JP2021004175A - Conformation disease treatment material, method of producing the same, and method of screening conformation disease treatment medicaments - Google Patents

Abstract

To provide a material usable for conformation disease treatment, a method of producing the same, and a method of screening conformation disease treatment medicaments.SOLUTION: The problem is solved by an oligomer protein in which a plurality of aberrant proteins causing conformation disease are cross-linked. The oligomer protein is produced by a method comprising: a provision step for providing aberrant proteins that cause conformation disease; a cross-link step of forming an oligomer by cross-linking the aberrant proteins; and a collection step of collecting the oligomer protein obtained by cross-linking.SELECTED DRAWING: Figure 2

Description

The present invention relates to a material for treating a conformational disease, a method for producing the same, and a method for screening a conformational disease drug.

Conformation disease or protein Conformation disease is a group of diseases in which the conformation of a specific host protein is changed to become sparingly soluble and accumulates in cells, tissues, or organs, resulting in a pathological condition. Disease cases have been reported (see Non-Patent Documents 1 to 3). This includes intractable neurological diseases such as Alzheimer's disease, chronic Lewy body disease, prion disease, polyglutamine disease, Parkinson's disease, and amyotrophic lateral sclerosis.
In conformational diseases, a specific protein takes a conformation rich in structural components called β-sheet structure, which is different from the original conformation, and becomes persistent. Among conformational diseases, a group of diseases in which proteins having such abnormal conformation accumulate in extracellular tissues to form a sparingly soluble substance (amyloid) is called amyloidosis.

On the other hand, there is also a group of diseases in which proteins having such abnormal conformations accumulate inside cells to form a poorly soluble substance called an “inclusion body”. The former includes various amyloidosis diseases such as familial amyloid polyneuropathy, AA amyloidosis, AL amyloidosis, dialysis amyloidosis, and senile amyloidosis, and the latter includes polyglutamine disease, Parkinson's disease, and muscle atrophy lateral sclerosis. Includes chronic amyloidosis, Huntington's disease, Pick's disease, multiple system atrophy, progressive amyloidosis, and frontotemporal dementia. In addition, Alzheimer's disease and prion's disease are found in both cases.

The causative proteins of these conformational diseases differ depending on the disease, and although there is no commonality in the primary structure of the proteins, it is considered that there are commonalities in the mechanism of abnormal conformation and the mechanism of promoting the deposition of abnormal proteins. It is possible to develop therapeutic compounds common to all conformational diseases or a plurality of conformational diseases (see Non-Patent Documents 4 to 5). In fact, there are many reports of drug development that are expected to show common efficacy in Alzheimer's disease, amyloidosis in general, and prion disease (see, for example, Non-Patent Documents 6 to 9). However, although the causes and mechanisms of action of these conformational diseases are gradually being elucidated, no drug that is decisively effective for prevention and treatment has been found so far.

In conformational diseases, inhibition of protein sparingly soluble aggregates and elimination of sparingly soluble aggregates are common drug discovery targets. Among the conformational diseases, Alzheimer's disease, which is being studied, is actively developing passive immunotherapy that administers antibodies against aggregated amyloid β and aggregated tau protein to patients, and some promising results have been reported. (See Non-Patent Documents 10-11). However, for each conformational disease, an antibody optimized for each aggregating protein needs to be selected, and the originally poorly soluble aggregate protein is not recognized as non-self, so only the aggregate protein is used. It is extremely difficult to produce a specific antibody to be recognized, much less a general-purpose development means for various poorly soluble cohesive proteins has been developed.

On the other hand, in active immunotherapy, there is an issue of ingenuity to induce safe and effective antibody production, such as how to prepare a protein as an antigen and which region of the protein is to be recognized. DNA vaccines, mucosal immunity, etc. have been developed to overcome these problems, but they do not necessarily specifically inhibit the formation of toxic aggregates. That is, it not only recognizes aggregate proteins, but also recognizes non-aggregated normal proteins that should be immune tolerant because the aggregate proteins are originally composed of their own proteins. It is also known that even if the formation of multimerized aggregates is inhibited, the formation of highly toxic oligomer aggregates cannot be inhibited (see Non-Patent Document 12).

Further, although various models have been proposed for the chemical structure of the poorly soluble aggregate, the exact chemical structure has not been elucidated due to its poorly soluble physical properties (see, for example, Non-Patent Document 13). Therefore, it is impossible to design drugs in in silico based on the chemical structure.

Here, to explain in more detail the prion disease known as a representative of the diseases caused by the abnormally aggregated protein, the prion disease is a disease in which the central nerve is affected by the prion, and the main body of the prion is the abnormally aggregated prion protein. It has been found that abnormally aggregated prion proteins are called abnormal prion proteins. Today, poorly soluble aggregate proteins that appear in other conformational diseases have been shown to behave similarly to (ie, disease-transmitting) aberrant prion proteins in prion disease, similar to prions. It is considered. Although the immunotherapy for Alzheimer's disease has been described above, some effects have been reported in a plurality of papers on the immunotherapy for prion protein even in prion disease (Non-Patent Document 14). However, none of them target normal prion proteins and none of them target inhibition of abnormal prion proteins. Therefore, as described above, the development of preventive and / or therapeutic agents for prion diseases targeting poorly soluble aggregated proteins also leads to the development of preventive and / or therapeutic agents for conformational diseases.

Robin W Carrell, David A Romes, The Lancet, 1997, No. 350, pp. 134-138 Tokuhiro Ishihara (supervised), Shuichi Ikeda (edited), "Basics and Clinical Practice of Amyloidosis", March 2005, published by Kanahara Publishing Co., Ltd., pp. 8-13 Takeshi Iwatsubo, Cell Engineering, 2001, Vol. 20, No. 11, pp. 1474-1477 Jarrett JT, Lansbury PT Jr, Cell, 1993, Vol. 73, pp. 1055-1558 Gervas F, Morissette C, KongX, Curr. Med. Chem. -Immune. , Endoc. & Metab. Agents, 2003, Volume 3, pp. 361-370 Watson JD, Lander DA, Selkoe JD, The Journal of Biological Chemistry, 1997, Vol. 50, pp. 31617-31624 Kisilevsky R, Nature Medicine, 1995, Volume 1, pp. 143-148 Zhu H, Yu J, Kindy MS, Molecular Medicine, 2001, Vol. 7, pp. 517-522 Katsumi Doura, Pharmacia, 2002, Vol. 7, pp. 635-639 Wisniewski T, Goni F. Neuron, 2015, Vol. 85, No. 6, pp. 1162-1176 Sevigny J, Chiao P, Bussiere T, et al. Nature, 2016, Vol. 537, No. 7618, pp. 50-56 Hara H, Ono F, Nakamura S, et al. Journal of Alzheimer's Disease. 2016, Vol. 54, No. 3, pp. 1047-1059 Shirai T, Saito M, Kobayashi A, et al. Structure, 2014, Vol. 22, No. 4, pp. 560-571 Goni F, Mathison CK, Yim L, et al. Vaccine, 2015, Vol. 33, No. 5, pp. 726-733

In view of the above background, an object of the present invention is to provide a therapeutic material that can be used for the treatment of conformational diseases, a method for producing the same, and a method for screening a conformational disease drug using the material. is there.

The present inventors have proceeded with research to solve the above problems, and have found that abnormal proteins that cause conformational diseases are cross-linked to each other in the process of aggregation to become oligomer proteins. Then, they have found that active immunization is possible by using the oligomer protein as an antigen, and completed the present invention.

One form of the invention is an oligomeric protein in which multiple aberrant proteins that cause conformational diseases are cross-linked.
In addition, another embodiment of the present invention includes a preparatory step for preparing an abnormal protein that causes conformational disease, a cross-linking step in which the abnormal protein is cross-linked to form an oligomer, and a cross-linked oligomer protein. Is a method for producing an oligomer protein, which comprises a recovery step of recovering.
In addition, another embodiment of the present invention comprises a contact step in which a candidate compound is brought into contact with the oligomer protein or the oligomer protein obtained by the above production method, and an activity and / or structure of the oligomer protein in which the candidate compound is brought into contact. A method for screening a conformational disease drug, which comprises a measurement step for measuring conformation disease, or a contact step in which a candidate compound is brought into contact with an abnormal protein that causes conformational disease, and the abnormal protein is cross-linked to obtain an oligomer. A method for screening a conformational disease drug, comprising a cross-linking step to form and a measuring step to measure the activity and / or structure of the cross-linked oligomer protein.

INDUSTRIAL APPLICABILITY According to the present invention, an oligomer protein useful for treating conformational diseases can be provided, and the oligomeric protein can be utilized for active immunity as an antigen for conformational diseases.
In addition, the oligomer protein is cross-linked at the interface of the abnormal protein, and can be utilized for producing an antibody that specifically recognizes the interface.
Further, the oligomer protein is contacted with a candidate compound for a conformational disease drug and the change in activity of the oligomer protein is measured, or the abnormal protein is contacted with a candidate compound for a conformational disease drug and the oligomer protein is contacted. By measuring changes in the forming activity of proteins, screening of conformational disease drugs may be possible.

It is a figure which shows the chemical structural formula of primulin. It is a drawing substitute photograph of a Western blot of an electrophoretic image showing the results of a test on the difference between the target of action of the abnormal prion protein cross-link reaction by primulin and the prion strain when primulin is added to the cell lysate of persistently infected prion cells. (Experimental example 1). It is a drawing substitute photograph of a Western blot of an electrophoretic image showing the result of a test on the action time dependence of an abnormal prion protein cross-link reaction by primulin using a cell lysate of a prion persistently infected cell (Experimental Example 2). FIG. 2 is a graph in which the amount of abnormal prion protein cross-linking reactant produced in the presence of primulin is plotted on the vertical axis and the reaction time is plotted on the horizontal axis in Experimental Example 2. It is a drawing substitute photograph of the Western blot of the electrophoretic image showing the result of evaluating the UV irradiation time dependence of the abnormal prion protein cross-link reaction using the cell lysate of the prion persistently infected cells cultured in the presence of primulin (experiment). Example 3). It is a drawing substitute photograph of the Western blot of the electrophoretic image which shows the result of having tested the effect on the normal prion protein by UV irradiation in the presence of primrin using the cell lysate of a prion non-infected cell (Experimental Example 4). It is a drawing substitute photograph of a Western blot of an electrophoretic image showing the result of testing the effect on β-actin by UV irradiation in the presence of primulin using a cell lysate of a prion non-infected cell (Experimental Example 4). It is a drawing substitute photograph of a Western blot of an electrophoretic image showing a test result of an abnormal prion protein cross-link reaction by UV irradiation in the presence of primulin using a brain extract solution (Experimental Example 5). It is a drawing substitute photograph of silver staining of the electrophoretic image of all proteins in a brain extract solution in the test result of Experimental Example 5. It is a figure which shows the chemical structural formula of the compound used in the test of Experimental Example 6. It is a drawing substitute photograph of a Western blot of an electrophoretic image showing a test result of an abnormal prion protein cross-link reaction by UV irradiation under a mixture of each compound (Experimental Example 6). It is a figure which shows the chemical structural formula of the compound used in the test of Experimental Example 7. It is a drawing substitute photograph of a Western blot of an electrophoretic image showing a test result of an abnormal prion protein cross-link reaction by UV irradiation under a mixture of each compound (Experimental Example 7). 6 is a drawing-substituting photograph of a Western blot of an electrophoretic image showing the result of verifying the effect of methylene blue found in Experimental Example 7 (Experimental Example 8). It is a figure which shows the chemical structural formula of the compound used in the test of Experimental Example 9. It is a drawing substitute photograph of a Western blot of an electrophoretic image showing a test result of an abnormal prion protein cross-link reaction by UV irradiation under a mixture of each compound (Experimental Example 9). It is a drawing substitute photograph of a Western blot of an electrophoretic image showing a test result of an abnormal prion protein cross-link reaction with ammonium persulfate (Experimental Example 10). It is a drawing substitute photograph of a Western blot of an electrophoretic image showing a test result of an abnormal prion protein cross-link reaction with ammonium persulfate and N, N, N', N'-tetramethylethylenediamine (Experimental Example 10). Western blot drawing-substituting photographs of electrophoretic images showing the results of testing the effects of compounds B (compB) and doxycycline (DOX), which are known to exhibit anti-prion activity, on abnormal prion protein cross-linking reactions. In addition, the vertical axis plots the signal intensity corresponding to the cross-link reaction product (dimer) in Western blot, and the horizontal axis plots the final concentration of the anti-prion compound added to the cell lysate (Experimental Example 11). It is a drawing-substituting photograph of a Western blot of an electrophoretic image showing the result of testing the effect of a urea solution on the generated abnormal prion protein cross-linking reaction product (Experimental Example 12). It is a drawing substitute photograph of the Western blot of the electrophoretic image which shows the result of having tested the abnormal prion protein cross-link reaction by UV irradiation when EDTA or copper ion was added (Experimental Example 13). It is a drawing substitute photograph of a Western blot of an electrophoretic image showing the result of an abnormal prion protein cross-linking reaction under the condition that valine, tyrosine, histidine, and tryptophan coexist and their amino acid concentrations are changed (Experimental Example 14). It is a drawing substitute photograph of a Western blot of an electrophoretic image showing the result of detecting an abnormal prion protein cross-linking reaction product by UV irradiation in the presence of primulin with various anti-prion protein antibodies (Experimental Example 15). It is a graph which showed the intensity ratio of the cross-link reaction product (dimer) signal with respect to the monomer signal of an abnormal protein for each antibody about the result of FIG. 23. It is a figure which shows the position of the epitope on the prion protein of the anti-prion protein antibody used in Experimental Example 15. It is a drawing substitute photograph of a Western blot of an electrophoretic image showing the result of detecting an abnormal protein when cross-linking was performed with a prion-infected cell lysate using glutaraldehyde, which is a general divalent cross-linking agent. Experimental example 16). Similar to the case of FIG. 26, it is a drawing-substituting photograph of a Western blot of an electrophoretic image showing the result of detecting the total prion protein without PK treatment (Experimental Example 16). Similar to the case of FIG. 26, it is a drawing-substituting photograph of a Western blot of an electrophoretic image showing the result of detecting β-actin (Experimental Example 16). FIG. 6 is a drawing-substituting photograph of a Western blot of an electrophoretographic image of an abnormal prion protein cross-linking reaction product purified after undergoing a strong protein denaturation treatment (Experimental Example 17).

Hereinafter, the present invention will be described in detail, but the present invention is not limited to the specific forms used in the description.

One embodiment of the invention is an oligomeric protein in which multiple aberrant proteins that cause conformational diseases are cross-linked.
The present inventors conducted research on conformational diseases focusing on the aggregation of abnormal proteins using prion disease as an example. As a result, the abnormal prion proteins were crossed by treating prion disease-infected cells with a specific compound or the like. It has been found that the formation of oligomers such as linked dimer, trimeric and tetramer is promoted. On the other hand, the normal prion protein did not promote cross-linking. In addition, this phenomenon occurred not only in prion-infected cells in solids, but also in cell lysate and prion-infected tissue extracts.
As a result of further research to identify the position of the cross-link, the present inventors have come up with the idea that it matches the interface shown in the abnormal prion protein structure model (Non-Patent Document 13). The present invention has been made based on such findings.

Conformation disease is a disease in which the conformation of a specific host protein is changed to become sparingly soluble and accumulates in cells, tissues, or organs, resulting in a pathological condition. Typically, intractable neurological diseases such as Alzheimer's disease, chronic Levy body disease, prion disease, polyglutamine disease, Parkinson's disease, and muscle atrophy lateral sclerosis, familial amyloid polyneuropathy, AA amyloidosis, and AL amyloidosis. , Dialysis amyloidosis, amyloidosis such as senile amyloidosis.

Cross-linking of aberrant proteins occurs, for example, by adding the following compounds:
Primulin, congo red, methylene blue, luciferin, curcumin derivatives (1,5-bis (2,3-dimethoxyphenyl) -1-hydroxypenta-1,4-dien-3-one and 1,3-bis (3,4-dimethoxyp)
Henyl) propane-1,3-dione, etc.), dye-based / luminescent compounds such as thioflavin T and compound B, radical generators such as ammonium persulfate, N, N, N', N'-tetramethylethylenediamine, etc.
Furthermore, cross-linking also occurs by irradiation with ultraviolet rays.
By using one or a combination of two or more of these, cross-linking of abnormal proteins can be generated.

The amount of the compound that causes cross-linking can be appropriately set depending on the type of compound, the type of conformational disease, the material used, the treatment method thereof, and the like. As an example, when primulin is used to cross-link aberrant proteins of prion disease, it is preferable to add 0.5 μM to 50 μM.

The oligomeric protein of the present embodiment is one in which abnormal proteins that cause conformational diseases are cross-linked to form multimers. Oligomers are typically dimers, trimers, tetramers, pentamers, hexamers, most of which are dimers. The body.

The number of residues of the monomer forming the oligomer protein of the present embodiment is not particularly limited, but it contains a cross-linking site and does not contain many amino acids other than the cross-linking site because of ease of handling and molecular design. It is preferably a fragment. Therefore, it may be fragmented by the enzyme treatment described later.
The number of residues of the monomer is not particularly limited as long as it contains a cross-linking site, but may be 5 or more, 8 or more, or 10 or more. Further, it may be 25 or less, 20 or less, and 15 or less.

The molecular weight of the oligomer protein of the present embodiment is not particularly limited, but may be 1000 or more, 1500 or more, 2000 or more, 5000 or less, 4000 or less, and may be. It may be 3000 or less.

The cross-linking sites of the oligomeric proteins of this embodiment occur at the interface of the aberrant protein that causes conformational disease. Although the structure of the abnormal protein that causes conformational diseases is still unclear, it is becoming clear, for example, a structural model of the abnormal prion protein has been proposed (Non-Patent Document 13). In the experiments of the present inventors, it was confirmed that the cross-linking site of the abnormal prion protein corresponds to interface 2 or interface 3 in the structural model. Further, in the case of an abnormal prion protein, the vicinity of amino acid residue 110 is involved. The neighborhood here is ± 5 residues, may be ± 3 residues, may be ± 2 residues, and may be ± 1 residues.

The method for producing an oligomer protein in which an abnormal protein causing conformational disease is cross-linked is shown below. The manufacturing method is another embodiment of the present invention.
The production method according to the present embodiment includes a preparatory step for preparing an abnormal protein that causes conformational disease, a cross-linking step in which the abnormal protein is cross-linked to form an oligomer, and a cross-linked oligomer protein. A method for producing an oligomeric protein, which comprises a recovery step.

The preparatory step prepares the aberrant protein that causes conformational disease.
The origin of the abnormal protein is not particularly limited, and may be derived from a human, an experimental animal such as a mouse or a rat, a cell, or an in vitro protein. Abnormal prion proteins can be obtained from the brains of infected patients with prion diseases, the brains of infected animals, infected cells, and the like.
Further, it may be a solid carrying the abnormal protein (for example, mouse, rat, etc.), or may be a cell lysate containing the abnormal protein.

When the oligomer protein is used as an antigen (vaccine), a denaturation step may be performed to denature (inactivate) the prepared protein.
Degeneration is carried out according to a conventional method. For example, aberrant proteins are denatured with protein denaturing agents such as sodium dodecyl sulfate (SDS), guanidine hydrochloride, guanidine thiocyanic acid, urea, and formic acid. The denaturation step may be performed on the abnormal protein after the cross-link step.

The cross-linking step is a step of performing a cross-linking treatment on an abnormal protein. The cross-linking is carried out by adding a compound that causes the cross-linking described above, irradiating with ultraviolet rays, or the like. The amount of the compound added and the amount of ultraviolet irradiation are appropriately set depending on the type of conformational disease, the type of the compound, the material used, and the treatment method thereof. Further, the wavelength of ultraviolet rays is not particularly limited, but may be, for example, 365 nm.
Whether or not it is cross-linked can be determined by, for example, enzymatically treating the protein with proteinase K or the like and detecting the molecular weight by SDS-PAGE. By cross-linking, an oligomer is formed, and a high molecular weight band representing a dimer, a trimer, or the like is observed in which the band is moved as compared with the monomer.

The step of recovering the cross-linked oligomeric protein can be performed according to a conventional method. At this time, it may be fragmented so as to remove other than the cross-link portion by enzyme treatment or the like. Further, the cross-link portion may be further isolated and generated.

The oligomer protein of the present embodiment obtained by the above method will be very useful for the treatment of conformational diseases. Active immunotherapy is possible by inactivating the oligomer protein and injecting it as an antigen.
In addition, by elucidating the interface structure of the cross-link site, it becomes possible to design an antibody or compound that specifically recognizes the interface, and it is possible to obtain a therapeutic or preventive drug that inhibits the cross-link.

In addition, another embodiment of the present invention comprises a step of contacting a candidate compound with the oligomer protein or the oligomer protein obtained by the above production method, and an activity and / or structure of the oligomer protein with which the candidate compound is contacted. A method for screening a conformational disease drug, including a step of measuring, or a contact step of contacting a candidate compound with an abnormal protein causing conformational disease, and cross-linking the abnormal protein to form an oligomer. A method for screening a conformational disease drug, comprising a cross-linking step and a measuring step of measuring the activity and / or structure of the cross-linked oligomer protein.

Cross-linked, conformational-causing oligomeric proteins are cross-linked to maintain the interface structure. Therefore, by contacting the candidate compound as a therapeutic drug with the oligomer protein and measuring the activity and / or structure of the oligomer protein, it is possible to directly understand whether or not the candidate compound has any effect on the conformational disease. be able to. Alternatively, by measuring the forming activity of the oligomer protein after contacting the candidate compound with the abnormal protein, it is possible to directly understand whether or not the candidate compound has any effect on the conformational disease. In particular, when the contact of the candidate compound causes a phenomenon such as loosening, bending, structural change, or decrease in cross-linking efficiency of the oligomer protein, the candidate compound has a conformational disease. It can be judged that the compound contributes to the elimination of the aggregation of the protein. Therefore, the screening method of the present embodiment makes it possible to perform a direct assay for a candidate compound for a conformational disease therapeutic agent.

Hereinafter, experiments related to the present invention will be described using an abnormal prion protein, which is a representative of abnormally aggregated proteins.

<Experimental example 1>
Using the cell lysate of prion-infected cells, it was observed that the abnormal prion protein cross-linking action of primulin was exhibited even in different prion strains. In addition, by exchanging the order of the cross-link reaction by the normal prion protein removal treatment and the primulin treatment, the cross-link reaction occurs in the abnormal prion protein among the molecular species of the prion protein, and the abnormal prion protein is partially degraded. It turned out to happen with or without it.

〔Method〕
As the prion persistently infected cells, ScN2a cells in which the scrapie prion RML strain was persistently infected with mouse neuroblastoma cells (Neuro2a), or N167 cells in which the 22L strain was persistently infected with Neuro2a were used. For cell culture, culture in a cell culture flask (bottom area 9.7 cm ² ), remove the culture supernatant when the cell density becomes confluent, and use phosphate buffer saline [PBS, pH 6.5] to remove the cells. Was washed. Remove the washing solution sufficiently, and use 0.5 mL of Lysis solution [0.5% sodium deoxycholate, 0.5% NP-40 (trade name, Wako Pure Chemical Industries, Ltd. octylphenyl ether), balance PBS]. The cells were lysed. The supernatant from which high molecular weight nucleic acid was removed by light centrifugation was used as a cell lysate. FIG. 1 shows the chemical structural formula of primulin, and the compound primulin (also known as direct hello-7, cas # 8064-60-6) was purchased from MP Bioscience and used without purification. Twelve samples of each cell lysate were prepared, and primulin was added to six samples. The final concentration is 0, 10, 2, 0.4, 0.08,
It is 0.016 μM. On the other hand, the remaining 6 samples were proteolytically treated. In the proteolysis treatment, proteinase K (PK: Proteinase K) derived from Tritiracium album was added to a 20 μL cell lysate to a final concentration of 10 μg / mL and reacted (proteolytically) at 37 ° C. for 30 minutes. Later, phenylmethanesulfonylfloride (PMSF) was added to a final concentration of 1 mM to stop the proteolytic reaction. Hereinafter, this proteolytic / termination reaction treatment will be referred to as PK treatment. A sample that was PK-treated after 30 minutes of primulin treatment and a sample that was primulin-treated for 30 minutes after PK treatment were prepared. After that, the abnormal prion protein (hereinafter referred to as PrPres) which is resistant to proteolysis treatment was recovered as a precipitate by centrifugation (9,000 × g, 1 minute), and SDS polyacrylamide gel was used for the total amount of the precipitate. Add 20 μL of buffer for electrophoresis (SDS-PAGE) [1% SDS (sodium dodecyl sulfate), 0.05% bromophenol blue, 4% glycerol, 1% β-mercaptoethanol, 25 mM Tris hydrochloride pH 6.8]. It became turbid and was subjected to heat denaturation treatment at 95 ° C. for 5 minutes to prepare a sample. Hereinafter, the sample at this stage will be referred to as an SDS-PAGE sample. SDS-PAGE samples were electrophoresed on a 15% Tris-glycine SDS-PAGE gel according to the method of Ishikawa, Doura et al. [Journal of General Virology 2004, Vol. 85, pp. 1785-1790], and PrPres was subjected to Western blotting. Detected. In Western blotting, the electrophoresed protein is blotting on nylon membrane (Millipore, PVDF membrane), a mouse monoclonal antibody against prion protein (SPI-BIO, SAF83 (5000-fold dilution)), and alkali. The prion protein was detected using a phosphatase-conjugated goat anti-mouse immunoglobulin antibody (manufactured by Promega (diluted 20000 times)) and a chemical luminescence detection reagent (manufactured by Amersham, CDP-Star detection reagent).

〔result〕
FIG. 2 shows the results of investigating the difference between the target of primulin's cross-linking action and the prion strain using cell lysates of N167 and ScN2a. Lanes 1 to 6 were PK-treated and then subjected to primulin, and lanes 7 to 12 were PK-treated and then PK-treated. The concentration of added primulin was 0 μM in lanes 1 and 7. Hereinafter, lanes 2 and 8, lanes 3 and 9, lanes 4 and 10, lanes 5 and 11, and lanes 6 and 12 are 10, 2, 0.4, 0.08, and 0.016 μM, respectively. The upper blot shows the results using N167, and the lower blot shows the results using ScN2a cell lysate. Abnormal prion protein cross-linking reactants were observed in a primulin concentration-dependent manner, regardless of prion strain. Regarding the order of primulin action and PK treatment, those who applied primulin and then PK treatment (lanes 7 to 12) were more likely than those who performed PK treatment and then applied primulin (lanes 1 to 6). There is a marked increase in the content of cross-linking reactants at the corresponding primulin concentrations. PrPres is a proteolytic resistant partial product in which the N-terminal region of the abnormal prion protein is cleaved by PK treatment. That is, PrPres and the abnormal prion protein differ in molecular size. The results of Example 1 show that the action of primulin tends to act on the abnormal prion protein that has not been cleaved, and that the action on PrPres is rather weak. Therefore, it can be said that this experimental example depicts biochemical differences other than the molecular size of the abnormal prion protein and PrPres on a Western blot.

<Experimental example 2>
The aberrant prion protein cross-link reaction with primulin is time-dependent.
〔Method〕
N167 prion persistently infected cells were cultured on a scale of 25 cm ² according to Experimental Example 1 to obtain 1 mL of cell lysate. Primulin was added to a final concentration of 10 μM, and 150 μL of the sample was taken at 0, 60, 120, 180, 240 and 360 minutes after the addition. The samples were frozen after collection, PK treatment was performed after all the samples were collected, PrPres was recovered by centrifugation, and SDS-PAGE samples were prepared. PrPress was detected according to Experimental Example 1. For comparison, the cell lysate containing no primulin was subjected to the same treatment to obtain a sample. PrPress was detected according to Experimental Example 1. The amount of the cross-link reactant generated from the obtained image was quantified from the pixel density, plotted on Excel, and fitted to an analytical formula assuming a primary reaction.

〔result〕
FIG. 3 shows the results of investigating the action time dependence of the cross-linking action of primulin using N167 cell lysate. Lanes 1 to 6 are the results in the presence of primulin, and lanes 7 to 12 are the results in the absence of primulin. The action time is 0 minutes for lanes 1 and 7, and lanes 2 and 8 below.
Lanes 3, 9, lanes 4, 10, lanes 5, 11, and lanes 6 and 12 are 60 minutes, 120 minutes, 180 minutes, 240 minutes, and 360 minutes, respectively. It can be seen that the formation of cross-link reactants is overwhelmingly promoted in the presence of primulin. Also, its action is reaction time dependent. FIG. 4 is a plot of the relative amount of the produced cross-link reactants on the vertical axis and the time on the horizontal axis, and the growth curve with respect to the time of the primary reaction is shown by a solid line. From the above, it can be said that the abnormal prion protein cross-link reaction by primulin is time-dependent.

<Experimental example 3>
Irradiation of primulin-treated cells with UV promotes the cross-linking reaction of abnormal prion proteins. That is, primulin is an interprotein cross-link photoresensitizer for abnormal prion proteins, and the abnormal prion protein cross-link reaction due to the combined use of primulin and UV irradiation is UV irradiation time-dependent. At the same time, it was found that UV irradiation promotes the abnormal prion protein cross-link reaction even in the absence of primulin.

〔Method〕
N167 prion persistently infected cells were cultured in a cell culture flask having a bottom area of 25 cm ² using a culture medium containing primulin at a final concentration of 2 μM or without primulin, and the cells were lysed with a lysis solution to 1 mL of the cell lysate. Got A UV lamp (Funakoshi, handy type; main wavelength 365 nm) was irradiated at a distance of 15 cm, and 195 μL of sample cell lysate was collected at 0, 15, 30, 45, and 60 minutes. The samples were frozen after collection, and after all the samples were collected, PK treatment was performed and PrPres was recovered by centrifugation to prepare SDS-PAGE samples. In order to make it easier to see the molecular weight of the cross-linking reactant, a treatment was performed to remove the sugar chain of the prion protein. That is, PNGase F derived from Flavobacteriia meningosepticum is added to the SDS-PAGE sample according to the instruction manual of NEB, and sugar chains on the protein contained in the SDS-PAGE sample are removed. The reaction was carried out. After a reaction time of 37 ° C. for 1 hour, a buffer for 5-fold concentrated SDS-PAGE was added, and a heat denaturation treatment at 95 ° C. for 5 minutes was added. Hereinafter, this sugar chain removal / reaction termination treatment will be referred to as a sugar chain removal treatment. The sample subjected to these treatments was electrophoresed in the same manner as in Experimental Example 1, and PrPres was detected by Western blotting.

〔result〕
FIG. 5 shows the results of examining the UV irradiation time dependence of the abnormal prion protein cross-link reaction using the lysate of N167 cells cultured in the presence or absence of primulin. Lanes 1 to 5 use lysates of cells cultured in the absence of primulin, and lanes 6 to 10 use lysates of cells cultured in the presence of primulin. The UV irradiation time is 0 minutes for lanes 1 and 6, and lanes 2 below.
7, lanes 3 and 8, lanes 4 and 9, lanes 5 and 10 are 15 minutes, 30 minutes, 45 minutes and 60 minutes, respectively.
First, the bands of the abnormal prion protein observed in FIGS. 2 and 3 so far are three types of molecular species in which sugar chains are modified at 0, 1, and 2 positions per prion protein molecule. However, in the result of the sugar chain removal treatment in Experimental Example 3, the band was converged to the band at 0 sugar chain modification as seen in lane 1. The molecular weight of this sugar-free chain-modified PrPres is 17 kDa (monomer size). On the other hand, in the UV-irradiated lane, clear bands are detected at 34 kDa (dimer size) and 51 kDa (trimer size). These bands are an integral multiple of 17 kDa and contain a strong protein denaturing agent or reducing agent during the heat denaturation treatment, which is consistent with the abnormal prion protein cross-linking reactant. Since these bands do not disappear during the sugar chain removal treatment, it is clear that the cross-link reaction does not occur at the sugar chain portion on the abnormal prion protein.

It can be confirmed that the abnormal prion protein cross-link reaction proceeds depending on UV irradiation regardless of the presence or absence of primulin. Due to the use of primulin and UV irradiation, almost no monomers derived from abnormal prion protein were detected in lanes 7 and 8, and dimers and trimers, which are abnormal prion protein cross-link reactants, were detected. It can be seen that is the main component of PrPress. However, when the accelerated reaction became excessive as seen in lanes 9 and 10, it was confirmed that the overall detection sensitivity of PrPress decreased. The cause of this optical bleaching effect is unknown.
From the above, it is shown that UV irradiation induces an abnormal prion protein cross-link reaction. The dimeric band observed in the abnormal prion protein cross-linking reaction is a fact that was often observed in PrPress detection experiments, but there has been no report that specifies the conditions of occurrence. Although experiments with commercially available crosslinkers have been reported, they are very common methods with purified proteins, such as in living cell lines or containing impurities, as observed here. Observation of crosslinks in cell lysates is unique. Furthermore, since the use of primulin significantly promotes the abnormal prion protein cross-link reaction by UV irradiation, it was clarified that primulin acts as a photosensitizer in the abnormal prion protein cross-link reaction.

<Experimental Example 4>
The abnormal prion protein cross-link reaction by primulin or UV irradiation as in the previous experimental examples does not act on the normal prion protein. In addition, β-actin, which is known to be in an equilibrium between aggregation and dissociation in cells, does not detect a cross-linking reactant under the conditions of Western blot detection.

〔Method〕
According to Experimental Example 1, prion-uninfected Neuro2a cells were cultured in a cell culture flask having a bottom area of 25 cm ² and lysed with Lysis solution to obtain 1 mL of cell lysate. It was taken into 6 tubes of 150 μL each and used as A, B, C, D, E and F. Primulin was added so that tubes C and D had a final concentration of 1 μM, and E and F had a final concentration of 5 μM. Tubes B, D and F were irradiated with UV for 30 minutes at a distance of 15 cm, while tubes A, C and E were stored in the dark. After completion of the irradiation reaction, a total of 12 12.5 μL reaction solutions were collected from each tube, two for normal prion protein detection and two for β-actin detection, to prepare SDS-PAGE samples. The sample for detecting the normal prion protein was subjected to sugar chain treatment according to Experimental Example 3, and the normal prion protein was detected according to Experimental Example 1. The sample for β-actin was not treated with a sugar chain, and β-actin, which is a target protein, was detected as in Experimental Example 1 except that an anti-β-actin antibody was used as the primary antibody.

〔result〕
The effects of abnormal prion proteins on normal prion proteins and β-actin under conditions that lead to a cross-linking reaction were investigated. FIG. 6 shows a normal prion protein that has been subjected to sugar chain treatment and has not been subjected to PK treatment, and FIG. 7 shows β-actin detected. Each of the lanes 1 to 6 in FIGS. 6 and 7 corresponds to the tubes A to F of the [method]. That is, lanes 1 and 2 are in the absence of primulin, and lanes 3, 4, and lanes 5 and 6 are 1 μM and 5 μM, respectively. In lane 6, which is the most severe reaction condition, a slight light bleaching effect was observed, but no cross-link reaction due to UV irradiation was observed regardless of the presence or absence of primulin. If a dimer is formed by cross-linking, it is expected that bands will be detected at 46 kDa and 80 kDa for the normal prion protein and β-actin, respectively. Therefore, in the examples so far, it was clarified that the formation of the cross-linking reaction product by the addition of primulin, UV irradiation, and the combined use thereof is a specific reaction in the abnormal prion protein.

<Experimental Example 5>
It was shown that an abnormal prion protein cross-link reaction was observed in UV irradiation treatment in the presence of primulin for a solution of a crude fraction containing an abnormal prion protein obtained from the brain of a prion-infected mouse.

〔Method〕
A mouse prion strain (22 L prion, 1% (w / v) PBS suspension) was inoculated into mice (ICR) intracerebrally, and the mice were bred until the terminal stage of illness, and then the brain was collected. The collected brain (260 mg) was placed in a crushing tube and frozen at −80 ° C. PBS (2340 μL) was added and crushed with a stainless steel stir bar to prepare 10% (w / v) brain homogenate, which was dispensed in 200 μL portions into a sealable tube. The following operations were performed on two of the tubes. 2 μL of Protenace E (10 mg / mL aqueous solution) in each tube
Was added and reacted at 37 ° C. for 30 minutes. Then, 4.1 μL of EDTA (0.5 M aqueous solution) and pH 8.0) were added to obtain a final concentration of 10 mM. 206 μL of PBS containing 4% sarcosyl was added, and after stirring, 0.83 μL of benzones was added and reacted at 37 ° C. for 30 minutes. To the reaction solution, add 33.5 μL of a 4% (w / v) aqueous solution of phosphotungstic acid (pH 7.4), 60% (w / v) iodoxanol 70 5.3 μL, and further add 4% (w / v). 57.2 μL of an aqueous phosphotungstic acid solution (pH 7.4) was added. Centrifugation (16100 xg, 90 minutes) was performed on each tube, and the collected supernatants were collected and then passed through a filtration membrane (filtration diameter 0.45 μm) to obtain a filtrate (2000 μL). The filtrate was dispensed into 4 tubes of 480 μL each. Primulin was added to three of them to a final concentration of 20 μM, and UV irradiation was performed at a distance of 10 cm for 30 minutes. The remaining one was an untreated sample. After the reaction, the iodoxanol content was increased by adding 480 μL of 0.3% PTA solution [2% (w / v) sarcosyl, 0.3% (w / v) phosphotungstic acid, PBS] to all the samples. After adjusting to 5.5% (w / v), centrifugation (16100 × g, 90 minutes) was performed, and the supernatant was discarded. Cleaning solution for each precipitate [17.
Add 200 μL of [PBS containing 5% (w / v) iodoxanol and 0.1% (w / v) sarcosyl], and after stirring, 16.2 μL of 4% (w / v) phosphotungstic acid aqueous solution (pH). 7.4) was added, centrifugation (16100 × g, 90 minutes) was carried out, the supernatant was discarded, and a precipitate was obtained. 200 μL of a washing solution was added to the precipitate, and PK treatment (final concentration 10 μg / mL, 37 ° C., 60 minutes) was performed. PMSF was added to a final concentration of 1 mM, and after stirring, 16.2 μL of a 4% (w / v) phosphotungstic acid aqueous solution (pH 7.4) was added, and centrifugation (16100 × g, 30 minutes) was performed. The supernatant was discarded to obtain a precipitate. 200 μL of the washing solution was added to the precipitate, and after stirring, 16.2 μL of a 4% (w / v) phosphotungstic acid aqueous solution (pH 7.4) was added, and centrifugation (16100 × g, 30 minutes) was performed. The supernatant was discarded to obtain a precipitate. After repeating this washing step twice, an SDS-PAGE sample was prepared according to Experimental Example 1, and further, a sugar chain treatment was performed according to Experimental Example 3. As a result of these treatments, one untreated sample and three UV irradiation-treated samples in the presence of primulin are obtained. Among them, one untreated sample (10 μL) and one UV-irradiated sample (10 μL) in the presence of primulin were used, and an amount equivalent to 1/200 of that was subjected to SDS-PAGE electrophoresis as in Experimental Example 1. Then, PrPres was detected by Western blotting. The remaining amount was subjected to SDS-PAGE electrophoresis, and the acrylamide gel was silver-stained according to the instructions of a protein silver-staining kit [(Silver-staining II kit Wako) Wako].

〔result〕
FIG. 8 shows the results of a cross-link reaction by UV treatment in the presence of primulin on prion-infected mouse brain samples. The formation of aberrant prion protein cross-linking reactants was also observed in mouse-infected brain samples by UV treatment in the presence of primulin. This result indicates that an abnormal prion protein cross-link reaction can occur regardless of the origin of the biomaterial. On the other hand, as shown in FIG. 9, since no noticeable band fluctuation was observed in silver staining, the contaminated protein in the crude fraction formed a cross-linking reaction unnecessarily with respect to UV treatment in the presence of primulin. It can be seen that is not occurring.

<Experimental example 6>
We searched for compounds that have the same or similar effects as primulin, and showed that Congo Red has the same efficacy as a sensitizer for the abnormal prion protein cross-link reaction.

〔Method〕
A cell lysate of N167 prion persistently infected cells was obtained according to Experimental Example 2, and 125 μL each was taken in 9 tubes and used as A, B, C, D, E, F, G, H, and I. The following compounds were added to tubes B to I to a final concentration of 50 μM. Lysis solution was added to tube A so that the amount was the same as that of other tubes. The added compounds are primulin, riboflavin, indocyanine green, evance blue, methyl violet, sudan black, fluoroscein and congo red for tubes B, C, D, E, F, G, H and I. UV irradiation was performed for 15 minutes at a distance of 7 cm with respect to the tubes A to I. Then, PK treatment, SDS-PAGE sample preparation, and sugar-free chain treatment were performed according to Experimental Example 3, and PrPres was detected according to Experimental Example 1.

〔result〕
The chemical structural formulas of the compounds used in FIG. 10 are shown in FIG. 11, and the experimental results of the abnormal prion protein cross-link reaction by UV irradiation under the mixture of various compounds are shown in FIG. Here, a study was conducted on a dye-based compound that is widely used. Each of the lanes 1 to 9 in FIG. 11 corresponds to the tubes A to I of [Method]. Similar to the experimental examples so far, in the comparison of lanes 1 and 2, primulin acts as a sensitizer for the abnormal prion protein cross-link reaction. In the comparison between lanes 1 and lanes 3 to 8, no remarkable effect was found. It was found that Congo-Red in lane 9 formed a cross-link reactant (multimer) more easily than primulin. However, due to the intensity of the reaction, it was considered difficult to control the reaction conditions.

<Experimental Example 7>
Similar to Experimental Example 6, another compound having the same or similar effect as primulin was searched for, and it was clarified that luciferin has an effect as a sensitizer for a similar abnormal prion protein cross-link reaction. became. On the other hand, the addition of methylene blue promoted the abnormal prion protein cross-link reaction, but no UV irradiation effect was observed.

〔Method〕
A cell lysate of N167 prion persistently infected cells was obtained according to Experimental Example 2, and 200 μL each was placed in 12 tubes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 And said. The following compounds were added to tubes 3 to 12 so as to have a final concentration of 50 μM. Lysis solution was added to tubes 1 and 2 in the same amount as the other tubes. The added compounds were primulin in tubes 3 and 4, methylene blue in tubes 5 and 6, luminol in tubes 7 and 8, luciferin in tubes 9 and 10, and α-cinnamic acid in tubes 11 and 12. UV irradiation was performed for 15 minutes at a distance of 7 cm to the tubes 2, 4, 6, 8, 10 and 12. Then, PK treatment, SDS-PAGE sample preparation, and sugar-free chain treatment were performed according to Experimental Example 3, and PrPres was detected according to Experimental Example 1.

〔result〕
The chemical structural formulas of the compounds used in FIG. 12 are shown, and FIG. 13 shows the experimental results of the abnormal prion protein cross-link reaction by UV irradiation under the mixture of compounds. In Experimental Example 7, a representative compound in which light is involved in the reaction was selected. Each lane 1-12 in FIG. 13 corresponds to tubes 1-12 of [Method]. Similar to the experimental examples so far, the comparison of lanes 1 and 2 showed the abnormal prion protein cross-linking effect by UV irradiation, and the comparison of lanes 3 and 4 showed that primulin sensitized the abnormal prion protein cross-linking reaction. It has been shown to act as an agent. Luciferin and methylene blue were characteristic of the newly investigated compounds. In the comparison of lanes 9 and 10, luciferin exhibited a sensitizing effect on the abnormal prion protein cross-link reaction by UV irradiation, similar to primulin. On the other hand, in the comparison of lanes 5 and 6, the intensities of the bands showing the multimers were the same. This means that methylene blue does not promote the abnormal prion protein cross-link reaction by UV irradiation, but signal enhancement of the band showing a remarkable multimer is observed in the comparison of lanes 1 and 5. That is, methylene blue causes an abnormal prion protein cross-link reaction in a manner different from that of primulin, congo-red, and luciferin.

<Experimental Example 8>
As a result of verifying the effect of methylene blue discovered in Experimental Example 7, it was shown that the methylene blue concentration of several μM to about 150 μM is suitable for the abnormal prion protein cross-link reaction by addition.

〔Method〕
A cell lysate of N167 prion persistently infected cells was obtained according to Experimental Example 2, and 160 μL of each was taken in eight tubes to make 1, 2, 3, 4, 5, 6, 7, and 8. The concentration of methylene blue added is 0 μM for tube 1 and 300, 150, 75, 38, 19, 9.5, 4.8 μM for tubes 2, 3, 4, 5, 6, 7, and 8, respectively. Then, PK treatment and SDS-PAGE sample preparation were performed according to Experimental Example 1, and PrPres was detected.

〔result〕
FIG. 14 shows the results of investigating the concentration dependence of the abnormal prion protein cross-link reaction using the N167 cell lysate of methylene blue. Each lane 1-12 in FIG. 14 corresponds to tubes 1-12 of [Method]. The intensity of the band indicating the dimer was the strongest in lanes 5 to 8, and the band intensity of the dimer decreased as the methylene blue concentration increased, but instead the band corresponding to the trimer became clearer in lane 3. It was recognized in ~ 5. However, in lane 2 where the methylene blue concentration was 300 μM, the band intensities of all the monomers and multimers were reduced.

<Experimental Example 9>
Similar to Experimental Examples 6 and 7, other compounds having the same or similar effects as primulin were searched for, 1,5-bis (2,3-dimethoxyphenyl) -1-hydroxypenta-1,4-diene. That -3-one or 1,3-bis (3,4-dimethoxyphenyl) propane-1,3-dione or thioflavin T has excellent efficacy as a sensitizer for similar aberrant prion protein crosslink reactions. Indicated.

[Method]
A cell lysate of N167 prion persistently infected cells was obtained according to Experimental Example 2, and 200 μL each was dispensed into a tube. The following compounds were added to the tube to a final concentration of 50 μM. Primulin for tube 2, 1,5-bis (2,3-dimethoxyphenyl) -1-hydroxypenta-1,4-dien-3-one for tube 3, and 1,3-bis (3,4) for tube 4. -Dimethoxyphenyl) propane-1,3-dione, and thioflavin T was added to tube 5. UV irradiation was performed for 40 minutes at a distance of 10 cm from the tubes 2, 3, 4, and 5. Then, PK treatment, SDS-PAGE sample preparation, and sugar-free chain treatment were performed according to Experimental Example 3, and PrPres was detected according to Experimental Example 1.

[result]
The chemical structural formulas of the compounds used in FIG. 15 are shown, and FIG. 16 shows the experimental results of the abnormal prion protein cross-link reaction by UV irradiation under the mixture of each compound. Each lane 1 in FIG.
~ 5 corresponds to tubes 1 to 5 of [Method]. Similar to the experimental examples so far, in the comparison of lanes 1 and 2, primulin acts as a sensitizer for the abnormal prion protein cross-link reaction. All of the newly investigated compounds are superior to primulin as a sensitizer for the abnormal prion protein cross-linking reaction, indicating that not only the cross-linking efficiency is good, but also the antigen epitope is hardly damaged by UV irradiation. Was done. In particular, lane 3 1,5-bis (2,3-dimethoxyphenyl) -1-hydroxypenta-1,4-dien-3-one and lane 4 1,3-bis (3,4-dimethoxyphenyl) propane-1. , 3-dione was excellent.

<Experimental Example 10>
It has been shown that common radical reactants also cause aberrant prion protein cross-linking reactions. It was also shown that the properties of the abnormal prion protein cross-linking reactant by the radical reactant are different from those of the abnormal prion protein cross-linking reactant obtained by UV irradiation in the presence of primulin.

[Method]
A cell lysate of N167 prion persistently infected cells was obtained according to Experimental Example 2, and 500 μL each was dispensed into eight tubes. Ammonium persulfate was added to tubes 2, 6, 3, 7, 4 and 8 at 0.05 mg, 0.5 mg and 5 mg, respectively, and tubes 6, 7 and 8 were further added with N, N, N'and N'-. 0.01 μL and 0.1 μL of tetramethylethylenediamine, respectively
1 μL was added. After reacting at 37 ° C. for 1 hour, the sample of each tube was divided into halves for PK treatment and non-treatment, and for PK treatment, PK treatment, SDS-PAGE sample preparation, and sugar desalination were performed according to Experimental Example 3. Chain processing was performed. For the non-treated product, PK treatment was not carried out, and SDS-PAGE sample preparation and sugar chain treatment were carried out according to Experimental Example 3 except for the PK treatment step. PrPress was detected for all the samples according to Experimental Example 1.

[result]
FIG. 17 shows the effect of adding ammonium persulfate on the abnormal prion protein with and without PK treatment. Samples 1 to 4 correspond to tubes 1 to 4 of [Method]. In lanes 3 and 7, which are samples supplemented with 0.5 mg of ammonium persulfate, a dimer-sized signal due to cross-linking of a remarkable abnormal prion protein was observed. FIG. 18 shows the effect of ammonium persulfate and N, N, N', N'-tetramethylethylenediamine added on the abnormal prion protein with and without PK treatment. Samples 5-8 correspond to tubes 5-8 of [Method]. Similar to FIG. 17, in lanes 3 and 7, which are samples to which 0.5 mg of ammonium persulfate and 0.1 μL of N, N, N', N'-tetramethylethylenediamine were added, a remarkable abnormal prion protein was cross-linked. A dimer-sized signal was observed. Therefore, it was shown that radical reactants also induce a cross-linking reaction of aberrant prion proteins.

<Experimental Example 11>
It is possible to classify anti-prion compounds by utilizing the abnormal prion protein cross-link reaction by UV irradiation, and the mechanism of action of the compounds can be considered using this index. On the contrary, this indicates that the abnormal prion protein cross-link reaction by UV irradiation can be used for the search for anti-prion compounds.

〔Method〕
A cell lysate of N167 prion persistently infected cells was obtained according to Experimental Example 2, and 180 μL each was taken in 10 tubes, which were designated as A, B, C, D, E, F, G, H, I, and J, and tube B. Compound B (compB: 4-[(E)-{2- [4- (1,), which is known to inhibit the structural conversion of normal prion protein to abnormal prion protein in C, D, and E. 3
-Oxazole-5-yl) phenyl] hydrazinylidene} methyl] pyridine), tubes G, H, I, J were added with doxycycline (DOX), which is known to relax the structure of aberrant prion proteins. The final concentrations of tubes B, G, tubes C, H, tubes D, I, tubes E, and J are 0.5, 2, respectively.
It is 10, 20 μM. Lysis solution was added to tubes A and F so as to have the same amount as the other tubes. After the addition, the tubes A to J were irradiated with UV for 30 minutes at a distance of 10 cm. After completion of the reaction, PK treatment was performed according to Experimental Example 3, a sample of SDS-PAGE was prepared, and after deglycanization treatment, PrPress was detected according to Experimental Example 1.

〔result〕
Compound B (compB) and doxycycline (DOX) are compounds known to exhibit anti-prion activity in previous reports. For example, Compound B was described in [Kawagoe K, Motoki K, Odagiri T, et al. 2004. Hydrazone derivative. WIPO Publication No. WO / 2004/087641, ka October 14, 2004].
i Y, Kawagoe K, Chen C, et al. Journal of Virology, 2007, Vol. 81, pp. 12889-12898] by inhibiting the structural conversion of normal prion proteins to abnormal prion proteins in cells and animals. It has been shown to exert anti-prion activity. Regarding doxycycline, [Tagliavini
F, Forloni G, Colombo L, et al. Journal of Molecular Biology 2000, Vol. 300, No. 5, pp. 1309-1322] has reported an action of relaxing the structure of abnormal prion proteins. In this experimental example, the effects of these two compounds on the abnormal prion protein cross-link reaction were tested. Lanes 1 to 10 of the Western blot in the upper part of FIG. 19 correspond to tubes A to J of [Method], respectively. The lower graph of FIG. 19 shows the intensity of the band of each lane corresponding to the dimer size of the Western blot. The concentration of the compound is shown on the horizontal axis. As is clear from the graph, when compound B was added, the signal intensity of the dimer band was enhanced in a concentration-dependent manner as observed with primulin in Experimental Example 1. On the other hand, when doxycycline was added, the signal intensity of the dimer band was attenuated depending on the addition concentration. Thus, the two anti-prion compounds can affect the outcome of the abnormal prion protein cross-link reaction by UV irradiation, and the difference in their actions between compound B and doxycycline is that of the abnormal prion protein cross-link reaction. It was shown to be reflected in the results. Therefore, it is possible to classify compounds known as anti-prion compounds and infer the mechanism of action using the difference in the effect on the abnormal prion protein cross-link reaction as an index. In addition, the results of this experimental example show that the abnormal prion protein cross-link reaction system can be used as a simple in vitro selection system for anti-prion candidate compounds.

<Experimental Example 12>
Abnormal prion protein cross-linking reactants produced by UV irradiation in the presence of primulin do not return to monomers by treatment with urea solution.

〔Method〕
Obtain a cell lysate of N167 prion persistently infected cells according to Experimental Example 2, take 200 μL each in 5 tubes, set A, B, C, D, and E, and add primulin to each tube so that the final concentration is 50 μM. After the addition, UV irradiation was performed for 15 minutes at a distance of 7 cm. Then, PK treatment was carried out according to Experimental Example 1 and a Precipitate of PrPres was obtained by centrifugation. The precipitate was dissolved in 8 μL of different aqueous urea solutions. The urea concentration of the urea solution is 0, 2, 4, 6, 8M in tubes A, B, C, D and E. After leaving to stand at room temperature for 10 minutes, a 5-fold concentration SDS-PAGE sample buffer was added to each tube to prepare a sample of SDS-PAGE, and PrPress was detected according to Experimental Example 1.

〔result〕
The properties of the abnormal prion protein cross-linking reactants observed in the experimental examples so far were investigated. FIG. 20 shows the results of investigating the effect of the urea solution on the abnormal prion protein cross-linking reactant. Each of the lanes 1 to 5 in FIG. 20 corresponds to the tubes A to E of the [method]. The signal intensity of the entire PrPress band in each lane tended to be attenuated by the presence of urea, but the degree of attenuation did not depend on the concentration of urea. The ratio of the signal intensities of the monomer band to the dimer band is constant over lanes 1-5. That is, no dissociation of the multimer into the monomer was observed even by the addition of the urea solution.
In rare cases, multimers may be detected in SDS-PAGE depending on the type of protein. In the experimental examples so far, 0.5% β-mercaptoethanol and 1% sodium dodecyl sulfate were already contained as a reducing agent and a protein solubilizing / denaturing agent in the sample buffer. In a normal protein, when this sample buffer is added and heated, it is completely solubilized as a monomer and detected at a position showing an appropriate molecular weight as a monomer. This is a very widely used experimental method. However, for a special protein, for example, transthyretin, a band with a molecular weight corresponding to a tetramer is detected in SDS-PAGE under the conditions of the experimental examples so far. However, by adding urea to the sample buffer [J
enne DE, Denzel K, Blatzinger P, et al. Proceedings of the National Academia of Sciences U.S.A. S. A. , 1996, Vol. 93, pp. 6302-6307], urea concentration 2.5
It is known that transthyretin is detected in a band having a molecular weight completely corresponding to a monomer at about M. This citation is an experiment using purified transthyretin and shows that the factor forming the multimer is due to the strong hydrogen bonds between the molecules. The results of this experimental example show that the aberrant prion protein cross-linking reactant induced by UV irradiation in the presence of primulin is not due to strong intermolecular hydrogen bonds. In addition, the fact that the abnormal prion protein cross-link reaction occurs in a contaminated system called prion persistently infected cell lysate and in the presence of a considerable surfactant in the lysate is different from the experimental conditions in the cited references. It is a major difference, reflecting the structural characteristics of the abnormally aggregated protein and the specificity of the reaction.

<Experimental Example 13>
As a result of investigating the effect of EDTA and copper ion on the abnormal prion protein cross-link reaction by UV irradiation, EDTA has no effect, but copper ion affects the abnormal prion protein and the abnormal prion protein cross-link reaction. Reduced efficiency.

〔Method〕
Obtain a cell lysate of N167 prion persistently infected cells according to Experimental Example 2, take 180 μL each in 16 tubes, add primulin to a final concentration of 10 μM, and add A, B, C, D, E, F. , G, H, I, J, K, L, M, N, O, P, EDTA-added lysis solution in tubes B, C, D, F, G, H, tubes J, K, L, N, O , P was added with a copper sulfate-added lysis solution. The final concentrations of tubes B, F, tubes C, G, tubes D, and H are 5000, 1000, and 200 μM, respectively, and tubes J, N, tubes K, O, and tubes L and P are 1000, 200, and 50 μM, respectively. is there. Lysis solution was added to tubes A, E, I, and M in the same amount as the other tubes. After the addition, tubes E, F, G, H, M, N, O and P were irradiated with UV for 15 minutes at a distance of 7 cm. Meanwhile, tubes A, B, C, D, I, J, K and L were stored in a cool and dark place. After the reaction, PK treatment was performed according to Experimental Example 1, a sample of SDS-PAGE was prepared, and PrPress was detected.

〔result〕
The region of the 51-91 amino acid sequence of the prion protein has been reported to bind to copper ions [Hornshaw MP, McDermott JR, Candy JM, Lake.
y JH. Biochemistry Biophysics Research Com
munication, 1995, Vol. 214, pp. 993-999]. This region is the part that is cleaved in the PK treatment that degrades the normal prion protein and turns the abnormal prion protein into a proteolytic enzyme resistant core (agreeing with PrPres) that shows its traces. In the abnormal prion protein cross-link reaction by primulin, UV irradiation, or a combination thereof, the cross-link reaction efficiency decreases in PrPres as observed in Experimental Example 1, and therefore, the cross-link reaction involves the 51-91 amino acid sequence. It has been suggested that indirect involvement, that is, auxiliary involvement when cross-linking occurs, may not be the area that receives direct cross-linking. Therefore, for the purpose of structural perturbation to the 51-91 amino acid sequence, a chelating agent and a copper ion were added, and an abnormal prion protein cross-link reaction was carried out. FIG. 21 shows the results of investigating the abnormal prion protein cross-link reaction by UV irradiation when EDTA or copper ions were added. Each of the lanes 1 to 16 in FIG. 21 corresponds to the tubes A to P of the [method]. EDTA did not affect the reaction in comparisons in lanes 1-4 and lanes 5-8. On the other hand, in the comparison in lanes 9 to 12 and lanes 13 to 16, the addition of copper ions showed a decrease in the signal intensity of the monomer band of the abnormal prion protein, and suppressed the abnormal prion protein cross-link reaction. Was done. Therefore, it is possible that the 51-91 amino acid sequence is indirectly involved in the efficiency of the abnormal prion protein cross-linking reaction. Reagents such as copper ions and EDTA cannot be used in a normal compound selection system using live cells because they cause cell damage, but the evaluation in an experimental system using a cell lysate as in this experimental example is abnormal. It is useful in evaluating reagents that act on prion proteins.

<Experimental Example 14>
The aberrant prion protein cross-link reaction is not inhibited by the addition of amino acids that have absorption properties in the UV wavelength region.

〔Method〕
A cell lysate of N167 prion persistently infected cells was obtained according to Experimental Example 2, taken in 16 tubes of 180 μL each, and primulin was added to a final concentration of 10 μM, and A, B, C, D, E, F were added. , G, H, I, J, K, L, M, N, O, P, valine for tubes B, C, D, tyrosine for tubes F, G, H, histidine for tubes J, K, L, tubes Tryptophan was added to N, O and P. The final concentrations of tubes B, F, J, N, tubes C, G, K, O, tubes D, H, L, and P are 100, 20, and 5 mM, respectively. Lysis solution was added to tubes A, E, I, and M in the same amount as the other tubes. After the addition, tubes A to P were irradiated with UV for 15 minutes at a distance of 7 cm. After the reaction, PK treatment was performed according to Experimental Example 1, a sample of SDS-PAGE was prepared, and PrPress was detected.

〔result〕
Although the solvent conditions and reaction conditions used are different from those of the inventor's experimental example, there is a report using purified protein and compound fluorescein for cross-linking by photoreaction [Webst].
er A, Britton D, App-Bologna A, Kemp G. Ana
Lytical Biochemistry, 1989, Vol. 179, pp. 154-157]. Although the reaction mode is not shown in the report, the intermolecular cross-linking of fibrinogen is shown in the reducing agent-free SDS-PAGE gel. It has also been shown that the photoinduced cross-linking of purified fibrinogen is inhibited by tryptophan and tyrosine, which are amino acids having absorption characteristics in the UV wavelength region at about 10 mM. Therefore, in this experimental example, it was tested whether or not the abnormal prion protein cross-link reaction was inhibited by coexisting an amino acid having absorption in the UV wavelength region.
FIG. 22 shows the results of the abnormal prion protein cross-link reaction under the condition that valine, tyrosine, histidine, and tryptophan coexist and their amino acid concentrations are changed. No inhibitory effect on the abnormal prion protein cross-link reaction was observed for any of the amino acids. This can be said to be a natural result, considering that the abnormal prion protein cross-linking reaction occurs specifically in the contaminating system. From the results in this experimental example, it is inferred that the cross-link in the abnormal prion protein is formed at a portion where the abnormal prion proteins, which are substrates, are temporally stable and located in the immediate vicinity.

<Experimental Example 15>
Analysis using the anti-prion protein antibody panel suggested that the cross-link reaction in the abnormal prion protein was related to the vicinity of amino acid residue 110. In the abnormal prion protein model (BH4 model) of Shirai et al. (Structure, 2014, Vol. 22, No. 4, pp. 560-571), this region is defined as interfaces 2 and 3 in which prion proteins interact with each other. It is in a position to be involved.

〔Method〕
A cell lysate of N167 prion persistently infected cells was obtained according to Experimental Example 2, primulin was added to a final concentration of 20 μM, and UV irradiation was performed for 15 minutes. After the reaction, PK treatment was performed according to Experimental Example 3, a sample of SDS-PAGE was prepared, and sugar chain treatment was performed to finally obtain a 90 μL SDS-PAGE sample. 4.8 μL of each was added to lanes 1 to 7, and SDS-PAGE gel electrophoresis and blotting to nylon membrane were performed according to Experimental Example 1. For the detection of PrPress signals, 1E4, 3C10, 6D11, 6H4, SAF70, SAF83, and M20 were used as primary antibodies against lanes 1, 2, 3, 4, 5, 6, and 7, respectively, and then with Experimental Example 1. The same was done. Among these antibodies, M20 used anti-goat IgG as a secondary antibody, and the rest used anti-mouse IgG.

〔result〕
In the experimental examples so far, SAF83 has been used as an antibody to detect PrPres monomer signals and multimer signals. The position of the epitope of SAF83 on the prion protein is on amino acid 126-164. By the way, when a cross-link reaction occurs, there is a high possibility that the antigen-antibody reaction between the epitope and the specific antibody in the vicinity including the site will be affected. Therefore, we searched for cross-linking sites by using various types of antibodies. FIG. 23 shows the results of detecting PrPress using various primary antibodies. In the detection using 1E4 and 3C10, the dimer signal detection rate was significantly lower than that in the case of using the other five types of antibodies. FIG. 24 is a graph showing the ratio of the dimer signal intensity to the monomer signal intensity obtained for the result of FIG. 23, and the ratio is shown on the vertical axis. It is clear that the detection rate using 1E4 and 3C10 significantly lowers the dimer signal detection rate than the case using the other five types of antibodies. The position of the epitope on the prion protein recognized by each antibody is shown in FIG. In both 1E4 and 3C10, the epitope is located in an extremely narrow region near amino acid residue 110, and from these results, it can be seen that the cross-link reaction in the abnormal prion protein is related to the vicinity of amino acid residue 110. Shown. According to Shirai et al.'S abnormal prion protein model (BH4 model) (Structure, 2014, Vol. 22, No. 4, pp. 560-571), this region is an interface 2 in which abnormal prion proteins interact with each other. It has been shown to be a region involved in and 3 and there is no contradiction even if a cross-link reaction due to UV irradiation occurs in this region.

<Experimental Example 16>
It was shown that cross-linking specific to aberrant prion proteins is not possible by cross-linking with a common divalent cross-linking agent such as glutaraldehyde.

〔Method〕
A cell lysate of N167 prion persistently infected cells was obtained according to Experimental Example 2, and 180 μL each was taken in 6 tubes, which were designated as A, B, C, D, E and F, and glutar in tubes C, D, E and F. Aldehyde was added. The final concentrations of tubes C and D and tubes E and F are 0.1 and 0.02%, respectively. Lysis solution was added to tubes A and B in the same amount as the other tubes. After the addition, tubes B, D and F were irradiated with UV for 30 minutes at a distance of 15 cm. After completion of the reaction, 10 μL of each tube was sampled for β-actin detection and 20 μL was sampled for total prion protein detection before PK treatment. The rest was PK-treated according to Experimental Example 3, a sample of SDS-PAGE was prepared, and after the sugar-free chain treatment, PrPress was detected according to Experimental Example 1. The detection of β-actin and total prion protein before PK treatment was carried out according to Experimental Example 4.

〔result〕
Glutaraldehyde is a compound having two aldehyde groups in a molecule, which is widely used for fixing tissues and the like. It was tested how the detection of prion protein and β-actin showed when cross-linking was performed in a contaminating system such as a cell lysate using a general cross-linking agent. Lanes 1 to 6 of FIGS. 26, 27, and 28 correspond to tubes A to F of [Method], respectively. 26, 27, and 28 show PrPres, total prion protein without PK treatment, and β-actin, respectively.

PrPress, shown as the 17 kDa band in FIG. 26, and the fragmented aberrant prion protein, shown as the 17 kDa band in FIG. 27, were detected in the presence of glutaraldehyde, as shown in FIGS. 5 and 6, respectively. The efficiency is significantly reduced. Further, the full-length prion protein of the abnormal type and the normal type shown as a band of 23 kDa in FIG. 27 has a reduced detection sensitivity in the presence of a low concentration of glutaraldehyde as shown in lanes 5 and 6. However, in lanes 3 to 6 of FIGS. 26 and 27, bands corresponding to dimers and trimers of abnormal prion proteins are not detected. On the other hand, β-actin is known as a protein that polymerizes and depolymerizes in cells, but a multimer band is not detected in lanes 3 to 6 of FIG. 28, and when a high concentration of glutaraldehyde is present, it depends on an antibody. The detection efficiency is low. As shown in lanes 5 and 6, if the concentration of glutaraldehyde is low, a monomeric signal is detected to the same extent as when glutaraldehyde is not added shown in lanes 1 and 2.
Therefore, specific cross-linking of abnormal prion proteins is not possible with a general divalent-reactive cross-linker method.

<Experimental example 17>
When guanidine thiocyanic acid treatment is performed in addition to sodium dodecyl sulfate addition heat treatment as a protein denaturing agent treatment necessary for the process of producing a therapeutic biological material or a therapeutic drug creation biological material from an abnormal prion protein cross-linking reaction product. However, the electrophoresis pattern showed that the cross-link reactants were unaffected.

〔Method〕
According to Experimental Example 2, a cell lysate of N167 prion persistently infected cells was obtained, and 1 mL each was taken in 12 tubes, and 6 of them were 1,3-bis (3,4-dimethoxyphenyl) propane-1,3. -Dione was added to a final concentration of 20 μM, and UV irradiation was performed for 80 minutes at a distance of 10 cm. The remaining 6 were stored in a cool and dark place. After completion of the reaction, 12 tubes were PK-treated according to Experimental Example 1 to prepare SDS-PAGE samples as follows. 15 μL of SDS-P after centrifugation of the cell lysate
It was dissolved in AGE sample buffer and heat-denatured at 95 ° C. for 5 minutes. Six UV-irradiated samples and six UV-irradiated samples were combined into one tube each to prepare a 90 μL solution. 60 μL of water was added to each sample, and 150 μL of chloroform, 600 μL of methanol, and 450 μL of water were added in this order to the stirred solution, and centrifugation was performed at 6000 × g for 10 minutes. The upper aqueous layer was removed, 450 μL of methanol was added thereto, and the mixture was centrifuged in the same manner. Then, the upper organic layer was removed to obtain a proteinaceous precipitate. 450 μL of methanol was added to the precipitate, the same centrifugation was performed, and the upper layer was discarded to wash the precipitate. The process of obtaining a protein precipitate from the above aqueous protein solution is called a chloroform-methanol precipitate. The washed precipitate was air-dried, and 20 μL of an 8M guanidine thiocyanate aqueous solution was added to the residue, and the mixture was reacted at 60 ° C. for 1 hour. Chloroform-methanol precipitation was performed on a total of 100 μL of a sample to which 80 μL of water was added after the reaction, 15 μL of SDS-PAGE sample buffer was added to the residue after air drying, and a part thereof was used as a sample for Western blotting. PrPress was detected according to 1.

〔result〕
The SDS-PAGE sample buffer contains the potent detergent sodium dodecyl sulfate. In addition, guanidine thiocyanate is known to be a potent denaturant of proteins. For the purpose of producing therapeutic biological materials or therapeutic biological materials, infectious abnormal prion proteins need to be sufficiently eliminated by strong protein denaturation at the same time. As long as it is used in a living body, it is necessary to prepare a test product in which the above-mentioned strong protein denaturant is sufficiently removed. By the series of methods shown in this experimental example, it is possible to remove the infectivity by thoroughly denaturing the protein and the denaturing agent used. In the results of Western blotting in FIG. 29, lane 1 was a sample without cross-linking, and lane 2 was cross-linked by UV irradiation in the presence of 1,3-bis (3,4-dimethoxyphenyl) propane-1,3-dione. A sample is shown. From this result, it was shown that the cross-linking reactant of the abnormal prion protein was not affected by the series of protein denaturation treatment and denaturing agent removal treatment.
When a known amount of recombinant prion protein was quantified as a standard, 48 μg of abnormal prion protein equivalent to 48 μg from 150 cm ² N167 prion persistently infected cultured cells was found in the sample corresponding to lane 1, and in the sample corresponding to lane 2. An abnormal prion protein equivalent to 52 μg was obtained from the same prion persistently infected cultured cells. Therefore, in the UV treatment in the presence of 1,3-bis (3,4-dimethoxyphenyl) propane-1,3-dione, the reaction does not cause loss of the abnormal prion protein. In addition, the densitometry of the band signal of lane 2 is shown on the right side of the figure. From this data, it is possible that a monomer and a dimer occur at a concentration ratio of 7: 3 under the reaction conditions of this experimental example. Shown.

<Experimental Example 18>
It is shown that cross-linking the abnormal prion protein itself is effective as a medicine. Further, it is shown that a protein denaturant of an abnormal prion protein cross-link reaction product (hereinafter referred to as a modified prion protein denaturant because the pathogenicity is lost by the protein denaturation treatment of Experimental Example 17) is effective as a medicine. Furthermore, it is shown that a pharmaceutical product prepared based on a modified prion protein cross-link is effective.

〔Method〕
In vitro prion amplification experiments, persistent prion-infected cells, and prion-infected mice induce the formation of abnormal prion protein cross-link reactants as shown in previous experimental examples by combining compound administration and electromagnetic irradiation at a certain wavelength. Then, it is investigated whether the formation of the abnormal prion protein cross-linking reaction itself has a therapeutic effect in an in vitro prion amplification experiment, prion persistently infected cells, and prion-infected mice. In an in vitro prion amplification experiment, the therapeutic effect is judged by whether or not the amplification of abnormal prion protein is inhibited. In persistently infected prion cells, the therapeutic effect is determined by whether or not the proliferation of abnormal prion protein is inhibited. In prion-infected mice, the therapeutic effect is determined by whether or not the period from the time of infection to the onset of the disease or the final stage of the disease is prolonged.

Further, based on the fraction containing the abnormal prion protein cross-linking reaction prepared from the mouse brain shown in Experimental Example 5, the protein denaturation treatment step and the protein recovery step shown in Experimental Example 17 were carried out for denaturation. A fraction containing a prion protein cross-link is prepared. This fraction is mixed with an adjuvant and the mixture is injected subcutaneously into the back of prion-infected mice for active immunization. The therapeutic effect is judged by whether the period from the onset of the prion infection to the onset of the disease or the terminal stage of the onset of the disease is extended. A control is a prion-infected mouse immunized with a fraction free of denatured prion protein crosslinks.
Furthermore, an antiserum obtained by immunizing a normal mouse with a fraction containing a denatured prion protein crosslinker and an antibody obtained by purifying the antibody from the antiserum were used in an in vitro prion amplification experimental system and a prion persistently infected cell supernatant. And prion-infected mice to examine the therapeutic effect. In an in vitro prion amplification experiment, the therapeutic effect is judged by whether or not the amplification of abnormal prion protein is inhibited. In persistently infected prion cells, the therapeutic effect is determined by whether or not the proliferation of abnormal prion protein is inhibited. In prion-infected mice, the therapeutic effect is determined by whether or not the period from the time of infection to the onset of the disease or the final stage of the disease is prolonged.

〔result〕
In the sample in which the abnormal prion protein cross-link reactant was formed by UV irradiation in the presence of primulin, the abnormal prion protein was obtained in the in vitro prion amplification experiment as compared with the control sample containing no abnormal prion protein cross-link reactant. It is expected that the amplification of the protein will be suppressed. In addition, in the prion persistently infected cells in which the abnormal prion protein cross-link reaction product was formed by UV irradiation in the presence of primulin, the proliferation of the abnormal prion protein was suppressed as compared with the prion persistently infected cells not subjected to the same treatment. It is expected to be done. Furthermore, in mice inoculated with prion into the back skin of shaved mice, administered with primulin to the inoculated area, and then irradiated with UV, compared to mice not subjected to the same treatment, from the time of infection to the onset or the final stage of onset of disease. It is expected that the period will be extended.

In addition, prion-infected mice that were actively immunized by mixing a fraction containing a denatured prion protein crosslink with an adjuvant and injecting the mixture subcutaneously into the back to immunize the fraction without the denatured prion protein crosslink. It is expected that the period from the time of infection to the onset of the disease or the final stage of the onset of the disease will be prolonged as compared with the prion-infected mice.
Furthermore, when an antiserum obtained by immunizing a normal mouse with a fraction containing a denatured prion protein cross-linker and an antibody obtained by purifying the antiserum are administered to an in vitro prion amplification experimental system, the control serum or It is expected that the amplification of abnormal prion protein is suppressed as compared with the case where the control antibody is administered. Anti-serum obtained by immunizing normal mice with a fraction containing a denatured prion protein cross-linker and an antibody obtained by purifying the anti-serum are administered to the supernatant of persistently infected prion cells to control serum or control antibody. It is expected that the growth of abnormal prion protein is suppressed as compared with the case of administration of. Furthermore, when an antiserum obtained by immunizing a normal mouse with a fraction containing a denatured prion protein crosslink and an antibody obtained by purifying the antiserum is administered to a prion peritoneal infected mouse, a control serum or a control antibody is obtained. It is expected that the period from the time of infection to the onset of illness or the final stage of illness will be longer than that of the administration of.

Claims (10)

An oligomeric protein in which multiple aberrant proteins that cause conformational diseases are cross-linked. The oligomeric protein according to claim 1, wherein the abnormal protein is a fragment of the protein. The oligomeric protein according to claim 1 or 2, wherein the crosslink occurs at the interface of the abnormal protein. The oligomer protein according to any one of claims 1 to 3, which is used as an antigen. Preparatory steps to prepare abnormal proteins that cause conformational diseases,
A method for producing an oligomer protein, which comprises a cross-linking step of subjecting the abnormal protein to an oligomer to form an oligomer, and a recovery step of recovering the cross-linked oligomer protein. The production method according to claim 5, further comprising a fragmentation step of enzymatically treating and fragmenting the abnormal protein. The production method according to claim 5 or 6, wherein the cross-link occurs at an interface present in the abnormal protein. The production method according to any one of claims 5 to 7, wherein the oligomer protein is used as an antigen. The contact step of contacting the candidate compound with the oligomer protein according to any one of claims 1 to 4 or the oligomer protein obtained by the production method according to any one of claims 5 to 7, and the above-mentioned. A measurement step, which measures the activity and / or structure of the oligomeric protein contacted with the candidate compound, is included.
Screening method for conformational disease drugs. Preparatory steps to prepare abnormal proteins that cause conformational diseases,
A contact step in which the candidate compound is brought into contact with the abnormal protein,
The aberrant protein contacted with the candidate compound is subjected to a cross-link treatment to form an oligomer, and a measurement step of measuring the activity and / or structure of the oligomer protein contacted with the candidate compound is included. Screening method for conformation disease drugs.

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