Screening method of deproteinizing enzyme inhibitor and inhibitor obtained through screening

Abstract

The invention relates to a screening method of a deproteinizing enzyme inhibitor and an inhibitor obtained through screening, and belongs to the technical field of biological medicine. The invention provides a method for efficiently screening a deproteinizing enzyme inhibitor, which comprises the following steps of: detecting fluorescence signal change after a compound to be detected and deproteinizing enzyme are incubated through a fluorescence polarization method by using an E. probe UFM1-Lys-TAMRA obtained through Lys-TAMRA fluorescence labeling as a substrate, calculating an inhibition ratio, and quickly screening a potential inhibitor. The compounds BAY 11-7082 and PR-619 screened by the method are used as deproteinization enzyme inhibition, wherein the BAY 11-7082 can be used for effectively reducing the level of intracellular Euclidinylation within the concentration range of 2.5-10 [mu] M. The invention solves the problem of lack of deproteinization enzyme inhibitors and high-throughput screening methods thereof, and provides a new tool and strategy for research of an EK modification system and development of drugs for treating related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for screening desmin inhibitors and the screened inhibitors. Background Art

[0002] Ufmylation is a newly discovered ubiquitin-like post-translational modification system with important biological functions. It dynamically regulates the modification and demodification of substrate proteins through a cascade reaction consisting of an E1 activating enzyme (UBA5), an E2 conjugating enzyme (UFC1), an E3 ligase (such as UFL1), and desufynylating enzymes (UFSPs). Unlike classic ubiquitination, ufmylation covalently binds to target proteins through the unique Ufm1 protein, playing a key role in endoplasmic reticulum (ER) stress response, DNA damage repair, DNA replication stress response, autophagy regulation, and cancer-related signaling pathways such as Hippo and NF-κB. As the "molecular switch" of ufmylation, the desufynylating enzymes UFSP1 and UFSP2 dynamically regulate the modification level by specifically hydrolyzing the isopeptide bond between Ufm1 and substrate proteins, making them important targets for intervention in aberrant ufmylation in pathological conditions. However, despite the increasingly prominent biological significance of UFSPs, there is still an extreme shortage of small molecule inhibitors developed for them, which seriously restricts the research on the mechanism of UFSP modification and the development of targeted drugs.

[0003] Current screening methods for desmin inhibitors primarily rely on virtual screening, which involves computer simulations to predict the binding ability of compounds to the active site of UFSPs. However, this approach has significant limitations: First, protein structural flexibility (such as the dynamic conformational changes of the active pocket) and solvent effects (such as the water network at the binding site) are difficult to accurately simulate, resulting in significant deviations between molecular docking results and actual binding activity. Second, virtual screening relies on high-quality three-dimensional protein structure databases, and the conformational dynamics of UFSPs may not be fully understood, further reducing the reliability of predictions. Furthermore, the low molecular diversity of reported virtual screening compound libraries and the incomplete understanding of the desmin mechanism have resulted in a relatively limited number of inhibitor types discovered through virtual screening. These limitations not only lead to inefficient inhibitor development but also hinder the translational application of desmin regulatory networks in disease treatment. Therefore, the development of a simple, sensitive, and large-scale desmin inhibitor detection platform that is suitable for large-scale screening is urgently needed to deepen basic research on desmin and promote the development of targeted therapies. Summary of the Invention

[0004] To address the above technical problems, the present invention provides a method for efficiently screening desucrose glycosylation inhibitors. The method uses the desucrose probe UFM1-Lys-TAMRA fluorescently labeled with Lys-TAMRA as a substrate, detects changes in the fluorescence signal after incubation of the test compound with desucrose glycosylation, calculates the inhibition rate, and rapidly screens potential inhibitors. Compounds BAY 11-7082 and PR-619 were screened by this method as desucrose glycosylation inhibitors. Compound BAY 11-7082 can be used to increase the level of desucrose modification in cells.

[0005] The first object of the present invention is to provide a use of the compound BAY 11-7082 as a desucrose inhibitor.

[0006] Furthermore, the structural formula of the compound BAY 11-7082 is

[0007]

[0008] Furthermore, the desutanease comprises UFSP1 and / or UFSP2.

[0009] In one embodiment of the present invention, the desucrose activating enzyme is UFSP1.

[0010] The second object of the present invention is to provide the use of the compound BAY 11-7082 in reducing the intracellular isotonic acid level.

[0011] Furthermore, the concentration of BAY 11-7082 is 2.5-10 μM.

[0012] The third object of the present invention is to provide the use of compound PR-619 as a desucrose catalytic enzyme inhibitor.

[0013] Furthermore, the structural formula of compound PR-619 is

[0014] Furthermore, the desutanease comprises UFSP1 and / or UFSP2.

[0015] In one embodiment of the present invention, the desucrose activating enzyme is UFSP1.

[0016] The fourth object of the present invention is to provide a desmin inhibitor comprising BAY 11-7082 and / or PR-619.

[0017] A fifth object of the present invention is to provide a drug for increasing the level of cytochrome modification, characterized in that the drug comprises BAY 11-7082 and / or PR-619.

[0018] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0019] A sixth object of the present invention is to provide a method for screening desucrose inhibitors, the screening method comprising the following steps:

[0020] Step S1, adding the compound to be screened and the desulphonating enzyme UFSP and / or UFSP2 to the fluorescence polarization reaction solution and incubating together;

[0021] Step S2, using the C-terminally Lys-TAMRA-labeled eugenol probe UFM1-Lys-TAMRA as a hydrolysis substrate for the deeugenol dehydrogenase UFSP1 and / or UFSP2, and measuring the mP value using a multifunctional microplate reader;

[0022] Step S3: calculating the inhibition rate of the drug to be screened, and screening for hydroxyurea inhibitors.

[0023] Furthermore, in step S1, the fluorescence polarization reaction solution includes PBS buffer, Tris buffer and HEPES buffer.

[0024] Furthermore, in step S1, the concentration of the compound to be screened is 1-100 μM.

[0025] Preferably, the concentration of the compound to be screened is 100 μM, accounting for 1 / 100 (v / v) of the total system.

[0026] Furthermore, in step S1, the final concentration of desucralose is 40-400 nM.

[0027] Preferably, the concentration of dextranase is 120 nM, accounting for 1 / 50 (v / v) of the total system.

[0028] Furthermore, in step S1, the compound to be screened and desucrose are incubated together at room temperature for 30 minutes.

[0029] Furthermore, in step S2, the concentration of the UFM1-Lys-TAMRA probe is 40-2000 nM.

[0030] Preferably, the concentration of the UFM1-Lys-TAMRA probe is 1 μM, accounting for 1 / 50 (v / v) of the total system.

[0031] Furthermore, in step S3, the calculation formula for the inhibition rate of the screened drug is as follows:

[0032]

[0033] Among them, mP 目标化合物 is the milli-deviation value of the drug to be screened; mP 阴性对照mP is the value of the milli-bias obtained when desaturase is added but no drug to be screened is added; 阳性对照 The values are the milli-deviation values obtained without adding desmin.

[0034] Furthermore, drugs with an inhibition rate of more than 50% are screened as candidate desminogen activator small molecule inhibitors.

[0035] Furthermore, the method further includes step S4, specifically verifying the inhibitory effect of the drug to be screened on desminogen activator.

[0036] Furthermore, the verification includes determination of cell cytotoxicity levels and / or inhibition curve determination.

[0037] Beneficial effects of the present invention:

[0038] This invention, through a screening method for desucrose catalytic enzyme inhibitors based on the ubiquitin probe UFM1-Lys-TAMRA, reveals new uses for the compounds BAY 11-7082 and PR-619 as desucrose catalytic enzyme inhibitors, expanding their application scenarios. Low concentrations of BAY 11-7082 can increase intracellular desucrose modification levels, offering advantages in both efficiency and potential safety. This screening method, based on fluorescence polarization technology, is simple to operate and highly sensitive, enabling high-throughput screening of specific desucrose catalytic enzyme inhibitors and accelerating drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0040] Figure 1 : is the concentration-polarization value curve of the probe UFM1-Lys-TAMRA in an embodiment of the present invention;

[0041] Figure 2 This is the reaction curve of the desulphonylase UFSP1 hydrolyzing UFM1-Lys-TAMRA in the embodiment of the present invention;

[0042] Figure 3 is the Z factor value of the fluorescence polarization high-throughput screening method in the embodiment of the present invention;

[0043] Figure 4 This is the result of high-throughput screening of UFSP1 inhibitors using fluorescence polarization in the examples of the present invention;

[0044] Figure 5 This is the result of high-throughput screening of UFSP2 inhibitors using fluorescence polarization in an embodiment of the present invention;

[0045] Figure 6The inhibition rate and IC of the compound BAY 11-7082 at the cellular level in the examples of the present invention are detected. 50 The measurement results of the values, where A is the Western blot result, B is the structural formula of BAY 11-7082, and C is the IC value of the compound for inhibiting UFSP1. 50 Value measurement results;

[0046] Figure 7 The detection of the inhibition rate of compound PR-619 at the cell level and IC 50 The values are measured, where A is the Western blot result, B is the structural formula of PR-619, and C is the IC value of the compound inhibiting UFSP1. 50 Value measurement results. DETAILED DESCRIPTION

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0048] The probe UFM1-Lys-TAMRA involved in the embodiment is disclosed in Chinese patent CN114507278A, a leucine probe UFM1-Lys-TAMRA and its synthesis method.

[0049] Example 1: Determination of the optimal reaction concentration of UFM1-Lys-TAMRA

[0050] 100 μM UFM1-Lys-TAMRA was diluted with fluorescence polarization reaction solution (30 mM HEPES, 150 mM NaCl, 0.05% Tween-20, 0.5 mg / mL BSA, pH = 7.5) to final concentrations of 5, 10, 20, 40, 80, 100, 200, 400, 800, 1000, 1250, 1500, 2000, and 2500 μmol / L, respectively, and added to a 96-well plate in sequence, 50 μL per well, with 3 replicates set up for each group, and the mP value was measured with a multifunctional microplate reader.

[0051] The experimental results showed that starting from 100 nmol / L, the mP values of different concentrations of UFM1-Lys-TAMRA in the experimental system remained basically consistent with minimal fluctuation, and basically remained between 125-135 ( Figure 1 In order to maintain good sensitivity and low background value of the experimental system, the optimal reaction concentration of UFM1-Lys-TAMRA was selected as 1 μM.

[0052] Example 2: Hydrolysis of UFM1-Lys-TAMRA Substrate by UFSP1

[0053] 250 μM UFSP1 was diluted to final concentrations of 5, 10, 20, 40, 80, 100, 120, 160, 200, 250, 300, and 400 nM, and added sequentially to a 96-well plate at 49 μL per well, with three replicates per group, and incubated at room temperature for 20 minutes. 100 μM UFM1-Lys-TAMRA (final concentration after addition to the system is 1 μM) was then added sequentially at 1 μL per well, and mP values were immediately measured using a multifunctional microplate reader.

[0054] The UFSP1 hydrolysis reaction curve shows that 120nM UFSP1 can fully hydrolyze UFM1-Lys-TAMRA substrate to Lys-TAMRA ( Figure 2 ).

[0055] Example 3: Z-factor values for fluorescence polarization high-throughput screening

[0056] The Z factor is a core parameter for evaluating the stability of a drug high-throughput screening model. Using a multifunctional microplate reader, the Z factor of this fluorescence polarization high-throughput screening model was calculated to be 0.74, which meets the basic requirement of a high-throughput screening model with a Z factor greater than 0.5 ( Figure 3 ).

[0057] Example 4: Operation steps of fluorescence polarization high-throughput screening method

[0058] 47.5 μL of fluorescence polarization working solution was added to a 96-well plate, followed by 0.5 μL of compound (final concentration 100 μM) and 1 μL of UFSP1 (final concentration 120 nM). After incubation at room temperature for 30 minutes, 1 μL of UFM1-Lys-TAMRA (final concentration 1 μM) was added. At the same time, a negative control group (wells with UFSP1 added) and a positive control group (wells without UFSP1 added) were set up, with 3 sets of replicates for each group. After adding the substrate, the mP value was immediately measured with a multifunctional microplate reader. The established fluorescence polarization high-throughput screening model was used to perform high-throughput screening of the deubiquitinase inhibitor compound library, and two compounds were successfully screened out that had good inhibitory effects on UFSP1 enzyme activity in vitro ( Figure 4 ).

[0059] 47.5 μL fluorescence polarization working solution was added to a 96-well plate, followed by 0.5 μL compound (final concentration 100 μM) and 1 μL UFSP2 (final concentration 120 nM). After incubation at room temperature for 30 minutes, 1 μL UFM1-Lys-TAMRA (final concentration 1 μM) was added. At the same time, a negative control group (wells with UFSP2 added) and a positive control group (wells without UFSP2 added) were set up, with 3 sets of replicates for each group. After adding the substrate, the mP value was immediately measured with a multifunctional microplate reader. The established fluorescence polarization high-throughput screening model was used to perform high-throughput screening of the deubiquitinase inhibitor compound library. Compounds BAY 11-7082 and PR-619 were also screened to have good inhibitory effects on UFSP2 enzyme activity in vitro ( Figure 5 ).

[0060] Example 5: Evaluation of inhibitory activity of lead compounds in vitro and in vivo

[0061] (1) Detection of intracellular cytosine kinase levels

[0062] Compounds at final concentrations of 2.5 μM, 5 μM, and 10 μM were added to 293T cells cultured in 12-well plates. The wells with DMSO were used as negative controls. After 12 hours of continuous culture, the cells were lysed, samples were prepared, and the overall cytotoxicity level of the cells was observed by Western blot.

[0063] (2)IC 50 Measurement of value

[0064] 10mM BAY 11-7082 was serially diluted (starting at 200μM, with a total of 11 dilutions) and added to a 96-well plate. 120nM UFSP1 was then added, with triplicate wells per group, and incubated at room temperature for 30 minutes. 30nM UFM1-Lys-TAMRA was then added to the 96-well plate, and mP values were immediately measured using a multi-function microplate reader.

[0065] Negative wells were set up with fluorescence polarization reaction solution (47.5 μL / well) + UFSP1 (6 μM, 1 μL / well) + DMSO (1 μL / well) + UFM1-Lys-TAMRA (100 μM, 0.5 μL / well). Positive wells were set up with fluorescence polarization reaction solution (48.5 μL / well) + DMSO (1 μL / well) + UFM1-Lys-TAMRA (100 μM, 0.5 μL / well). The inhibition curve of BAY 11-7082 was fitted and the IC was calculated. 50 value.

[0066] 10mM PR-619 was serially diluted (starting at 200μM, with a total of 11 dilutions) and added to a 96-well plate. 120nM UFSP1 was then added, with triplicate wells per group, and incubated at room temperature for 30 minutes. 30nM UFM1-Lys-TAMRA was then added to the 96-well plate, and mP values were immediately measured using a multi-microplate reader.

[0067] Negative wells were set up with fluorescence polarization reaction solution (47.5 μL / well) + UFSP1 (6 μM, 1 μL / well) + DMSO (1 μL / well) + UFM1-Lys-TAMRA (100 μM, 0.5 μL / well). Positive wells were set up with fluorescence polarization reaction solution (48.5 μL / well) + DMSO (1 μL / well) + UFM1-Lys-TAMRA (100 μM, 0.5 μL / well). The inhibition curve of PR-619 was fitted and the IC was calculated. 50 value.

[0068] Among them, the results of intracellular isotonic acid level detection of BAY 11-7082 are as follows Figure 6 As shown in A, IC 50 The results of the value determination are as follows Figure 6 As shown in Figure C. The results of the intracellular cytotoxicity test of PR-619 are shown in Figure Figure 7 As shown in A, IC 50 The results of the value determination are as follows Figure 7 As shown in C. It can be seen that BAY 11-7082 has the effect of inhibiting the activity of desminogen activator in cells, while the inhibitory effect of PR-619 in cells is not obvious. 50 value was 4.8 μM, while the IC 50 The value is 11.96μM, which is slightly higher than that of PR-619. It is speculated that this may be due to the relatively high toxicity of PR-619, which has a certain impact on cells.

[0069] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of compound BAY 11-7082 as a desmin inhibitor.

2. The use according to claim 1, characterized in that: The desucralose enzyme includes UFSP1 and / or UFSP2.

3. Use of compound BAY 11-7082 in reducing intracellular isotonic acid levels.

4. The use according to claim 3, characterized in that: The concentration of BAY 11-7082 was 2.5-10 μM.

5. Use of compound PR-619 as a desmin inhibitor.

6. The use according to claim 5, characterized in that: The desucralose enzyme includes UFSP1 and / or UFSP2.

7. A desmin inhibitor characterized by: The desmin inhibitors include BAY 11-7082 and / or PR-619.

8. A drug for increasing the level of cytochrome modification, characterized in that: The drugs include BAY 11-7082 and / or PR-619.

9. The drug according to claim 8, characterized in that: The drug further includes a pharmaceutically acceptable carrier.

10. A method for screening desmin inhibitors, characterized in that: The screening method comprises the following steps: Step S1, adding the compound to be screened and the desulphonating enzyme UFSP and / or UFSP2 to the fluorescence polarization reaction solution and incubating together; Step S2, using the C-terminally Lys-TAMRA-labeled eugenol probe UFM1-Lys-TAMRA as a hydrolysis substrate for the deeugenol dehydrogenase UFSP1 and / or UFSP2, and measuring the mP value using a multifunctional microplate reader; Step S3: calculating the inhibition rate of the drug to be screened, and screening for hydroxyurea inhibitors.

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