WO2022126633A1

WO2022126633A1 - Application of compound

Info

Publication number: WO2022126633A1
Application number: PCT/CN2020/137714
Authority: WO
Inventors: 张志刚; 屠洁
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2020-12-17
Filing date: 2020-12-18
Publication date: 2022-06-23
Also published as: CN112546051A

Abstract

An application of a compound, the compound being used to enhance in vitro the activity of γ-secretase. The compound is N-[4-[[(3-methoxypyrazinyl)amino]sulfonyl]phenyl]-3-(5-nitro-2-thienyl)-2-acrylamide, the structural formula of the compound is represented by formula (I), and the compound can significantly increase the activity of γ-secretase in an in vitro cell model, thereby reducing the difficulty of detecting and analyzing γ-secretase, and the present invention can provide good conditions for subsequent large-scale drug screening work that further use γ-secretase as a target.

Description

application of a compound

technical field

The invention belongs to the field of biotechnology, and relates to the application of a compound, in particular to the compound N-[4-[[(3-methoxypyrazinyl)amino]sulfonyl]phenyl]-3-(5-nitro - Use of 2-thienyl)-2-acrylamide.

Background technique

Alzheimer's disease (AD) is the most common cause of dementia, resulting in loss of memory, cognition, reasoning, judgment and orientation. AD is characterized by the presence of extracellular amyloid plaques, intracellular neurofibrillary tangles, in addition to the loss of synapses and neurons in the brain. The major component of amyloid plaques is β-amyloid (Aβ), a 4 kDa peptide.

The accumulation of Aβ is considered to be an early and critical step in the pathogenesis of Alzheimer's disease (AD). A[beta] causes a cascade of toxic and inflammatory events that ultimately lead to neuronal death and cognitive impairment. A[beta] peptides are derived from the proteolysis of amyloid precursor protein (APP). The APP protein is a transmembrane protein composed of a large extracellular domain and a short cytoplasmic tail, and the Aβ fragment includes part of the extracellular and transmembrane domains of APP.

APP can be processed by either of two routes, the non-amyloid source and the amyloid source pathway. Most APP is processed through the non-amyloid-derived pathway, whereby the protease α-secretase cleaves APP in the Aβ domain to release a large soluble N-terminal fragment (sAPPα) and a non-amyloid-derived C-terminal fragment ( C83), this fragment is further processed by γ-secretase to generate a 22-24 residue peptide (p3). In the amyloidogenic pathway, APP is cleaved by β-secretase (BACE1), which forms the shorter N-terminal domain (sAPPβ) and the amyloidogenic C-terminus (C99), and this C99 fragment subsequently Cleaved by γ-secretase. Gamma-secretase is a protease formed by complexes of the following proteins: Presenilin-1 (PS-1), Nicastrin, PEN-2 and APH-1. Proteolytic hydrolysis of APP intermediates by gamma-secretase yields A[beta] peptides of various lengths (A[beta]37, A[beta]38, A[beta]39, A[beta]40, A[beta]42). Among these peptides, A[beta]42 is the least soluble, most aggregated substance and the major constituent of toxic oligomers and amyloid plaques in AD brain. Therefore, inhibiting the activity of γ-secretase in vivo is considered to be an important way to treat Alzheimer's disease. The existing published reports are also exploring inhibitors of γ-secretase activity in vivo and related drugs.

Currently in the development process targeting γ-secretase, the most commonly used cell models include in vitro cell lines and primary cells. The activity of γ-secretase in in vitro cell lines is very low, and it is difficult to detect by conventional biochemical methods. It is necessary to enrich the γ-secretase by ultracentrifugation and other methods before detection, which greatly increases the economic and time cost. The activity of γ-secretase is high in primary cells (eg, primary neurons), however, the process of isolating primary cells is complicated, the cycle is long, the number of primary cells isolated is also limited, and the materials are difficult to obtain. The use of progeny cells in large-scale drug screening. How to enhance the activity of γ-secretase in in vitro cell models is of great significance for large-scale drug screening work targeting γ-secretase.

SUMMARY OF THE INVENTION

In view of the deficiencies in the prior art, the present invention provides an application of a compound to solve the problem that the activity of γ-secretase in an in vitro cell model is low, which is not conducive to the detection and analysis of γ-secretase.

In order to achieve the above object of the invention, the present invention provides the application of a compound, the compound is N-[4-[[(3-methoxypyrazinyl)amino]sulfonyl]phenyl]-3-(5 -Nitro-2-thienyl)-2-acrylamide, the structural formula of the compound is the following formula (I):

Among them, the compound is used to enhance the activity of γ-secretase in vitro.

Specifically, the compound is dissolved in an organic solvent to form a compound solution, and then the compound solution is added to the culture medium of in vitro cultured cells to enhance the activity of cellular γ-secretase.

Specifically, in the compound solution, the concentration of the compound is 0.5 mmol/L to 1 mmol/L.

Specifically, the organic solvent is dimethyl sulfoxide.

Specifically, the compound solution is added to the culture medium of the cells cultured in vitro, so that the concentration of the compound in the cell culture medium is 0.5 μmol/L˜2 μmol/L.

The application of a compound provided in the embodiments of the present invention is used to enhance the activity of γ-secretase in vitro. The compound (N-[4-[[(3-methoxypyrazinyl)amino]sulfonyl]phenyl]-3-(5-nitro-2-thienyl)-2-acrylamide) is capable of in vitro In the cell model, the activity of γ-secretase is significantly enhanced, thereby reducing the difficulty of detection and analysis of γ-secretase, and can provide good conditions for further large-scale drug screening work targeting γ-secretase.

Description of drawings

Fig. 1 is the normalized statistical chart of the activity detection results of γ-secretase in three cell membrane protein samples of Example 1;

Fig. 2 is the immunoblotting representation of the Flag of three cell protein samples of Example 2;

Figure 3 is a schematic representation of the immunoblotting of AICD of three cellular protein samples of Example 2;

FIG. 4 is a normalized statistical graph of the gray value of the AICD strip in FIG. 3 .

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the invention shown in the drawings and described with reference to the drawings are merely exemplary and the invention is not limited to these embodiments.

Here, it should also be noted that, in order to avoid obscuring the present invention due to unnecessary details, only the structures and/or processing steps closely related to the solution according to the present invention are shown in the drawings, and the related structures and/or processing steps are omitted. Other details not relevant to the invention.

The embodiments of the present invention provide the use of a compound for enhancing the activity of γ-secretase in vitro. Wherein, the Chinese name of the compound is N-[4-[[(3-methoxypyrazinyl)amino]sulfonyl]phenyl]-3-(5-nitro-2-thienyl)-2 -Acrylamide, the English name is necrosulfonamide, the CAS number is 1360614-48-7, the molecular formula is C ₁₈ H ₁₅ N ₅ O ₆ S ₂ , the structural formula of the compound is as follows (I):

In the process of studying the mechanism of programmed cell necroptosis (necroptosis), scientists discovered that necroptosis is regulated by the RIPK1-RIPK3-MLKL signaling pathway. Cells are treated with stimulatory factors, such as tumor necrosis factor TNFα, which can trigger programmed cell necrosis: RIPK1 kinase is activated, and then activated RIPK1 interacts with its downstream RIPK3 kinase. RIPK1 activates by phosphorylating RIPK3, while Activated RIPK3 is autophosphorylated so that more RIPK3 protein is activated by phosphorylation. Activated RIPK3 binds to its downstream MLKL protein and phosphorylates it. Phosphorylated MLKL undergoes oligomerization to form oligomers, and at the same time, MLKL oligomers are translocated in the cell from the cytoplasm to the cell membrane, and these oligomers form ion channels on the cell membrane. Pores, the massive formation of these pores causes the cell membrane to rupture, causing cell death. The study found that the compound necrosulfonamide can specifically bind to the human MLKL protein and inhibit the programmed cell necrosis mediated by it.

The present invention discovers a new function of the compound necrosulfonamide, that is, the compound necrosulfonamide can significantly enhance the activity of γ-secretase in vitro.

For the application of the compound of formula (I) (necrosulfonamide, hereinafter referred to as compound (I)), specifically, the compound (I) is dissolved in an organic solvent to form a compound solution, and then the compound solution is added to in vitro cultured cells in the culture medium to enhance the activity of cellular γ-secretase.

In a preferred solution, in the compound solution, the concentration of the compound (I) is formulated to be 0.5 mmol/L to 1 mmol/L. Wherein, the organic solvent is dimethyl sulfoxide.

In a preferred solution, the compound solution is added to the culture medium of in vitro cultured cells, so that the concentration (ie working concentration) of compound (1) in the cell culture medium is 0.5 μmol/L to 2 μmol/L.

Based on the solutions provided in the above examples, the compound (I) can significantly enhance the activity of γ-secretase in an in vitro cell model, thereby reducing the difficulty of detecting and analyzing γ-secretase, and can provide a basis for further use of γ-secretase in the future. It provides good conditions for large-scale drug screening of targets.

Example 1: Detection of γ-secretase activity by direct method

(1) Spread the human embryonic kidney cells HEK293 cells in a 10 cm culture dish, and culture the cells with 10 ml of culture medium for 24 hours to make the cells grow to about 60% full bottom.

(2), add a solution of necrosulfonamide (solvent is dimethyl sulfoxide) with a concentration of 1 mM into the HEK293 culture medium of step (1). The amount of the necrosulfonamide solution added was controlled so that the concentration of necrosulfonamide in the culture medium was 0.5 μM.

(3) Incubate the sample of step (2) overnight (about 16 hours), and then supplement the sample with 10 ml of fresh cell culture medium containing the same concentration of necrosulfonamide and incubate for another 3 hours.

(4) Discard the supernatant of the sample incubated in step (3), collect the cells with a cell scraper, and extract the cell membrane protein by ultracentrifugation (because the γ-secretase is located on the membrane), which is recorded as "membrane protein sample" NSA0.5 μM”, and then use the γ-secretase fluorescent substrate detection kit to detect the activity of γ-secretase in the membrane protein sample to obtain the activity test data of NSA0.5 μM of the membrane protein sample.

(5), referring to the process of the above steps (1)-(4), the amount of the necrosulfonamide solution added in the step (2) is controlled so that the concentration of necrosulfonamide in the culture solution is 1 μM, correspondingly, in the step (4) The extracted cell membrane protein is recorded as the membrane protein sample "NSA1 μM", and the activity test data of the membrane protein sample NSA1 μM is obtained after testing according to step (4).

(6) As a comparison, referring to the process of the above steps (1)-(4), in the step (2), the necrosulfonamide solution with a concentration of 0 was added to the culture solution, that is, only the solvent dimethyl sulfoxide was added. , Correspondingly, the cell membrane protein extracted in step (4) is marked as the membrane protein sample "DMSO", and the activity test data of the sample DMSO is obtained after testing according to step (4).

FIG. 1 is a normalized statistical chart of the detection results of the activity of γ-secretase in three cell membrane protein samples of the present embodiment, and the activity of DMSO in the blank control sample is denoted as 1. FIG. It can be seen from Figure 1 that the γ-secretase activity of the membrane protein sample NSA0.5 μM with the working concentration of necrosulfonamide of 0.5 μM has a greater increase than that of the control sample DMSO, while the working concentration of necrosulfonamide is 1 μM. The γ-secretase activity was significantly improved compared to the control sample DMSO.

Example 2: Detection of γ-secretase activity by indirect method

APP and Notch1 are the two most studied protein substrates among the many substrates of γ-secretase. Therefore, the activity of γ-secretase can be indirectly reflected by detecting the amount of the product generated after γ-secretase cleaves the substrate.

(1) Construct HEK293 stably transfected cell lines expressing the APPSwedish mutant and the Notch1 protein excised from the extracellular segment, in which a Flag tag is attached to the carbon terminus (C terminus) of the Notch1 protein excised from the extracellular segment. The cell lines were named APPswe-HEK293 and N1ΔE-Flag-HEK293, respectively. The two cell lines were plated in 6-well plates, and 3 ml of culture medium per well was cultured for 24 hours to allow the cells to grow to about 60% confluence.

(2), add the solution of necrosulfonamide (the solvent is dimethyl sulfoxide) with a concentration of 1 mM into the APPswe-HEK293 and N1ΔE-Flag-HEK293 culture solutions of step (1), respectively. Among them, the amount of necrosulfonamide solution added was controlled so that the concentration of necrosulfonamide in the culture medium was 1 μM.

(3), incubate the sample of step (2) overnight (about 16 hours), then supplement the sample with 3 ml of fresh cell culture medium containing the same concentration of necrosulfonamide and incubate for another 3 hours.

(4) Discard the supernatant of the samples incubated in step (3), put the 6-well plate on ice, add 200 μL of protein lysis solution (containing protease inhibitors) to each well, and collect with a cell scraper after ice bathing for 20 minutes. The protein sample, centrifuged to take the supernatant, which is the total protein extracted, marked as protein sample "NSA1μM"; after the protein concentration was determined, the product AICD produced by γ-secretase cleavage of APP (corresponding to cell line APPswe-HEK293) was detected by immunoblotting And the amount of product NICD produced by γ-secretase cleaving Notch1 (corresponding to cell line N1ΔE-Flag-HEK293) (because NICD and Flag tags are fused together, the amount of Flag represents the amount of NICD), to obtain the test data of protein sample NSA1μM .

(5) Referring to the process of the above steps (1)-(4), the amount of the necrosulfonamide solution added in the step (2) is controlled so that the concentration of necrosulfonamide in the culture solution is 2 μM, correspondingly, the step (4) extracts The total protein is recorded as the protein sample "NSA2μM", and the test data of the protein sample NSA2μM is obtained after testing according to step (4).

(6) As a comparison, referring to the process of the above steps (1)-(4), in the step (2), the necrosulfonamide solution with a concentration of 0 was added to the culture solution, that is, only the solvent dimethyl sulfoxide was added. , correspondingly, the total protein extracted in step (4) is marked as protein sample "DMSO", and the test data of protein sample DMSO is obtained after testing according to step (4).

Fig. 2 is a schematic diagram of immunoblotting of Flag (ie, NICD) of three cell protein samples in this example, and β-actin is used as an internal reference protein for immunoblotting. Figure 3 is a schematic representation of the immunoblotting of AICD of three cellular protein samples of this example. It can be known from Figure 2 and Figure 3 that, compared with the control sample DMSO, after the APPswe-HEK293 and N1ΔE-Flag-HEK293 cells were treated with the compound Necrosulfonamide in the protein sample NSA1 μM and the protein sample NSA2 μM, the γ-secretase cleavage product Flag (ie NICD ) and AICD were significantly increased, indicating that the activity of γ-secretase was significantly improved.

FIG. 4 is a normalized statistical graph of the gray value of the AICD band in FIG. 3 , in which the activity of the blank control sample DMSO is denoted as 1. It can be seen from Figure 4 that the protein level of AICD in the protein sample with a working concentration of 1 μM of necrosulfonamide of NSA1 μM and a protein sample with a working concentration of 2 μM of NSA2 μM compared to the control sample DMSO was significantly increased, indicating the activity of γ-secretase. with a significant improvement.

In summary, the present invention combines the compound (N-[4-[[(3-methoxypyrazinyl)amino]sulfonyl]phenyl]-3-(5-nitro-2-thienyl)- 2-acrylamide) is used to enhance the activity of γ-secretase in in vitro cell models, thereby reducing the difficulty of detection and analysis of γ-secretase, which can be used for subsequent large-scale drug screening work targeting γ-secretase Offers good conditions.

The above are only specific embodiments of the present application. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can also be made. It should be regarded as the protection scope of this application.

Claims

Application of a compound, the compound is N-[4-[[(3-methoxypyrazinyl)amino]sulfonyl]phenyl]-3-(5-nitro-2-thienyl)- 2-acrylamide, the structural formula of the compound is the following formula (I):

Among them, the compound is used to enhance the activity of γ-secretase in vitro.
The application according to claim 1, wherein the compound is dissolved in an organic solvent to form a compound solution, and then the compound solution is added to the culture medium of in vitro cultured cells to enhance the activity of cellular γ-secretase.
The application according to claim 2, wherein, in the compound solution, the concentration of the compound is 0.5 mmol/L to 2 mmol/L.
The application according to claim 2, wherein the organic solvent is dimethyl sulfoxide.
The application according to any one of claims 2-4, wherein the compound solution is added to the culture medium of in vitro cultured cells, so that the concentration of the compound in the cell culture medium is 0.5 μmol/L～1 μmol /L.