Conformationally fixed amyloid β42 derivative

Abstract

To provide an amyloid β42 derivative applicable to development of an antibody capable of specifically recognizing the toxic turn conformation identical to that of human wild-type amyloid β42, a reagent for quick diagnosis of ultra-early Alzheimer's disease, a compound that binds to amyloid β42 with toxic conformation and alleviates the toxicity, and development of antibody pharmaceuticals.SOLUTION: Design and synthesis of a conformationally fixed amyloid β42 derivative, having a stereostructure in which the 28th and 42nd amino acid residues are crosslinked and a turn structure between β-sheet structures formed by the amino acid sequence is fixed at the 22nd glutamic acid and 23rd aspartic acid residues, and in which the amino acid sequence other than at the 28th and 42nd positions is identical to that of wild-type amyloid β42, yielded a derivative showing extremely high neurotoxicity and immediate and lasting β-sheet structure formation and aggregation capability.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to derivatives of amyloid β42 that are intramolecularly crosslinked and fixed so as to maintain a toxic turn structure in the sequence of the wild-type amyloid β42 peptide. [Background technology]

[0002] Alzheimer's disease (AD) is a neurodegenerative disorder. Currently, few effective treatments are available. Therefore, elucidating the pathogenesis of AD and establishing a fundamental treatment are highly desired. In recent years, it has become clear that amyloid beta protein (Aβ), which accumulates in senile plaques, plays a key role in the pathogenesis of AD, and its aggregation is toxic to neurons. AD is characterized by the accumulation of Aβ, primarily consisting of 40 or 42 amino acids (hereafter referred to as Aβ40 and Aβ42, respectively). It has been found that this is the etiology of AD. However, Aβ42 is thought to play a more important role in the pathogenesis of AD than Aβ40 due to its high aggregation tendency and neurotoxicity. However, therapeutic AD treatments, such as anti-Aβ antibodies and anti-tau antibodies, have so far met with limited success. Currently, only drugs designed to slow the progression of AD (e.g., Aricept and Memantine) have been approved. This failure may be due to treatment being initiated after the disease has progressed. Early diagnosis and appropriate treatment may potentially enable treatment of AD. Initially, removal of Aβ aggregates was thought to be effective in AD treatment, due to the neuronal death surrounding senile plaques (Aβ aggregates). However, because the amount of senile plaques (Aβ aggregates) is poorly correlated with AD pathology, metastable Aβ oligomers, rather than Aβ aggregates, are now considered to be the toxic entity. Here, "metastable" refers to the ability to exist in this state for approximately 24 hours, allowing for confirmation of the Aβ oligomer structure by mass spectrometry or evaluation of the toxicity of Aβ oligomers in cell-based assays. However, little is known about which of the numerous Aβ oligomer species are responsible for cytotoxicity or the higher-order structure they adopt. Furthermore, these Aβ oligomers exist in equilibrium between monomers and amyloid fibrils, making it impossible to isolate and characterize pure oligomers.

[0003] Some Aβ oligomers are thought to be particularly important for inducing synaptic toxicity in the early stages of AD and for inducing hyperphosphorylation of tau protein, which ultimately leads to neuronal loss. Based on analyses of Aβ42 using systematic proline substitution, solid-state NMR, and electron spin resonance spectroscopy, the inventor, Irie et al., proposed a dimer and trimer model (Figure 1, bottom) characterized by a turn structure between the β-sheet structures in the central part of the amino acid sequence [near glutamic acid residue at position 22 (E22) and aspartic acid residue at position 23 (D23)] (hereinafter referred to as the "toxic turn structure" because it is responsible for cytotoxicity; Figure 1, top left) and a hydrophobic core at the C-terminus (Figure 1, top right). Specifically, we believe that Aβ oligomers with a toxic turn structure and those with a certain molecular weight (e.g., 20-mer or greater) are necessary for toxicity (ACS Chem. Neurosci. 2017, 8 (4), 807-816). Furthermore, Irie et al. have proposed the "toxic conformation theory" (Non-Patent Document 1), which states that Aβ42 forms toxic oligomers by bending particularly in the central region between the intermolecular β-sheet structures (near positions 22 and 23) (bottom right of Figure 1). Here, "toxicity" refers to cell growth inhibitory activity, cell death, inhibition of long-term potentiation (LTP) (synaptic toxicity or neurotoxicity), etc. The amino acid sequence of human wild-type Aβ42 (WT-Aβ42) is shown in SEQ ID NO: 1 and Figure 1 in the Sequence Listing.

[0004] According to the toxic conformation theory, the formation of a turn structure in the central region of Aβ42 (particularly the amino acid sequence from positions 20 to 25, centered around positions 22 and 23) can be understood as efficiently promoting the oxidation of the sulfur atom of the methionine residue at position 35 (M35) by the phenoxy radical of the tyrosine residue at position 10 (Y10) generated by reaction with copper ions, etc., and stabilizing the resulting cation radical with the carboxylate anion at the C-terminus. As a result, the hydrophobicity of the uncharged C-terminal core of Aβ42 increases, making it more susceptible to aggregation, leading to the formation of dimers and trimers. On the other hand, the neutral radical at the C-terminus of Aβ42 remains within the oligomers, which is thought to enable it to exert oxidative stress on cells for a long period of time. Aβ42 also forms turn structures at the glycine residue at position 25 (G25) and the serine residue at position 26 (S26) (Figure 1, upper right). However, these turn structures have been shown to be significantly less toxic than Aβ42 with turn structures at positions 22 (E22) and 23 (D23). Therefore, the toxic turn structures at positions 22 and 23 are thought to be significantly involved in the cytotoxicity of Aβ42. In the toxic turn structure, both the carboxyl groups at E22 and D23 face outward (cis configuration) relative to the main chain. Through these studies, Irie et al. have identified key structural factors in Aβ42 that contribute to Aβ42 aggregation and neuronal toxicity, and have developed Aβ42 derivatives locked into potentially harmful conformations (see Patent Document 1).

[0005] Irie et al. have previously prepared a derivative of an Aβ peptide (E22P-Aβ9-35) consisting of residues 9 to 35 of Aβ42, in which E22 is replaced with proline, conjugated to a carrier protein. Using this derivative as an antigen, they developed the conformation-specific antibody 24B3 (also referred to as the "IBL-102 antibody"). The 24B3 antibody is an anti-amyloid β monoclonal antibody that was initially thought to specifically recognize Aβ42, which has a toxic turn structure (see Patent Document 2). The 24B3 antibody binds strongly to E22P-Aβ and its dimer model, but has low binding affinity to WT-Aβ42 monomers, which lack the toxic turn structure. This monoclonal antibody has achieved some success in diagnosing AD using human cerebrospinal fluid (see Non-Patent Documents 1 and 2). A sandwich ELISA using the 24B3 antibody and an existing N-terminal antibody was jointly developed by Irie et al. and the Immunobiology Institute. Analysis of cerebrospinal fluid from AD patients, with the cooperation of Professor Takahiko Tokuda of Kyoto Prefectural University of Medicine (currently at the National Institutes for Quantum and Radiological Science and Technology), revealed that the ratio of toxic conformers (the amount of Aβ molecular species with toxic turns) to total Aβ42 was significantly higher in AD patients than in non-AD patients. This ratio also tended to be higher in patients with idiopathic normal pressure hydrocephalus, suggesting that it could be an indicator of progression to AD (collaboration with Associate Professor Madoka Nakajima of Juntendo University). Furthermore, since the 24B3 antibody inhibited the neuronal toxicity of Aβ42 more potently than several commercially available antibodies, Irie et al., in collaboration with the Chiba University Graduate School of Medicine, investigated the therapeutic effects of the antibody on AD model mice (Tg2576). The elevated plus maze and nest-building tests demonstrated improved cognitive function. The sandwich ELISA using the 24B3 antibody was released as a research reagent by the Immunobiology Institute in November 2016 and is used by many researchers both in Japan and overseas.

[0006] Subsequent X-ray cocrystal structure analysis revealed that the 24B3 antibody specifically recognizes and binds to the E22P-Aβ derivative and its dimer model, which are thought to have the toxic turn structure described above. However, the binding mode to the toxic turn structure of WT-Aβ42 in vivo remains unknown. This is partly due to the use of the E22P-Aβ derivative, a proline-substituted form not present in the human brain, as the antigen (hapten). Therefore, to develop antibodies more useful for AD diagnosis, especially at an early stage, it was considered desirable to develop an Aβ42 derivative with the same amino acid sequence as WT-Aβ42 at the toxic turn without proline substitution or lactam formation between the 22 and 23 side chains.

[0007] In a subsequent study, Irie et al. systematically synthesized derivatives in which two positions, mainly E22 and D23, were substituted with cysteine residues (C), and then DMSO-oxidized each to form intramolecular disulfide (SS) bonds. As a result, they developed an Aβ42 derivative with a cross-linked structure in which both the leucine residue at position 17 (L17) and the lysine residue at position 28 (K28) were substituted with cysteine residues to form intramolecular SS bonds [L17C,K28C-17,28-SS-Amyloid-β42, hereafter referred to as "17,28-SS-Aβ42(CC)" (SEQ ID NO: 2)]. Hereafter, the Aβ42 derivative with a cross-linked structure in which the toxic turn structure is fixed by the intramolecular SS bond will be referred to as "SS-Aβ42." This 17,28-SS-Aβ42(CC) has the same amino acid sequence as WT-Aβ42 at E22 and D23, which form the toxic turn structure of WT-Aβ42 and the four residues before and after (positions 18-27), but E22 does not contain a proline substitution, which is not present in vivo, and the toxic turn conformation is stably fixed without forming a lactam between the side chains of E22 and D23. 17,28-SS-Aβ42(CC)[17,28-SS-Aβ42] was found to exhibit aggregation and neurotoxicity comparable to or greater than E22P-Aβ42, which had previously been highly aggregative and had the highest neurotoxicity (see Patent Document 3 and Non-Patent Document 3). In addition to 17,28-SS-Aβ(CC), derivatives in which L17 is substituted with a homocysteine residue and K28 with a cysteine residue to form an intramolecular SS bond [17,28-SS-Aβ(hC-C)] and derivatives in which L17 is substituted with a cysteine residue and K28 with a homocysteine residue to form an intramolecular SS bond [17,28-SS-Aβ(C-hC)] have also been shown to exhibit high aggregation and neurotoxicity (see Patent Document 4). Hereinafter, Aβ42 derivatives that form an intramolecular SS bond between positions 17 and 28 will be collectively referred to as "17,28-SS-Aβ42." [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-265189 [Patent Document 2] WO2016 / 092865 International Publication [Patent Document 3] Patent Publication No. 2021-123551 (Patent No. 7357354) [Patent Document 4] Patent Publication No. 2022-155700 [Non-patent literature]

[0009] [Non-Patent Document 1] Irie, K., Biosci.Biotechnol.Biochem.2020,84(1),1-16. [Non-patent document 2] Kazuhiro Irie, Journal of the Society of Synthetic Organic Chemistry, 2019,77(12),1201-1208. [Non-patent document 3] Y. Matsushima,et.al.,Chemical Communications,2020,56(29),4118-4121 Summary of the Invention [Problem to be solved by the invention]

[0010] Solid-state NMR analysis of the three-dimensional structure of WT-Aβ42 aggregates revealed that, as shown in Figure 2, an intramolecular ionic bond between the lysine residue at position 28 (K28) and the alanine residue at position 42 (A42) causes the C-terminal side to bend in the opposite direction to the toxic turn at positions 22 and 23, forming an intermolecular β-sheet structure (Xiao, Y. et al., Nat. Struct. Mol. Biol. 2015, 22, 499-505. Colvin, MT et al., J. Am. Chem. Soc. 2016, 138, 9663-9674. Walti, MA et al., Proc. Natl. Acad. Sci. USA 2016, 113, E4976-E4984. Note that the "a" in Walti's name is an alternating vowel). On the other hand, as shown in the figure, WT-Aβ40 is two residues shorter at the C-terminus than WT-Aβ42. Therefore, the amino group of the lysine residue at position 28 does not form an intramolecular ionic bond with the carboxyl group of the valine residue at position 40, but instead forms an intramolecular ionic bond with the carboxyl group of the aspartic acid residue at position 23. As a result, the C-terminus is linearly extended, forming an intermolecular β-sheet structure (Petkova, AT et al., Proc. Natl. Acad. Sci. USA 2002, 99, 16742-16747.). These structural differences strongly support the existence of a toxic turn structure in WT-Aβ42 and explain why WT-Aβ40, which exhibits little toxicity, is less likely to form a toxic turn structure.

[0011] In addition, a recent report on the structural analysis of WT-Aβ42 aggregates in the brains of AD patients using cryo-electron microscopy (Cryo-EM) revealed that divalent metal ions coordinate to the carboxylates of E22 and D23, stabilizing the toxic turn, as shown in Figure 3. Among the structural analyses, Type-I revealed that the amino group of K28 and the carboxyl group of A42 are in close intramolecular proximity (Yang, Y. et al., Science 2022, 375, 167-172). Based on this, it is suggested that turn formation at E22 and D23 is promoted by ionic bond formation between the amino group of K28 and the carboxyl group of A42. In Type-II, shown in the same figure, K28 and A42 of WT-Aβ42 form intermolecular ionic bonds with neighboring A42 and K28 of WT-Aβ42, respectively. In the case of Type-II, it is thought that it is easy to form mature fibrils and that it is difficult to exist for long periods in the toxic oligomeric state.In contrast, Type-I, because adjacent WT-Aβ42 molecules do not form intermolecular ionic bonds between K28 and A42, is thought to have a conformation that is difficult to form mature fibrils and can exist for long periods in the toxic oligomeric state.

[0012] In Aβ42 derivatives with intramolecular SS bonds at positions 17 and 28, such as the aforementioned 17,28-SS-Aβ42, strong SS bonds (covalent bonds) are formed in the regions surrounding the toxic turn structure at positions 22 and 23 in the central region (position 17 on the N-terminal side and position 28 on the C-terminal side). This results in an extended C-terminal structure like WT-Aβ40, which is thought to differ from the natural three-dimensional structure of WT-Aβ42.

[0013] In light of the above, the inventors have been working to develop conformationally locked derivatives that more closely resemble the toxic conformation of WT-Aβ42. As a result, they have come up with the idea of creating derivatives with the same fixed toxic turn structure at E22 and D23 as WT-Aβ42 by appropriately substituting K28 and A42 of WT-Aβ42 with other amino acid residues and cross-linking between positions 28 and 42. This aims to provide Aβ42 derivatives with stable conformations that exhibit even higher levels of aggregation and neuronal toxicity. [Means for solving the problem]

[0014] In other words, the conformationally fixed amyloid β42 derivative of the present invention (hereinafter referred to as the "conformationally fixed Aβ42 derivative") has a three-dimensional structure in which the amino acid residues at positions 28 and 42 in the 42-amino acid amyloid β (Aβ42) are cross-linked, thereby fixing the turn structure near the glutamic acid residue at position 22 and the aspartic acid residue at position 23 between the β-sheet structure, and is characterized in that all amino acid sequences except for the amino acid residues at positions 28 and 42 are the same as those of WT-Aβ42.

[0015] Based on the above considerations, we predict that similar to the C-terminal bend in WT-Aβ42 aggregates that forms a toxic turn structure centered on E22 and D23, the present invention will result in a conformationally locked Aβ42 derivative in which the C-terminal bend structure is formed by cross-linking the amino acid residues at positions 28 and 42, thereby strongly locking the toxic turn structure centered on E22 and D23. As a result, the conformationally locked Aβ42 derivative will have a conformation that is more likely to form intermolecular β-sheets and will exhibit more stable aggregation and cytotoxicity than WT-Aβ42. Such conformationally locked Aβ42 derivatives of the present invention will be extremely useful compounds for the generation of conformation-specific antibodies, which will contribute greatly not only to the development of antibodies that can specifically detect toxic oligomers but also to the development of antibody drugs for human use.

[0016] Among such conformationally restricted Aβ42 derivatives, those specifically established in the present invention are those in which the lysine residue at position 28 (K28) is substituted with a cysteine residue (C28) or a homocysteine residue (hC28), and the alanine residue at position 42 (A42) is substituted with a cysteine residue (C28) or a homocysteine residue (hC28), forming an intramolecular disulfide (SS) bond between the amino acid residues at positions 28 and 42.

[0017] The conformationally restricted Aβ42 derivatives include a derivative in which both K28 and A42 are substituted with cysteine residues [K28C,A42C-28,42-SS-Amyloid-β42, hereinafter referred to as "28,42-SS-Aβ42(CC)" (SEQ ID NO: 3)], a derivative in which K28 is substituted with a homocysteine residue [homocysteine (Hcy), hereinafter referred to as "hC"] and the A42 residue is substituted with a cysteine residue [K28hC-A42C-28,42-SS-Amyloid-β42, hereinafter referred to as "28,42-SS-Aβ42(hC-C)" (SEQ ID NO: 4)], and Amyloid-β42 includes a derivative in which K28 is substituted with a cysteine residue and A42 is substituted with a homocysteine residue [K28C,A42hC-28,42-SS-Amyloid-β42, hereinafter referred to as "28,42-SS-Aβ42(C-hC)" (SEQ ID NO: 5)], and a derivative in which both K28 and A42 are substituted with homocysteine residues [K28hC,A42hC-28,42-SS-Amyloid-β42, hereinafter referred to as "28,42-SS-Aβ42(hC-hC)" (SEQ ID NO: 6)]. These are collectively referred to as "28,42-SS-Aβ42." The amino acid sequence of 28,42-SS-Aβ42 is identical to that of wild-type Aβ42 except for positions 28 and 42.

[0018] The conformationally locked Aβ42 derivative of the present invention, "28,42-SS-Aβ42," was tested as described below. It exhibited cytotoxicity at 1 / 1000th the concentration of WT-Aβ42 and was far more cytotoxic than 17,28-SS-Aβ42 described in Patent Document 3. Furthermore, CD spectroscopy of 28,42-SS-Aβ42 suggested that it formed a β-sheet structure immediately after dissolution in PBS buffer and remained stable for a long period of time. Aggregation studies in PBS buffer also suggested that it formed a β-sheet structure immediately after dissolution. Furthermore, the addition of a small amount of 28,42-SS-Aβ42 to WT-Aβ42 dramatically increased the aggregation rate and fluorescence intensity, indicating that 28,42-SS-Aβ42 already formed a toxic oligomeric structure (a toxic turn structure at E22 and D23) from the beginning. Furthermore, electron microscopy of the aggregates in PBS buffer revealed that 28,42-SS-Aβ42 ultimately formed prominent fibrils.

[0019] These results demonstrate that 28,42-SS-Aβ42 is a useful molecular target discovery tool for addressing the amyloid hypothesis. Furthermore, using this hapten to generate conformation-specific antibodies is likely to yield antibodies capable of specifically and sensitively detecting toxic oligomers present in human cerebrospinal fluid and plasma. Humanization of these antibodies could potentially lead to antibody drugs superior to the recently approved lecanemab. Furthermore, natural organic compounds (including functional ingredients in foods) that inhibit 28,42-SS-Aβ42 aggregation are likely to be useful as AD preventative drugs. Therefore, this invention could also serve as a valuable tool molecule for developing small molecule compounds and nucleic acid drugs that specifically act on toxic oligomers. [Effects of the Invention]

[0020] The present invention provides an Aβ42 derivative that is fixed in a toxic conformation similar to that of WT-Aβ42 aggregates by cross-linking the amino acid residues at positions 28 and 42 in the Aβ42 derivative. The Aβ42 derivative exhibits extremely high cytotoxicity and immediate aggregability at physiological concentrations and can stably maintain an oligomeric state for a long period of time. Therefore, it can be provided as an antibody that can be used as a reagent or diagnostic agent for ultra-early AD diagnosis, and as a substance that will be more useful than ever before for the development of therapeutic drugs. [Brief explanation of the drawings]

[0021] [Figure 1] Diagram showing the toxic conformational theory of Aβ42. [Figure 2] FIG. 1 shows the three-dimensional structure analysis of Aβ42 aggregates and Aβ40 aggregates by solid-state NMR. [Figure 3] Cryo-electron microscopy shows the three-dimensional structure of Aβ42 aggregates in the brains of AD patients. [Figure 4] FIG. 1 shows the design of 28,42-SS-Aβ42 in one embodiment of the present invention. [Figure 5] FIG. 1 shows the results of a toxicity test (MTT assay) of each Aβ42 on THP1 cells. [Figure 6] FIG. 1 shows the results of a toxicity test (MTT assay) of each Aβ42 on SH-SY5Y cells. [Figure 7] A diagram showing the CD spectra of each Aβ42. [Figure 8] FIG. 1 shows the results of aggregation tests of each Aβ42 using Th-T fluorescence method. [Figure 9] FIG. 1 shows the results of a test of the effect of conformationally restricted Aβ42 derivatives on the aggregation ability of WT-Aβ42. [Figure 10] FIG. 1 shows the results of morphological observation of each Aβ42 aggregate by transmission electron microscope (TEM). DETAILED DESCRIPTION OF THE INVENTION

[0022] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. First, as examples of Aβ42 derivatives that have the same toxic turn structure at the central region E22 and D23 as WT-Aβ42 and are fixed in a toxic conformation as WT-Aβ42, four Aβ42 derivatives, 28,42-SS-Aβ42, were designed in which K28 and A42 were replaced with cysteine or homocysteine residues, respectively, to form SS bonds at positions 28 and 42, and cross-linking was performed between positions 28 and 42. These 28,42-SS-Aβ42 fragments were prepared by microwave-assisted Fmoc solid-phase synthesis followed by intramolecular crosslinking via DMSO oxidation. The synthesis method follows the procedure reported by Matsushima et al. (Matsushima, Y., Yanagita, RC, Irie, K., Chem. Commun. 2020, 56, 4118-4121).

[0023] Specifically, each Aβ42 derivative was synthesized using the Fmoc strategy (Fmoc solid-phase peptide synthesis) on a fully automated microwave peptide synthesizer (Biotage, Initiator + Alstra). After chain elongation, each peptide resin was deprotected and cleaved from the resin using a trifluoroacetic acid-based cocktail. The crude peptide was oxidized with 10% DMSO in water containing 0.1% NH4OH to form an intramolecular disulfide bond. The oxidized crude peptide was purified by HPLC. The purified peptides were analyzed by high-resolution electrospray ionization-time-of-flight mass spectrometry (HR-ESI-qTOF-MS), and mass spectral data were consistent with the calculated values.

[0024] The predicted three-dimensional structure of 28,42-SS-Aβ42(C-C) is such that, as shown in the lower part of Fig. 4, the C-terminal side is bent outward and fixed by an intramolecular S-S bond between positions 28 and 42, thereby having a conformation that fixes the toxic turn structures at E22 and D23. For comparison, the three-dimensional structure deduced by solid-state NMR and cryo-electron microscopy of WT-Aβ42 is shown again in the upper part of the same figure. 28,42-SS-Aβ42(C-C) is presumed to be very similar to the structure of WT-Aβ42 in which the amino group of K28 and the carboxy group of A42 are bent outward and fixed by an intramolecular ionic bond, and the toxic turn structures at E22 and D23 are fixed. From this, it is considered that, similar to WT-Aβ42, in 28,42-SS-Aβ42(C-C) as well, the coordination of metal ions to the carboxylate anions of E22 and D23 stabilizes the toxic turn structure and makes it easier to form intermolecular β-sheets. Although not shown in the figure, it is expected that 28,42-SS-Aβ42(hC-C), 28,42-SS-Aβ42(C-hC), and 28,42-SS-Aβ42(hC-hC) also tend to adopt a similar toxic three-dimensional structure.

Example

[0025] <Cytotoxicity test (MTT assay) against THP1 cells> First, the neurotoxicity of 28,42-SS-Aβ42 against THP-1 cells, a type of immune system cell, was evaluated using an MTT assay. Four types of 28,42-SS-Aβ42 were evaluated: 28,42-SS-Aβ42 (CC), 28,42-SS-Aβ42 (hC-C), 28,42-SS-Aβ42 (C-hC), and 28,42-SS-Aβ42 (hC-hC). For comparison, WT-Aβ42 and 17,28-SS-Aβ42 (17,28-SS-Aβ42 (CC)) (Patent Document 3) were used. The experimental procedure was as follows: THP-1 cells (TIB-202, obtained from the American Type Culture Collection, USA) were cultured in suspension in RPMI 1640 medium supplemented with 10% FBS (Fetal Bovine Serum). THP-1 cells were differentiated by treatment with 100 nM PMA (phorbol 12-myristate-13-acetate, Sigma-Aldrich, USA) in 5% FBS / RPMI 1640 for 48 hours. After 48 hours, the PMA-containing medium was replaced with fresh 5% FBS / RPMI 1640 for 24 hours. For cell differentiation in a 96-well plate (IWAKI, Japan), 25,000 THP-1 cells were seeded per well. Cell viability was assessed using the MTT Cell Count Kit (Nacalai Tesque, Japan) according to the manufacturer's protocol. Each Aβ42 sample, prepared in 400 μM DMSO after HFIP (hexafluoro-2-propanol) treatment, was diluted to final concentrations of 0.1, 0.3, 1, 3, 10, 30, and 100 nM in 1% FBS / RPMI 1640. For the control sample, DMSO was mixed with 1% FBS / RPMI 1640 to a final concentration of 0.25%. 100 μL of conditioned medium containing each Aβ42 sample at the above concentrations was added to differentiated THP-1 cells and incubated at 37°C for 24 hours. The medium containing each Aβ42 sample was replaced with 110 μL of solution (10 μL MTT and 100 μL 1% FBS / RPMI 1640), and the cells were further incubated at 37°C for 3 hours. 100 μL of MTT solubilization solution was added to each well and mixed by pipetting.The absorbance at 570 nm was measured using a plate reader (Infinite M200). Cell viability was calculated by setting the absorbance obtained with THP-1 cells incubated in 0.25% DMSO / 1% FBS / RPMI 1640 medium as 100%. This experiment was performed three times, and the values were averaged. Error bars indicate standard deviation (SD). Figure 5(a) shows the results of an MTT assay comparing 28,42-SS-Aβ42 (hC-C) with WT-Aβ42 and 17,28-SS-Aβ42. Figure 5(b) shows the results of an MTT assay for 28,42-SS-Aβ42 (hC-C), 28,42-SS-Aβ42 (CC), 28,42-SS-Aβ42 (C-hC), and 28,42-SS-Aβ42 (hC-hC).

[0026] As a result of the MTT assay on THP1 cells, as shown in Fig. 5(a), it was found that 28,42-SS-Aβ42 (hC-C) showed significant cytotoxicity against THP-1 cells even at a concentration about 1 / 1000 (100 pM) compared to WT-Aβ42. Also, it showed overwhelmingly higher cytotoxicity compared to 17,28-SS-Aβ42. Further, from Fig. 5(b), it was found that the cytotoxicity of 28,42-SS-Aβ42 (C-C) and 28,42-SS-Aβ42 (C-hC) was the highest, followed by 28,42-SS-Aβ42 (hC-C) and 28,42-SS-Aβ42 (hC-hC) in order of decreasing cytotoxicity, but all four types of 28,42-SS-Aβ42 showed higher cytotoxicity than WT-Aβ42 and 17,28-SS-Aβ42. So far, the amyloid hypothesis has been somewhat questioned because the cytotoxicity shown by Aβ42 in vitro was more than 100 times the physiological concentration, but this test strongly suggests that Aβ42 may adopt a conformation that shows toxicity at the pM (picomolar concentration) level through interactions with biomolecules and coordination with metal ions in vivo. In particular, the four types of 28,42-SS-Aβ42, which are conformation-fixed Aβ42 derivatives, are effective as target-searching molecular tools for approaching the essence of the amyloid hypothesis. If stereospecific antibodies are prepared using these as haptens, it is highly likely that antibodies capable of specifically and sensitively detecting toxic oligomers present in human cerebrospinal fluid and plasma can be obtained. Also, by humanizing the antibody, there is a possibility of becoming an antibody drug that surpasses recently approved lecanemab. Among the four types of 28,42-SS-Aβ42, in particular, three types, 28,42-SS-Aβ42 (C-C), 28,42-SS-Aβ42 (C-hC), and 28,42-SS-Aβ42 (hC-C), were shown to be particularly promising by the MTT assay using THP1 cells.

[0027] <Cytotoxicity test (MTT assay) against SH-SY5Y cells> Next, the cytotoxicity of the four types of 28,42-SS-Aβ42 against SH-SY5Y cells, a human nervous system cell line, was evaluated using an MTT assay. The evaluation subjects and controls were the same as those used in the MTT assay for THP1 cells described above. First, SH-SY5Y cells were suspended in medium (a 1:1 mixture of E-MEM and Ham's F12 medium containing 10% FBS) and plated at a nearly confluent concentration (2.5 x 10) in a 96-well plate (Biocoat Collagen I Cellware, Corning, USA) (100 μL / well). 4Cells were seeded at 100 μL / well. The 96-well plate was then incubated at 37°C for 24 hours. Each HFIP-treated Aβ42 sample, prepared as a 400 μM DMSO solution, was diluted in a 1:1 mixture of E-MEM and Ham's F12 supplemented with 1% FBS to final concentrations of 0.0176, 0.088, 0.44, 2.2, and 11 μM. As a control, DMSO was added to the medium to a final concentration of less than 0.25%. 10 μL of the resulting peptide solution was added to 100 μL of medium and incubated at 37°C for 24 hours. Then, 15 μL / well of the dye solution from the MTT Cell Titer 96 Non-Radioactive Cell Proliferation Assay Kit (Promega, USA) was added to the cells and incubated at 37°C for 4 hours. Next, 100 μL / well of solubilization / stop solution was added to the cells. The resulting cell lysates were then incubated overnight at room temperature in the dark and then measured at 570 nm using a microplate reader (Multiskan FC). The absorbance obtained upon addition of solvent (0.25% DMSO) was set as 100%. Figure 6(a) shows the results of an MTT assay comparing 28,42-SS-Aβ42 (hC-C) with WT-Aβ42 and 17,28-SS-Aβ42. Figure 6(b) also shows the results of an MTT assay for 28,42-SS-Aβ42 (hC-C), 28,42-SS-Aβ42 (CC), 28,42-SS-Aβ42 (C-hC), 28,42-SS-Aβ42 (hC-hC), and WT-Aβ42.

[0028] Results of the MTT assay on SH-SY5Y cells. From Figure 6(a), 28,42-SS-Aβ42(hC-C) showed significantly higher cytotoxicity against SH-SY5Y cells compared to WT-Aβ42 and higher cytotoxicity than 17,28-SS-Aβ42. Also, from Figure 6(b), the cytotoxicity of 28,42-SS-Aβ42(hC-C) and 28,42-SS-Aβ42(C-hC) was the highest, followed by 28,42-SS-Aβ42(C-C) and 28,42-SS-Aβ42(hC-hC) showing comparable high cytotoxicity, but all four types of 28,42-SS-Aβ42 showed much higher cytotoxicity than WT-Aβ42.

[0029] From the results of the MTT assay on THP1 cells and SH-SY5Y cells, it was found that the cytotoxicity of the four types of 28,42-SS-Aβ42 was significantly higher than that of WT-Aβ42 and 17,28-SS-Aβ42, and the cytotoxicity was higher against immune cells than against nerve cells. In particular, it was found that 28,42-SS-Aβ42(hC-C) and 28,42-SS-Aβ42(C-hC), two derivatives in which either position 28 or 42 was substituted with a cysteine residue and the other was substituted with a homocysteine residue, showed extremely high cytotoxicity.

[0030] <CD Spectrum Measurement Test> CD spectra were measured for 28,42-SS-Aβ42(C-C) and 28,42-SS-Aβ42(hC-C). The comparison samples were WT-Aβ42 and 17,28-SS-Aβ42 [17,28-SS-Aβ42(C-C)]. A 0.1 mm quartz cell was used for CD spectrum measurement. Each sample of Aβ42 dissolved at 200 μM in 0.15% NH4OH was diluted 10-fold with PBS buffer mixed with 50 mM sodium phosphate and 100 mM NaCl (pH 7.4) to a final concentration of 20 mM and incubated at 37 °C. After 0, 2, 4, 6, 24, and 48 hours, an equal-volume aliquot (200 μL) of the diluted solution was loaded into the quartz cell, and after subtracting the spectrum of the solvent, the CD spectrum was recorded at 190 - 260 nm. The CD spectra of WT-Aβ42 and 17,28-SS-Aβ42 are shown in Figs. 7(a) and (b), and the CD spectra of 28,42-SS-Aβ42(C-C) and 28,42-SS-Aβ42(hC-C) are shown in Figs. 6(c) and (d).

[0031] In the CD spectrum, the absorption spectrum (the trough of the waveform) near 220 nm indicates the presence of the β-sheet structure of Aβ42. In WT-Aβ42, the presence of a prominent β-sheet structure was confirmed after 6 hours. On the other hand, in 17,28-SS-Aβ42, a prominent β-sheet structure was observed in just 2 hours. In contrast, for 28,42-SS-Aβ42(C-C) and 28,42-SS-Aβ42(hC-C), it was suggested that they formed a β-sheet structure immediately after dissolution in PBS buffer (0 hour) and that it existed stably over a long period of time.

[0032] <Aggregation test by Th-T fluorescence method> The aggregation ability of four types of 28,42-SS-Aβ42 [28,42-SS-Aβ42 (CC), 28,42-SS-Aβ42 (hC-C), 28,42-SS-Aβ42 (C-hC), and 28,42-SS-Aβ42 (hC-hC)] was assessed by thioflavin T (Th-T) fluorescence. The controls were wild-type Aβ42 and 17,28-SS-Aβ42 [17,28-SS-Aβ42 (CC)]. For the experiment, 390 μL of PBS buffer containing 50 mM sodium phosphate and 100 mM NaCl (pH 7.4) was dispensed into a 1.5 mL tube, and 10 μL of 1 mM Th-T solution was added. A 100 μL aliquot of each Aβ42 sample, dissolved at 100 μM in 0.15% NH4OH, was added to a tube of PBS / Th-T solution to a final concentration of 10 μM. The samples were dispensed into a black 96-well plate (100 μL / well) and incubated at room temperature (23°C). Fluorescence intensity was automatically measured every 10 minutes for 24 hours using a microplate reader (Fluoroskan Ascent, Thermo Fisher Scientific, USA) at an excitation wavelength of 430 nm and an emission wavelength of 485 nm. For each sample, net fluorescence was measured by subtracting the fluorescence intensity of the solvent containing no Aβ42 as a blank. Figure 8(a) shows the results of the Th-T fluorescence assay comparing 28,42-SS-Aβ42 (CC) and 28,42-SS-Aβ42 (hC-C) with WT-Aβ42 and 17,28-SS-Aβ42. Furthermore, the results of aggregation assays using the Th-T fluorescence method for four types of 28,42-SS-Aβ42 [28,42-SS-Aβ42 (CC), 28,42-SS-Aβ42 (hC-C), 28,42-SS-Aβ42 (C-hC), and 28,42-SS-Aβ42 (hC-hC)] and WT-Aβ42 are shown in Figure 8(b).

[0033] In the aggregation assay, the fluorescence intensity of WT-Aβ42 gradually increased after dissolution in PBS buffer, peaked at approximately 7 hours, and then gradually decreased. The fluorescence intensity of 17,28-SS-Aβ42 peaked approximately 1 hour after dissolution in PBS buffer, then gradually decreased, reaching a similar level to that of WT-Aβ42 after approximately 8–10 hours. Thereafter, the fluorescence intensity gradually decreased, similar to that of WT-Aβ42. In contrast, 28,42-SS-Aβ42 (CC) and 28,42-SS-Aβ42 (hC-C) showed high fluorescence intensity immediately after dissolution in PBS buffer, confirming the formation of β-sheet structure immediately after dissolution. 28,42-SS-Aβ42 (CC) peaked at approximately 3 hours and then gradually decreased, but maintained a higher fluorescence intensity than WT-Aβ42 and 17,28-SS-Aβ42. For 28,42-SS-Aβ42(hC-C), the fluorescence intensity peaked immediately after dissolution in PBS buffer and then gradually decreased. After 3 hours, the fluorescence intensity remained relatively high, although it was higher than that of WT-Aβ42 and 17,28-SS-Aβ42 and lower than that of 28,42-SS-Aβ42(CC). 28,42-SS-Aβ42(hC-C) and 28,42-SS-Aβ42(C-hC) showed the fastest aggregation rate and highest aggregation tendency, while 28,42-SS-Aβ42(CC) and 28,42-SS-Aβ42(hC-hC) showed the next fastest aggregation rate and highest fluorescence intensity, with similar trends.

[0034] In particular, 28,42-SS-Aβ42 (hC-C) and 28,42-SS-Aβ42 (C-hC) readily formed oligomers after dissolution in PBS buffer, suggesting that they may be useful tools for developing small molecules and nucleic acid drugs that specifically target toxic oligomers. Furthermore, 28,42-SS-Aβ42 (hC-C) and 28,42-SS-Aβ42 (C-hC) exhibited the highest cytotoxicity and aggregation potential, followed by 28,42-SS-Aβ42 (CC) and 28,42-SS-Aβ42 (hC-hC). These results suggest that the appropriate distance of the intramolecular disulfide bonds in 28,42-SS-Aβ42 significantly affects cytotoxicity and aggregation potential.

[0035] Furthermore, in response to this result, in order to examine the effect of 28,42-SS-Aβ42(hC-C) on the aggregation of WT-Aβ42, samples were prepared by adding small amounts [molar ratios of 10:1 (10%) and 50:1 (2%)] of 28,42-SS-Aβ42(hC-C) to WT-Aβ42, and aggregation tests were performed using the Th-T fluorescence method (Figure 9). As a result, even when only a very small amount of 28,42-SS-Aβ42(hC-C) was added, the aggregation rate and fluorescence intensity of WT-Aβ42 increased dramatically. This result supports the idea that 28,42-SS-Aβ42(hC-C) forms the toxic turn structure at E22 and D23 from the beginning and acts as an aggregation nucleus.

[0036] <Morphological Observation of Aβ42 Aggregates by Transmission Electron Microscope (TEM)> Regarding the aggregates of 28,42-SS-Aβ42(C-C), 28,42-SS-Aβ42(hC-C), WT-Aβ42, and 17,28-SS-Aβ42 [17,28-SS-Aβ42(C-C)], the fibril formation process was morphologically observed by TEM. In the experiment, a sample solution prepared by adding 20 μM of each Aβ42 aggregate to PBS buffer (mixed with 50 mM sodium phosphate and 100 mM NaCl, pH 7.4) was incubated for 48 hours, and then observed by TEM [JEM-1400Plus (product name), manufactured by JEOL Ltd. (Japan)]. After centrifuging each sample solution (4°C, 15,000 rpm, 10 minutes), the supernatant was removed, and the pellet was resuspended in water (20 μL) using a vortex for 1 minute and then centrifuged using a tabletop microcentrifuge. The sample suspension (5 μL) was incubated on a 400-mesh carbon-coated copper grid [thickness: 30 - 40 nm, EM Fine Grid (product name), manufactured by Nisshin EM Co., Ltd. (Japan)] for 5 minutes and negatively stained twice with 2% uranyl acetate [manufactured by Merck & Co., Inc. (USA)]. The stained samples were morphologically observed by TEM (Figure 10).

[0037] Morphological observations by TEM revealed that, compared with WT-Aβ42 aggregates, the aggregates of Aβ42 derivatives (crosslinked forms) that form intramolecular disulfide bonds, 28,42-SS-Aβ42 (CC), 28,42-SS-Aβ42 (hC-C), and 17,28-SS-Aβ42, all ultimately formed prominent fibrils.

[0038] These results suggest that 28,42-SS-Aβ42 derivatives, which are conformationally restricted Aβ42 derivatives in which the toxic turn structure at E22 and D23 is fixed by cross-linking between positions 28 and 42, are far superior to the conventional 17,28-SS-Aβ42 in terms of high cytotoxicity, rapid and stable β-sheet structure formation, aggregate formation, and fibril formation. These results suggest that these derivatives will be valuable targets for the development of novel antibodies against the toxic turn structure of Aβ42.

[0039] It should be noted that the present invention is not limited to 28,42-SS-Aβ42, which are the four types of conformationally restricted Aβ42 derivatives used in this example. In addition, any conformationally restricted Aβ42 derivative that has the same amino acid sequence as WT-Aβ42 except for positions 28 and 42 and in which positions 28 and 42 are cross-linked by an appropriate method can be expected to produce effects similar to those of the above-described examples. Therefore, the present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the present invention. [Industrial Applicability]

[0040] The conformationally restricted Aβ42 derivatives of the present invention are extremely useful for the development of antibodies that recognize the same toxic turn structure as wild-type Aβ42, and for the development of rapid diagnostic reagents, test agents, and antibody drugs for very early stage AD based on these antibodies.

Claims

1. In amyloid β42, which consists of 42 amino acid residues, A conformationally fixed amyloid β42 derivative characterized in that the amino acid residues at positions 28 and 42 are cross-linked, and a turn structure consisting of a glutamic acid residue at position 22 and an aspartic acid residue at position 23 located between the β-sheet forming regions has a fixed conformation, and the entire amino acid sequence except for the amino acid residues at positions 28 and 42 has the same amino acid sequence as wild-type amyloid β42.

2. 2. The conformationally fixed amyloid β42 derivative according to claim 1, wherein the lysine residue at position 28 is substituted with a cysteine residue or a homocysteine residue, the alanine residue at position 42 is substituted with a cysteine residue or a homocysteine residue, and the turn structure is fixed by forming an intramolecular disulfide bond between the amino acid residues at positions 28 and 42.

3. 3. The conformationally restricted amyloid β42 derivative according to claim 2, wherein the lysine residue at position 28 is substituted with a homocysteine residue and the alanine residue at position 42 is substituted with a cysteine residue.

4. 3. The conformationally restricted amyloid β42 derivative according to claim 2, wherein the lysine residue at position 28 is substituted with a cysteine residue and the alanine residue at position 42 is substituted with a homocysteine residue.

5. 3. The conformationally restricted amyloid β42 derivative according to claim 2, wherein the lysine residue at position 28 and the alanine residue at position 42 are both substituted with cysteine residues.

6. 3. The conformationally restricted amyloid β42 derivative according to claim 2, wherein the lysine residue at position 28 and the alanine residue at position 42 are both substituted with homocysteine residues.

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