Amyloid β-Targeted Inhibitory Peptides for Alzheimer’s Disease: Current State and Future Perspectives

Safura Jokar¹ • Saeedeh Khazaei² • Xaniar Esmailzadeh Gameshgoli³ • Mona Khafaji⁴ • Behnam Yarani⁵ • Mohammad Sharifzadeh⁶ • Davood Beiki⁷ • Omid Bavi⁸

¹Department of Nuclear Pharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; ²Department of Pharmaceutical Biomaterials, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; ³Colloids and Polymers Physics Group, Particle Physics Department, Universidade de Santiago de Compostela, Santiago de Compostela, Spain; ⁴Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran, Iran; ⁵Division of Nanobiotechnology, Department of Life Sciences Engineering, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Iran; ⁶Department of Toxicology and Pharmacology, Faculty of Pharmacy; Toxicology and Poisoning Research Centre, Tehran University of Medical Sciences, Tehran, Iran; ⁷Research Center for Nuclear Medicine, Tehran University of Medical Sciences, Tehran, Iran; ⁸Department of Mechanical and Aerospace Engineering, Shiraz University of Technology, Shiraz, Iran

Abstract: Alzheimer’s disease is the most common irreversible neurodegenerative disorder. To date, there is no cure for Alzheimer’s disease. While multiple pathological mechanisms have been proposed for the onset and progression of Alzheimer’s disease, the hypothesis that attracted much attention is the amyloid hypothesis. The senile plaques that accumulate in the brain of Alzheimer’s disease patients are predominantly composed of beta amyloid (Aβ). Aβ deposition in the brain is thought to occur years before the emergence of clinical symptoms. The overproduction, aggregation, and fibrillation of Aβ, combined with reduced clearance, eventually lead to amyloid plaque formation and subsequent neurotoxicity. Hence, inhibition of Aβ aggregation and the promotion of Aβ clearance have been actively explored as therapeutic strategies for Alzheimer’s disease. This chapter provides an overview of the current knowledge on one such strategy, Aβ-targeted inhibitory peptides.

Keywords: Aβ aggregation in Alzheimer’s disease; biopanning; inhibitory peptides for Alzheimer’s disease; peptide–nanostructure conjugates; peptidomimetics

INTRODUCTION

Alzheimer’s disease is an age-dependent disorder that is the fifth leading cause of death in people aged 65 years and older. It is estimated that over 50 million people worldwide suffer from Alzheimer’s disease, and this figure is set to increase to 152 million by 2050 with a financial burden of 1.1 trillion US dollars by 2050 (1–3). Several hypotheses, including the amyloid, cholinergic (4), and Tau protein hypotheses have been proposed to explain the pathophysiology and etiology of Alzheimer’s disease (5). Because of the presence of Aβ in the brain tissue, cerebrospinal fluid, and plasma, the amyloid cascade hypothesis is the most widely accepted. The amyloid cascade hypothesis states that neurodegeneration in Alzheimer’s disease is the result of amyloid plaque and neurofibrillary tangle formations (6, 7). The overproduction, clearance failure, aggregation, and fibrillation of Aβ eventually leads to amyloid plaque formation. These factors also contribute to neuroinflammation and cell death. Aβ deposition in the brain is likely to be the first pathological incident that occurs years before the emergence of clinical symptoms. Aβ is produced through the proteolytic cleavage of the amyloid precursor protein (APP), a transmembrane glycoprotein, which is made up of a cytoplasmic domain with 55 amino acids and a long extracellular domain with 590–680 amino acids (8). APP cleavage by the proteases β- and γ-secretases produce Aβ fragments of varying size depending on the cleavage site (9), of which Aβ₄₀ (about 90%) and Aβ₄₂ (about 5–10%) are the most prevalent (Figure 1). Aβ₄₂ is more toxic than Aβ₄₀. After production, the Aβ peptides aggregate to form amyloid deposits. There are different aggregation forms such as low molecular weight oligomers, protofibrils, as well as mature fibrils that eventually come together to form amyloid deposits in the brain parenchyma and cerebrovascular spaces (10, 11).

Figure 1 Amyloid-β fibrillation and neuronal damage. First, amyloid precursor protein is cleaved by β and γ secretases, respectively. A peptide fragment of 39-42 amino acid is formed depending on the site of cleavage. After cleavage, Aβ monomers start to self-assemble to form soluble toxic aggregates, and finally into insoluble fibrils, which subsequently cause synaptic dysfunction and neuronal death.

Therefore, inhibition of Aβ aggregation and the promotion of Aβ clearance have been investigated as therapeutic strategies for Alzheimer’s disease. Some of these strategies include the use of metal chelators (12), peptides (13), organic molecules (14), and biomolecules (15, 16). Peptides are considered a better option than small molecule-based compounds because of their high affinity for Aβ and low toxicity (17). Although natural amino acid-based peptides are effective inhibitors of Aβ aggregation, they are prone to faster enzymatic degradation and show a tendency for self-assembly into fibrils during administration (15). To overcome these problems, modified peptides have been generated (18) with D-amino acids, retro-inverso cyclization, fluorination, as well as N-methylation of the ester bond (19). With this knowledge, peptides could be potential candidates for inhibiting Aβ conformational transitions, self-assembly, and toxicity against neurons, and promotion of the pathways of the nontoxic fibrillation and early diagnosis of Alzheimer’s disease (20). This chapter provides an overview of the therapeutic potential peptides as Aβ aggregation inhibitors.

PEPTIDIC INHIBITORS

Luhrs and co-workers first experimentally described the structure of the Aβ₄₂ fibril (Figure 2, right) (21). At least four specific structural sites for interaction have been identified on the Aβ fibril (22): (i), hydrophobic regions of Ala30–Val36, and Leu17–Ala21 residues from the C and N-terminal β-sheets respectively; (ii), hydrophilic part using electrostatic interactions between Asp23 and Lys28 residues; (iii), central cleft in the interior of the U-shaped turn; and (iv), Glu22 ladder between the side chains of the Glu22 residues of the adjacent β-strands (Figure 2, bottom right). The formation of the salt-bridge between Asp23 and Lys28 is an essential β-sheet conformation stabilizer. Moreover, it might stimulate the oligomerization of Aβ via stabilizing the Val24–Asn27 turn (23). The hydrophobic residue of Met35 in the C-terminus domain could support fibril stability by hydrophobic interactions. The Met35 binding site can potentially inhibit protein-protein interactions and prevent amyloid fibril formation (24). These sites are probably critical regions in the initiation of Aβ nucleation, conformational transition promotion, and fibril formation. The residues ¹⁶KLVFFA²¹ (Figure 2) of the central hydrophobic core (CHC) region is a critical nucleation site, or self-recognition sequence. The Ile41 and Ala₄₂ residues can modulate Aβ₄₂ oligomer formation (25) by interacting with the N and C-terminus of Aβ₄₂ (26). Figure 2, left, shows the binding sites of the most common inhibitory peptides, such as LPFFD and KVLFF, on the Aβ₄₀ fibril structure.

Figure 2 The structure of Aβ. Right, schematic of the Aβ₄₂ amyloid fibril. Left, binding sites of inhibitory peptides of LPFFD and KVLFF on Aβ₄₀ fibril.

Peptide inhibitors are generally divided into Aβ-based peptide inhibitors and non-Aβ-based peptide inhibitors. A list of select Aβ inhibitory peptides are presented in Table 1.

TABLE 1 A select list of inhibitory peptides against Aβ peptide aggregation

No	Name/sequence of inhibitor peptide	Configuration	Type	Therapeutic findings	Ref.
1	RR (RYYAAFFARR)	L-enantiomer	Rational design based on hydrophobic core and salt bridge region	Inhibition of Aβ40 fibrillation and decrease of Aβ40 induced toxicity (in vitro)	(27)
2	D-(PGKLVYA) and D(KKLVFFARRRRA)	D-enantiomer	CHC-derived sequence	Inhibition of Aβ aggregation Increase life time in an animal model of expressing Aβ42	(28)
3	CP-2 (lLwHsK)	Cyclic d,l-α-peptide	Screening of the randomly library members (500)	Nontoxic Stabilize small Aβ oligomers Disassemble formed Aβ fibrils	(29)
4	D3D3 (D-RPRTRLHTHRNRRPRvTRLHTHRNR)	D-enantiomer	Not derived from Aβ sequence	More inhibitory effect against Aβ42 oligomerization than D3 peptide Formation of nontoxic amorphous instead of toxic oligomers (In vivo)	(30)
5	RD2 (DPTLHTHNRRRR-NH2)	D-enantiomer	Not derived from Aβ sequence	Good binding affinity to Aβ Reduce Aβ fibrillation formation High stability in mouse plasma High bioavailability (In vivo)	(31, 32)
6	AOEP2 (FDYKAEFMPWDT)	D-enantiomer	A mimotope of the Aβ oligomer Selected by phage display	Targeting of all forms of Aβ The considrable reduction of TNF-α	(33)
7	LPYFD-amide	L-enantiomer	CHC-derived sequence	Neuroprotective Decrease tau aggregation (In vivo)	(34)
8	Ac-LPFFN-NH2	L-enantiomer	CHC-derived sequence	Inhibit of Aβ40 aggregation Stabilize the α-helical conformation of Aβ40 Prolong the fibril formation	(35)
9	D-4F peptide	D-enantiomer	Not derived from Aβ sequence	Inhibit Aβ deposition Improve cognitive function	(36)
10	Diazirine-equipped cyclo-KLVF(b-Ph)F	cyclic	CHC-derived sequence	Inhibit Aβ aggregation Prevent Aβ42 induced toxicity	(37)
11	Attached flavin to an Aβ-binding peptide	cyclic	CHC-derived sequence	Inhibition of Aβ aggregation Decrease of the aggregation potency and Aβ induced neurotoxicity	(38)
12	ZAb3 affibody	L-enantiomer	Selected by phage display technique	Inhibit Aβ aggregation Disassemble preformed oligomers	(39)
13	H102 (HKQLPFFEED)	L-enantiomer	CHC-derived sequence	Inhibit Aβ42 fibrillization	(40)
14	Th-NT, Th-CT, and Th-SC	Trehalose conjugated peptides, L-enantiomer	CHC-derived sequence	Inhibit fibril formation	(41)
15	Fc-KLVFF	Ferrocene- conjugated peptide, L-enantiomer	CHC-derived sequence	Inhibit Aβ aggregation	(42)
16	N-methylated Aβ(32–37)	L-enantiomer	C-terminal fragment	Inhibits Aβ aggregation Improve Drosophila longevity Increase locomotion	(43)
17	D-Trp-Aib	D-enantiomer	Rational design	Inhibits Aβ oligomerization Decrease cerebral amyloid deposits in transgenic model Improves cognitive activity (in vivo)	(44)
18	GABA-FPLIAIMA	D-enantiomer	C-terminal fragment	Inhibit and reduce the Aβ aggregation	(22)
19	Tyr(Allyl-RCM)-Xaa-Gly(Allyl-RCM) and ‘Gly(Allyl-RCM)-Xaa-Tyr(Allyl-RCM)	cyclic	CHC-derived sequence	Inhibit significantly the Aβ42 aggregation	(45)
20	D(KLVFW)- aminobutyric acid (Aib)	D-enantiomer	CHC-derived sequence	High specific interaction with Aβ42 monomers and oligomers Remarkable inhibition of A Aβ42 fibril formation No cytotoxic effects	(46)

Aβ-based peptide inhibitors

These are based on the structure of the C-terminal fragments (CTFs) and the CHC sequences of the Aβ peptide. They bind to the Aβ peptide at specific sites and prevent its assembly into amyloid fibrils. Peptides consisting of D-enantiomeric amino acids exhibit greater stability against proteases and show a higher binding affinity for Aβ compared with their L-enantiomeric counterparts. Moreover, D-peptides inhibit Aβ aggregation in animal models (28). Retro-inverso peptides are a special class of modified peptides that contain D-amino acids and reversed NH and CO groups in the peptide bonds. These peptides could keep the same spatial position in the side chain of the residues and preserve the desirable 3D structure compared to unchanged L-peptides (47). They also displayed advantages in terms of Aβ aggregation inhibition, higher proteolytic stability, lower self-assembly, and better blood-brain-barrier (BBB) permeability when compared with L-peptides in an animal model (48, 49). Fluorinated hydrophobic valine or phenylalanine in the LVFFA-based peptides can considerably delay the formation of Aβ aggregation. Fluorinated amino acids can also inhibit Aβ aggregation (50). Modification of amide functional groups with a methyl group is another strategy in the development of new inhibitors. N-methylated amide groups could enhance the peptide’s solubility in aqueous solutions and decrease Aβ-induced toxicity. Cyclic peptides have a higher inhibitory activity than acyclic derivatives (51). Because of their high enzymatic resistance, they are degraded slowly. Residues of lysine and glutamic acid have been known to be effective stabilizing and enhancing agents of Aβ fibrillation due to their ability to improve surface tension. In contrast, arginine residues have been reported as aggregation inhibitors or destabilizers (chaotropes) (52).

The ¹⁶KLVFF²⁰-based peptides play a crucial role in disrupting Aβ aggregation by binding to full-length Aβ peptides and preventing fibril formation (53, 54). Ac-LVFFARK-NH2 (LK7), designed by adding arginine and lysine to KLVFF, induced a dose-dependent inhibition on Aβ₄₂ fibrillation; however, it was cytotoxic due to high self-assembling properties (55). When conjugated with poly (lactic-co-glycolic acid) nanoparticles (NPs), the LK7-PLGA-NPs complex resulted in the elimination of the LK7 self-assembly feature while inhibiting Aβ₄₂ fibrillation (55). Binding β-cyclodextrin to LK7 (56) improved LK7 peptide solubility, inhibited its tendency to self-aggregate, improved its binding to Aβ, and inhibited Aβ aggregation. Head-to-tail cyclization of LK7 peptide also resulted in a decrease in self-assembly of the LK7, an increase in binding affinity to the Aβ₄₀ peptide, and proteolytic stability in serum. This derivative also can stabilize the Aβ₄₀ secondary structure and inhibit Aβ₄₀ –mediated cytotoxicity. Another derivative of LK7 peptide is Ac-LVFFARKHH-NH2 (LK7-HH), in which LK7 has been conjugated to the HH ligand as a chelator for reducing reactive oxygen species (ROS) production and capturing free and complexed ions of Cu²⁺ (57). This chelator also improved the anti-aggregate effects of LK7 on Aβ peptide and reduced its self-aggregation properties.

Proline and aspartic acid were exchanged for valine and alanine, respectively, in KVLFFA (58, 59). The derived peptide, referred to as 5-mer iAβ5 with sequence LPFFD, inhibited Aβ aggregation, neurotoxicity, and reduced plaque load (58). Due to the lack of a proton on the secondary substituted nitrogen in the peptide bond of proline residue, it could inhibit the formation of the intramolecular hydrogen bonds into fibrils. Since these small peptides are prone to faster enzymatic degradation and have reduced BBB permeability in vivo, iAβ5p was modified by N-methylation between Pro and Phe residues to improve its stability (60). The results from in vitro and in vivo studies showed that it has the same inhibitory activity as the parental iAβ5 peptide against amyloid fibril formation and neurotoxicity but with improved protease resistance. Also, molecular dynamics simulations show that this peptide has more durable binding and enhanced activity against Aβ₄₀ aggregation in comparison to the iAβ5 peptide. In a similar study, the RIVFF sequence was produced by residue mutations of lysine16 (K) to arginine (R) and leucine17 (L) to isoleucine (I) on the KLVFF segment (61). The results indicated that this peptide could self-aggregate into β-sheet structures by reducing the surface tension of water and at higher concentrations (>250 μM) enhanced the Aβ-induced cytotoxicity.

The peptide D-GRKKRRQRRR-GGGG-DVEFRH (Aβ₁₋₆ A2V-TAT) was investigated in vivo (62, 63). It was generated by modifying the N-terminal fragment of ¹DAEFRH⁶ through mutation of alanine in position 2 to valine and conjugating with the HIV protein transduction domain GRKKRRQRRR (TAT). The resulting peptide showed strong anti-amyloidogenic effects in vitro and Aβ aggregation inhibition in mouse models of Alzheimer’s disease (64). The KLVFWAK motif was designed based on the ¹⁶KLVFFAE²² sequence with mutations introduced at phenylalanine and glutamic acid residues to tryptophan and lysine respectively to enhance solubility and disrupt self-assembly via electrostatic repulsion. Results showed that the designed motif could only target the C-terminal region of Aβ oligomers. The designed motif exhibited a lower self-aggregation tendency in comparison to other KLVFF-related sequences. Moreover, it demonstrated a higher binding affinity to Aβ aggregates and fibrils than monomers (65).

RGKLVFFGR (OR1) and RGKLVFFGR-NH (OR2) are retro-inverso peptides (66), designed by the addition of arginine (R) and glycine (G) to the KLVFF sequence. They exhibit high solubility and stability against enzymes. However, only the OR2 peptide showed inhibitory effects on Aβ oligomer formation and cytotoxicity. OR2 was modified to HN-rGklvffGr-Ac (RI-OR2) by acetylation of the C-terminal residue (49). The result illustrated that the peptide has a high resistance to proteolysis, while maintaining the same inhibitory activity in vivo. In a follow-up study, the RI-OR2 peptide was attached to the TAT peptide to improve its permeability into cells and the BBB (48). The results showed the peptide was able to decrease Aβ aggregation, plaque levels, and oxidative damages as well as increase the number of young neurons in the brain.

³¹IIGLMVGGVVIA⁴² and ³⁹VVIA⁴² sequences were designed based on the C-terminal domain of Aβ₄₂ (67). The ³⁹VVIA⁴² sequences could interact with Aβ₄₂ monomers and smaller oligomers at several sites, specifically at the N-terminal domain. At micromolar concentrations, the VVIA-NH₂ peptide inhibited Aβ₄₂ aggregation, exhibited less toxicity, and protected synaptic activity. However, these effects were not observed for the acetylated Ac-VVIA sequence (68). The non-acetylated VVIA-NH₂ sequence particularly interacts with the C-terminal domain while the Ac-VVIA peptide has a dispersed binding distribution (68). The Ac-³²IGLMVG³⁷-NH₂ sequence, a hexapeptide from the C-terminal fragment, has been shown to have a moderate efficacy with less toxicity (69).

O-acyl isopeptide and NMe-b-Ala26 (70) were derived from the full-length Aβ sequence with modification of an ester bond at the Gly25-Ser26 moiety and an N-methyl amide-β-Ala26, respectively. O-acyl isopeptide inhibited Aβ₄₂ fibrillation at equimolar concentrations through an inhibitory mechanism distinct from any other peptidic inhibitors reported previously. Also, this derivative was more soluble than Aβ₄₂ peptides and rapidly decomposed to Aβ₄₂ monomers under physiological conditions through an O-to-N acyl rearrangement reaction whereas NMe-b-Ala26 showed higher chemical stability at physiological conditions.

Non-Aβ-based peptide inhibitors

Carnosine, a natural imidazole-containing dipeptide is a metal ion chelator (71). It inhibits the fibrillation and toxicity of amyloidogenic species such as glycated α-Crystallin, Aβ peptide, and prions. This peptide also inhibits the intramolecular salt bridge formation, which is vital to the stability and elongation of fibrils (71). Peptide D1, QSHYRHISPAQV (72), is another non-Aβ peptide that reduces Aβ aggregation and Aβ-associated cytotoxicity at high concentrations. N-methylated proprietary peptides such as D-NH₂ (SEN304) and SEN1576 can inhibit Aβ-associated toxicity in vivo (73). Furthermore, SEN304 is a more potent inhibitor than customized versions of the KLVFF peptide. These peptides could interfere with the nucleation of Aβ, convert them into non-toxic forms, and eliminate toxic oligomers.

PEPTIDE LIBRARY SCREENING

There are many screening approaches to identify target-specific ligands (74, 75). Phage display is one such efficient high-throughput screening method that allows the screening of a wide variety of peptide libraries to identify specific peptide sequences against the desired target (76, 77). Wang et al. synthesized a linear peptide with sequence PYRWQLWWHNWS selected based on the screening of a randomized 12-mer peptide library against the target Aβ_1–10 sequence (78). After screening, specific phages were selected and their binding affinity to Aβ_1–10 was evaluated by real-time biomolecular interaction analysis. This peptide could specifically bind to Aβ_1–10, inhibit the aggregation of Aβ into plaques, and reduce Aβ_1–42 induced-apoptosis. Furthermore, it illustrated a protective effect against Aβ_1–42-induced memory and learning impairments in animal models (59).

Larbanoix et al. utilized the phage display method to discover a linear hexapeptide against Aβ_1–42 aggregation (79). Two of the selected clones, Pep1: LIAIMA and Pep2: IFALMG, corresponding to fragment ³¹IIGLMV³⁶ from Aβ_1–42 peptide, demonstrated the highest binding affinities to Aβ_1–42 with Kd values in the micromolar range. Their specific interactions with Aβ_1–42 plaques were identified by immunohistochemistry on harvested brain tissue from an animal model of Alzheimer’s disease. The peptides did not induce any toxicity in neurons in vitro. Moreover, the thioflavin T aggregation assay indicated that the designed peptides could suppress the amyloid fibril formation.

In 2010, a random heptapeptide library (XX–P–XXXX) on T7 phage was reported by Kawasaki et al. (80). The library was designed based on the LPFFD sequence XX-P-XXXX, where P is proline, and X is any amino acid. After the fifth-round of biopanning against Aβ_1–42 soluble oligomers, eight new peptides containing arginine residues were obtained. The peptide with the strongest affinity to Aβ (RGPRGRV) suppressed the formation of 37–48 kDa oligomers and maintained the monomeric form of Aβ_1–42 for up to 24 h. In follow-up studies, to assess the effect of the peptide length on the inhibition of soluble oligomers formation, random libraries containing 3-residue and 4-residue peptides were prepared by phage display and evaluated. The results demonstrated that the 3-residue peptides could not significantly inhibit oligomers formation because of their smaller size. In contrast, the 4-residue peptide with the RFRK sequence inhibited the soluble oligomer formation like the heptapeptide (RGPRGRV). It also showed a slight decrease on Aβ fibrillation (81), similar to the inhibitory activity of the N-Methylated Peptide (SEN304), against Aβ₄₂ aggregation (25, 82, 83). Tsuji-Ueno et al. utilized the all-steps-all-combinations (ASAC) method to explore Aβ₄₂-binding peptide aptamers. The identified peptides from the primary and secondary libraries showed a weak binding affinity to Aβ₄₂ (Kd values in the μ m range) (84). To further improve the peptide aptamers, Gautam et al. applied the mRNA display technique and paired-peptide library method. The library was assembled by a random shuffling method on selected peptide blocks taken from the formed primary and secondary peptide libraries by Tsuji-Ueno et al. (84). They reported two peptides with high binding affinity to Aβ₄₂ (Kd in the nM range) which significantly inhibited the Aβ₄₂ aggregation (85). The improved peptide aptamers, P84 (CGILDPIPWGGSGGSCGILDPIPW) and P131 (GCPCIGIIGGSGGSDCSSDLTPS), where GGSGGS is the linker sequence, demonstrated a higher binding affinity for the Aβ₄₂ peptide (Kd values in the nanomolar range) compared to the primary and secondary Aβ₄₂-binding peptides (86). The results showed that both peptides could inhibit the Aβ₄₂ aggregation and result in the reduction of the cytotoxic effects of Aβ₄₂ fibrils and Aβ₄₂ oligomers in PC12 cells; P84 showed better efficacy than P131 on the cell line.

Groen and co-workers employed mirror-image phage display to identify selective and high-affinity D-peptide ligands for Aβ_1–42. The D-enantiomer Aβ_1–42 was used as a target for selection from a randomized 12- amino acid peptide library with more than 1 billion different peptides. After six rounds of biopanning, they identified a specific D-enantiomeric peptide, RPRTRLHTHRNR, called D3 (73). The D3 ligand inhibited Aβ aggregation, and dissolved pre-formed Aβ fibrils. Additionally, D3 ligand could disaggregate pre-existing amyloid plaques in the brain and result in an increase in the amount of Aβ monomeric form, which has high clearance from the brain (87). FITC fluorescence data demonstrated that Aβ–D3 clearance might have been associated with pericytes, which have a major role in the clearance of different Aβ_40/42 species (88, 89). Glial fibrillary acidic protein (GFAP) staining of astrocytes and CD11b staining of microglia in brain sections revealed that the D3 significantly decreased the amount of plaque-related inflammation markers (active astrocytes and microglia) around the Aβ plaques in comparison to the untreated animals. In addition to the anti-inflammatory properties, this peptide ligand could drastically reduce the Aβ plaque load in brain tissue of transgenic APP-PSD mice after a 30-day treatment with administration of 9 mg D3 per day per mouse. Computational simulation studies demonstrated strong electrostatic interactions between the arginine-rich D3 and negatively charged groups of Aβ nonamer; D3 binding to Aβ nonamer could change the topology of the Aβ oligomers by inducing a twist in them and consequently promote the formation of Aβ nonfibrillar aggregations (73, 90, 91).

Luo et al. applied peptoid chemistry, N-substituted glycine oligomers as a class of peptidomimetics, to develop and improve selective high-affinity ligands for Aβ₄₂ (92). They constructed an on-bead peptoid library of 38,416 unique peptoids. After screening for Aβ₄₂-selective peptoid ligands, the IAM1 ligand and its dimeric form were selected and further evaluated. IAM1 peptide showed about 10-fold more affinity for Aβ₄₂-binding than for Aβ₄₀, and inhibited Aβ₄₂ aggregation in vitro. The dimeric derivative (IAM1)₂ demonstrated a 7.4-fold higher affinity for Aβ₄₂ (60 nM) than the monomeric form. Moreover, (IAM1)₂ demonstrated neuroprotective effects on primary hippocampal neurons against Aβ₄₂-induced toxicity.

Due to the considerable similarities between the self-assembly of cyclic d,l-α-peptides and amyloid structures, it is possible such peptides can bind to Aβ non-toxic forms and stabilize them (29). Richman et al. described the cyclic peptide CP-2, cyclo-[l–J–w– H–s–K]s (J denotes l-norleucine), by screening a 6-residue library of head-to-tail cyclic d,l-α-peptides consisting of residues Lys, Glu, Ser, Leu, Trp, and His using a one-bead-one-peptide combinatorial approach (29, 93). The selected peptide strongly interacted with Aβ₄₀/Aβ₄₂ and prevented their assembly, entirely disassembled Aβ₄₀ fibrils, and protected PC12 cells against Aβ₄₀/Aβ₄₂-induced toxicity, without having any toxic effects of its own. NMR spectroscopy revealed that the CP-2 peptide, in a self-assembled form, interacted with monomeric and low-oligomeric structures of Aβ₄₀ and induced weak α-helix structures during the initial stage of Aβ₄₀ aggregation and subsequently promoted the conformational transition shift from a more toxic antiparallel β-sheet conformation to the less toxic parallel β-sheet.

In another study, Acerra et al. utilized an intracellular protein-fragment complementation assay (PCA) methodology for the screening of selective high-affinity peptides to Aβ (94). The Aβ_25–35 sequence, known to self-assemble into toxic fibrils (95), was inserted into one half of the murine dihydrofolate reductase enzyme as a target, and the Aβ29–35 sequence-based peptide was inserted on the other half of the enzyme (96). After the screening of primary and secondary libraries, two new targeting peptides L2P1, FSKATSN, and L2P2, PVKATTA were selected. These peptides shared no homology with the starting template Aβ_29–35. The results showed that all selected peptides could bind Aβ₄₂, inhibit fibril formation, and disaggregate pre-formed fibrils. To further improve the metabolic stability of selected peptides from primary and secondary libraries, their retro-inverso (RI) analogs were evaluated (86). All RI peptide ligands, such as KAR-R1, L2P1a-RI, L2P1b-RI, L2P2a-RI, and L2P2b-RI, inhibited Aβ fibrillation and disaggregated pre-formed fibrils, and reduced Aβ₄₂-induced toxicity in PC-12 cells.

THE CURRENT STATE AND FUTURE DIRECTIONS OF Aβ INHIBITORY PEPTIDES IN ALZHEIMER’S DISEASE

A wide range of peptide-based inhibitors has been evaluated in cellular and animal models as new therapeutic compounds for inhibition of Aβ aggregation. While experimental studies generated promising results, only a few of these inhibitory peptides have been successful enough to enter clinical trials. NAP or Davunetide peptide with NAPVSIPQ sequence, derived from the activity-dependent neuroprotective protein (ADNP), was reported in 2003 by Gozes et al. (97). NAP was able to inhibit Aβ aggregation, disassemble pre-formed fibrils, and protect the neuronal cells from Aβ induced toxicity. Though NAP demonstrated benefits in phase II clinical trials for mild cognitive impairment, it failed in a phase III trial (98–100). PPI-1019 peptide (APAN), with a sequence of D-(H-[(Me-L)-VFFL]NH₂), is an N-methylated peptide which is derived from the D-enantiomeric Cholyl-LVFFA-NH₂ that could inhibit Aβ aggregation and the induce toxicity in experimental studies (101). The phases I and II clinical trials of APAN was completed in patients with mild-moderate Alzheimer’s disease in 2005 (NCT00100282, NCT00100334), but the outcome of this study is still unknown (https://clinicaltrials.gov/, last assessed 28 October 2020). Other reported inhibitory peptides including, D3 (102), D-Trp-Aib−OH (44), D-4F (36), TAT-R1-OR2 (48), NL-RI-OR2-TAT90 (103), and R1-OR2 (49) have shown considerable efficacy in preclinical trials, but they have not yet entered clinical trials.

The reality is that, to date, there is no cure for Alzheimer’s disease. Only optimism remains. Therefore, it is necessary to discover potential peptides for testing in clinical trials. The current inhibitory peptides have certain limitations such as poor BBB permeability and high cytotoxicity. To overcome these problems and further improve the inhibitory activity, a number of studies have focused on peptide–nanostructure conjugates (PNCs) approach that provides an opportunity to increase the capabilities of both these classes of materials (55, 104, 105). Nanostructures could be considered as a potential vehicle to overcome poor BBB permeability and bring hope for neurodegenerative diseases therapy due to their size and various surface modifications (106). As an example, multivalent inhibitors can be developed against Aβ aggregation by decorating gold nanoparticles with VVIA and LPFFD (107). The PNCs approach gives a fascinating insight into the fields of diagnosis and treatment, and provides new opportunities for the design of high-performance peptides (108, 109).

CONCLUSION

Despite a better understanding of the pathogenic mechanisms of Alzheimer’s disease, finding efficient therapeutic compounds to prevent or halt the progression of Alzheimer’s disease continues to be a challenge. Aβ aggregation inhibition-based approaches are being developed with the aim to stop disease progression. While the reported inhibitory peptides have considerable advantages over other compounds, and experimental evidence has been encouraging, bench-to-bedside has not yet become a reality. Therefore, adequate knowledge of binding interactions of these peptides with their biological targets, the ligand-target complex, is required to design more accurate therapeutic biomolecules. Peptide inhibitors have unique properties, particularly, high selectivity, low accumulation in tissues, low side-effects and toxicity, and different chemical and biological synthesis routes when compared with other compounds. As researchers continue to focus on rational design, characterization, optimization, and interaction between the inhibitor and the Aβ peptide complex, more peptide inhibitors are expected to succeed in clinical trials.

Conflict of Interest: The authors declare no potential conflicts of interest with respect to research, authorship and/or publication of this chapter.

Copyright and Permission Statement: We confirm that the materials included in this chapter do not violate copyright laws. Where relevant, appropriate permissions have been obtained from the original copyright holder(s). All original sources have been appropriately acknowledged and/or referenced.

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