Thiophilic interaction chromatography of Alzheimer's β‐amyloid peptides

Abstract: Alzheimer's disease is characterized by a progressive formation of senile plaques in the brain, the major constituent of which is β‐amyloid (Aβ) peptide, a proteolytic product of the transmembrane β‐amyloid precursor protein (APP). Prior to the measurement of levels of the Aβ peptide for diagnostic purposes, this peptide must be isolated from the myriad of proteins resident in the human serum. Thiophilic interaction chromatography is an effective method for the isolation of proteins and peptides containing clusters of aromatic residues such as tryptophan, phenylalanine and tyrosine. The purpose of the present study was to develop a protocol for binding and recovery of Aβ peptides (1–38), (1–40) and (1–42) to T‐gels by varying T‐gel type and elution conditions such as the salt concentration and type of eluent. We established the minimal salt concentration necessary for the binding of the Aβ(1–40) peptide to the 3S‐gel; binding at that concentration was subsequently compared with that of model proteins, lysozyme and α‐chymotrypsin and this methodology was extended to 2S‐gels and PyS. β‐Amyloid peptide (1–40) showed a remarkably strong affinity for all three types of T‐gels in comparison to lysozyme and α‐chymotrypsin and was found to bind best to 2S‐gel.     

Design and characterization of a membrane permeable N‐methyl amino acid‐containing peptide that inhibits Aβ1–40 fibrillogenesis

Abstract: Alzheimer's disease, Huntington's disease and prion diseases are part of a growing list of diseases associated with formation of β‐sheet containing fibrils. In a previous publication, we demonstrated that the self‐association of the Alzheimer's β‐amyloid (Aβ) peptide is inhibited by peptides homologous to the central core domain of Aβ, but containing N‐methyl amino acids at alternate positions. When these inhibitor peptides are arrayed in an extended, β‐strand conformation, the alternating position of N‐methyl amino acids gives the peptide two distinct faces, one exhibiting a normal pattern of peptide backbone hydrogen bonds, but the other face having limited hydrogen‐bonding capabilities due to the replacement of the amide protons by N‐methyl groups. Here, we demonstrate, through two‐dimensional NMR and circular dichroic spectroscopy, that a pentapeptide with two N‐methyl amino acids, Aβ16–20m or Ac‐K(Me)LV(Me)FF‐NH₂, does indeed have the intended structure of an extended β‐strand. This structure is remarkably stable to changes in solvent conditions and resists denaturation by heating, changes in pH (from 2.5 to 10.5), and addition of denaturants such as urea and guanindine‐HCl. We also show that this peptide, despite its hydrophobic composition, is highly water soluble, to concentrations > 30 mm , in contrast to the nonmethylated congener, Aβ16–20 (Ac‐KLVFF‐NH₂). The striking water solubility, in combination with the hydrophobic composition of the peptide, suggested that the peptide might be able to pass spontaneously through cell membranes and model phospholipid bilayers such as unilamellar vesicles. Thus, we also demonstrate that this peptide is indeed able to pass spontaneously through both synthetic phospholipid bilayer vesicles and cell membranes. Characterization of the biophysical properties of the Aβ16–20m peptide may facilitate the application of this strategy to other systems as diverse as the HIV protease and chemokines, in which there is dimerization through β‐strand domains.     

Specific binding of amyloid‐β‐protein to IMR‐32 neuroblastoma cell membrane

In flow cytometry using two detecting methods, we have found that amyloid‐β‐protein(1–40) [Aβ(1–40)] has high affinity to IMR‐32 neuroblastoma cell membrane when it is aggregated to form β‐sheet conformation, whereas random coil small Aβ‐species has low affinity. The difference in the binding ability to the cell membranes well accounts for the cytotoxicity of Aβ(1–40); namely, aggregated β‐sheet Aβ(1–40) gives cytotoxicity higher than random coil Aβ(1–40). Specific binding between Aβ(1–40) and ganglioside GM1 of the raft‐like domain in lipid membrane is suggested from a surface plasmon resonance (SPR) experiment.     

Inhibition of β‐amyloid toxicity by short peptides containing N‐methyl amino acids

Abstract: Single N‐methyl amino acid‐containing peptides related to the central hydrophobic region β_16–20 (Lys‐Leu‐Val‐Phe‐Phe) of the β‐amyloid protein are able to reduce the cytotoxicity of natural β_1–42 in PC12 cell cultures. N‐methyl phenylalanine analogs yield statistically significant increments in cell viability (Student's t‐test < 0.01%) and are nontoxic in the same assay. These promising results indicate that these peptide molecules could be a starting point for the development of potential therapeutic compounds for the treatment of Alzheimer's disease.     

Protective effects of β‐sheet breaker α/β‐hybrid peptide against amyloid β‐induced neuronal apoptosis in vitro

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid‐β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid‐β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β‐sheet breaker α/β‐hybrid peptide (BSBHp) and the underlying mechanisms against Aβ₄₀‐induced neurotoxicity in human neuroblastoma SH‐SY5Y cells. Cells were pretreated with the peptide Aβ₄₀ to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca²⁺, and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ₄₀‐induced toxicity by retaining cell viability, suppressing generation of ROS, Ca²⁺ levels, and effectively protects neuronal apoptosis by suppressing pro‐apoptotic protein Bax and up‐regulating antiapoptotic protein Bcl‐2. These results suggest that α/β‐hybrid peptide has neuroprotective effects against Aβ₄₀‐induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.     

Synthesis and 11C‐labelling of (E,E)‐1‐(3′,4′‐dihydroxystyryl)‐4‐(3′‐methoxy‐4′‐hydroxystyryl) benzene for PET imaging of amyloid deposits†

Carboxylic acid derivatives of the amyloid‐binding dye Congo red do not enter the brain well and are thus unable to serve as in vivo amyloid‐imaging agents. A neutral amyloid probe, (E,E)‐1‐(3′,4′‐dihydroxystyryl)‐4‐(3′‐methoxy‐4′‐hydroxystyryl)benzene ( 3 ), devoid of any carboxylate groups has been designed and synthesized via a 12‐step reaction sequence with a total yield of 30%. The unsymmetric compound 3 has also been labelled with C‐11 via [¹¹C]methyl iodide ([¹¹C]CH₃I) methylation of a symmetric 4,4′‐dimesyl protected precursor followed by deprotection. Preliminary evaluation indicated that compound 3 selectively stained plaques and neurofibrillary tangles in post‐mortem AD brain, and exhibited good binding affinity (K_i=38±8 nM) for Aβ(1–40) fibrils in vitro. In vivo pharmacokinetic studies indicated that [¹¹C] 3 exhibited higher brain uptake than its carboxylic acid analogs and good clearance from normal control mouse brain. [¹¹C] 3 also exhibited specific in vivo binding to pancreatic amyloid deposits in the NOR‐beta transgenic mouse model. These results justify further investigation of 3 and similar derivatives as surrogate markers for in vivo quantitation of amyloid deposits. Copyright © 2002 John Wiley & Sons, Ltd.     

Conformational properties of hybrid peptides containing α‐ and ω‐amino acids*

Abstract: This review briefly surveys the conformational properties of guest ω‐amino acid residues when incorporated into host α‐peptide sequences. The results presented focus primarily on the use of β‐ and γ‐residues in αω sequences. The insertion of additional methylene groups into peptide backbones enhances the range of accessible conformations, introducing additional torsional variables. A nomenclature system, which permits ready comparisons between α‐peptides and hybrid sequences, is defined. Crystal structure determination of hybrid peptides, which adopt helical and β‐hairpin conformations permits the characterization of backbone conformational parameters for β‐ and γ‐residues inserted into regular α‐polypeptide structures. Substituted β‐ and γ‐residues are more limited in the range of accessible conformation than their unsubstituted counterparts. The achiral β,β‐disubstituted γ‐amino acid, gabapentin, is an example of a stereochemically constrained residue in which the torsion angles about the C^β–C^γ (θ₁) and C^α–C^β (θ₂) bonds are restricted to the gauche conformation. Hybrid sequences permit the design of novel hydrogen bonded rings in peptide structures.     

Significantly different contact patterns between Aβ40 and Aβ42 monomers involving the N‐terminal region

Aβ42 peptide, with two additional residues at C‐terminus, aggregates much faster than Aβ40. We performed equilibrium replica‐exchange molecular dynamics simulations of their monomers using our residue‐specific force field. Simulated ³J_HNHα‐coupling constants agree excellently with experimental data. Aβ40 and Aβ42 have very similar local conformational features, with considerable β‐strand structures in the segments: A2‐H6 ( A ), L17‐A21 ( B ), A30‐V36 ( C ) of both peptides and V39‐I41 ( D ) of Aβ42. Both peptides have abundant A‐B and B‐C contacts, but Aβ40 has much more contacts between A and C than Aβ42, which may retard its aggregation. Only Aβ42 has considerable A ‐ B ‐ C ‐ D topology. Decreased probability of A ‐ C contact in Aβ42 relates to the competition from C ‐ D contact. Increased A ‐ C contact probability may also explain the slower aggregation of A2T and A2V mutants of Aβ42.     

Synthesis and secondary structural studies of penta(acetyl-Hmb)Aβ(1–40)

Abstract: The Fmoc solid phase synthesis of Aβ(1–40), a strongly aggregating peptide found in Alzheimer’s disease brain, was performed using 2-hydroxy-4-methoxybenzyl (Hmb) backbone amide protection. Hmb-Gly residues were incorporated using N^α-Fmoc-Hmb-Gly-OH rather than N,O-bisFmoc-Hmb-Gly-OPfp. Amino acid acylation of the sterically hindered Hmb-amino acids was monitored using ‘semi-on-line’ MALDI-TOF-MS in a novel application of this technique which significantly simplified the successful incorporation of these residues. Standard coupling conditions in N,N-dimethylformamide (DMF) were used throughout the synthesis. Comparative structural studies of acetyl-Hmb-protected and native Aβ(1–40) were performed to investigate the structural basis of Hmb-mediated disaggregation. The incorporation of backbone amide protection was observed by circular dichroism spectroscopy and gel electrophoresis to strongly affect the solution structure of Aβ(1–40). Despite the reported structure-breaking activity of Hmb groups, penta(acetyl-Hmb)Aβ(1–40) was found to adopt both α-helix and intermolecular β-sheet conformations. In 100% TFE a mixed α-helix/random coil structure was formed by the protected peptide indicating reduced α-helical propensity relative to Aβ(1–40). The protected peptide formed β-sheet structures in aqueous buffer. Gel electrophoresis indicated that, unlike native Aβ(1–40), penta(acetyl-Hmb)Aβ(1–40) did not form large aggregate species.     

Structure‐based derivation of peptide inhibitors to target TGF‐β1 receptor for the suppression of hypertrophic scarring fibroblast activation

The intermolecular recognition and interaction between human transforming growth factor β‐1 (TGF‐β1) and its cognate receptor TβRII have been implicated in the pathological condition of hypertrophic scarring (HS). Here, we attempted to rationally derive peptide inhibitors from the complex interface of TGF‐β1 with TβRII to disrupt such interaction for the suppression of fibroblast activation involved in HS. A synthetic strategy that integrated computational design and fluorescence‐based assay was described to examine the structural basis and energetic property of TGF‐β1–TβRII crystal structure, from which a small peptide segment in the complex binding site was stripped artificially. Molecular dynamics simulations revealed that the linear peptide possesses a large intrinsic disorder that would incur considerable entropy penalty upon binding to TβRII; the peptide segment was then extended and cyclized by introducing a disulfide bond across its terminal residues that were premutated to cysteine. Normal mode analysis indicated that, as expected, the peptide flexibility was largely reduced upon the cyclization, and thus, the entropy penalty was minimized substantially, consequently promoting the spontaneous binding of peptide to TβRII. Fluorescence polarization assay confirmed that all linear peptides are typical non‐binders of TβRII (K_d = ND), while the designed cyclic peptides exhibit moderate or high affinity with K_d at micromolar level.     

Aggregation of Aβ Alzheimer's disease-related peptide studied by dynamic light scattering

The aggregation behavior of the major component of Alzheimer's disease-related, amyloid peptides, Aβ-(1–40) and Aβ-(1–42), was studied in solution using dynamic light scattering. With most solvents employed, we found fibrils coexisting with oligomeric Aβ species. Pronounced differences were observed in aggregation of Aβ-(1–40) and (1–42) sequences in acetonitrile-water mixtures. Cofactors such as Zn²⁺ were found to induce deaggregation of Aβ instead of aggregation. The results indicated that the initial state of the peptide immediately after synthesis is rather poorly defined. Using freezing instead of lyophilization after the final peptide synthesis step, may partially relieve these problems.     

From β‐N‐tert‐butyloxycarbonyl N‐carboxyanhydrides to β‐aminoacid derivatives

Abstract: β‐N‐tert‐butyloxycarbonyl‐N‐carboxyanhydrides are very reactive β‐amino acid derivatives. They react cleanly and smoothly with different nucleophiles like aminoesters, enolates, N‐methyl‐d ‐glucamine, amidoximes to afford in good to excellent yields peptides, β‐amino ketocompounds, β‐aminosugars and functionalized disubstituted 1,2,4‐oxadiazoles.     

A New Series of 1,3‐Dihidro‐Imidazo[1,5‐c]thiazole‐5,7‐Dione Derivatives: Synthesis and Interaction with Aβ(25‐35) Amyloid Peptide

Deposition of senile plaques composed of fibrillar aggregates of Aβ‐amyloid peptide is a characteristic hallmark of Alzheimer’s disease. A widely employed approach in the study of anti‐Alzheimer agents involves the identification of substances able to prevent amyloid aggregation, or to disaggregate the amyloid fibrils through a direct structural interaction with the soluble or aggregated forms of the peptide. Here, we report the synthesis of a set of 1,3‐dihydro‐3,6‐disubstituted‐imidazo[1,5‐c]thiazole‐5,7‐dione derivatives supporting different alkyl, aryl and alkylamine side chains. The ability of these compounds to interact with the Aβ(25‐35) peptide was evaluated using circular dichroism, nuclear magnetic resonance and thioflavin fluorescence spectroscopy. A molecular model for Aβ(25‐35)–ligand interactions was calculated by molecular docking procedures. Our data show that the ability of the synthesized compounds to modify the structural behaviour of Aβ(25‐35) varies as a function of the overall structural features of the ligands rather contributions from specific individual substituents.     

Fibril formation by amyloid-β proteins may involve β-helical protofibrils

Abstract: We have proposed that amyloid fibrils contain subunits (protofibrils) that are formed from β-strands wound into continuous 2–3 nm-diameter β-helices. Subsequent lateral aggregation of the β-helices to form the widely observed 5–12 nm-diameter fibrils could be promoted by hydrophobic residues on the exterior of the postulated β-helix. A number of short peptide fragments of the amyloid-β (Aβ) proteins, such as Aβ34–42 [LMVGGVVIA], the nine-residue, carboxyl-terminal portion of Aβ1–42, can also form amyloid fibrils. In the present study, it was found that a β-helix formed from Aβ34–42 accounts for features suggested by published rotational resonance solid-state NMR data, including an anomalous conformation about the Gly-37–Gly-38 region and exaggerated pleating. An analogue of Aβ34–42 was synthesized in which the hydrophobic groups on the exterior of the postulated β-helix were replaced with glutamates, giving LEVGGVEIE. The analogue was completely soluble at pH 7, but at pH 2.5 it produced 2–2.5 nm-diameter fibrils which did not associate into larger-diameter bundles. The results of this study support the proposal that amyloid fibrils are formed from β-helical subunits.     

Effects of transthyretin on thyroxine and β‐amyloid removal from cerebrospinal fluid in mice

Transthyretin (TTR) is a binding protein for the thyroid hormone thyroxine (T₄), retinol and β‐amyloid peptide. TTR aids the transfer of T₄ from the blood to the cerebrospinal fluid (CSF), but also prevents T₄ loss from the blood‐CSF barrier. It is, however, unclear whether TTR affects the clearance of β‐amyloid from the CSF. This study aimed to investigate roles of TTR in β‐amyloid and T₄ efflux from the CSF. Eight‐week‐old 129sv male mice were anaesthetized and their lateral ventricles were cannulated. Mice were infused with artificial CSF containing ¹²⁵I‐T₄/³H‐mannitol, or ¹²⁵I‐Aβ40/³H‐inulin, in the presence or absence of TTR. Mice were decapitated at 2, 4, 8, 16, 24 minutes after injection. The whole brain was then removed and divided into different regions. The radioactivities in the brain were determined by liquid scintillation counting. At baseline, the net uptake of ¹²⁵I‐T₄ into the brain was significantly higher than that of ¹²⁵I‐Aβ40, and the half time for efflux was shorter (¹²⁵I‐T₄, 5.16; ³H‐mannitol, 7.44; ¹²⁵I‐Aβ40, 8.34; ³H‐inulin, 10.78 minutes). The presence of TTR increased the half time for efflux of ¹²⁵I‐T₄ efflux, and caused a noticeable increase in the uptake of ¹²⁵I‐T₄ and ¹²⁵I‐Aβ40 in the choroid plexus, whilst uptakes of ³H‐mannitol and ³H‐inulin remained similar to control experiments. This study indicates that thyroxine and amyloid peptide effuse from the CSF using different transporters. TTR binds to thyroxine and amyloid peptide to prevent the loss of thyroxine from the brain and redistribute amyloid peptide to the choroid plexus.     

Identification of the molecular interaction site of amyloid β peptide by using a fluorescence assay

Abstract: β‐Amyloid peptides (Aβ) are the main protein components of neuritic plaques and are important in the pathogenesis of Alzheimer's disease. It is reported that Aβ itself is not toxic; however, it becomes toxic to neuronal cells once it has aggregated into amyloid fibrils by peptide–peptide interactions. In this study, to specify the molecular mechanism of aggregation, a novel fluorescence assay was designed. For this purpose, possible partial peptides (38 types of 5‐mer) were synthesized on solid‐phase. The molecular interactions were examined by a fluorescence probe possessing Lys‐Leu‐Val‐Phe‐Phe (KLVFF) as a molecular recognition site. KLVFF is known to be a minimum sequence for formation of the Aβ aggregate. A specific interaction was observed between labeled and immobilized KLVFF. It suggests that the aggregation of Aβ was controlled by the recognition of KLVFF itself by hydrophobic and electrostatic interactions.     

Integrin α5β1: a new purification strategy based on immobilized peptides

Abstract: Novel efficient and robust affinity chromatography material: There are several strategies known for the purification of integrins by affinity chromatography, but the disadvantages of common strategies like insufficient selectivity or compelling conditions for the elution still require alternatives. A new strategy, based on the immobilized C‐terminally modified peptide Ac‐Gly‐Ala‐c‐(Cys^SS‐Arg‐Arg‐Glu‐Thr‐Ala‐Trp‐Ala‐Cys^SS)‐Gly‐Ala‐O(CH₂CH₂O)₂CH₂CH₂‐NH₂ allows for the affinity purification of the integrin α₅β₁. While RGD peptides have been proven in the past to be inappropriate for selective purification of integrins by affinity chromatography, the new peptide can be efficiently used for selective enrichment of the integrin α₅β₁. It is a specific ligand of the target protein, but does not contain an RGD sequence. The application of well‐characterized affinity chromatography material with a site‐specifically immobilized peptide allows to obtain integrin α₅β₁ in a single chromatography step without contamination by other integrins. This process combines the advantages of a selective and monospecific protein‐ligand recognition with mild elution conditions and a low sensitivity of the immobilized ligand with respect to column regeneration.     

Entrapping unusual folding characteristics of the β‐Ala residues in a model peptide: Boc‐β‐Ala‐Aib‐β‐Ala‐NHCH3

Abstract: Crystal structure analysis of a model peptide: Boc‐β‐Ala‐Aib‐β‐Ala‐NHCH₃ (β‐Ala: 3‐amino propionic acid; Aib: α‐aminoisobutyric acid) revealed distinct conformational preferences for folded [φ≈136°, µ ≈ ?62°, ψ ≈100°] and semifolded [φ ≈ 83°, µ ≈ ?177°, ψ ≈ ?117°] structures of the N‐ and C‐terminus β‐Ala residues, respectively. The overall folded conformation is stabilized by unusual N_i···H‐N_i+1 and nonconventional C–H···O intramolecular hydrogen bonding interactions.     

6‐Ethyl‐N,N′‐bis(3‐hydroxyphenyl)[1,3,5]triazine‐2,4‐diamine (RS‐0466) Enhances the Protective Effect of Brain‐Derived Neurotrophic Factor on Amyloid β‐Induced Cytotoxicity in Cortical Neurones

Abstract: Amyloid β peptide in the senile plaques of patients with Alzheimer's disease is considered to be responsible for the pathology of Alzheimer's disease. We have previously reported that 6‐ethyl‐N,N′‐bis(3‐hydroxyphenyl)[1,3,5]triazine‐2,4‐diamine, RS‐0466, is capable of significantly inhibiting amyloid β‐induced cytotoxicity in HeLa cells. To determine various profiles of RS‐0466, we investigated whether RS‐0466 would enhance the neuroprotective effect of brain‐derived neurotrophic factor on amyloid β_1–42‐induced cytotoxicity in rat cortical neurones. Consistent with previous observations, brain‐derived neurotrophic factor ameliorated amyloid β_1–42‐induced cytotoxicity. Furthermore, co‐application of RS‐0466 enhanced the neuroprotective effect of brain‐derived neurotrophic factor. RS‐0466 also reversed amyloid β_1–42‐induced decrease of brain‐derived neurotrophic factor‐triggered phosphorylated Akt. These results raise the possibility that RS‐0466 or one of its derivatives has potential to enhance the neuroprotective effect of brain‐derived neurotrophic factor, and could serve as a therapeutic agent for patients with Alzheimer's disease.     

Synthesis of a non‐peptidic PET tracer designed for α5β1 integrin receptor

Arginine–glycine–aspartic acid (RGD)‐containing peptides have been traditionally used as PET probes to noninvasively image angiogenesis, but recently, small selective molecules for α₅β₁ integrin receptor have been developed with promising results. Sixty‐one antagonists were screened, and tert‐butyl (S)‐3‐(2‐((3R,5S)‐1‐(3‐(1‐(2‐fluoroethyl)‐1H‐1,2,3‐triazol‐4‐yl)propanoyl)‐5‐((pyridin‐2‐ylamino)methyl)pyrrolidin‐3‐yloxy)acetamido)‐2‐(2,4,6‐trimethylbenzamido)propanoate (FPMt) was selected for the development of a PET tracer to image the expression of α₅β₁ integrin receptors. An alkynyl precursor (PMt) was initially synthesized in six steps, and its radiolabeling was performed according to the azide–alkyne copper(II)‐catalyzed Huisgen's cycloaddition by using 1‐azido‐2‐[¹⁸F]fluoroethane ([¹⁸F]12). Different reaction conditions between PMt and [¹⁸F]12 were investigated, but all of them afforded [¹⁸F]FPMt in 15 min with similar radiochemical yields (80–83%, decay corrected). Overall, the final radiopharmaceutical ([¹⁸F]FPMt) was obtained after a synthesis time of 60–70 min in 42–44% decay‐corrected radiochemical yield. Copyright © 2014 John Wiley & Sons, Ltd.