. (1991) Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol. 82, 239−259. (180) Goedert, M., Spillantini, M. G., Cairns, N. J., and Crowther, R. A. (1992) Tau proteins of Alzheimer paired helical filaments: abnormal phosphorylation of all six brain isoforms. Neuron 8, 159− 168. (181) Lee, V. M., Balin, B. J., Otvos, L., Jr., and Trojanowski, J. Q. (1991) A68: a major subunit of paired helical filaments and derivatized forms of normal Tau. Science 251, 675−678. (182) Wischik, C. M., Novak, M., Edwards, P. C., Klug, A., Tichelaar, W., and Crowther, R. A. (1988) Structural characterization of the core of the paired helical filament of Alzheimer disease. Proc. Natl. Acad. Sci. U. S. A. 85, 4884−4888. (183) Wischik, C. M., Novak, M., Thogersen, H. C., Edwards, P. C., Runswick, M. J., Jakes, R., Walker, J. E., Milstein, C., Roth, M., and Klug, A. (1988) Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease. Proc. Natl. Acad. Sci. U. S. A. 85, 4506−4510. (184) Lai, R. Y., Gertz, H. N., Wischik, D. J., Xuereb, J. H., Mukaetova-Ladinska, E. B., Harrington, C. R., Edwards, P. C., Mena, R., Paykel, E. S., Brayne, C., et al. (1995) Examination of phosphorylated tau protein as a PHF-precursor at early stage Alzheimer’s disease. Neurobiol. Aging 16, 433−445. (185) Wischik, C. M., Harrington, C. R., and Storey, J. M. D. (2014) Tau-aggregation inhibitor therapy for Alzheimer’s disease. Biochem. Pharmacol. 88, 529−539. (186) Wischik, C. M., Edwards, P. C., Lai, R. Y., Gertz, H. N., Xuereb, J. H., Paykel, E. S., Brayne, C., Huppert, F. A., MukaetovaT


Review

ACS Chemical Neuroscience Ladinska, E. B., Mena, R., et al. (1995) Quantitative analysis of tau protein in paired helical filament preparations: implications for the role of tau protein phosphorylation in PHF assembly in Alzheimer’s disease. Neurobiol. Aging 16, 409−417. (187) Morozova, O. A., March, Z. M., Robinson, A. S., and Colby, D. W. (2013) Conformational features of tau fibrils from Alzheimer’s disease brain are faithfully propagated by unmodified recombinant protein. Biochemistry 52, 6960−6967. (188) Schneider, A., Biernat, J., von Bergen, M., Mandelkow, E., and Mandelkow, E. M. (1999) Phosphorylation that detaches tau protein from microtubules (Ser262, Ser214) also protects it against aggregation into Alzheimer paired helical filaments. Biochemistry 38, 3549−3558. (189) Wischik, C. M., Edwards, P. C., Lai, R. Y., Roth, M., and Harrington, C. R. (1996) Selective inhibition of Alzheimer diseaselike tau aggregation by phenothiazines. Proc. Natl. Acad. Sci. U. S. A. 93, 11213−11218. (190) Clavaguera, F., Lavenir, I., Falcon, B., Frank, S., Goedert, M., and Tolnay, M. (2013) ″Prion-like″ templated misfolding in tauopathies. Brain Pathol. 23, 342−349. (191) Clavaguera, F., Bolmont, T., Crowther, R. A., Abramowski, D., Frank, S., Probst, A., Fraser, G., Stalder, A. K., Beibel, M., Staufenbiel, M., Jucker, M., Goedert, M., and Tolnay, M. (2009) Transmission and spreading of tauopathy in transgenic mouse brain. Nat. Cell Biol. 11, 909−913. (192) Frost, B., Jacks, R. L., and Diamond, M. I. (2009) Propagation of tau misfolding from the outside to the inside of a cell. J. Biol. Chem. 284, 12845−12852. (193) Congdon, E. E., Kim, S., Bonchak, J., Songrug, T., Matzavinos, A., and Kuret, J. (2008) Nucleation-dependent tau filament formation: the importance of dimerization and an estimation of elementary rate constants. J. Biol. Chem. 283, 13806−13816. (194) Kolarova, M., García-Sierra, F., Bartos, A., Ricny, J., and Ripova, D. (2012) Structure and pathology of tau protein in Alzheimer disease. Int. J. Alzheimer's Dis. 2012, 1−13. (195) von Bergen, M., Friedhoff, P., Biernat, J., Heberle, J., Mandelkow, E.-M., and Mandelkow, E. (2000) Assembly of τ protein into Alzheimer paired helical filaments depends on a local sequence motif (306VQIVYK311) forming β structure. Proc. Natl. Acad. Sci. U. S. A. 97, 5129−5134. (196) von Bergen, M., Barghorn, S., Li, L., Marx, A., Biernat, J., Mandelkow, E. M., and Mandelkow, E. (2001) Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure. J. Biol. Chem. 276, 48165−48174. (197) Margittai, M., and Langen, R. (2004) Template-assisted filament growth by parallel stacking of tau. Proc. Natl. Acad. Sci. U. S. A. 101, 10278−10283. (198) Daebel, V., Chinnathambi, S., Biernat, J., Schwalbe, M., Habenstein, B., Loquet, A., Akoury, E., Tepper, K., Müller, H., Baldus, M., Griesinger, C., Zweckstetter, M., Mandelkow, E., Vijayan, V., and Lange, A. (2012) β-Sheet core of tau paired helical filaments revealed by solid-state NMR. J. Am. Chem. Soc. 134, 13982−13989. (199) Sawaya, M. R., Sambashivan, S., Nelson, R., Ivanova, M. I., Sievers, S. A., Apostol, M. I., Thompson, M. J., Balbirnie, M., Wiltzius, J. J., McFarlane, H. T., Madsen, A. O., Riekel, C., and Eisenberg, D. (2007) Atomic structures of amyloid cross-β spines reveal varied steric zippers. Nature 447, 453−457. (200) Ganguly, P., Do, T. D., Larini, L., LaPointe, N. E., Sercel, A. J., Shade, M. F., Feinstein, S. C., Bowers, M. T., and Shea, J. E. (2015) Tau assembly: the dominant role of PHF6 (VQIVYK) in microtubule binding region repeat R3. J. Phys. Chem. B 119, 4582−4593. (201) Goedert, M., and Jakes, R. (2005) Mutations causing neurodegenerative tauopathies. Biochim. Biophys. Acta, Mol. Basis Dis. 1739, 240−250. (202) Przybyla, M., Stevens, C. H., van der Hoven, J., Harasta, A., Bi, M., Ittner, A., van Hummel, A., Hodges, J. R., Piguet, O., Karl, T., Kassiou, M., et al. (2016) Disinhibition-like behaviour in a P301S

mutant tau transgenic mouse model of frontotemporal dementia. Neurosci. Lett. 631, 24−29. (203) Dammers, C., Yolcu, D., Kukuk, L., Willbold, D., Pickhardt, M., Mandelkow, E., Horn, A. H. C., Sticht, H., Malhis, M. N., Will, N., Schuster, J., and Funke, S. A. (2016) Selection and characterization of tau binding D-enantiomeric peptides with potential for therapy of Alzheimer disease. PLoS One 11, e0167432. (204) von Bergen, M., Friedhoff, P., Biernat, J., Heberle, J., Mandelkow, E. M., and Mandelkow, E. (2000) Assembly of τ protein into Alzheimer paired helical filaments depends on a local sequence motif (306VQIVYK311) forming β structure. Proc. Natl. Acad. Sci. U. S. A. 97, 5129−5134. (205) Goux, W. J., Kopplin, L., Nguyen, A. D., Leak, K., Rutkofsky, M., Shanmuganandam, V. D., Sharma, D., Inouye, H., and Kirschner, D. A. (2004) The formation of straight and twisted filaments from short tau peptides. J. Biol. Chem. 279, 26868−26875. (206) Boxer, A. L., Lang, A. E., Grossman, M., Knopman, D. S., Miller, B. L., Schneider, L. S., Doody, R. S., Lees, A., Golbe, L. I., Williams, D. R., Corvol, J. C., Ludolph, A., Burn, D., Lorenzl, S., Litvan, I., Roberson, E. D., Höglinger, G. U., Koestler, M., Jack, C. R., Jr., Van Deerlin, V., Randolph, C., Lobach, I. V., Heuer, H. W., Gozes, I., Parker, L., Whitaker, S., Hirman, J., Stewart, A. J., Gold, M., and Morimoto, B. H. (2014) Davunetide in patients with progressive supranuclear palsy: a randomised, double-blind, placebo-controlled phase 2/3 trial. Lancet Neurol. 13, 676−685. (207) Sievers, S. A., Karanicolas, J., Chang, H. W., Zhao, A., Jiang, L., Zirafi, O., Stevens, J. T., Münch, J., Baker, D., and Eisenberg, D. (2011) Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation. Nature 475, 96−100. (208) Liu, F., and Gong, C. X. (2008) Tau exon 10 alternative splicing and tauopathies. Mol. Neurodegener. 3, 8. (209) Siddiqua, A., Luo, Y., Meyer, V., Swanson, M. A., Yu, X., Wei, G., Zheng, J., Eaton, G. R., Ma, B., Nussinov, R., Eaton, S. S., and Margittai, M. (2012) Conformational basis for asymmetric seeding barrier in filaments of three- and four-repeat tau. J. Am. Chem. Soc. 134, 10271−10278. (210) Chemerovski-Glikman, M., Frenkel-Pinter, M., Abu-Mokh, A., Mdah, R., Gazit, E., and Segal, D. (2017) Inhibition of the aggregation and toxicity of the minimal amyloidogenic fragment of tau by its prosubstituted analogues. Chem. - Eur. J. 23, 9618−9624. (211) Seidler, P. M., Boyer, D. R., Rodriguez, J. A., Sawaya, M. R., Cascio, D., Murray, K., Gonen, T., and Eisenberg, D. S. (2018) Structure-based inhibitors of tau aggregation. Nat. Chem. 10, 170− 176. (212) Zheng, J., Liu, C., Sawaya, M. R., Vadla, B., Khan, S., Woods, R. J., Eisenberg, D., Goux, W. J., and Nowick, J. S. (2011) Macrocyclic β-sheet peptides that inhibit the aggregation of a tau-protein-derived hexapeptide. J. Am. Chem. Soc. 133, 3144−3157. (213) Wang, C. K., Northfield, S. E., Huang, Y. H., Ramos, M. C., and Craik, D. J. (2016) Inhibition of tau aggregation using a naturallyoccurring cyclic peptide scaffold. Eur. J. Med. Chem. 109, 342−349. (214) Richman, M., Wilk, S., Chemerovski, M., Wärmländer, S. K. T. S., Wahlström, A., Gräslund, A., and Rahimipour, S. (2013) In vitro and mechanistic studies of an antiamyloidogenic self-assembled cyclic D, L-α-peptide architecture. J. Am. Chem. Soc. 135, 3474−3484. (215) Arai, T., Sasaki, D., Araya, T., Sato, T., Sohma, Y., and Kanai, M. (2014) A cyclic KLVFF-derived peptide aggregation inhibitor induces the formation of less-toxic off-pathway amyloid-β oligomers. ChemBioChem 15, 2577−2583. (216) Kino, R., Araya, T., Arai, T., Sohma, Y., and Kanai, M. (2015) Covalent modifier-type aggregation inhibitor of amyloid-β based on a cyclo-KLVFF motif. Bioorg. Med. Chem. Lett. 25, 2972−2975. (217) Arai, T., Araya, T., Sasaki, D., Taniguchi, A., Sato, T., Sohma, Y., and Kanai, M. (2014) Rational design and identification of a nonpeptidic aggregation inhibitor of amyloid-β based on a pharmacophore motif obtained from cyclo[-Lys-Leu-Val-Phe-Phe-]. Angew. Chem., Int. Ed. 53, 8236−8239. (218) Luo, Y., Vali, S., Sun, S., Chen, X., Liang, X., Drozhzhina, T., Popugaeva, E., and Bezprozvanny, I. (2013) Aβ42-binding peptoids as U


Review


protein α-synuclein associated with Parkinson’s disease. Nat. Chem. 7, 913−920. (236) Oller-Salvia, B., Sánchez-Navarro, M., Giralt, E., and Teixidó, M. (2016) Blood-brain barrier shuttle peptides: an emerging paradigm for brain delivery. Chem. Soc. Rev. 45, 4690−4707. (237) Liu, F. F., Ji, L., Dong, X. Y., and Sun, Y. (2009) Molecular insight into the inhibition effect of trehalose on the nucleation and elongation of amyloid β-peptide oligomers. J. Phys. Chem. B 113, 11320−11329. (238) Liu, R., Barkhordarian, H., Emadi, S., Park, C. B., and Sierks, M. R. (2005) Trehalose differentially inhibits aggregation and neurotoxicity of beta-amyloid 40 and 42. Neurobiol. Dis. 20, 74−81. (239) Airoldi, C., Cardona, F., Sironi, E., Colombo, L., Salmona, M., Silva, A., Nicotra, F., and La Ferla, B. (2011) cis-Glyco-fused benzopyran compounds as new amyloid-β peptide ligands. Chem. Commun. 47, 10266−10268. (240) Miura, Y., Yasuda, K., Yamamoto, K., Koike, M., Nishida, Y., and Kobayashi, K. (2007) Inhibition of Alzheimer amyloid aggregation with sulfated glycopolymers. Biomacromolecules 8, 2129−2134. (241) Di Domizio, J., Zhang, R., Stagg, L. J., Gagea, M., Zhuo, M., Ladbury, J. E., and Cao, W. (2012) Binding with nucleic acids or glycosaminoglycans converts soluble protein oligomers to amyloid. J. Biol. Chem. 287, 736−747. (242) Ariga, T., Miyatake, T., and Yu, R. K. (2010) Role of proteoglycans and glycosaminoglycans in the pathogenesis of Alzheimer’s disease and related disorders: amyloidogenesis and therapeutic strategies–a review. J. Neurosci. Res. 88, 2303−2315. (243) Dorgeret, B., Khemtémourian, L., Correia, I., Soulier, J. L., Lequin, O., and Ongeri, S. (2011) Sugar-based peptidomimetics inhibit amyloid β-peptide aggregation. Eur. J. Med. Chem. 46, 5959− 5969. (244) Kaffy, J., Brinet, D., Soulier, J. L., Khemtémourian, L., Lequin, O., Taverna, M., Crousse, B., and Ongeri, S. (2014) Structure-activity relationships of sugar-based peptidomimetics as modulators of amyloid β-peptide early oligomerization and fibrillization. Eur. J. Med. Chem. 86, 752−758. (245) Kaffy, J., Brinet, D., Soulier, J. L., Correia, I., Tonali, N., Fera, K. F., Iacone, Y., Hoffmann, A. R., Khemtémourian, L., Crousse, B., Taylor, M., Allsop, D., Taverna, M., Lequin, O., and Ongeri, S. (2016) Designed glycopeptidomimetics disrupt protein-protein interactions mediating amyloid β-peptide aggregation and restore neuroblastoma cell viability. J. Med. Chem. 59, 2025−2040. (246) Vidu, A., Dufau, L., Bannwarth, L., Soulier, J. L., Sicsic, S., Piarulli, U., Reboud-Ravaux, M., and Ongeri, S. (2010) Toward the first nonpeptidic molecular tong inhibitor of wild-type and mutated HIV-1 protease dimerization. ChemMedChem 5, 1899−1906. (247) Sinopoli, A., Giuffrida, A., Tomasello, M. F., Giuffrida, M. L., Leone, M., Attanasio, F., Caraci, F., De Bona, P., Naletova, I., Saviano, M., Copani, A., Pappalardo, G., and Rizzarelli, E. (2016) Ac-LPFFDTh: A trehalose-conjugated peptidomimetic as a strong suppressor of amyloid-β oligomer formation and cytotoxicity. ChemBioChem 17, 1541−1549. (248) Autiero, I., Langella, E., and Saviano, M. (2013) Insights into the mechanism of interaction between trehalose-conjugated betasheet breaker peptides and Aβ(1−42) fibrils by molecular dynamics simulations. Mol. BioSyst. 9, 2835−2841. (249) Frenkel-Pinter, M., Richman, M., Belostozky, A., Abu-Mokh, A., Gazit, E., Rahimipour, S., and Segal, D. (2016) Selective inhibition of aggregation and toxicity of a tau-derived peptide using its glycosylated analogues. Chem. - Eur. J. 22, 5945−5952. (250) Goyal, D., Shuaib, S., Mann, S., and Goyal, B. (2017) Rationally designed peptides and peptidomimetics as inhibitors of amyloid-β(Aβ) aggregation: potential therapeutics of Alzheimer’s disease. ACS Comb. Sci. 19, 55−80.

amyloid aggregation inhibitors and detection ligands. ACS Chem. Neurosci. 4, 952−962. (219) Kokkoni, N., Stott, K., Amijee, H., Mason, J. M., and Doig, A. J. (2006) N-methylated peptide inhibitors of β-amyloid aggregation and toxicity. optimization of the inhibitor structure. Biochemistry 45, 9906−9918. (220) Hiramatsu, H., Ochiai, H., and Komuro, T. (2016) Effects of N-methylated amyloid-β30−40 peptides on the fibrillation of amyloidβ1−40. Chem. Biol. Drug Des. 87, 425−433. (221) Etienne, M. A., Aucoin, J. P., Fu, Y., McCarley, R. L., and Hammer, R. P. (2006) Stoichiometric inhibition of amyloid β-protein aggregation with peptides containing alternating α,α-disubstituted amino acids. J. Am. Chem. Soc. 128, 3522−3523. (222) Wu, H., Li, Y., Bai, G., Niu, Y., Qiao, Q., Tipton, J. D., Cao, C., and Cai, J. (2014) γ-AApeptide-based small-molecule ligands that inhibit Aβ aggregation. Chem. Commun. 50, 5206−5208. (223) Wei, C.-W., Peng, Y., Zhang, L., Huang, Q., Cheng, M., Liu, Y.-N., and Li, J. (2011) Synthesis and evaluation of ferrocenoyl pentapeptide (Fc-KLVFF) as an inhibitor of Alzheimer’s Aβ1−42 fibril formation in vitro. Bioorg. Med. Chem. Lett. 21, 5818−5821. (224) Wei, C.-W., Li, J., and Liu, Y.-N. (2013) Electrochemical properties and self-assembly of ferrocene modified hydrophobic pentapeptide fragment of β-amyloid. Chin. J. Inorg. Chem. 29, 45−49. (225) Li, X., Wei, C., Liu, X., and Liu, Y. (2010) Synthesis of ferrocenoyl-peptide and its inhibition for β-amyloid peptide. Chin. J. Org. Chem. 30, 1492−1496. (226) Rocha, S., Cardoso, I., Borner, H., Pereira, M. C., Saraiva, M. J., and Coelho, M. (2009) Design and biological activity of β-sheet breaker peptide conjugates. Biochem. Biophys. Res. Commun. 380, 397−401. (227) De Bona, P., Giuffrida, M. L., Caraci, F., Copani, A., Pignataro, B., Attanasio, F., Cataldo, S., Pappalardo, G., and Rizzarelli, E. (2009) Design and synthesis of new trehalose-conjugated pentapeptides as inhibitors of Aβ(1−42) fibrillogenesis and toxicity. J. Pept. Sci. 15, 220−228. (228) Liu, F., Iqbal, K., Grundke-Iqbal, I., Hart, G. W., and Gong, C. X. (2004) O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A. 101, 10804−10809. (229) Liu, F., Shi, J., Tanimukai, H., Gu, J., Gu, J., Grundke-Iqbal, I., Iqbal, K., and Gong, C.-X. (2009) Reduced O-GlcNAcylation links lower brain glucose metabolism and tau pathology in Alzheimer’s disease. Brain 132, 1820−1832. (230) Yuzwa, S. A., Shan, X., Macauley, M. S., Clark, T., Skorobogatko, Y., Vosseller, K., and Vocadlo, D. J. (2012) Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation. Nat. Chem. Biol. 8, 393−399. (231) Yuzwa, S. A., Shan, X., Jones, B. A., Zhao, G., Woodward, M. L., Li, X., Zhu, Y., McEachern, E. J., Silverman, M. A., Watson, N. V., Gong, C. X., and Vocadlo, D. J. (2014) Pharmacological inhibition of O-GlcNAcase (OGA) prevents cognitive decline and amyloid plaque formation in bigenic tau/APP mutant mice. Mol. Neurodegener. 9, 42. (232) Chun, Y. S., Park, Y., Oh, H. G., Kim, T. W., Yang, H. O., Park, M. K., and Chung, S. (2015) O-GlcNAcylation promotes nonamyloidogenic processing of amyloid-β protein precursor via inhibition of endocytosis from the plasma membrane. J. Alzheimer's Dis. 44, 261−275. (233) Chen, P. Y., Lin, C. C., Chang, Y. T., Lin, S. C., and Chan, S. I. (2002) One O-linked sugar can affect the coil-to-β structural transition of the prion peptide. Proc. Natl. Acad. Sci. U. S. A. 99, 12633−12638. (234) Yuzwa, S. A., Cheung, A. H., Okon, M., McIntosh, L. P., and Vocadlo, D. J. (2014) O-GlcNAc modification of tau directly inhibits its aggregation without perturbing the conformational properties of tau monomers. J. Mol. Biol. 426, 1736−1752. (235) Marotta, N. P., Lin, Y. H., Lewis, Y. E., Ambroso, M. R., Zaro, B. W., Roth, M. T., Arnold, D. B., Langen, R., and Pratt, M. R. (2015) O-GlcNAc modification blocks the aggregation and toxicity of the V


Peptides, Peptidomimetics, and CarbohydrateâPeptide Conjugates as

Recommend Documents

Peptides, Peptidomimetics, and CarbohydrateâPeptide Conjugates as