Atsuhiko Taniguchi

“Click Peptide“: pH‐Triggered in Situ Production and Aggregation of Monomer Aβ1–42

Into neutral: We demonstrate the unique features of a pH click peptide based on an O‐acyl isopeptide method. Under acidic conditions, the click peptide remains in a monomeric form. Upon increase of the pH to 7.4, the click peptide is quickly able to convert into Aβ1–42 through an O‐to‐N intramolecular acyl migration. Further study using this pH click peptide would elucidate the pathological role of Aβ1–42 in Alzheimers disease.

Attenuation of the Aggregation and Neurotoxicity of Amyloid‐β Peptides by Catalytic Photooxygenation

Alzheimers disease (AD), a progressive severe neurodegenerative disorder, is currently incurable, despite intensive efforts worldwide. Herein, we demonstrate that catalytic oxygenation of amyloid-β peptides (Aβ) might be an effective approach to treat AD. Aβ1-42 was oxygenated under physiologically-relevant conditions (pH 7.4, 37 °C) using a riboflavin catalyst and visible light irradiation, with modifications at the Tyr(10) , His(13) , His(14) , and Met(35) residues. The oxygenated Aβ1-42 exhibited considerably lower aggregation potency and neurotoxicity compared with native Aβ. Photooxygenation of Aβ can be performed even in the presence of cells, by using a selective flavin catalyst attached to an Aβ-binding peptide; the Aβ cytotoxicity was attenuated in this case as well. Furthermore, oxygenated Aβ1-42 inhibited the aggregation and cytotoxicity of native Aβ.

Controlled Production of Amyloid β Peptide from a Photo-Triggered, Water-Soluble Precursor “Click Peptide“

In biological experiments, poor solubility and uncontrolled assembly of amyloid β peptide (Aβ) 1–42 pose significant obstacles to establish an experiment system that clarifies the function of Aβ1–42 in Alzheimers disease (AD). Herein, as an experimental tool to overcome these problems, we developed a water‐soluble photo‐“click peptide” with a coumarin‐derived photocleavable protective group that is based on an O‐acyl isopeptide method. The click peptide had nearly 100‐fold higher water solubility than Aβ1–42 and did not self‐assemble, as the isomerized structure in its peptide backbone drastically changed the conformation that was derived from Aβ1–42. Moreover, the click peptide afforded Aβ1–42 quickly under physiological conditions (pH 7.4, 37 °C) by photoirradiation followed by an O–N intramolecular acyl migration. Because the in situ production of intact Aβ1–42 from the click peptide could improve the difficulties in handling Aβ1–42 caused by its poor solubility and highly aggregative nature, this click peptide strategy would provide a reliable experiment system for investigating the pathological function of Aβ1–42 in AD.

Rational Design and Identification of a Non‐Peptidic Aggregation Inhibitor of Amyloid‐β Based on a Pharmacophore Motif Obtained from cyclo[‐Lys‐Leu‐Val‐Phe‐Phe‐]

Inhibition of pathogenic protein aggregation may be an important and straightforward therapeutic strategy for curing amyloid diseases. Small-molecule aggregation inhibitors of Alzheimers amyloid-β (Aβ) are extremely scarce, however, and are mainly restricted to dye- and polyphenol-type compounds that lack drug-likeness. Based on the structure-activity relationship of cyclic Aβ16-20 (cyclo-[KLVFF]), we identified unique pharmacophore motifs comprising side-chains of Leu(2), Val(3), Phe(4), and Phe(5) residues without involvement of the backbone amide bonds to inhibit Aβ aggregation. This finding allowed us to design non-peptidic, small-molecule aggregation inhibitors that possess potent activity. These molecules are the first successful non-peptidic, small-molecule aggregation inhibitors of amyloids based on rational molecular design.

Switchable photooxygenation catalysts that sense higher-order amyloid structures

Proteins can misfold into amyloid structures that are associated with diseases; however, the same proteins often have important biological roles. To degrade selectively the amyloid form without affecting the fraction of functional protein is, therefore, an attractive goal. Here we report target-state-dependent photooxygenation catalysts that are active only when bound to the cross-β-sheet structure that is characteristic of pathogenic aggregated amyloid proteins. We show these catalysts can selectively oxygenate the amyloid form of amyloid β-protein (Aβ) 1-42 in the presence of non-amyloid off-target substrates. Furthermore, photooxygenation with a catalyst that bears an Aβ-binding peptide attenuated the Aβ pathogenicity in the presence of cells. We also show that selective photooxygenation is generally applicable to other amyloidogenic proteins (amylin, insulin, β2-microglobulin, transthyretin and α-synuclein) and does not affect the physiologically functional non-aggregate states of these proteins. This is the first report of an artificial catalyst that can be selectively and reversibly turned on and off depending on the structure and aggregation state of the substrate protein.

Design, synthesis, and biophysical properties of a helical Aβ1–42 analog: Inhibition of fibrillogenesis and cytotoxicity

The aggregation of amyloid beta-peptide (Abeta) into beta-sheet-rich aggregates is a crucial step in the etiology of Alzheimers disease. Helical forms of Abeta have been suggested to be intermediates in the aggregation process of the peptide in aqueous phase, micelles and membranes. A stable helical Abeta analog would be useful to investigate the role of helical intermediates in fibrillization by Abeta. Here we designed a helical analog by simply cross-linking the Cys residues of A30C, G37C-Abeta1-42 with 1,6-bismaleimidohexane. The analog assumed a weak alpha-helical conformation in model membranes mimicking lipid raft microdomains of neuronal membranes under conditions in which the wild-type Abeta1-42 formed a beta-sheet, indicating the cross-linking locally induced a helical conformation. Furthermore, addition of equimolar helical Abeta analog significantly reduced the amyloid formation and cytotoxicity by Abeta1-42. Thus, our helical Abeta1-42 is not only a model peptide to investigate the role of helical intermediates in fibrillization by Abeta, but also an inhibitor of Abeta-induced cytotoxicity.

'Click peptide' using production of monomer Aβ from the O-acyl isopeptide: application to assay system of aggregation inhibitors and cellular cytotoxicity.

The O-acyl isopeptide of Aβ1-42 (1), possessing an ester bond at the Gly(25)-Ser(26) sequence, is a water-soluble and non-aggregative precursor molecule and is capable of production of monomer Aβ1-42. The SDS-PAGE result showed that the Aβ1-42, produced from 1, adopted monomeric state at first and then self-assembled to oligomer. The oligomeric state was stabilized by nordihydroguaiaretic acid. The Thioflavin-T (ThT) fluorescence intensity derived from Aβ1-42 (generated from 1) was suppressed by various aggregation inhibitors. Finally, 1 could generate Aβ1-42 via the O-to-N acyl migration under cellular medium conditions and the produced Aβ1-42 exhibited cytotoxicity against PC12 cells. These results suggest that the click peptide system, which enables us to predominantly produce monomer Aβ1-42 under physiological conditions, would be adoptable to various biochemical and biophysical experiments including cellular system to investigate the functions of Aβ1-42.

Synthesis of amyloid β peptide 1–42 (E22Δ) click peptide: pH-triggered in situ production of its native form

Amyloid beta peptide (Abeta) 1-42 is known to be involved in the onset of Alzheimers disease (AD). We developed a click peptide of Abeta1-42 as a useful tool for AD research on the basis of an O-acyl isopeptide method. The click peptide quickly produced intact Abeta1-42 via a pH-dependent O-to-N intramolecular acyl migration (pH-click). Herein, a click peptide (26-O-acyl isoAbeta1-42 (E22Delta)) of a new mutant Abeta1-42 (E22Delta) was synthesized. The mutant click peptide was more water-soluble than Abeta1-42 (E22Delta). Moreover it quantitatively converted to the native peptide under physiological conditions (pH 7.4, 37 degrees C). CD analyses showed a conformational change from a random-coil structure of the click peptide to a beta-sheet structure of the in situ produced Abeta1-42 (E22Delta). This click peptide is a useful precursor of a mutant Abeta1-42 to establish an experiment system for investigating the properties of the mutant.

Asparagine-selective cleavage of peptide bonds through hypervalent iodine-mediated Hofmann rearrangement in neutral aqueous solution

Amide bonds of peptides and proteins are generally unreactive toward hydrolysis, but backbone amide bond cleavage at a specific amino acid-site in an aqueous neutral solution at mild temperature could have many applications. Chemical cleavage methods that complement enzymatic digestion should facilitate the determination of primary structures for peptides and proteins, especially for substrates containing unnatural amino acids and/or chemical modifications that are resistant to enzymatic hydrolysis. As a new entry of site-selective chemical peptide bond cleavage, an asparagine-selective method using diacetoxyiodobenzene (DIB) is described herein. DIB-mediated Hofmann rearrangement at the primary amide moiety of an Asn side chain afforded a five-membered N-acylurea intermediate that was successively hydrolyzed into two peptide fragments. The Asn-selective peptide bond cleavage proceeded in aqueous neutral solution at 37 °C for various oligopeptides (20 examples) with a protected N-terminal, including a disulfide bond-containing peptide, biologically active peptides, and [Pyr11]Aβ11–40, which is associated with Alzheimer’s disease. An unnatural peptide sequence comprising D-amino acids was also successfully cleaved as well. Moreover, this method was used to determine oxidation sites of a photo-oxidized Aβ3–16 derivative that was resistant to enzymatic cleavage.

Discovery of a Human Neuromedin U Receptor 1-Selective Hexapeptide Agonist with Enhanced Serum Stability

Neuromedin U (NMU) activates two NMU receptors (NMUR1 and NMUR2) and is a useful antiobesity drug lead. We report discovery of a hexapeptide agonist, 2-thienylacetyl-Trp1-Phe(4-F)2-Arg3-Pro4-Arg5-Asn6-NH2 (4). However, the NMUR1 selectivity and serum stability of this agonist were unsatisfactory. Through a structure-activity relationship study focused on residue 2 of agonist 4, serum stability, and pharmacokinetic properties, we report here the discovery of a novel NMUR1 selective hexapeptide agonist 7b that suppresses body weight gain in mice.