JP7357354B2 - Amyloid β42 cross-linked analog peptide - Google Patents

Description

The present invention relates to an analog peptide that is a derivative of the wild-type amyloid β42 peptide sequence in which a toxic turn structure is intramolecularly crosslinked and fixed.

Alzheimer's disease (AD) is a type of neurodegenerative disease, but there are currently few effective treatments, and there is a strong desire to elucidate the onset mechanism of AD and establish a fundamental treatment method. In recent years, amyloid-β protein (Aβ) (hereinafter also referred to as "amyloid-β peptide (Aβ)") plays an important role in the onset of AD, and its aggregation (oligomerization) causes neuronal cell toxicity. It has been found that this shows that Initially, it was thought that removing Aβ aggregates, which are amyloid fibrils, would be effective in treating AD because nerve cells around senile plaques (Aβ aggregates) were dying. However, since there is little correlation between the amount of senile plaques and AD pathology, it has come to be considered that metastable Aβ oligomers, rather than amyloid fibrils, are the main cause of toxicity. However, the reality is chaotic. For example, oligomers of 12-mer, 24-mer, and even 100-mer have been reported, with dimers and trimers as basic units, but it is unclear which oligomers are involved in cytotoxicity and how they are related to each other. Little is known about whether it has such a higher-order structure. Furthermore, these oligomers are in an equilibrium state between monomers and fibrils, making it impossible to isolate and determine the structure of pure oligomers.

AD is characterized by the accumulation of amyloid β protein (hereinafter referred to as "Aβ40" and "Aβ42", the same shall apply hereinafter), which mainly consists of 40 or 42 amino acid residues, and is the cause of AD. It has been found that Aβ42 is more aggregating and neurotoxic than Aβ40, but Aβ40 is approximately 10 times more abundant in body fluids than Aβ42. Recently, oligomeric species of Aβ are thought to be particularly important for inducing hyperphosphorylation of tau protein, which exhibits synaptic toxicity in the early onset of AD and ultimately leads to neuronal loss. The present inventors have so far analyzed Aβ42 using a systematic proline substitution method, solid-state NMR method, electron spin resonance method, etc., and have found that the central region (glutamic acid at position 22 (denoted as Glu22 or E22) proposed a dimer and trimer model characterized by a turn structure (hereinafter referred to as a "toxic turn") in the vicinity of aspartic acid (denoted as Asp23 or B22) and a hydrophobic core at the C-terminus. (See Figure 1). In this toxic turn structure, the residues (carboxy groups) of Glu22 and Asp23 both face outward with respect to the main chain (cis configuration). Among these, the present inventors determined that Aβ42, which assumes a harmful conformation, was not found to be toxic by itself, but by clarifying the structural factors of Aβ42 that are key to Aβ42 aggregation and the expression of neuronal cytotoxicity. Analog peptides have been discovered (see Patent Document 1).

Furthermore, the present inventor has proposed a "toxic conformation theory" (see FIG. 2) in which toxic oligomers are formed by bending the central portion of Aβ42 (near Glu22 and Asp23), which is particularly toxic. Here, the amino acid sequence of human wild type Aβ42 is as shown in SEQ ID NO: 1 of the sequence listing and FIG. 2. According to the toxic conformation theory, the turn shape in the central part of Aβ42 is due to the oxidation of the sulfur atom of methionine (Met35) at position 35 by the phenoxy radical of tyrosine (Tyr10) at position 10 generated by reaction with copper ions, etc. It can be understood that the generated cation radical can be efficiently promoted and stabilized by the carboxylate anion at the C-terminus, and as a result, the hydrophobicity of the C-terminal core that has lost its charge increases, making it easier to aggregate, and the We believe that while the oligomerization of oligomers and trimers occurs, the neutral radical at the C-terminus exists within the oligomer, making it possible to apply oxidative stress to cells over a long period of time. .

Therefore, the present inventors developed a conformation-specific antibody "24B3" using a 9- to 35-residue peptide (E22P-Aβ9-35) in which Glu22 is replaced with proline as an antigen, using human cerebrospinal fluid. It has achieved a certain degree of success in AD diagnosis (see Non-Patent Documents 1 and 2). The 24B3 antibody strongly binds to E22P-Aβ and its dimer model by immunizing mice with E22P-Aβ9-35, which mimics the toxic turn structure of Aβ42, bound to a carrier protein. , is a monoclonal antibody that has low binding ability to wild-type Aβ monomer, which does not have many toxic turn structures.

Since the 24B3 antibody recognized the toxic turn structure very specifically, a sandwich ELISA with an existing N-terminal antibody was jointly developed by the present inventors and the Institute of Immunobiology, and Professor Tokuda of Kyoto Prefectural University of Medicine As a result of analyzing the cerebrospinal fluid of AD patients with the cooperation of found. It was also found that this rate tends to be high in patients with idiopathic normal pressure hydrocephalus, and it has been suggested that this rate may be an indicator for determining the transition to AD. Furthermore, since the 24B3 antibody suppressed Aβ42 neuronal cytotoxicity more strongly than several commercially available antibodies, the present inventors, in collaboration with Chiba University Graduate School of Medicine, investigated its therapeutic effect on AD model mice (Tg2576). As a result, cognitive function improvement effects were observed in the elevated plus maze and nest building tests. Sandwich ELISA using the 24B3 antibody was sold as a research reagent by the Institute of Immunobiology in November 2016, and is used by many researchers in Japan and abroad.

Japanese Patent Application Publication No. 2006-265289

Irie, K. , Biosci. Biotechnol. Biochem. 2019, 84, 1-16. Kazuhiro Irie, Journal of the Society of Organic Synthetic Chemistry, 2019, 77, 1201-1208.

However, in order to apply the 24B3 antibody to ultra-early AD diagnosis using plasma, improvements were needed in terms of sensitivity and specificity. One of the reasons for this is thought to be that E22P-Aβ analogue, which is a proline substitute that does not originally exist in the human brain, is used as an antigen (hapten). Furthermore, the E22P-Aβ analogs do not have a tightly fixed conformation around the toxic turn, which can pose problems in the development of ideal antibodies. From this, in order to develop antibodies that are more useful for AD diagnosis, especially for very early diagnosis, it is necessary to use the 24B3 antibody as an antigen at the position of the toxic turn in order to increase the specificity and sensitivity of the 24B3 antibody to the toxic turn of Aβ oligomers. It would be desirable to develop an Aβ42 analog that has a wild-type amino acid sequence over several residues before and after the toxic turn, without proline substitution or lactam formation between side chains.

Recently, the present inventors, in collaboration with Chiba University, succeeded in creating homozygous knock-in mice by crossing chimeric mice with a knock-in human E22P-Aβ sequence that mimics the toxic turn structure of Aβ42. did. At 6 months of age, this mouse exhibited abnormal behavior in the novel object recognition test compared to wild-type mice, and while Aβ trimers were detected in brain slice extracts, monomers were hardly detected. I couldn't. This is an interesting result since trimers of Aβ have been reported to exhibit synaptic toxicity. On the other hand, no loss of nerve cells was observed, and the symptoms were mild. E22P-Aβ42 tends to adopt a toxic turn structure, and forms oligomers more quickly than wild-type Aβ42 in in vitro tests. Nevertheless, the fact that no serious AD symptoms were shown supports that Aβ oligomer alone has low neuronal cytotoxicity and exhibits synaptic toxicity. Following Aβ accumulation, tau phosphorylation is required for the development of AD, and Aβ oligomers may act as a bridge.

The present invention was made with attention to these points, and the main purpose is to fix the toxic turn of wild-type Aβ42 and to achieve extremely high aggregation and neuronal cell toxicity, based on the results of previous research. The present invention provides an intramolecularly cross-linked analog peptide of Aβ42, which has a stable conformation exhibiting the following, and has an amino acid sequence similar to that of wild-type Aβ42 over several residues before and after the toxic turn.

That is, the amyloid β42 cross-linked analog peptide of the present invention (hereinafter referred to as "Aβ42 analog" or "cross-linked Aβ42 analog") has amino acid residues at the 17th position and 28th position in the amyloid β protein (Aβ42), which has 42 amino acid residues. has a three-dimensional structure in which the amino acid residues at positions 22 and 23 have a fixed turn structure between the β-sheet structures according to the amino acid sequence, and at least position 18, which includes this turn structure, It is characterized by having the same amino acid sequence as the wild-type amyloid β42 protein at the 27th amino acid residue from the amino acid residue of .

As a result of the research, Aβ42 analogues with a structure in which amino acid residues at positions 17 and 28 are cross-linked, amino acid residues at positions 22 and 23, which form the toxic turn of Aβ42, and 4 residues each before and after the turn structure. 4 residues on the N-terminal side and 4 residues on the C-terminal side (positions 18 to 27) are the amino acid sequence of wild type Aβ42, especially glutamic acid at position 22 (Glu22) and aspartic acid at position 23 (Asp23). ) was not artificially substituted with proline, and it was revealed that the three-dimensional structure of the toxic turn was stably fixed in a state where no lactam was formed between the side chains. The Aβ42 analog of the present invention, in which the amino acid residues at positions 17 and 28 are intramolecularly cross-linked, has an aggregation property equivalent to that of E22P-Aβ42, which has the highest aggregation property and has the highest neurotoxicity among the currently known ones. and was found to be neurotoxic. Considering that the 24B3 antibody is obtained from this E22P-Aβ42, the cross-linked Aβ42 analog of the present invention can be used as an anti-Aβ42 antibody (at the 22nd position of Aβ42) as a reagent or test agent for the rapid diagnosis of AD, which is superior to the 24B3 antibody. This can be said to contribute to the development of antibodies that recognize the fixed toxic turn structure at position 23 and 23.

In the research related to the present invention, the earliest and most useful cross-linked Aβ42 analog was one in which the leucine residue at position 17 (Leu17) and the lysine residue at position 28 (Lys28) were both replaced with cysteine residue (Cys). , is an Aβ42 analog in which the toxic turn structure at positions 22 and 23 is fixed by an intramolecular disulfide bond between the amino acid residues at positions 17 and 28. More specifically, the present invention provides amino acid residues in which the amino acid residues from position 1 to position 16 and from amino acid residue position 29 to position 42 have the same amino acid sequence as the wild-type amyloid β42 protein. It is something that Among them, the amino acid residues at positions 17 and 28 (Leu17 and Lys28) were substituted with cysteine residues (Cys), and there was an intramolecular disulfide bond between them, resulting in a cross-linked Aβ42 analog (L17C-K28C-17,28-S- S-Aβ42) was found to have extremely good test results for aggregation and neuronal toxicity (details of test results, etc. will be described later). In addition, methods for intramolecular crosslinking between the 17th and 28th positions of Aβ42 analogs include crosslinking based on disulfide bonds by substitution with homocysteine (L17homoC-K28homoC-Aβ42), crosslinking by metathesis polymerization reaction, and click reaction. Crosslinking can be mentioned. It is thought that it is possible to create antibodies specific to the toxic turn structure of wild type Aβ42 if the amino acid sequence of the surrounding region of the fixed toxic turn structure as wide as possible becomes the same as that of wild type Aβ42. In the present invention, a crosslink is formed between Leu17 and Lys28, so in order to have the same sequence as wild-type Aβ42 in as wide a region as possible, at least the toxic turn structure sandwiched by this crosslink and the surrounding positions 18 to 27 It is desirable that the amino acid sequence spanning the positions is the same as that of wild-type Aβ42. Furthermore, if all amino acid residues other than positions 17 and 28 are made to have the same sequence as wild-type Aβ42, a cross-linked Aβ42 analog having an amino acid sequence extremely similar to wild-type Aβ42 and a toxic turn structure can be obtained. In addition, in the present invention, the amino acid sequence on the N-terminal side of position 17 and the amino acid sequence on the C-terminal side of position 28 do not necessarily have to be the same amino acid sequence as wild-type Aβ42; This does not preclude the possibility of other mutations or additions, substitutions, or deletions of amino acids.

As described above, according to the present invention, the center of wild-type Aβ42, which exhibits high neuronal cytotoxicity, can be used as a substance useful for the development of antibodies that can be used as reagents or diagnostic agents for very early AD diagnosis. It is possible to provide new cross-linked Aβ42 analogs with fixed turn structures and faster aggregation properties.

The figure which shows the estimated model of Aβ dimer and trimer. A diagram showing the toxic conformation theory of Aβ42 and the amino acid sequence of wild-type Aβ42. A diagram showing the structural relationship between a highly toxic conformation and a less toxic conformation of Aβ42. A model diagram and a list of cross-linked Aβ42 analogs synthesized as an embodiment of the present invention. SH-SY5Y human neuroblastoma cytotoxicity test results for Aβ42 analogs 1 to 3 (A), aggregation ability test results (B), and transmission electron microscopy (TEM) of aggregates of Aβ42 analogs 1 and 2 and wild-type Aβ42. ) Photograph of fibril formation by analysis (C). Figures showing SH-SY5Y human neuroblastoma cytotoxicity test results (A) and aggregation ability test results (B) for Aβ42 analogs 4 to 6. A diagram showing HPLC profiles of analogs 1 to 6 and HR-ESI-qTOF-MS data. A diagram showing HPLC profiles of analogs 7 to 10 and HR-ESI-qTOF-MS data. SH-SY5Y human neuroblastoma cytotoxicity test results for Aβ42 analogs 7 to 10 (A), aggregation ability test results (B), and transmission electron microscopy (TEM) of aggregates of Aβ42 analogs 1 and 2 and wild-type Aβ42. ) Photograph of fibril formation by analysis (C). A diagram showing the neuronal cytotoxicity test results of Aβ42 analog 8, wild-type Aβ42, and E22P-Aβ42. A diagram showing the aggregation ability test results of Aβ42 analog 8, wild type Aβ42, and E22P-Aβ42.

Hereinafter, one embodiment of the present invention will be described with reference to the drawings.
First, in order to create a cross-linked Aβ42 analog that has the same amino acid sequence as around the toxic turn structure of wild-type Aβ42 and in which this toxic turn structure is fixed, we first created an analog in which intramolecular disulfide bonds of Aβ42 were formed. I tried to create it. It has been previously known that when two amino acid residues of Aβ42 are substituted with cysteine (Cys) residues at appropriate positions, they are oxidized to form an intramolecular disulfide (SS) bond. For example, it has been reported that Aβ40 and Aβ42 analogs with intramolecular disulfide bonds at positions 21 and 30 do not form fibrils but form soluble oligomers (Sandberg, ML et. al., Proc. Natl. Acad. Sci. USA, 2010, 107, 15595-15600.). It has also been reported that a salt bridge substitute (surrogate) of Aβ42 was synthesized at the 23rd and 28th positions by an amide bond, and an analog fixed in a less toxic conformation was also created (M. Yamamoto, et al. al., Bioorg. Med. Chem., 2019, 27, 888-893.). However, as far as the present inventors know, there has been no systematic attempt to fix the neuron-cytotoxic three-dimensional structure of Aβ42 by such a method.

Here again, FIG. 3 shows the structural relationship between the highly toxic conformation and the less toxic conformation of Aβ42. The turn positions are different between these cars, with the former forming turns at 22nd and 23rd positions, and the latter forming turns at 25th and 26th positions. Therefore, in this embodiment, 10 types of cross-linked Aβ42 analogs with a fixed highly toxic conformation were synthesized. Figure 4 shows model diagrams of these synthesized cross-linked Aβ42 analogs (the left figure shows the structure of the cross-linked Aβ42 analog, and the right figure shows the conformation of the Aβ42 unit in the aggregate determined by solid-state NMR) and their This is a list. First, three types of cross-linked Aβ42 analogs (analogs 1 to 3) were synthesized. Analog 1 is a wild-type Aβ42 cross-linked at positions 21 and 24 as a toxic turn model in which the side chains of Glu22 and Asp23 are outside the ring. Analog 2 is a fibril model in which the 19th and 30th positions are crosslinked, and the carbon atoms at the 19th and 30th positions are located close to each other. Since Tyr10 and Met35 are essential for inducing neuronal cell toxicity, analog 3 was created by crosslinking positions 10 and 35 as a negative control. For analogs 1-3, homocysteine (homoCys) residues were used for substitution and molecular dynamics simulations were used to optimize each conformation.

These analogs 1 to 3 were synthesized using the Fmoc strategy (Fmoc solid phase peptide synthesis method) with a fully automatic microwave peptide synthesizer (manufactured by Biotage, Initiator + Alstra (product name)) (synthesis method The same applies to analogs 4 to 10, which will be described later). After completion of chain extension, each peptide resin was deprotected and cleaved from the resin. The crude peptide was oxidized with 10% DMSO in water containing _NH4OH (0.1%) to form intramolecular disulfide bonds. Each crude oxidized peptide was purified by HPLC. Satisfactory mass spectrum data were obtained for the purified peptide by high-resolution electrospray ionization-time-of-flight mass spectrometry (HR-ESI-qTOF-MS).

First, the neuronal cytotoxicity of Aβ42 analogs 1-3 on SH-SY5Y human neuroblastoma cells was evaluated as one of the typical models for neuronal cell culture. Wild type Aβ42 (WT-Aβ42) is used for comparison. Cell viability after 24 hours of incubation at 37°C was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, and is shown in Figure 5(A). The results are shown below. Below, the numbers 1 to 10 shown in bold in each figure are the numbers of the Aβ42 model (see the list in FIG. 4). The absorbance obtained after addition of solvent (0.1% _NH4OH ) was taken as 100% ( ^* p<0.05 vs. solvent). As a result, analog 1, a toxic turn model, exhibited significant neuronal cytotoxicity comparable to wild-type Aβ42. In contrast, the fibril model, analog 2, was less toxic compared to wild-type Aβ42, and the negative control, analog 3, was completely inactive. The aggregation ability of analogs 1-3 was evaluated by Thioflavin T (Th-T) fluorescence method. Figure 5(B) shows the results of a Th-T fluorescence test of wild-type Aβ42 (25 μM) and analogs 1 to 3 at 37°C. The data are expressed as mean±standard deviation (n=8). As shown in the figure, their aggregation ability estimated by Th-T fluorescence, which depends on the formation of β-sheet structures, correlated well with neuronal cytotoxicity. As shown in FIG. 5(C), fibril formation in the aggregates of analog 1 and analog 2 and wild-type Aβ42 was confirmed by transmission electron microscopy (TEM) analysis. A photograph (scale bar is 50 nm (magnification: 50k)) from TEM analysis of the aggregates after incubation for 48 hours at 37°C is shown. These data suggest that cross-linking at positions 22 and 23 fixes Aβ42 into a toxic conformation, such as E22K, D22E-Aβ42-lactam, and that the Aβ42 fibril conformation exhibits weak neuronal cytotoxicity. are doing. This result is consistent with the recent hypothesis that fibrils are not the major cause of neuronal cytotoxicity.

Based on the results of analogs 1 to 3, we created three cross-linked Aβ42s in which the amino acid residues at positions 21 and 24 were replaced with Cys or homoCys residues to optimize the toxic turn at positions 22 and 23 of Aβ42. Analogs (Analogs 4 to 6) were synthesized (Tables 4 to 6 in Figure 4). Similar to analogs 1 to 3, the neuronal toxicity of analogs 4 to 6 was evaluated by the MTT test (FIG. 6(A)), and the aggregation ability was evaluated by the Th-T fluorescence test (FIG. 6(B)). Analog 1, which is a toxic turn model, and wild type Aβ42 were used for comparison. The conditions for the MTT test are the same as those for analogs 1 to 3. Regarding the conditions of the Th-T fluorescence test, the incubation temperature was room temperature (23 °C), the concentration of wild-type Aβ42 was 10 μM, and the data are expressed as mean ± standard deviation (n = 4), and every 10 minutes. It differs from analogs 1 to 3 Th-T fluorescence tests in that it automatically observes These results suggested that analog 1 had the highest aggregation and neurotoxicity, and that analogs 4 to 6, which were cross-linked with a combination of Cys and homoCys, could not improve the activity of analog 1. This may be due to the fact that smaller cyclic peptides are not flexible enough to adopt optimal conformations with strong neuronal cytotoxicity and aggregation ability.

Therefore, we further designed four cross-linked Aβ42 analogs (analogs 7 to 10) in which the three-dimensional structure of Aβ42 was fixed (Tables 7 to 10 in Figure 4), and Glu22 and Asp23 are connected to two Cys involved in intramolecular disulfide bonds. It was synthesized so that it was located in the center of the residue. Previous reports have suggested that amino acid residues from positions 15 to 21 are involved in the intermolecular α-sheet structure of Aβ42 aggregates. Furthermore, it has been demonstrated that His14, Glu22, Asp23, Gly33, Gly37, and Gly38 play important roles in Aβ42 aggregation (F. Hsu, et. al., ACS Omega 2018, 3, 8401-8407.) . Although data are not shown here, Aβ42 analogs fixed in a different conformation, bridged at positions 20 and 25 by Cys residues, are in fact less neurotoxic than analogs 4 and 7. It turns out that.

These analogs 7 to 10 were synthesized and characterized using the method described above with slight modifications. That is, since there is a high possibility that an aromatic ring tert-butyl group will be introduced during the deprotection of the protecting group, a pseudoproline type dipeptide was used in place of Fmoc-Asp and Fmoc-Ser at the 7th and 8th positions. Pseudoproline is also an Fmoc-protected oxazolidine or thiazolidine consisting of two amino acid residues, which prevents aggregation during peptide synthesis by immobilizing β-sheets. Here, FIG. 7 shows the HPLC profiles and HR-ESI-qTOF-MS data of analogs 2, 4, and 7 to 10.

The results of the neuronal cytotoxicity test against SH-SY5Y human neuroblastoma cells of Analogs 7 to 10 and, for comparison, Analog 4 and wild-type Aβ42 are shown in FIG. 9(A). The test method is similar to the neurotoxicity test for analogs 1-3. As a result, analog 8 cross-linked by an S-S bond between positions 17 and 28 showed significantly higher neuronal activity compared to wild-type Aβ42, especially at low concentrations (0.04, 0.2, 1 μM). Showed toxicity. The neuronal cytotoxicity of analog 4 and analog 7 cross-linked at positions 21 and 24 and 19 and 26, respectively, was almost equivalent to that of wild-type Aβ42. On the other hand, analog 9 and analog 10, which were cross-linked at positions 15 and 30 and positions 30 and 32, respectively, did not exhibit neuronal cytotoxicity (data for analog 10 are not shown).

We also tested the aggregation ability of analogs 7 to 10, analog 4, and wild-type Aβ42 by Th-T fluorescence and TEM analysis (FIGS. 9(B) and (C)). In addition, in the same figure (C), only the photographs of each aggregate of analog 4, 7, 8 and wild type Aβ42 are shown. In particular, since the aggregation ability of analog 8 (L17C-K28C-17,28-SS-Aβ42) was extremely high, it was decided that the Th-T fluorescence test would be automatically observed every 10 minutes. Analog 8, which showed the highest neuronal cytotoxicity, aggregated more rapidly than wild type Aβ42. Analog 4 and Analog 7 aggregated moderately. These three analogs formed typical fibrils after 24 hours of incubation, as shown in Figure (C). On the other hand, with non-neuronal cytotoxic analogs 9 and 10, neither fluorescence intensity nor significant aggregates were observed.

These results indicate that the aggregation ability and neuronal cytotoxicity of Aβ42 can be modulated by intramolecular disulfide bonds. Analog 8 (SEQ ID NO: 2 in the sequence listing), which has an intramolecular disulfide bond between positions 17 and 28, has the highest neuronal cytotoxicity and aggregation property, and has the highest neuronal cytotoxicity and aggregation property compared to wild-type Aβ42. When compared with E22P-Aβ42, an Aβ42 analog, which had higher neuronal cytotoxicity, the neuronal cytotoxicity of analog 8 was almost equivalent to that of E22P-Aβ42 (FIG. 10). Interestingly, analog 8 showed very high neuronal cytotoxicity comparable to E22P-Aβ42 at low concentrations (0.04, 0.2, 1 μM), but its neurotoxicity was lower at higher concentrations (above 5 μM). It was weaker than wild type Aβ42. It is believed that this difference in neuronal cytotoxicity may be caused by the higher aggregation ability of analog 8 at 5 μM. Such concentration-dependent aggregation ability was investigated by Th-T fluorescence test (FIG. 11). The results clearly show that E22P-Aβ42 did not exhibit significant fluorescence at 5 μM, suggesting that analog 8 rapidly aggregated to form less toxic fibrils even at 5 μM.

The above results demonstrated that intramolecular disulfide bridges to Aβ42 are a reliable and simple method for controlling the tertiary structure of Aβ42. Toxic turn structure at positions 22 and 23 and 4 residues before and after it (including positions 22 and 23 forming the toxic turn structure, 4 residues on the N-terminal side and 4 residues on the C-terminal side, positions 18 to 27) Analog 8, an innovative derivative that has the amino acid sequence of wild-type Aβ42 in ) but is cross-linked at positions 17 and 28 to fix its toxic turn structure, is a promising candidate for the development of new antibodies against the toxic turn of Aβ42. It is a potential target molecule. Analog 8 has the same amino acid sequence as wild-type Aβ42, with a fixed turn structure formed at positions 22 and 23, and an amino acid sequence other than positions 17 and 28, where Cys is substituted to form a disulfide bond, i.e. It can be said that it is a cross-linked Aβ analog having the same amino acid sequence as wild-type Aβ42 on both the inside and outside of the cross-linked portion including the toxic turn structure. If a conformation-specific antibody is created using this analog 8 as a hapten, it will be possible to specifically and sensitively detect an antibody that recognizes and binds to the same toxic turn structure as wild-type Aβ42, that is, a toxic oligomer present in the human brain. It becomes possible to develop antibodies that can However, although the high aggregation ability of analog 8 may be a problem, recent studies have shown that dimer and trimer models of Aβ40 form metastable oligomers of approximately 20 mers in at least 24 hours. (Y. Irie, et. al., ACS Chem. Neurosci. 2017, 8, 807-816) , 182.). It is believed that dimerization or trimerization of analog 8 is effective in overcoming its high aggregation and may be a useful tool for the preparation of antibodies for AD diagnosis and treatment. Therefore, by creating dimeric and trimeric models of analog 8, a path was opened to the development of low-molecular-weight compounds that specifically act on toxic oligomers. Until now, there have been examples of synthesis of derivatives in which intramolecular disulfide bonds are formed in Aβ, but the present invention is the first example in which the molecules have been designed and synthesized by focusing on the toxic turn structure around the 22nd and 23rd positions. be.

In addition, the present invention provides that the amino acid sequence of positions 22 and 23, which form the toxic turn structure of wild-type Aβ42, and four residues each before and after the same sequence as the wild type, but that the toxic turn structure is fixed. It is important that it is an Aβ42 analog, and for that purpose, the amino acid residue at position 17 and the amino acid residue at position 28 must be in a cross-linked state. It is not essential that the amino acid residue at position 28 is replaced with a Cys residue to form an intramolecular disulfide bond, and analog 8 is an example of the present invention. In addition, for example, Aβ42 in which the amino acid residues at positions 17 and 28 are substituted with homoCys residues to form an intramolecular disulfide bond, metathesis polymerization reaction or click reaction between the amino acid residues at positions 17 and 28, etc. The present invention also includes Aβ42 analogs in a state where crosslinks are formed by reaction. In addition, the present invention is not limited to the embodiments described above, and various modifications can be made without departing from the spirit of the present invention.

By using the Aβ42 cross-linked analog peptide of the present invention as a target, it is possible to develop antibodies that recognize the same toxic turn structure as wild-type Aβ42, and to develop reagents and test drugs for rapid diagnosis of very early AD based on the antibodies. It is extremely useful for such purposes.

Claims (3)

In amyloid β42 protein of 42 amino acid residues,
The amino acid residue at position 17 and the amino acid residue at position 28 are cross-linked,
The amino acid residue at position 22 and the amino acid residue at position 23 have a three-dimensional structure in which the turn structure between the β-sheet structures according to the amino acid sequence is fixed, and at least the amino acid residues from position 18 to position 27, which include the turn structure, An amyloid β42 cross-linked analog peptide characterized by having the same amino acid sequence as the wild-type amyloid β42 protein in terms of amino acid residues. A leucine residue at position 17 and a lysine residue at position 28 are both replaced with cysteine residues, and the turn structure is fixed by an intramolecular disulfide bond between the amino acid residues at positions 17 and 28. 1. The amyloid β cross-linked analog peptide according to 1. Claim 1 or 2, wherein the amino acid residues from position 1 to position 16 and from amino acid residue position 29 to position 42 have the same amino acid sequence as the wild-type amyloid β42 protein. amyloid β42 cross-linked analog peptide described in .

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