Aβ drug screening targets and screening method

Abstract

Anti-Aβ drug screening targets and a drug screening method. The screening method uses different Aβ aggregates prepared by simulating the in vivo environment and conditions as targets, to more accurately and effectively screen anti-Aβ candidate drugs; the Aβ targets involved in the screening method are aggregated and incubated under conditions approaching different in vivo microenvironments, and do not require labeling or modification; in addition, the screened drug molecules do not require any modification or labeling, and are not limited to any specific drug class, being widely applicable to different drug types, such as antibodies, peptides and small molecules, truly reflecting the interactions between targets and these drug molecules, greatly reducing the likelihood of problems such as false positives, false negatives or incorrect binding modes.

Description

Aβ drug screening targets and screening methods

[0001] Priority Declaration

[0002] The present invention claims priority from Chinese patent application No. 2024102819552, filed on March 12, 2024, entitled “Anti-Aβ Drug Screening Targets and Screening Methods.” The present invention incorporates the entire text of the aforementioned Chinese patent application.

[0003] This application claims priority to PCT patent application number PCTCN2025074897, filed January 24, 2025, entitled “Benzoheterocyclic compounds and their applications.” The present application incorporates the entirety of the aforementioned patent application. Technical Field

[0004] The present invention relates to the field of biomedicine, and in particular to anti-Aβ drug screening targets and screening methods. Background Art

[0005] As the aging population worsens, the economic burden and social problems caused by Alzheimer's disease (AD) are becoming increasingly severe, becoming one of the greatest challenges facing humanity. The aggregation of amyloid β-protein (Aβ) is the most important hypothesis for the development of AD, and preventing Aβ aggregation has been proven to be an effective treatment for AD. Existing studies have demonstrated that soluble Aβ polymers are the primary source of toxicity, playing a significant role in the development and progression of AD by affecting cell membrane ion channels, generating oxidative stress, and activating glial cells to trigger inflammatory responses. Aβ exists in multiple forms in the human body, primarily Aβ42 and Aβ40. Aβ42 has become a research focus due to its greater toxicity and increased susceptibility to aggregation. However, other forms of Aβ, such as N-terminally truncated and modified N3pE, are also believed to be highly correlated with AD pathology. Therefore, targeting the clearance of Aβ in the brain is the most widely studied mechanism of action for drugs. Aβ monoclonal antibodies are currently the most extensively validated and only approved novel AD treatment in patients. Numerous trials have shown that Aβ monoclonal antibodies can clear Aβ plaques and, to a certain extent, mitigate cognitive and functional decline. Although some monoclonal antibodies have been discontinued due to poor blood-brain barrier passage, insignificant efficacy, and adverse events such as cerebral edema or microbleeds, more than a dozen Aβ monoclonal antibodies are currently in various stages of clinical trials.

[0006] Various drug development approaches targeting Aβ essentially use Aβ prepared under specific conditions as a drug target. Screening for drug molecules that specifically bind to various Aβ species requires a diverse array of Aβ polymers, representing diverse sequences, aggregation levels, and mixed ratios. Aβ preparation methods can generally be categorized into three types. The first involves extracting and purifying Aβ from in vivo sources. This method maximizes the availability of a wide range of Aβ aggregates that closely resemble the in vivo state. However, extraction from postmortem AD patient brains is clearly impractical and cannot be used for drug screening. Transgenic mouse-derived Aβ, due to genotype constraints, is difficult to reflect the complex Aβ types found in the human brain. Furthermore, the extraction and purification process is complex and laborious, and the quality of the extraction is easily affected by various conditions, making it difficult to apply to large-scale drug screening. The second method involves extracting and purifying Aβ expressed in transfected cells. This method is more efficient than the first method in obtaining sufficient Aβ, but requires rigorous sterile biological culture and manipulation, complex plasmid construction, and inefficiently obtains multiple Aβ sequences and mixed forms simultaneously. The third approach involves the use of artificially synthesized Aβ. Solid-phase peptide synthesis technology is well established, allowing for rapid, easy, and highly purified production of a wide variety of Aβ sequences. However, there remains a lack of standardized methods for using these Aβ sequences to prepare various Aβ multimers for drug screening. The inventors discovered that numerous methods for preparing Aβ aggregates have been reported in various publications, but most rely on simple buffer or culture medium conditions, lacking systematic comparison and analysis of the morphology and aggregation of Aβ multimers prepared under different conditions. Unlike other common protein targets, Aβ exhibits relatively well-defined binding sites and a defined three-dimensional structure. The polymorphism of Aβ significantly increases the difficulty of studying it as a target. Due to the presence of various Aβ species with varying lengths and terminal modifications in vivo, the aggregation properties of these Aβ species reported in prior art vary significantly. Different incubation conditions can also significantly affect Aβ aggregation behavior. Furthermore, the Aβ aggregation process itself exhibits dynamic aggregation with varying degrees, morphologies, and sizes, resulting in significant heterogeneity. Consequently, the success rate of anti-Aβ drug development is extremely low. Summary of the Invention

[0007] The object of the present invention is to provide a method for preparing Aβ aggregates.

[0008] Another object of the present invention is to provide a target for screening anti-Aβ drugs.

[0009] Another object of the present invention is to provide a method for screening anti-Aβ drugs.

[0010] In order to solve the above technical problems, the first aspect of the present invention provides a method for preparing Aβ aggregates, which comprises the following steps:

[0011] S1, dissolving Aβ lyophilized powder and drying to remove the solvent to obtain Aβ peptide film;

[0012] S2, mixing the Aβ peptide film and a buffer to obtain an Aβ solution with a concentration of 0.2-100 μM;

[0013] S3, incubating the Aβ solution to obtain Aβ aggregates; and

[0014] In step S1, the Aβ lyophilized powder is Aβ40, Aβ42, N3pE42, Aβ38 or a combination thereof;

[0015] In step S2, the buffer is selected from at least one of Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium elements, HBS buffer containing 8-12 mM HEPES and 140-160 mM NaCl, artificial cerebrospinal fluid (aCSF), DMEM, FBS, endosome simulation fluid, and lysosome simulation fluid;

[0016] In step S3, the incubation temperature is 30-43° C., the incubation speed is 0-2500 rpm, and the incubation time is not less than one day.

[0017] In some preferred embodiments, in step S1, the solvent is hexafluoroisopropanol.

[0018] In some preferred embodiments, in step S3, the incubation time is 1-7 days.

[0019] In some preferred embodiments, in step S3, the incubation is performed in a constant temperature homogenizer, and the rotation speed of the constant temperature homogenizer is 0-2500 rpm.

[0020] In some preferred embodiments, the concentration of the Aβ solution is 0.5-50 uM, more preferably 1-20 uM.

[0021] In some preferred embodiments, the buffer is Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium elements, HBS buffer containing 8-12 mM HEPES and 140-160 mM NaCl, or artificial cerebrospinal fluid (aCSF).

[0022] In some preferred embodiments, the endosome simulation solution is a mixture of a sodium citrate buffer solution with a pH of 5.5 and a concentration of 18-22 mM, a NaCl solution with a concentration of 75-85 mM, and a KCl solution with a concentration of 18-22 mM.

[0023] In some preferred embodiments, the lysosome simulation solution is a mixture of a sodium citrate buffer solution with a concentration of 18-22 mM and a pH of 4.5, a NaCl solution with a concentration of 45-55 mM, and a KCl solution with a concentration of 45-55 mM.

[0024] In some preferred embodiments, the lyophilized Aβ powder is a combination of Aβ40 and Aβ42, or a combination of Aβ40 and N3pE42, or a combination of Aβ40, Aβ42, and N3pE42. Preferably, the molar percentage of Aβ40 in the combination is not less than 50%. In some preferred embodiments, the molar ratio of Aβ40 to the other Aβ in the combination is (5-9):(1-5).

[0025] In some preferred embodiments, the Aβ freeze-dried powder is a combination of Aβ40 and Aβ42, wherein the molar ratio of Aβ40 to Aβ42 is 9:1, 8:2, 7:3, 6:4 or 5:5.

[0026] In some preferred embodiments, the Aβ lyophilized powder is a combination of Aβ40 and N3pE42, wherein the molar ratio of Aβ40 to N3pE42 is 9: 1. In some preferred embodiments, the Aβ lyophilized powder is a combination of Aβ40, Aβ42, and N3pE42, wherein the molar ratio of Aβ40, Aβ42, and N3pE42 is 5:4:1.

[0027] In some preferred embodiments, the incubation temperature is 36-41° C. and the incubation speed is 1500-2500 rpm.

[0028] The second aspect of the present invention provides the use of Aβ aggregates as a target for screening anti-Aβ drugs, wherein the Aβ aggregates are prepared by the method described in the first aspect of the present invention.

[0029] The third aspect of the present invention provides a target for screening anti-Aβ drugs, wherein the target is Aβ aggregates, and the Aβ aggregates are prepared by the method described in the first aspect of the present invention.

[0030] The fourth aspect of the present invention provides an anti-Aβ drug screening method, which comprises the steps of: screening anti-Aβ drugs using the anti-Aβ drug screening target described in the third aspect of the present invention.

[0031] In some preferred embodiments, the method includes the steps of screening for drugs that can disaggregate Aβ aggregates, or can bind to Aβ aggregates, or can inhibit the polymerization of Aβ aggregates, wherein the drug is selected from at least one of antibodies, polypeptides and small molecule compounds.

[0032] In some preferred embodiments, drugs that can disaggregate Aβ aggregates in brain-like micro-organs or inhibit the aggregation of Aβ into aggregates in brain-like micro-organs are screened.

[0033] In some preferred embodiments, the brain-like micro-organs are formed by inducing differentiation of hPSCs carrying an APP mutation of the Swedish type.

[0034] In some preferred embodiments, the Swedish-type mutation is K595N and / or M596L.

[0035] The fifth aspect of the present invention provides an anti-Aβ drug, which is obtained by screening using the method described in the fourth aspect of the present invention.

[0036] In some preferred embodiments, the compound is a compound of formula I,

[0037] In a sixth aspect, the present invention provides the use of a compound of formula I for non-therapeutic in vitro (i) inhibiting the formation of Aβ polymers; (ii) promoting the disaggregation of Aβ polymers; and / or (iii) binding to Aβ polymers;

[0038] In a seventh aspect, the present invention provides a method for screening anti-Aβ drugs, comprising the steps of contacting the drug to be screened with a brain-like micro-organ.

[0039] In some preferred embodiments, the method includes the step of screening drugs that can (i) inhibit the formation of Aβ polymers; (ii) promote the disaggregation of Aβ polymers; and / or (iii) bind to Aβ polymers in brain-like micro-organs.

[0040] In some preferred embodiments, the brain-like micro-organs are formed by inducing differentiation of hPSCs carrying an APP mutation of the Swedish type.

[0041] In some preferred embodiments, the Swedish-type mutation is K595N and / or M596L.

[0042] Compared with the prior art, the present invention has at least the following advantages:

[0043] (1) The screening method provided by the present invention uses different Aβ aggregates prepared by simulating the in vivo environment and conditions as targets to more accurately and effectively screen for anti-Aβ candidate drugs;

[0044] (2) The screening method provided by the present invention involves incubating the aggregation of Aβ targets under different microenvironmental conditions close to those in vivo, without the need for any labeling or modification, and the drug molecules screened also do not need any modification or labeling;

[0045] (3) The screening method provided by the present invention can more intuitively screen and obtain anti-Aβ drug candidates with good activity;

[0046] (4) The screening method provided by the present invention is not limited to a certain type of drug, but can be widely applied to different types of drugs such as antibodies, peptides, and small molecules. It can truly reflect the interaction between the target and these drug molecules, greatly reducing the probability of false positives, false negatives, or incorrect binding patterns.

[0047] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] One or more embodiments are exemplarily described by the figures in the corresponding drawings, and these exemplified descriptions do not constitute limitations on the embodiments.

[0049] FIG1 is a representative AFM image of a typical Aβ polymer according to an embodiment of the present invention (left: Aβ42 oligomer; right: Aβ42 plaque);

[0050] FIG2 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (50 μM, 37° C., 2000 rpm, HBS incubation for 24 h);

[0051] FIG3 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (10 μM, 37° C., 2000 rpm, HBS incubation for 24 h);

[0052] FIG4 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (2 uM, 37° C., 2000 rpm, HBS incubation for 24 h);

[0053] FIG5 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (50 μM, 37° C., 2000 rpm, incubated in HBS for 1 week);

[0054] FIG6 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (50 μM, 37° C., 2000 rpm, aCSF incubation for 24 h);

[0055] FIG7 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (10 uM, 37° C., 2000 rpm, aCSF incubation for 24 h);

[0056] FIG8 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (2 uM, 37° C., 2000 rpm, aCSF incubation for 24 h);

[0057] FIG9 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (50 uM, 37° C., 2000 rpm, aCSF incubation for 1 week);

[0058] FIG10 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (10 uM, 37° C., 2000 rpm, aCSF incubation for 1 week);

[0059] FIG11 is a representative AFM image according to an embodiment of the present invention (Aβ40 / 42=5 / 5uM, 37° C., 2000 rpm, aCSF incubation for 24 h);

[0060] FIG12 is a representative AFM image according to an embodiment of the present invention (Aβ40 / 42=5 / 5uM, 37° C., 2000 rpm, aCSF incubation for 1 week);

[0061] FIG13 is a representative example of monitoring Aβ aggregation dynamics by ThT according to an embodiment of the present invention, wherein the incubation conditions of Aβ aggregates are 25uM Aβ42+40uM ThT, DPBS, 37° C.;

[0062] FIG14 is an example of ThT binding to Aβ aggregates according to an embodiment of the present invention, wherein the Aβ aggregate incubation conditions are 5uM Aβ42 incubated in DPBS at 37°C for 78h, 40uM ThT)

[0063] FIG15 is an AFM screening of compounds that can inhibit Aβ polymerization according to an embodiment of the present invention, wherein the Aβ polymer incubation conditions are 10 mM HCl + 150 mM NaCl, 37° C., and incubation for 48 h;

[0064] FIG16 is an AFM screening of compounds that can inhibit Aβ polymerization according to an embodiment of the present invention, wherein the Aβ polymer incubation conditions are DPBS, 37° C., and incubation for 78 h;

[0065] Figure 17 shows the aggregation observed by AFM before and after the addition of compounds according to an embodiment of the present invention, wherein the left side shows 25uM N3pE42 aggregates (DPBS, 70h), and the right side shows 25uM N3pE42 aggregates (DPBS, 70h) + 40uM compound 1, and then incubated for 90h.

[0066] FIG18 is a representative AFM image of Aβ38 (10 μM, 37° C., 2000 rpm, aCSF incubation for 24 h) according to an embodiment of the present invention;

[0067] FIG19 is a representative AFM image of Aβ38 (2 μM, 41° C., 2000 rpm, aCSF incubation for 24 h) according to an embodiment of the present invention;

[0068] FIG20 is a representative AFM image of Aβ38 (2 μM, 41° C., 2000 rpm, incubated in Lysosome simulation solution for 24 h) according to an embodiment of the present invention;

[0069] FIG21 is a representative AFM image of Aβ42 (10 μM, 37° C., 0 rpm, incubated in HBS for 24 h) according to an embodiment of the present invention;

[0070] FIG22 is a representative AFM image of Aβ40 (50 μM, 37° C., 0 rpm, incubated in HBS for 24 h) according to an embodiment of the present invention;

[0071] FIG23 is a representative AFM image of Aβ40 (50 μM, 37° C., 0 rpm, incubated in HBS for 1 week) according to an embodiment of the present invention;

[0072] FIG. 24 is a graph showing the effect of the compounds in the examples of the present invention in inhibiting Aβ levels. DETAILED DESCRIPTION

[0073] The success rate of developing anti-Aβ drugs in the existing technology is low. The inventors unexpectedly discovered that the morphology, size, and aggregation state of the target Aβ aggregates used will affect the efficiency of the screening method and whether the screened drugs are effective. The incubation conditions used for Aβ aggregates used in the existing technology are different and differ greatly from the in vivo environment. The inventors believe that this factor is one of the reasons for the low success rate of anti-Aβ drug development. Furthermore, the inventors simulated different microenvironments in the human brain to prepare different Aβ aggregates, including DPBS, HBS, aCSF, etc. that simulate the neutral extracellular environment and buffer solutions that simulate the endosome / lysosome internal environment, and simulated common human body temperature, the temperature of superficial parts of the brain, the temperature of deep areas of the brain, etc. at different temperatures such as 37°C, 38.5°C, and 41°C. Different speed conditions such as 0-2000rpm were used to simulate various different conditions such as human rest, movement, and blood flow. Under the above-mentioned different conditions, Aβ aggregates of each single sequence were incubated for binding specificity screening, and aggregates of other Aβ sequences were mixed and incubated with Aβ40 as the main body to simulate complex Aβ aggregates in vivo for broad screening.

[0074] Preparation method of Aβ aggregates

[0075] The present invention relates to a method for preparing Aβ aggregates, comprising the steps of: S1, dissolving Aβ freeze-dried powder in hexafluoroisopropanol, and drying and removing the hexafluoroisopropanol to obtain an Aβ peptide film; S2, mixing the Aβ peptide film and a buffer to obtain an Aβ solution with a concentration of 0.2-100 μM; and S3, incubating the Aβ solution to obtain Aβ aggregates.

[0076] As Aβ lyophilized powder, one or more Aβ subtypes can be selected, such as Aβ40, Aβ42, Aβ38, N3pE42, or a combination thereof. The sequences of these Aβ subtypes are all known and commercially available. For example, it can be an Aβ40 multimer, an Aβ42 multimer, or a multimer formed by any two or all three of Aβ40, Aβ42, Aβ38, and N3pE42. When multiple Aβ subtypes are mixed and incubated, preferably, at least one subtype is Aβ40, and more preferably, Aβ40 is used as the main component. In some embodiments of the present invention, a mixture of Aβ40 and other Aβ is used for co-incubation to simulate different physiological and pathological states in the brain, for example, a mixture of Aβ40 and Aβ42, or a mixture of Aβ40 and N3pE42, or a mixture of Aβ40 and Aβ38, or a mixture of Aβ40, Aβ42, and N3pE42. When multiple Aβ isoforms are mixed and incubated, the molar percentage of Aβ40 is preferably not less than 50%. In some embodiments of the present invention, the molar ratio of Aβ40 to other Aβ is (5-9):(1-5), for example (molar ratios in parentheses): Aβ40 / 42 (9:1), Aβ40 / 42 (8:2), Aβ40 / 42 (7:3), Aβ40 / 42 (6:4), Aβ40 / 42 (5:5), Aβ40 / N3pE42 (9:1), Aβ40 / 42 / N3pE42 (5:4:1), Aβ40 / 42 / N3pE42 (5:4:1), etc.

[0077] As a buffer, it is optionally a buffer that simulates different microenvironments in the human brain, preferably at least one of Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium elements, HBS buffer containing 8-12mM HEPES and 140-160mM NaCl, artificial cerebrospinal fluid (aCSF), DMEM, FBS, endosome simulation fluid and lysosome simulation fluid. In some embodiments of the invention, Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium elements, artificial cerebrospinal fluid (aCSF) or HBS buffer containing 8-12mM HEPES and 140-160mM NaCl is used as a buffer.

[0078] The incubation temperature is 36-41°C, preferably 37°C, 38.5°C, or 41°C, to simulate normal human body temperature, superficial brain temperature, and deep brain temperature, respectively. The incubation speed is 0-2500 rpm, for example, 0 or 2000 rpm to simulate resting and moving states, respectively.

[0079] In the present invention, the term "Aβ aggregate" refers to a polymer formed by the aggregation of one or more β-amyloid protein monomers.

[0080] Uses of Aβ Aggregates

[0081] The present invention also relates to the use of Aβ aggregates prepared by the above-mentioned method as targets for screening anti-Aβ drugs. Due to the polymorphism of Aβ aggregates, Aβ aggregates prepared by different methods are essentially different, similar to different targets. Compared with Aβ aggregates obtained in classical literature, Aβ aggregates prepared by the present method can be more intuitive and rapid in screening for effective candidate drugs. AFM images show that the screened drugs have a disaggregating effect on Aβ aggregates, a property of candidate drugs that cannot be directly determined by other methods.

[0082] Anti-Aβ drug screening targets

[0083] The present invention also relates to a target for anti-Aβ drug screening, which is the Aβ aggregates prepared by the above-mentioned method. Representative AFM images show that the Aβ aggregates obtained by the present invention are significantly different from typical Aβ monomers, typical Aβ oligomers, typical Aβ fibers, and typical Aβ plaques, representing distinct targets. The terms "typical Aβ monomers," "typical Aβ oligomers," "typical Aβ fibers," and "typical Aβ plaques" refer to typical Aβ monomers, typical Aβ oligomers, typical Aβ fibers, and typical Aβ plaques prepared by the methods described in the comparative examples of the present invention (methods in classic literature).

[0084] Anti-Aβ drug screening method

[0085] The present invention also relates to a method for screening anti-Aβ drugs, comprising the steps of screening for anti-Aβ drugs using the aforementioned anti-Aβ drug screening target. Preferably, the method screens for drugs that can disaggregate Aβ aggregates, bind to Aβ aggregates, or inhibit the aggregation of Aβ aggregates. The types of the aforementioned drugs are not limited and may include antibodies, peptides, and small molecule compounds.

[0086] Anti-Aβ drugs

[0087] The present invention also relates to an anti-Aβ drug screened by the method of the present invention, as shown in the following formula I:

[0088] The present invention also confirms through SPR experiments and ThT competitive binding experiments that the compound of formula I can bind to Aβ, and the binding effect is equivalent to that of Flutemetamol and AZD4694.

[0089] The present invention also relates to the in vitro non-therapeutic use of the compound of formula I for (i) inhibiting the formation of Aβ polymers; (ii) promoting the disaggregation of Aβ polymers; and / or (iii) binding to Aβ polymers.

[0090] In a preferred embodiment of the present invention, the "anti-Aβ drug" refers to a drug that can disaggregate Aβ polymers or prevent the formation of Aβ polymers.

[0091] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0092] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of this application.

[0093] Unless otherwise indicated, the term "or" means and is used interchangeably with the term "and / or."

[0094] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0095] Example 1

[0096] In this example, Aβ polymers were prepared. The specific method is as follows:

[0097] (1) Preparation and storage of Aβ peptide film: HFIP (hexafluoroisopropanol) was added to the artificially synthesized Aβ freeze-dried powder Aβ40, Aβ42, and Aβ38 (purchased from Gill Biochemical) to prepare a 1 mM solution, and the solution was incubated at room temperature for 30 minutes. 10 μl of the solution was dispensed into 0.5 ml low-adsorption microcentrifuge tubes, and the tubes were placed in a fume hood overnight to allow HFIP to evaporate. The tubes were then transferred to a vacuum centrifuge (SpeedVac) and dried for 1 hour to remove residual HFIP and moisture to obtain the Aβ peptide film. The tubes were sealed with a desiccant and stored at -20°C for later use.

[0098] (2) Preparation of Aβ DMSO stock solution

[0099] The Aβ peptide film was taken out and equilibrated to room temperature, and anhydrous DMSO (dimethyl sulfoxide) was added to prepare a 5 mM stock solution. After vortexing for 30 seconds, it was sonicated for 10 minutes. The stock solution was used as the starting material for preparing various Aβ aggregates.

[0100] (3) Preparation of single-sequence Aβ aggregates under conditions close to those in vivo:

[0101] The 5 mM Aβ DMSO stock solution prepared in (2) was taken and HBS was added to prepare a 100 μM Aβ solution. The solution was then diluted to 50 μM with HBS buffer and transferred to 37°C and incubated at 2000 rpm. After incubation for 24 hours, the aggregation morphology was determined by AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 2 shows the AFM morphology of Aβ40 and Aβ42.

[0102] Example 2

[0103] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0104] The 5 mM Aβ DMSO stock solution prepared in (2) was taken and HBS was added to prepare a 100 μM Aβ solution. The solution was then diluted to 10 μM with HBS buffer and transferred to 37°C and incubated at 2000 rpm. After incubation for 24 hours, the aggregation morphology was determined by AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 3 shows the AFM morphology of Aβ40 and Aβ42.

[0105] Example 3

[0106] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0107] The 5 mM Aβ DMSO stock solution prepared in (2) was taken and HBS was added to prepare a 100 μM Aβ solution. The solution was then diluted to 2 μM with HBS buffer and transferred to 37°C and incubated at 2000 rpm. After incubation for 24 hours, the aggregation morphology was determined by AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 4 shows the AFM morphology of Aβ40 and Aβ42.

[0108] Example 4

[0109] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0110] The 5 mM Aβ DMSO stock solution prepared in (2) was taken and HBS was added to prepare a 100 μM Aβ solution. The solution was then diluted to 50 μM with HBS buffer and transferred to 37°C and incubated at 2000 rpm. After incubation for 1 week, the aggregation morphology was determined by AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 5 shows the AFM morphology of Aβ40 and Aβ42.

[0111] Example 5

[0112] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0113] The 5 mM Aβ DMSO stock solution prepared in (2) was added to aCSF to prepare a 100 μM Aβ solution, which was then diluted to 50 μM with aCSF buffer. The solution was transferred to 37°C and incubated at 2000 rpm for 24 hours before use. The aggregation morphology was determined by AFM, and Aβ aggregates with corresponding aggregation degrees and morphologies were obtained. Figure 6 shows the AFM morphology of Aβ40 and Aβ42.

[0114] Example 6

[0115] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0116] The 5 mM Aβ DMSO stock solution prepared in (2) was taken and added to aCSF to prepare a 100 μM Aβ solution. The solution was then diluted to 10 μM with aCSF buffer and incubated at 37°C and 2000 rpm for 24 hours before use. The aggregation morphology was determined by AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 7 shows the AFM morphology of Aβ40 and Aβ42.

[0117] Example 7

[0118] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0119] The 5 mM Aβ DMSO stock solution prepared in (2) was added to aCSF to prepare a 100 μM Aβ solution, which was then diluted to 2 μM with aCSF buffer. The solution was transferred to 37°C and incubated at 2000 rpm for 24 hours before use. The aggregation morphology was determined by AFM, and Aβ aggregates with corresponding aggregation degrees and morphologies were obtained. Figure 8 shows the AFM morphology of Aβ40 and Aβ42.

[0120] Example 8

[0121] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0122] The 5 mM Aβ DMSO stock solution prepared in (2) was added to aCSF to prepare a 100 μM Aβ solution, which was then diluted to 50 μM with aCSF buffer. The solution was transferred to 37°C and incubated at 2000 rpm for 1 week before use. The aggregation morphology was determined by AFM, and Aβ aggregates with corresponding aggregation degrees and morphologies were obtained. Figure 9 shows the AFM morphology of Aβ40 and Aβ42.

[0123] Example 9

[0124] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0125] The 5 mM Aβ DMSO stock solution prepared in (2) was added to aCSF to prepare a 100 μM Aβ solution, which was then diluted to 10 μM with aCSF buffer. The solution was transferred to 37°C and incubated at 2000 rpm for 1 week before use. The aggregation morphology was determined by AFM, and Aβ aggregates with corresponding aggregation degrees and morphologies were obtained. Figure 10 shows the AFM morphology of Aβ40 and Aβ42.

[0126] Example 10

[0127] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0128] Using Aβ40 as a background, Aβ42 was mixed with Aβ40 at a specific ratio and added to aCSF to create a 5µM:5µM solution. The solution was then transferred to 37°C and incubated at 2000 rpm for 24 hours before use. The aggregation morphology was determined by AFM, yielding Aβ aggregates of corresponding aggregation degree and morphology. Figure 11 shows the AFM morphology of Aβ40 and Aβ42.

[0129] Example 11

[0130] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0131] Using Aβ40 as a background, Aβ42 was mixed with Aβ40 at a specific ratio and added to aCSF to create a 5µM:5µM solution. The solution was then transferred to 37°C and incubated at 2000 rpm for one week before use. AFM was used to determine the aggregation morphology and obtain Aβ aggregates of the corresponding aggregation degree and morphology. Figure 12 shows the AFM morphology of Aβ40 and Aβ42.

[0132] Example 12

[0133] In this example, Aβ multimers were prepared by referring to the method for forming typical Aβ plaques in classic literature.

[0134] The 5 mM Aβ DMSO stock solution prepared in (2) was prepared by adding a mixture of 10 mM HCl + 150 mM NaCl to a 100 μM Aβ solution, which was then diluted to 5 μM with a mixture of 10 mM HCl + 150 mM NaCl. The solution was transferred to 37°C and incubated at 0 rpm for 48 hours before use. The aggregation morphology was determined by AFM, and Aβ aggregates with the corresponding aggregation degree and morphology were obtained. The left image of Figure 15 shows the AFM morphology of Aβ42.

[0135] Example 13

[0136] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0137] The 5 mM Aβ DMSO stock solution prepared in (2) was added to DPBS to prepare a 100 μM Aβ solution, which was then diluted to 25 μM with DPBS buffer and incubated at 37°C, 0 rpm. After 78 hours of incubation, the solution was used to determine its aggregation morphology using AFM, and Aβ aggregates with the corresponding aggregation degree and morphology were obtained. The left image in Figure 16 shows the AFM morphology of Aβ42.

[0138] Example 14

[0139] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0140] The 5 mM DMSO stock solution of Aβ38 prepared in (2) was taken and added to aCSF to prepare a 100 μM Aβ solution. The solution was then diluted to 10 μM with aCSF buffer and incubated at 37°C and 2000 rpm for 24 hours before use. The aggregation morphology was determined by AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 18 shows the AFM morphology of Aβ38.

[0141] Example 15

[0142] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0143] The 5 mM DMSO stock solution of Aβ38 prepared in (2) was added to aCSF to prepare a 100 μM Aβ solution, which was then diluted to 2 μM with aCSF buffer. The solution was transferred to 41°C and incubated at 2000 rpm for 24 hours before use. The aggregation morphology was determined by AFM, and Aβ aggregates with the corresponding aggregation degree and morphology were obtained. Figure 19 shows the AFM morphology of Aβ38.

[0144] Example 16

[0145] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0146] The 5 mM DMSO stock solution of Aβ38 prepared in (2) was added to a 100 μM Aβ solution prepared by lysosomes, and then diluted to 2 μM with lysosome buffer. The mixture was transferred to 41°C and incubated at 2000 rpm for 24 hours before use. The aggregation morphology was determined by AFM, and Aβ aggregates with the corresponding aggregation degree and morphology were obtained. Figure 20 shows the AFM morphology of Aβ38.

[0147] Example 17

[0148] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0149] The 5 mM DMSO stock solution of Aβ42 prepared in (2) was taken and HBS was added to prepare a 100 μM Aβ solution. The solution was then diluted to 10 μM with HBS buffer and incubated at 37°C and 0 rpm. After incubation for 24 hours, the aggregation morphology was determined using AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 21 shows the AFM morphology of Aβ42.

[0150] Compared with the samples incubated at 2000 rpm under the same conditions (Figure 3 right), the degree of polymerization of the samples incubated at 0 rpm was slightly lower.

[0151] Example 18

[0152] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0153] The 5 mM DMSO stock solution of Aβ40 prepared in (2) was taken and HBS was added to prepare a 100 μM Aβ solution. The solution was then diluted to 50 μM with HBS buffer and incubated at 37°C and 0 rpm. After incubation for 24 hours, the aggregation morphology was determined using AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 22 shows the AFM morphology of Aβ40.

[0154] Compared with the samples incubated at 2000 rpm under the same conditions (Figure 2 left), the degree of aggregation of the samples incubated at 0 rpm was significantly lower.

[0155] Example 19

[0156] The method for preparing Aβ multimers in this example is substantially the same as that in Example 1, except that the incubation conditions are partially adjusted.

[0157] The 5 mM DMSO stock solution of Aβ40 prepared in (2) was taken and HBS was added to prepare a 100 μM Aβ solution. The solution was then diluted to 50 μM with HBS buffer and incubated at 37°C and 0 rpm. After incubation for 1 week, the aggregation morphology was determined using AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 22 shows the AFM morphology of Aβ40.

[0158] Compared with the samples incubated at 2000 rpm under the same conditions (Figure 5 left), the degree of polymerization of the samples incubated at 0 rpm was significantly lower.

[0159] The following comparative examples provide methods for preparing Aβ in classical literature.

[0160] Comparative Example 1

[0161] Preparation of typical Aβ monomers

[0162] The Aβ stock solution prepared in Example 1 was added with ice water to a final concentration of 100 μM Aβ, and vortexed for 15 seconds before being used immediately as Aβ monomer.

[0163] Comparative Example 2

[0164] Preparation of typical Aβ oligomers

[0165] Cold F12 medium was added to the Aβ stock solution to dilute Aβ to 100 μM, vortexed for 15 seconds, and transferred to 4°C for incubation for 24 hours to obtain typical Aβ oligomers, as shown in the left figure of Figure 1.

[0166] Comparative Example 3

[0167] Preparation of typical Aβ fibers

[0168] 10 mM HCl was added to the Aβ stock solution to dilute Aβ to 100 uM, vortexed for 15 seconds, and transferred to 37°C for incubation for 24 hours to obtain typical Aβ fibers. Typical Aβ fibers formed slender and curved fibrous aggregates.

[0169] Comparative Example 4

[0170] Preparation of typical Aβ plaques

[0171] 10 mM HCl + 50-100 mM NaCl was added to the Aβ stock solution to make the Aβ concentration 100 uM, vortexed for 15 seconds, transferred to 37°C and incubated for 24 hours to obtain Aβ plaques, as shown in the right figure of Figure 1.

[0172] As shown in Figures 1, 2-12, 15, 16, and 18-23 above, the morphology of the Aβ aggregates prepared by incubation under the conditions of the extracellular matrix environment (physiological salt concentration, neutral) in the human brain in Examples 1-14 is different from the typical Aβ plaques in the comparative examples. As shown in Figures 2, 3, 5, 6, and 9, the aggregation morphology of Aβ40 tends to form fibrous aggregates, while Aβ42 tends to form small spherical aggregates and irregular clumps formed by the accumulation thereof. The morphology of the Aβ40 and Aβ42 aggregates of the present invention is significantly different from that of their respective comparative examples in the classic literature. The two also have different aggregation rates. References to Aβ40 at 100 μM only occasionally produce short fibers, while at physiological salt concentrations, 50 μM and even 10 μM Aβ40 can form fibrillar aggregates within 24 hours (Figures 2, 3, and 6). The morphology of these fibers is also different from that of Aβ40 and Aβ42 in the literature (The Journal of Biological Chemistry, 2002, 277(35), 32046-32053). Therefore, drug screening using these aggregates separately will lead to different screening results, similar to drug screening for different targets. The screening of Aβ aggregates prepared in a microenvironment close to the in vivo microenvironment used in the present invention has higher research value and application prospects.

[0173] Test Example 1

[0174] The formation of Aβ aggregates was detected by ThT binding assay.

[0175] ThT can bind to Aβ aggregates and thus exhibit fluorescence enhancement. The following experiments were performed to monitor the degree of Aβ aggregation and the binding of ThT to Aβ aggregates.

[0176] a) Monitoring the kinetics of Aβ aggregation: The starting Aβ solution (5-100 μM) prepared according to the method of Example 14 was immediately added to a 96-well plate. A certain amount of ThT (5-100 μM) was also added. The fluorescence value was measured regularly using a fluorescence spectrophotometer (BioTek, Cytation3) to monitor the aggregation process (Ex / Em: 440 nm / 490 nm). Three replicates were set up for each group. Statistical analysis was performed using GraphPad Prism. See Figure 13.

[0177] b) Fluorescence Changes in Aβ Aggregates after Addition of ThT: Aβ aggregates prepared in Example 14 were added to a 96-well plate at an Aβ concentration of 5-100 μM. Fluorescence was measured using a fluorescence spectrophotometer (BioTek, Cytation3). ThT (final concentration 5-100 μM) was added and incubated for 10-30 minutes. Fluorescence was then measured again (Ex / Em: 440 nm / 490 nm). Three replicates were performed for each group, and statistical analysis was performed using GraphPad Prism. As shown in Figure 14 below, fluorescence significantly increased after the addition of ThT to Aβ aggregates.

[0178] Test Example 2

[0179] The morphology of each prepared Aβ aggregate was monitored by atomic force microscopy (AFM).

[0180] a) Fill a syringe with ultrapure water and filter through a 0.02 mm filter. Discard the first 1–2 mL and use 0.02 mm filtered water for all subsequent steps.

[0181] b) Each Aβ sample was diluted to 10 uM or samples with a final concentration lower than 10 uM were directly used for preparing test samples on mica.

[0182] c) Use tape to remove the top one to four layers of mica to reveal a clean, flat mica surface.

[0183] d) Drop the sample onto mica and let it sit for 30 seconds. Rinse with 200-400 μl of water and blow dry with compressed air.

[0184] e) Place in a clean cabinet at room temperature (cover can be used to prevent dust) until analysis.

[0185] f) Scanning was performed using the intelligent tapping mode (peakforce) of a Bruker FastScan atomic force microscope and an SNL-A probe, with at least four areas scanned to ensure representative sampling.

[0186] g) NanoScope software was used for data processing and image presentation.

[0187] Example 14

[0188] In this example, the ability of compound 1 to bind to Aβ was determined by SPR.

[0189] The interaction between the compounds and Aβ was measured using a Biacore 8K surface plasmon resonance instrument. The system temperature was set at 25°C, the flow rate was set at 10 μL / min, channel 2 (FC-2) was designated as the coupled protein channel, and channel 1 (FC-1) was designated as the control channel to detect nonspecific binding reactions and to subtract background signals.

[0190] Pretreatment - Selection of coupling pH: Without activating the chip surface, Aβ samples (Gill Biochemical, Cat#52487) were dissolved in sodium acetate buffer at different pH values ​​(pH = 4.0, 4.5, 5.0, 5.5) to impart different amounts of positive charge. The protein samples were then flowed over the chip surface at a flow rate of 10 μL / min, and the appropriate pH conditions were determined by the binding curve.

[0191] Aβ coupling: CM5 chip was selected, and protein coupling was performed according to the amino coupling method. The coupling steps are as follows:

[0192] (1) Activation: Inject NHS / EDC mixture (0.1 M NHS and 0.4 M EDC, mixed in a 1:1 volume ratio before use and used immediately) at a flow rate of 10 μL / min to activate the carboxyl groups of dextran on the chip surface;

[0193] (2) Coupling: A 50 μg / mL Aβ solution was prepared in the buffer solution with the pH value determined in the pretreatment (preparation method refers to Alzheimer's Disease and Frontotemporal Dementia 2010, 610, 13-32) and injected at a flow rate of 10 μL / min until the immobilization level reached approximately 3000 RU;

[0194] (3) Blocking: Block with 1M ethanolamine hydrochloride to complete the protein coupling process.

[0195] Binding assay: The analyte to be tested was dissolved in DMSO to a 10 mM concentration and then diluted to the desired concentration series with PBS-P+ (Cytiva). The sample was injected at a flow rate of 30 μL / min for 5 minutes, with binding (kon) lasting 60 seconds and dissociation (koff) lasting 60 seconds. The results were analyzed after solvent correction, and the reference channel sensorgram was subtracted from the sample sensorgram.

[0196] In an SPR experiment, when the analytical compound solution flows onto the chip surface, the analyte binds to the protein conjugate on the chip surface, causing a change in the chip surface mass, thereby causing a change in the resonance signal. The interaction result is represented by a sensorgram, where the ordinate represents the binding level of the small molecule to the protein, which is continuously recorded as the response signal (RU), and the abscissa represents time. The dissociation equilibrium constant (KD) obtained by fitting is shown in Table 1 below for some relevant examples. The analyte concentrations are 3.91, 7.81, 15.63, 31.25, 62.50, and 125.0 μM, respectively. The kinetic parameters are obtained by assuming a 1:1 binding simulation. As can be seen from the table, compound 1, reference substance 1, and reference substance 2 are all able to bind to Aβ.

[0197] Table 1. Results of the ability of compounds to bind to Aβ

[0198] Example 15

[0199] In this example, the binding of the compound to Aβ was determined by ThT competition binding assay.

[0200] Aβ42 aggregates prepared in Example 14 (incubated in DPBS at 37°C for 60 h) were added to a 96-well plate. A 1 mM solution of the standard compound in DMSO and an equal volume of DMSO without the standard compound were added, respectively. The Aβ42 concentration was 5 μM and the standard compound concentration was 40 μM. Fluorescence values ​​(Ex / Em: 440 nm / 490 nm) were measured at 37°C using a fluorescence spectrophotometer (BioTek, Cytation3). ThT (final concentration 40 μM) was then added to each well and fluorescence values ​​(Ex / Em: 440 nm / 490 nm) were measured. Three replicates were performed for each group, and statistical analysis was performed using GraphPad Prism.

[0201] In the thioflavin T (ThT) competitive binding assay, compound 1 competed with thioflavin T (ThT) for binding to Aβ aggregates. The compound itself had no obvious fluorescence under the assay conditions. However, after the compound was incubated with Aβ aggregates and then ThT was added, its fluorescence was significantly lower than that of the control group without the compound, indicating that compound 1 can compete with ThT for binding to Aβ aggregates.

[0202] Table 2. Results of the competition between compounds and ThT for Aβ binding

[0203] AFM was used to observe the inhibitory effect of the added active compound on Aβ aggregation and / or the disaggregation effect on Aβ aggregates.

[0204] Inhibition of Aβ aggregation: After Aβ monomers were co-incubated with the compound for a certain period of time, the aggregation of the two groups was observed using AFM. Representative implementation results are shown in Figures 15 and 16.

[0205] Test Example 3

[0206] Typical Aβ plaques and the Aβ aggregates prepared in the present invention were used to screen active Compound 1.

[0207] Under typical Aβ plaque formation conditions (10 mM HCl + 150 mM NaCl, 37°C), even when the Aβ concentration was reduced from 100 uM to 5 uM, incubation with Compound 1 only slightly inhibited Aβ aggregation, as shown in Figure 15. This result indicates that Aβ aggregates prepared under solution conditions that typically form Aβ plaques are not effective targets for screening Compound 1.

[0208] However, when incubated under conditions closer to the in vivo environment provided in Example 13 of the present invention (DPBS, 37°C, 0 rpm), even at a higher concentration (25 uM), the same concentration of compound 1 can significantly inhibit the aggregation of Aβ. As shown in the right figure in Figure 16, co-incubation of compound 1 with Aβ can significantly inhibit the formation of Aβ aggregates, indicating that the Aβ aggregates of the present invention can be used as a target for screening to obtain active compound 1, and the results are more intuitive.

[0209] In addition, the inventors also found that after incubating the Aβ aggregates prepared in the examples with the compounds for a certain period of time and comparing the aggregation before and after the addition of the compounds using AFM, compound 1 can also significantly promote the disaggregation of Aβ aggregates. Representative implementation results are shown in Figure 17.

[0210] Example 18: Method for Screening Drugs Using Brain-like Micro-organs

[0211] In this embodiment, brain-like micro-organs were constructed with reference to the patent application with publication number WO 2023 / 143637 Al.

[0212] hPSCs carrying the APP swedish mutation were digested into single cells with Accutase and seeded at 9,000 cells / well in 96-well round-bottom ultra-low attachment plates containing EB Formation Medium (Stem Cell Technologies) to form embryoid bodies (EBs). On day 5, the EBs were transferred to 24-well ultra-low attachment plates containing Induction Medium (Stem Cell Technologies) for a further two days of induction culture. They were then embedded in Matrigel and expanded in Expansion Medium (Stem Cell Technologies) for another three days. On day 10, the organoids were transferred to Maturation Medium and cultured on a rocking platform for long-term maturation. Strict protocols were adhered to throughout the process to ensure the stability and reproducibility of organoid development. Brain organoids of similar size and growth status around day 60 were selected and evenly distributed across 24-well plates. Compounds were added at the desired concentrations. After seven days of treatment, supernatants were collected and assayed for Aβ levels by ELISA.

[0213] As shown in FIG24 , the exemplary compounds can significantly inhibit Aβ levels at concentrations of 1-100 μM, and the inhibitory effect gradually increases with increasing compound concentration, showing a significant dose effect.

[0214] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and that in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. A method for preparing Aβ aggregates, characterized in that: The method for preparing Aβ aggregates comprises the steps of: S1, dissolving Aβ lyophilized powder and drying to remove the solvent to obtain Aβ peptide film; S2, mixing the Aβ peptide film and a buffer to obtain an Aβ solution with a concentration of 0.2-100 μM; S3, incubating the Aβ solution to obtain Aβ aggregates; and In step S1, the Aβ lyophilized powder is Aβ40, Aβ42, N3pE42, Aβ38 or a combination thereof; In step S2, the buffer is selected from at least one of Dulbecco's phosphate buffered saline (PBS) without calcium and magnesium elements, HBS buffer containing 8-12 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and 140-160 mM NaCl, artificial cerebrospinal fluid, DMEM, FBS, endosome simulation fluid, and lysosome simulation fluid; In step S3, the incubation temperature is 30-43° C., the incubation time is not less than 1 day, and the incubation speed is 0-2500 rpm.

2. The method according to claim 1, characterized in that In step S3, the incubation time is 1-7 days.

3. The method according to claim 1, characterized in that In step S3, the incubation is performed in a constant temperature homogenizer, and the rotation speed of the constant temperature homogenizer is 0-2500 rpm.

4. The method according to claim 1, wherein The buffer solution is Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium elements, HBS buffer solution containing 8-12 mM HEPES and 140-160 mM NaCl, or artificial cerebrospinal fluid (aCSF).

5. An anti-Aβ drug screening target, characterized in that: The target is Aβ aggregate, and the Aβ aggregate is prepared by the method according to any one of claims 1 to 4.

6. Use of Aβ aggregates as a target for screening anti-Aβ drugs, characterized in that: The Aβ aggregates are prepared by the method according to any one of claims 1 to 4.

7. A method for screening anti-Aβ drugs, characterized in that: The method comprises the step of screening the anti-Aβ drug using the target according to claim 5 .

8. The method according to claim 7, characterized in that Screening a drug that can disaggregate Aβ aggregates or inhibit Aβ from forming aggregates, wherein the drug is selected from at least one of an antibody, a polypeptide and a small molecule compound.

9. The method according to claim 8, characterized in that Screen for drugs that can disaggregate Aβ aggregates in brain-like microorgans or inhibit the aggregation of Aβ in brain-like microorgans.

10. The method according to claim 9, characterized in that The brain-like micro-organs are formed by inducing differentiation of hPSCs carrying an APP mutation of the Swedish type.

11. The method according to claim 10, characterized in that The Swedish type mutations are K595N and / or M596L.

12. An anti-Aβ drug, characterized in that The anti-Aβ drug is obtained by the method according to any one of claims 7 to 10.

13. Use of a compound of formula I, characterized in that For non-therapeutic in vitro (i) inhibiting the formation of Aβ polymers; (ii) promoting the disaggregation of Aβ polymers; and / or (iii) binding to Aβ polymers; 14. A method for screening anti-Aβ drugs, characterized in that: The method comprises the steps of: contacting the drug to be screened with the brain-like micro-organ.

15. The method according to claim 14, characterized in that The method includes the steps of screening drugs that can (i) inhibit the formation of Aβ polymers; (ii) promote the disaggregation of Aβ polymers; and / or (iii) bind to Aβ polymers in brain-like micro-organs.

16. The method according to claim 15, characterized in that The brain-like micro-organs are formed by inducing differentiation of hPSCs carrying an APP mutation of the Swedish type.

17. The method according to claim 16, characterized in that The Swedish type mutations are K595N and / or M596L.

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