JP2006265189A - beta-AMYLOID PEPTIDE AND METHOD FOR SCREENING THERAPEUTIC AGENT OR PROPHYLACTIC AGENT FOR ALZHEIMER'S DISEASE USING THE SAME - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an Aβ (β amyloid peptide) elucidating structural factors of aggregation of the Aβ and expression of neural cytotoxicity and to provide a method for screening a therapeutic agent or a prophylactic agent for AD (Alzheimer's disease) utilizing the Aβ. <P>SOLUTION: An Aβ42 is composed of 42 amino acid residues. The Aβ42 in which a turn structure between β sheet structures based on amino acid sequences before and after the 22- and 23-positions and further the 38- and 39-positions is introduced into the 22- and 23-positions and the 38- and 39-positions is prepared. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a β amyloid peptide considered to be a causative agent of Alzheimer's disease, and a screening method for a therapeutic agent for Alzheimer's disease using the β amyloid peptide.

  Alzheimer's disease (AD) is a kind of neurodegenerative disease, but there are few effective treatment methods at present, and elucidation of the pathogenesis of AD and establishment of a fundamental treatment method are strongly desired. Neuropathological features of AD patients' brain include senile plaques, which are spherical structures mainly found in the cerebral neocortex, and neurofibrillary tangles, which are fibrous inclusions that appear in the hippocampus and cerebral neocortex. Is recognized. Senile plaques have been attracting attention by many researchers because they are highly disease-specific to AD patients and are frequently observed from early AD. So far, it has been found that senile plaques are aggregates of β-amyloid peptide (Aβ) mainly composed of amino acids of 40 and 42 residues. As a precursor protein, amyloid precursor protein having 770 amino acid residues ( A single-pass membrane protein named amyloid precursor protein (APP) has been identified. That is, Aβ is produced when APP is cleaved by β-secretase outside the cell membrane and then cleaved by γ-secretase inside the cell membrane. However, since it functions, it can be said that Aβ is one of the normal metabolites of APP.

Aβ mainly consists of 40 residues of Aβ40 and 42 residues of Aβ42. Physiologically, Aβ40 and Aβ42 are present in a ratio of about 10: 1, but the general idea is that Aβ42, which has a particularly high aggregation ability, aggregates to form a nucleus, and Aβ40 aggregates around it to form senile plaques. Is. Since Aβ causes neuronal cell death, it is considered to be a causative agent of AD (amyloid hypothesis). Its neurotoxicity is closely related to the aggregating activity, but the aggregate itself has low toxicity, and Aβ It has recently been considered that oligomeric structures are toxic bodies. However, the oligomeric structure of Aβ that produces toxicity has not been clarified so far. On the other hand, it has been clarified that Aβ aggregates by taking an intermolecular β sheet structure and expresses neurocytotoxicity, and inhibits neurotoxicity by utilizing the property of Aβ taking β sheet conformation. An examination method for substances has been considered (see Patent Document 1). Although this document describes that there is a direct correlation between the β-sheet structure of Aβ and neurotoxicity, no specific data has been disclosed, and the mechanism of cytotoxicity is not disclosed. There is no mention about. In addition, most of the research subjects so far have been directed to Aβ40, which is relatively easy to synthesize including the above-mentioned literature, and Aβ42, which is only about 1/10 of the amount of Aβ40 produced and difficult to synthesize, has not received much attention. . In recent years, the application of Aβ42 antibodies to therapy has been attempted, but since these antibodies are produced using normal Aβ42 as an antigen, they are practically used for side effects such as encephalitis. The current situation is that it has not been realized.
Japanese Patent Laid-Open No. 06-294798

  In the present invention, Aβ42 is not neurotoxic, but Aβ42 has a specific conformation, so that the aggregation activity is increased. The main object of the present invention is to provide a screening method for beneficial AD therapeutics or preventives using Aβ42, and to provide Aβ42 having a harmful conformation by clarifying the structural factor of Aβ42. Is.

  The present inventors have reported a mutation in the Aβ sequence in familial AD (familial Alzheimer's disease: FAD) accompanied by a genetic mutation, in particular, a mutation in the Aβ sequence in which Aβ aggregates are abnormally deposited on the cerebral blood vessel wall. By focusing on the above, and chemically synthesizing these Aβ40 and Aβ42 mutants with high purity and examining their neurotoxicity, aggregating activity and secondary structure, new findings on the aggregation mechanism of Aβ42 and the neurocytotoxic expression mechanism To obtain the present invention.

  That is, the β amyloid peptide according to the present invention is a β amyloid peptide composed of 42 amino acid residues, and has a turn structure between β sheet structures based on the amino acid sequences before and after the 22 and 23 amino acid residues. It is characterized by. Here, the wild-type amino acid sequence of β-amyloid peptide (Aβ42) having 42 amino acids is represented by SEQ ID NO: 1 in the Sequence Listing, and the amino acid sequence of wild-type β-amyloid peptide (Aβ40) having 40 amino acids for comparison. Are shown in SEQ ID NO: 2, respectively.

  Wild-type Aβ40 and Aβ42 both exhibit aggregating activity and neurotoxicity, but Aβ42 has a higher aggregating activity, higher neuronal toxicity and longer duration than Aβ40. The present inventors have found that the difference in secondary structure between Aβ40 and Aβ42 is in the C-terminal region. That is, although the C-terminal 2 residues of Aβ42 are involved in the formation of an intramolecular β-sheet structure, the C-terminal region of Aβ40 does not have a β-sheet structure. Since this difference was suggested to be a critical difference regarding the aggregation activity and neurotoxicity of Aβ40 and Aβ42, the present invention is particularly a β amyloid peptide (Aβ42) consisting of 42 amino acid residues, It is a β amyloid peptide characterized in that the amino acid residues at positions 22 and 23 and the amino acid residues at positions 38 and 39 each form a turn structure between β sheet structures according to the amino acid sequences before and after them.

  In particular, in the above-mentioned two present inventions, amino acid residues that are likely to have a turn structure of an intramolecular β-sheet structure at positions 22, 23, 38, and 39 of Aβ42 are those near the N-terminus of the turn (position 22, 38) is any one amino acid residue selected from proline, serine, lysine, arginine, aspartic acid, threonine, glutamine, glycine, asparagine, alanine and glutamic acid, and the amino acid residue adjacent to it (position 23) , Position 39) is preferably any one amino acid residue selected from asparagine, aspartic acid, glycine, arginine, serine, cysteine, tyrosine, histidine, glutamic acid, threonine, and lysine.

  In particular, a turn structure based on a sequence of two amino acid residues is likely to occur when the side close to the N-terminus of the turn is a proline residue. Therefore, for Aβ42 according to the present invention, the glutamic acid residue at position 22 is proline. Those substituted with a residue are preferred.

  In particular, Aβ42, which can rationally explain the mechanism of Aβ's strong aggregation activity and neurotoxicity, and is extremely useful for the creation of AD therapeutics or preventives, has a glutamic acid residue at position 22 and a glycine residue at position 38. Are optimally substituted with a proline residue.

  As described above, Aβ can be obtained by synthesis as will be described in detail later, exhibits high aggregation activity and neurotoxicity, and provides a new model of Aβ42 aggregation and toxicity expression that has never been seen before. Therefore, it greatly contributes to the development of therapeutic or preventive drugs (including antibodies) for AD.

  Therefore, by using Aβ42 according to the present invention described above, it becomes possible to screen a substance that is an active ingredient of an AD therapeutic agent or a preventive agent. That is, the screening method according to the present invention is a method characterized by including a step of bringing a test substance into contact with any one of the above Aβ42s and measuring the aggregation activity of the Aβ42.

  Other screening methods for AD therapeutic agents or preventive agents according to the present invention include a step of contacting a test substance with any one of the above Aβ42 and measuring the neurotoxicity by the Aβ42. It is a method to do.

  The β-amyloid peptide (Aβ42) of the present invention represents a rational new Aβ42 aggregation model and neurocytotoxic expression model that is considered to be the etiology of Alzheimer's disease (AD), and Aβ42 itself causes AD. Instead, it suggests that AD may be caused by taking a specific conformation such as prion protein. Therefore, screening using this Aβ42 can provide a new and innovative path to the development of AD therapeutics or preventives based on the structure of Aβ42.

Hereinafter, the present invention will be described in detail with reference to the drawings.
<Aβ42 Aggregation Model> FIG. 1 shows two types of β-amyloid peptides (Aβ40, Aβ42, both of which are produced by cleaving γ-secretase from the amyloid precursor protein APP penetrating the cell membrane. It is a model figure which shows the variation | mutation of the A (beta) arrangement | sequence in familial Alzheimer's disease (familial Alzheimer's disease: FAD). In the figure, each amino acid is represented by one letter. In FAD having a mutation near the center in the sequence of Aβ, the alanine residue at position 21 is mutated to a glycine residue (A21G-Aβ) family (Flemish), and the glutamic acid residue at position 22 is glutamine. Mutated to residue (E22Q-Aβ) Family (Dutch), Glutamic acid residue at position 22 is mutated to lysine residue (E22K-Aβ) Family (Italian), Glutamic acid residue at position 22 is glycine There are families that are mutated to residues (E22G-Aβ) (Arctic), and aspartic acid residues at position 23 are mutated to asparagine residues (D23N-Aβ). Therefore, five types of mutant Aβ40 and Aβ42 (A21G, E22Q, E22K, E22G, D23N) found in these FADs were chemically synthesized with high purity by the method shown in the examples described later, and these mutant neurons Toxicity was examined by the MTT method using PC12 cells. As shown in FIG. 2, the Aβ40 and Aβ42 mutants at positions 22 and 23 both showed higher toxicity than the wild-type. In addition, the toxicity of the Aβ42 mutant was 50 to 200 times higher than that of the corresponding Aβ40 mutant. These results suggested that the Aβ42 mutant may be a cause of FAD. Next, the aggregation activity of each mutant was examined by quantifying the supernatant after centrifugation by HPLC. As shown in FIG. 3 (a), E22G-Aβ42 (Arctic) and D23N-Aβ42 (Iowa ) Was almost the same as the wild type, whereas E22K-Aβ42 (Italian) and E22Q-Aβ4 (Dutch) aggregated significantly more than the wild type. This result shows that abnormal deposition of aggregates of Aβ is observed in the brains of Italian and Dutch family FAD patients (Science, 248, 1124-1126 (1990), Alzheimer's Rep., 2, S28 (1999)). Match well. Further, when these aggregates were photographed with an electron microscope, fibrils (microfibers) similar to the wild type were observed in all the mutants. From the above results, it was clarified that a high correlation was observed between the neurotoxicity and the aggregation activity in the mutants at positions 22 and 23 of Aβ. On the other hand, A21G-Aβ42 (Flemish) showed almost the same level of neurotoxicity as that of the wild type, but its aggregation ability was low. Therefore, fibril formation of Aβ is not always necessary for the expression of neurotoxicity. This suggests a possible result.

  By the way, in order for Aβ to aggregate, it is considered important to have a β sheet structure at a specific site (Int. J. Exp. Clin. Invest., 5, 121-142 (1998)). As a result of conducting secondary structure analysis by Fourier transform infrared spectroscopy (FT-IR) for the samples before and after the aggregation of each of the Aβ42 mutants, it was found that the β sheet content was increased by aggregation in any sample. It was. On the other hand, considering the sequences of the 2 amino acid residues at positions 22 and 23 of each of the mutants that showed high aggregating activity, both were well observed in the turn structure that turned the intramolecular β-sheet structure back. It was found to be a sequence (J. Mol. Biol., 115, 135-175 (1977)). Therefore, in the same manner as the above-mentioned various FAD mutants, the proline residue which is the amino acid residue which is most likely to have a turn structure and the valine residue which is the amino acid residue which is most difficult to take a turn structure are each positioned at the 22nd position. Aβ42 (E22P-Aβ42, E22V-Aβ42) substituted with a glutamic acid residue, and Aβ40 (E22P-Aβ40) in which the glutamic acid residue at position 22 was substituted with a proline residue were synthesized, and their aggregating activities (FIG. 3 ( b)) and neurotoxicity (see FIG. 2) were examined. As a result, E22P-Aβ42 and E22P-Aβ40 showed extremely high aggregation activity and toxicity compared to the wild type, whereas E22V-Aβ42 showed neither aggregation activity nor toxicity. From the above results, it was found that the turn structures at the 22 and 23 positions of Aβ are strongly involved in the aggregation and neurotoxicity of Aβ.

  Therefore, for Aβ42, 34 types of mutants in which amino acid residues at each position were substituted with proline residues in order were prepared, and the aggregation activity of each mutant was examined. The result is shown in FIG. As is clear from the figure, among the mutants at positions 15 to 32, only the mutant at position 22 (E22P-Aβ42) showed a much higher and faster aggregation activity than the wild type. It was found that the mutants at positions 21 to 24 and positions 24 to 32 were less likely to aggregate. That is, it can be said that the site where the aggregation activity is lower than that of the wild type has a β sheet structure, and conversely, the site where the aggregation activity does not decrease has a turn structure. From this, it was found that the 15th to 21st positions and the 24th to 32nd positions of Aβ each have a β sheet structure, and the 22nd and 23rd positions have a turn structure. Similarly, from the examination of the site where the decrease in the aggregation activity was observed and the site where the aggregation activity was not observed, Aβ 35-position to 37-position and 40-position to 42-position have β-sheet structure, and 33-position and 34-position. , And positions 38 and 39 were found to be turn structures. Based on the above results, FIG. 5A shows the Aβ42 aggregation model of the present invention having turn structures at the 22nd and 23rd positions, the 33rd and 34th positions, the 38th and 39th positions, respectively. In the figure, a plate-like arrow represents a part having a β sheet structure. Recent studies have revealed that the C-terminus of Aβ40 does not form a β-sheet (J. Mol. Biol., 335, 833-842 (2004)). It can be said that the critical difference in the secondary structure is whether or not a β sheet is present at the C-terminal. In addition, the ease of taking a turn structure by the arrangement of two amino acid residues is not limited to the case where the side close to the N-terminal of the turn is a proline residue. That is, as shown in FIG. 5 (b), combinations of two amino acid residues that are likely to have a turn structure are listed, and in the above-mentioned Aβ42 aggregation model, the amino acid residue on the side close to the N-terminus of the site having the turn structure (first position) In addition to proline residues, any of serine, lysine, arginine, aspartic acid, threonine, glutamine, glycine, asparagine, alanine, and glutamic acid residues can be applied to the group. On the other hand, the amino acid residue (second position) closer to the C-terminus of the turn includes any of the residues of asparagine, aspartic acid, glycine, arginine, serine, cysteine, tyrosine, histidine, glutamic acid, threonine, and lysine. Can be applied.

<Aβ42 Aggregation and Neurocytotoxic Expression Mechanism> Increased oxidative stress is characteristically observed in the brain of AD patients. The cause is that oxygen molecules are reduced by Aβ in the presence of metal catalysts such as Cu (II), Zn (II), Fe (III), etc. to generate hydrogen peroxide (H ₂ O ₂ ), and lipids and proteins In other words, neurotoxicity is manifested by damaging biomolecules such as. It has been reported that the mechanism involves both the tyrosyl radical at position 10 of Aβ and the methionine radical at position 35 (a radical generated on the sulfur atom of the methionine residue) (Brain Res Bull., 50, 133-141 (1999), Biochemistry, 43, 560-568 (2004), etc.) are still unclear. Further, as described above, most of the studies so far have been directed to Aβ40, and the difference in aggregation ability and cytotoxicity between Aβ42 and Aβ40 could not be rationally explained.

However, with the Aβ42 aggregation model shown in FIG. 5 (a), the possibility of explaining the difference in aggregation and cytotoxicity between Aβ42 and Aβ40 was considered. Therefore, first, the influence of the turn formation at positions 22 and 23 of Aβ42 on the expression of Aβ42 neurotoxicity was examined. For this purpose, the aforementioned E22K-Aβ42 (Italian), E22Q-Aβ42 (Dutch), E22G-Aβ42 (Arctic), E22P-Aβ42, E22V-Aβ42, wild type (Wild-type) having a mutation in the glutamic acid residue at position 22 ) Aβ42 was used to examine their production of H ₂ O ₂ . As shown in FIG. 6 (a), E22K-Aβ42, E22Q-Aβ42, E22G-Aβ42, and E22P-Aβ42 produced H ₂ O ₂ more than twice that of wild-type Aβ42, whereas E22V-Aβ42 Only a lower amount of H ₂ O _{2 was} produced than wild type Aβ42. Further, even in electron spin resonance (ESR) spectroscopic analysis using PBN (N-tert-butyl-α-phenylnitrone) as a trap reagent, as shown in FIG. After time, each mutant except E22V-Aβ42 showed an increase in signal intensity compared to wild-type Aβ42, while E22V-Aβ42 showed a decrease compared to wild-type Aβ42. A characteristic quadruple line was observed in the body (FIG. 6C). These results indicate that there is a high correlation between H ₂ O ₂ production, radical formation, and neurocytotoxicity. From this, the turn structure at positions 22 and 23 of Aβ42 is a neuronal cell through radical formation. A conclusion is drawn that it is essential for the development of toxicity. Although not described in detail, the present inventors have confirmed that a turn structure exists at the 22-position and the 23-position by solid-state NMR for the fibril of E22K-Aβ42. It can be said that it is well supported.

Next, the effects of the tyrosine residue at position 10 (Tyr-10) and the methionine residue at position 35 (Met-35) on neuronal toxicity were examined. For this purpose, an Aβ42 mutant (Y10F-Aβ42) in which a tyrosine residue at position 10 (Tyr-10) is substituted with a phenylalanine residue, and a methionine residue at position 35 (Met-35) as norvaline (nV; 2-amino Radical production was measured for Aβ42 mutant (M35nV-Aβ42) substituted with pentanoic acid) and Aβ42 mutant (Y10F, M35nV-Aβ42) substituted with phenylalanine residue and norvaline at positions 10 and 35, respectively. Norvaline is employed because the methyl group has hydrophobicity similar to that of the S-methyl group. As shown in FIG. 7 (a), each of the mutants Y10F-Aβ42, M35nV-Aβ42, Y10F, M35nV-Aβ42 showed extremely low production of H ₂ O ₂ compared to wild-type Aβ42. Also, in ESR spectroscopic analysis, as shown in FIG. 7 (b), these mutants showed a decrease in signal intensity compared to the wild type after incubation for 24 hours. Furthermore, as shown in FIG. 7 (c), a significant decrease in the neurotoxicity of each mutant against PC12 cells was also observed. From these results, it is shown that the tyrosyl radical at the 10th position and the methionine radical at the 35th position play an extremely important role with respect to the neurotoxicity via the Aβ42 radical. On the other hand, according to experiments using Aβ25-36, it is considered that the radical cation of the oxidized sulfur atom of Met-35 is too short to cause oxidative stress effectively (J. Am. Chem. Soc 122, 10224-10225 (2000)).

By the way, the Aβ42 aggregation model described above has a structure in which the β-sheet region from the 35th position to the 37th position and the region from the 40th position to the 42nd position are separated by the turn structure at the 38th and 39th positions in the C-terminal region. is doing. From this, as shown in the C-terminal structural model of FIG. 8 (a), an intramolecular antiparallel β sheet was formed between positions 35 and 42 of Aβ42, and Met-35 was oxidized. This suggests that the reaction between the sulfur atom and the C-terminal carboxylate anion is possible. Thus, Aβ42 (amide) obtained by converting a C-terminal carboxylic acid into an amide was used to examine the radical productivity and neurotoxicity. Aβ42 (amide) produced H ₂ O ₂ (FIG. 8 (b)), radical production (FIG. 8 (c)), and neurotoxicity (FIG. 8 (d)) were significantly higher than wild type Aβ42. Declined. This result strongly suggests that the C-terminal carboxylate anion and the methionine radical at position 35 through the intramolecular antiparallel β-sheet in the C-terminal region of Aβ42 are essential for the expression of Aβ42 neurocytotoxicity. Here, in the case of Aβ40, since it does not have a β-sheet structure in the C-terminal region, the methionine radical at position 35 is unstable and exhibits only low neurocytotoxicity. Moreover, since the radical generation and neurotoxicity expression levels of the rarely-occurring 43-residue Aβ peptide (Aβ43) are low (see FIGS. (B) and (c)), the length of the C-terminal region of Aβ42 is It can be said that it is extremely important for the development of neurotoxicity through generation.

  As described above, it is recognized that there is a correlation between the toxicity of Aβ to PC12 cells and the aggregation mechanism, but it is necessary to clarify the specific mechanism of how aggregation is related to neurotoxicity based on radical formation. There is. Then, next, the radical production ability and the aggregation ability were examined for all the Aβ mutants substituted at the 10-position, 35-position and 42-position. As shown in FIG. 9 (a), it was found that each of the mutants Y10F-Aβ42, M35nV-Aβ42, Y10F, M35nV-Aβ42 has a slower aggregation compared to wild-type Aβ42. This suggests that the 10th tyrosyl radical and the 35th methionine radical play an important role in the aggregation of Aβ42. Aβ42 (amide) and Aβ43 had the same reaction rate as wild type Aβ42 (however, the molar concentration of the water-soluble peptide in the equilibrium state of Aβ42 (amide) and Aβ43 was 2.2 ± 0, respectively. .22, 2.3 ± 0.10 μM, higher than the same molar concentration of wild-type Aβ of 0.85 ± 0.04 μM). This suggests that the thermodynamic stability of fibrils of Aβ42 (amide) and Aβ43 is lower than that of wild type Aβ42. This result shows that the oligomer of Aβ42 becomes extremely stable due to the S—O bond between the oxidized sulfur atom of Met-35 and the carboxylate anion at the C-terminus (Ala-42). .

  According to the C-terminal structural model of Aβ42 described above (FIG. 8A), only Aβ42 having a core at the C-terminus can accumulate radicals in oligomers or fibrils. It means being permanently damaged. In order to prove this hypothesis, the amount of radicals produced by Aβ42 and Aβ40 was measured over time by ESR (light, a 6-fold signal was observed under the conditions of 37 ° C.). As shown, an observable level of signal was observed for Aβ42 after 12.5 hours of incubation, and the signal intensity increased to a maximum after 24 hours of incubation. In contrast, Aβ40 did not show a significant level of signal even after 24 hours of incubation. On the other hand, in the aggregation activity test (25 μM) at a concentration lower than the concentration measured by ESR (165 μM), as shown in FIG. 9 (c), 90% or more of Aβ42 aggregated after 16 hours of incubation. In contrast, Aβ40 did not show significant aggregation even after 24 hours of incubation. Further, as shown in FIG. 8 (d), the neurocytotoxicity of Aβ40 was extremely low. From the above, it is clear that Aβ40 cannot stabilize radical generation by oligomer or fibril formation, and as a result, Aβ42 oligomer or fibril can continue to release radicals, whereas Aβ42 is less neurotoxic than Aβ42. became.

  Based on the above results, a mechanism model of Aβ42 aggregation and neurotoxicity expression is shown in FIG. That is, first, Aβ42 oxidizes the phenolic hydroxyl group of the 10-position tyrosine residue (10-Tyr) by chelating a small amount of metal ion to N-terminal histidine. Then, the turn structures at the 22nd and 23rd positions of Aβ42 bring the tyrosyl radical generated by oxidation closer to the sulfur atom of Met-35, thereby generating a methionine radical ((a) in the figure). The methionine radicals bind to the C-terminal carboxylate anion through intramolecular β-sheets at positions 35 to 37 and 40 to 42, which are turned at positions 38 and 39, and bind to the C-terminal of Aβ42. The region is stabilized ((b) in the figure). As a result, a core is formed at the C-terminus of Aβ42 to induce oligomerization or fibril formation of Aβ42, and radical species that are continuously generated through equilibration of Aβ42 monomer are stabilized by the Aβ42 oligomer ( (C) in the figure. This means that Aβ42 having a β-sheet structure at the C-terminus is very likely to aggregate and exhibits neurotoxicity for a long time compared to Aβ40 having no β-sheet structure at the C-terminus. Match well.

  <Screening Method for AD Therapeutic Agent or Preventive Agent> By using the Aβ42 aggregation model as described above and a neurocytotoxic expression model based thereon, screening of a substance containing a component that becomes a therapeutic agent or a preventive agent for AD is performed. Is possible. Examples of the screening method include a method for measuring the aggregation activity of Aβ42, and a method for measuring the neurotoxicity of Aβ42. Here, as Aβ42 that can be used, those in which some amino acid residues are substituted, such as the above-mentioned various mutants having a turn structure at positions 22 and 23, and positions 38 and 39, are suitable. However, the peptide having the most easy turn structure and the most suitable for this screening is Aβ42 (E22P, G38P-Aβ42) in which a proline residue is introduced at both positions 22 and 38 as shown in SEQ ID NO: 3. It is.

  As a screening method based on measuring the aggregation activity using such Aβ42, for example, a test substance and thioflavin T which is a fluorescent dye are added to Aβ42 and incubated to screen a substance which decreases the fluorescence intensity. The method (Th-T assay; Anal. Biochem., 177, 244-249 (1989), J. Biol. Chem., 278, 46179-46187 (2003)) can be exemplified. Specific test substance candidates include natural product-derived substances represented by foods, microbial metabolites, and plant extracts.

  Moreover, as a screening method based on measuring neurocytotoxicity using the above Aβ42, a random screening using an RNA aptamer can be exemplified. In this case, it can be expected that the three-dimensional structure of the inhibitor that reduces neurotoxicity is clarified and a low-molecular-weight organic compound that mimics it is designed as an AD therapeutic or prophylactic.

  In addition to the screening methods described above, for example, a low molecular weight compound that mimics the turn structure of either one or both of positions 22 and 38 in Aβ42 can be designed, and a monoclonal antibody can be produced using that as a hapten. It is considered to be a useful method for the creation of AD therapeutic agents or preventive agents.

  <Synthesis of β Amyloid Peptide (Aβ) Mutant> The various Aβ42 mutants described above, except for Aβ42 (amide), were prepared by solid phase synthesis (Fmoc method (Biochem. Biophys. Res. Commun., 295, 306-311 ( 2002), J. Biol. Chem., 279, 52781-52788 (2004), Biochem. Biophys. Res. Commun., 294, 5-10 (2002), J. Biol. Chem., 278, 46179-46187 ( Based on 2003))), it was synthesized by supplying Fmoc amino acid stepwise to 0.1 mmol of Fmoc-Ala-PEG-PS resin in a Pioneer (trade name) peptide synthesizer (manufactured by Applied Biosystems). For the synthesis of Aβ42 (amide), Fmoc-PAL-PEG-PS resin was used. Aβ43 was purchased from Peptide Institute, Inc. (Osaka, Japan). After completion of peptide chain extension, each peptide-resin complex was treated as a mixture containing TFA solution for final deprotection and release from the resin. The crude peptide precipitated with diethyl ether was purified under basicity by HPLC. The obtained Aβ42 was freeze-dried and the purity was confirmed by HPLC (> 98%). Satisfactory data was obtained for the purified Aβ42 using a MALDI-TOF-MS analyzer (Applied Biosystems). That is, the difference between the calculated molecular weight and the theoretical value was less than 1 mass unit. This synthesis process is shown in FIG.

Production of <in vitro hydrogen peroxide assay> _H 2 _{O 2} was measured in accordance with the specifications with a color metric _H 2 _{O 2} assay kit (OXIS Co.). Each Aβ variant was dissolved in 5 mM sodium phosphate buffer solution (containing 150 mM NaCl, pH 7.4) at 165 μM and incubated at 37 ° C. for 4, 8, 16, 24, 48 hours. 100 μl of each solution was added to 1 ml of a color reagent containing xylenol orange in an acidic solution of sorbitol and ammonium iron sulfate, and the absorbance was measured at 560 nm. Note that background values other than peptides were subtracted.

  <ESR spectroscopic analysis> Each Aβ variant is 5 mM sodium phosphate buffer solution containing 50 mM PBN (manufactured by Aldrich) purified by repeated recrystallization in ethyl acetate / hexane, containing 150 mM NaCl, pH 7. In 4), it was diluted to 165 μM. In order to suppress the influence of an excessive metal catalyst reaction, the buffer solution was stirred at room temperature for 48 hours in the presence of Chelex-100 resin (Aldrich). Prior to the addition of Aβ, deferoxamine mesylate was added to the buffer to a concentration of 2 mM. The resulting peptide solution was incubated at 37 degrees for 24 or 48 hours. In preparation for ESR analysis, 400 μl of each solution was placed in an ESR flat cell for a suitable time. ESR spectroscopic analysis was performed at 37 ° C. at room temperature with an EMX ESR spectroscopic analyzer (Burker). The measurement parameters are as follows. Microwave output: 20 mW, microwave frequency: 9.8 GHz, modulation frequency: 100 kHz, amplitude modulation: 1.0 G, conversion time: 40.96 ms, scan: 100 times (room temperature) or 200 times (37 ° C.). The radical intensity integration was performed after removing the background spectrum.

  <Evaluation of cell survival> Mitochondrial function of PC12 cells derived from rat adrenal pheochromocytoma was evaluated by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) reduction assay. . Experimental methods are described in known literature (Biochem. Biophys. Res. Commun., 295, 306-311 (2002), J. Biol. Chem., 279, 52781-52788 (2004), Biochem. Biophys. Res. Commun., 294, 5-10 (2002), J. Biol. Chem., 278, 46179-46187 (2003)).

  <Aggregation Test> The aggregation rate of each Aβ mutant was evaluated by a sedimentation assay. Experimental methods are described in known literature (Biochem. Biophys. Res. Commun., 295, 306-311 (2002), J. Biol. Chem., 279, 52781-52788 (2004), Biochem. Biophys. Res. Commun., 294, 5-10 (2002), J. Biol. Chem., 278, 46179-26187 (2003)). The 220 nm absorption region was integrated and expressed as a percentage of the control. The thermodynamic stability of Aβ fibrils was evaluated by measuring the molar concentration of soluble Aβ in equilibrium using the Micro BCA protein assay (Pierce) according to its specifications.

  Note that the present invention is not limited to the above-described embodiments, and can of course be modified as appropriate without departing from the spirit of the present invention.

The figure which shows the model where Aβ is cut out from APP The figure which shows the neurocytotoxic activity test result of A (beta) 42 variant The figure which shows the aggregation activity test result of A (beta) 42 variant The figure which shows the aggregation activity test result of an A (beta) 42 proline introduction mutant The figure which shows the aggregation model of A (beta) 42 The figure showing the various test results which show the influence which the 22nd and 23rd turn formation of A (beta) 42 mutant has on neurocytotoxic expression The figure showing the various test results which show the influence which it has on neurocytotoxicity of Tyr-10 and Met-35 of A (beta) 42 mutant The figure showing various test results showing the relationship between C-terminal model of Aβ42 mutant and radical generation and neurotoxicity The figure showing the various test results which show the relationship between radical production | generation and aggregation activity of A (beta) 42 variant Model diagram showing the mechanism of Aβ42 aggregation and neurotoxicity expression Schematic process diagram showing the synthesis method of Aβ mutant

Explanation of symbols

Aβ ... β amyloid peptide APP ... Amyloid precursor protein

Claims (7)

A β-amyloid peptide comprising 42 amino acid residues, wherein the amino acid residues at positions 22 and 23 form a turn structure between β-sheet structures according to the amino acid sequences before and after the amino acid sequence. The β-amyloid peptide according to claim 1, wherein the amino acid residues at positions 38 and 39 form a turn structure between β-sheet structures based on the amino acid sequences before and after them. Any one amino acid residue selected from proline, serine, lysine, arginine, aspartic acid, threonine, glutamine, glycine, asparagine, alanine, and glutamic acid, of the two amino acid residues constituting the turn structure. The amino acid residue which is a group and is adjacent thereto is any one amino acid residue selected from asparagine, aspartic acid, glycine, arginine, serine, cysteine, tyrosine, histidine, glutamic acid, threonine, and lysine. Or β-amyloid peptide according to 2. The β-amyloid peptide according to claim 1, wherein the glutamic acid residue at position 22 is substituted with a proline residue. The β-amyloid peptide according to claim 2, wherein both the glutamic acid residue at position 22 and the glycine residue at position 38 are substituted with proline residues. A method for screening a substance which is an active ingredient of an Alzheimer's disease therapeutic agent or preventive agent, wherein the test substance is brought into contact with the β amyloid peptide according to any one of claims 1 to 5, and the aggregation activity of the β amyloid peptide is measured. A method comprising the step of measuring. A method for screening a substance which is an active ingredient of an Alzheimer's disease therapeutic agent or preventive drug, comprising contacting a test substance with the β-amyloid peptide according to any one of claims 1 to 5 and causing neurocytotoxicity by the β-amyloid peptide A method comprising the step of measuring.

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